[ { "id": "authors:7sxh5-9ty59", "collection": "authors", "collection_id": "7sxh5-9ty59", "cite_using_url": "https://authors.library.caltech.edu/records/7sxh5-9ty59", "type": "article", "title": "N-degron pathways", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "An N-degron is a degradation signal whose main determinant is a \"destabilizing\" N-terminal residue of a protein. Specific N-degrons, discovered in 1986, were the first identified degradation signals in short-lived intracellular proteins. These N-degrons are recognized by a ubiquitin-dependent proteolytic system called the Arg/N-degron pathway. Although bacteria lack the ubiquitin system, they also have N-degron pathways. Studies after 1986 have shown that all 20 amino acids of the genetic code can act, in specific sequence contexts, as destabilizing N-terminal residues. Eukaryotic proteins are targeted for the conditional or constitutive degradation by at least five N-degron systems that differ both functionally and mechanistically: the Arg/N-degron pathway, the Ac/N-degron pathway, the Pro/N-degron pathway, the fMet/N-degron pathway, and the newly named, in this perspective, GASTC/N-degron pathway (GASTC = Gly, Ala, Ser, Thr, Cys). I discuss these systems and the expanded terminology that now encompasses the entire gamut of known N-degron pathways.", "doi": "10.1073/pnas.2408697121", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences", "publication_date": "2024-09-24", "series_number": "39", "volume": "121", "issue": "39", "pages": "e2408697121" }, { "id": "authors:9tc7z-e4h36", "collection": "authors", "collection_id": "9tc7z-e4h36", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230530-441768000.69", "type": "article", "title": "Deletions of DNA in cancer and their possible uses for therapy", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Lewis", "given_name": "Kim", "clpid": "Lewis-Kim" }, { "family_name": "Chen", "given_name": "Shun\u2010Jia", "orcid": "0000-0002-5489-4930", "clpid": "Chen-Shun-Jia" } ], "abstract": "Despite advances in treatments over the last decades, a uniformly reliable and free of side effects therapy of human cancers remains to be achieved. During chromosome replication, a premature halt of two converging DNA replication forks would cause incomplete replication and a cytotoxic chromosome nondisjunction during mitosis. In contrast to normal cells, most cancer cells bear numerous DNA deletions. A homozygous deletion permanently marks a cell and its descendants. Here, we propose an approach to cancer therapy in which a pair of sequence-specific roadblocks is placed solely at two cancer-confined deletion sites that are located ahead of two converging replication forks. We describe this method, termed \"replication blocks specific for deletions\" (RBSD), and another deletions-based approach as well. RBSD can be expanded by placing pairs of replication roadblocks on several different chromosomes. The resulting simultaneous nondisjunctions of these chromosomes in cancer cells would further increase the cancer-specific toxicity of RBSD.", "doi": "10.1002/bies.202300051", "pmcid": "PMC11102808", "issn": "0265-9247", "publisher": "Wiley-Blackwell", "publication": "Bioessays", "publication_date": "2023-07", "series_number": "7", "volume": "45", "issue": "7", "pages": "e2300051" }, { "id": "authors:3tx3g-5jr71", "collection": "authors", "collection_id": "3tx3g-5jr71", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230518-332288000.5", "type": "article", "title": "Dieter Wolf (1941\u20132023): a life dedicated to understanding protein quality control and the ubiquitin\u2010proteasome system", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Finley", "given_name": "Daniel", "orcid": "0000-0003-2076-5863", "clpid": "Finley-Daniel" }, { "family_name": "Goldberg", "given_name": "Alfred L.", "orcid": "0000-0002-8319-5856", "clpid": "Goldberg-Alfred-L" }, { "family_name": "Rapoport", "given_name": "Tom A.", "orcid": "0000-0001-9911-4216", "clpid": "Rapoport-Tom-A" }, { "family_name": "Pfirrmann", "given_name": "Thorsten", "orcid": "0000-0002-9474-9535", "clpid": "Pfirrmann-Thorsten" } ], "abstract": "Professor Dieter H. Wolf, a scientist of vision and depth, a role model for his students and colleagues, and a long-standing member of the Editorial Advisory Board of The EMBO Journal, sadly passed away on February 16th, 2023, in Stuttgart-Vaihingen, Germany.", "doi": "10.15252/embj.2023114222", "issn": "0261-4189", "publisher": "European Molecular Biology Organization", "publication": "EMBO Journal", "publication_date": "2023-06-01", "series_number": "11", "volume": "42", "issue": "11", "pages": "Art. No. e114222" }, { "id": "authors:76g6j-4e449", "collection": "authors", "collection_id": "76g6j-4e449", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220726-998191000", "type": "article", "title": "Crystal structure of the Ate1 arginyl-tRNA-protein transferase and arginylation of N-degron substrates", "author": [ { "family_name": "Kim", "given_name": "Bong Heon", "clpid": "Kim-Bong-Heon" }, { "family_name": "Kim", "given_name": "Min Kyung", "clpid": "Kim-Min-Kyung" }, { "family_name": "Oh", "given_name": "Sun Joo", "clpid": "Oh-Sun-Joo" }, { "family_name": "Nguyen", "given_name": "Kha The", "orcid": "0000-0001-9191-8754", "clpid": "Nguyen-Kha-The" }, { "family_name": "Kim", "given_name": "Jun Hoe", "orcid": "0000-0001-5866-9952", "clpid": "Kim-Jun-Hoe" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Hwang", "given_name": "Cheol-Sang", "orcid": "0000-0002-0105-5957", "clpid": "Hwang-Cheol-Sang" }, { "family_name": "Song", "given_name": "Hyun Kyu", "orcid": "0000-0001-5684-4059", "clpid": "Song-Hyun-Kyu" } ], "abstract": "N-degron pathways are proteolytic systems that target proteins bearing N-terminal (Nt) degradation signals (degrons) called N-degrons. Nt-Arg of a protein is among Nt-residues that can be recognized as destabilizing ones by the Arg/N-degron pathway. A proteolytic cleavage of a protein can generate Arg at the N terminus of a resulting C-terminal (Ct) fragment either directly or after Nt-arginylation of that Ct-fragment by the Ate1 arginyl-tRNA-protein transferase (R-transferase), which uses Arg-tRNA\u1d2c\u02b3\u1d4d as a cosubstrate. Ate1 can Nt-arginylate Nt-Asp, Nt-Glu, and oxidized Nt-Cys* (Cys-sulfinate or Cys-sulfonate) of proteins or short peptides. Ate1 genes of fungi, animals, and plants have been cloned decades ago, but a three-dimensional structure of Ate1 remained unknown. A detailed mechanism of arginylation is unknown as well. We describe here the crystal structure of the Ate1 R-transferase from the budding yeast Kluyveromyces lactis. The 58-kDa R-transferase comprises two domains that recognize, together, an acidic Nt-residue of an acceptor substrate, the Arg residue of Arg-tRNA\u1d2c\u02b3\u1d4d, and a 3\u2032-proximal segment of the tRNA\u1d2c\u02b3\u1d4d moiety. The enzyme's active site is located, at least in part, between the two domains. In vitro and in vivo arginylation assays with site-directed Ate1 mutants that were suggested by structural results yielded inferences about specific binding sites of Ate1. We also analyzed the inhibition of Nt-arginylation activity of Ate1 by hemin (Fe\u00b3\u207a-heme), and found that hemin induced the previously undescribed disulfide-mediated oligomerization of Ate1. Together, these results advance the understanding of R-transferase and the Arg/N-degron pathway.", "doi": "10.1073/pnas.2209597119", "pmcid": "PMC9351520", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2022-08-02", "series_number": "31", "volume": "119", "issue": "31", "pages": "Art. No. e2209597119" }, { "id": "authors:rxc8w-1pe53", "collection": "authors", "collection_id": "rxc8w-1pe53", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20211019-154957670", "type": "article", "title": "Aminopeptidases trim Xaa-Pro proteins, initiating their degradation by the Pro/N-degron pathway", "author": [ { "family_name": "Chen", "given_name": "Shun-Jia", "orcid": "0000-0002-5489-4930", "clpid": "Chen-Shun-Jia" }, { "family_name": "Kim", "given_name": "Leehyeon", "orcid": "0000-0002-1266-7828", "clpid": "Kim-Leehyeon" }, { "family_name": "Song", "given_name": "Hyun Kyu", "orcid": "0000-0001-5684-4059", "clpid": "Song-Hyun-Kyu" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "N-degron pathways are proteolytic systems that recognize proteins bearing N-terminal (Nt) degradation signals (degrons) called N-degrons. Our previous work identified Gid4 as a recognition component (N-recognin) of the Saccharomyces cerevisiae proteolytic system termed the proline (Pro)/N-degron pathway. Gid4 is a subunit of the oligomeric glucose-induced degradation (GID) ubiquitin ligase. Gid4 targets proteins through the binding to their Nt-Pro residue. Gid4 is also required for degradation of Nt-Xaa-Pro (Xaa is any amino acid residue) proteins such as Nt-[Ala-Pro]-Aro10 and Nt-[Ser-Pro]-Pck1, with Pro at position 2. Here, we show that specific aminopeptidases function as components of the Pro/N-degron pathway by removing Nt-Ala or Nt-Ser and yielding Nt-Pro, which can be recognized by Gid4-GID. Nt-Ala is removed by the previously uncharacterized aminopeptidase Fra1. The enzymatic activity of Fra1 is shown to be essential for the GID-dependent degradation of Nt-[Ala-Pro]-Aro10. Fra1 can also trim Nt-[Ala-Pro-Pro-Pro] (stopping immediately before the last Pro) and thereby can target for degradation a protein bearing this Nt sequence. Nt-Ser is removed largely by the mitochondrial/cytosolic/nuclear aminopeptidase Icp55. These advances are relevant to eukaryotes from fungi to animals and plants, as Fra1, Icp55, and the GID ubiquitin ligase are conserved in evolution. In addition to discovering the mechanism of targeting of Xaa-Pro proteins, these insights have also expanded the diversity of substrates of the Pro/N-degron pathway.", "doi": "10.1073/pnas.2115430118", "pmcid": "PMC8639330", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2021-10-26", "series_number": "43", "volume": "118", "issue": "43", "pages": "Art. No. e2115430118" }, { "id": "authors:0xjfa-py570", "collection": "authors", "collection_id": "0xjfa-py570", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201124-103211964", "type": "article", "title": "The Arg/N-degron pathway targets transcription factors and regulates specific genes", "author": [ { "family_name": "Vu", "given_name": "Tri T. M.", "orcid": "0000-0002-1098-5646", "clpid": "Vu-Tri-T-M" }, { "family_name": "Mitchell", "given_name": "Dylan C.", "clpid": "Mitchell-Dylan-C" }, { "family_name": "Gygi", "given_name": "Steven P.", "orcid": "0000-0001-7626-0034", "clpid": "Gygi-Steven-P" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The Arg/N-degron pathway targets proteins for degradation by recognizing their N-terminal or internal degrons. Our previous work produced double-knockout (2-KO) HEK293T human cell lines that lacked the functionally overlapping UBR1 and UBR2 E3 ubiquitin ligases of the Arg/N-degron pathway. Here, we studied these cells in conjunction with RNA-sequencing, mass spectrometry (MS), and split-ubiquitin binding assays. 1) Some mRNAs, such as those encoding lactate transporter MCT2 and \u03b2-adrenergic receptor ADRB2, are strongly (\u223c20-fold) up-regulated in 2-KO cells, whereas other mRNAs, including those encoding MAGEA6 (a regulator of ubiquitin ligases) and LCP1 (an actin-binding protein), are completely repressed in 2-KO cells, in contrast to wild-type cells. 2) Glucocorticoid receptor (GR), an immunity-modulating transcription factor (TF), is up-regulated in 2-KO cells and also physically binds to UBR1, strongly suggesting that GR is a physiological substrate of the Arg/N-degron pathway. 3) PREP1, another TF, was also found to bind to UBR1. 4) MS-based analyses identified \u223c160 proteins whose levels were increased or decreased by more than 2-fold in 2-KO cells. For example, the homeodomain TF DACH1 and the neurofilament subunits NF-L (NFEL) and NF-M (NFEM) were expressed in wild-type cells but were virtually absent in 2-KO cells. 5) The disappearance of some proteins in 2-KO cells took place despite up-regulation of their mRNAs, strongly suggesting that the Arg/N-degron pathway can also modulate translation of specific mRNAs. In sum, this multifunctional proteolytic system has emerged as a regulator of mammalian gene expression, in part through conditional targeting of TFs that include ATF3, GR, and PREP1.", "doi": "10.1073/pnas.2020124117", "pmcid": "PMC7733807", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2020-12-08", "series_number": "49", "volume": "117", "issue": "49", "pages": "31094-31104" }, { "id": "authors:z6ts0-15367", "collection": "authors", "collection_id": "z6ts0-15367", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200721-100007126", "type": "article", "title": "The ATF3 Transcription Factor Is a Short-Lived Substrate of the Arg/N-Degron Pathway", "author": [ { "family_name": "Vu", "given_name": "Tri T. M.", "orcid": "0000-0002-1098-5646", "clpid": "Vu-Tri-T-M" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The Arg/N-degron pathway targets proteins for degradation by recognizing their specific N-terminal residues or, alternatively, their non-N-terminal degrons. In mammals, this pathway is mediated by the UBR1, UBR2, UBR4, and UBR5 E3 ubiquitin ligases, and by the p62 regulator of autophagy. UBR1 and UBR2 are sequelogous, functionally overlapping, and dominate the targeting of Arg/N-degron substrates in examined cell lines. We constructed, here, mouse strains in which the double mutant [UBR1^(\u2013/\u2013)UBR2^(\u2013/\u2013)] genotype can be induced conditionally, in adult mice. We also constructed human [UBR1^(\u2013/\u2013)UBR2^(\u2013/\u2013)] HEK293T cell lines that unconditionally lack UBR1/UBR2. ATF3 is a basic leucine zipper transcription factor that regulates hundreds of genes and can act as either a repressor or an activator of transcription. Using the above double-mutant mice and human cells, we found that the levels of endogenous, untagged ATF3 were significantly higher in both of these [UBR1^(\u2013/\u2013)UBR2^(\u2013/\u2013)] settings than in wild-type cells. We also show, through chase-degradation assays with [UBR1^(\u2013/\u2013)UBR2^(\u2013/\u2013)] and wild-type human cells, that the Arg/N-degron pathway mediates a large fraction of ATF3 degradation. Furthermore, we used split-ubiquitin and another protein interaction assay to detect the binding of ATF3 to both UBR1 and UBR2, in agreement with the UBR1/UBR2-mediated degradation of endogenous ATF3. Full-length 24 kDa ATF3 binds to \u223c100 kDa fragments of 200 kDa UBR1 and UBR2 but does not bind (in the setting of interaction assays) to full-length UBR1/UBR2. These and other binding patterns, whose mechanics remain to be understood, may signify a conditional (regulated) degradation of ATF3 by the Arg/N-degron pathway.", "doi": "10.1021/acs.biochem.0c00514", "pmcid": "PMC7669821", "issn": "0006-2960", "publisher": "American Chemical Society", "publication": "Biochemistry", "publication_date": "2020-08-04", "series_number": "30", "volume": "59", "issue": "30", "pages": "2796-2812" }, { "id": "authors:4ba5k-cdy56", "collection": "authors", "collection_id": "4ba5k-cdy56", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200609-091521870", "type": "article", "title": "Recognition of nonproline N-terminal residues by the Pro/N-degron pathway", "author": [ { "family_name": "Dong", "given_name": "Cheng", "orcid": "0000-0002-2891-8759", "clpid": "Dong-Cheng" }, { "family_name": "Chen", "given_name": "Shun-Jia", "orcid": "0000-0002-5489-4930", "clpid": "Chen-Shun-Jia" }, { "family_name": "Melnykov", "given_name": "Artem", "orcid": "0000-0002-4733-4249", "clpid": "Melnykov-A-V" }, { "family_name": "Weirich", "given_name": "Sara", "clpid": "Weirich-S" }, { "family_name": "Sun", "given_name": "Kelly", "clpid": "Sun-Kelly" }, { "family_name": "Jeltsch", "given_name": "Albert", "clpid": "Jeltsch-A" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Min", "given_name": "Jinrong", "clpid": "Min-Jinrong" } ], "abstract": "Eukaryotic N-degron pathways are proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, and to target these proteins for degradation by the 26S proteasome or autophagy. GID4, a subunit of the GID ubiquitin ligase, is the main recognition component of the proline (Pro)/N-degron pathway. GID4 targets proteins through their Nt-Pro residue or a Pro at position 2, in the presence of specific downstream sequence motifs. Here we show that human GID4 can also recognize hydrophobic Nt-residues other than Pro. One example is the sequence Nt-IGLW, bearing Nt-Ile. Nt-IGLW binds to wild-type human GID4 with a K_d of 16 \u03bcM, whereas the otherwise identical Nt-Pro\u2013bearing sequence PGLW binds to GID4 more tightly, with a K_d of 1.9 \u03bcM. Despite this difference in affinities of GID4 for Nt-IGLW vs. Nt-PGLW, we found that the GID4-mediated Pro/N-degron pathway of the yeast Saccharomyces cerevisiae can target an Nt-IGLW\u2013bearing protein for rapid degradation. We solved crystal structures of human GID4 bound to a peptide bearing Nt-Ile or Nt-Val. We also altered specific residues of human GID4 and measured the affinities of resulting mutant GID4s for Nt-IGLW and Nt-PGLW, thereby determining relative contributions of specific GID4 residues to the GID4-mediated recognition of Nt-Pro vs. Nt-residues other than Pro. These and related results advance the understanding of targeting by the Pro/N-degron pathway and greatly expand the substrate recognition range of the GID ubiquitin ligase in both human and yeast cells.", "doi": "10.1073/pnas.2007085117", "pmcid": "PMC7322002", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2020-06-23", "series_number": "25", "volume": "117", "issue": "25", "pages": "14158-14167" }, { "id": "authors:z7h4z-0q312", "collection": "authors", "collection_id": "z7h4z-0q312", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200505-133829988", "type": "article", "title": "Five enzymes of the Arg/N-degron pathway form a targeting complex: The concept of superchanneling", "author": [ { "family_name": "Oh", "given_name": "Jang-Hyun", "orcid": "0000-0002-2053-7468", "clpid": "Oh-Jang-Hyun" }, { "family_name": "Hyun", "given_name": "Ju-Yeon", "orcid": "0000-0002-5526-9651", "clpid": "Hyun-Ju-Yeon" }, { "family_name": "Chen", "given_name": "Shun-Jia", "orcid": "0000-0002-5489-4930", "clpid": "Chen-Shun-Jia" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The Arg/N-degron pathway targets proteins for degradation by recognizing their N-terminal (Nt) residues. If a substrate bears, for example, Nt-Asn, its targeting involves deamidation of Nt-Asn, arginylation of resulting Nt-Asp, binding of resulting (conjugated) Nt-Arg to the UBR1-RAD6 E3-E2 ubiquitin ligase, ligase-mediated synthesis of a substrate-linked polyubiquitin chain, its capture by the proteasome, and substrate's degradation. We discovered that the human Nt-Asn\u2013specific Nt-amidase NTAN1, Nt-Gln\u2013specific Nt-amidase NTAQ1, arginyltransferase ATE1, and the ubiquitin ligase UBR1-UBE2A/B (or UBR2-UBE2A/B) form a complex in which NTAN1 Nt-amidase binds to NTAQ1, ATE1, and UBR1/UBR2. In addition, NTAQ1 Nt-amidase and ATE1 arginyltransferase also bind to UBR1/UBR2. In the yeast Saccharomyces cerevisiae, the Nt-amidase, arginyltransferase, and the double-E3 ubiquitin ligase UBR1-RAD6/UFD4-UBC4/5 are shown to form an analogous targeting complex. These complexes may enable substrate channeling, in which a substrate bearing, for example, Nt-Asn, would be captured by a complex-bound Nt-amidase, followed by sequential Nt modifications of the substrate and its polyubiquitylation at an internal Lys residue without substrate's dissociation into the bulk solution. At least in yeast, the UBR1/UFD4 ubiquitin ligase interacts with the 26S proteasome, suggesting an even larger Arg/N-degron\u2013targeting complex that contains the proteasome as well. In addition, specific features of protein-sized Arg/N-degron substrates, including their partly sequential and partly nonsequential enzymatic modifications, led us to a verifiable concept termed \"superchanneling.\" In superchanneling, the synthesis of a substrate-linked poly-Ub chain can occur not only after a substrate's sequential Nt modifications, but also before them, through a skipping of either some or all of these modifications within a targeting complex.", "doi": "10.1073/pnas.2003043117", "pmcid": "PMC7245096", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2020-05-19", "series_number": "20", "volume": "117", "issue": "20", "pages": "10778-10788" }, { "id": "authors:xy4s4-ttj37", "collection": "authors", "collection_id": "xy4s4-ttj37", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20200103-103302869", "type": "article", "title": "Evolution of Substrates and Components of the Pro/N-Degron Pathway", "author": [ { "family_name": "Chen", "given_name": "Shun-Jia", "orcid": "0000-0002-5489-4930", "clpid": "Chen-Shun-Jia" }, { "family_name": "Melnykov", "given_name": "Artem", "orcid": "0000-0002-4733-4249", "clpid": "Melnykov-Artem-V" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Gid4, a subunit of the ubiquitin ligase GID, is the recognition component of the Pro/N-degron pathway. Gid4 targets proteins in particular through their N-terminal (Nt) proline (Pro) residue. In Saccharomyces cerevisiae and other Saccharomyces yeasts, the gluconeogenic enzymes Fbp1, Icl1, and Mdh2 bear Nt-Pro and are conditionally destroyed by the Pro/N-degron pathway. However, in mammals and in many non-Saccharomyces yeasts, for example, in Kluyveromyces lactis, these enzymes lack Nt-Pro. We used K. lactis to explore evolution of the Pro/N-degron pathway. One question to be addressed was whether the presence of non-Pro Nt residues in K. lactis Fbp1, Icl1, and Mdh2 was accompanied, on evolutionary time scales (S. cerevisiae and K. lactis diverged \u223c150 million years ago), by a changed specificity of the Gid4 N-recognin. We used yeast-based two-hybrid binding assays and protein-degradation assays to show that the non-Pro (Ala) Nt residue of K. lactis Fbp1 makes this enzyme long-lived in K. lactis. We also found that the replacement, through mutagenesis, of Nt-Ala and the next three residues of K. lactis Fbp1 with the four-residue Nt-PTLV sequence of S. cerevisiae Fbp1 sufficed to make the resulting \"hybrid\" Fbp1 a short-lived substrate of Gid4 in K. lactis. We consider a blend of quasi-neutral genetic drift and natural selection that can account for these and related results. To the best of our knowledge, this work is the first study of the ubiquitin system in K. lactis, including development of the first protein-degradation assay (based on the antibiotic blasticidin) suitable for use with this organism.", "doi": "10.1021/acs.biochem.9b00953", "pmcid": "PMC7286083", "issn": "0006-2960", "publisher": "American Chemical Society", "publication": "Biochemistry", "publication_date": "2020-02-04", "series_number": "4", "volume": "59", "issue": "4", "pages": "582-593" }, { "id": "authors:dbhp0-rte90", "collection": "authors", "collection_id": "dbhp0-rte90", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190725-112336927", "type": "article", "title": "Gid10 as an alternative N-recognin of the Pro/N-degron pathway", "author": [ { "family_name": "Melnykov", "given_name": "Artem", "orcid": "0000-0002-4733-4249", "clpid": "Melnykov-A-V" }, { "family_name": "Chen", "given_name": "Shun-Jia", "orcid": "0000-0002-5489-4930", "clpid": "Chen-Shun-Jia" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "In eukaryotes, N-degron pathways (formerly \"N-end rule pathways\") comprise a set of proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal degradation signals called N-degrons, thereby causing degradation of these proteins by the 26S proteasome or autophagy. Gid4, a subunit of the GID ubiquitin ligase in the yeast Saccharomyces cerevisiae, is the recognition component (N-recognin) of the GID-mediated Pro/N-degron pathway. Gid4 targets proteins by recognizing their N-terminal Pro residues or a Pro at position 2, in the presence of distinct adjoining sequence motifs. Under conditions of low or absent glucose, cells make it through gluconeogenesis. When S. cerevisiae grows on a nonfermentable carbon source, its gluconeogenic enzymes Fbp1, Icl1, Mdh2, and Pck1 are expressed and long-lived. Transition to a medium containing glucose inhibits the synthesis of these enzymes and induces their degradation by the Gid4-dependent Pro/N-degron pathway. While studying yeast Gid4, we identified a similar but uncharacterized yeast protein (YGR066C), which we named Gid10. A screen for N-terminal peptide sequences that can bind to Gid10 showed that substrate specificities of Gid10 and Gid4 overlap but are not identical. Gid10 is not expressed under usual (unstressful) growth conditions, but is induced upon starvation or osmotic stresses. Using protein binding analyses and degradation assays with substrates of GID, we show that Gid10 can function as a specific N-recognin of the Pro/N-degron pathway.", "doi": "10.1073/pnas.1908304116", "pmcid": "PMC6689949", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2019-08-06", "series_number": "32", "volume": "116", "issue": "32", "pages": "15914-15923" }, { "id": "authors:e0q7b-9yd87", "collection": "authors", "collection_id": "e0q7b-9yd87", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190513-150129586", "type": "article", "title": "On the cause of sleep: Protein fragments, the concept of sentinels, and links to epilepsy", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The molecular-level cause of sleep is unknown. In 2012, we suggested that the cause of sleep stems from cumulative effects of numerous intracellular and extracellular protein fragments. According to the fragment generation (FG) hypothesis, protein fragments (which are continually produced through nonprocessive cleavages by intracellular, intramembrane, and extracellular proteases) can be beneficial but toxic as well, and some fragments are eliminated slowly during wakefulness. We consider the FG hypothesis and propose that, during wakefulness, the degradation of accumulating fragments is delayed within natural protein aggregates such as postsynaptic densities (PSDs) in excitatory synapses and in other dense protein meshworks, owing to an impeded diffusion of the \u223c3,000-kDa 26S proteasome. We also propose that a major function of sleep involves a partial and reversible expansion of PSDs, allowing an accelerated destruction of PSD-localized fragments by the ubiquitin/proteasome system. Expansion of PSDs would alter electrochemistry of synapses, thereby contributing to a decreased neuronal firing during sleep. If so, the loss of consciousness, a feature of sleep, would be the consequence of molecular processes (expansions of protein meshworks) that are required for degradation of protein fragments. We consider the concept of FG sentinels, which signal to sleep-regulating circuits that the levels of fragments are going up. Also discussed is the possibility that protein fragments, which are known to be overproduced during an epileptic seizure, may contribute to postictal sleep and termination of seizures. These and related suggestions, described in the paper, are compatible with current evidence about sleep and lead to testable predictions.", "doi": "10.1073/pnas.1904709116", "pmcid": "PMC6561186", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2019-05-28", "series_number": "22", "volume": "116", "issue": "22", "pages": "10773-10782" }, { "id": "authors:pn60k-tme35", "collection": "authors", "collection_id": "pn60k-tme35", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20190108-090142944", "type": "article", "title": "N-degron and C-degron pathways of protein degradation", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "This perspective is partly review and partly proposal. N-degrons and C-degrons are degradation signals whose main determinants are, respectively, the N-terminal and C-terminal residues of cellular proteins. N-degrons and C-degrons include, to varying extents, adjoining sequence motifs, and also internal lysine residues that function as polyubiquitylation sites. Discovered in 1986, N-degrons were the first degradation signals in short-lived proteins. A particularly large set of C-degrons was discovered in 2018. We describe multifunctional proteolytic systems that target N-degrons and C-degrons. We also propose to denote these systems as \"N-degron pathways\" and \"C-degron pathways.\" The former notation replaces the earlier name \"N-end rule pathways.\" The term \"N-end rule\" was introduced 33 years ago, when only some N-terminal residues were thought to be destabilizing. However, studies over the last three decades have shown that all 20 amino acids of the genetic code can act, in cognate sequence contexts, as destabilizing N-terminal residues. Advantages of the proposed terms include their brevity and semantic uniformity for N-degrons and C-degrons. In addition to being topologically analogous, N-degrons and C-degrons are related functionally. A proteolytic cleavage of a subunit in a multisubunit complex can create, at the same time, an N-degron (in a C-terminal fragment) and a spatially adjacent C-degron (in an N-terminal fragment). Consequently, both fragments of a subunit can be selectively destroyed through attacks by the N-degron and C-degron pathways.", "doi": "10.1073/pnas.1816596116", "pmcid": "PMC6329975", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2019-01-08", "series_number": "2", "volume": "116", "issue": "2", "pages": "358-366" }, { "id": "authors:2nbxf-gth90", "collection": "authors", "collection_id": "2nbxf-gth90", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20181108-125308990", "type": "article", "title": "Formyl-methionine as an N-degron of a eukaryotic N-end rule pathway", "author": [ { "family_name": "Kim", "given_name": "Jeong-Mok", "orcid": "0000-0002-7223-248X", "clpid": "Kim-Jeong-Mok" }, { "family_name": "Seok", "given_name": "Ok-Hee", "clpid": "Seok-Ok-Hee" }, { "family_name": "Ju", "given_name": "Shinyeong", "orcid": "0000-0001-5483-4690", "clpid": "Ju-Shinyeong" }, { "family_name": "Heo", "given_name": "Ji-Eun", "clpid": "Heo-Ji-Eun" }, { "family_name": "Yeom", "given_name": "Jeonghun", "clpid": "Yeom-Jeonghun" }, { "family_name": "Kim", "given_name": "Da-Som", "clpid": "Kim-Da-Som" }, { "family_name": "Yoo", "given_name": "Joo-Yeon", "orcid": "0000-0001-5942-3989", "clpid": "Yoo-Joo-Yeon" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Lee", "given_name": "Cheolju", "orcid": "0000-0001-8482-4696", "clpid": "Lee-Cheolju" }, { "family_name": "Hwang", "given_name": "Cheol-Sang", "orcid": "0000-0002-0105-5957", "clpid": "Hwang-Cheol-Sang" } ], "abstract": "In bacteria, nascent proteins bear the pretranslationally generated N-terminal (Nt) formyl-methionine (fMet) residue. Nt-fMet of bacterial proteins is a degradation signal, termed fMet/N-degron. In contrast, proteins synthesized by cytosolic ribosomes of eukaryotes were presumed to bear unformylated Nt-Met. Here we found that the yeast formyltransferase Fmt1, although imported into mitochondria, could also produce Nt-formylated proteins in the cytosol. Nt-formylated proteins were strongly up-regulated in stationary phase or upon starvation for specific amino acids. This up-regulation strictly required the Gcn2 kinase, which phosphorylates Fmt1 and mediates its retention in the cytosol. We also found that the Nt-fMet residues of Nt-formylated proteins act as fMet/N-degrons, and identified the Psh1 ubiquitin ligase as the recognition component of this eukaryotic fMet/N-end rule pathway, which destroys Nt-formylated proteins.", "doi": "10.1126/science.aat0174", "pmcid": "PMC6551516", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2018-11-30", "series_number": "6418", "volume": "362", "issue": "6418", "pages": "Art. No. eaat0174" }, { "id": "authors:y009t-fmd75", "collection": "authors", "collection_id": "y009t-fmd75", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180416-140609766", "type": "article", "title": "Understanding the Pro/N-end rule pathway", "author": [ { "family_name": "Dougan", "given_name": "David A.", "clpid": "Dougan-D-A" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Regulated destruction of proteins underlies just about everything a cell does. A structural study of human Gid4, the N-recognin of the Pro/N-end rule pathway that targets proteins through their N-terminal proline, illuminates the recognition of substrates by this proteolytic system.", "doi": "10.1038/s41589-018-0045-0", "issn": "1552-4450", "publisher": "Nature Publishing Group", "publication": "Nature Chemical Biology", "publication_date": "2018-05", "series_number": "5", "volume": "14", "issue": "5", "pages": "415-416" }, { "id": "authors:t3t9b-waq47", "collection": "authors", "collection_id": "t3t9b-waq47", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20171113-111614515", "type": "article", "title": "A reference-based protein degradation assay without global translation inhibitors", "author": [ { "family_name": "Oh", "given_name": "Jang-Hyun", "orcid": "0000-0002-2053-7468", "clpid": "Oh-Jang-Hyun" }, { "family_name": "Chen", "given_name": "Shun-Jia", "orcid": "0000-0002-5489-4930", "clpid": "Chen-Shun-Jia" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Although it is widely appreciated that the use of global translation inhibitors, such as cycloheximide, in protein degradation assays may result in artefacts, these inhibitors continue to be employed, owing to the absence of robust alternatives. We describe here the promoter reference technique (PRT), an assay for protein degradation with two advantageous features: a reference protein and a gene-specific inhibition of translation. In PRT assays, one measures, during a chase, the ratio of a test protein to a long-lived reference protein, a dihydrofolate reductase (DHFR). The test protein and DHFR are coexpressed, in the yeast Saccharomyces cerevisiae, on a low-copy plasmid from two identical PTDH3 promoters containing additional, previously developed DNA elements. Once transcribed, these elements form 5\u2032-RNA aptamers that bind to the added tetracycline, which represses translation of aptamer-containing mRNAs. The selectivity of repression avoids a global inhibition of translation. This selectivity is particularly important if a component of a relevant proteolytic pathway (e.g. a specific ubiquitin ligase) is itself short-lived. We applied PRT to the Pro/N-end rule pathway, whose substrates include the short-lived Mdh2 malate dehydrogenase. Mdh2 is targeted for degradation by the Gid4 subunit of the GID ubiquitin ligase. Gid4 is also a metabolically unstable protein. Through analyses of short-lived Mdh2 as a target of short-lived Gid4, we illustrate the advantages of PRT over degradation assays that lack a reference and/or involve cycloheximide. In sum, PRT avoids the use of global translation inhibitors during a chase and also provides a \"built-in\" reference protein.", "doi": "10.1074/jbc.M117.814236", "pmcid": "PMC5766948", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "2017-12-29", "series_number": "52", "volume": "292", "issue": "52", "pages": "21457-21465" }, { "id": "authors:vrpfp-30632", "collection": "authors", "collection_id": "vrpfp-30632", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170628-083941867", "type": "article", "title": "The Ubiquitin System, Autophagy, and Regulated Protein Degradation", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "This brief disquisition about the early history of studies on regulated protein degradation introduces several detailed reviews about the ubiquitin system and autophagy.", "doi": "10.1146/annurev-biochem-061516-044859", "issn": "0066-4154", "publisher": "Annual Reviews", "publication": "Annual Review of Biochemistry", "publication_date": "2017-06", "volume": "86", "pages": "123-128" }, { "id": "authors:fcaj5-f7b59", "collection": "authors", "collection_id": "fcaj5-f7b59", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170518-073119611", "type": "article", "title": "Control of Hsp90 chaperone and its clients by N-terminal acetylation and the N-end rule pathway", "author": [ { "family_name": "Oh", "given_name": "Jang-Hyun", "orcid": "0000-0002-2053-7468", "clpid": "Oh-Jang-Hyun" }, { "family_name": "Hyun", "given_name": "Ju-Yeon", "orcid": "0000-0002-5526-9651", "clpid": "Hyun-Ju-Yeon" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "We found that the heat shock protein 90 (Hsp90) chaperone system of the yeast Saccharomyces cerevisiae is greatly impaired in naa10\u0394 cells, which lack the NatA N^\u03b1-terminal acetylase (Nt-acetylase) and therefore cannot N-terminally acetylate a majority of normally N-terminally acetylated proteins, including Hsp90 and most of its cochaperones. Chk1, a mitotic checkpoint kinase and a client of Hsp90, was degraded relatively slowly in wild-type cells but was rapidly destroyed in naa10\u0394 cells by the Arg/N-end rule pathway, which recognized a C terminus-proximal degron of Chk1. Diverse proteins (in addition to Chk1) that are shown here to be targeted for degradation by the Arg/N-end rule pathway in naa10\u0394 cells include Kar4, Tup1, Gpd1, Ste11, and also, remarkably, the main Hsp90 chaperone (Hsc82) itself. Protection of Chk1 by Hsp90 could be overridden not only by ablation of the NatA Nt-acetylase but also by overexpression of the Arg/N-end rule pathway in wild-type cells. Split ubiquitin-binding assays detected interactions between Hsp90 and Chk1 in wild-type cells but not in naa10\u0394 cells. These and related results revealed a major role of Nt-acetylation in the Hsp90-mediated protein homeostasis, a strong up-regulation of the Arg/N-end rule pathway in the absence of NatA, and showed that a number of Hsp90 clients are previously unknown substrates of the Arg/N-end rule pathway.", "doi": "10.1073/pnas.1705898114", "pmcid": "PMC5465900", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2017-05-30", "series_number": "22", "volume": "114", "issue": "22", "pages": "E4370-E4379" }, { "id": "authors:znw58-vcp71", "collection": "authors", "collection_id": "znw58-vcp71", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20170130-100756755", "type": "article", "title": "An N-end rule pathway that recognizes proline and destroys gluconeogenic enzymes", "author": [ { "family_name": "Chen", "given_name": "Shun-Jia", "orcid": "0000-0002-5489-4930", "clpid": "Chen-Shun-Jia" }, { "family_name": "Wu", "given_name": "Xia", "clpid": "Wu-Xia" }, { "family_name": "Wadas", "given_name": "Brandon", "clpid": "Wadas-Brandon-C" }, { "family_name": "Oh", "given_name": "Jang-Hyun", "orcid": "0000-0002-2053-7468", "clpid": "Oh-Jang-Hyun" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Cells synthesize glucose if deprived of it, and destroy gluconeogenic enzymes upon return to glucose-replete conditions. We found that the Gid4 subunit of the ubiquitin ligase GID in the yeast Saccharomyces cerevisiae targeted the gluconeogenic enzymes Fbp1, Icl1, and Mdh2 for degradation. Gid4 recognized the N-terminal proline (Pro) residue and the ~5-residue-long adjacent sequence motifs. Pck1, the fourth gluconeogenic enzyme, contains Pro at position 2; Gid4 directly or indirectly recognized Pro at position 2 of Pck1, contributing to its targeting. These and related results identified Gid4 as the recognition component of the GID-based proteolytic system termed the Pro/N-end rule pathway. Substrates of this pathway include gluconeogenic enzymes that bear either the N-terminal Pro residue or a Pro at position 2, together with adjacent sequence motifs.", "doi": "10.1126/science.aal3655", "pmcid": "PMC5457285", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2017-01-27", "series_number": "6323", "volume": "355", "issue": "6323", "pages": "Art. No. eaal3655" }, { "id": "authors:9sh4d-mt676", "collection": "authors", "collection_id": "9sh4d-mt676", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160815-140416336", "type": "article", "title": "Analyzing N-terminal Arginylation Through the Use of Peptide Arrays and Degradation Assays", "author": [ { "family_name": "Wadas", "given_name": "Brandon", "orcid": "0000-0003-2796-2384", "clpid": "Wadas-Brandon-C" }, { "family_name": "Piatkov", "given_name": "Konstantin", "clpid": "Piatkov-Konstantin-I" }, { "family_name": "Brower", "given_name": "Christopher", "orcid": "0000-0003-0289-5541", "clpid": "Brower-Christopher-S" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "N\u03b1-terminal arginylation (Nt-arginylation) of proteins is mediated by the Ate1 arginyltransferase (R-transferase), a component of the Arg/N-end rule pathway. This proteolytic system recognizes proteins containing N terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. The definitively identified (canonical) residues that are Nt-arginylated by R-transferase are N-terminal Asp, Glu and (oxidized) Cys. Over the last decade, several publications suggested (i) that Ate1 can also arginylate non-canonical N-terminal residues; (ii) that Ate1 is capable of arginylating not only alpha-amino groups of N-terminal residues but also gamma-carboxyl groups of internal (non-N-terminal) Asp and Glu; and (iii) that some isoforms of Ate1 are specific for substrates bearing N-terminal Cys residues. In the present study, we employed arrays of immobilized 11-residue peptides and pulse-chase assays to examine the substrate specificity of mouse R-transferase. We show that amino acid sequences immediately downstream of canonical (Nt-arginylatable) N-terminal residue of a substrate, particularly a residue at position 2, can affect the rate of Nt-arginylation by R-transferase and thereby the rate of degradation of a substrate protein. We also show that the four major isoforms of mouse R transferase have similar Nt-arginylation specificities in vitro, contrary to the claim about specificity of some Ate1 isoforms for N terminal Cys. In addition, we found no evidence for a significant activity of the Ate1 R-transferase toward previously invoked non-canonical N-terminal or internal amino acid residues. Together, our results raise technical concerns about earlier studies that invoked non-canonical arginylation specificities of Ate1.", "doi": "10.1074/jbc.M116.747956", "pmcid": "PMC5076509", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "2016-09-30", "series_number": "40", "volume": "291", "issue": "40", "pages": "Art. No. 20976" }, { "id": "authors:1at7j-9gp90", "collection": "authors", "collection_id": "1at7j-9gp90", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160706-082440341", "type": "article", "title": "Degradation of Serotonin N-Acetyltransferase, a Circadian Regulator, by the N-end Rule Pathway", "author": [ { "family_name": "Wadas", "given_name": "Brandon", "orcid": "0000-0003-2796-2384", "clpid": "Wadas-Brandon-C" }, { "family_name": "Borjigin", "given_name": "Jimo", "clpid": "Borjigin-Jimo" }, { "family_name": "Huang", "given_name": "Zheping", "clpid": "Huang-Zheping" }, { "family_name": "Oh", "given_name": "Jang-Hyun", "orcid": "0000-0002-2053-7468", "clpid": "Oh-Jang-Hyun" }, { "family_name": "Hwang", "given_name": "Cheol-Sang", "orcid": "0000-0002-0105-5957", "clpid": "Hwang-Cheol-Sang" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Serotonin N-acetyltransferase (AANAT) converts serotonin to N-acetylserotonin (NAS), a distinct biological regulator and the immediate precursor of melatonin, a circulating hormone that influences circadian processes, including sleep. N-terminal sequences of AANAT enzymes vary among vertebrates. Mechanisms that regulate the levels of AANAT are incompletely understood. Previous findings were consistent with the possibility that AANAT may be controlled through its degradation by the N-end rule pathway. By expressing the rat and human AANATs and their mutants not only in mammalian cells but also in the yeast Saccharomyces cerevisiae, and by taking advantage of yeast genetics, we show here that two complementary forms of rat AANAT are targeted for degradation by two complementary branches of the N-end rule pathway. Specifically, the N\u03b1 terminally acetylated (Nt-acetylated) Ac-AANAT is destroyed through the recognition of its Nt acetylated N terminal Met residue by the Ac/N-end rule pathway, whereas the non Nt acetylated AANAT is targeted by the Arg/N end rule pathway, which recognizes the unacetylated N-terminal Met-Leu sequence of rat AANAT. We also show, by constructing lysine to arginine mutants of rat AANAT, that its degradation is mediated by polyubiquitylation of its Lys residue(s). Human AANAT, whose N-terminal sequence differs from that of rodent AANATs, is longer lived than its rat counterpart, and appears to be refractory to degradation by the N-end rule pathway. Together, these and related results indicate both a major involvement of the N-end rule pathway in the control of rodent AANATs and substantial differences in the regulation of rodent and human AANATs that stem from differences in their N terminal sequences.", "doi": "10.1074/jbc.M116.734640", "pmcid": "PMC5016120", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "2016-08-12", "series_number": "33", "volume": "291", "issue": "33", "pages": "17178-17196" }, { "id": "authors:mp90e-04y80", "collection": "authors", "collection_id": "mp90e-04y80", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160218-082640962", "type": "article", "title": "Degradation of the Separase-cleaved Rec8, a Meiotic Cohesin Subunit, by the N-end Rule Pathway", "author": [ { "family_name": "Liu", "given_name": "Yu-Jiao", "clpid": "Liu-Yu-Jiao" }, { "family_name": "Liu", "given_name": "Chao", "orcid": "0000-0001-7049-493X", "clpid": "Liu-Chao" }, { "family_name": "Chang", "given_name": "ZeNan", "clpid": "Chang-ZeNan" }, { "family_name": "Wadas", "given_name": "Brandon", "orcid": "0000-0003-2796-2384", "clpid": "Wadas-Brandon-C" }, { "family_name": "Brower", "given_name": "Christopher S.", "orcid": "0000-0003-0289-5541", "clpid": "Brower-Christopher-S" }, { "family_name": "Song", "given_name": "Zhen-Hua", "clpid": "Song-Zhen-Hua" }, { "family_name": "Xu", "given_name": "Zhi-Liang", "clpid": "Xu-Zhi-Liang" }, { "family_name": "Shang", "given_name": "Yong-Liang", "clpid": "Shang-Yong-Liang" }, { "family_name": "Liu", "given_name": "Wei-Xiao", "clpid": "Liu-Wei-Xiao" }, { "family_name": "Wang", "given_name": "Li-Na", "clpid": "Wang-Li-Na" }, { "family_name": "Dong", "given_name": "Wen", "clpid": "Dong-Wen" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Hu", "given_name": "Rong-Gui", "clpid": "Hu-Rong-Gui" }, { "family_name": "Li", "given_name": "Wei", "orcid": "0000-0003-2543-2558", "clpid": "Li-Wei" } ], "abstract": "The Ate1 arginyltransferase (R-transferase) is a component of the N-end rule pathway, which recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. Ate1 arginylates N-terminal Asp, Glu, or (oxidized) Cys. The resulting N-terminal Arg is recognized by ubiquitin ligases of the N-end rule pathway. In the yeast Saccharomyces cerevisiae, the separase-mediated cleavage of the Scc1/Rad21/Mcd1 cohesin subunit generates a C-terminal fragment that bears N-terminal Arg and is destroyed by the N-end rule pathway without a requirement for arginylation. In contrast, the separase-mediated cleavage of Rec8, the mammalian meiotic cohesin subunit, yields a fragment bearing N-terminal Glu, a substrate of the Ate1 R-transferase. Here we constructed and used a germ cell-confined Ate1\u2212/\u2212 mouse strain to analyze the separase-generated C-terminal fragment of Rec8. We show that this fragment is a short-lived N-end rule substrate, that its degradation requires N-terminal arginylation, and that male Ate1\u2212/\u2212 mice are nearly infertile, due to massive apoptotic death of Ate1\u2212/\u2212 spermatocytes during the metaphase of meiosis I. These effects of Ate1 ablation are inferred to be caused, at least in part, by the failure to destroy the C-terminal fragment of Rec8 in the absence of N-terminal arginylation.", "doi": "10.1074/jbc.M116.714964", "pmcid": "PMC4817174", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "2016-04-01", "series_number": "14", "volume": "291", "issue": "14", "pages": "7426-7438" }, { "id": "authors:yp81w-t1j67", "collection": "authors", "collection_id": "yp81w-t1j67", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160509-130856265", "type": "article", "title": "Formyl-methionine as a degradation signal at the N-termini of bacterial proteins", "author": [ { "family_name": "Piatkov", "given_name": "Konstantin", "clpid": "Piatkov-Konstantin-I" }, { "family_name": "Vu", "given_name": "Tri", "orcid": "0000-0002-1098-5646", "clpid": "Vu-Tri-T-M" }, { "family_name": "Hwang", "given_name": "Cheol-Sang", "orcid": "0000-0002-0105-5957", "clpid": "Hwang-Cheol-Sang" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "In bacteria, all nascent proteins bear the pretranslationally formed N-terminal formyl-methionine (fMet) residue. The fMet residue is cotranslationally deformylated by a ribosome-associated deformylase. The formylation of N-terminal Met in bacterial proteins is not strictly essential for either translation or cell viability. Moreover, protein synthesis by the cytosolic ribosomes of eukaryotes does not involve the formylation of N-terminal Met. What, then, is the main biological function of this metabolically costly, transient, and not strictly essential modification of N\u2011terminal Met, and why has Met formylation not been eliminated during bacterial evolution? One possibility is that the similarity of the formyl and acetyl groups, their identical locations in N\u2011terminally formylated (Nt\u2011formylated) and Nt-acetylated proteins, and the recently discovered proteolytic function of Nt-acetylation in eukaryotes might also signify a proteolytic role of Nt\u2011formylation in bacteria. We addressed this hypothesis about fMet\u2011based degradation signals, termed fMet/N-degrons, using specific E. coli mutants, pulse-chase degradation assays, and protein reporters whose deformylation was altered, through site-directed mutagenesis, to be either rapid or relatively slow. Our findings strongly suggest that the formylated N-terminal fMet can act as a degradation signal, largely a cotranslational one. One likely function of fMet/N-degrons is the control of protein quality. In bacteria, the rate of polypeptide chain elongation is nearly an order of magnitude higher than in eukaryotes. We suggest that the faster emergence of nascent proteins from bacterial ribosomes is one mechanistic and evolutionary reason for the pretranslational design of bacterial fMet/N\u2011degrons, in contrast to the cotranslational design of analogous Ac/N\u2011degrons in eukaryotes.", "doi": "10.15698/mic2015.10.231", "pmcid": "PMC4745127", "issn": "2311-2638", "publisher": "Shared Science Publishers", "publication": "Microbial Cell", "publication_date": "2015-10", "series_number": "10", "volume": "2", "issue": "10", "pages": "376-393" }, { "id": "authors:esanb-nzg83", "collection": "authors", "collection_id": "esanb-nzg83", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150316-112138335", "type": "article", "title": "Control of mammalian G protein signaling by N-terminal acetylation and the N-end rule pathway", "author": [ { "family_name": "Park", "given_name": "Sang-Eun", "clpid": "Park-Sang-Eun" }, { "family_name": "Kim", "given_name": "Jeong-Mok", "orcid": "0000-0002-7223-248X", "clpid": "Kim-Jeong-Mok" }, { "family_name": "Seok", "given_name": "Ok-Hee", "clpid": "Seok-Ok-Hee" }, { "family_name": "Cho", "given_name": "Hanna", "clpid": "Cho-Hanna" }, { "family_name": "Wadas", "given_name": "Brandon", "orcid": "0000-0003-2796-2384", "clpid": "Wadas-Brandon-C" }, { "family_name": "Kim", "given_name": "Seon-Young", "clpid": "Kim-Seon-Young" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Hwang", "given_name": "Cheol-Sang", "orcid": "0000-0002-0105-5957", "clpid": "Hwang-Cheol-Sang" } ], "abstract": "Rgs2, a regulator of G proteins, lowers blood pressure by decreasing signaling through G\u03b1q. Human patients expressing Met-Leu-Rgs2 (ML-Rgs2) or Met-Arg-Rgs2 (MR-Rgs2) are hypertensive relative to people expressing wild-type Met-Gln-Rgs2 (MQ-Rgs2). We found that wild-type MQ-Rgs2 and its mutant, MR-Rgs2, were destroyed by the Ac/N-end rule pathway, which recognizes N\u03b1-terminally acetylated (Nt-acetylated) proteins. The shortest-lived mutant, ML-Rgs2, was targeted by both the Ac/N-end rule and Arg/N-end rule pathways. The latter pathway recognizes unacetylated N-terminal residues. Thus, the Nt-acetylated Ac-MX-Rgs2 (X = Arg, Gln, Leu) proteins are specific substrates of the mammalian Ac/N-end rule pathway. Furthermore, the Ac/N-degron of Ac-MQ-Rgs2 was conditional, and Teb4, an endoplasmic reticulum (ER) membrane-embedded ubiquitin ligase, was able to regulate G protein signaling by targeting Ac-MX-Rgs2 proteins for degradation through their N^\u03b1-terminal acetyl group.", "doi": "10.1126/science.aaa3844", "pmcid": "PMC4748709", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2015-03-13", "series_number": "6227", "volume": "347", "issue": "6227", "pages": "1249-1252" }, { "id": "authors:30jfy-4qw73", "collection": "authors", "collection_id": "30jfy-4qw73", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20141105-093127516", "type": "article", "title": "Liat1, an arginyltransferase-binding protein whose evolution among primates involved changes in the numbers of its 10-residue repeats", "author": [ { "family_name": "Brower", "given_name": "Christopher S.", "clpid": "Brower-C-S" }, { "family_name": "Rosen", "given_name": "Connor E.", "clpid": "Rosen-C-E" }, { "family_name": "Jones", "given_name": "Richard H.", "clpid": "Jones-R-H" }, { "family_name": "Wadas", "given_name": "Brandon C.", "clpid": "Wadas-B-C" }, { "family_name": "Piatkov", "given_name": "Konstantin I.", "clpid": "Piatkov-K-I" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The arginyltransferase Ate1 is a component of the N-end rule pathway, which recognizes proteins containing N-terminal degradation signals called N-degrons, polyubiquitylates these proteins, and thereby causes their degradation by the proteasome. At least six isoforms of mouse Ate1 are produced through alternative splicing of Ate1 pre-mRNA. We identified a previously uncharacterized mouse protein, termed Liat1 (ligand of Ate1), that interacts with Ate1 but does not appear to be its arginylation substrate. Liat1 has a higher affinity for the isoforms Ate1^(1A7A) and Ate1^(1B7A). Liat1 stimulated the in vitro N-terminal arginylation of a model substrate by Ate1. All examined vertebrate and some invertebrate genomes encode proteins sequelogous (similar in sequence) to mouse Liat1. Sequelogs of Liat1 share a highly conserved \u223c30-residue region that is shown here to be required for the binding of Liat1 to Ate1. We also identified non-Ate1 proteins that interact with Liat1. In contrast to Liat1 genes of nonprimate mammals, Liat1 genes of primates are subtelomeric, a location that tends to confer evolutionary instability on a gene. Remarkably, Liat1 proteins of some primates, from macaques to humans, contain tandem repeats of a 10-residue sequence, whereas Liat1 proteins of other mammals contain a single copy of this motif. Quantities of these repeats are, in general, different in Liat1 of different primates. For example, there are 1, 4, 13, 13, 17, and 17 repeats in the gibbon, gorilla, orangutan, bonobo, neanderthal, and human Liat1, respectively, suggesting that repeat number changes in this previously uncharacterized protein may contribute to evolution of primates.", "doi": "10.1073/pnas.1419587111", "pmcid": "PMC4246273", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2014-11-18", "series_number": "46", "volume": "111", "issue": "46", "pages": "E4936-E4945" }, { "id": "authors:6s8mz-15d54", "collection": "authors", "collection_id": "6s8mz-15d54", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140613-125640695", "type": "article", "title": "Discovery of the Biology of the Ubiquitin System", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "This article, about the discovery of the biology of the ubiquitin system, describes my laboratory's main contribution to date, and stems, in part, from previous historical summaries. Another antecedent is the interview given in 2006 to Dr Istvan Hargittai, a distinguished Hungarian chemist. It described my life and science, including the early years in Moscow (Russia), the 1977 emigration from the former Soviet Union to the United States, and scientific work that ensued.", "doi": "10.1001/jama.2014.5549", "issn": "0098-7484", "publisher": "American Medical Association", "publication": "JAMA : the journal of the American Medical Association", "publication_date": "2014-05-21", "series_number": "19", "volume": "311", "issue": "19", "pages": "1969-1970" }, { "id": "authors:2sjx5-gx317", "collection": "authors", "collection_id": "2sjx5-gx317", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140225-100028331", "type": "article", "title": "Calpain-generated natural protein fragments as short-lived substrates of the N-end rule pathway", "author": [ { "family_name": "Piatkov", "given_name": "Konstantin I.", "clpid": "Piatkov-K-I" }, { "family_name": "Oh", "given_name": "Jang-Hyun", "clpid": "Oh-Jang-Hyun" }, { "family_name": "Liu", "given_name": "Yuan", "clpid": "Liu-Yuan" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Calpains are Ca^(2+)-dependent intracellular proteases. We show here that calpain-generated natural C-terminal fragments of proteins that include G protein\u2013coupled receptors, transmembrane ion channels, transcriptional regulators, apoptosis controllers, kinases, and phosphatases (Phe-GluN2a, Lys-Ica512, Arg-Ankrd2, Tyr-Grm1, Arg-Atp2b2, Glu-Bak, Arg-Igfbp2, Glu-I\u03baB\u03b1, and Arg-c-Fos), are short-lived substrates of the Arg/N-end rule pathway, which targets destabilizing N-terminal residues. We also found that the identity of a fragment's N-terminal residue can change during evolution, but the residue's destabilizing activity is virtually always retained, suggesting selection pressures that favor a short half-life of the calpain-generated fragment. It is also shown that a self-cleavage of a calpain can result in an N-end rule substrate. Thus, the autoprocessing of calpains can control them by making active calpains short-lived. These and related results indicate that the Arg/N-end rule pathway mediates the remodeling of oligomeric complexes by eliminating protein fragments that are produced in these complexes through cleavages by calpains or other nonprocessive proteases. We suggest that this capability of the Arg/N-end rule pathway underlies a multitude of its previously known but mechanistically unclear functions.", "doi": "10.1073/pnas.1401639111", "pmcid": "PMC3948289", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2014-03-04", "series_number": "9", "volume": "111", "issue": "9", "pages": "E817-E826" }, { "id": "authors:95v5k-pr368", "collection": "authors", "collection_id": "95v5k-pr368", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20140102-103647381", "type": "article", "title": "The N-Terminal Methionine of Cellular Proteins as a Degradation Signal", "author": [ { "family_name": "Kim", "given_name": "Heon-Ki", "clpid": "Kim-Heon-Ki" }, { "family_name": "Kim", "given_name": "Ryu-Ryun", "clpid": "Kim-Ryu-Ryun" }, { "family_name": "Oh", "given_name": "Jang Hyun", "clpid": "Oh-Jang-Hyun" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Hwang", "given_name": "Cheol-Sang", "orcid": "0000-0002-0105-5957", "clpid": "Hwang-Cheol-Sang" } ], "abstract": "The Arg/N-end rule pathway targets for degradation proteins that bear specific unacetylated N-terminal residues while the Ac/N-end rule pathway targets proteins through their N^\u03b1-terminally acetylated (Nt-acetylated) residues. Here, we show that Ubr1, the ubiquitin ligase of the Arg/N-end rule pathway, recognizes unacetylated N-terminal methionine if it is followed by a hydrophobic residue. This capability of Ubr1 expands the range of substrates that can be targeted for degradation by the Arg/N-end rule pathway because virtually all nascent cellular proteins bear N-terminal methionine. We identified Msn4, Sry1, Arl3, and Pre5 as examples of normal or misfolded proteins that can be destroyed through the recognition of their unacetylated N-terminal methionine. Inasmuch as proteins bearing the Nt-acetylated N-terminal methionine residue are substrates of the Ac/N-end rule pathway, the resulting complementarity of the Arg/N-end rule and Ac/N-end rule pathways enables the elimination of protein substrates regardless of acetylation state of N-terminal methionine in these substrates.", "doi": "10.1016/j.cell.2013.11.031", "pmcid": "PMC3988316", "issn": "0092-8674", "publisher": "Elsevier", "publication": "Cell", "publication_date": "2014-01-16", "series_number": "1", "volume": "156", "issue": "1", "pages": "158-169" }, { "id": "authors:42vdk-rv035", "collection": "authors", "collection_id": "42vdk-rv035", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20130813-094316614", "type": "article", "title": "Ubiquitin Reference Technique and Its Use in Ubiquitin-Lacking Prokaryotes", "author": [ { "family_name": "Piatkov", "given_name": "Konstantin", "clpid": "Piatkov-K-I" }, { "family_name": "Graciet", "given_name": "Emmanuelle", "clpid": "Graciet-E" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "In a pulse-chase assay, the in vivo degradation of a protein is measured through a brief labeling of cells with, for example, a radioactive amino acid, followed by cessation of labeling and analysis of cell extracts prepared at different times afterward (\"chase\"), using immunoprecipitation, electrophoresis and autoradiography of a labeled protein of interest. A conventional pulse-chase assay is fraught with sources of data scatter, as the efficacy of labeling and immunoprecipitation can vary, and sample volumes can vary as well. The ubiquitin reference technique (URT), introduced in 1996, addresses these problems. In eukaryotes, a DNA-encoded linear fusion of ubiquitin to another protein is cleaved by deubiquitylases at the ubiquitin-protein junction. A URT assay uses a fusion in which the ubiquitin moiety is located between a downstream polypeptide (test protein) and an upstream polypeptide (a long-lived reference protein). The cotranslational cleavage of a URT fusion by deubiquitylases after the last residue of ubiquitin produces, at the initially equimolar ratio, a test protein with a desired N-terminal residue and a reference protein containing C-terminal ubiquitin moiety. In addition to being more accurate than pulse-chases without a reference, URT makes it possible to detect and measure the degradation of a test protein during the pulse (before the chase). Because prokaryotes, including Gram-negative bacteria such as, for example, Escherichia coli and Vibrio vulnificus, lack the ubiquitin system, the use of URT in such cells requires ectopic expression of a deubiquitylase. We describe designs and applications of plasmid vectors that coexpress, in bacteria, both a URT-type fusion and Ubp1, a deubiquitylase of the yeast Saccharomyces cerevisiae. This single-plasmid approach extends the accuracy-enhancing URT assay to studies of protein degradation in prokaryotes.", "doi": "10.1371/journal.pone.0067952", "pmcid": "PMC3692480", "issn": "1932-6203", "publisher": "Public Library of Science", "publication": "PLoS ONE", "publication_date": "2013-06-25", "series_number": "6", "volume": "8", "issue": "6", "pages": "Art. No. e67952" }, { "id": "authors:qtdvy-6z785", "collection": "authors", "collection_id": "qtdvy-6z785", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20130716-134300224", "type": "article", "title": "Control of Protein Quality and Stoichiometries by N-Terminal\n Acetylation and the N-End Rule Pathway", "author": [ { "family_name": "Shemorry", "given_name": "Anna", "clpid": "Shemorry-A" }, { "family_name": "Hwang", "given_name": "Cheol-Sang", "orcid": "0000-0002-0105-5957", "clpid": "Hwang-Cheol-Sang" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "N^\u03b1-terminal acetylation of cellular proteins was recently discovered to create specific degradation signals termed Ac/N-degrons and targeted by the Ac/N-end rule pathway. We show that Hcn1, a subunit of the APC/C ubiquitin ligase, contains an Ac/N-degron that is repressed by Cut9, another APC/C subunit and the ligand of Hcn1. Cog1, a subunit of the Golgi-associated COG complex, is also shown to contain an Ac/N-degron. Cog2 and Cog3, direct ligands of Cog1, can repress this degron. The subunit decoy technique was used to show that the long-lived endogenous Cog1 is destabilized and destroyed via its activated (unshielded) Ac/N-degron if the total level of Cog1 increased in a cell. Hcn1 and Cog1 are the first examples of protein regulation through the physiologically relevant transitions that shield and unshield natural Ac/N-degrons. This mechanistically straightforward circuit can employ the demonstrated conditionality of Ac/N-degrons to regulate subunit stoichiometries and other aspects of protein quality control.", "doi": "10.1016/j.molcel.2013.03.018", "pmcid": "PMC3665649", "issn": "1097-2765", "publisher": "Elsevier", "publication": "Molecular Cell", "publication_date": "2013-05-23", "series_number": "4", "volume": "50", "issue": "4", "pages": "540-551" }, { "id": "authors:r4a1e-2jr41", "collection": "authors", "collection_id": "r4a1e-2jr41", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20130530-133709518", "type": "article", "title": "Neurodegeneration-Associated Protein Fragments as Short-Lived Substrates of the N-End Rule Pathway", "author": [ { "family_name": "Brower", "given_name": "Christopher S.", "clpid": "Brower-C-S" }, { "family_name": "Piatkov", "given_name": "Konstantin I.", "clpid": "Piatkov-K-I" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Protein aggregates are a common feature of neurodegenerative syndromes. Specific protein fragments were found to be aggregated in disorders including Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease. Here, we show that the natural C-terminal fragments of Tau, TDP43, and \u03b1-synuclein are short-lived substrates of the Arg/N-end rule pathway, a processive proteolytic system that targets proteins bearing \"destabilizing\" N-terminal residues. Furthermore, a natural TDP43 fragment is shown to be metabolically stabilized in Ate1\u2212/\u2212 fibroblasts that lack the arginylation branch of the Arg/N-end rule pathway, leading to accumulation and aggregation of this fragment. We also found that a fraction of A\u03b242, the Alzheimer's disease-associated fragment of APP, is N-terminally arginylated in the brains of 5xFAD mice and is degraded by the Arg/N-end rule pathway. The discovery that neurodegeneration-associated natural fragments of TDP43, Tau, \u03b1-synuclein, and APP can be selectively destroyed by the Arg/N-end rule pathway suggests that this pathway counteracts neurodegeneration.", "doi": "10.1016/j.molcel.2013.02.009", "pmcid": "PMC3640747", "issn": "1097-2765", "publisher": "Elsevier", "publication": "Molecular Cell", "publication_date": "2013-04-25", "series_number": "2", "volume": "50", "issue": "2", "pages": "161-171" }, { "id": "authors:rmrqr-nee95", "collection": "authors", "collection_id": "rmrqr-nee95", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20130211-154805502", "type": "article", "title": "The Auto-Generated Fragment of the Usp1 Deubiquitylase Is a Physiological Substrate of the N-End Rule Pathway", "author": [ { "family_name": "Piatkov", "given_name": "Konstantin I.", "clpid": "Piatkov-K-I" }, { "family_name": "Colnaghi", "given_name": "Luca", "clpid": "Colnaghi-L" }, { "family_name": "B\u00e9k\u00e9s", "given_name": "Miklos", "clpid": "B\u00e9k\u00e9s-M" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Huang", "given_name": "Tony T.", "clpid": "Huang-Tony-T" } ], "abstract": "Deamidation of N-terminal Gln by the Ntaq1 Nt^Q-amidase is a part of the Arg/N-end rule pathway, a ubiquitin-dependent proteolytic system. Here we identify Gln-Usp1^(Ct), the C-terminal fragment of the autocleaved Usp1 deubiquitylase, as the first physiological Arg/N-end rule substrate that is targeted for degradation through deamidation of N-terminal Gln. Usp1 regulates genomic stability, in part through the deubiquitylation of monoubiquitylated PCNA, a DNA polymerase processivity factor. The autocleaved Usp1 remains a deubiquitylase because its fragments remain associated with Uaf1, an enhancer of Usp1 activity, until the Gln-Usp1^(Ct) fragment is selectively destroyed by the Arg/N-end rule pathway. We also show that metabolic stabilization of Gln-Usp1^(Ct) results in a decreased monoubiquitylation of PCNA and in a hypersensitivity of cells to ultraviolet irradiation. Thus, in addition to its other functions in DNA repair and chromosome segregation, the Arg/N-end rule pathway regulates genomic stability through the degradation-mediated control of the autocleaved Usp1 deubiquitylase.", "doi": "10.1016/j.molcel.2012.10.012", "pmcid": "PMC3889152", "issn": "1097-2765", "publisher": "Elsevier", "publication": "Molecular Cell", "publication_date": "2012-12-28", "series_number": "6", "volume": "48", "issue": "6", "pages": "926-933" }, { "id": "authors:3pqpz-b0563", "collection": "authors", "collection_id": "3pqpz-b0563", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120831-090649691", "type": "article", "title": "Augmented generation of protein fragments during wakefulness as the molecular cause of sleep: A hypothesis", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Despite extensive understanding of sleep regulation, the molecular-level cause and function of sleep are unknown. I suggest that they originate in individual neurons and stem from increased production of protein fragments during wakefulness. These fragments are transient parts of protein complexes in which the fragments were generated. Neuronal Ca^(2+) fluxes are higher during wakefulness than during sleep. Subunits of transmembrane channels and other proteins are cleaved by Ca^(2+)-activated calpains and by other nonprocessive proteases, including caspases and secretases. In the proposed concept, termed the fragment generation (FG) hypothesis, sleep is a state during which the production of fragments is decreased (owing to lower Ca^(2+) transients) while fragment-destroying pathways are upregulated. These changes facilitate the elimination of fragments and the remodeling of protein complexes in which the fragments resided. The FG hypothesis posits that a proteolytic cleavage, which produces two fragments, can have both deleterious effects and fitness-increasing functions. This (previously not considered) dichotomy can explain both the conservation of cleavage sites in proteins and the evolutionary persistence of sleep, because sleep would counteract deleterious aspects of protein fragments. The FG hypothesis leads to new explanations of sleep phenomena, including a longer sleep after sleep deprivation. Studies in the 1970s showed that ethanol-induced sleep in mice can be strikingly prolonged by intracerebroventricular injections of either Ca^(2+) alone or Ca^(2+) and its ionophore. These results, which were never interpreted in connection to protein fragments or the function of sleep, may be accounted for by the FG hypothesis about molecular causation of sleep.", "doi": "10.1002/pro.2148", "pmcid": "PMC3527701", "issn": "0961-8368", "publisher": "Wiley", "publication": "Protein Science", "publication_date": "2012-11", "series_number": "11", "volume": "21", "issue": "11" }, { "id": "authors:brwnm-0qs67", "collection": "authors", "collection_id": "brwnm-0qs67", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120817-134304319", "type": "article", "title": "The N-end rule pathway counteracts cell death by destroying proapoptotic protein fragments", "author": [ { "family_name": "Piatkov", "given_name": "Konstantin I.", "clpid": "Piatkov-K-I" }, { "family_name": "Brower", "given_name": "Christopher S.", "clpid": "Brower-C-S" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "In the course of apoptosis, activated caspases cleave \u223c500 to \u223c1,000 different proteins in a mammalian cell. The dynamics of apoptosis involve a number of previously identified, caspase-generated proapoptotic protein fragments, defined as those that increase the probability of apoptosis. In contrast to activated caspases, which can be counteracted by inhibitor of apoptosis proteins, there is little understanding of antiapoptotic responses to proapoptotic protein fragments. One possibility is the regulation of proapoptotic fragments through their selective degradation. The previously identified proapoptotic fragments Cys-RIPK1, Cys-TRAF1, Asp-BRCA1, Leu-LIMK1, Tyr-NEDD9, Arg-BID, Asp-BCL_XL, Arg-BIM_EL, Asp-EPHA4, and Tyr-MET bear destabilizing N-terminal residues. Tellingly, the destabilizing nature (but not necessarily the actual identity) of N-terminal residues of proapoptotic fragments was invariably conserved in evolution. Here, we show that these proapoptotic fragments are short-lived substrates of the Arg/N-end rule pathway. Metabolic stabilization of at least one such fragment, Cys-RIPK1, greatly augmented the activation of the apoptosis-inducing effector caspase-3. In agreement with this understanding, even a partial ablation of the Arg/N-end rule pathway in two specific N-end rule mutants is shown to sensitize cells to apoptosis. We also found that caspases can inactivate components of the Arg/N-end rule pathway, suggesting a mutual suppression between this pathway and proapoptotic signaling. Together, these results identify a mechanistically specific and functionally broad antiapoptotic role of the Arg/N-end rule pathway. In conjunction with other apoptosis-suppressing circuits, the Arg/N-end rule pathway contributes to thresholds that prevent a transient or otherwise weak proapoptotic signal from reaching the point of commitment to apoptosis.", "doi": "10.1073/pnas.1207786109", "pmcid": "PMC3390858", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2012-07-03", "series_number": "27", "volume": "109", "issue": "27", "pages": "E1839-E1847" }, { "id": "authors:n3cw1-gfq89", "collection": "authors", "collection_id": "n3cw1-gfq89", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120614-103921398", "type": "article", "title": "The Ubiquitin System, an Immense Realm", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Among the functions of intracellular proteolysis are the elimination of misfolded or otherwise abnormal proteins; the maintenance of amino acid pools in cells affected by stresses, such as starvation; and the generation of protein fragments that act as hormones, antigens, or other effectors. Many intracellular proteins are either conditionally or constitutively short-lived, with in vivo half-lives that can be as brief as a few minutes. In some cases, a proteolytic pathway targets and destroys a protein cotranslationally, i.e., an emerging polypeptide chain can be degraded while it is still a ribosome-associated\npeptidyl-tRNA (1, 2). The regulated and processive degradation of intracellular proteins is carried out largely by the ubiquitin (Ub)- proteasome system (Ub system), in conjunction with molecular chaperones, autophagy, and lysosomal proteolysis. Other mediators of\nintracellular protein degradation include proteases such as caspases, calpains, and separases. These and other nonprocessive proteases can function as \"upstream\" components of the Ub system, generating protein fragments that are targeted and degraded to short peptides by Ub-mediated pathways. Proteins that are damaged, misfolded, or otherwise abnormal are often recognized as such and selectively destroyed by the Ub system. Physiologically\nimportant exceptions include conformationally perturbed proteins and/or their aggregates that are harmful but cannot be efficaciously repaired or removed. The resulting proteotoxicity underlies both aging and specific diseases,\nincluding neurodegeneration.", "doi": "10.1146/annurev-biochem-051910-094049", "issn": "0066-4154", "publisher": "Annual Reviews", "publication": "Annual Review of Biochemistry", "publication_date": "2012-07", "volume": "81", "pages": "167-176" }, { "id": "authors:j3t96-2zk72", "collection": "authors", "collection_id": "j3t96-2zk72", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120227-084239400", "type": "book_section", "title": "Three decades of studies to understand the functions of the ubiquitin family", "book_title": "Ubiquitin family modifiers and the proteasome: reviews and protocols", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "contributor": [ { "family_name": "Dohmen", "given_name": "R. J\u00fcrgen", "clpid": "Dohmen-R-J" }, { "family_name": "Scheffner", "given_name": "Martin", "clpid": "Scheffner-M" } ], "abstract": "Many intracellular proteins are metabolically unstable or can\nbecome unstable during their lifetime in a cell. The in vivo half-lives\nof specific proteins range from less than a minute to many days.\nAmong the functions of intracellular proteolysis are the elimination\nof misfolded or otherwise abnormal proteins; maintenance of\namino acid pools in cells affected by stresses such as starvation; and\ngeneration of protein fragments that act as hormones, antigens, or\nother effectors. One major function of proteolytic pathways is the\nselective destruction of proteins whose concentrations must vary\nwith time and alterations in the state of a cell. Short in vivo half-lives\nof such proteins provide a way to generate their spatial gradients\nand to rapidly adjust their concentration or subunit\ncomposition through changes in the rate of their degradation.\nThe regulated (and processive) degradation of intracellular proteins\nis carried out largely by the ubiquitin\u2013proteasome system (Ub\nsystem), in conjunction with autophagy-lysosome pathways. Other\ncontributors to intracellular proteolysis include cytosolic and\nnuclear proteases, such as caspases, calpains, and separases. They\noften function as \"upstream\" components of the Ub system, which\ndestroys protein fragments that had been produced by these (nonprocessive)\nproteases. Ub, a 76-residue protein, mediates selective\nproteolysis through its enzymatic conjugation to proteins that contain\nprimary degradation signals (degrons (1)), thereby marking\nsuch proteins for degradation by the 26S proteasome, an ATPdependent\nmultisubunit protease. Ub conjugation involves the\nformation of a poly-Ub chain that is linked (in most cases) to the \u03b5-amino group of an internal Lys residue in a substrate protein.\nUb is a \"secondary\" degron, in that Ub is conjugated to proteins\nthat contain primary degradation signals. Ub has nonproteolytic\nfunctions as well. The design of the Ub system is summarized in\nFig. 1.", "doi": "10.1007/978-1-61779-474-2_1", "isbn": "9781617794742", "publisher": "Humana Press", "place_of_publication": "New York", "publication_date": "2012", "pages": "1-11" }, { "id": "authors:3db69-v9f85", "collection": "authors", "collection_id": "3db69-v9f85", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20111025-084945445", "type": "article", "title": "Ubiquitin Ligases of the N-End Rule Pathway: Assessment of Mutations in UBR1 That Cause the Johanson-Blizzard Syndrome", "author": [ { "family_name": "Hwang", "given_name": "Cheol-Sang", "orcid": "0000-0002-0105-5957", "clpid": "Hwang-Cheol-Sang" }, { "family_name": "Sukalo", "given_name": "Maja", "clpid": "Sukalo-M" }, { "family_name": "Batygin", "given_name": "Olga", "clpid": "Batygin-Olga" }, { "family_name": "Addor", "given_name": "Marie-Claude", "clpid": "Addor-M-C" }, { "family_name": "Brunner", "given_name": "Han", "clpid": "Brunner-H" }, { "family_name": "Perez Aytes", "given_name": "Antonio", "clpid": "Perez-Aytes-A" }, { "family_name": "Mayerle", "given_name": "Julia", "clpid": "Mayerle-J" }, { "family_name": "Song", "given_name": "Hyun Kyu", "clpid": "Song-Hyun-Kyu" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Zenker", "given_name": "Martin", "clpid": "Zenker-M" } ], "abstract": "Background: Johanson-Blizzard syndrome (JBS; OMIM 243800) is an autosomal recessive disorder that includes congenital exocrine pancreatic insufficiency, facial dysmorphism with the characteristic nasal wing hypoplasia, multiple malformations, and frequent mental retardation. Our previous work has shown that JBS is caused by mutations in human UBR1, which encodes one of the E3 ubiquitin ligases of the N-end rule pathway. The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. One class of degradation signals (degrons) recognized by UBR1 are destabilizing N-terminal residues of protein substrates.\nMethodology/Principal Findings: Most JBS-causing alterations of UBR1 are nonsense, frameshift or splice-site mutations that abolish UBR1 activity. We report here missense mutations of human UBR1 in patients with milder variants of JBS. These single-residue changes, including a previously reported missense mutation, involve positions in the RING-H2 and UBR domains of UBR1 that are conserved among eukaryotes. Taking advantage of this conservation, we constructed alleles of the yeast Saccharomyces cerevisiae UBR1 that were counterparts of missense JBS-UBR1 alleles. Among these yeast Ubr1 mutants, one of them (H160R) was inactive in yeast-based activity assays, the other one (Q1224E) had a detectable but weak activity, and the third one (V146L) exhibited a decreased but significant activity, in agreement with manifestations of JBS in the corresponding JBS patients.\nConclusions/Significance: These results, made possible by modeling defects of a human ubiquitin ligase in its yeast counterpart, verified and confirmed the relevance of specific missense UBR1 alleles to JBS, and suggested that a residual activity of a missense allele is causally associated with milder variants of JBS.", "doi": "10.1371/journal.pone.0024925", "pmcid": "PMC3172311", "issn": "1932-6203", "publisher": "Public Library of Science", "publication": "PLoS ONE", "publication_date": "2011-09-13", "series_number": "9", "volume": "6", "issue": "9", "pages": "Art.No. e24925" }, { "id": "authors:gqxrr-nyv08", "collection": "authors", "collection_id": "gqxrr-nyv08", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20110819-091924451", "type": "article", "title": "The N-end rule pathway and regulation by proteolysis", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. Degradation signals (degrons) that are targeted by the N-end rule pathway include a set called N-degrons. The main determinant of an N-degron is a destabilizing N-terminal residue of a protein. In eukaryotes, the N-end rule pathway is a part of the ubiquitin system and consists of two branches, the Ac/N-end rule and the Arg/N-end rule pathways. The Ac/N-end rule pathway targets proteins containing N^\u03b1-terminally acetylated (Nt-acetylated) residues. The Arg/N-end rule pathway recognizes unacetylated N-terminal residues and involves N-terminal arginylation. Together, these branches target for degradation a majority of cellular proteins. For example, more than 80% of human proteins are cotranslationally Nt-acetylated. Thus, most proteins harbor a specific degradation signal, termed ^(Ac)N-degron, from the moment of their birth. Specific N-end rule pathways are also present in prokaryotes and in mitochondria. Enzymes that produce N-degrons include methionine-aminopeptidases, caspases, calpains, Nt-acetylases, Nt-amidases, arginyl-transferases, and leucyl-transferases. Regulated degradation of specific proteins by the N-end rule pathway mediates a legion of physiological functions, including the sensing of heme, oxygen, and nitric oxide; selective elimination of misfolded proteins; the regulation of DNA repair, segregation, and condensation; the signaling by G proteins; the regulation of peptide import, fat metabolism, viral and bacterial infections, apoptosis, meiosis, spermatogenesis, neurogenesis, and cardiovascular development; and the functioning of adult organs, including the pancreas and the brain. Discovered 25 years ago, this pathway continues to be a fount of biological insights.", "doi": "10.1002/pro.666", "pmcid": "PMC3189519", "issn": "0961-8368", "publisher": "Wiley", "publication": "Protein Science", "publication_date": "2011-08", "series_number": "8", "volume": "20", "issue": "8", "pages": "1298-1345" }, { "id": "authors:snq5f-0xg71", "collection": "authors", "collection_id": "snq5f-0xg71", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20101220-134450477", "type": "article", "title": "The N-end rule pathway is mediated by a complex of the RING-type Ubr1 and HECT-type Ufd4 ubiquitin ligases", "author": [ { "family_name": "Hwang", "given_name": "Cheol-Sang", "orcid": "0000-0002-0105-5957", "clpid": "Hwang-Cheol-Sang" }, { "family_name": "Shemorry", "given_name": "Anna", "clpid": "Shemorry-A" }, { "family_name": "Auerbach", "given_name": "Daniel", "clpid": "Auerbach-D" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Substrates of the N-end rule pathway are recognized by the Ubr1 E3 ubiquitin ligase through their destabilizing amino-terminal residues. Our previous work showed that the Ubr1 E3 and the Ufd4 E3 together target an internal degradation signal (degron) of the Mgt1 DNA repair protein. Ufd4 is an E3 enzyme of the ubiquitin-fusion degradation (UFD) pathway that recognizes an N-terminal ubiquitin moiety. Here we show that the RING-type Ubr1 E3 and the HECT-type Ufd4 E3 interact, both physically and functionally. Although Ubr1 can recognize and polyubiquitylate an N-end rule substrate in the absence of Ufd4, the Ubr1\u2013Ufd4 complex is more processive in that it produces a longer substrate-linked polyubiquitin chain. Conversely, Ubr1 can function as a polyubiquitylation-enhancing component of the Ubr1\u2013Ufd4 complex in its targeting of UFD substrates. We also found that Ubr1 can recognize the N-terminal ubiquitin moiety. These and related advances unify two proteolytic systems that have been studied separately for two decades.", "doi": "10.1038/ncb2121", "pmcid": "PMC3003441", "issn": "1465-7392", "publisher": "Nature Publishing Group", "publication": "Nature Cell Biology", "publication_date": "2010-12", "series_number": "12", "volume": "12", "issue": "12", "pages": "1177-1185" }, { "id": "authors:1kvg5-3f930", "collection": "authors", "collection_id": "1kvg5-3f930", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20100615-141115251", "type": "article", "title": "Mouse Dfa Is a Repressor of TATA-box Promoters and Interacts with the Abt1 Activator of Basal Transcription", "author": [ { "family_name": "Brower", "given_name": "Christopher S.", "clpid": "Brower-C-S" }, { "family_name": "Veiga", "given_name": "Lucia", "clpid": "Veiga-L" }, { "family_name": "Jones", "given_name": "Richard H.", "clpid": "Jones-R-H" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Our study of the mouse Ate1 arginyltransferase, a component of the N-end rule pathway, has shown that Ate1 pre-mRNA is produced from a bidirectional promoter that also expresses, in the opposite direction, a previously uncharacterized gene (Hu, R. G., Brower, C. S., Wang, H., Davydov, I. V., Sheng, J., Zhou, J., Kwon, Y. T., and Varshavsky, A. (2006) J. Biol. Chem. 281, 32559\u201332573). In this work, we began analyzing this gene, termed Dfa (divergent from Ate1). Mouse Dfa was found to be transcribed from both the bidirectional P_(Ate1/Dfa) promoter and other nearby promoters. The resulting transcripts are alternatively spliced, yielding a complex set of Dfa mRNAs that are present largely, although not exclusively, in the testis. A specific Dfa mRNA encodes, via its 3\u2032-terminal exon, a 217-residue protein termed Dfa^A. Other Dfa mRNAs also contain this exon. DfaA is sequelogous (similar in sequence) to a region of the human/mouse HTEX4 protein, whose physiological function is unknown. We produced an affinity-purified antibody to recombinant mouse DfaA that detected a 35-kDa protein in the mouse testis and in several cell lines. Experiments in which RNA interference was used to down-regulate Dfa indicated that the 35-kDa protein was indeed Dfa^A. Furthermore, Dfa^A was present in the interchromatin granule clusters and was also found to bind to the Ggnbp1 gametogenetin-binding protein-1 and to the Abt1 activator of basal transcription that interacts with the TATA-binding protein. Given these results, RNA interference was used to probe the influence of Dfa levels in luciferase reporter assays. We found that Dfa^A acts as a repressor of TATA-box transcriptional promoters.", "doi": "10.1074/jbc.M110.118638", "pmcid": "PMC2878062", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "2010-05-28", "series_number": "22", "volume": "285", "issue": "22", "pages": "17218-17234" }, { "id": "authors:d72pn-2as98", "collection": "authors", "collection_id": "d72pn-2as98", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20100301-145500742", "type": "article", "title": "N-Terminal Acetylation of Cellular Proteins Creates Specific Degradation Signals", "author": [ { "family_name": "Hwang", "given_name": "Cheol-Sang", "orcid": "0000-0002-0105-5957", "clpid": "Hwang-Cheol-Sang" }, { "family_name": "Shemorry", "given_name": "Anna", "clpid": "Shemorry-A" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The retained N-terminal methionine (Met) residue of a nascent protein is often N-terminally acetylated (Nt-acetylated). Removal of N-terminal Met by Met-aminopeptidases frequently leads to Nt-acetylation of the resulting N-terminal alanine (Ala), valine (Val), serine (Ser), threonine (Thr), and cysteine (Cys) residues. Although a majority of eukaryotic proteins (for example, more than 80% of human proteins) are cotranslationally Nt-acetylated, the function of this extensively studied modification is largely unknown. Using the yeast Saccharomyces cerevisiae, we found that the Nt-acetylated Met residue could act as a degradation signal (degron), targeted by the Doa10 ubiquitin ligase. Moreover, Doa10 also recognized the Nt-acetylated Ala, Val, Ser, Thr, and Cys residues. Several examined proteins of diverse functions contained these N-terminal degrons, termed ^(Ac)N-degrons, which are a prevalent class of degradation signals in cellular proteins.", "doi": "10.1126/science.1183147", "pmcid": "PMC4259118", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2010-02-19", "series_number": "5968", "volume": "327", "issue": "5968", "pages": "973-977" }, { "id": "authors:1ca60-xaj70", "collection": "authors", "collection_id": "1ca60-xaj70", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20100127-092828613", "type": "article", "title": "Ablation of Arginylation in the Mouse N-End Rule Pathway: Loss of Fat, Higher Metabolic Rate, Damaged Spermatogenesis, and Neurological Perturbations", "author": [ { "family_name": "Brower", "given_name": "Christopher S.", "clpid": "Brower-C-S" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "In the N-end rule pathway of protein degradation, the destabilizing activity of N-terminal Asp, Glu or (oxidized) Cys residues requires their conjugation to Arg, which is recognized directly by pathway's ubiquitin ligases. N-terminal arginylation is mediated by the Ate1 arginyltransferase, whose physiological substrates include the Rgs4, Rgs5 and Rgs16 regulators of G proteins. Here, we employed the Cre-lox technique to uncover new physiological functions of N-terminal arginylation in adult mice. We show that postnatal deletion of mouse Ate1 (its unconditional deletion is embryonic lethal) causes a rapid decrease of body weight and results in early death of ~15% of Ate1-deficient mice. Despite being hyperphagic, the surviving Ate1-deficient mice contain little visceral fat. They also exhibit an increased metabolic rate, ectopic induction of the Ucp1 uncoupling protein in white fat, and are resistant to diet-induced obesity. In addition, Ate1-deficient mice have enlarged brains, an enhanced startle response, are strikingly hyperkinetic, and are prone to seizures and kyphosis. Ate1-deficient males are also infertile, owing to defects in Ate1^(\u2212/\u2212) spermatocytes. The remarkably broad range of specific biological processes that are shown here to be perturbed by the loss of N-terminal arginylation will make possible the dissection of regulatory circuits that involve Ate1 and either its known substrates, such as Rgs4, Rgs5 and Rgs16, or those currently unknown.", "doi": "10.1371/journal.pone.0007757", "pmcid": "PMC2773024", "issn": "1932-6203", "publisher": "Public Library of Science", "publication": "PLoS ONE", "publication_date": "2009-11-13", "series_number": "11", "volume": "4", "issue": "11", "pages": "Art. No. e7757" }, { "id": "authors:wrjdf-kq640", "collection": "authors", "collection_id": "wrjdf-kq640", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20090828-231031940", "type": "article", "title": "The N-end rule pathway controls multiple functions during Arabidopsis shoot and leaf development", "author": [ { "family_name": "Graciet", "given_name": "Emmanuelle", "clpid": "Graciet-E" }, { "family_name": "Walter", "given_name": "Franziska", "clpid": "Walter-Franziska" }, { "family_name": "\u00d3 Maoil\u00e9idigh", "given_name": "Diarmuid", "clpid": "\u00d3-Maoil\u00e9idigh-D" }, { "family_name": "Pollmann", "given_name": "Stephan", "clpid": "Pollmann-S" }, { "family_name": "Meyerowitz", "given_name": "Elliot M.", "orcid": "0000-0003-4798-5153", "clpid": "Meyerowitz-E-M" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Wellmer", "given_name": "Frank", "clpid": "Wellmer-F" } ], "abstract": "The ubiquitin-dependent N-end rule pathway relates the in vivo half-life of a protein to the identity of its N-terminal residue. This proteolytic system is present in all organisms examined and has been shown to have a multitude of functions in animals and fungi. In plants, however, the functional understanding of the N-end rule pathway is only beginning. The N-end rule has a hierarchic structure. Destabilizing activity of N-terminal Asp, Glu, and (oxidized) Cys requires their conjugation to Arg by an arginyl\u2013tRNA\u2013protein transferase (R-transferase). The resulting N-terminal Arg is recognized by the pathway's E3 ubiquitin ligases, called \"N-recognins.\" Here, we show that the Arabidopsis R-transferases AtATE1 and AtATE2 regulate various aspects of leaf and shoot development. We also show that the previously identified N-recognin PROTEOLYSIS6 (PRT6) mediates these R-transferase-dependent activities. We further demonstrate that the arginylation branch of the N-end rule pathway plays a role in repressing the meristem-promoting BREVIPEDICELLUS (BP) gene in developing leaves. BP expression is known to be excluded from Arabidopsis leaves by the activities of the ASYMMETRIC LEAVES1 (AS1) transcription factor complex and the phytohormone auxin. Our results suggest that AtATE1 and AtATE2 act redundantly with AS1, but independently of auxin, in the control of leaf development.", "doi": "10.1073/pnas.0906404106", "pmcid": "PMC2726413", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2009-08-11", "series_number": "32", "volume": "106", "issue": "32", "pages": "13618-13623" }, { "id": "authors:f5jbv-w8333", "collection": "authors", "collection_id": "f5jbv-w8333", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20090828-112810807", "type": "article", "title": "Glutamine-Specific N-Terminal Amidase, a Component of the N-End Rule Pathway", "author": [ { "family_name": "Wang", "given_name": "Haiqing", "clpid": "Wang-Haiqing" }, { "family_name": "Piatkov", "given_name": "Konstantin I.", "clpid": "Piatkov-K-I" }, { "family_name": "Brower", "given_name": "Christopher S.", "clpid": "Brower-C-S" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Deamidation of N-terminal Gln by Nt^Q-amidase, an N-terminal amidohydrolase, is a part of the N-end rule pathway of protein degradation. We detected the activity of Nt^Q-amidase, termed Ntaq1, in mouse tissues, purified Ntaq1 from bovine brains, identified its gene, and began analyzing this enzyme. Ntaq1 is highly conserved among animals, plants, and some fungi, but its sequence is dissimilar to sequences of other amidases. An earlier mutant in the Drosophila Cg8253 gene that we show here to encode Nt^Q-amidase has defective long-term memory. Other studies identified protein ligands of the uncharacterized human C8orf32 protein that we show here to be the Ntaq1 Nt^Q-amidase. Remarkably, \"high-throughput\" studies have recently solved the crystal structure of C8orf32 (Ntaq1). Our site-directed mutagenesis of Ntaq1 and its crystal structure indicate that the active site and catalytic mechanism of Nt^Q-amidase are similar to those of transglutaminases.", "doi": "10.1016/j.molcel.2009.04.032", "pmcid": "PMC2749074", "issn": "1097-2765", "publisher": "Elsevier", "publication": "Molecular Cell", "publication_date": "2009-06-26", "series_number": "6", "volume": "34", "issue": "6", "pages": "686-695" }, { "id": "authors:nrafk-8z197", "collection": "authors", "collection_id": "nrafk-8z197", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20090827-155821961", "type": "article", "title": "Two proteolytic pathways regulate DNA repair by cotargeting the Mgt1 alkylguanine transferase", "author": [ { "family_name": "Hwang", "given_name": "Cheol-Sang", "orcid": "0000-0002-0105-5957", "clpid": "Hwang-Cheol-Sang" }, { "family_name": "Shemorry", "given_name": "Anna", "clpid": "Shemorry-A" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "O^6-methylguanine (O^6meG) and related modifications of guanine in double-stranded DNA are functionally severe lesions that can be produced by many alkylating agents, including N-methyl-N\u2032-nitro-N-nitrosoguanidine (MNNG), a potent carcinogen. O^6meG is repaired through its demethylation by the O^6-alkylguanine-DNA alkyltransferase (AGT). This protein is called Mgmt (or MGMT) in mammals and Mgt1 in the yeast Saccharomyces cerevisiae. AGT proteins remove methyl and other alkyl groups from an alkylated O^6 in guanine by transferring the adduct to an active-site cysteine residue. The resulting S-alkyl-Cys of AGT is not restored back to Cys, so repair proteins of this kind can act only once. We report here that S. cerevisiae Mgt1 is cotargeted for degradation, through a degron near its N terminus, by 2 ubiquitin-mediated proteolytic systems, the Ubr1/Rad6-dependent N-end rule pathway and the Ufd4/Ubc4-dependent ubiquitin fusion degradation (UFD) pathway. The cotargeting of Mgt1 by these pathways is synergistic, in that it increases not only the yield of polyubiquitylated Mgt1, but also the processivity of polyubiquitylation. The N-end rule and UFD pathways comediate both the constitutive and MNNG-accelerated degradation of Mgt1. Yeast cells lacking the Ubr1 and Ufd4 ubiquitin ligases were hyperresistant to MNNG but hypersensitive to the toxicity of overexpressed Mgt1. We consider ramifications of this discovery for the control of DNA repair and mechanisms of substrate targeting by the ubiquitin system.", "doi": "10.1073/pnas.0812316106", "pmcid": "PMC2650122", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2009-02-17", "series_number": "7", "volume": "106", "issue": "7", "pages": "2142-2147" }, { "id": "authors:fq2bc-6yw80", "collection": "authors", "collection_id": "fq2bc-6yw80", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:VARjbc08", "type": "article", "title": "Discovery of cellular regulation by protein degradation", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "What follows is a story of some of the lab's adventures mentioned above, including the inventions of new biochemical and genetic methods. This account stems, in part, from previous descriptions of the early history of the Ub field (31,32). Another antecedent is an interview I gave to Dr. Istvan Hargittai, a distinguished Hungarian chemist. It describes my life and science, including the early years in Moscow, the 1977 escape from the former Soviet Union, the essentially accidental hiring of me by MIT, and the work that ensued (33). The narrative below borrows from these sources, and mentions our more recent contributions as well.", "doi": "10.1074/jbc.X800009200", "pmcid": "PMC3259866", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "2008-12-12", "series_number": "50", "volume": "283", "issue": "50", "pages": "34469-34489" }, { "id": "authors:tsqr1-10892", "collection": "authors", "collection_id": "tsqr1-10892", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:HWApnas08", "type": "article", "title": "Regulation of peptide import through phosphorylation of Ubr1, the ubiquitin ligase of the N-end rule pathway", "author": [ { "family_name": "Hwang", "given_name": "Cheol-Sang", "orcid": "0000-0002-0105-5957", "clpid": "Hwang-Cheol-Sang" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Substrates of the N-end rule pathway include proteins with destabilizing N-terminal residues. These residues are recognized by E3 ubiquitin ligases called N-recognins. Ubr1 is the N-recognin of the yeast Saccharomyces cerevisiae. Extracellular amino acids or short peptides up-regulate the peptide transporter gene PTR2, thereby increasing the capacity of a cell to import peptides. Cup9 is a transcriptional repressor that down-regulates PTR2. The induction of PTR2 by peptides or amino acids involves accelerated degradation of Cup9 by the N-end rule pathway. We report here that the Ubr1 N-recognin, which conditionally targets Cup9 for degradation, is phosphorylated in vivo at multiple sites, including Ser300 and Tyr277. We also show that the type-I casein kinases Yck1 and Yck2 phosphorylate Ubr1 on Ser300, and thereby make possible (\"prime\") the subsequent (presumably sequential) phosphorylations of Ubr1 on Ser296, Ser292, Thr288, and Tyr277 by Mck1, a kinase of the glycogen synthase kinase 3 (Gsk3) family. Phosphorylation of Ubr1 on Tyr277 by Mck1 is a previously undescribed example of a cascade-based tyrosine phosphorylation by a Gsk3-type kinase outside of autophosphorylation. We show that the Yck1/Yck2-mediated phosphorylation of Ubr1 on Ser300 plays a major role in the control of peptide import by the N-end rule pathway. In contrast to phosphorylation on Ser300, the subsequent (primed) phosphorylations, including the one on Tyr277, have at most minor effects on the known properties of Ubr1, including regulation of peptide import. Thus, a biological role of the rest of Ubr1 phosphorylation cascade remains to be identified.", "doi": "10.1073/pnas.0808891105", "pmcid": "PMC2614737", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2008-12-09", "series_number": "49", "volume": "105", "issue": "49", "pages": "19188-19193" }, { "id": "authors:jn5py-sja73", "collection": "authors", "collection_id": "jn5py-sja73", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:VARnsmb08", "type": "article", "title": "The N-end rule at atomic resolution", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway, ubiquitin-dependent in eukaryotes, is also present in prokaryotes, which lack the ubiquitin system. An illuminating new study presents the crystal structure of a bacterial N-end rule recognition component in complex with a peptide containing a cognate degradation signal.", "doi": "10.1038/nsmb1208-1238", "issn": "1545-9985", "publisher": "Nature Publishing Group", "publication": "Nature Structural & Molecular Biology", "publication_date": "2008-12", "series_number": "12", "volume": "15", "issue": "12", "pages": "1238-1240" }, { "id": "authors:c9yn1-7c083", "collection": "authors", "collection_id": "c9yn1-7c083", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:XIAjbc08b", "type": "article", "title": "Amino acids induce peptide uptake via accelerated degradation of CUP9, the transcriptional repressor of the PTR2 peptide transporter", "author": [ { "family_name": "Xia", "given_name": "Zanxian", "clpid": "Xia-Zanxian" }, { "family_name": "Turner", "given_name": "Glenn C.", "clpid": "Turner-G-C" }, { "family_name": "Hwang", "given_name": "Cheol-Sang", "orcid": "0000-0002-0105-5957", "clpid": "Hwang-Cheol-Sang" }, { "family_name": "Byrd", "given_name": "Christopher", "clpid": "Byrd-C" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Multiple pathways link expression of PTR2, the transporter of di- and tripeptides in the yeast Saccharomyces cerevisiae, to the availability and quality of nitrogen sources. Previous work has shown that induction of PTR2 by extracellular amino acids requires, in particular, SSY1 and PTR3. SSY1 is structurally similar to amino acid transporters, but functions as a sensor of amino acids. PTR3 acts downstream of SSY1. Expression of the PTR2 peptide transporter is induced not only by amino acids but also by dipeptides with destabilizing N-terminal residues. These dipeptides bind to UBR1, the ubiquitin ligase of the N-end rule pathway, and allosterically accelerate the UBR1-dependent degradation of CUP9, a transcriptional repressor of PTR2. UBR1 targets CUP9 through its internal degron. Here we demonstrate that the repression of PTR2 by CUP9 requires TUP1 and SSN6, the corepressor proteins that form a complex with CUP9. We also show that the induction of PTR2 by amino acids is mediated by the UBR1-dependent acceleration of CUP9 degradation that requires both SSY1 and PTR3. The acceleration of CUP9 degradation is shown to be attained without increasing the activity of the N-end rule pathway toward substrates with destabilizing N-terminal residues. We also found that GAP1, a general amino acid transporter, strongly contributes to the induction of PTR2 by Trp. While several aspects of this complex circuit remain to be understood, our findings establish new functional links between the amino acids-sensing SPS system, the CUP9-TUP1-SSN6 repressor complex, the PTR2 peptide transporter and the UBR1-dependent N-end rule pathway.", "doi": "10.1074/jbc.M803980200", "pmcid": "PMC2570885", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "2008-10-24", "series_number": "43", "volume": "283", "issue": "43", "pages": "28958-28968" }, { "id": "authors:9cyyg-hf949", "collection": "authors", "collection_id": "9cyyg-hf949", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:XIAjbc08", "type": "article", "title": "Substrate-binding sites of UBR1, the ubiquitin ligase of the N-end rule pathway", "author": [ { "family_name": "Xia", "given_name": "Zanxian", "clpid": "Xia-Zanxian" }, { "family_name": "Webster", "given_name": "Ailsa", "clpid": "Webster-A" }, { "family_name": "Du", "given_name": "Fangyong", "clpid": "Du-Fangyong" }, { "family_name": "Piatkov", "given_name": "Konstantin", "clpid": "Piatkov-K" }, { "family_name": "Ghislain", "given_name": "Michel", "clpid": "Ghislain-M" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Substrates of a ubiquitin-dependent proteolytic system called the N-end rule pathway include proteins with destabilizing N-terminal residues. N-recognins, the pathway's ubiquitin ligases, contain three substrate-binding sites. The type-1 site is specific for basic N-terminal residues (Arg, Lys, His). The type-2 site is specific for bulky hydrophobic N-terminal residues (Trp, Phe, Tyr, Leu, Ile). We show here that the type-1/2 sites of UBR1, the sole N-recognin of the yeast Saccharomyces cerevisiae, are located in the first ~700 residues of the 1,950-residue UBR1. These sites are distinct in that they can be selectively inactivated by mutations, identified through a genetic screen. Mutations inactivating the type-1 site are in the previously delineated ~70 residue UBR motif characteristic of N-recognins. Fluorescence polarization and surface plasmon resonance were used to determine that UBR1 binds, with Kd of ~1 microM, to either type-1 or type-2 destabilizing N-terminal residues of reporter peptides, but does not bind to a stabilizing N-terminal residue such as Gly. A third substrate-binding site of UBR1 targets an internal degron of CUP9, a transcriptional repressor of peptide import. We show that the previously demonstrated in vivo dependence of CUP9 ubiquitylation on the binding of (cognate) dipeptides to the type-1/2 sites of UBR1 can be reconstituted in a completely defined in vitro system. We also found that purified UBR1 and CUP9 interact nonspecifically, and that specific binding (which involves, in particular, the binding by cognate dipeptides to the UBR1's type-1/2 sites) can be restored either by a chaperone such as EF1A or through macromolecular crowding.", "doi": "10.1074/jbc.M802583200", "pmcid": "PMC2527112", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "2008-08-29", "series_number": "35", "volume": "283", "issue": "35", "pages": "24011-24028" }, { "id": "authors:ajqc6-a6z95", "collection": "authors", "collection_id": "ajqc6-a6z95", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150112-114337627", "type": "article", "title": "Enzymatic N-terminal Addition of Noncanonical Amino Acids to Peptides and Proteins", "author": [ { "family_name": "Connor", "given_name": "Rebecca E.", "clpid": "Connor-R-E" }, { "family_name": "Piatkov", "given_name": "Konstantin", "clpid": "Piatkov-K-I" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Tirrell", "given_name": "David A.", "orcid": "0000-0003-3175-4596", "clpid": "Tirrell-D-A" } ], "abstract": "A chromatographic assay was used to identify ten noncanonical amino acids that can be appended to the N terminus of peptides and proteins by the L,F-transferase of Escherichia coli. A model protein substrate, E. coli dihydrofolate reductase, was modified with p-ethynylphenylalanine and conjugated to azide\u2013biotin and azide\u2013polyethylene glycol\u2013fluorescein probes (see scheme).", "doi": "10.1002/cbic.200700605", "issn": "1439-4227", "publisher": "Wiley", "publication": "ChemBioChem", "publication_date": "2008-02-15", "series_number": "3", "volume": "9", "issue": "3", "pages": "366-369" }, { "id": "authors:n5xjg-c3w52", "collection": "authors", "collection_id": "n5xjg-c3w52", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:HURpnas08", "type": "article", "title": "The N-end rule pathway is a sensor of heme", "author": [ { "family_name": "Hu", "given_name": "Rong-Gui", "clpid": "Hu-Rong-Gui" }, { "family_name": "Wang", "given_name": "Haiqing", "clpid": "Wang-Haiqing" }, { "family_name": "Xia", "given_name": "Zanxian", "clpid": "Xia-Zanxian" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The conjugation of arginine, by arginyl-transferase, to N-terminal aspartate, glutamate or oxidized cysteine is a part of the N-end rule pathway of protein degradation. We report that arginyl-transferase of either the mouse or the yeast Saccharomyces cerevisiae is inhibited by hemin (Fe3+-heme). Furthermore, we show that hemin inhibits arginyl-transferase through a redox mechanism that involves the formation of disulfide between the enzyme's Cys-71 and Cys-72 residues. Remarkably, hemin also induces the proteasome-dependent degradation of arginyl-transferase in vivo, thus acting as both a \"stoichiometric\" and \"catalytic\" down-regulator of the N-end rule pathway. In addition, hemin was found to interact with the yeast and mouse E3 ubiquitin ligases of the N-end rule pathway. One of substrate-binding sites of the yeast N-end rule's ubiquitin ligase UBR1 targets CUP9, a transcriptional repressor. This site of UBR1 is autoinhibited but can be allosterically activated by peptides that bear destabilizing N-terminal residues and interact with two other substrate-binding sites of UBR1. We show that hemin does not directly occlude the substrate-binding sites of UBR1 but blocks the activation of its CUP9-binding site by dipeptides. The N-end rule pathway, a known sensor of short peptides, nitric oxide, and oxygen, is now a sensor of heme as well. One function of the N-end rule pathway may be to coordinate the activities of small effectors, both reacting to and controlling the redox dynamics of heme, oxygen, nitric oxide, thiols, and other compounds, in part through conditional degradation of specific transcription factors and G protein regulators.", "doi": "10.1073/pnas.0710568105", "pmcid": "PMC2224235", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2008-01-08", "series_number": "1", "volume": "105", "issue": "1", "pages": "76-81" }, { "id": "authors:bwefx-f1811", "collection": "authors", "collection_id": "bwefx-f1811", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:SCHNUiai07", "type": "article", "title": "Listeriolysin O Secreted by Listeria monocytogenes into the Host Cell Cytosol Is Degraded by the N-End Rule Pathway", "author": [ { "family_name": "Schnupf", "given_name": "Pamela", "clpid": "Schnupf-P" }, { "family_name": "Zhou", "given_name": "Jianmin", "clpid": "Zhou-Jianmin" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Portnoy", "given_name": "Daniel A.", "clpid": "Portnoy-D-A" } ], "abstract": "The intracellular pathogen Listeria monocytogenes escapes from a phagosomal compartment into the cytosol by secreting the pore-forming cytolysin listeriolysin O (LLO). During the proliferation of L. monocytogenes bacteria in the mammalian cell cytosol, the secreted LLO is targeted for degradation by the ubiquitin system. We report here that LLO is a substrate of the ubiquitin-dependent N-end rule pathway, which recognizes LLO through its N-terminal Lys residue. Specifically, we demonstrated by reverse-genetic and pharmacological methods that LLO was targeted for degradation by the N-end rule pathway in reticulocyte extracts and mouse NIH 3T3 cells and after its secretion by intracellular bacteria into the mouse cell cytosol. Replacing the N-terminal Lys of LLO with a stabilizing residue such as Val increased the in vivo half-life of LLO but did not strongly affect the intracellular growth or virulence of L. monocytogenes. Nevertheless, this replacement decreased the virulence of L. monocytogenes by nearly twofold, suggesting that a destabilizing N-terminal residue of LLO may stem from positive selection during the evolution of this and related bacteria. A double mutant strain of L. monocytogenes in which upregulated secretion of LLO was combined with a stabilizing N-terminal residue was severely toxic to infected mammalian cells, resulting in reduced intracellular growth of bacteria and an ~100-fold-lower level of virulence. In summary, we showed that LLO is degraded by the N-end rule pathway and that the degradation of LLO can reduce the toxicity of L. monocytogenes during infection, a property of LLO that may have been selected for its positive effects on fitness during the evolution of L. monocytogenes.", "doi": "10.1128/IAI.00164-07", "pmcid": "PMC2168281", "issn": "0019-9567", "publisher": "American Society for Microbiology", "publication": "Infection and Immunity", "publication_date": "2007-11", "series_number": "11", "volume": "75", "issue": "11", "pages": "5135-5147" }, { "id": "authors:tn5jp-73t17", "collection": "authors", "collection_id": "tn5jp-73t17", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:VARpnas07", "type": "article", "title": "Targeting the absence: Homozygous DNA deletions as immutable signposts for cancer therapy", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Many cancers harbor homozygous DNA deletions (HDs). In contrast to other attributes of cancer cells, their HDs are immutable features that cannot change during tumor progression or therapy. I describe an approach, termed deletion-specific targeting (DST), that employs HDs (not their effects on RNA/protein circuits, but deletions themselves) as the targets of cancer therapy. The DST strategy brings together both existing and new methodologies, including the ubiquitin fusion technique, the split-ubiquitin assay, zinc-finger DNA-recognizing proteins and split restriction nucleases. The DST strategy also employs a feedback mechanism that receives input from a circuit operating as a Boolean OR gate and involves the activation of split nucleases, which destroy DST vector in normal (nontarget) cells. The logic of DST makes possible an incremental and essentially unlimited increase in the selectivity of therapy. If DST strategy can be implemented in a clinical setting, it may prove to be curative and substantially free of side effects.", "doi": "10.1073/pnas.0706546104", "pmcid": "PMC1986591", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2007-09-18", "series_number": "38", "volume": "104", "issue": "38", "pages": "14935-14940" }, { "id": "authors:nh9nf-t4139", "collection": "authors", "collection_id": "nh9nf-t4139", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:TASjbc07", "type": "article", "title": "Biochemical and Genetic Studies of UBR3, a Ubiquitin Ligase with a Function in Olfactory and Other Sensory Systems", "author": [ { "family_name": "Tasaki", "given_name": "Takafumi", "clpid": "Tasaki-Takafumi" }, { "family_name": "Sohr", "given_name": "Reinhard", "clpid": "Sohr-Reinhard" }, { "family_name": "Xia", "given_name": "Zanxian", "clpid": "Xia-Zanxian" }, { "family_name": "Hellweg", "given_name": "Rainer", "clpid": "Hellweg-Rainer" }, { "family_name": "H\u00f6rtnagl", "given_name": "Heidi", "clpid": "H\u00f6rtnagl-Heidi" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Kwon", "given_name": "Yong Tae", "clpid": "Kwon-Yong-Tae" } ], "abstract": "Our previous work identified E3 ubiquitin ligases, termed UBR1-UBR7, that contain the ~70-residue UBR box, a motif important for the targeting of N-end rule substrates. In this pathway, specific N-terminal residues of substrates are recognized as degradation signals by UBR box-containing E3s that include UBR1, UBR2, UBR4, and UBR5. The other E3s of this set, UBR3, UBR6, and UBR7, remained uncharacterized. Here we describe the cloning and analyses of mouse UBR3. The similarities of UBR3 to the UBR1 and UBR2 E3s of the N-end rule pathway include the RING and UBR domains. We show that HR6A and HR6B, the E2 enzymes that bind to UBR1 and UBR2, also interact with UBR3. However, in contrast to UBR1 and UBR2, UBR3 does not recognize N-end rule substrates. We also constructed UBR3-lacking mouse strains. In the 129SvImJ background, UBR3-/- mice died during embryogenesis, whereas the C57BL/6 background UBR3-/- mice exhibited neonatal lethality and suckling impairment that could be partially rescued by litter size reduction. The adult UBR3-/- mice had female-specific behavioral anosmia. Cells of the olfactory pathway were found to express beta-galactosidase (LacZ) that marked the deletion/disruption UBR3- allele. The UBR3-specific LacZ expression was also prominent in cells of the touch, vision, hearing, and taste systems, suggesting a regulatory role of UBR3 in sensory pathways, including olfaction. By analogy with functions of the UBR domain in the N-end rule pathway, we propose that the UBR box of UBR3 may recognize small compounds that modulate the targeting, by this E3, of its currently unknown substrates.", "doi": "10.1074/jbc.M701894200", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "2007-06-22", "series_number": "25", "volume": "282", "issue": "25", "pages": "18510-18520" }, { "id": "authors:qe58w-4v927", "collection": "authors", "collection_id": "qe58w-4v927", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:HURjbc06", "type": "article", "title": "Arginyltransferase, Its Specificity, Putative Substrates, Bidirectional Promoter, and Splicing-derived Isoforms", "author": [ { "family_name": "Hu", "given_name": "Rong-Gui", "clpid": "Hu-Rong-Gui" }, { "family_name": "Brower", "given_name": "Christopher S.", "clpid": "Brower-Christopher-S" }, { "family_name": "Wang", "given_name": "Haiqing", "clpid": "Wang-Haiqing" }, { "family_name": "Davydov", "given_name": "Ilia V.", "clpid": "Davydov-Ilia-V" }, { "family_name": "Sheng", "given_name": "Jun", "clpid": "Sheng-Jun" }, { "family_name": "Zhou", "given_name": "Jianmin", "clpid": "Zhou-Jianmin" }, { "family_name": "Kwon", "given_name": "Yong Tae", "clpid": "Kwon-Yong-Tae" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Substrates of the N-end rule pathway include proteins with destabilizing N-terminal residues. Three of them, Asp, Glu, and (oxidized) Cys, function through their conjugation to Arg, one of destabilizing N-terminal residues that are recognized directly by the pathway's ubiquitin ligases. The conjugation of Arg is mediated by arginyltransferase, encoded by ATE1. Through its regulated degradation of specific proteins, the arginylation branch of the N-end rule pathway mediates, in particular, the cardiovascular development, the fidelity of chromosome segregation, and the control of signaling by nitric oxide. We show that mouse ATE1 specifies at least six mRNA isoforms, which are produced through alternative splicing, encode enzymatically active arginyltransferases, and are expressed at varying levels in mouse tissues. We also show that the ATE1 promoter is bidirectional, mediating the expression of both ATE1 and an oppositely oriented, previously uncharacterized gene. In addition, we identified GRP78 (glucose-regulated protein 78) and protein-disulfide isomerase as putative physiological substrates of arginyltransferase. Purified isoforms of arginyltransferase that contain the alternative first exons differentially arginylate these proteins in extract from ATE1-/- embryos, suggesting that specific isoforms may have distinct functions. Although the N-end rule pathway is apparently confined to the cytosol and the nucleus, and although GRP78 and protein-disulfide isomerase are located largely in the endoplasmic reticulum, recent evidence suggests that these proteins are also present in the cytosol and other compartments in vivo, where they may become N-end rule substrates.", "doi": "10.1074/jbc.M604355200", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "2006-10-27", "series_number": "43", "volume": "281", "issue": "43", "pages": "32559-32573" }, { "id": "authors:vy84d-ydc22", "collection": "authors", "collection_id": "vy84d-ydc22", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:ANJpnas06", "type": "article", "title": "Impaired neurogenesis and cardiovascular development in mice lacking the E3 ubiquitin ligases UBR1 and UBR2 of the N-end rule pathway", "author": [ { "family_name": "An", "given_name": "Jee Young", "clpid": "An-Jee-Young" }, { "family_name": "Seo", "given_name": "Jai Wha", "clpid": "Seo-Jai-Wha" }, { "family_name": "Tasaki", "given_name": "Takafumi", "clpid": "Tasaki-Takafumi" }, { "family_name": "Lee", "given_name": "Min Jae", "clpid": "Lee-Min-Jae" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Kwon", "given_name": "Yong Tae", "clpid": "Kwon-Y-T" } ], "abstract": "The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. A subset of degradation signals recognized by the N-end rule pathway comprises the signals, called N-degrons, whose determinants include destabilizing N-terminal residues. Our previous work identified a family of at least four mammalian E3 ubiquitin ligases, including UBR1 and UBR2, that share the UBR box and recognize N-degrons. These E3 enzymes mediate the multifunctional N-end rule pathway, but their individual roles are just beginning to emerge. Mutations of UBR1 in humans are the cause of Johanson\u2013Blizzard syndrome. UBR1 and UBR2 are 46% identical and appear to be indistinguishable in their recognition of N-degrons. UBR1\u2013/\u2013 mice are viable but have defects that include pancreatic insufficiency, similarly to UBR1\u2013/\u2013 human patients with Johanson\u2013Blizzard syndrome. UBR2\u2013/\u2013 mice are inviable in some strain backgrounds and are defective in male meiosis. To examine functional relationships between UBR1 and UBR2, we constructed mouse strains lacking both of these E3s. We report here that UBR1\u2013/\u2013UBR2\u2013/\u2013 embryos die at midgestation, with defects in neurogenesis and cardiovascular development. These defects included reduced proliferation as well as precocious migration and differentiation of neural progenitor cells. The expression of regulators such as D-type cyclins and Notch1 was also altered in UBR1\u2013/\u2013UBR2\u2013/\u2013 embryos. We conclude that the functions of UBR1 and UBR2 are significantly divergent, in part because of differences in their expression patterns and possibly also because of differences in their recognition of protein substrates that contain degradation signals other than N-degrons.", "doi": "10.1073/pnas.0601700103", "pmcid": "PMC1458857", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2006-04-18", "series_number": "16", "volume": "103", "issue": "16", "pages": "6212-6217" }, { "id": "authors:2v8nh-r1s34", "collection": "authors", "collection_id": "2v8nh-r1s34", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:GRApnas06a", "type": "article", "title": "Aminoacyl-transferases and the N-end rule pathway of prokaryotic/eukaryotic specificity in a human pathogen", "author": [ { "family_name": "Graciet", "given_name": "Emmanuelle", "clpid": "Graciet-E" }, { "family_name": "Hu", "given_name": "Rong-Gui", "clpid": "Hu-Rong-Gui" }, { "family_name": "Piatkov", "given_name": "Konstantin", "clpid": "Piatkov-K" }, { "family_name": "Rhee", "given_name": "Joon Haeng", "clpid": "Rhee-Joon-Haeng" }, { "family_name": "Schwartz", "given_name": "Erich M.", "clpid": "Schwartz-E-M" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Primary destabilizing N-terminal residues (Ndp) are recognized directly by the targeting machinery. The recognition of secondary destabilizing N-terminal residues (Nds) is preceded by conjugation of an Ndp residue to Nds of a polypeptide substrate. In eukaryotes, ATE1-encoded arginyl-transferases (RD,E,C*-transferases) conjugate Arg (R), an Ndp residue, to Nds residues Asp (D), Glu (E), or oxidized Cys residue (C*). Ubiquitin ligases recognize the N-terminal Arg of a substrate and target the (ubiquitylated) substrate to the proteasome. In prokaryotes such as Escherichia coli, Ndp residues Leu (L) or Phe (F) are conjugated, by the aat-encoded Leu/Phe-transferase (L/FK,R-transferase), to N-terminal Arg or Lys, which are Nds in prokaryotes but Ndp in eukaryotes. In prokaryotes, substrates bearing the Ndp residues Leu, Phe, Trp, or Tyr are degraded by the proteasome-like ClpAP protease. Despite enzymological similarities between eukaryotic RD,E,C*-transferases and prokaryotic L/FK,R-transferases, there is no significant sequelogy (sequence similarity) between them. We identified an aminoacyl-transferase, termed Bpt, in the human pathogen Vibrio vulnificus. Although it is a sequelog of eukaryotic RD,E,C*-transferases, this prokaryotic transferase exhibits a \"hybrid\" specificity, conjugating Ndp Leu to Nds Asp or Glu. Another aminoacyl-transferase, termed ATEL1, of the eukaryotic pathogen Plasmodium falciparum, is a sequelog of prokaryotic L/FK,R-transferases (Aat), but has the specificity of eukaryotic RD,E,C*-transferases (ATE1). Phylogenetic analysis suggests that the substrate specificity of R-transferases arose by two distinct routes during the evolution of eukaryotes.", "doi": "10.1073/pnas.0511224103", "pmcid": "PMC1413915", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2006-02-28", "series_number": "9", "volume": "103", "issue": "9", "pages": "3078-3083" }, { "id": "authors:aqw7v-key86", "collection": "authors", "collection_id": "aqw7v-key86", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150319-070411230", "type": "article", "title": "Deficiency of UBR1, a ubiquitin ligase of the N-end rule pathway, causes pancreatic dysfunction, malformations and mental retardation (Johanson-Blizzard syndrome)", "author": [ { "family_name": "Zenker", "given_name": "Martin", "clpid": "Zenker-Martin" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Johanson-Blizzard syndrome (OMIM 243800) is an autosomal recessive disorder that includes congenital exocrine pancreatic insufficiency, multiple malformations such as nasal wing aplasia, and frequent mental retardation1. We mapped the disease-associated locus to chromosome 15q14\u201321.1 and identified mutations, mostly truncating ones, in the gene UBR1 in 12 unrelated families with Johanson-Blizzard syndrome. UBR1 encodes one of at least four functionally overlapping E3 ubiquitin ligases of the N-end rule pathway, a conserved proteolytic system whose substrates include proteins with destabilizing N-terminal residues. Pancreas of individuals with Johanson-Blizzard syndrome did not express UBR1 and had intrauterine-onset destructive pancreatitis. In addition, we found that Ubr1-/- mice, whose previously reported phenotypes include reduced weight and behavioral abnormalities, had an exocrine pancreatic insufficiency, with impaired stimulus-secretion coupling and increased susceptibility to pancreatic injury. Our findings indicate that deficiency of UBR1 perturbs the pancreas' acinar cells and other organs, presumably owing to metabolic stabilization of specific substrates of the N-end rule pathway.", "doi": "10.1038/ng1681", "issn": "1061-4036", "publisher": "Nature Publishing Group", "publication": "Nature Genetics", "publication_date": "2005-12", "series_number": "12", "volume": "37", "issue": "12", "pages": "1345-1350" }, { "id": "authors:jdz5q-wee59", "collection": "authors", "collection_id": "jdz5q-wee59", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150325-092008023", "type": "article", "title": "The N-end rule pathway as a nitric oxide sensor controlling the levels of multiple regulators", "author": [ { "family_name": "Hu", "given_name": "Rong-Gui", "clpid": "Hu-Rong-Gui" }, { "family_name": "Sheng", "given_name": "Jun", "clpid": "Sheng-Jun" }, { "family_name": "Xu", "given_name": "Zhenming", "clpid": "Xu-Zhenming" }, { "family_name": "Takahashi", "given_name": "Terry T.", "clpid": "Takahashi-Terry-T" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The conjugation of arginine to proteins is a part of the N-end rule pathway of protein degradation. Three amino (N)-terminal residues\u2014aspartate, glutamate and cysteine\u2014are arginylated by ATE1-encoded arginyl-transferases. Here we report that oxidation of N-terminal cysteine is essential for its arginylation. The in vivo oxidation of N-terminal cysteine, before its arginylation, is shown to require nitric oxide. We reconstituted this process in vitro as well. The levels of regulatory proteins bearing N-terminal cysteine, such as RGS4, RGS5 and RGS16, are greatly increased in mouse ATE1^-/- embryos, which lack arginylation. Stabilization of these proteins, the first physiological substrates of mammalian N-end rule pathway, may underlie cardiovascular defects in ATE1^-/- embryos. Our findings identify the N-end rule pathway as a new nitric oxide sensor that functions through its ability to destroy specific regulatory proteins bearing N-terminal cysteine, at rates controlled by nitric oxide and apparently by oxygen as well.", "doi": "10.1038/nature04027", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2005-10-13", "series_number": "7061", "volume": "437", "issue": "7061", "pages": "981-989" }, { "id": "authors:evgj9-7qf93", "collection": "authors", "collection_id": "evgj9-7qf93", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:TASmcb05.969", "type": "article", "title": "A Family of Mammalian E3 Ubiquitin Ligases That Contain the UBR Box Motif and Recognize N-Degrons", "author": [ { "family_name": "Tasaki", "given_name": "Takafumi", "clpid": "Tasaki-Takafumi" }, { "family_name": "Mulder", "given_name": "Lubbertus C. F.", "clpid": "Mulder-L-C-F" }, { "family_name": "Iwamatsu", "given_name": "Akihiro", "clpid": "Iwamatsu-A" }, { "family_name": "Lee", "given_name": "Min Jae", "clpid": "Lee-Min-Jae" }, { "family_name": "Davydov", "given_name": "Ilia V.", "clpid": "Davydov-I-V" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Muesing", "given_name": "Mark", "clpid": "Muesing-M" }, { "family_name": "Kwon", "given_name": "Yong Tae", "clpid": "Kwon-Yong-Tae" } ], "abstract": "A subset of proteins targeted by the N-end rule pathway bear degradation signals called N-degrons, whose determinants include destabilizing N-terminal residues. Our previous work identified mouse UBR1 and UBR2 as E3 ubiquitin ligases that recognize N-degrons. Such E3s are called N-recognins. We report here that while double-mutant UBR1\u2013/\u2013 UBR2\u2013/\u2013 mice die as early embryos, the rescued UBR1\u2013/\u2013 UBR2\u2013/\u2013 fibroblasts still retain the N-end rule pathway, albeit of lower activity than that of wild-type fibroblasts. An affinity assay for proteins that bind to destabilizing N-terminal residues has identified, in addition to UBR1 and UBR2, a huge (570 kDa) mouse protein, termed UBR4, and also the 300-kDa UBR5, a previously characterized mammalian E3 known as EDD/hHYD. UBR1, UBR2, UBR4, and UBR5 shared a ~70-amino-acid zinc finger-like domain termed the UBR box. The mammalian genome encodes at least seven UBR box-containing proteins, which we propose to call UBR1 to UBR7. UBR1\u2013/\u2013 UBR2\u2013/\u2013 fibroblasts that have been made deficient in UBR4 as well (through RNA interference) were significantly impaired in the degradation of N-end rule substrates such as the Sindbis virus RNA polymerase nsP4 (bearing N-terminal Tyr) and the human immunodeficiency virus type 1 integrase (bearing N-terminal Phe). Our results establish the UBR box family as a unique class of E3 proteins that recognize N-degrons or structurally related determinants for ubiquitin-dependent proteolysis and perhaps other processes as well.", "doi": "10.1128/MCB.25.16.7120-7136.2005", "pmcid": "PMC1190250", "issn": "0270-7306", "publisher": "American Society for Microbiology", "publication": "Molecular and Cellular Biology", "publication_date": "2005-08-15", "series_number": "16", "volume": "25", "issue": "16", "pages": "7120-7136" }, { "id": "authors:myk7w-z4w06", "collection": "authors", "collection_id": "myk7w-z4w06", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20111004-142209812", "type": "article", "title": "RECQL4, mutated in the Rothmund\u2013Thomson and RAPADILINO syndromes, interacts with ubiquitin ligases UBR1 and UBR2 of the N-end rule pathway", "author": [ { "family_name": "Yin", "given_name": "Jinhu", "clpid": "Yin-Jinhu" }, { "family_name": "Kwon", "given_name": "Yong Tae", "clpid": "Kwon-Yong-Tae" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Wang", "given_name": "Weidong", "clpid": "Wang-Weidong" } ], "abstract": "The Rothmund\u2013Thomson syndrome (growth retardation, skin and bone defects, predisposition to cancer) and the RAPADILINO syndrome are caused by mutations in the RECQL4 gene. The 133\u2005kDa RECQL4 is a putative DNA helicase, a member of the family that includes the BLM and WRN helicases. The latter are mutated, respectively, in the Bloom and Werner syndromes, whose manifestations include predisposition to cancer. Using antibodies to human RECQL4, we found that the bulk of RECQL4 was present in a cytoplasmic extract of HeLa cells, in contrast to the largely nuclear BLM and WRN helicases. However, in untransformed WI-38 fibroblasts, RECQL4 was found to be largely nuclear, and was present at significantly lower total levels than in transformed HeLa cells. RECQL4 from HeLa cells was isolated as a stable complex with UBR1 and UBR2. These 200\u2005kDa proteins are ubiquitin ligases of the N-end rule pathway, whose substrates include proteins with destabilizing N-terminal residues. The functions of this proteolytic pathway include the regulation of peptide import, chromosome stability, meiosis, apoptosis and cardiovascular development. Although the known role of UBR1 and UBR2 is to mediate polyubiquitylation (and subsequent degradation) of their substrates, the UBR1/2-bound RECQL4 was not ubiquitylated in vivo, and was a long-lived protein in HeLa cells. The isolated RECQL4\u2013UBR1/2 complex had a DNA-stimulated ATPase activity, but was inactive in DNA-based assays for helicases and translocases, the assays in which the BLM helicase was active. We discuss ramifications of these results, possible functions of RECQL4, and the involvement of the N-end rule pathway.", "doi": "10.1093/hmg/ddh269", "issn": "0964-6906", "publisher": "Oxford University Press", "publication": "Human Molecular Genetics", "publication_date": "2004-10-15", "series_number": "20", "volume": "13", "issue": "20", "pages": "2421-2430" }, { "id": "authors:tna4t-rkx96", "collection": "authors", "collection_id": "tna4t-rkx96", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:KWOmcb03", "type": "article", "title": "Female Lethality and Apoptosis of Spermatocytes in Mice Lacking the UBR2 Ubiquitin Ligase of the N-End Rule Pathway", "author": [ { "family_name": "Kwon", "given_name": "Yong Tae", "clpid": "Kwon-Yong-Tae" }, { "family_name": "Xia", "given_name": "Zanxian", "clpid": "Xia-Zanxian" }, { "family_name": "An", "given_name": "Jee Young", "clpid": "An-Jee-Young" }, { "family_name": "Tasaki", "given_name": "Takafumi", "clpid": "Tasaki-Takafumi" }, { "family_name": "Davydov", "given_name": "Ilia V.", "clpid": "Davydov-Ilia-V" }, { "family_name": "Seo", "given_name": "Jai Wha", "clpid": "Seo-Jai-Wha" }, { "family_name": "Sheng", "given_name": "Jun", "clpid": "Sheng-Jun" }, { "family_name": "Xie", "given_name": "Youming", "clpid": "Xie-Youming" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Substrates of the ubiquitin-dependent N-end rule pathway include proteins with destabilizing N-terminal residues. UBR1-/- mice, which lacked the pathway's ubiquitin ligase E3\u03b1, were viable and retained the N-end rule pathway. The present work describes the identification and analysis of mouse UBR2, a homolog of UBR1. We demonstrate that the substrate-binding properties of UBR2 are highly similar to those of UBR1, identifying UBR2 as the second E3 of the mammalian N-end rule pathway. UBR2-/- mouse strains were constructed, and their viability was found to be dependent on both gender and genetic background. In the strain 129 (inbred) background, the UBR2-/- genotype was lethal to most embryos of either gender. In the 129/B6 (mixed) background, most UBR2-/- females died as embryos, whereas UBR2-/- males were viable but infertile, owing to the postnatal degeneration of the testes. The gross architecture of UBR2-/- testes was normal and spermatogonia were intact as well, but UBR2-/- spermatocytes were arrested between leptotene/zygotene and pachytene and died through apoptosis. A conspicuous defect of UBR2-/- spermatocytes was the absence of intact synaptonemal complexes. We conclude that the UBR2 ubiquitin ligase and, hence, the N-end rule pathway are required for male meiosis and spermatogenesis and for an essential aspect of female embryonic development.", "doi": "10.1128/MCB.23.22.8255-8271.2003", "pmcid": "PMC262401", "issn": "0270-7306", "publisher": "American Society for Microbiology", "publication": "Molecular and Cellular Biology", "publication_date": "2003-11-15", "series_number": "22", "volume": "23", "issue": "22", "pages": "8255-8271" }, { "id": "authors:1e0ay-z5m55", "collection": "authors", "collection_id": "1e0ay-z5m55", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150325-153608289", "type": "article", "title": "The N-end rule and regulation of apoptosis", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The ubiquitin-dependent N-end rule pathway targets proteins for degradation through their destabilizing N-terminal residues. This pathway is known to control the import of peptides, chromosome stability and cardiovascular development. A new report identifies yet another function of the N-end rule pathway: the regulation of apoptosis through degradation of Drosophila melanogaster DIAP1.", "doi": "10.1038/ncb0503-373", "issn": "1465-7392", "publisher": "Nature Publishing Group", "publication": "Nature Cell Biology", "publication_date": "2003-05", "series_number": "5", "volume": "5", "issue": "5", "pages": "373-376" }, { "id": "authors:qqsba-rhv67", "collection": "authors", "collection_id": "qqsba-rhv67", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150325-152811477", "type": "article", "title": "UFD4 lacking the proteasome-binding region catalyses ubiquitination but is impaired in proteolysis", "author": [ { "family_name": "Xie", "given_name": "Youming", "clpid": "Xie-Youming" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The ubiquitin system recognizes degradation signals of protein substrates through E3\u2013E2 ubiquitin ligases, which produce a substrate-linked multi-ubiquitin chain. Ubiquitinated substrates are degraded by the 26S proteasome, which consists of the 20S protease and two 19S particles. We previously showed that UBR1 and UFD4, two E3 ligases of the yeast Saccharomyces cerevisiae, interact with specific proteasomal subunits. Here we advance this analysis for UFD4 and show that it interacts with RPT4 and RPT6, two subunits of the 19S particle. The 201-residue amino-terminal region of UFD4 is essential for its binding to RPT4 and RPT6. UFD4 \u0394N, which lacks this N-terminal region, adds ubiquitin to test substrates with apparently wild-type activity, but is impaired in conferring short half-lives on these substrates. We propose that interaction of a targeted substrate with the 26S proteasome involves contacts of specific proteasomal subunits with the substrate-bound ubiquitin ligase, with the substrate-linked multi-ubiquitin chain and with the substrate itself. This multiple-site binding may function to slow down dissociation of the substrate from the proteasome and to facilitate the unfolding of substrate through ATP-dependent movements of the chaperone subunits of the 19S particle.", "doi": "10.1038/ncb889", "issn": "1465-7392", "publisher": "Nature Publishing Group", "publication": "Nature Cell Biology", "publication_date": "2002-12", "series_number": "12", "volume": "4", "issue": "12", "pages": "1003-1007" }, { "id": "authors:nh11f-sq826", "collection": "authors", "collection_id": "nh11f-sq826", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:DUFpnas02", "type": "article", "title": "Pairs of dipeptides synergistically activate the binding of substrate by ubiquitin ligase through dissociation of its autoinhibitory domain", "author": [ { "family_name": "Du", "given_name": "Fangyong", "clpid": "Du-Fangyong" }, { "family_name": "Navarro-Garcia", "given_name": "Federico", "clpid": "Navarro-Garcia-F" }, { "family_name": "Xia", "given_name": "Zanxian", "clpid": "Xia-Zanxian" }, { "family_name": "Tasaki", "given_name": "Takafumi", "clpid": "Tasaki-Takafumi" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Protein degradation by the ubiquitin (Ub) system controls the concentrations of many regulatory proteins. The degradation signals (degrons) of these proteins are recognized by the system's Ub ligases (complexes of E2 and E3 enzymes). Two substrate-binding sites of UBR1, the E3 of the Wend rule pathway in the yeast Saccharomyces cerevisiae, recognize basic (type 1) and bulky hydrophobic (type 2) N-terminal residues of proteins or short peptides. A third substrate-binding site of UBR1 targets CUP9, a transcriptional repressor of the peptide transporter PTR2, through an internal (non-N-terminal) degron of CUP9. Previous work demonstrated that dipeptides with destabilizing N-terminal residues allosterically activate UBR1, leading to accelerated in vivo degradation of CUP9 and the induction of PTR2 expression. Through this positive feedback, S. cerevisiae can sense the presence of extracellular peptides and react by accelerating their uptake. Here, we show that dipeptides with destabilizing N-terminal residues cause dissociation of the C-terminal autoinhibitory domain of UBR1 from its N-terminal region that contains all three substrate-binding sites. This dissociation, which allows the interaction between UBR1 and CUP9, is strongly increased only if both type 1- and type 2-binding sites of UBR1 are occupied by dipeptides. An aspect of autoinhibition characteristic of yeast UBR1 also was observed with mammalian (mouse) UBR1. The discovery of autoinhibition in Ub ligases of the UBR family indicates that this regulatory mechanism may also control the activity of other Ub ligases.", "doi": "10.1073/pnas.172527399", "pmcid": "PMC137845", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2002-10-29", "series_number": "22", "volume": "99", "issue": "22", "pages": "14110-14115" }, { "id": "authors:zj5pg-4r183", "collection": "authors", "collection_id": "zj5pg-4r183", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20141114-160435229", "type": "article", "title": "An Essential Role of N-Terminal Arginylation in Cardiovascular Development", "author": [ { "family_name": "Kwon", "given_name": "Yong Tae", "clpid": "Kwon-Yong-Tae" }, { "family_name": "Kashina", "given_name": "Anna S.", "clpid": "Kashina-A-S" }, { "family_name": "Davydov", "given_name": "Ilia V.", "clpid": "Davydov-I-V" }, { "family_name": "Hu", "given_name": "Rong-Gui", "clpid": "Hu-Rong-Gui" }, { "family_name": "An", "given_name": "Jee Young", "clpid": "An-Jee-Young" }, { "family_name": "Seo", "given_name": "Jai Wha", "clpid": "Seo-Jai-Wha" }, { "family_name": "Du", "given_name": "Fangyong", "clpid": "Du-Fangyong" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The enzymatic conjugation of arginine to the N-termini of proteins is a part of the ubiquitin-dependent N-end rule pathway of protein degradation. In mammals, three N-terminal residues\u2014aspartate, glutamate, and cysteine\u2014are substrates for arginylation. The mouseATE1 gene encodes a family of Arg-tRNA-protein transferases (R-transferases) that mediate N-terminal arginylation. We constructed ATE1-lacking mouse strains and found thatATE1 \u2212/\u2212 embryos die with defects in heart development and in angiogenic remodeling of the early vascular plexus. Through biochemical analyses, we show that N-terminal cysteine, in contrast to N-terminal aspartate and glutamate, is oxidized before its arginylation by R-transferase, suggesting that the arginylation branch of the N-end rule pathway functions as an oxygen sensor.", "doi": "10.1126/science.1069531", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2002-07-05", "series_number": "5578", "volume": "297", "issue": "5578", "pages": "96-99" }, { "id": "authors:gew04-j8m51", "collection": "authors", "collection_id": "gew04-j8m51", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:KWOmcb01", "type": "article", "title": "Construction and Analysis of Mouse Strains Lacking the Ubiquitin Ligase UBR1 (E3\u03b1) of the N-End Rule Pathway", "author": [ { "family_name": "Kwon", "given_name": "Yong Tae", "clpid": "Kwon-Yong-Tae" }, { "family_name": "Xia", "given_name": "Zanxian", "clpid": "Xia-Xanxian" }, { "family_name": "Davydov", "given_name": "Ilia V.", "clpid": "Davydov-I-V" }, { "family_name": "Lecker", "given_name": "Stewart H.", "clpid": "Lecker-S-H" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In the yeast Saccharomyces cerevisiae, the UBR1-encoded ubiquitin ligase (E3) of the N-end rule pathway mediates the targeting of substrate proteins in part through binding to their destabilizing N-terminal residues. The functions of the yeast N-end rule pathway include fidelity of chromosome segregation and the regulation of peptide import. Our previous work described the cloning of cDNA and a gene encoding the 200-kDa mouse UBR1 (E3\u03b1). Here we show that mouse UBR1, in the presence of a cognate mouse ubiquitin-conjugating (E2) enzyme, can rescue the N-end rule pathway in ubr1\u0394 S. cerevisiae. We also constructed UBR1-/- mouse strains that lacked the UBR1 protein. UBR1-/- mice were viable and fertile but weighed significantly less than congenic +/+ mice. The decreased mass of UBR1-/- mice stemmed at least in part from smaller amounts of the skeletal muscle and adipose tissues. The skeletal muscle of UBR1-/- mice apparently lacked the N-end rule pathway and exhibited abnormal regulation of fatty acid synthase upon starvation. By contrast, and despite the absence of the UBR1 protein, UBR1-/- fibroblasts contained the N-end rule pathway. Thus, UBR1-/- mice are mosaics in regard to the activity of this pathway, owing to differential expression of proteins that can substitute for the ubiquitin ligase UBR1 (E3\u03b1). We consider these UBR1-like proteins and discuss the functions of the mammalian N-end rule pathway.", "doi": "10.1128/MCB.21.23.8007-8021.2001", "pmcid": "PMC99968", "issn": "0270-7306", "publisher": "American Society for Microbiology", "publication": "Molecular and Cellular Biology", "publication_date": "2001-12-01", "series_number": "23", "volume": "21", "issue": "23", "pages": "8007-8021" }, { "id": "authors:cc78h-k7v74", "collection": "authors", "collection_id": "cc78h-k7v74", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150331-072449879", "type": "article", "title": "Degradation of a cohesin subunit by the N-end rule pathway is essential for chromosome stability", "author": [ { "family_name": "Rao", "given_name": "Hai", "clpid": "Rao-Hai" }, { "family_name": "Uhlmann", "given_name": "Frank", "clpid": "Uhlmann-Frank" }, { "family_name": "Nasmyth", "given_name": "Kim", "clpid": "Nasmyth-Kim" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Cohesion between sister chromatids is established during DNA replication and depends on a protein complex called cohesin. At the metaphase\u2013anaphase transition in the yeast Saccharomyces cerevisiae, the ESP1-encoded protease separin cleaves SCC1, a subunit of cohesin with a relative molecular mass of 63,000 (M_r 63K). The resulting 33K carboxy-terminal fragment of SCC1 bears an amino-terminal arginine\u2014a destabilizing residue in the N-end rule. Here we show that the SCC1 fragment is short-lived (t_(1/2)\u2248 2 min), being degraded by the ubiquitin/proteasome-dependent N-end rule pathway. Overexpression of a long-lived derivative of the SCC1 fragment is lethal. In ubr1\u0394 cells, which lack the N-end rule pathway, we found a highly increased frequency of chromosome loss. The bulk of increased chromosome loss in ubr1\u0394 cells is caused by metabolic stabilization of the ESP1-produced SCC1 fragment. This fragment is the first physiological substrate of the N-end rule pathway that is targeted through its N-terminal residue. A number of yeast proteins bear putative cleavage sites for the ESP1 separin, suggesting other physiological substrates and functions of the N-end rule pathway.", "doi": "10.1038/35073627", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2001-04-19", "series_number": "6831", "volume": "410", "issue": "6831", "pages": "955" }, { "id": "authors:zg153-4ve19", "collection": "authors", "collection_id": "zg153-4ve19", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:XIEpnas01", "type": "article", "title": "RPN4 is a ligand, substrate, and transcriptional regulator of the 26S proteasome: A negative feedback circuit", "author": [ { "family_name": "Xie", "given_name": "Youming", "clpid": "Xie-Youming" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The RPN4 (SON1, UFD5) protein of the yeast Saccharomyces cerevisiae is required for normal levels of intracellular proteolysis. RPN4 is a transcriptional activator of genes encoding proteasomal subunits. Here we show that RPN4 is required for normal levels of these subunits. Further, we demonstrate that RPN4 is extremely short-lived (t(1/2) approximate to 2 min), that it directly interacts with RPN2, a subunit of the 26S proteasome, and that rpn4 Delta cells are perturbed in their cell cycle. The degradation signal of RPN4 was mapped to its N-terminal region, outside the transcription-activation domains of RPN4. The ability of RPN4 to augment the synthesis of proteasomal subunits while being metabolically unstable yields a negative feedback circuit in which the same protein up-regulates the proteasome production and is destroyed by the assembled active proteasome.", "doi": "10.1073/pnas.071022298", "pmcid": "PMC30606", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2001-03-13", "series_number": "6", "volume": "98", "issue": "6", "pages": "3056-3061" }, { "id": "authors:fjmx6-dmq70", "collection": "authors", "collection_id": "fjmx6-dmq70", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150513-100923361", "type": "article", "title": "The ubiquitin system", "author": [ { "family_name": "Hershko", "given_name": "Avram", "clpid": "Hershko-Avram" }, { "family_name": "Ciechanover", "given_name": "Aaron", "clpid": "Ciechanover-Aaron" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "It has been often stated that until recently the ubiquitin system was thought to be mainly a 'garbage disposal' for the removal of abnormal or damaged proteins. This statement is certainly not true for those who have been interested in the selective and regulated degradation of proteins in cells. The dynamic turnover of cellular proteins was discovered in the pioneering studies of Rudolf Schoenheimer in the 1930s, when he first used isotopically labeled compounds for biological studies. Between 1960 and1970 it became evident that protein degradation in animal cells is highly selective, and is important in the control of specific enzyme concentrations. The molecular mechanisms responsible for this process, however, remained unknown. Some imaginative models have been proposed to account for the selectivity of protein degradation, such as one suggesting that all cellular proteins are rapidly engulfed into the lysosome, but only short-lived proteins are degraded in the lysosome, whereas long-lived proteins escape back to the cytosol.", "doi": "10.1038/80384", "issn": "1078-8956", "publisher": "Nature Publishing Group", "publication": "Nature Medicine", "publication_date": "2000-10", "series_number": "10", "volume": "6", "issue": "10", "pages": "1073-1081" }, { "id": "authors:9hx9c-s0172", "collection": "authors", "collection_id": "9hx9c-s0172", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20141112-112135305", "type": "article", "title": "Detecting and Measuring Cotranslational Protein Degradation in Vivo", "author": [ { "family_name": "Turner", "given_name": "Glenn C.", "clpid": "Turner-G-C" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Nascent polypeptides emerging from the ribosome and not yet folded may at least transiently present degradation signals similar to those recognized by the ubiquitin system in misfolded proteins. The ubiquitin sandwich technique was used to detect and measure cotranslational protein degradation in living cells. More than 50 percent of nascent protein molecules bearing an amino-terminal degradation signal can be degraded cotranslationally, never reaching their mature size before their destruction by processive proteolysis. Thus, the folding of nascent proteins, including abnormal ones, may be in kinetic competition with pathways that target these proteins for degradation cotranslationally.", "doi": "10.1126/science.289.5487.2117", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2000-09-22", "series_number": "5487", "volume": "289", "issue": "5487", "pages": "2117-2120" }, { "id": "authors:9nzv9-k6p20", "collection": "authors", "collection_id": "9nzv9-k6p20", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20180803-074725065", "type": "article", "title": "The Ubiquitin System and the N-End Rule Pathway", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Turner", "given_name": "Glenn", "clpid": "Turner-Glenn-C" }, { "family_name": "Du", "given_name": "Fangyong", "clpid": "Du-Fangyong" }, { "family_name": "Xie", "given_name": "Youming", "clpid": "Xie-Youming" } ], "abstract": "Eukaryotes contain a highly conserved multienzyme system which covalently links a small protein, ubiquitin, to a variety of intracellular proteins that bear degradation signals recognized by this system. The resulting ubiquitin-protein conjugates are degraded by the 26S proteasome, an ATP-dependent protease. Pathways that involve ubiquitin play major roles in a huge variety of processes, including cell differentiation, cell cycle, and responses to stress. In this article we briefly review the design of the ubiquitin system, and describe two recent advances, the finding that ubiquitin ligases interact with specific components of the 26S proteasome, and the demonstration that peptides accelerate their uptake into cells by activating the N-end rule pathway, one of several proteolytic pathways of the ubiquitin system.", "doi": "10.1515/BC.2000.101", "issn": "1431-6730", "publisher": "De Gruyter", "publication": "Biological Chemistry", "publication_date": "2000-09", "series_number": "9-10", "volume": "381", "issue": "9-10", "pages": "779-789" }, { "id": "authors:y4ptb-xp542", "collection": "authors", "collection_id": "y4ptb-xp542", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150330-105345474", "type": "article", "title": "Peptides accelerate their uptake by activating a ubiquitin-dependent proteolytic pathway", "author": [ { "family_name": "Turner", "given_name": "Glenn C.", "clpid": "Turner-Glenn-C" }, { "family_name": "Du", "given_name": "Fangyong", "clpid": "Du-Fangyong" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Protein degradation by the ubiquitin system controls the intracellular concentrations of many regulatory proteins. A protein substrate of the ubiquitin system is conjugated to ubiquitin through the action of three enzymes, E1, E2 and E3, with the degradation signal (degron) of the substrate recognized by E3. The resulting multi-ubiquitylated substrate is degraded by the 26S proteasome. Here we describe the physiological regulation of a ubiquitin-dependent pathway through allosteric modulation of its E3 activity by small compounds. Ubr1, the E3 enzyme of the N-end rule pathway (a ubiquitin-dependent proteolytic system) in Saccharomyces cerevisiae mediates the degradation of Cup9, a transcriptional repressor of the peptide transporter Ptr2. Ubr1 also targets proteins that have destabilizing amino-terminal residues. We show that the degradation of Cup9 is allosterically activated by dipeptides with destabilizing N-terminal residues. In the resulting positive feedback circuit, imported dipeptides bind to Ubr1 and accelerate the Ubr1-dependent degradation of Cup9, thereby de-repressing the expression of Ptr2 and increasing the cell's capacity to import peptides. These findings identify the physiological rationale for the targeting of Cup9 by Ubr1, and indicate that small compounds may regulate other ubiquitin-dependent pathways.", "doi": "10.1038/35014629", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2000-06-01", "series_number": "6786", "volume": "405", "issue": "6786", "pages": "579" }, { "id": "authors:56ynm-2kf07", "collection": "authors", "collection_id": "56ynm-2kf07", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:KWOmcb00", "type": "article", "title": "Altered Activity, Social Behavior, and Spatial Memory in Mice Lacking the NTAN1p Amidase and the Asparagine Branch of the N-End Rule Pathway", "author": [ { "family_name": "Kwon", "given_name": "Yong Tae", "clpid": "Kwon-Yong-Tae" }, { "family_name": "Balogh", "given_name": "Seth A.", "clpid": "Balogh-S-A" }, { "family_name": "Davydov", "given_name": "Ilia V.", "clpid": "Davydov-I-V" }, { "family_name": "Kashina", "given_name": "Anna S.", "clpid": "Kashina-A-S" }, { "family_name": "Yoon", "given_name": "Jeong Kyo", "clpid": "Yoon-Jeong-Kyo" }, { "family_name": "Xie", "given_name": "Youming", "clpid": "Xie-Youming" }, { "family_name": "Gaur", "given_name": "Arti", "clpid": "Gaur-A" }, { "family_name": "Hyde", "given_name": "Lynn", "clpid": "Hyde-L" }, { "family_name": "Denenberg", "given_name": "Victor H.", "clpid": "Denenberg-V-H" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. N-terminal asparagine and glutamine are tertiary destabilizing residues, in that they are enzymatically deamidated to yield secondary destabilizing residues aspartate and glutamate, which are conjugated to arginine, a primary destabilizing residue. N-terminal arginine of a substrate protein is bound by the Ubr1-encoded E3alpha , the E3 component of the ubiquitin-proteasome-dependent N-end rule pathway. We describe the construction and analysis of mouse strains lacking the asparagine-specific N-terminal amidase (NtN-amidase), encoded by the Ntan1 gene. In wild-type embryos, Ntan1 was strongly expressed in the branchial arches and in the tail and limb buds. The Ntan1-/- mouse strains lacked the NtN-amidase activity but retained glutamine-specific NtQ-amidase, indicating that the two enzymes are encoded by different genes. Among the normally short-lived N-end rule substrates, only those bearing N-terminal asparagine became long-lived in Ntan1-/- fibroblasts. The Ntan1-/- mice were fertile and outwardly normal but differed from their congenic wild-type counterparts in spontaneous activity, spatial memory, and a socially conditioned exploratory phenotype that has not been previously described with other mouse strains.", "pmcid": "PMC85783", "issn": "0270-7306", "publisher": "American Society for Microbiology", "publication": "Molecular and Cellular Biology", "publication_date": "2000-06-01", "series_number": "11", "volume": "20", "issue": "11", "pages": "4135-4148" }, { "id": "authors:6wjyd-rqg89", "collection": "authors", "collection_id": "6wjyd-rqg89", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:DAVjbc00", "type": "article", "title": "RGS4 is arginylated and degraded by the N-end rule pathway in vitro", "author": [ { "family_name": "Davydov", "given_name": "Ilia V.", "clpid": "Davydov-Ilia-V" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. We used an expression-cloning screen to search for mouse proteins that are degraded by the ubiquitin/proteasome-dependent N-end rule pathway in a reticulocyte lysate. One substrate thus identified was RGS4, a member of the RGS family of GTPase-activating proteins that down-regulate specific G proteins. A determinant of the RGS4 degradation signal (degron) was located at the N terminus of RGS4, because converting cysteine 2 to either glycine, alanine, or valine completely stabilized RGS4. Radiochemical sequencing indicated that the N-terminal methionine of the lysate-produced RGS4 was replaced with arginine. Since N-terminal arginine is a destabilizing residue not encoded by RGS4 mRNA, we conclude that the degron of RGS4 is generated through the removal of N-terminal methionine and enzymatic arginylation of the resulting N-terminal cysteine. RGS16, another member of the RGS family, was also found to be an N-end rule substrate. RGS4 that was transiently expressed in mouse L cells was short-lived in these cells. However, the targeting of RGS4 for degradation in this in vivo setting involved primarily another degron, because N-terminal variants of RGS4 that were stable in reticulocyte lysate remained unstable in L cells.", "doi": "10.1074/jbc.M001605200", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "2000-04-26", "series_number": "30", "volume": "275", "issue": "30", "pages": "22931-22941" }, { "id": "authors:1xdmg-emc20", "collection": "authors", "collection_id": "1xdmg-emc20", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:XIEpnas00", "type": "article", "title": "Physical association of ubiquitin ligases and the 26S proteasome", "author": [ { "family_name": "Xie", "given_name": "Youming", "clpid": "Xie-Youming" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The ubiquitin (Ub) system recognizes degradation signals of the target proteins through the E3 components of E3-E2 Ub ligases. A targeted substrate bears a covalently linked multi-Ub chain and is degraded by the ATP-dependent 26S proteasome, which consists of the 20S core protease and two 19S particles. The latter mediate the binding and unfolding of a substrate protein before its transfer to the interior of the 20S core. It is unclear how a targeted substrate is delivered to the 26S proteasome, inasmuch as Rpn10p, the only known proteasomal subunit that binds multi-Ub chains, has been found to he not essential for degradation of many proteins in the yeast Saccharomyces cerevisiae. Here we show that Ubr1p and Ufd4p, the E3 components of two distinct Ub ligases, directly interact with the 26S proteasome. Specifically, Ubr1p is shown to bind to the Rpn2p, Rpt1p, and Rpt6p proteins of the 19S particle, and Ufd4p is shown to bind to Rpt6p. These and related results suggest that a substrate-bound Ub ligase participates in the delivery of substrates to the proteasome, because of affinity between the ligase's E3 component and specific proteins of the 19S particle.", "doi": "10.1073/pnas.060025497", "pmcid": "PMC15957", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "2000-03-14", "series_number": "6", "volume": "97", "issue": "6", "pages": "2497-2502" }, { "id": "authors:9de2j-dmb46", "collection": "authors", "collection_id": "9de2j-dmb46", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:KWOjbc99", "type": "article", "title": "Bivalent Inhibitor of the N-end Rule Pathway", "author": [ { "family_name": "Kwon", "given_name": "Yong Tae", "clpid": "Kwon-Yong-Tae" }, { "family_name": "L\u00e9vy", "given_name": "Fr\u00e9d\u00e9ric", "clpid": "L\u00e9vy-Fr\u00e9d\u00e9ric" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Ubr1p, the recognition (E3) component of the Saccharomyces cerevisiae N-end rule pathway, contains at least two substrate-binding sites. The type 1 site is specific for N-terminal basic residues Arg, Lys, and His. The type 2 site is specific for N-terminal bulky hydrophobic residues Phe, Leu, Trp, Tyr, and Ile. Previous work has shown that dipeptides bearing either type 1 or type 2 N-terminal residues act as weak but specific inhibitors of the N-end rule pathway. We took advantage of the two-site architecture of Ubr1p to explore the feasibility of bivalent N-end rule inhibitors, whose expected higher efficacy would result from higher affinity of the cooperative (bivalent) binding to Ubr1p. The inhibitor comprised mixed tetramers of beta-galactosidase that bore both N-terminal Arg (type 1 residue) and N-terminal Leu (type 2 residue) but that were resistant to proteolysis in vivo. Expression of these constructs in S. cerevisiae inhibited the N-end rule pathway much more strongly than the expression of otherwise identical beta-galactosidase tetramers whose N-terminal residues were exclusively Arg or exclusively Leu. In addition to demonstrating spatial proximity between the type 1 and type 2 substrate-binding sites of Ubr1p, these results provide a route to high affinity inhibitors of the N-end rule pathway.", "doi": "10.1074/jbc.274.25.18135", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "1999-06-18", "series_number": "25", "volume": "274", "issue": "25", "pages": "18135-18139" }, { "id": "authors:6fyhb-vbm94", "collection": "authors", "collection_id": "6fyhb-vbm94", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:DUNmbc99", "type": "article", "title": "Detection of Transient In Vivo Interactions between Substrate and Transporter during Protein Translocation into the Endoplasmic Reticulum", "author": [ { "family_name": "D\u00fcnnwald", "given_name": "Martin", "clpid": "D\u00fcnnwald-Martin" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Johnsson", "given_name": "Nils", "clpid": "Johnsson-Nils" } ], "abstract": "The split-ubiquitin technique was used to detect transient protein interactions in living cells. Nub, the N-terminal half of ubiquitin (Ub), was fused to Sec62p, a component of the protein translocation machinery in the endoplasmic reticulum of Saccharomyces cerevisiae. Cub, the C-terminal half of Ub, was fused to the C terminus of a signal sequence. The reconstitution of a quasi-native Ub structure from the two halves of Ub, and the resulting cleavage by Ub-specific proteases at the C terminus of Cub, serve as a gauge of proximity between the two test proteins linked to Nub and Cub. Using this assay, we show that Sec62p is spatially close to the signal sequence of the prepro-alpha-factor in vivo. This proximity is confined to the nascent polypeptide chain immediately following the signal sequence. In addition, the extent of proximity depends on the nature of the signal sequence. Cub fusions that bore the signal sequence of invertase resulted in a much lower Ub reconstitution with Nub-Sec62p than otherwise identical test proteins bearing the signal sequence of prepro-alpha-factor. An inactive derivative of Sec62p failed to interact with signal sequences in this assay. These in vivo findings are consistent with Sec62p being part of a signal sequence-binding complex.", "doi": "10.1091/mbc.10.2.329", "pmcid": "PMC25172", "issn": "1059-1524", "publisher": "American Society for Cell Biology", "publication": "Molecular Biology of the Cell", "publication_date": "1999-02", "series_number": "2", "volume": "10", "issue": "2", "pages": "329-344" }, { "id": "authors:fk1xk-sm430", "collection": "authors", "collection_id": "fk1xk-sm430", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:KWOmcb99", "type": "article", "title": "Alternative Splicing Results in Differential Expression, Activity, and Localization of the Two Forms of Arginyl-tRNA-Protein Transferase, a Component of the N-End Rule Pathway", "author": [ { "family_name": "Kwon", "given_name": "Yong Tae", "clpid": "Kwon-Yong-Tae" }, { "family_name": "Kashina", "given_name": "Anna S.", "clpid": "Kashina-Anna-S" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The underlying ubiquitin-dependent proteolytic system, called the N-end rule pathway, is organized hierarchically: N-terminal aspartate and glutamate (and also cysteine in metazoans) are secondary destabilizing residues, in that they function through their conjugation, by arginyl-tRNA-protein transferase (R-transferase), to arginine, a primary destabilizing residue. We isolated cDNA encoding the 516-residue mouse R-transferase, ATE1p, and found two species, termed Ate1-1 and Ate1-2. The Ate1 mRNAs are produced through a most unusual alternative splicing that retains one or the other of the two homologous 129-bp exons, which are adjacent in the mouse Ate1 gene. Human ATE1 also contains the alternative 129-bp exons, whereas the plant (Arabidopsis thaliana) and fly (Drosophila melanogaster) Ate1 genes encode a single form of ATE1p. A fusion of ATE1-1p with green fluorescent protein (GFP) is present in both the nucleus and the cytosol, whereas ATE1-2p-GFP is exclusively cytosolic. Mouse ATE1-1p and ATE1-2p were examined by expressing them in ate1Delta Saccharomyces cerevisiae in the presence of test substrates that included Asp-beta gal (beta -galactosidase) and Cys-beta gal. Both forms of the mouse R-transferase conferred instability on Asp-beta gal (but not on Cys-beta gal) through the arginylation of its N-terminal Asp, the ATE1-1p enzyme being more active than ATE1-2p. The ratio of Ate1-1 to Ate1-2 mRNA varies greatly among the mouse tissues; it is ~0.1 in the skeletal muscle, ~0.25 in the spleen, ~3.3 in the liver and brain, and ~10 in the testis, suggesting that the two R-transferases are functionally distinct.", "doi": "10.1128/mcb.19.1.182", "pmcid": "PMC83877", "issn": "0270-7306", "publisher": "American Society for Microbiology", "publication": "Molecular and Cellular Biology", "publication_date": "1999-01", "series_number": "1", "volume": "19", "issue": "1", "pages": "182-193" }, { "id": "authors:591c5-s5334", "collection": "authors", "collection_id": "591c5-s5334", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201125-124911297", "type": "article", "title": "Recent studies of the ubiquitin system and the N-end rule pathway", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "[no abstract]", "issn": "0073-0874", "publisher": "Wiley-Liss", "publication": "Harvey lectures", "publication_date": "1999", "volume": "96", "pages": "93-116" }, { "id": "authors:9amaz-q5111", "collection": "authors", "collection_id": "9amaz-q5111", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:KWOpnas98", "type": "article", "title": "The mouse and human genes encoding the recognition component of the N-end rule pathway", "author": [ { "family_name": "Kwon", "given_name": "Yong Tae", "clpid": "Kwon-Yong-Tae" }, { "family_name": "Reiss", "given_name": "Yuval", "clpid": "Reiss-Yuval" }, { "family_name": "Fried", "given_name": "Victor A.", "clpid": "Fried-Victor-A" }, { "family_name": "Hershko", "given_name": "Avram", "clpid": "Hershko-Avram" }, { "family_name": "Yoon", "given_name": "Jeong Kyo", "clpid": "Yoon-Jeong-Kyo" }, { "family_name": "Gonda", "given_name": "David K.", "clpid": "Gonda-David-K" }, { "family_name": "Sangan", "given_name": "Pitchai", "clpid": "Sangan-Pitchai" }, { "family_name": "Copeland", "given_name": "Neal G.", "clpid": "Copeland-Neal-G" }, { "family_name": "Jenkins", "given_name": "Nancy A.", "clpid": "Jenkins-Nancy-A" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. The N-end rule pathway is one proteolytic pathway of the ubiquitin system. The recognition component of this pathway, called N-recognin or E3, binds to a destabilizing N-terminal residue of a substrate protein and participates in the formation of a substrate-linked multiubiquitin chain. We report the cloning of the mouse and human Ubr1 cDNAs and genes that encode a mammalian N-recognin called E3 alpha. Mouse UBR1p (E3 alpha) is a 1,757-residue (200-kDa) protein that contains regions of sequence similarity to the 225-kDa Ubr1p of the yeast Saccharomyces cerevisiae. Mouse and human UBR1p have apparent homologs in other eukaryotes as well, thus defining a distinct family of proteins, the UBR family. The residues essential for substrate recognition by the yeast Ubr1p are conserved in the mouse UBR1p. The regions of similarity among the UBR family members include a putative zinc finger and RING-H2 finger, another zinc-binding domain. Ubr1 is located in the middle of mouse chromosome 2 and in the syntenic 15q15-q21.1 region of human chromosome 15. Mouse Ubr1 spans approximate to 120 kilobases of genomic DNA and contains approximate to 50 exons. Ubr1 is ubiquitously expressed in adults, with skeletal muscle and heart being the sites of highest expression. In mouse embryos, the Ubr1 expression is highest in the branchial arches and in the tail and limb buds. The cloning of Ubr1 makes possible the construction of Ubr1-lacking mouse strains, a prerequisite for the functional understanding of the mammalian N-end rule pathway.", "doi": "10.1073/pnas.95.14.7898", "pmcid": "PMC20901", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1998-07-07", "series_number": "14", "volume": "95", "issue": "14", "pages": "7898-7903" }, { "id": "authors:tf36z-5cq91", "collection": "authors", "collection_id": "tf36z-5cq91", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:VARpnas98", "type": "article", "title": "Codominant interference, antieffectors, and multitarget drugs", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The insufficient selectivity of drugs is a bane of present-day therapies. This problem is significant for antibacterial drugs, difficult for antivirals, and utterly unsolved for anticancer drugs, which remain ineffective against major cancers, and in addition cause severe side effects. The problem may be solved if a therapeutic agent could have a multitarget, combinatorial selectivity, killing, or otherwise modifying, a cell if and only if it contains a predetermined set of molecular targets and larks another predetermined set of targets. An earlier design of multitarget drugs [Varshavsky, A. (1995) Proc. Natl. Acad. Sci. USA 92, 3663-3667] was confined to macromolecular reagents such as proteins, with the attendant difficulties of intracellular delivery and immunogenicity. I now propose a solution to the problem of drug selectivity that is applicable to small (less than or equal to 1 kDa) drugs. Two ideas, codominant interference and antieffectors, should allow a therapeutic regimen to possess combinatorial selectivity, in which the number of positively and negatively sensed macromolecular targets can be two, three, or more. The nature of the effector and interference moieties in a multitarget drug determines its use: selective killing of cancer cells or, for example, the inhibition of a neurotransmitter-inactivating enzyme in a specific subset of the enzyme-containing cells. The in vivo effects of such drugs would be analogous to the outcomes of the Boolean operations \"and,\" \"or,\" and combinations thereof. I discuss the logic and applications of the antieffector and interference/codominance concepts, and the attendant problem of pharmacokinetics.", "pmcid": "PMC19261", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1998-03-03", "series_number": "5", "volume": "95", "issue": "5", "pages": "2094-2099" }, { "id": "authors:q81az-y4q35", "collection": "authors", "collection_id": "q81az-y4q35", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210211-162600012", "type": "article", "title": "Ump1p Is Required for Proper Maturation of the 20S Proteasome and Becomes Its Substrate upon Completion of the Assembly", "author": [ { "family_name": "Ramos", "given_name": "Paula C.", "clpid": "Ramos-Paula-C" }, { "family_name": "H\u00f6ckendorff", "given_name": "J\u00f6rg", "clpid": "H\u00f6ckendorff-J\u00f6rg" }, { "family_name": "Johnson", "given_name": "Erica S.", "clpid": "Johnson-Erica-S" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Dohmen", "given_name": "R. J\u00fcrgen", "clpid": "Dohmen-R-J\u00fcrgen" } ], "abstract": "We report the discovery of a short-lived chaperone that is required for the correct maturation of the eukaryotic 20S proteasome and is destroyed at a specific stage of the assembly process. The S. cerevisiae Ump1p protein is a component of proteasome precursor complexes containing unprocessed \u03b2 subunits but is not detected in the mature 20S proteasome. Upon the association of two precursor complexes, Ump1p is encased and is rapidly degraded after the proteolytic sites in the interior of the nascent proteasome are activated. Cells lacking Ump1p exhibit a lack of coordination between the processing of \u03b2 subunits and proteasome assembly, resulting in functionally impaired proteasomes. We also show that the propeptide of the Pre2p/Doa3p \u03b2 subunit is required for Ump1p's function in proteasome maturation.", "doi": "10.1016/s0092-8674(00)80942-3", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "1998-02-20", "series_number": "4", "volume": "92", "issue": "4", "pages": "489-499" }, { "id": "authors:vjba6-88k12", "collection": "authors", "collection_id": "vjba6-88k12", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210211-162600155", "type": "article", "title": "The N-end rule pathway controls the import of peptides through degradation of a transcriptional repressor", "author": [ { "family_name": "Byrd", "given_name": "Christopher", "clpid": "Byrd-Christopher" }, { "family_name": "Turner", "given_name": "Glenn C.", "clpid": "Turner-Glenn-C" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Ubiquitin\u2010dependent proteolytic systems underlie many processes, including the cell cycle, cell differentiation and responses to stress. One such system is the N\u2010end rule pathway, which targets proteins bearing destabilizing N\u2010terminal residues. Here we report that Ubr1p, the main recognition component of this pathway, regulates peptide import in the yeast Saccharomyces cerevisiae through degradation of Cup9p, a 35 kDa homeodomain protein. Cup9p was identified using a screen for mutants that bypass the previously observed requirement for Ubr1p in peptide import. We show that Cup9p is a short\u2010lived protein (t_\u00bd \u223c5 min) whose degradation requires Ubr1p. Cup9p acts as a repressor of PTR2, a gene encoding the transmembrane peptide transporter. In contrast to engineered N\u2010end rule substrates, which are recognized by Ubr1p through their destabilizing N\u2010terminal residues, Cup9p is targeted by Ubr1p through an internal degradation signal. The Ubr1p\u2010Cup9p\u2010Ptr2p circuit is the first example of a physiological process controlled by the N\u2010end rule pathway. An earlier study identified Cup9p as a protein required for an aspect of resistance to copper toxicity in S.cerevisiae. Thus, one physiological substrate of the N\u2010end rule pathway functions as both a repressor of peptide import and a regulator of copper homeostasis.", "doi": "10.1093/emboj/17.1.269", "pmcid": "PMC1170377", "issn": "1460-2075", "publisher": "European Molecular Biology Organization", "publication": "EMBO Journal", "publication_date": "1998-01-01", "series_number": "1", "volume": "17", "issue": "1", "pages": "269-277" }, { "id": "authors:b9g6e-jwx55", "collection": "authors", "collection_id": "b9g6e-jwx55", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210211-162600310", "type": "article", "title": "The ubiquitin system", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Eukaryotes contain a highly conserved multi-enzyme system that covalently links ubiquitin to a variety of intracellular proteins that bear degradation signals recognized by this system. The resulting ubiquitin-protein conjugates are degraded by the 26S proteasome, a large ATP-dependent protease. Pathways that involve ubiquitin underlie a multitude of processes, including cell differentiation, the cell cycle and responses to stress.", "doi": "10.1016/s0968-0004(97)01122-5", "issn": "0968-0004", "publisher": "Cell Press", "publication": "Trends in Biochemical Sciences", "publication_date": "1997-10", "series_number": "10", "volume": "22", "issue": "10", "pages": "383-387" }, { "id": "authors:tqrx1-h8a41", "collection": "authors", "collection_id": "tqrx1-h8a41", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210211-162600429", "type": "article", "title": "The N-end rule pathway of protein degradation", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N\u2010end rule relates the in vivo half\u2010life of a protein to the identity of its N\u2010terminal residue. Similar but distinct versions of the N\u2010end rule operate in all organisms examined, from mammals to fungi and bacteria. In eukaryotes, the N\u2010end rule pathway is a part of the ubiquitin system. Ubiquitin is a 76\u2010residue protein whose covalent conjugation to other proteins plays a role in many biological processes, including cell growth and differentiation. I discuss the current understanding of the N\u2010end rule pathway.", "doi": "10.1046/j.1365-2443.1997.1020301.x", "issn": "1356-9597", "publisher": "Wiley", "publication": "Genes to Cells", "publication_date": "1997-01", "series_number": "1", "volume": "2", "issue": "1", "pages": "13-28" }, { "id": "authors:xqyag-m5t15", "collection": "authors", "collection_id": "xqyag-m5t15", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:GRIjbc96", "type": "article", "title": "A Mouse Amidase Specific for N-terminal Asparagine: the gene, the enzyme, and their function in the N-end rule pathway", "author": [ { "family_name": "Grigoryev", "given_name": "Sergei", "clpid": "Grigoryev-Sergei" }, { "family_name": "Stewart", "given_name": "Albert E.", "clpid": "Stewart-Albert-E" }, { "family_name": "Kwon", "given_name": "Yong Tae", "clpid": "Kwon-Yong-Tae" }, { "family_name": "Arfin", "given_name": "Stuart M.", "clpid": "Arfin-Stuart-M" }, { "family_name": "Bradshaw", "given_name": "Ralph A.", "clpid": "Bradshaw-Ralph-A" }, { "family_name": "Jenkins", "given_name": "Nancy A.", "clpid": "Jenkins-Nancy-A" }, { "family_name": "Copeland", "given_name": "Neal G.", "clpid": "Copeland-Neal-G" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In both fungi and mammals, the tertiary destabilizing N-terminal residues asparagine and glutamine function through their conversion, by enzymatic deamidation, into the secondary destabilizing residues aspartate and glutamate, whose destabilizing activity requires their enzymatic conjugation to arginine, one of the primary destabilizing residues. We report the isolation and analysis of a mouse cDNA and the corresponding gene (termed Ntan1) that encode a 310-residue amidohydrolase (termed NtN-amidase) specific for N-terminal asparagine. The ~17-kilobase pair Ntan1 gene is located in the proximal region of mouse chromosome 16 and contains 10 exons ranging from 54 to 177 base pairs in length. The ~1.4-kilobase pair Ntan1 mRNA is expressed in all of the tested mouse tissues and cell lines and is down-regulated upon the conversion of myoblasts into myotubes. The Ntan1 promoter is located ~500 base pairs upstream of the Ntan1 start codon. The deduced amino acid sequence of mouse NtN-amidase is 88% identical to the sequence of its porcine counterpart, but bears no significant similarity to the sequence of the NTA1-encoded N-terminal amidohydrolase of the yeast Saccharomyces cerevisiae, which can deamidate either N-terminal asparagine or glutamine. The expression of mouse NtN-amidase in S. cerevisiae nta1Delta was used to verify that NtN-amidase retains its asparagine selectivity in vivo and can implement the asparagine-specific subset of the N-end rule. Further dissection of mouse Ntan1, including its null phenotype analysis, should illuminate the functions of the N-end rule, most of which are still unknown.", "doi": "10.1074/jbc.271.45.28521", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "1996-11-08", "series_number": "45", "volume": "271", "issue": "45", "pages": "28521-28532" }, { "id": "authors:88cy4-hem10", "collection": "authors", "collection_id": "88cy4-hem10", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:VARpnas96", "type": "article", "title": "The N-end rule: Functions, mysteries, uses", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Similar but distinct versions of the N-end rule operate in all organisms examined, from mammals to fungi and bacteria. In eukaryotes, the N-end rule pathway is a part of the ubiquitin system. I discuss the mechanisms and functions of this pathway, and consider its applications.", "doi": "10.1073/pnas.93.22.12142", "pmcid": "PMC37957", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1996-10-29", "series_number": "22", "volume": "93", "issue": "22", "pages": "12142-12149" }, { "id": "authors:qtcvr-8zg83", "collection": "authors", "collection_id": "qtcvr-8zg83", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210211-162600585", "type": "article", "title": "Cdc48p interacts with Ufd3p, a WD repeat protein required for ubiquitin-mediated proteolysis in Saccharomyces cerevisiae", "author": [ { "family_name": "Ghislain", "given_name": "Michel", "clpid": "Ghislain-Michel" }, { "family_name": "Dohmen", "given_name": "R. J\u00fcrgen", "clpid": "Dohmen-R-J\u00fcrgen" }, { "family_name": "L\u00e9vy", "given_name": "Fr\u00e9d\u00e9ric", "clpid": "L\u00e9vy-Fr\u00e9d\u00e9ric" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "A library of random 10 residue peptides fused to the N\u2010terminus of a reporter protein was screened in the yeast Saccharomyces cerevisiae for sequences that can target the reporter for degradation by the N\u2010end rule pathway, a ubiquitin (Ub)\u2010dependent proteolytic system that recognizes potential substrates through binding to their destabilizing N\u2010terminal residues. One of the N\u2010terminal sequences identified by this screen was used in a second screen for mutants incapable of degrading the corresponding reporter fusion. A mutant thus identified had an abnormally low content of free Ub. This mutant was found to be allelic to a previously isolated mutant in a Ub\u2010dependent proteolytic system distinct from the N\u2010end rule pathway. We isolated the gene involved, termed UFD3, which encodes an 80 kDa protein containing tandem repeats of a motif that is present in many eukaryotic proteins and called the WD repeat. Both co\u2010immunoprecipitation and two\u2010hybrid assays demonstrated that Ufd3p is an in vivo ligand of Cdc48p, an essential ATPase required for the cell cycle progression and the fusion of endoplasmic reticulum membranes. Further, we showed that, similarly to Ufd3p, Cdc48p is also required for the Ub\u2010dependent proteolysis of test substrates. The discovery of the Ufd3p\u2013Cdc48p complex and the finding that this complex is a part of the Ub system open up a new direction for studies of the function of Ub in the cell cycle and membrane dynamics.", "doi": "10.1002/j.1460-2075.1996.tb00869.x", "pmcid": "PMC452226", "issn": "0261-4189", "publisher": "European Molecular Biology Organization", "publication": "EMBO Journal", "publication_date": "1996-09-01", "series_number": "18", "volume": "15", "issue": "18", "pages": "4884-4899" }, { "id": "authors:2egr2-f3n37", "collection": "authors", "collection_id": "2egr2-f3n37", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:LEVpnas96", "type": "article", "title": "Using ubiquitin to follow the metabolic fate of a protein", "author": [ { "family_name": "L\u00e9vy", "given_name": "Fr\u00e9d\u00e9ric", "clpid": "L\u00e9vy-Fr\u00e9d\u00e9ric" }, { "family_name": "Johnsson", "given_name": "Nils", "clpid": "Johnsson-Nils" }, { "family_name": "R\u00fcmenapf", "given_name": "Tillmann", "clpid": "R\u00fcmenapf-Tillmann" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "We describe a method that can be used to produce equimolar amounts of two or more specific proteins in a cell. In this approach, termed the ubiquitin/protein/reference (UPR) technique, a reference protein and a protein of interest are synthesized as a polyprotein separated by a ubiquitin moiety. This tripartite fusion is cleaved, cotranslationally or nearly so, by ubiquitin-specific processing proteases after the last residue of ubiquitin, producing equimolar amounts of the protein of interest and the reference protein bearing a C-terminal ubiquitin moiety. In applications such as pulse-chase analysis, the UPR technique can compensate for the scatter of immunoprecipitation yields, sample volumes, and other sources of sample-to-sample variation. In particular, this method allows a direct comparison of proteins' metabolic stabilities from the pulse data alone. We used UPR to examine the N-end rule (a relation between the in vivo half-life of a protein and the identity of its N-terminal residue) in L cells, a mouse cell line. The increased accuracy afforded by the UPR technique underscores insufficiency of the current \"half-life\" terminology, because in vivo degradation of many proteins deviates from first-order kinetics. We consider this problem and discuss other applications of UPR.", "doi": "10.1073/pnas.93.10.4907", "pmcid": "PMC39378", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1996-05-14", "series_number": "10", "volume": "93", "issue": "10", "pages": "4907-4912" }, { "id": "authors:y9h4j-d7j16", "collection": "authors", "collection_id": "y9h4j-d7j16", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210211-152331529", "type": "article", "title": "An Essential Yeast Gene Encoding a Homolog of Ubiquitin-activating Enzyme", "author": [ { "family_name": "Dohmen", "given_name": "R. J\u00fcrgen", "clpid": "Dohmen-R-J\u00fcrgen" }, { "family_name": "Stappen", "given_name": "Reiner", "clpid": "Stappen-Reiner" }, { "family_name": "McGrath", "given_name": "John P.", "clpid": "McGrath-John-P" }, { "family_name": "Forrov\u00e1", "given_name": "Helena", "clpid": "Forrov\u00e1-Helena" }, { "family_name": "Kolarov", "given_name": "Jordan", "clpid": "Kolarov-Jordan" }, { "family_name": "Goffeau", "given_name": "Andr\u00e9", "clpid": "Goffeau-Andr\u00e9" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Ubiquitin (Ub) activation by the Ub-activating (E1) enzyme is the initial and essential step common to all of the known processes that involve post-translational conjugation of Ub to itself or other proteins. The \"activated\" Ub, linked via a thioester bond to a specific cysteine residue of E1 enzyme, can be transferred to a cysteine residue in one of several Ub-conjugating (E2) enzymes, which catalyze the formation of isopeptide bonds between the C-terminal glycine of Ub and lysine residues of acceptor proteins. In the yeast Saccharomyces cerevisiae, a 114-kDa E1 enzyme is encoded by an essential gene termed UBA1 (McGrath, J. P., Jentsch, S., and Varshavsky, A.(1991) EMBO J. 10, 227-236). We describe the isolation and analysis of another essential gene, termed UBA2, that encodes a 71-kDa protein with extensive sequence similarities to both the UBA1-encoded yeast E1 and E1 enzymes of other organisms. The regions of similarities between Uba1p and Uba2p encompass a putative ATP-binding site as well as a sequence that is highly conserved between the known E1 enzymes and contains the active-site cysteine of E1. This cysteine is shown to be required for an essential function of Uba2p, suggesting that Uba2p-catalyzed reactions involve a transient thioester bond between Uba2p and either Ub or another protein. Uba2p is located largely in the nucleus. The putative nuclear localization signal of Uba2p is near its C terminus. The Uba1p (E1 enzyme) and Uba2p cannot complement each others essential functions even if their subcellular localization is altered by mutagenesis. Uba2p appears to interact with itself and several other S. cerevisiae proteins with apparent molecular masses of 52, 63, 87, and 120 kDa. Uba2p is multiubiquitinated in vivo, suggesting that at least a fraction of Uba2p is metabolically unstable. Uba2p is likely to be a component of the Ub system that functions as either an E2 or E1/E2 enzyme.", "doi": "10.1074/jbc.270.30.18099", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "1995-07-28", "series_number": "30", "volume": "270", "issue": "30", "pages": "18099-18109" }, { "id": "authors:ysksk-7rv52", "collection": "authors", "collection_id": "ysksk-7rv52", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210211-152331656", "type": "article", "title": "Yeast N-terminal Amidase: a new enzyme and component of the N-end rule pathway", "author": [ { "family_name": "Baker", "given_name": "Rohan T.", "clpid": "Baker-Rohan-T" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Tertiary destabilizing N-terminal residues asparagine and glutamine function through their conversion, by enzymatic deamidation, into the secondary destabilizing residues aspartate and glutamate, whose activity requires their enzymatic conjugation to arginine, one of the primary destabilizing residues. We isolated a Saccharomyces cerevisiae gene, termed NTA1, that encodes an amidase (Nt-amidase) specific for N-terminal asparagine and glutamine. Alterations at the putative active-site cysteine of the 52-kDa Nt-amidase inactivate the enzyme. Null nta1 mutants are viable but unable to degrade N-end rule substrates that bear N-terminal asparagine or glutamine. The effects of overexpressing Nt-amidase and other components of the N-end rule pathway suggest interactions between these components and the existence of a multienzyme targeting complex.", "doi": "10.1074/jbc.270.20.12065", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "1995-05-19", "series_number": "20", "volume": "270", "issue": "20", "pages": "12065-12074" }, { "id": "authors:smd2s-zg179", "collection": "authors", "collection_id": "smd2s-zg179", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:VARpnas95", "type": "article", "title": "Codominance and toxins: A path to drugs of nearly unlimited selectivity", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The effectiveness of drugs is often limited by their insufficient selectivity. I propose designs of therapeutic agents that address this problem. The key feature of these reagents, termed comtoxins (codominance-mediated toxins), is their ability to utilize codominance, a property characteristic of many signals in proteins, including degradation signals (degrons) and nuclear localization signals. A comtoxin designed to kill cells that express intracellular proteins P1 and P2 but to spare cells that lack P1 and/or P2 is a multidomain fusion containing a cytotoxic domain and two degrons placed within or near two domains P1* and P2* that bind, respectively, to pi and P2. In a cell containing both P1 and P2, these proteins would bind to the P1* and P2* domains of the comtoxin and sterically mask the nearby (appropriately positioned) degrons, resulting in a long-lived and therefore toxic drug. By contrast, in a cell lacking P1 and/or P2, at least one of the comtoxin's degrons would be active (unobstructed), yielding a short-lived and therefore nontoxic drug. A comtoxin containing both a degron and a nuclear localization signal can be designed to kill exclusively cells that contain P1 but lack P2. Analogous strategies yield comtoxins sensitive to the presence (or absence) of more than two proteins in a cell. Also considered is a class of comtoxins in which a toxic domain is split by a flexible insert containing binding sites for the target proteins. The potentially unlimited, combinatorial selectivity of comtoxins may help solve the problem of side effects that bedevils present-day therapies, for even nonselective delivery of a comtoxin would not affect cells whose protein \"signatures\" differ from the targeted one.", "doi": "10.1073/pnas.92.9.3663", "pmcid": "PMC42021", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1995-04-25", "series_number": "9", "volume": "92", "issue": "9", "pages": "3663-3667" }, { "id": "authors:5m00q-zp830", "collection": "authors", "collection_id": "5m00q-zp830", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210211-152331800", "type": "article", "title": "Methotrexate Inhibits Proteolysis of Dihydrofolate Reductase by the N-end Rule Pathway", "author": [ { "family_name": "Johnston", "given_name": "Jennifer A.", "clpid": "Johnston-Jennifer-A" }, { "family_name": "Johnson", "given_name": "Erica S.", "clpid": "Johnson-Erica-S" }, { "family_name": "Waller", "given_name": "Patrick R. H.", "clpid": "Waller-Patrick-R-H" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In eukaryotes, the N-end rule pathway is a ubiquitin-dependent, proteasome-based system that targets and processively degrades proteins bearing certain N-terminal residues. Arg-DHFR, a modified dihydrofolate reductase bearing an N-terminal arginine (destabilizing residue in the N-end rule), is short lived in ATP-supplemented reticulocyte extract. It is shown here that methotrexate, which is a folic acid analog and high affinity ligand of DHFR, inhibits the degradation but not ubiquitination of Arg-DHFR by the N-end rule pathway. The degradation of other N-end rule substrates is not affected by methotrexate. We discuss implications of these results for the mechanism of proteasome-mediated protein degradation.", "doi": "10.1074/jbc.270.14.8172", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "1995-04-07", "series_number": "14", "volume": "270", "issue": "14", "pages": "8172-8178" }, { "id": "authors:f32kc-jem77", "collection": "authors", "collection_id": "f32kc-jem77", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210211-152331936", "type": "article", "title": "The N-end Rule", "author": [ { "family_name": "Varshavsky", "given_name": "A. J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the in vivo half-life1 of a protein to the identity of its N-terminal residue (Varshavsky 1992). Similar but distinct versions of the N-end rule have been shown to operate in all organisms examined, from mammals to fungi and bacteria. I summarize the current understanding of the N-end rule pathway and describe some of the recent methods that utilize the N-end rule. \n\nFeatures of a protein that confer metabolic instability are called degradation signals, or degrons (Varshavsky 1991). The essential component of one degron, the first to be identified, is a destabilizing N-terminal residue of a protein (Bachmair et al. 1986). This signal is called the N-degron. The N-end rule (defined above) results from the existence of N-degrons containing different destabilizing residues (Varshavsky 1992). In eukaryotes, the N-degron comprises two determinants: a destabilizing N-terminal residue and an internal lysine (or lysines) of a substrate. The lysine residue is the site of formation of a multiubiquitin chain, which is required for the degradation of at least some N-end rule substrates (Bachmair and Varshavsky 1989; Hill et al. 1993; Dohmen et al. 1994). Ubiquitin (Ub) is a 76-residue protein whose covalent conjugation to other proteins (often in the form of a multi-Ub chain) plays a role in a number of processes, primarily through routes that involve protein degradation (for review, see Finley and Chau 1991; Gottesman and Maurizi 1992; Hershko and Ciechanover 1992; Jentsch 1992; Varshavsky 1992, 1995a; Parsell and Lindquist 1993; Vierstra 1993; Ciechanover 1994; Gonda 1994; Hochstrasser 1995).", "doi": "10.1101/sqb.1995.060.01.051", "issn": "0091-7451", "publisher": "Cold Spring Harbor Laboratory", "publication": "Cold Spring Harbor Symposia on Quantitative Biology", "publication_date": "1995-01-01", "volume": "60", "pages": "461-478" }, { "id": "authors:29w1a-w6t11", "collection": "authors", "collection_id": "29w1a-w6t11", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:JOHpnas94", "type": "article", "title": "Split ubiquitin as a sensor of protein interactions in vivo", "author": [ { "family_name": "Johnsson", "given_name": "Nils", "clpid": "Johnsson-Nils" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "We describe an assay for in vivo protein interactions. Protein fusions containing ubiquitin, a 76-residue, single-domain protein, are rapidly cleaved in vivo by ubiquitin-specific proteases, which recognize the folded conformation of ubiquitin. When a C-terminal fragment of ubiquitin (C-ub) is expressed as a fusion to a reporter protein, the fusion is cleaved only if an N-terminal fragment of ubiquitin (N-ub) is also expressed in the same cell. This reconstitution of native ubiquitin from its fragments, detectable by the in vivo cleavage assay, is not observed with a mutationally altered N-ub. However, if C-ub and the altered N-ub are each linked to polypeptides that interact in vivo, the cleavage of the fusion containing C-ub is restored, yielding a generally applicable assay for kinetic and equilibrium aspects of in vivo protein interactions. This method, termed USPS (ubiquitin-based split-protein sensor), makes it possible to monitor a protein-protein interaction as a function of time, at the natural sites of this interaction in a living cell.", "doi": "10.1073/pnas.91.22.10340", "pmcid": "PMC45015", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1994-10-25", "series_number": "22", "volume": "91", "issue": "22", "pages": "10340-10344" }, { "id": "authors:xzwbm-91p35", "collection": "authors", "collection_id": "xzwbm-91p35", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150121-092636074", "type": "article", "title": "Degradation of G\u03b1 by the N-End Rule Pathway", "author": [ { "family_name": "Madura", "given_name": "Kiran", "clpid": "Madura-Kiran" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the in vivo half-life of a protein to the identity of its amino-terminal residue. Overexpression of targeting components of the N-end rule pathway in Saccharomyces cerevisiae inhibited the growth of haploid but not diploid cells. This ploidy-dependent toxicity was shown to result from enhanced degradation of Gpa1, the alpha subunit (G\u03b1) of a heterotrimeric guanine nucleotide-binding protein (G protein) that regulates cell differentiation in response to mating pheromones. Sst2, a protein whose absence renders cells hypersensitive to pheromone, was essential for degradation of G alpha but not other N-end rule substrates, suggesting the involvement of an indirect, or trans-, targeting mechanism. G\u03b1 degradation by the N-end rule pathway adds another regulatory dimension to the multitude of signaling functions mediated by G proteins.", "doi": "10.1126/science.8073290", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "1994-09-02", "series_number": "5177", "volume": "265", "issue": "5177", "pages": "1454-1458" }, { "id": "authors:9jks8-2ew61", "collection": "authors", "collection_id": "9jks8-2ew61", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210210-151525258", "type": "article", "title": "Ubiquitin-assisted dissection of protein transport across membranes", "author": [ { "family_name": "Johnsson", "given_name": "Nils", "clpid": "Johnsson-Nils" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "We describe a new way to analyze targeting in protein translocation. A fusion in which ubiquitin (Ub) is positioned between a signal sequence and a reporter domain is cleaved by Ub-specific proteases (UBPs) in the cytosol unless the fusion can 'escape' into a compartment such as the endoplasmic reticulum (ER). The critical step involves rapid folding of the newly formed Ub moiety, which precludes its translocation and makes possible its cleavage by UBPs. However, if a sufficiently long spacer is present between the signal sequence and Ub, then by the time the Ub polypeptide emerges from the ribosome, the latter is already docked at the transmembrane channel, allowing the translocation of both the Ub and reporter domains of the fusion into the ER. We show that Ub fusions can be used as in vivo probes for kinetic and stochastic aspects of targeting in protein translocation, for distinguishing directly between cotranslational and posttranslational translocation, and for comparing the strengths of different signal sequences. This method should also be applicable to non-ER translocation.", "doi": "10.1002/j.1460-2075.1994.tb06559.x", "pmcid": "PMC395143", "issn": "0261-4189", "publisher": "European Molecular Biology Organization", "publication": "EMBO Journal", "publication_date": "1994-06-01", "series_number": "11", "volume": "13", "issue": "11", "pages": "2686-2698" }, { "id": "authors:rjhsv-hyz20", "collection": "authors", "collection_id": "rjhsv-hyz20", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150120-134752646", "type": "article", "title": "Heat-Inducible Degron: A Method for Constructing Temperature-Sensitive Mutants", "author": [ { "family_name": "Dohmen", "given_name": "R. J\u00fcrgen", "clpid": "Dohmen-R-J\u00fcrgen" }, { "family_name": "Wu", "given_name": "Peipei", "clpid": "Wu-Peipei" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "A temperature-sensitive (ts) mutant retains the function of a gene at a low (permissive) temperature but not at a high (nonpermissive) temperature. Arg-DHFR, a dihydrofolate reductase bearing an amino-terminal (N-terminal) arginine, is long-lived in the yeast Saccharomyces cerevisiae, even though arginine is a destabilizing residue in the N-end rule of protein degradation. A ts derivative of Arg-DHFR was identified that is long-lived at 23\u00b0C but rapidly degraded by the N-end rule pathway at 37\u00b0C. Fusions of ts Arg-DHFR to either Ura3 or Cdc28 of S. cerevisiae confer ts phenotypes specific for these gene products. Thus, Arg-DHFR^ts is a heat-inducible degradation signal that can be used to produce ts mutants without a search for ts mutations.", "doi": "10.1126/science.8122109", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "1994-03-04", "series_number": "5151", "volume": "263", "issue": "5151", "pages": "1273-1276" }, { "id": "authors:tjveh-wpt66", "collection": "authors", "collection_id": "tjveh-wpt66", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150122-121838530", "type": "article", "title": "A yeast protein similar to bacterial two-component regulators", "author": [ { "family_name": "Ota", "given_name": "Irene M.", "clpid": "Ota-Irene-M" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Many bacterial signaling pathways involve a two-component design. In these pathways, a sensor kinase, when activated by a signal, phosphorylates its own histidine, which then serves as a phosphoryl donor to an aspartate in a response regulator protein. The Sln1 protein of the yeast Saccharomyces cerevisiae has sequence similarities to both the histidine kinase and the response regulator proteins of bacteria. A missense mutation in SLN1 is lethal in the absence but not in the presence of the N-end rule pathway, a ubiquitin-dependent proteolytic system. The finding of SLN1 demonstrates that a mode of signal transduction similar to the bacterial two-component design operates in eukaryotes as well.", "doi": "10.1126/science.8211183", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "1993-10-22", "series_number": "5133", "volume": "262", "issue": "5133", "pages": "566-569" }, { "id": "authors:k2z6b-eq991", "collection": "authors", "collection_id": "k2z6b-eq991", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120308-111115401", "type": "article", "title": "The N-End Rule in Escherichia coli: Cloning and Analysis of\n the Leucyl, Phenylalanyl-tRNA-Protein Transferase Gene aat", "author": [ { "family_name": "Shrader", "given_name": "Thomas E.", "clpid": "Shrader-Thomas-E" }, { "family_name": "Tobias", "given_name": "John W.", "clpid": "Tobias-John-W" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Distinct versions of the N-end rule operate in bacteria, fungi, and mammals. We report the cloning and analysis of aat, the Escherichia coli gene that encodes leucyl, phenylalanyl-tRNA-protein transferase (L/F-transferase), a component of the bacterial N-end rule pathway. L/F-transferase is required for the degradation of N-end rule substrates bearing an N-terminal arginine or lysine. The aat gene maps to the 19-min region of the E. coli chromosome and encodes a 234-residue protein whose sequence lacks significant similarities to sequences in data bases. In vitro, L/F-transferase catalyzes the posttranslational conjugation of leucine or phenylalanine to the N termini of proteins that bear an N-terminal arginine or lysine. However, the isolation and sequence analysis of a \u03b2-galactosidase variant engineered to expose an N-terminal arginine in vivo revealed the conjugation of leucine but not of phenylalanine to the N terminus of the \u03b2-galactosidase variant. Thus, the specificity of L/F-transferase in vivo may be greater than that in vitro. The aat gene is located approximately 1 kb from clpA, which encodes a subunit of ATP-dependent protease Clp. Although both aat and clpA are required for the degradation of certain N-end rule substrates, their nearly adjacent genes are convergently transcribed. The aat gene lies downstream of an open reading frame that encodes a homolog of the mammalian multidrug resistance P glycoproteins.", "doi": "10.1128/jb.175.14.4364-4374.1993", "pmcid": "PMC204876", "issn": "0021-9193", "publisher": "American Society for Microbiology", "publication": "Journal of Bacteriology", "publication_date": "1993-07", "series_number": "14", "volume": "175", "issue": "14", "pages": "4364-4374" }, { "id": "authors:qnje6-5rg64", "collection": "authors", "collection_id": "qnje6-5rg64", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210210-151525353", "type": "article", "title": "N-recognin/Ubc2 interactions in the N-end rule pathway", "author": [ { "family_name": "Madura", "given_name": "Kiran", "clpid": "Madura-Kiran" }, { "family_name": "Dohmen", "given_name": "R. J\u00fcrgen", "clpid": "Dohmen-R-J\u00fcrgen" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In the yeast Saccharomyces cerevisiae, substrates of the N-end rule pathway are targeted for degradation by a complex that includes the 225-kDa N-recognin, encoded by UBR1, and the 20-kDa ubiquitin-conjugating enzyme encoded by UBC2. We report that both physical stability and functional activity of the N-recognin.Ubc2 complex require the presence of a highly acidic 23-residue region at the C terminus of Ubc2. Ubc2-C88A, an inactive variant of Ubc2 in which the active-site Cys-88 has been replaced by Ala, is shown to retain the affinity for N-recognin. Expression of Ubc2-C88A inhibits the N-end rule pathway, apparently as a result of competition between Ubc2 and Ubc2-C88A for binding to N-recognin. The two-hybrid (interaction cloning) technique was used to identify a approximately 170-residue C-terminal fragment of the 1,950-residue N-recognin as a Ubc2-interacting domain. We also show that the level of UBR1 mRNA decreases upon overexpression of UBC2. This effect of UBC2 is observed with cells whose UBR1 is expressed from an unrelated promoter but is not observed if UBR1 contains a frameshift mutation, or if the Ubc2 protein lacks its C-terminal acidic region. The N-recognin.Ubc2 complex appears to regulate the expression of N-recognin through changes in the metabolic stability of its mRNA.", "doi": "10.1016/s0021-9258(19)50306-4", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "1993-06-05", "series_number": "16", "volume": "268", "issue": "16", "pages": "12046-12054" }, { "id": "authors:q0tqy-4mw52", "collection": "authors", "collection_id": "q0tqy-4mw52", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210210-151525426", "type": "article", "title": "Ubiquitin-specific proteases of Saccharomyces cerevisiae. Cloning of UBP2 and UBP3, and functional analysis of the UBP gene family", "author": [ { "family_name": "Baker", "given_name": "Rohan T.", "clpid": "Baker-Rohan-T" }, { "family_name": "Tobias", "given_name": "John W.", "clpid": "Tobias-John-W" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "In eukaryotes, both natural and engineered ubiquitin (Ub) fusions to itself or other proteins are cleaved by processing proteases after the last (Gly76) residue of ubiquitin. YUH1 and UBP1, the genes for two ubiquitin-specific proteases of the yeast Saccharomyces cerevisiae, have been cloned previously and shown to encode nonhomologous proteins. Using an Escherichia coli-based genetic screen, we have isolated two other yeast genes for ubiquitin-specific proteases, named UBP2 and UBP3. Ubp2 (1,264 residues), Ubp3 (912 residues), and the previously cloned Ubp1 (809 residues) are largely dissimilar except for two short regions containing Cys and His which encompass their putative active sites. Neither of these proteases has sequence similarities to Yuh1. Both Ubp2 and the previously identified Ubp1 cleave in vitro at the C terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their size, poly-Ub being the exception. However, both Ubp1 and Ubp2 are also capable of cleaving poly-Ub when coexpressed with it in E. coli, suggesting that such cleavage is largely cotranslational. Although inactive in E. coli extracts, Ubp3 was active with all of the tested ubiquitin fusions except poly-Ub when coexpressed with them in E. coli. Null yuh1 ubp1 ubp2 ubp3 quadruple mutants are viable and retain the ability to deubiquitinate ubiquitin fusions, indicating the presence of at least one more ubiquitin-specific processing protease in S. cerevisiae.", "doi": "10.1016/s0021-9258(18)50100-9", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "1992-11-15", "series_number": "32", "volume": "267", "issue": "32", "pages": "23364-23375" }, { "id": "authors:y7dbd-7dy57", "collection": "authors", "collection_id": "y7dbd-7dy57", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210210-151525504", "type": "article", "title": "The N-end rule", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Since its discovery in 1986, distinct versions of the N-end rule have been shown to operate in all organisms examined, from animals and plants to yeast and bacteria. This review considers intracellular protein degradation with an emphasis on the N-end rule.", "doi": "10.1016/0092-8674(92)90285-k", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "1992-05-29", "series_number": "5", "volume": "69", "issue": "5", "pages": "725-735" }, { "id": "authors:cg4e8-b8y63", "collection": "authors", "collection_id": "cg4e8-b8y63", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:OTApnas92", "type": "article", "title": "A gene encoding a putative tyrosine phosphatase suppresses lethality of an N-end rule-dependent mutant", "author": [ { "family_name": "Ota", "given_name": "Irene M.", "clpid": "Ota-Irene-M" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In the yeast Saccharomyces cerevisiae, mutational inactivation of the N-end rule pathway is neither lethal nor phenotypically conspicuous. We have used a \"synthetic lethal\" screen to isolate a mutant that requires the N-end rule pathway for viability. An extragenic suppressor of this mutation was cloned and found to encode a 750-residue protein with strong sequence similarities to protein phosphotyrosine phosphatases. This heat-inducible gene was named PTP2. Null ptp2 mutants grow slowly, are hypersensitive to heat, and are viable in either the presence or absence of the N-end rule pathway. We discuss possible connections between dephosphorylation of phosphotyrosine in proteins and the N-end rule pathway of protein degradation.", "doi": "10.1073/pnas.89.6.2355", "pmcid": "PMC48656", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1992-03-15", "series_number": "6", "volume": "89", "issue": "6", "pages": "2355-2359" }, { "id": "authors:m40j0-gvv11", "collection": "authors", "collection_id": "m40j0-gvv11", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210210-151525582", "type": "article", "title": "Ubiquitin as a degradation signal", "author": [ { "family_name": "Johnson", "given_name": "Erica S.", "clpid": "Johnson-Erica-S" }, { "family_name": "Bartel", "given_name": "Bonnie", "clpid": "Bartel-Bonnie" }, { "family_name": "Seufert", "given_name": "Wolfgang", "clpid": "Seufert-Wolfgang" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "For many short\u2010lived eukaryotic proteins, conjugation to ubiquitin, yielding a multiubiquitin chain, is an obligatory pre\u2010degradation step. The conjugated ubiquitin moieties function as a 'secondary' signal for degradation, in that their posttranslational coupling to a substrate protein is mediated by amino acid sequences of the substrate that act as a primary degradation signal. We report that the fusion protein ubiquitin\u2010\u2010proline\u2010\u2010beta\u2010galactosidase (Ub\u2010P\u2010beta gal) is short\u2010lived in the yeast Saccharomyces cerevisiae because its N\u2010terminal ubiquitin moiety functions as an autonomous, primary degradation signal. This signal mediates the formation of a multiubiquitin chain linked to Lys48 of the N\u2010terminal ubiquitin in Ub\u2010P\u2010beta gal. The degradation of Ub\u2010P\u2010beta gal is shown to require Ubc4, one of at least seven ubiquitin\u2010conjugating enzymes in S.cerevisiae. Our findings provide the first direct evidence that a monoubiquitin moiety can function as an autonomous degradation signal. This generally applicable, cis\u2010acting signal can be used to manipulate the in vivo half\u2010lives of specific intracellular proteins.", "doi": "10.1002/j.1460-2075.1992.tb05080.x", "pmcid": "PMC556480", "issn": "0261-4189", "publisher": "European Molecular Biology Organization", "publication": "EMBO Journal", "publication_date": "1992-02", "series_number": "2", "volume": "11", "issue": "2", "pages": "497-505" }, { "id": "authors:9z5zs-99870", "collection": "authors", "collection_id": "9z5zs-99870", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210210-151525693", "type": "article", "title": "The N-end rule in bacteria", "author": [ { "family_name": "Tobias", "given_name": "John W.", "clpid": "Tobias-John-W" }, { "family_name": "Shrader", "given_name": "Thomas E.", "clpid": "Shrader-Thomas-E" }, { "family_name": "Rocap", "given_name": "Gabrielle", "clpid": "Rocap-Gabrielle" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the in vivo half-life of a protein to the identity of its amino-terminal residue. Distinct versions of the N-end rule operate in all eukaryotes examined. It is shown that the bacterium Escherichia coli also has the N-end rule pathway. Amino-terminal arginine, lysine, leucine, phenylalanine, tyrosine, and tryptophan confer 2-minute half-lives on a test protein; the other amino-terminal residues confer greater than 10-hour half-lives on the same protein. Amino-terminal arginine and lysine are secondary destabilizing residues in E. coli because their activity depends on their conjugation to the primary destabilizing residues leucine or phenylalanine by leucine, phenylalanine-transfer RNA-protein transferase. The adenosine triphosphate-dependent protease Clp (Ti) is required for the degradation of N-end rule substrates in E. coli.", "doi": "10.1126/science.1962196", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "1991-11-29", "series_number": "5036", "volume": "254", "issue": "5036", "pages": "1374-1377" }, { "id": "authors:3f5d0-he036", "collection": "authors", "collection_id": "3f5d0-he036", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:DOHpnas91", "type": "article", "title": "The N-End Rule is Mediated by the UBC2(RAD6) Ubiquitin-Conjugating Enzyme", "author": [ { "family_name": "Dohmen", "given_name": "R. J\u00fcrgen", "clpid": "Dohmen-R-J\u00fcrgen" }, { "family_name": "Madura", "given_name": "Kiran", "clpid": "Madura-Kiran" }, { "family_name": "Bartel", "given_name": "Bonnie", "clpid": "Bartel-Bonnie" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the in vivo half-life of a protein to the identity of its amino-terminal residue. Distinct versions of the N-end rule operate in all organisms examined, from mammals to bacteria. We show that UBC2(RAD6), one of at least seven ubiquitin-conjugating enzymes in the yeast Saccharomyces cerevisiae, is essential for multiubiquitination and degradation of the N-end rule substrates. We also show that UBC2 is physically associated with UBR1, the recognition component of the N-end rule pathway. These results indicate that some of the UBC2 functions, which include DNA repair, induced mutagenesis, sporulation, and regulation of retrotransposition, are mediated by protein degradation via the N-end rule pathway.", "doi": "10.1073/pnas.88.16.7351", "pmcid": "PMC52293", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1991-08-15", "series_number": "16", "volume": "88", "issue": "16", "pages": "7351-7355" }, { "id": "authors:3wwa0-0z645", "collection": "authors", "collection_id": "3wwa0-0z645", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210210-151525763", "type": "article", "title": "Cloning and functional analysis of the ubiquitin-specific protease gene UBP1 of Saccharomyces cerevisiae", "author": [ { "family_name": "Tobias", "given_name": "John W.", "clpid": "Tobias-John-W" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "In eukaryotes, both natural and engineered fusions of ubiquitin to itself or other proteins are cleaved by processing proteases after the last (Gly76) residue of ubiquitin. Using the method of sib selection, and taking advantage of the fact that bacteria such as Escherichia coli lack ubiquitin-specific enzymes, we have cloned a gene, named UBP1, of the yeast Saccharomyces cerevisiae that encodes a ubiquitin-specific processing protease. With the exception of polyubiquitin, the UBP1 protease cleaves at the carboxyl terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their size or the presence of an amino-terminal ubiquitin extension. These properties of UBP1 distinguish it from the previously cloned yeast protease YUH1, which deubiquitinates relatively short ubiquitin fusions but is virtually inactive with longer fusions such as ubiquitin-beta-galactosidase. The amino acid sequence of the 809-residue UBP1 lacks significant similarities to other known proteins, including the 236-residue YUH1 protease. Null ubp1 mutants are viable, and retain the ability to deubiquitinate ubiquitin-beta-galactosidase, indicating that the family of ubiquitin-specific proteases in yeast is not limited to UBP1 and YUH1.", "doi": "10.1016/s0021-9258(18)99059-9", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "1991-06-25", "series_number": "18", "volume": "266", "issue": "18", "pages": "12021-12028" }, { "id": "authors:1dtn7-prp50", "collection": "authors", "collection_id": "1dtn7-prp50", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:HOCpnas91", "type": "article", "title": "The short-lived MAT\u03b12 transcriptional regulator is ubiquitinated in vivo", "author": [ { "family_name": "Hochstrasser", "given_name": "Mark", "clpid": "Hochstrasser-Mark" }, { "family_name": "Ellison", "given_name": "Michael J.", "clpid": "Ellison-Michael-J" }, { "family_name": "Chau", "given_name": "Vincent", "clpid": "Chau-Vincent" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The substrates of ubiquitin-dependent proteolytic pathways include both damaged or otherwise abnormal proteins and undamaged proteins that are naturally short-lived. Few specific examples of the latter class have been identified, however. Previous work has shown that the cell type-specific MAT-alpha-2 repressor of the yeast Saccharomyces cerevisiae is an extremely short-lived protein. We now demonstrate that alpha-2 is conjugated to ubiquitin in vivo. More than one lysine residue of alpha-2 can be joined to ubiquitin, and some of the ubiquitin moieties form a Lys48-linked multiubiquitin chain. Overexpression of degradation-impaired ubiquitin variants was used to show that at least a significant fraction of alpha-2 degradation is dependent on its ubiquitination.", "doi": "10.1073/pnas.88.11.4606", "pmcid": "PMC51714", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1991-06-01", "series_number": "11", "volume": "88", "issue": "11", "pages": "4606-4610" }, { "id": "authors:za0pr-h4027", "collection": "authors", "collection_id": "za0pr-h4027", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:BAKpnas91", "type": "article", "title": "Inhibition of the N-end rule pathway in living cells", "author": [ { "family_name": "Baker", "given_name": "Rohan T.", "clpid": "Baker-Rohan-T" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule relates the metabolic stability of a protein to the identity of its amino-terminal residue. Previous work, using amino acid derivatives such as dipeptides to inhibit N-end rule-mediated protein degradation in an extract from mammalian reticulocytes, has demonstrated the existence of specific N-end-recognizing proteins in this in vitro system. We now show that these nontoxic amino acid derivatives, when added to growing cells of the yeast Saccharomyces cerevisiae, are able to inhibit the degradation of proteins by the N-end rule pathway in vivo. Moreover, this inhibition is shown to be selective for the two distinct classes of destabilizing amino-terminal residues in substrates of the N-end rule pathway.", "doi": "10.1073/pnas.88.4.1090", "pmcid": "PMC50962", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1991-02-15", "series_number": "4", "volume": "88", "issue": "4", "pages": "1090-1094" }, { "id": "authors:dn085-by896", "collection": "authors", "collection_id": "dn085-by896", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210210-151525821", "type": "article", "title": "Naming a targeting signal", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "A staggering variety of potential fates awaits a protein emerging from a ribosome. The presence (or absence) of certain amino acid sequences in a nascent protein constrains\nthe set of metabolic fates that actually befall it by making some fates more probable than others. These sequences, known as targeting signals, can determine the spatial destination of a protein, its folding, posttranslational modifications, and metabolic stability. The understanding of targeting signals has grown enormously over the last fifteen years (reviewed by Blobel, 1980; Wold, 1981; Creighton, 1984; Walter and Lingappa, 1987; Randall et al., 1987; Pfeffer and Rothman, 1987; Burgess and Kelly, 1987; Verner and Schatz, 1988; Gierasch, 1989; Hart1 and Neupert, 1990). Unfortunately, these advances have not been accompanied by a clarification and simplification of terminology. For instance, signals that enable a protein to translocate across the plane of a membrane are called signal sequences, topogenic sequences, targeting sequences, transit sequences, presequences, translocation sequences, or leader sequences. Ad hoc, often semantically cumbersome terms are also used to denote other targeting signals. Yet most of the transformations that proteins undergo can be grouped into five distinct classes of transitions:\n\n* Translocation of a protein across the plane of a membrane\nseparating two compartments. \n\n* Transfer of a protein between compartments without crossing the plane of a membrane (vesicle-mediated transport). \n\n* Hydrolysis of some or all of the peptide bonds in a protein.\n\n* Chemical modifications of a protein that do not involve\nhydrolysis of peptide bonds. \n\n* Conformational modifications of a protein, including its\nfolding and oligomerization.", "doi": "10.1016/0092-8674(91)90202-a", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "1991-01-11", "series_number": "1", "volume": "64", "issue": "1", "pages": "13-15" }, { "id": "authors:nr0ce-awg31", "collection": "authors", "collection_id": "nr0ce-awg31", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210210-151525895", "type": "article", "title": "UBA 1: an essential yeast gene encoding ubiquitin-activating enzyme", "author": [ { "family_name": "McGrath", "given_name": "John P.", "clpid": "McGrath-John-P" }, { "family_name": "Jentsch", "given_name": "Stefan", "clpid": "Jentsch-Stefan" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "All known functions of ubiquitin are mediated through its covalent attachment to other proteins. The post\u2010translational formation of ubiquitin\u2013protein conjugates is preceded by an ATP\u2010requiring step in which the carboxyl terminus of ubiquitin is adenylated and subsequently joined, through a thiolester bond, to a cysteine residue in the ubiquitin\u2010activating enzyme, also known as E1. We report the isolation and functional analysis of the gene (UBA1) for the ubiquitin\u2010activating enzyme of the yeast Saccharomyces cerevisiae. UBA1 encodes a 114 kd protein whose amino acid sequence contains motifs characteristic of nucleotide\u2010binding sites. Expression of catalytically active UBA1 protein in E. coli, which lacks the ubiquitin system, confirmed that the yeast UBA1 gene encodes a ubiquitin\u2010activating enzyme. Deletion of the UBA1 gene is lethal, demonstrating that the formation of ubiquitin\u2013protein conjugates is essential for cell viability.", "doi": "10.1002/j.1460-2075.1991.tb07940.x", "pmcid": "PMC452634", "issn": "0261-4189", "publisher": "European Molecular Biology Organization", "publication": "EMBO Journal", "publication_date": "1991-01", "series_number": "1", "volume": "10", "issue": "1", "pages": "227-236" }, { "id": "authors:8sv4m-9sj92", "collection": "authors", "collection_id": "8sv4m-9sj92", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210129-142402554", "type": "article", "title": "Cis-trans recognition and subunit-specific degradation of short-lived proteins", "author": [ { "family_name": "Johnson", "given_name": "Erica S.", "clpid": "Johnson-Erica-S" }, { "family_name": "Gonda", "given_name": "David K.", "clpid": "Gonda-David-K" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The N-end rule, a code that relates the metabolic stability of a protein to the identity of its ammo-terminal residue, is universal in that different versions of the N-end rule operate in mammals, yeast and bacteria (unpublished data). The N-end rule-based degradation signal comprises a destabilizing amino-terminal residue and a specific internal lysine residue. We now show that, in a multisubunit protein, these two determinants can be located on different subunits and still target the protein for destruction. Moreover, in this case (trans recognition) only the subunit that bears the lysine determinant is actually degraded. Thus an oligomeric protein can contain both short-lived and long-lived subunits. These insights have functional and practical implications.", "doi": "10.1038/346287a0", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "1990-07-19", "series_number": "6281", "volume": "346", "issue": "6281", "pages": "287-291" }, { "id": "authors:c8bw0-n7428", "collection": "authors", "collection_id": "c8bw0-n7428", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210129-142402686", "type": "article", "title": "In vivo degradation of a transcriptional regulator: The yeast \u03b12 repressor", "author": [ { "family_name": "Hochstrasser", "given_name": "Mark", "clpid": "Hochstrasser-Mark" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Metabolic instability is characteristic of regulatory proteins whose in vivo concentrations must vary as a function of time. The cell type-specific \u03b12 repressor of the yeast S. cerevisiae is shown here to have a half-life of only \u223c5 min. Each of the two structural domains of \u03b12 carries a sequence that can independently target a normally long-lived protein for rapid destruction. Moreover, these two degradation signals are shown to operate via distinct mechanisms. Mutants deficient in the degradation of \u03b12 have been isolated and found to have a number of additional defects, indicating that the pathways responsible for \u03b12 turnover include components with multiple functions. Finally, we demonstrate that a short-lived subunit of an oligomeric protein can be degraded in vivo without destabilizing other, long-lived subunits of the same protein. This subunit-specific degradation makes possible a novel type of posttranslational remodeling in which a heteromeric protein could be functionally modified by selective, degradation-mediated replacement of its subunits.", "doi": "10.1016/0092-8674(90)90481-s", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "1990-05-18", "series_number": "4", "volume": "61", "issue": "4", "pages": "697-708" }, { "id": "authors:a69mh-krn45", "collection": "authors", "collection_id": "a69mh-krn45", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210129-142402779", "type": "article", "title": "Cloning and functional analysis of the arginyl-tRNA-protein transferase gene ATE1 of Saccharomyces cerevisiae", "author": [ { "family_name": "Balzi", "given_name": "Elisabetta", "clpid": "Balzi-Elisabetta" }, { "family_name": "Choder", "given_name": "Mordechai", "clpid": "Choder-Mordechai" }, { "family_name": "Chen", "given_name": "Weining", "clpid": "Chen-Weining" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Goffeau", "given_name": "Andre", "clpid": "Goffeau-Andre" } ], "abstract": "Aminoacyl-tRNA-protein transferases (Arg-transferases) catalyze post-translational conjugation of specific amino acids to the amino termini of acceptor proteins. A function of these enzymes in eukaryotes has been shown to involve the conjugation of destabilizing amino acids to the amino termini of short-lived proteins, these reactions being a part of the N-end rule pathway of protein degradation (Gonda, D. K., Bachmair, A., W\u00fcnning, I., Tobias, J. W., Lane, W. S., and Varshavsky, A. (1989) J. Biol. Chem. 264, 16700-16712). We have cloned the ATE1 gene of the yeast Saccharomyces cerevisiae which encodes arginyl-tRNA-protein transferase. ATE1 gives rise to a approximately 1.6-kilobase mRNA and codes for a 503-residue protein. Expression of the yeast ATE1 gene in Escherichia coli, which lacks Arg-transferases, was used to show that the ATE1 protein possesses the Arg-transferase activity. Null ate1 mutants are viable but lack the Arg-transferase activity and are unable to degrade those substrates of the N-end rule pathway that start with residues recognized by the Arg-transferase.", "doi": "10.1016/s0021-9258(19)39136-7", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "1990-05-05", "series_number": "13", "volume": "265", "issue": "13", "pages": "7464-7471" }, { "id": "authors:s0arq-n3s65", "collection": "authors", "collection_id": "s0arq-n3s65", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210129-142402869", "type": "article", "title": "Universality and Structure of the N-end Rule", "author": [ { "family_name": "Gonda", "given_name": "David K.", "clpid": "Gonda-David-K" }, { "family_name": "Bachmair", "given_name": "Andreas", "clpid": "Bachmair-Andreas" }, { "family_name": "W\u00fcnning", "given_name": "Ingrid", "clpid": "W\u00fcnning-Ingrid" }, { "family_name": "Tobias", "given_name": "John W.", "clpid": "Tobias-John-W" }, { "family_name": "Lane", "given_name": "William S.", "clpid": "Lane-William-S" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Our previous work has shown that, in the yeast Saccharomyces cerevisiae, any of the eight stabilizing amino-terminal residues confers a long (> 20 h) half-life on a test protein \u03b2-galactosidase (\u03b2 gal), whereas 12 destabilizing amino-terminal residues confer on \u03b2 gal half-lives from less than 3 min to 30 min. We now show that an analogous single-residue code (the N-end rule) operates in an in vitro system derived from mammalian reticulocytes. We also show that the N-end rule has a hierarchical structure. Specifically, amino-terminal Glu and Asp (and also Cys in reticulocytes) are secondary destabilizing residues in that they are destabilizing through their ability to be conjugated to primary destabilizing residues such as Arg. Amino-terminal Gln and Asn are tertiary destabilizing residues in that they are destabilizing through their ability to be converted, via selective deamidation, into secondary destabilizing residues Glu and Asp. Furthermore, in reticulocytes, distinct types of the N-end-recognizing activity are shown to be specific for three classes of primary destabilizing residues: basic (Arg, Lys, His), bulky hydrophobic (Phe, Leu, Trp, Tyr), and small uncharged (Ala, Ser, Thr). Features of the N-end rule in reticulocytes suggest that the exact form of the N-end rule may depend on the cell's physiological state, thereby providing a mechanism for selective destruction of preexisting proteins upon cell differentiation.", "doi": "10.1016/s0021-9258(19)84762-2", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "1989-10-05", "series_number": "28", "volume": "264", "issue": "28", "pages": "16700-16712" }, { "id": "authors:wsbww-x9946", "collection": "authors", "collection_id": "wsbww-x9946", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210129-142402941", "type": "article", "title": "The yeast STE6 gene encodes a homologue of the mammalian multidrug resistance P-glycoprotein", "author": [ { "family_name": "McGrath", "given_name": "John P.", "clpid": "McGrath-John-P" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "MAMMALIAN tumours displaying multidrug resistance overexpress a plasma membrane protein (P-glycoprotein), which is encoded by the MDR1 gene and apparently functions as an energy-dependent drug efflux pump. Tissue-specific expression of MDR1 and other members of the MDR gene family has been observed in normal cells, suggesting a role for P-glycoproteins in secretion. We have isolated a gene from the yeast Saccharomyces cerevisiae that encodes a protein very similar to mammalian P-glycoproteins. Deletion of this gene resulted in sterility of MATa, but not of MAT\u03b1 cells. Subsequent analysis revealed that the yeast P-glycoprotein is the product of the STE6 gene, a locus previously shown to be required in MATa cells for production of a-factor pheromone. Our findings suggest that the STE6 protein functions to export the hydrophobic a-factor lipopeptide in a manner analogous to the efflux of hydrophobic cytotoxic drugs catalysed by the related mammalian P-glycoprotein. Thus, the evolutionarily conserved family of MDR-like genes, including the hlyB gene of Escherichia coli and the STE6 gene of S. cerevisiae, encodes components of secretory pathways distinct from the classical, signal sequence-dependent protein translocation system.", "doi": "10.1038/340400a0", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "1989-08-03", "series_number": "6232", "volume": "340", "issue": "6232", "pages": "400-404" }, { "id": "authors:4s1nj-zx346", "collection": "authors", "collection_id": "4s1nj-zx346", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210129-142403046", "type": "article", "title": "A DNA binding protein that recognizes oligo(dA)\u2022oligo(dT) tracts", "author": [ { "family_name": "Winter", "given_name": "Edward", "clpid": "Winter-Edward" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Oligo(dA)\u2022oligo(dT) tracts are common intergenic sequences in many organisms. In the yeast Saccharomyces cerevisiae, these sequences have been shown to influence transcription of adjacent genes. We have purified an oligo(dA)\u2022oligo(dT)\u2010binding protein from S. cerevisiae and cloned its gene. This protein, which has been named datin, requires at least 9\u201011 bp of oligo(dA)\u2022oligo(dT) DNA for high affinity binding. The gene for datin (the DAT gene) encodes a 248\u2010residue protein which contains a number of repeated sequence motifs. Datin purified from yeast corresponds to the N\u2010terminal half of the DAT gene product. Null mutants in the DAT gene are viable but phenotypically distinguishable from congenic wild\u2010type strains. We discuss unusual structural features and biochemical properties of datin in relation to its possible functions.", "doi": "10.1002/j.1460-2075.1989.tb03583.x", "pmcid": "PMC401036", "issn": "0261-4189", "publisher": "European Molecular Biology Organization", "publication": "EMBO Journal", "publication_date": "1989-06", "series_number": "6", "volume": "8", "issue": "6", "pages": "1867-1877" }, { "id": "authors:4hbgy-zyf49", "collection": "authors", "collection_id": "4hbgy-zyf49", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210129-142403105", "type": "article", "title": "The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis", "author": [ { "family_name": "Finley", "given_name": "Daniel", "clpid": "Finley-Daniel" }, { "family_name": "Bartel", "given_name": "Bonnie", "clpid": "Bartel-Bonnie" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Three of the four yeast ubiquitin genes encode hybrid proteins which are cleaved to yield ubiquitin and previously unidentified ribosomal proteins. The transient association between ubiquitin and these proteins promotes their incorporation into nascent ribosomes and is required for efficient ribosome biogenesis. These results suggest a novel 'chaperone' function for ubiquitin, in which its covalent association with other proteins promotes the formation of specific cellular structures.", "doi": "10.1038/338394a0", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "1989-03-30", "series_number": "6214", "volume": "338", "issue": "6214", "pages": "394-401" }, { "id": "authors:te37s-8aw24", "collection": "authors", "collection_id": "te37s-8aw24", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210129-142403306", "type": "article", "title": "The degradation signal in a short-lived protein", "author": [ { "family_name": "Bachmair", "given_name": "Andreas", "clpid": "Bachmair-Andreas" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Our previous work has shown that the amino-terminal residue of a short-lived protein is a distinct component of the protein's degradation signal. To define the complete signal, otherwise identical dihydrofolate reductase test proteins bearing different extensions and either a \"stabilizing\" or a \"destabilizing\" amino-terminal residue were expressed in the yeast S. cerevisiae and their in vivo half-lives compared. The amino-terminal degradation signal is shown to comprise two distinct determinants. One, discovered previously, is the protein's amino-terminal residue. The second determinant, identified in the present work, is a specific lysine residue whose function in the degradation signal is not dependent on the unique amino acid sequences in the vicinity of the residue. The mechanistic significance of the second determinant is illuminated by the finding that in a targeted, short-lived protein, a chain of branched ubiquitin-ubiquitin conjugates is confined to a lysine residue that has been identified in the present work as the second determinant of the degradation signal.", "doi": "10.1016/0092-8674(89)90635-1", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "1989-03-24", "series_number": "6", "volume": "56", "issue": "6", "pages": "1019-1032" }, { "id": "authors:4gf5m-j5q44", "collection": "authors", "collection_id": "4gf5m-j5q44", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210129-142403198", "type": "article", "title": "A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein", "author": [ { "family_name": "Chau", "given_name": "Vincent", "clpid": "Chau-Vincent" }, { "family_name": "Tobias", "given_name": "John W.", "clpid": "Tobias-John-W" }, { "family_name": "Bachmair", "given_name": "Andreas", "clpid": "Bachmair-Andreas" }, { "family_name": "Marriott", "given_name": "David", "clpid": "Marriott-David" }, { "family_name": "Ecker", "given_name": "David J.", "clpid": "Ecker-David-J" }, { "family_name": "Gonda", "given_name": "David K.", "clpid": "Gonda-David-K" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The ubiquitin-dependent degradation of a test protein \u03b2-galactosidase (\u03b2gal) is preceded by ubiquitination of beta gal. The many (from 1 to more than 20) ubiquitin moieties attached to a molecule of beta gal occur as an ordered chain of branched ubiquitin-ubiquitin conjugates in which the carboxyl-terminal Gly\u2077\u2076 of one ubiquitin is jointed to the internal Lys\u2074\u2078 of an adjacent ubiquitin. This multiubiquitin chain is linked to one of two specific Lys residues in \u03b2gal. These same Lys residues have been identified by molecular genetic analysis as components of the aminoterminal degradation signal in \u03b2gal. The experiments with ubiquitin mutated at its Lys\u2074\u2078 residue indicate that the multiubiquitin chain in a targeted protein is essential for the degradation of the protein.", "doi": "10.1126/science.2538923", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "1989-03-24", "series_number": "4898", "volume": "243", "issue": "4898", "pages": "1576-1583" }, { "id": "authors:bps1z-7cg83", "collection": "authors", "collection_id": "bps1z-7cg83", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201125-154026315", "type": "article", "title": "Targeting of proteins for degradation", "author": [ { "family_name": "Varshavsky", "given_name": "A.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Bachmair", "given_name": "A.", "clpid": "Bachmair-Andreas" }, { "family_name": "Finley", "given_name": "D.", "clpid": "Finley-D" }, { "family_name": "Gonda", "given_name": "D. K.", "clpid": "Gonda-D-K" }, { "family_name": "W\u00fcnning", "given_name": "I.", "clpid": "W\u00fcnning-I" } ], "abstract": "[no abstract]", "issn": "0740-7378", "publisher": "Butterworths", "publication": "Biotechnology (Reading, Mass.)", "publication_date": "1989", "volume": "13", "pages": "109-43" }, { "id": "authors:5eeqj-66687", "collection": "authors", "collection_id": "5eeqj-66687", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210129-142403433", "type": "article", "title": "The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme", "author": [ { "family_name": "Goebl", "given_name": "Mark G.", "clpid": "Goebl-Mark-G" }, { "family_name": "Yochem", "given_name": "John", "clpid": "Yochem-John" }, { "family_name": "Jentsch", "given_name": "Stefan", "clpid": "Jentsch-Stefan" }, { "family_name": "McGrath", "given_name": "John P.", "clpid": "McGrath-John-P" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Byers", "given_name": "Breck", "clpid": "Byers-Breck" } ], "abstract": "Mutants in the gene CDC34 of the yeast Saccharomyces cerevisiae are defective in the transition from G1 to the S phase of the cell cycle. This gene was cloned and shown to encode a 295-residue protein that has substantial sequence similarity to the product of the yeast RAD6 gene. The RAD6 gene is required for a variety of cellular functions including DNA repair and was recently shown to encode a ubiquitin-conjugating enzyme. When produced in Escherichia coli, the CDC34 gene product catalyzed the covalent attachment of ubiquitin to histones H2A and H2B in vitro, demonstrating that the CDC34 protein is another distinct member of the family of ubiquitin-conjugating enzymes. The cell cycle function of CDC34 is thus likely to be mediated by the ubiquitin-conjugating activity of its product.", "doi": "10.1126/science.2842867", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "1988-09-09", "series_number": "4871", "volume": "241", "issue": "4871", "pages": "1331-1335" }, { "id": "authors:k2v65-xwt80", "collection": "authors", "collection_id": "k2v65-xwt80", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210129-142403364", "type": "article", "title": "Mapping protein-DNA interactions in vivo with formaldehyde: Evidence that histone H4 is retained on a highly transcribed gene", "author": [ { "family_name": "Solomon", "given_name": "Mark J.", "clpid": "Solomon-Mark-J" }, { "family_name": "Larsen", "given_name": "Pamela L.", "clpid": "larsen-Pamela-L" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "We have used formaldehyde-mediated protein-DNA crosslinking within intact cells to examine the in vivo chromatin structure of the D. melanogaster heat shock protein 70 (hsp70) genes. In agreement with previous in vitro studies, we find that the heat shock-mediated transcriptional induction of the hsp70 genes perturbs their chromatin structure, resulting in fewer proteinDNA contacts crosslinkable in vivo by formaldehyde. However, contrary to earlier in vitro evidence that histones may be absent from actively transcribed genes, we show directly, by immunoprecipitation of in vivo-crosslinked chromatin fragments, that at least histone H4 remains bound to hsp70 DNA in vivo, irrespective of its rate of transcription. The formaldehyde-based in vivo mapping techniques described in this work are generally applicable, and can be used both to probe proteinDNA interactions within specific genes and to determine the genomic location of specific chromosomal proteins.", "doi": "10.1016/s0092-8674(88)90469-2", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "1988-06-17", "series_number": "6", "volume": "53", "issue": "6", "pages": "937-947" }, { "id": "authors:m53g7-0mv19", "collection": "authors", "collection_id": "m53g7-0mv19", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210129-142403546", "type": "article", "title": "Hypersensitivity to heavy water: A new conditional phenotype", "author": [ { "family_name": "Bartel", "given_name": "Bonnie", "clpid": "Bartel-Bonnie" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Wild-type strains of the yeast S. cerevisiae can grow on media containing 90% D\u2082O. Using chemical mutagenesis we obtained a number of strains that grow on H\u2082O-containing media but not on otherwise identical media containing 90% D\u2082O. The frequency of these D\u2082O-sensitive (ds) mutants is comparable to the frequency of conventional temperature-sensitive (ts) mutants in the same mutagenized sample, and the ds mutations are distributed over a large number of complementation groups. Furthermore, most ds mutants do not display other conditional phenotypes, such as heat, cold, or osmotic sensitivity. Conversely, of 17 cell division cycle is mutants tested, only 2 are also ds. Thus, the ds technique should be useful for producing conditional mutations in genes that are not amenable to the is and cs approaches, and also for generating alternative conditional (ds) alleles in many other genes. In addition, the ds technique should make it possible to generate conditional (ds) mutants in homeothermic animals, thereby extending the advantages of conditional phenotypes to mammalian and avian genetics.", "doi": "10.1016/0092-8674(88)90435-7", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "1988-03-25", "series_number": "6", "volume": "52", "issue": "6", "pages": "935-941" }, { "id": "authors:43t5m-r3492", "collection": "authors", "collection_id": "43t5m-r3492", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201201-103332052", "type": "article", "title": "N-end rule of selective protein turnover: mechanistic aspects and functional implications", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Bachmair", "given_name": "Andreas", "clpid": "Bachmair-Andreas" }, { "family_name": "Finley", "given_name": "Daniel", "clpid": "Finley-Daniel" } ], "abstract": "In both bacterial and eukaryotic cells, relatively long-lived proteins, whose half-lives are close to or exceed the cell generation time, coexist with proteins whose half-lives can be less than 1% of the cell generation time. Rates of intra-cellular protein degeneration are a function of the cell's physiological state, and appear to be controlled differentially for individual proteins. In particular, damaged and some otherwise abnormal proteins are metabolically unstable in vivo. Although the specific functions of selective protein degradation are in most cases still unknown, it is clear that many regulatory proteins are extremely short-lived in vivo. Metabolic instability of such proteins allows for rapid adjustments of their intracellular concentrations through regulated changes in rates of their synthesis or degradation.", "doi": "10.1042/bst0150815", "issn": "0300-5127", "publisher": "Portland Press Ltd.", "publication": "Biochemical Society Transactions", "publication_date": "1987-10-01", "series_number": "5", "volume": "15", "issue": "5", "pages": "815-816" }, { "id": "authors:5drew-49a02", "collection": "authors", "collection_id": "5drew-49a02", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:PECmcb87", "type": "article", "title": "Transcriptionally inactive oocyte-type 5S RNA genes of Xenopus laevis are complexed with TFIIIA in vitro", "author": [ { "family_name": "Peck", "given_name": "Lawrence J.", "clpid": "Peck-Lawrence-J" }, { "family_name": "Millstein", "given_name": "Larry", "clpid": "Millstein-Larry" }, { "family_name": "Eversole-Cire", "given_name": "Pamela", "clpid": "Eversole-Cire-Pamela" }, { "family_name": "Gottesfeld", "given_name": "Joel M.", "clpid": "Gottesfeld-Joel-M" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "An extract from whole oocytes of Xenopus laevis was shown to transcribe somatic-type 5S RNA genes approximately 100-fold more efficiently than oocyte-type 5S RNA genes. This preference was at least 10-fold greater than the preference seen upon microinjection of 5S RNA genes into oocyte nuclei or upon in vitro transcription in an oocyte nuclear extract. The approximately 100-fold transcriptional bias in favor of the somatic-type 5S RNA genes observed in vitro in the whole oocyte extract was similar to the transcriptional bias observed in developing Xenopus embryos. We also showed that in the whole oocyte extract, a promoter-binding protein required for 5S RNA gene transcription, TFIIIA, was bound both to the actively transcribed somatic-type 5S RNA gene and to the largely inactive oocyte-type 5S RNA genes. These findings suggest that the mechanism for the differential expression of 5S RNA genes during Xenopus development does not involve differential binding of TFIIIA to 5S RNA genes.", "doi": "10.1128/mcb.7.10.3503", "pmcid": "PMC368002", "issn": "0270-7306", "publisher": "American Society for Microbiology", "publication": "Molecular and Cellular Biology", "publication_date": "1987-10", "series_number": "10", "volume": "7", "issue": "10", "pages": "3503-3510" }, { "id": "authors:7mf1g-0r085", "collection": "authors", "collection_id": "7mf1g-0r085", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:SOLmcb87", "type": "article", "title": "A nuclease-hypersensitive region forms de novo after chromosome replication", "author": [ { "family_name": "Solomon", "given_name": "Mark J.", "clpid": "Solomon-Mark-J" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Regular nucleosome arrays in eucaryotic chromosomes are punctuated at specific locations, such as active promoters and replication origins, by apparently nucleosome-free sites, also called nuclease-hypersensitive, or exposed, regions. The -400-base pair-exposed region within simian virus 40 (SV40) chromosomes is present in approximately 20% of the chromosomes in lytically infected cells and encompasses the replication origin, transcriptional enhancer, and both late and early SV40 promoters. We report that nearly all SV40 chromosomes lacked the exposed region during replication and that newly formed chromosomes acquired the exposed region of the same degree as did bulk SV40 chromosomes within 1 h after replication. Furthermore, a much lower but significant level of exposure was detectable in late SV40 replication intermediates, indicating that formation of the exposed region could start within minutes after passage of the replication fork.", "doi": "10.1128/mcb.7.10.3822", "pmcid": "PMC368040", "issn": "0270-7306", "publisher": "American Society for Microbiology", "publication": "Molecular and Cellular Biology", "publication_date": "1987-10", "series_number": "10", "volume": "7", "issue": "10", "pages": "3822-3825" }, { "id": "authors:k2ary-ncn68", "collection": "authors", "collection_id": "k2ary-ncn68", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210122-162657916", "type": "article", "title": "The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme", "author": [ { "family_name": "Jentsch", "given_name": "Stefan", "clpid": "Jentsch-Stefan" }, { "family_name": "McGrath", "given_name": "John P.", "clpid": "McGrath-John-P" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The RAD6 gene of the yeast Saccharomyces cerevisiae is required for a variety of cellular functions including DNA repair. The discovery that the RAD6 gene product can catalyse the covalent attachment of ubiquitin to other proteins suggests that the multiple functions of the RAD6 protein are mediated by its ubiquitin-conjugating activity.", "doi": "10.1038/329131a0", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "1987-09-10", "series_number": "6135", "volume": "329", "issue": "6135", "pages": "131-134" }, { "id": "authors:42c6e-2kr84", "collection": "authors", "collection_id": "42c6e-2kr84", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210119-114432603", "type": "article", "title": "The yeast ubiquitin genes: a family of natural gene fusions", "author": [ { "family_name": "\u00d6zkaynak", "given_name": "Engin", "clpid": "\u00d6zkaynak-Engin" }, { "family_name": "Finley", "given_name": "Daniel", "clpid": "Finley-Daniel" }, { "family_name": "Solomon", "given_name": "Mark J.", "clpid": "Solomon-Mark-J" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Ubiquitin is a 76\u2010residue protein highly conserved among eukaryotes. Conjugation of ubiquitin to intracellular proteins mediates their selective degradation in vivo. We describe a family of four ubiquitin\u2010coding loci in the yeast Saccharomyces cerevisiae. UB11, UB12 and UB13 encode hybrid proteins in which ubiquitin is fused to unrelated ('tail') amino acid sequences. The ubiquitin coding elements of UB11 and UB12 are interrupted at identical positions by non\u2010homologous introns. UB11 and UB12 encode identical 52\u2010residue tails, whereas UB13 encodes a different 76\u2010residue tail. The tail amino acid sequences are highly conserved between yeast and mammals. Each tail contains a putative metal\u2010binding, nucleic acid\u2010binding domain of the form Cys\u2010X2\u20104\u2010Cys\u2010X2\u201015\u2010Cys\u2010X2\u20104\u2010Cys, suggesting that these proteins may function by binding to DNA. The fourth gene, UB14, encodes a polyubiquitin precursor protein containing five ubiquitin repeats in a head\u2010to\u2010tail, spacerless arrangement. All four ubiquitin genes are expressed in exponentially growing cells, while in stationary\u2010phase cells the expression of UB11 and UB12 is repressed. The UB14 gene, which is strongly inducible by starvation, high temperatures and other stresses, contains in its upstream region strong homologies to the consensus 'heat shock box' nucleotide sequence. Elsewhere we show that the essential function of the UB14 gene is to provide ubiquitin to cells under stress.", "doi": "10.1002/j.1460-2075.1987.tb02384.x", "pmcid": "PMC553949", "issn": "0261-4189", "publisher": "European Molecular Biology Organization", "publication": "EMBO Journal", "publication_date": "1987-05", "series_number": "5", "volume": "6", "issue": "5", "pages": "1429-1439" }, { "id": "authors:r359e-7r495", "collection": "authors", "collection_id": "r359e-7r495", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210122-162657728", "type": "article", "title": "The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses", "author": [ { "family_name": "Finley", "given_name": "Daniel", "clpid": "Finley-Daniel" }, { "family_name": "\u00d6zkaynak", "given_name": "Engin", "clpid": "\u00d6zkaynak-Engin" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Conjugation of ubiquitin to intracellular proteins mediates their selective degradation in eukaryotes. In the yeast Saccharomyces cerevisiae, four distinct ubiquitin-coding loci have been described. UBI1, UBI2, and UBI3 each encode hybrid proteins in which ubiquitin is fused to unrelated sequences. The fourth gene, UBI4, contains five ubiquitin-coding elements in a head-to-tail arrangement, and thus encodes a polyubiquitin precursor protein. A precise, oligonucleotide-directed deletion of UBI4 was constructed in vitro and substituted in the yeast genome in place of the wild-type allele. ubi4 deletion mutants are viable as vegetative cells, grow at wild-type rates, and contain wild-type levels of free ubiquitin under exponential growth conditions. However, although ubi4/UBI4 diploids can form four initially viable spores, the two ubi4 spores within the ascus lose viability extremely rapidly, apparently a novel phenotype in yeast. Furthermore, ubi4/ubi4 diploids are sporulation-defective. ubi4 mutants are also hypersensitive to high temperatures, starvation, and amino acid analogs. These three conditions, while diverse in nature, are all known to induce stress proteins. Expression of the UBI4 gene is similarly induced by either heat stress or starvation. These results indicate that UBI4 is specifically required for the resistance of cells to stress, and that ubiquitin is an essential component of the stress response system.", "doi": "10.1016/0092-8674(87)90711-2", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "1987-03-27", "series_number": "6", "volume": "48", "issue": "6", "pages": "1035-1046" }, { "id": "authors:m3ck0-6xj67", "collection": "authors", "collection_id": "m3ck0-6xj67", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210122-162658053", "type": "book_section", "title": "Electrophoretic assay for DNA-binding proteins", "book_title": "Methods in Enzymology", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "This chapter describes a generally applicable assay for specific DNA-binding proteins in crude extracts. The assay is based on gel electrophoretic separation of protein\u2013DNA complexes from each other and from free DNA. This assay employed in several laboratories to detect a variety of DNA-binding proteins. In spite of relatively brief experience with the electrophoretic assay as a detection tool, its exceptionally high sensitivity has already made this assay the method of choice in many experimental settings. The electrophoretic assay has been successfully applied to the detection of both highly sequence-specific, nonabundant DNA-binding proteins and abundant DNA-binding proteins of relatively low nucleotide sequence specificity. After briefly describing some of the alternative techniques suitable for the detection of DNA-binding proteins in crude extracts, it discusses the electrophoretic assay, its recent modifications, and consider other applications of the assay. Although nondenaturing electrophoretic systems have existed for almost as long as the method of gel electrophoresis itself, the arrival of powerful analytical applications of this technology is quite recent and other useful variations of the theme are still to come.", "doi": "10.1016/s0076-6879(87)51044-8", "isbn": "9780121820527", "publisher": "Academic Press", "publication_date": "1987", "pages": "551-565" }, { "id": "authors:66400-gfq87", "collection": "authors", "collection_id": "66400-gfq87", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:SNApnas86", "type": "article", "title": "Post-separation detection of nucleic acids and proteins by neutron activation", "author": [ { "family_name": "Snapka", "given_name": "Robert M.", "clpid": "Snapka-Robert-M" }, { "family_name": "Kwok", "given_name": "Kwan", "clpid": "Kwok-Kwan" }, { "family_name": "Bernard", "given_name": "John A.", "clpid": "Bernard-John-A" }, { "family_name": "Harling", "given_name": "Otto K.", "clpid": "Harling-Otto-K" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "We describe approaches to neutron activation analysis and their application to post-separation autoradiographic detection of biological compounds. Specifically, we have extended the use of a \"direct-labeling\" method to the post-separation detection of DNA after gel electrophoresis and to the detection of nucleotides separated by TLC. In addition, we describe a more generally applicable \"indirect-labeling\" method in which separated compounds of interest are selectively bound to ligands containing highly neutron-activatable elements, such as manganese (55Mn), europium (151Eu), or dysprosium (164Dy), and then irradiated with thermal neutrons. This method is illustrated with nucleotides separated by TLC and with proteins separated by polyacrylamide gel electrophoresis. In contrast to the direct-labeling approach, the indirect-labeling method can be adapted to detect any class of substances for which a highly neutron-activatable, selectively binding ligand is available. The theoretically achievable sensitivity of the indirect-labeling method is in the attomole (10-18 mol) range.", "doi": "10.1073/pnas.83.23.8939", "pmcid": "PMC387049", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1986-12-01", "series_number": "23", "volume": "83", "issue": "23", "pages": "8939-8942" }, { "id": "authors:h784q-0ye08", "collection": "authors", "collection_id": "h784q-0ye08", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210122-162658169", "type": "article", "title": "In vivo half-life of a protein is a function of its amino-terminal residue", "author": [ { "family_name": "Bachmair", "given_name": "Andreas", "clpid": "Bachmair-Andreas" }, { "family_name": "Finley", "given_name": "Daniel", "clpid": "Finley-Daniel" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "When a chimeric gene encoding a ubiquitin-beta-galactosidase fusion protein is expressed in the yeast Saccharomyces cerevisiae, ubiquitin is cleaved off the nascent fusion protein, yielding a deubiquitinated beta-galactosidase (beta gal). With one exception, this cleavage takes place regardless of the nature of the amino acid residue of beta gal at the ubiquitin-beta gal junction, thereby making it possible to expose different residues at the amino-termini of the otherwise identical beta gal proteins. The beta gal proteins thus designed have strikingly different half-lives in vivo, from more than 20 hours to less than 3 minutes, depending on the nature of the amino acid at the amino-terminus of beta gal. The set of individual amino acids can thus be ordered with respect to the half-lives that they confer on beta gal when present at its amino-terminus (the \"N-end rule\"). The currently known amino-terminal residues in long-lived, noncompartmentalized intracellular proteins from both prokaryotes and eukaryotes belong exclusively to the stabilizing class as predicted by the N-end rule. The function of the previously described posttranslational addition of single amino acids to protein amino-termini may also be accounted for by the N-end rule. Thus the recognition of an amino-terminal residue in a protein may mediate both the metabolic stability of the protein and the potential for regulation of its stability.", "doi": "10.1126/science.3018930", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "1986-10-10", "series_number": "4773", "volume": "234", "issue": "4773", "pages": "179-186" }, { "id": "authors:evk77-zca03", "collection": "authors", "collection_id": "evk77-zca03", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210122-162658310", "type": "article", "title": "Enhancement of immunoblot sensitivity by heating of hydrated filters", "author": [ { "family_name": "Swerdlow", "given_name": "Paul S.", "clpid": "Swerdlow-Paul-S" }, { "family_name": "Finley", "given_name": "Daniel", "clpid": "Finley-Daniel" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Immunoblots of either dot or Western type were exposed to heat before reaction with antibody. Dramatic increases in immunoblot sensitivity were seen for certain antigen-antibody pairs after heating of either dry or hydrated nitrocellulose filters at or above 100\u00b0C. Heating of filters in the hydrated state improved the linearity of immunodetection and produced the highest signal-to-noise ratio. This treatment greatly increased immunoblot sensitivity with several peptide-generated antibodies, whereas decreased sensitivity was seen with antibodies against native proteins. Heating of hydrated filters after antigen immobilization is thus a potentially powerful way to increase the sensitivity of immunoblot analysis for antibodies that preferentially recognize epitopes in denatured proteins.", "doi": "10.1016/0003-2697(86)90166-1", "issn": "0003-2697", "publisher": "Elsevier", "publication": "Analytical Biochemistry", "publication_date": "1986-07", "series_number": "1", "volume": "156", "issue": "1", "pages": "147-153" }, { "id": "authors:r6m6s-cvd90", "collection": "authors", "collection_id": "r6m6s-cvd90", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:SOLpnas86", "type": "article", "title": "A mammalian high mobility group protein recognizes any stretch of six A\u00b7T base pairs in duplex DNA", "author": [ { "family_name": "Solomon", "given_name": "Mark J.", "clpid": "Solomon-Mark-J" }, { "family_name": "Strauss", "given_name": "Francois", "clpid": "Strauss-Francois" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "\u03b1-Protein is a high mobility group protein originally purified from African green monkey cells based on its affinity for the 172-base-pair repeat of monkey \u03b1-satellite DNA. We have used DNase I footprinting to identify 50 \u03b1-protein binding sites on simian virus 40 DNA and thereby to determine the DNA binding specificity of this mammalian nuclear protein. \u03b1-Protein binds with approximately equal affinity to any run of six or more A\u00b7T base pairs in duplex DNA, to many, if not all, runs of five A\u00b7T base pairs, and to a small number of other sequences within otherwise (A+T)-rich regions. Unlike well characterized sequence-specific DNA binding proteins such as bacterial repressors, \u03b1-protein makes extensive contacts within the minor groove of B-DNA. These and related findings indicate that, rather than binding to a few specific DNA sequences, \u03b1-protein recognizes a configuration of the minor groove characteristic of short runs of A\u00b7T base pairs. We discuss possible functions of \u03b1-protein and the similarities in DNA recognition by \u03b1-protein and the antibiotic netropsin.", "doi": "10.1073/pnas.83.5.1276", "pmcid": "PMC323058", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1986-03-01", "series_number": "5", "volume": "83", "issue": "5", "pages": "1276-1280" }, { "id": "authors:vqt40-27807", "collection": "authors", "collection_id": "vqt40-27807", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:GROpnas86", "type": "article", "title": "Isolation and characterization of DNA sequences amplified in multidrug-resistant hamster cells", "author": [ { "family_name": "Gros", "given_name": "Philippe", "clpid": "Gros-Philippe" }, { "family_name": "Croop", "given_name": "James", "clpid": "Croop-James" }, { "family_name": "Roninson", "given_name": "Igor", "clpid": "Roninson-Igor" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Housman", "given_name": "David E.", "clpid": "Housman-David-E" } ], "abstract": "The mechanism by which mammalian cells acquire resistance to chemotherapeutic agents has been investigated by using molecular genetic techniques, LZ and C5, two independently derived multidrug-resistant Chinese hamster cell lines, share specific amplified DNA sequences. We demonstrate that commonly amplified DNA sequences reside in a contiguous domain of approximate to 120 kilobases (kb). We report the isolation of this DNA domain in cosmid clones and show that the level of amplification of the domain is correlated with the level of resistance in multidrug-resistant cell lines. The organization of the amplified domain was deduced by a unique approach utilizing in-gel hybridization of cloned DNA with amplified genomic DNA. We show that the entire cloned region is amplified in adriamycin-resistant LZ cells and independently derived, colchicine-resistant C5 cells. A mRNA species of 5 kb is encoded by a gene located within the boundaries of this region. Genomic sequences homologous to the 5-kb mRNA span over 75 kb of the amplified DNA segment. The level of expression of this mRNA in multidrug-resistant cells is correlated with the degree of gene amplification and the degree of drug resistance. Our results strongly suggest that the 5-kb mRNA species plays a role in the mechanism of multidrug resistance common to the LZ and C5 cell lines.", "doi": "10.1073/pnas.83.2.337", "pmcid": "PMC322853", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1986-01-15", "series_number": "2", "volume": "83", "issue": "2", "pages": "337-341" }, { "id": "authors:rf7rk-c1e93", "collection": "authors", "collection_id": "rf7rk-c1e93", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210122-162658489", "type": "article", "title": "In vivo effects of cis- and trans-diamminedichloroplatinum(II) on SV40 chromosomes: differential repair, DNA-protein crosslinking, and inhibition of replication", "author": [ { "family_name": "Ciccarelli", "given_name": "Richard B.", "clpid": "Ciccarelli-Richard-B" }, { "family_name": "Solomon", "given_name": "Mark J.", "clpid": "Solomon-Mark-J" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Lippard", "given_name": "Stephen J.", "clpid": "Lippard-Stephen-J" } ], "abstract": "The mechanism of action of the antitumor drug cis-diamminedichloroplatinum(II), cis-DDP, was investigated by using the ~5200 base pair (bp) chromosome of simian virus 40 (SV40) as an in vivo chromatin model. Comparative studies were also carried out with the clinically ineffective isomer trans-DDP. Although 14 times more trans- than cis-DDP in the culture medium is required to inhibit SV40 DNA replication in SV40-infected green monkey CV-1 cells, the two isomers are equally effective at inhibiting replication when equimolar amounts are bound to SV40 DNA in vivo. Since both isomers are transported into CV-1 cells at similar rates, differential uptake cannot account for the greater ability of cis-DDP to inhibit SV40 DNA replication. Rather, this result is explained by the finding that cis-DDP-DNA adducts accumulate continuously over the incubation period, whereas trans-DDP binding to DNA reaches a maximum at 6 h and thereafter decreases dramatically. We suggest that the different accumulation behavior of cis-DDP and trans-DDP on DNA is due to their differential repair in CV-1 cells. A variety of non-histone proteins, including SV40 capsid proteins but virtually no histones, are cross-linked to SV40 DNA in vivo by either cis- or trans-DDP. More DNA-protein cross-links are formed by trans-DDP than by cis-DDP at equivalent amounts of DNA-bound platinum. Since the cis-DDP analogue dichloro(ethylenediamine)platinum(II) inhibits SV40 DNA replication as efficiently as cis- and /trans-DDP but does not form DNA-protein cross-links, platinum-mediated DNA-protein cross-linking is not directly responsible for the inhibition of DNA replication. We discuss these findings in relation to the known cytotoxic and antitumor properties of cis-DDP.", "doi": "10.1021/bi00347a005", "issn": "0006-2960", "publisher": "American Chemical Society", "publication": "Biochemistry", "publication_date": "1985-12-17", "series_number": "26", "volume": "24", "issue": "26", "pages": "7533-7540" }, { "id": "authors:b3zfz-24755", "collection": "authors", "collection_id": "b3zfz-24755", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:SOLpnas85", "type": "article", "title": "Formaldehyde-mediated DNA-protein crosslinking: A probe for in vivo chromatin structures", "author": [ { "family_name": "Solomon", "given_name": "Mark J.", "clpid": "Solomon-Mark-J" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Formaldehyde (HCHO) produces DNA-protein crosslinks both in vitro and in vivo. Simian virus 40 (SV40) chromosomes that have been fixed by prolonged incubation with HCHO either in vitro or in vivo (within SV40-infected cells) can be converted to nearly protein-free DNA by limit-digestion with Pronase in the presence of NaDodSO4. The remaining Pronase-resistant DNA-peptide adducts retard the DNA upon gel electrophoresis, allowing resolution of free and crosslink-containing DNA. Though efficiently crosslinking histones to DNA within nucleosomes both in vitro and in vivo, HCHO does not crosslink either purified lac repressor to lac operator-containing DNA or an (A + T)-DNA-binding protein ( -protein) to its cognate DNA in vitro. Furthermore, a protein that does not bind to DNA, such as serum albumin, is not crosslinked to DNA by HCHO even at extremely high protein concentrations. These properties of HCHO as a DNA-protein crosslinker are used to probe the distribution of nucleosomes in vivo. We show that there are no HCHO-crosslinkable DNA-protein contacts in a subset of SV40 chromosomes in vivo within a 325-base-pair stretch that spans the \"exposed\" (nuclease-hypersensitive) region of the SV40 chromosome. This replication origin-proximal region has been found previously to lack nucleosomes in a subset of isolated SV40 chromosomes. We discuss other applications of the HCHO technique, including the possibility of obtaining base-resolution in vivo nucleosome \"footprints.\"", "doi": "10.1073/pnas.82.19.6470", "pmcid": "PMC390738", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1985-10-01", "series_number": "19", "volume": "82", "issue": "19", "pages": "6470-6474" }, { "id": "authors:yen4c-9ax33", "collection": "authors", "collection_id": "yen4c-9ax33", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210122-162658617", "type": "article", "title": "Preferential localization of variant nucleosomes near the 5'-end of the mouse dihydrofolate reductase gene", "author": [ { "family_name": "Barsoum", "given_name": "James", "clpid": "Barsoum-James" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "We have probed the structure of nucleosomes within the 31-kilobase pair long, transcriptionally active gene for dihydrofolate reductase (DHFR) in mouse cells which contain multiple copies of the DHFR gene. We found that the distribution of electrophoretically variant nucleosomes within the DHFR gene is highly nonuniform: variant DHFR nucleosomes are abundant within and in the immediate vicinity of the approximately 200-base pair (bp) long first DHFR exon, and decrease by at least 10-fold within two nucleosomes upstream and downstream from this region. The nonuniformly distributed variant DHFR mononucleosomes are of two electrophoretically distinguishable discrete types. One corresponds to a mononucleosome containing a approximately 180-bp DNA fragment and possibly also histone H1 and high mobility group proteins. The other type of variant DHFR mononucleosome contains a approximately 146-bp DNA fragment, and its changes in relative content within the DHFR gene closely parallel those of the 180-bp variant mononucleosome. Several lines of evidence are consistent with the interpretation that the electrophoretically variant approximately 146-bp (core) mononucleosome species corresponds to diubiquitinated DHFR nucleosomes. We discuss possible causal relationships between the observed nonuniform distribution of variant nucleosomes within the DHFR gene and the DHFR gene transcription.", "doi": "10.1016/s0021-9258(17)39663-1", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "1985-06-25", "series_number": "12", "volume": "260", "issue": "12", "pages": "7688-7697" }, { "id": "authors:wx07d-48q55", "collection": "authors", "collection_id": "wx07d-48q55", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201125-154027285", "type": "article", "title": "Mammalian cell cycle mutant defective in intracellular protein degradation and ubiquitin-protein conjugation", "author": [ { "family_name": "Ciechanover", "given_name": "A.", "clpid": "Ciechanover-Aaron" }, { "family_name": "Finley", "given_name": "D.", "clpid": "Finley-D" }, { "family_name": "Varshavsky", "given_name": "A.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Ubiquitin, a 76 residue protein, occurs in eukaryotic cells either free or covalently joined via its carboxyl terminus to epsilon-amino groups of lysine residues in a wide variety of protein species. Previous work has shown that ubiquitin-protein conjugates are preferred substrates in vitro for a non-lysosomal ATP-dependent proteolytic pathway, suggesting that ubiquitin may function as a signal for attack by proteinases specific for ubiquitin-protein conjugates. One strategy to define the potential significance of the ubiquitin-dependent proteolytic pathway is to identify conditional mutants in the pathway. ts85 is a mouse derived cell-cycle mutant which has been shown to lose uH2A, a specific ubiquitin-histone H2A conjugate, at the nonpermissive temperature. We show that the loss of uH2A from ts85 cells is due to reduced ubiquitin-protein conjugation. We further show that the reduced conjugation is due to the specific thermolability of ubiquitin activating enzyme, E1, one of the three enzymic components of the ubiquitin-protein ligase system. We therefore proceeded to test whether the degradation of short-lived proteins is also temperature-sensitive in ts85 cells. Indeed, while more than 70% of the prelabeled abnormal (amino acid analog-containing) proteins or puromycyl peptides are degraded within 4 hours at the permissive temperature in the mutant (ts85), wild type (FM3A), and revertant (ts85R-MN3) cells, less than 15% of these proteins are degraded in ts85 cells at the nonpermissive temperature. In contrast, the rate of degradation of these proteins does not change significantly in either wild-type or revertant cells between permissive and nonpermissive temperatures. Degradation of normal short-lived proteins is also specifically temperature-sensitive in ts85 cells. Immunochemical analysis shows a strong and specific reduction in ubiquitin-protein conjugate levels in vivo at the nonpermissive temperature in ts85 cells. Taken together, our in vitro and in vivo findings with ts85 cells demonstrate that the degradation of the bulk of short-lived proteins in this higher eukaryotic cell is accomplished through a ubiquitin-mediated pathway.", "issn": "0361-7742", "publisher": "Wiley Liss", "publication": "Progress in clinical and biological research", "publication_date": "1985", "volume": "180", "pages": "17-31" }, { "id": "authors:nj6aw-71408", "collection": "authors", "collection_id": "nj6aw-71408", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210119-120945572", "type": "article", "title": "The yeast ubiquitin gene: head-to-tail repeats encoding a polyubiquitin precursor protein", "author": [ { "family_name": "\u00d6zkaynak", "given_name": "Engin", "clpid": "\u00d6zkaynak-Engin" }, { "family_name": "Finley", "given_name": "Daniel", "clpid": "Finley-Daniel" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Ubiquitin, a 76-residue protein, occurs in cells either free or covalently joined to a variety of protein species, from chromosomal histones to cytoplasmic proteins. Conjugation of ubiquitin to proteolytic substrates is essential for the selective degradation of intracellular proteins in higher eukaryotes. We show here that a protein homologous to human ubiquitin exists in the yeast Saccharomyces cerevisiae, and that yeast extracts conjugate human ubiquitin to a variety of endogenous proteins in an ATP-dependent reaction. We have isolated the S. cerevisiae ubiquitin gene and found it to contain six consecutive ubiquitin-coding repeats in a head-to-tail arrangement. This apparently unique gene organization suggests that yeast ubiquitin is generated by processing of a precursor protein in which several exact repeats of the ubiquitin amino acid sequence are joined directly via Gly\u2013Met peptide bonds between the last and first residues of mature ubiquitin, respectively. Ubiquitin-coding yeast DNA repeats are restricted to a single genomic locus; although the sequenced repeats differ in up to 27 of 228 bases per repeat, they encode identical amino acid sequences. As this predicted amino acid sequence differs in only 3 of 76 residues from that of ubiquitin in higher eukaryotes, ubiquitin is apparently the most conserved of known proteins.", "doi": "10.1038/312663a0", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "1984-12-13", "series_number": "5995", "volume": "312", "issue": "5995", "pages": "663-666" }, { "id": "authors:5h8z6-vqx67", "collection": "authors", "collection_id": "5h8z6-vqx67", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210122-162658719", "type": "article", "title": "A protein binds to a satellite DNA repeat at three specific sites that would be brought into mutual proximity by DNA folding in the nucleosome", "author": [ { "family_name": "Strauss", "given_name": "Fran\u00e7ois", "clpid": "Strauss-Fran\u00e7ois" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Using a generally applicable assay for specific DNA-binding proteins in crude extracts, we have detected and purified an HMG-like nuclear protein from African green monkey cells that preferentially binds to the 172 bp repeat of \u03b1-satellite DNA (\u03b1-DNA). DNAase I footprinting with the purified protein detects three specific binding sites (I\u2013III) per \u03b1-DNA repeat. Site II is 145 bp (one core nucleosome length) from site III on the adjacent \u03b1-DNA repeat, while site I lies midway between sites II and III. In the \u03b1-nucleosome phasing frame corresponding with this arrangement, sites I\u2013III would be brought into mutual proximity by DNA folding in the nucleosome. This phasing frame is identical with the preferred frame detected previously in isolated chromatin. Our results suggest that this new and abundant protein recognizes a family of short, related nucleotide sequences found not only in \u03b1-DNA but also throughout the genome, and that functions of this protein are mediated through its nucleosome-positioning activity. Such nucleosome-positioning proteins may underlie the sequence specificity of both nucleosome arrangements and higher order chromatin structures.", "doi": "10.1016/0092-8674(84)90424-0", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "1984-07", "series_number": "3", "volume": "37", "issue": "3", "pages": "889-901" }, { "id": "authors:smgcf-qv008", "collection": "authors", "collection_id": "smgcf-qv008", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210119-114432302", "type": "article", "title": "Amplification of specific DNA sequences correlates with multi-drug resistance in Chinese hamster cells", "author": [ { "family_name": "Roninson", "given_name": "Igor B.", "clpid": "Roninson-Igor-B" }, { "family_name": "Abelson", "given_name": "Herbert T.", "clpid": "Abelson-Herbert-T" }, { "family_name": "Housman", "given_name": "David E.", "clpid": "Housman-David-E" }, { "family_name": "Howell", "given_name": "Neil", "clpid": "Howell-Neil" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Mammalian cells selected for resistance to certain cytotoxic drugs frequently develop cross-resistance to a broad spectrum of other drugs unrelated in structure to the original selective agent. This phenomenon constitutes a major problem in cancer chemotherapy. Multi-drug resistance arises from decreased intracellular drug accumulation, apparently due to an alteration of the plasma membrane. The observation of double minute chromosomes or homogeneously staining regions in some of the multi-drug-resistant cell lines suggests that gene amplification underlies this phenomenon. We have used the technique of DNA renaturation in agarose gels to detect, compare and clone amplified DNA sequences in Adriamycin- and colchicine-resistant sublines of Chinese hamster cells. We show that both Adriamycin- and colchicine-resistant cells contain amplified DNA fragments, some of which are amplified in both of these independently derived cell lines. Furthermore, loss of the multi-drug resistance phenotype on growth in the absence of drugs correlates with the loss of amplified DNA. These results strongly suggest that the DNA sequences which are amplified in common in multi-drug-resistant cell lines include the gene(s) responsible for a common mechanism of multi-drug resistance in these cells. We have cloned one of the commonly amplified DNA fragments and show that the degree of amplification of this fragment in the cells correlates with the degree of their drug resistance.", "doi": "10.1038/309626a0", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "1984-06-14", "series_number": "5969", "volume": "309", "issue": "5969", "pages": "626-628" }, { "id": "authors:f5tpx-yw957", "collection": "authors", "collection_id": "f5tpx-yw957", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210122-162658866", "type": "article", "title": "Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85", "author": [ { "family_name": "Ciechanover", "given_name": "Aaron", "clpid": "Ciechanover-Aaron" }, { "family_name": "Finley", "given_name": "Daniel", "clpid": "Finley-Daniel" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "We have shown that covalent conjugation of ubiquitin to proteins is temperature-sensitive in the mouse cell cycle mutant ts85 due to a specifically thermolabile ubiquitin-activating enzyme (accompanying paper). We show here that degradation of short-lived proteins is also temperature sensitive in ts85, in contrast to wild-type and revertant cells. While more than 70% of the prelabeled abnormal proteins (containing amino acid analogs) or puromycyl peptides are degraded within 4 hr at the permissive temperature in both ts85 and wild-type cells, less than 15% are degraded in ts85 cells at the nonpermissive temperature. Degradation of abnormal proteins and puromycyl peptides in both ts85 cells and wild-type cells is nonlysosomal and ATP-dependent. Immunochemical analysis shows a strong and specific reduction in the levels of in vivo labeled ubiquitin-protein conjugates at the nonpermissive temperature in ts85 cells. Degradation of normal, shortlived proteins is also specifically temperature sensitive in ts85. We suggest that the contribution of ubiquitin-independent pathways to the degradation of short-lived proteins in this higher eucaryotic cell is no more than 10%, and possibly less.", "doi": "10.1016/0092-8674(84)90300-3", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "1984-05", "series_number": "1", "volume": "37", "issue": "1", "pages": "57-66" }, { "id": "authors:17eky-8z796", "collection": "authors", "collection_id": "17eky-8z796", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210122-162659002", "type": "article", "title": "Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85", "author": [ { "family_name": "Finley", "given_name": "Daniel", "clpid": "Finley-Daniel" }, { "family_name": "Ciechanover", "given_name": "Aaron", "clpid": "Ciechanover-Aaron" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Ubiquitin, a 76 residue protein, occurs in eucaryotic cells either free or covalently joined to a variety of protein species. Previous work suggested that ubiquitin may function as a signal for attack by proteinases specific for ubiquitin-protein conjugates. We show that the mouse cell line ts85, a previously isolated cell cycle mutant, is temperature-sensitive in ubiquitin-protein conjugation, and that this effect is due to the specific thermolability of the ts85 ubiquitin-activating enzyme (E1). From E1 thermoinactivation kinetics in mixed (wild-type plus ts85) extracts, and from copurification of the determinant of E1 thermolability with E1 in ubiquitin-affinity chromatography, we conclude that the determinant of E1 thermolability is contained within the E1 polypeptide. ts85 cells fail to degrade otherwise short-lived intracellular proteins at the nonpermissive temperature (accompanying paper), demonstrating that degradation of the bulk of short-lived proteins in this higher eucaryotic cell proceeds through a ubiquitin-dependent pathway. We discuss possible roles of ubiquitin-dependent pathways in DNA transactions, the cell cycle, and the heat shock response.", "doi": "10.1016/0092-8674(84)90299-x", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "1984-05", "series_number": "1", "volume": "37", "issue": "1", "pages": "43-55" }, { "id": "authors:9p73n-dhe21", "collection": "authors", "collection_id": "9p73n-dhe21", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201201-102219067", "type": "article", "title": "The ubiquitin-mediated proteolytic pathway and mechanisms of energy\u2010dependent intracellular protein degradation", "author": [ { "family_name": "Ciechanover", "given_name": "Aaron", "clpid": "Ciechanover-Aaron" }, { "family_name": "Finley", "given_name": "Daniel", "clpid": "Finley-Daniel" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "In this review we briefly describe the lysosomal system, consider the evidence for multiplicity of protein degradation pathways in vivo, discuss in detail the ubiquitin\u2010mediated pathway of intracellular ATP\u2010dependent protein degradation, and also the possible significance of ubiquitin\u2010histone conjugates in chromatin. For detailed discussions of the various characteristics and physiological roles of intracellular protein breakdown, the reader is referred to earlier reviews [1\u20137] and reports of recent symposia [8\u201310]. Information on the ubiquitin system prior to 1981 was described in an earlier review [11]. Hershko has briefly reviewed more recent information [12].", "doi": "10.1002/jcb.240240104", "issn": "0730-2312", "publisher": "Wiley", "publication": "Journal of Cellular Biochemistry", "publication_date": "1984", "series_number": "1", "volume": "24", "issue": "1", "pages": "27-53" }, { "id": "authors:48t44-0se12", "collection": "authors", "collection_id": "48t44-0se12", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210119-114432693", "type": "article", "title": "Do stalled replication forks synthesize a specific alarmone?", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Potential causes of premature arrest of a replication fork in vivo include an encounter with a chemical lesion in the DNA, inhibition of one of the essential enzymes of the fork, and spontaneous failure of the fork due to its finite degree of processivity. I suggest that a premature arrest of either a eukaryotic or prokaryotic replication fork induces it to enter a different state in which the fork synthesizes a specific signal nucleotide (\"alarmone\"). One function of the postulated new alarmone would be to increase the probability of re-initiation of DNA replication, either in cis (at an origin proximal to a site of the fork arrest) or in trans (at many different origins). An additional, mechanistically related function of the postulated alarmone could be to increase the probability of re-assembly of an arrested fork beyond an otherwise impassable DNA lesion. In case of multiple fork arrests, an alarmone-mediated increase in the probability of replicon reinitiation (disproportionate DNA replication) would result in gene amplification at many different loci, thereby increasing the probability of cell's survival in a cytotoxic medium. Other likely functions of a fork-produced alarmone may include stimulation of DNA repair pathways including excision repair. I review the experimental evidence which although indirect, is consistent with the idea of a fork-produced alarmone and specifically with the possibility that the postulated alarmone is diadenosine 5', 5\u2032\u2032\u2032P\u00b9, P\u2074-tetraphosphate (Ap\u2084A) or a closely related adenylated nucleotide. The proposed hypothesis leads to specific, testable predictions; it also provides a unifying explanation for several hitherto unconnected observations on DNA replication, repair and amplification.", "doi": "10.1016/0022-5193(83)90228-x", "issn": "0022-5193", "publisher": "Elsevier", "publication": "Journal of Theoretical Biology", "publication_date": "1983-12-21", "series_number": "4", "volume": "105", "issue": "4", "pages": "707-714" }, { "id": "authors:2dh4d-w4s46", "collection": "authors", "collection_id": "2dh4d-w4s46", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:SNApnas83", "type": "article", "title": "Loss of unstably amplified dihydrofolate reductase genes from mouse cells is greatly accelerated by hydroxyurea", "author": [ { "family_name": "Snapka", "given_name": "Robert M.", "clpid": "Snapka-Robert-M" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Previous work has shown that mammalian cells that carry unstably amplified genes for dihydrofolate reductase (DHFR) gradually lose the amplified DHFR genes when grown in the absence of the DHFR inhibitor methotrexate (MTX). Unstably amplified genes occur on small acentric chromosomes called double minutes (DMs) or even smaller chromatin fragments, in contrast to stably amplified genes, which reside in centromere-containing chromosomes. We have found that the rate of loss of the unstably amplified DHFR genes can be greatly increased by growing the cells in the presence of a nonlethal concentration of hydroxyurea. For example, in one MTX-resistant subline studied, approximate to 90% of the original DHFR gene dosage is lost in 25-30 cell doublings in the absence of MTX. The same degree of loss is achieved, however, in <4 doublings if cells are grown in the presence of 50 \u03bcM hydroxyurea. This new effect of hydroxyurea does not appear to be due to changes in plating efficiency or selective cytotoxicity. In particular, no increase in cell death occurs at 50 \u03bcM hydroxyurea, and cells continue to multiply, albeit 1/2 to 2/3 as fast as in the absence of hydroxyurea. The ability to selectively accelerate the loss of amplified genes from mammalian cells as shown in the present work may have important implications both for the problem of drug resistance in cancer chemotherapy and for curing mammalian cells of extrachromosomally maintained DNA genomes of pathogenic viruses.", "doi": "10.1073/pnas.80.24.7533", "pmcid": "PMC389986", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1983-12-15", "series_number": "24", "volume": "80", "issue": "24", "pages": "7533-7537" }, { "id": "authors:afydv-pkv19", "collection": "authors", "collection_id": "afydv-pkv19", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210119-114432865", "type": "article", "title": "Nucleosome arrangement in green monkey \u03b1-satellite chromatin: Superimposition of non-random and apparently random patterns", "author": [ { "family_name": "Wu", "given_name": "Kun Chi", "clpid": "Wu-Kun-Chi" }, { "family_name": "Strauss", "given_name": "Fran\u00e7ois", "clpid": "Strauss-Fran\u00e7ois" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "We have studied the structure of tandemly repetitive \u03b1-satellite chromatin (\u03b1-chromatin) in African green monkey cells (CV-1 line), using restriction endonucleases and staphylococcal nuclease as probes.", "doi": "10.1016/s0022-2836(83)80228-9", "issn": "0022-2836", "publisher": "Elsevier", "publication": "Journal of Molecular Biology", "publication_date": "1983-10-15", "series_number": "1", "volume": "170", "issue": "1", "pages": "93-117" }, { "id": "authors:q0fkv-vt431", "collection": "authors", "collection_id": "q0fkv-vt431", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210119-114432779", "type": "article", "title": "Diadenosine 5\u2032, 5\u2032\u2032\u2032-P\u00b9, P\u2074-tetraphosphate: a pleiotropically acting alarmone?", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "In 1966, Zamecnik and his colleagues found an unusual dinucleotide, diadenosine 5', 5\"'-P\u00b9, P\u2074-tetraphosphate (Apa), synthesized in an in vitro system consisting of ATP, Mg\u00b2\u207a, L-lysine, and purified E. coli lysyl-tRNA synthetase\n(Zamecnik et al., BBRC 24, 91-97, 1966). Ap\u2084A is\nsynthesized in vitro by many, though possibly not all,\naminoacyl-tRNA synthetases (aa-tRNA synthetases)\nthrough aminoacyladenylate as an obligatory intermediate\n(reaction 3) (Goerlich et al., Eur. J. Biochem. 726, 13%\n142, 1982; Brevet et al., JBC 257, 14611-14615, 1982).", "doi": "10.1016/0092-8674(83)90526-3", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "1983-10", "series_number": "3", "volume": "34", "issue": "3", "pages": "711-712" }, { "id": "authors:ww3jb-74124", "collection": "authors", "collection_id": "ww3jb-74124", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:BARpnas83", "type": "article", "title": "Mitogenic hormones and tumor promoters greatly increase the incidence of colony-forming cells bearing amplified dihydrofolate reductase genes", "author": [ { "family_name": "Barsoum", "given_name": "James", "clpid": "Barsoum-James" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Previous work has shown that the presence of a phorbol ester tumor promoter, phorbol 12-myristate 13-acetate (PMA), during a single-step selection for methotrexate (MTX)-resistant mouse 3T6 cells results in an up to 100-fold increase in the incidence of MTX-resistant, colony-forming cells. MTX resistance of most of these cells is due to amplification of the gene for dihydrofolate reductase (DHFR), the target enzyme for MTX. We show here that other active, noncytotoxic phorbol ester tumor promoters, such as phorbol 12, 13-didecanoate and 20-phorbol 12, 13-butyrate, at their optimal concentrations (approximate to 0.1 \u00b5M) are approximately equal to PMA in increasing the incidence of MTX-resistant 3T6 colonies. Mezerein, a potent second-stage tumor promoter, but a weak complete promoter, increases the incidence of MTX resistance up to 350-fold, the strongest effect for any of the agents so far tested. PMA analogs that are inactive as tumor promoters, such as phorbol or phorbol 12, 13, 20-triacetate, have no effect on the incidence of MTX-resistant 3T6 colonies. Anthralin, a nonphorbol tumor promoter, is approximate to 40% as active as PMA in the MTX selection assay. Remarkably, the hormones insulin, arginine vasopressin, and epidermal growth factor, all of which are mitogenic for 3T6 cells, also exert a strong PMA-like effect on the incidence of MTX-resistant 3T6 colonies under conditions of MTX selection. The effect of insulin at its optimal concentration (approximate to 1 \u00b5g/ml) is approximate to 70% that of PMA. Although the effect of PMA on the incidence of MTX-resistant 3T6 colonies does not significantly depend on the initial density of seeded cells or volume of the medium added, the analogous effect of insulin is strongly influenced by these parameters. Mevalonic acid, arachidonic acid, thymidine, caffeine, and nicotine, all of which are known to influence patterns of DNA synthesis in mammalian cells, were tested at their highest noncytotoxic concentrations and failed to produce any significant effect on the incidence of MTX-resistant 3T6 colonies. We discuss possible mechanisms of hormone- and tumor promoter-facilitated gene amplification in mammalian cells, relationship of mitogenic hormones to tumor promoters, and also implications of our findings for the problem of drug resistance in cancer chemotherapy.", "doi": "10.1073/pnas.80.17.5330", "pmcid": "PMC384249", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1983-09-01", "series_number": "17", "volume": "80", "issue": "17", "pages": "5330-5334" }, { "id": "authors:seapy-jjf12", "collection": "authors", "collection_id": "seapy-jjf12", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210122-150600293", "type": "article", "title": "Affinity of HMG17 for a mononucleosome is not influenced by the presence of ubiquitin-H2A semihistone but strongly depends on DNA fragment size", "author": [ { "family_name": "Swerdlow", "given_name": "Paul S.", "clpid": "Swerdlow-Paul-S" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "We have used a two-dimensional (deoxyribonucleoprotein \u2192DNA) electrophoretic binding assay to study the interaction of the purified high mobility group protein HMG17 with isolated HeLa mononucleosomes as a function of their DNA fragment size and the presence of ubiquitin-H2A semihistone. No significant differences between affinities of HMG17 for ubiquitinated and non-ubiquitinated core mononucleosomes were observed. In striking contrast, the apparent affinity of HMG17 for a mononucleosome increases more than 100-fold upon an increase of the length of the mononucleosomal DNA fragment by as few as 3 to 5 bp over the core DNA length (\u223d146 bp). We suggest that the magnitude of this effect is sufficient to explain the preferential binding of HMG17 in vitro to mononucleosomes derived from actively transcribed genes.", "doi": "10.1093/nar/11.2.387", "pmcid": "PMC325721", "issn": "0305-1048", "publisher": "Oxford University Press", "publication": "Nucleic Acids Research", "publication_date": "1983-01-25", "series_number": "2", "volume": "11", "issue": "2", "pages": "387-401" }, { "id": "authors:48kzq-e2589", "collection": "authors", "collection_id": "48kzq-e2589", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201125-154028200", "type": "article", "title": "Acquisition and loss of amplified genes: dramatic effects of hormones, tumor promoters and cytotoxic drugs", "author": [ { "family_name": "Varshavsky", "given_name": "A.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Barsoum", "given_name": "J.", "clpid": "Barsoum-J" }, { "family_name": "Roninson", "given_name": "I.", "clpid": "Roninson-I" }, { "family_name": "Snapka", "given_name": "R.", "clpid": "Snapka-R" } ], "abstract": "We discuss our recent findings in three related areas of the gene amplification field. 1) We have found that tumor-promoting phorbol esters, nonphorbol tumor promoters, and most significantly, mitogenic hormones, such as insulin, vasopressin, and epidermal growth factor (EGF), greatly increase the incidence of methotrexate (MTX) resistance in 3T6 cells under condition of MTX selection. Most of these MTX-resistant cells bear amplified dihydrofolate reductase (DHFR) genes. 2) We have discovered that when mouse cells bearing unstably amplified DHFR genes are grown in the presence of nonlethal concentrations of hydroxyurea (HU), the rate of loss of the DHFR genes from these cells is greatly increased. 3) We have developed a new method for detection and mapping of homologous, repeated and amplified DNA sequences, and have used this method to detect and clone amplified DNA fragments in mammalian cells resistant simultaneously to a number of different drugs.", "publisher": "Japan Scientific Societies Press", "publication": "Princess Takamatsu symposia", "publication_date": "1983", "volume": "14", "pages": "235-254" }, { "id": "authors:vta1v-qwq28", "collection": "authors", "collection_id": "vta1v-qwq28", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210122-150600550", "type": "article", "title": "Cellular and SV40 Chromatin: Replication, Segregation, Ubiqiritination, Nuclease-hypersensitive Sites, HMG-containing Nueleosomes, and Heterochromatin-specific Protein", "author": [ { "family_name": "Varshavsky", "given_name": "A.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Levinger", "given_name": "L.", "clpid": "Levinger-Louis" }, { "family_name": "Sundin", "given_name": "O.", "clpid": "Sundin-Olof-H" }, { "family_name": "Barsoum", "given_name": "J.", "clpid": "Barsoum-James" }, { "family_name": "\u00d6zkaynak", "given_name": "E.", "clpid": "\u00d6zkaynak-E" }, { "family_name": "Swerdlow", "given_name": "P.", "clpid": "Swerdlow-Paul-S" }, { "family_name": "Finley", "given_name": "D.", "clpid": "Finley-D" } ], "abstract": "During lytic infection, SV40 DNA exists as a minichromosome within the host nucleus and is replicated in a manner similar to that of the cellular genome. The 5.2-kb circular, double-stranded SV40 DNA directs site-specific synthesis of a single replication \"bubble,\" which is then enlarged through movement of both replication forks (for review, see DePamphilis and Wassarman 1980). Separate, covalently closed, circular SV40 DNA molecules appear approximately 5 minutes after the forks meet halfway around the genome (Levine et al. 1970). We have recently discovered a new class of SV40 replicative intermediates participating in these latest stages of SV40 DNA replication (Sundin and Varshavsky 1980, 1981). All members of this class are catenated dimers, two circular molecules of SV40 DNA linked topologically by one or more intertwining events. The catenated SV40 DNA dimers comprise three specific families and occur as minichromosomes. They are all rapidly processed in vivo to mature supercoiled SV40 DNA I. We have also found conditions for selective arrest of SV40 replication in vivo at the stage of catenated chromosomes (Sundin and Varshavsky 1981). \n\nSV40 is the first example of a circular, double-stranded replicon that uses multiply intertwined catenated dimers as segregation intermediates. It is possible that analogous mechanisms operate during the final stages of replication in a variety of other eukaryotic and prokaryotic replicons. Some of the early studies on singly intertwined catenated dimers in mitochondrial, SV40, and other DNAs are those done by Rush et al. (1970), Jaenisch and Levine (1972), Novick et al. (1973), and Kupersztoch and Helinski (1973). \n\nWhat follows is a condensed account of the \"DNA-level\" work on multiply intertwined catenated dimers (Sundin and Varshavsky 1980, 1981), as well as our more recent data on the chromatin organization of SV40 chromosomes during the segregation of daughter DNA molecules.", "doi": "10.1101/sqb.1983.047.01.061", "issn": "0091-7451", "publisher": "Cold Spring Harbor Laboratory", "publication": "Cold Spring Harbor Symposia on Quantitative Biology", "publication_date": "1983", "volume": "47", "pages": "511-528" }, { "id": "authors:pb3bd-bs343", "collection": "authors", "collection_id": "pb3bd-bs343", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:LEVpnas82", "type": "article", "title": "Protein D1 preferentially binds A+T-rich DNA in vitro and is a component of Drosophila melanogaster nucleosomes containing A+T-rich satellite DNA", "author": [ { "family_name": "Levinger", "given_name": "Louis", "clpid": "Levinger-Louis" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Our previous work [Levinger, L. & Varshavsky, A. (1982) Cell 28, 375-385] has shown that D1, a 50-kilodalton chromosomal protein of Drosophila melanogaster, is specifically associated with isolated nucleosomes that contain a complex A+T-rich satellite DNA with buoyant density of 1.688 g/ml. We show here that D1 is also a component of nucleosomes containing a simple-sequence, pure A+T satellite DNA, buoyant density 1.672 g/ml. Furthermore, using a modification of a protein blotting technique in which proteins are not exposed to dodecyl sulfate denaturation, we have found that D1 preferentially binds to A+T-rich double-stranded DNA in vitro, and it is apparently the only abundant nuclear protein in cultured D. melanogaster cells that possesses this property. Synthetic poly[(A-T)]\u00b7 poly[d(A-T)] and poly(dA)\u00b7 poly(dT) duplexes effectively compete in vitro with A+T-rich D. melanogaster satellite DNAs for binding to D1, whereas total Escherichia coli DNA is an extremely poor competitor. These findings strongly suggest that D1 is a specific component of A+T-rich, tandemly repeated, heterochromatic regions, which constitute up to 15-20% of the total D. melanogaster genome. Possible functions of D1 protein include compaction of A+T-rich heterochromatin and participation in microtubule--centromere interactions in mitosis. In addition, D1 may prevent nonspecific binding to A+T-rich satellite DNA of other nuclear proteins that have a preference for AT-DNA, such as RNA polymerase or regulatory proteins, and may also participate in the higher-order chromatin organization outside tandemly repetitive regions by binding to nonrandomly positioned stretches of A+T-rich DNA.", "doi": "10.1073/pnas.79.23.7152", "pmcid": "PMC347296", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1982-12-01", "series_number": "23", "volume": "79", "issue": "23", "pages": "7152-7156" }, { "id": "authors:93za1-7er33", "collection": "authors", "collection_id": "93za1-7er33", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210122-150600418", "type": "article", "title": "On the chromatin structure of the amplified, transcriptionally active gene for dihydrofolate reductase in mouse cells", "author": [ { "family_name": "Barsoum", "given_name": "James", "clpid": "Barsoum-James" }, { "family_name": "Levinger", "given_name": "Louis", "clpid": "Levinger-Louis" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "The method for two-dimensional hybridization mapping of nucleosomes (Levinger, L., Barsoum, J., and Varshavsky, A. (1981) J. Mol. Biol. 146, 287-304) was used to analyze chromatin structure of the gene for dihydrofolate reductase (DHF reductase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase (EC 1.5.1.3)) in L5178Y-R mouse cells. The DHF reductase gene in these cells is amplified about 350-fold as a result of selection for resistance to methotrexate. Dramatic overproduction of DHF reductase mRNA in L5178Y-R cells suggests that most of the DHF reductase genes in these cells are transcribed. We report that all major mononucleosomal species resolvable by two-dimensional fractionation are detected by both DHF reductase- and satellite DNA-specific hybridization probes. Although the DHF reductase and satellite hybridization patterns differ somewhat from each other and from the total mononucleosomal pattern, their overall similarity is very high. In particular, no large differences in the abundance of mononucleosomes containing high mobility group non-histone proteins (HMG) 14 and 17 are seen between the DHF reductase and satellite chromatin regions under a wide variety of conditions for chromatin isolation, digestion, and fractionation. Possible interpretations of the apparent lack of selectivity of HMG-chromatin interactions in this system are discussed. We also found that the amplified DHF reductase genes possess a wide range of nucleosomal repeat lengths close to that in the bulk chromatin. In contrast, the range of nucleosomal repeat lengths in the satellite chromatin is much narrower than in both DHF reductase and bulk chromatin.", "doi": "10.1016/s0021-9258(18)34667-2", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "1982-05-10", "series_number": "9", "volume": "257", "issue": "9", "pages": "5274-5282" }, { "id": "authors:wczyr-vpg62", "collection": "authors", "collection_id": "wczyr-vpg62", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:BOYjvir82", "type": "article", "title": "New way to isolate simian virus 40 nucleoprotein complexes from infected cells: use of a thiol-specific reagent", "author": [ { "family_name": "Boyce", "given_name": "Frederick M.", "clpid": "Boyce-Frederick-M" }, { "family_name": "Sundin", "given_name": "Olof", "clpid": "Sundin-Olof" }, { "family_name": "Barsoum", "given_name": "James", "clpid": "Barsoum-James" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "A new method for the isolation of simian virus 40 nucleoprotein complexes from nuclei of lytically infected cells is described. The method is based on the addition of a thiol-specific reagent, 5'5'-dithiobis(2-nitrobenzoic acid), to lysis and extraction buffers. By inhibiting an uncoating activity during simian virus 40 extraction, 5'5'-dithiobis (2-nitrobenzoic acid) allows the use of efficient extraction buffers, such as one containing Triton X-100 and EDTA, for the isolation of native simian virus 40 minichromosomes and virion-type structures. Use of the method is illustrated by following encapsidation of simian virus 40 minichromosomes in a pulse-chase experiment. Since 5'5'-dithiobis (2-nitrobenzoic acid) is an inhibitor of many different enzymes, the 5',5'-dithiobis (2-nitrobenzoic acid) extraction technique may be useful for the isolation of not only papovaviruses but also other viruses and possibly cellular chromatin.", "doi": "10.1128/jvi.42.1.292-296.1982", "pmcid": "PMC256070", "issn": "0022-538X", "publisher": "American Society for Microbiology", "publication": "Journal of Virology", "publication_date": "1982-04", "series_number": "1", "volume": "42", "issue": "1", "pages": "292-296" }, { "id": "authors:86mar-acv91", "collection": "authors", "collection_id": "86mar-acv91", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210122-150600167", "type": "article", "title": "Selective arrangement of ubiquitinated and D1 protein-containing nucleosomes within the drosophila genome", "author": [ { "family_name": "Levinger", "given_name": "Louis", "clpid": "Levinger-Louis" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "We have used a new approach, two-dimensional hybridization mapping of nucleosomes, to compare the structures of mononucleosomes from different regions of the Drosophila melanogaster genome. Approximately one in two nucleosomes of the transcribed copia and heat-shock 70 (hsp 70) genes in nonshocked cultured cells contains ubiquitin-H2A (uH2A) semihistone, a covalent conjugate of histone H2A and a small protein, ubiquitin. In striking contrast, less than one in 25 nucleosomes of tandemly repeated, nontranscribed 1.688 satellite DNA contains uH2A, suggesting that most of the nucleosomal uH2A is located in transcribed genes. Approximately 25% of all nucleosomes are ubiquitinated in nonsynchronized cultured Drosophila cells. The hsp 70 genes in nonshocked cells occur in nucleosomes, are greatly enriched in uH2A and are not digested preferentially by staphylococcal nuclease. In contrast, the same genes in chromatin from heat-shocked cells are highly sensitive to staphylococcal nuclease and no longer possess nucleosomal organization recognizable with this probe. Histone ubiquitination in transcribed nucleosomes may prevent formation of higher order chromosomal structures by modifying nucleosome-nucleosome interactions. The observed loss of nucleosomal organization in very actively transcribed genes, such as the hsp 70 genes in shocked cells, may be related to the recent finding that ubiquitin conjugates are substrates for the cytoplasmic ATP-dependent proteolytic system. We have also found that 1.688 satellite mononucleosomes contain a specific \u223c50,000 dalton nonhistone protein, D1, in addition to being extremely under-ubiquitinated. D1 may be involved in formation of the highly compact structure of satellite heterochromatin.", "doi": "10.1016/0092-8674(82)90355-5", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "1982-02", "series_number": "2", "volume": "28", "issue": "2", "pages": "375-385" }, { "id": "authors:rrjkp-qa893", "collection": "authors", "collection_id": "rrjkp-qa893", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210122-150600047", "type": "article", "title": "Heat-shock proteins of Drosophila are associated with nuclease-resistant, high-salt-resistant nuclear structures", "author": [ { "family_name": "Levinger", "given_name": "Louis", "clpid": "Levinger-Louis" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Proteins produced in cultured Drosophila cells during the heat-shock response (HSPs) were recently shown by autoradiography to be confined in large measure to the cell nucleus. We report here that nuclear HSPs are not associated with nucleosomes solubilizes by treatment with staphylococcal nuclease at low ionic strength nor are HSPs released by extraction with high salt, which solubilized most of the remaining histones and DNA. Possible functions of nuclear HSPs are discussed.", "doi": "10.1083/jcb.90.3.793", "pmcid": "PMC2111893", "issn": "0021-9525", "publisher": "Rockefeller University Press", "publication": "Journal of Cell Biology", "publication_date": "1981-09", "series_number": "3", "volume": "90", "issue": "3", "pages": "793-796" }, { "id": "authors:j0y7a-1kj22", "collection": "authors", "collection_id": "j0y7a-1kj22", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210122-150559952", "type": "article", "title": "Arrest of segregation leads to accumulation of highly intertwined catenated dimers: Dissection of the final stages of SV40 DNA replication", "author": [ { "family_name": "Sundin", "given_name": "Olof", "clpid": "Sundin-Olof" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "When SV40-infected cells are placed into hypertonic medium, newly synthesized DNA accumulates as form C catenated dimers. These molecules consist of two supercoiled monomer circles of SV40 DNA interlocked by one or more topological intertwinings and are seen as transiently labeled intermediates during normal replication. Form C catenated dimers represent pure segregation intermediates, replicative DNA structures in which DNA synthesis is complete but which still require topological separation of the two daughter circles. Hypertonic shock seems to block selectively a type II topoisomerase activity involved in disentangling the two circles. This is reflected in the fact that form C catenated dimers that accumulate during the block are highly intertwined with catenation linkage numbers up to C_L = 20. While initiation of replication is also inhibited by hypertonic treatment, ongoing SV40 DNA synthesis is not affected, and replication is free to proceed from the earliest cairns structure through to form C catenated dimers. The block to segregation is rapidly and completely released by shifting the cells back to normal medium. A much slower recovery of DNA segregation takes place on prolonged incubation in hypertonic medium, perhaps because of some cellular homeostatic mechanism. The results of this work lead to a detailed view of the final stages of SV40 DNA replication.", "doi": "10.1016/0092-8674(81)90173-2", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "1981-09", "series_number": "3", "volume": "25", "issue": "3", "pages": "659-669" }, { "id": "authors:688tc-fyc12", "collection": "authors", "collection_id": "688tc-fyc12", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:VARpnas81", "type": "article", "title": "On the possibility of metabolic control of replicon \"misfiring\": Relationship to emergence of malignant phenotypes in mammalian cell lineages", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Constraints of a multireplicon chromosomal organization and of the necessity to maintain constant gene dosages demand that each origin of replication in a eukaryotic cell \"fire\" (initiate replication) only once per cell cycle. The central idea of this work is that a low probability of an extra (\"illegitimate\") round of DNA replication (called below \"replicon misfiring\") within any given chromosomal domain could be increased by certain substances of either intra- or extracellular origin. The term \"firone\" is proposed for such a substance. It is shown that existence of firones could greatly speed up evolution of cellular systems under selection pressure, a developing tumor being one example of such a system. Experimentally testable predictions of the firone hypothesis are discussed.", "doi": "10.1073/pnas.78.6.3673", "pmcid": "PMC319633", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1981-06", "series_number": "6", "volume": "78", "issue": "6", "pages": "3673-3677" }, { "id": "authors:3xnvh-w9c14", "collection": "authors", "collection_id": "3xnvh-w9c14", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210122-150559856", "type": "article", "title": "Two-dimensional hybridization mapping of nucleosomes: Comparison of DNA and protein patterns", "author": [ { "family_name": "Levinger", "given_name": "Louis", "clpid": "Levinger-Louis" }, { "family_name": "Barsoum", "given_name": "James", "clpid": "Barsoum-James" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "A new approach was developed for analysis of complex mixtures of staphylococcal nuclease-produced nucleosomes, consisting of two-dimensional electrophoretic fractionation of nucleosomes (low ionic strength electrophoresis of deoxyribo-nucleoproteins in the first dimension followed by DNA electrophoresis in the second dimension), with subsequent electrophoretic transfer of nucleosomal DNA from polyacrylamide gels to DBM paper for hybridization with specific DNA probes. Hybridization was accompanied by parallel two-dimensional analysis of protein composition of mononucleosomes from HeLa chromatin. The major results are: \n\n1. (1) The H1-lacking mononucleosomes containing proteins HMG 14 and HMG 17 migrate as a broad band in the first dimension, and their DNA fragments form an identifiable subset of the total mononucleosomal DNA pattern. \n\n2. (2) Hybridization of a highly repetitive human DNA fragment to fractionated HeLa mononucleosomal DNA lights up all major mononucleosomal DNA spots, suggesting that transcriptionally inactive regions of HeLa chromatin contain both A24 semihistone and HMG proteins. \n\n3. (3) Hybridization of cloned ribosomal DNA to fractionated HeLa mononucleosomal DNA also lights up all major DNA spots. The regions corresponding to A24- and HMG-containing mononucleosomes, however, appear to be under-represented in the hybridization pattern. \n\n4. (4) A fragment of highly repetitive rat DNA hybridizes to a subset of the total oligonucleosomal DNA pattern from rat 14B chromatin, strongly suggesting that the range of nucleosomal DNA repeat lengths within regions of chromatin homologous to this highly repetitive probe is much narrower than that in bulk chromatin. \n\nOur findings indicate that specific interactions of HMG proteins with transcriptionally active regions of chromatin (Weisbrod et al., 1980) are superimposed over a significant background of apparently non-specific HMG chromatin interactions. We suggest that the process of transcription itself is responsible for \"saturation\" of a chromatin region with HMG proteins in vitro.", "doi": "10.1016/0022-2836(81)90389-2", "issn": "0022-2836", "publisher": "Elsevier", "publication": "Journal of Molecular Biology", "publication_date": "1981-03-05", "series_number": "3", "volume": "146", "issue": "3", "pages": "287-304" }, { "id": "authors:1zhxc-3gs43", "collection": "authors", "collection_id": "1zhxc-3gs43", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210122-150559204", "type": "article", "title": "Terminal stages of SV40 DNA replication proceed via multiply intertwined catenated dimers", "author": [ { "family_name": "Sundin", "given_name": "Olof", "clpid": "Sundin-Olof" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "We have identified a new class of SV40 replicative intermediates which consists of a least 20 discrete DNA species. All members of this class are catenated dimers, two circular molecules of SV40 duplex DNA linked topologically by one or more intertwining events. Most of these molecules are linked by several intertwining events, and the range of linkage states observed runs from L = 1 to L = 10. A catenated dimer with a given linkage state is assigned to one of three distinct families (A, B or C) depending on the open or covalently closed nature of its two circular components: in form A catenated dimers, both circles are nicked or gapped; in form B, one of the circles is supercoiled; and in form C, both circles are supercoiled. Members of all three of these families are found in SV40 chromatin pulse-labeled with \u00b3H-thymidine, and together they comprise 10\u201320% of the total replicative form SV40 DNA, appearing as a discrete series of electrophoretically resolved bands superimposed upon a continuous smear of growing cairns structures. The distribution of linkage states varies between the families, A being the most intertwined and C the least intertwined. Upon a chase with cold thymidine, label is lost rapidly from all these catenated DNA species. We suggest that the sequence A \u2192 B \u2192 C \u2192 mature monomeric supercoiled SV40 DNA represents the final stages of SV40 replication, and that a special enzyme activity exists in vivo to uncatenate the SV40 daughter chromosomes.", "doi": "10.1016/0092-8674(80)90118-x", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "1980-08", "series_number": "1", "volume": "21", "issue": "1", "pages": "103-114" }, { "id": "authors:43ns9-bng32", "collection": "authors", "collection_id": "43ns9-bng32", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:LEVpnas80", "type": "article", "title": "High-resolution fractionation of nucleosomes: minor particles, \"whiskers,\" and separation of mononucleosomes containing and lacking A24 semihistone", "author": [ { "family_name": "Levinger", "given_name": "Louis", "clpid": "Levinger-Louis" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Staphylococcal nuclease digests of HeLa chromatin fractionated on low ionic strength nucleoprotein gels have been further analyzed by second-dimension DNA and protein gel electrophoresis. In vivo radioactive labeling of chromatin components and use of longer gels allowed a higher sensitivity and resolution than has been previously reported for this approach. A number of nonhistone protein spots and about 20 DNA spots can be detected in the mononucleosomal region of the second-dimension gel. In particular, there are three DNA spots identical in DNA size that correspond to three discrete kinds of core mononucleosomes resolved on the first-dimension nucleoprotein gel. Analysis of protein composition shows that the most rapidly migrating particle contains all four core histones but no A24 semihistone (A24 is a covalent conjugate of histone H2A and a specific nonhistone protein, ubiquitin), whereas the other two core mononucleosomes contain A24 semihistone. Thus, one can now quantitatively separate the A24-lacking core mononucleosomes from those containing A24, making it possible to directly address the question of whether A24 is associated with nucleosomes containing a specific subset of DNA sequences. Additional features of two-dimensional nucleoprotein-DNA patterns are \"whiskers,\" which run slower than core mononucleosomes in the nucleoprotein dimension and both faster and slower than core-length DNA in the DNA dimension. In more extensive digests, \"secondary whiskers\" are observed, which run faster than core mononucleosomes in both dimensions and appear to coincide with previously described subnucleosomal particles SN7 and SN8 [Bakayev, V., Bakayeva, T. & Varshavsky, A. (1977) Cell 11, 619-629]. Possible mechanisms of whisker formation are discussed.", "doi": "10.1073/pnas.77.6.3244", "pmcid": "PMC349591", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1980-06", "series_number": "6", "volume": "77", "issue": "6", "pages": "3244-3248" }, { "id": "authors:k4vv4-nac91", "collection": "authors", "collection_id": "k4vv4-nac91", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210122-150559088", "type": "article", "title": "Staphylococcal nuclease makes a single non-random cut in the simian virus 40 viral minichromosome", "author": [ { "family_name": "Sundin", "given_name": "Olof", "clpid": "Sundin-Olof" }, { "family_name": "Varshavsky", "given_name": "Alexander", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "When compact simian virus 40 (SV40) minichromosomes are treated with staphylococcal nuclease at 0 \u00b0C under limit-digest conditions, about one-third of the minichromosomes remain resistant to nuclease, a third of them are nicked, while the remaining third suffer one and only one double-stranded cut. Results show that each cleaved minichromosome is cut only once and afterwards becomes resistant to further fragmentation. This is in marked contrast to the action of staphylococcal nuclease at 37 \u00b0C, which leads to a rapid fragmentation of all minichromosomes to oligo- and mononucleosomes. \n\nThe SV40 linear DNA III produced by low-temperature nuclease digestion of minichromosomes was redigested with single-cut restriction endonucleases. By this mapping procedure it was determined that the location of the staphylococcal nuclease cut is neither unique nor random; it occurs at a number of discrete sites on the DNA, half of all cuts being concentrated at the origin of replication and nearby in the \"late\" portion of the SV40 genome. Control experiments have shown that when staphylococcal nuclease digests naked SV40 DNA at 0 \u00b0C it does not \"hesitate\" after the first cut. Although initial cuts in the purified DNA are non-random in location, their distribution is quite different from that generated by a low-temperature nuclease digestion of compact SV40 minichromosomes. Possible interpretations of these results are discussed in view of the recent finding that a specific region of the SV40 genome is uniquely exposed in the minichromosome (Varshavsky et al., 1978, 1979; Scott & Wigmore, 1978).", "doi": "10.1016/0022-2836(79)90274-2", "issn": "0022-2836", "publisher": "Elsevier", "publication": "Journal of Molecular Biology", "publication_date": "1979-08-15", "series_number": "3", "volume": "132", "issue": "3", "pages": "535-546" }, { "id": "authors:mcjnj-8ag05", "collection": "authors", "collection_id": "mcjnj-8ag05", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210122-150556684", "type": "article", "title": "A stretch of \"late\" SV40 viral DNA about 400 bp long which includes the origin of replication is specifically exposed in SV40 minichromosomes", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Sundin", "given_name": "Olof", "clpid": "Sundin-Olof" }, { "family_name": "Bohn", "given_name": "Michael", "clpid": "Bohn-Michael" } ], "abstract": "Examination of DNA fragments produced from either formaldehyde-fixed or unfixed SV40 minichromosomes by multiple-cut restriction endonucleases has led to the following major results: \n\nExhaustive digestion of unfixed minichromosomes with Hae III generated all ten major limit-digest DNA fragments as well as partial cleavage products. In striking contrast to this result, Hae III acted on formaldehyde-fixed minichromosomes to yield only one of the limit-digest fragments, F, which is located in the immediate vicinity of the origin of replication, spanning nucleotides 5169 and 250 on the DNA sequence map of Reddy et al. (1978). This 300 base pair (bp) fragment was released as naked DNA from formaldehyde-fixed, Hae III-digested minichromosomes following treatment either by pronase-SDS or by SDS alone. In the latter case, the remainder of the minichromosome retained its compact configuration as assayed by both sedimentational and electrophoretic methods. In minichromosomes, the F fragment is therefore not only accessible to Hae III at its ends, but is also neither formaldehyde crosslinked into any SDS-resistant nucleoprotein structure nor topologically \"locked\" within the compact minichromosomal particle. This same fragment was preferentially produced during the early stages of digestion of unfixed minichromosomes with Hae III, and its final yield in the exhaustive Hae III digest was significantly higher than that of other limit-digest fragments. Similar results were obtained upon digestion of either unfixed or formaldehyde-fixed minichromosomes with Alu I. In particular, of approximately twenty major limit-digest DNA fragments, only two fragments (F and P, encompassing nucleotides 5146 to 190, and 190 to 325, respectively) were produced by Alu I from the formaldehyde-fixed minichromosomes. All other restriction endonucleases tested (Mbo I, Mbo II, Hind III, Hin II+III and Hinf I), for which there are no closely spaced recognition sequences in the above mentioned regions of the SV40 genome, did not produce any significant amount of limit-digest DNA fragments from formaldehyde-fixed minichromosomes. \n\nThese findings, taken together with our earlier data on the preferential exposure of the origin of replication in SV40 minichromosomes (Varshavsky, Sundin and Bohn, 1978), strongly suggest that a specific region of the \"late\" SV40 DNA approximately 400 bp long is uniquely exposed in the compact minichromosome. It is of interest that, in addition to the origin of replication, this region contains binding sites for T antigen (Tjian, 1977), specific tandem repeated sequences and apparently also the promoters for synthesis of late SV40 mRNAs (Fiers et al., 1978; Reddy et al. (1978)).", "doi": "10.1016/0092-8674(79)90021-7", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "1979-02", "series_number": "2", "volume": "16", "issue": "2", "pages": "453-466" }, { "id": "authors:ctpdq-93418", "collection": "authors", "collection_id": "ctpdq-93418", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210119-135308672", "type": "article", "title": "SV40 viral minichromosome: preferential exposure of the origin of replication as probed by restriction endonucleases", "author": [ { "family_name": "Varshavsky", "given_name": "A. J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Sundin", "given_name": "O. H.", "clpid": "Sundin-Olof-H" }, { "family_name": "Bohn", "given_name": "M. J.", "clpid": "Bohn-Michael-J" } ], "abstract": "Isolated SV40 minichromosomes [1\u20133] were treated with different single-cut restriction endonucleases to probe the arrangement of nucleosomes in relation to the SV40 DNA sequence. While Eco RI and Bam HI each cut 22\u201327% of the SV40 minichromosomes under limit-digest conditions, Bgl I, which cuts SV40 DNA at or very near the origin of replication [4,5], cleaves 90\u201395% of the minichromosomes in a preparation. Similar results were obtained with minichromosomes which had been fixed with formaldehyde before endonuclease treatment. One possible interpretation of these findings is that the arrangement of nucleosomes in the compact SV40 minichromosomes is nonrandom at least with regard to sequences near the origin of DNA replication.", "doi": "10.1093/nar/5.10.3469", "pmcid": "PMC342688", "issn": "0305-1048", "publisher": "Oxford University Press", "publication": "Nucleic Acids Research", "publication_date": "1978-10", "series_number": "10", "volume": "5", "issue": "10", "pages": "3469-3478" }, { "id": "authors:keada-pnv58", "collection": "authors", "collection_id": "keada-pnv58", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210119-135308449", "type": "article", "title": "On the structure of cellular and viral chromatin", "author": [ { "family_name": "Varshavsky", "given_name": "A. J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Bakayev", "given_name": "V. V.", "clpid": "Bakayev-V-V" }, { "family_name": "Bakayeva", "given_name": "T. G.", "clpid": "Bakayeva-T-G" }, { "family_name": "Chumackov", "given_name": "P. M.", "clpid": "Chumackov-P-M" }, { "family_name": "Schmatchenko", "given_name": "V. V.", "clpid": "Schmatchenko-V-V" }, { "family_name": "Georgiev", "given_name": "G.P.", "clpid": "Georgiev-Georgiy-P" } ], "abstract": "Some of the recent experimental data obtained in our laboratory are briefly reviewed. 1. A mild staphylococcal nuclease digestion of either chromatin or nuclei from mouse Ehrlich tumour cells results in chromatin subunits (mononucleosomes) of three discrete kinds. The smallest mononucleosome (MN\u2081 contains all histones except H1 and a DNA fragment 140 base pairs long. The intermediate mononucleosome (MN\u2082) contains all five histones and a DNA fragment 170 base pairs long. The third mononucleosome (MN\u2083) also contains all five histones, but its associated DNA is longer and somewhat heterogeneous in size (180-200 base pairs). Most of the MN\u2083 particles are rapidly converted to mononucleosomes MN\u2082 and MN\u2081 by nuclease digestion. However, there exists a relatively nuclease-resistant subpopulation of the MN\u2083 mononucleosomes. These 200 base-pair MN\u2083 particles contain not only histones but also non-histone proteins and are significantly more resistant to nuclease then even the smaller mononucleosomes MN\u2081 and MN\u2082. 2. Nuclease digestion of hen erythrocyte nuclei or chromatin, in which histone H1 is partially replaced by histone H5 produces the mononucleosomes MN\u2081 and two electrophoretically resolvable kinds of MN\u2082 mononucleosomes, one containing histone H1 and the other one histone H5. A relatively nuclease-resistant subset of the mononucleosomes MN\u2083 is preferentially accumulated at later stages of the digestion. 3. Although pancreatic DNase (DNase I) and spleen acid DNase (DNase II) attack the DNA in chromatin in a manner different from that of staphylococcal nuclease, the deoxyribonucleoprotein (DNP) products of digestion are similar for all three enzymes under identical solvent conditions, as revealed by gel electrophoresis of the DNP at low ionic strength. 4. There are eight major kinds of staphylococcal nuclease-produced soluble subnucleosomes (i.e. particles smaller than the mononucleosomes). In particular, the subnucleosome SN\u2081 is a set of naked double-stranded DNA fragments ca. 20 base pairs long. Subnucleosome SN\u2082 is a complex of a specific highly basic non-histone protein and a DNA fragment ca. 27 base pairs long. Subnucleosomes SN\u2087 and SN\u2088 each contain all of the histones except H1 and DNA fragments ca. 100 and 120 base pairs long, respectively. 5. Nuclease digestion of isolated mono- and dinucleosomes does not produce all of the subnucleosomes. These and related findings indicate that the cleavages required to generate these subnucleosomes result from some aspect of chromatin structure which is lost upon digestion to mono- or dinucleosomes. Nuclease digestion of isolated minichromosomes of Simian virus 40 (SV40) (which contain all five histones including H1) produces mononucleosomes MN\u2081 and MN\u2082 but does not produce some of the subnucleosomes or the relatively nuclease-resistant subset of the MN\u2083 mononucleosomes. 6. The rate of sedimentation of the SV40 minichromosomes (ca. 60S) under 'physiological' ionic conditions (\u03bc \u2248 0.15) is about two times higher than that in a low ionic strength buffer (\u03bc \u2248 0.005). Occurrence of the compact state of the minichromosome critically depends upon the presence of histone H1 and can be irreversibly fixed by treatment with formaldehyde.", "doi": "10.1098/rstb.1978.0024", "issn": "0080-4622", "publisher": "The Royal Society", "publication": "Philosophical Transactions of the Royal Society of London. B, Biological Sciences", "publication_date": "1978-05-11", "series_number": "997", "volume": "283", "issue": "997", "pages": "275-285" }, { "id": "authors:7htcz-anp50", "collection": "authors", "collection_id": "7htcz-anp50", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210119-135308547", "type": "article", "title": "A technique of low-pH gel electrophoresis of chromosomal proteins which does not require preliminary removal of DNA", "author": [ { "family_name": "Shmatchenko", "given_name": "V. V.", "clpid": "Shmatchenko-V-V" }, { "family_name": "Varshavsky", "given_name": "A. J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Fractionation of chromosomal proteins, in particular, of histones, by acetic acid-urea polyacrylamide gel electrophoresis usually requires preliminary removal of DNA from deoxyribonucleoprotein samples to obtain good separation of proteins. We have found that this difficulty can be overcome by addition of cetyltrimethylammonium bromide (CTAB) to the gel and electrode buffers. Since CTAB can readily diffuse into polyacrylamide gels two-dimensional fractionation becomes possible; that is, deoxyribonucleoprotein particles are fractionated in the first dimension followed by immersion of a gel in a CTAB solution and then low-pH gel electrophoresis of proteins in the second dimension.", "doi": "10.1016/0003-2697(78)90271-3", "issn": "0003-2697", "publisher": "Elsevier", "publication": "Analytical Biochemistry", "publication_date": "1978-03", "series_number": "1", "volume": "85", "issue": "1", "pages": "42-46" }, { "id": "authors:nhjcm-w7v29", "collection": "authors", "collection_id": "nhjcm-w7v29", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210119-135308326", "type": "article", "title": "On the Structure of Eukaryotic, Prokaryotic, and Viral Chromatin", "author": [ { "family_name": "Varshavsky", "given_name": "A. J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Bakayev", "given_name": "V. V.", "clpid": "Bakayev-V-V" }, { "family_name": "Nedospasov", "given_name": "S. A.", "clpid": "Nedospasov-S-A" }, { "family_name": "Georgiev", "given_name": "G. P.", "clpid": "Georgiev-Georgiy-P" } ], "abstract": "In this paper we summarize our recent experimental findings obtained in three different but mutually interdependent lines of research, namely, in studies on the structure of the eukaryotic (mouse), bacterial (Escherichia coli), and viral (SV40) chromatin.", "doi": "10.1101/sqb.1978.042.01.049", "issn": "0091-7451", "publisher": "Cold Spring Harbor Laboratory", "publication": "Cold Spring Harbor Symposia on Quantitative Biology", "publication_date": "1978", "series_number": "1", "volume": "42", "issue": "1", "pages": "457-473" }, { "id": "authors:ses30-pzw90", "collection": "authors", "collection_id": "ses30-pzw90", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201218-095603050", "type": "article", "title": "Compact form of SV40 viral minichromosome is resistant to nuclease: possible implications for chromatin structure", "author": [ { "family_name": "Varshavsky", "given_name": "A. J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Nedospasov", "given_name": "S. A.", "clpid": "Nedospasov-S-A" }, { "family_name": "Schmatchenko", "given_name": "V. V.", "clpid": "Schmatchenko-V-V" }, { "family_name": "Bakayev", "given_name": "V. V.", "clpid": "Bakayev-V-V" }, { "family_name": "Chumackov", "given_name": "P. M.", "clpid": "Chumackov-P-M" }, { "family_name": "Georgiev", "given_name": "G. P.", "clpid": "Georgiev-G-P" } ], "abstract": "We report two new findings bearing on the \"supranucleosomal\" level of the structure of the Simian Virus 40 mini-chromosome. I) Isolated SV40 minichromosone which contains all five histones including HI /I exists in solutionunder approximately physiological ionic conditions as a compact roughly spherical particle\u223c300 \u00c5 in diameter which is capable of fitting within the virus capsid. In spite of such a compact conformation of the minichromosome individual nucleosomes can be readily visualized within the particle. Compact state of SV40 minichromosome depends on both the presence of histone HI and maintenance of approximately physiologicalionic strength of solution (\u03bc\u22480.15). Removal of HI results in a conversion of the compact minichromosome into an extended (circular beaded) structure. 2) The compact form of the SV40 minichromosome in contrast to its circular beaded form is virtually completely resistant to staphylococcal nuclease, strongly suggesting that in particular nucleasesensitive, parts of the internucleosomal DNA regions are not exposed on the outside of the compact SV40 minichromosome. On the other hand, DNase I which is known to attack both inter- and intranucleosomal DNA in the chromatin /2, 3/ readily digests the compact form of the SV40 minichromosome.Possible models of the compact minichromosome and implications for higher order structures of the cellular chromatin are discussed.", "doi": "10.1093/nar/4.10.3303", "pmcid": "PMC342655", "issn": "0305-1048", "publisher": "Oxford University Press", "publication": "Nucleic Acids Research", "publication_date": "1977-10", "series_number": "10", "volume": "4", "issue": "10", "pages": "3303-3325" }, { "id": "authors:60mag-vvh97", "collection": "authors", "collection_id": "60mag-vvh97", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201218-095602925", "type": "article", "title": "Histone-like proteins in the purified Escherichia coli deoxyribonucleoprotein", "author": [ { "family_name": "Varshavsky", "given_name": "A. J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Nedospasov", "given_name": "S. A.", "clpid": "Nedospasov-S-A" }, { "family_name": "Bakayev", "given_name": "V. V.", "clpid": "Bakayev-V-V" }, { "family_name": "Bakayeva", "given_name": "T. G.", "clpid": "Bakayeva-T-G" }, { "family_name": "Georgiev", "given_name": "G. P.", "clpid": "Georgiev-G-P" } ], "abstract": "Analysis of E.coli chromosomes isolated under conditions similar to those used for isolation of eukaryotic chromatin has shown that: 1) The proteins of highly purified E.coli deoxyribonucleoprotein are mainly in addition to RNA polymerase two specific histone-like proteins of apparent molecular weight of 17,000 and 9,000 (proteins 1 and 2, respectively). 2) Proteins 1 and 2 occur in approximately equal molar amounts in the isolated E.coli chromosome, and their relative content corresponds to one molecule of protein 1 plus one molecule of protein 2 per 150\u2013200 base pairs of DNA. 3) There are no long stretches of naked DNA in the purified E.coli deoxyribonucleoprotein suggesting a fairly uniform distribution of the proteins 1 and 2 along DNA. 4; The protein 2 is apparently identical to the DNA-binding protein HU which was isolated previously /1/ from extracts of E.coli cells. 5) Digestion of the isolated E.coli chromosomes with staphylococcal nuclease proceeds through discrete deoxyribonucleoprotein intermediates (in particular, at \u223c120 base pairs) which contain both proteins 1 and 2. However, since no repeating multimer structure was observed so far in nuclease digests of the E.coli chromosome, it seems premature to draw definite conclusions about possible similarities between the nucleosomal organization of the eukaryotic chromatin and the E.coli chromatin structure.", "doi": "10.1093/nar/4.8.2725", "pmcid": "PMC342604", "issn": "0305-1048", "publisher": "Oxford University Press", "publication": "Nucleic Acids Research", "publication_date": "1977-08", "series_number": "8", "volume": "4", "issue": "8", "pages": "2725-2746" }, { "id": "authors:gtppg-mdx91", "collection": "authors", "collection_id": "gtppg-mdx91", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201218-095602789", "type": "article", "title": "Nucleosomes and subnucleosomes: heterogeneity and composition", "author": [ { "family_name": "Bakayev", "given_name": "V. V.", "clpid": "Bakayev-V-V" }, { "family_name": "Bakayeva", "given_name": "T. G.", "clpid": "Bakayeva-T-G" }, { "family_name": "Varshavsky", "given_name": "A. J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "Previous studies (Varshavsky, Bakayev and Georgiev, 1976a) have shown that chromatin subunits (mononucleosomes) and their oligomers in a mild staphylococcal nuclease digest of chromatin display a heterogeneous content of histone H1. We now report that a mild staphylococcal nuclease digest of either chromatin or nuclei from mouse Ehrlich tumor cells contains mononucleosomes of three discrete kinds. The smallest mononucleosome (MN\u2081) contains all histones except H1 and a DNA fragment 140 base pairs (bp) long. The intermediate mononucleosome (MN\u2082) contains all five histones and a DNA fragment 170 bp long. The third mononucleosome (MN\u2083) also contains all five histones, but its DNA fragment is longer and more heterogeneous in size (180\u2013200 bp). Most of the MN\u2083 particles are rapidly converted by nuclease into mononucleosomes MN\u2081 and MN\u2082. There exists, however, a relatively nuclease-resistant subpopulation of the MN\u2083 mononucleosomes. These 200 bp MN\u2081 particles contain not only histones but also nonhistone proteins, and are significantly more resistant to nuclease than the bulk of MN\u2083 particles and the smaller mononucleosomes MN\u2081 and MN\u2082. \n\nThere are eight major kinds of staphylococcal nuclease-produced soluble subnucleosomes (SN). The SN\u2081 is a set of naked double-stranded DNA fragments \u223c20 bp long. The SN\u2082 is a complex of a specific basic nonhistone protein (molecular weight \u223c16,000 daltons) and a DNA fragment \u223c27 bp long. The SN\u2083 contains histone H4, the above-mentioned specific nonhistone protein and a DNA fragment \u223c27 bp long. The SN4 contains histones H2a, H2b, H4 and a DNA fragment \u223c45 bp long. The SN5 contains histones H2a, H2b, H3 and a DNA fragment \u223c55 bp long. The SN6 is a complex of histone H1 and a DNA fragment \u223c35 bp long. Subnucleosomes SN\u2087 and SN\u2088 each contain all the histones except H1, and DNA fragments \u223c100 and \u223c120 bp long, respectively. \n\nNuclease digestion of isolated mono- or dinucleosomes does not produce some of the subnucleosomes. These and related findings indicate that the cleavage required to generate these subnucleosomes result from some aspect of chromatin structure which is lost upon digestion to mono- and dinucleosomes.", "doi": "10.1016/0092-8674(77)90079-4", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "1977-07", "series_number": "3", "volume": "11", "issue": "3", "pages": "619-629" }, { "id": "authors:kwcvb-ab307", "collection": "authors", "collection_id": "kwcvb-ab307", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210203-074258460", "type": "article", "title": "Free DNA stretches in histone H1-depleted chromatin and their possible relation to chromomere structure", "author": [ { "family_name": "Varshavsky", "given_name": "A. J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Georgiev", "given_name": "G. P.", "clpid": "Georgiev-Georgiy-P" } ], "abstract": "Oligomers of chromatin subunits (oligonucleosomes) were prepared by a mild digestion of chromatin with staphylococcal nuclease followed by a purification of a high molecular weight material (hexanucleosomes and larger DNP particles) by gel chromatography. The main finding is that a mild removal of histone H1 from the oligonucleosome preparation by treatment with tRNA in the absence of any significant hydrodynamic shearing leads to the formation of free DNA molecules which constitute 5\u20136% of the total oligonucleosomal DNA. \n\nThe size of nucleosome-free DNA stretches in H1-depleted hydrodynamically sheared chromatin is about 6000 base pairs and their content is apparently 10\u201312% of the total DNA. These and related findings are discussed in terms of the previously proposed 'asymmetric hairpin' model of DNA packing in chromatin. Different kinds of the asymmetric hairpin are considered and ambiguities in interpretations of experimental data are pointed out.", "doi": "10.1007/bf00357206", "issn": "0301-4851", "publisher": "Springer", "publication": "Molecular Biology Reports", "publication_date": "1976-09", "series_number": "1", "volume": "3", "issue": "1", "pages": "27-38" }, { "id": "authors:6j3bm-sfm72", "collection": "authors", "collection_id": "6j3bm-sfm72", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201216-124003102", "type": "article", "title": "Minichromosome of simian virus 40: presence of histone HI", "author": [ { "family_name": "Varshavsky", "given_name": "A. J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Bakayev", "given_name": "V. V.", "clpid": "Bakayev-V-V" }, { "family_name": "Chumackov", "given_name": "P. M.", "clpid": "Chumackov-P-M" }, { "family_name": "Georgiev", "given_name": "G. P.", "clpid": "Georgiev-G-P" } ], "abstract": "In contrast to conclusions of previous studies /I\u20133/ claiming the absence of histone HI from the SV40 and polyoma viral minichromosomes we have found that a preparation of purified SV40 minichromosomes does contain histone HI. The content of HI in relation to other four histones in the SV40 minichromosomes is close to that in the cellular chromatin. Histone HI in the isolated SV40 minichromosomes is bound apparently to internucleosomal DNA stretches as was shown already for HI in the cellular chromatin /4/. \n\nIn addition it was found that more than 90% of the purified SV40 minichromosomes migrated as a single discrete deoxyribonucleoprotein band upon agarose gel electrophoresis.", "doi": "10.1093/nar/3.8.2101", "pmcid": "PMC343065", "issn": "0305-1048", "publisher": "Oxford University Press", "publication": "Nucleic Acids Research", "publication_date": "1976-08", "series_number": "8", "volume": "3", "issue": "8", "pages": "2101-2114" }, { "id": "authors:mrpf1-zez84", "collection": "authors", "collection_id": "mrpf1-zez84", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201216-124002591", "type": "article", "title": "Studies on Chromatin. Free DNA in Sheared Chromatin", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Bakayev", "given_name": "Valery V.", "clpid": "Bakayev-Valery-V" }, { "family_name": "Ilyin", "given_name": "Yurii V.", "clpid": "Ilyin-Yurii-V" }, { "family_name": "Bayev", "given_name": "Alexey A., Jr.", "clpid": "Bayev-Alexey-A-Jr" }, { "family_name": "Georgiev", "given_name": "Georgii P.", "clpid": "Georgiev-Georgii-P" } ], "abstract": "Chromatin which has been hydrodynamically sheared in a low\u2010ionic\u2010strength buffer lacking divalent cations (I = 0.005 M) contains a heterogeneous set of deoxyribonucleoprotein particles but no molecules of free DNA. The main finding is that a transference of sheared chromatin to 1\u20132 mM MgCl\u2082 or to 0.1\u20130.2 M NaCl results in appearance of completely free DNA molecules. A salt\u2010induced rearrangement of DNA\u2010bound histories but not a partial loss of them is responsible for the observed phenomenon. Formation of free DNA molecules is accompanied by aggregation of the majority of remaining deoxyribonucleoprotein particles. \n\nThe percentage of free DNA molecules in the chromatin which was sheared to an average DNA length of about 400 base pairs is increased from zero in the initial sample to 8\u20139% in 1 mM MgCl\u2082 and further to \u223c 25% of the total DNA in 0.15 M NaCl, 2 mM MgCl\u2082. Free DNA molecules in the sheared chromatin are observed not only upon isopycnic banding of formaldehyde\u2010fixed deoxyribonucleoproteins in CsCl gradients but also in non\u2010ionic metrizamide gradients with either fixed or unfixed deoxyribonucleoprotein samples. The process of free DNA formation is a reversible one; its direction and the equilibrium state depend in particular on the ionic conditions of the medium.", "doi": "10.1111/j.1432-1033.1976.tb10510.x", "issn": "0014-2956", "publisher": "Wiley", "publication": "European Journal of Biochemistry", "publication_date": "1976-07", "series_number": "2", "volume": "66", "issue": "2", "pages": "211-223" }, { "id": "authors:8cxmj-ahm43", "collection": "authors", "collection_id": "8cxmj-ahm43", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201216-124002783", "type": "article", "title": "Heterogeneity of chromatin subunits in vitro and location of histone H1", "author": [ { "family_name": "Varshavsky", "given_name": "A. J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Bakayev", "given_name": "V. V.", "clpid": "Bakayev-V-V" }, { "family_name": "Georgiev", "given_name": "G. P.", "clpid": "Georgiev-G-P" } ], "abstract": "Chromatin subunits (\"nucleosomes\") which were purified by sucrose gradient centrifugation of a staphylococcal nuclease digest of chromatic, have been studied. We found that such a preparation contains nucleosomes of two discrete types which can be separated from each other by polyacrylamide gel electrophoresis. Nucleosome of the first type contains all five histones and a DNA segment of approximately 200 base pairs long, whereas nucleosome of the second type lacks his\u2014tone H1 and its DNA segment is approximately 170 base pairs long, i.e., about 30 base pairs shorter than the DNA segment of the nucleosome of the first type. Purified dimer of the nucleosome also can be fractionated by gel electrophoresis into three discrete bands which correspond to dinucleosomes containing two molecules of histone H1, one and no H1. These and related findings strongly suggest that the H1 molecule is bound to a short (approximately 30 base pairs) terminal stretch of the nucleosomal DNA segment which can be removed by nuclease (possibly in the form of H1-DNA complex) without any significant disturbance of main structural features of the nucleosome.", "doi": "10.1093/nar/3.2.477", "pmcid": "PMC342917", "issn": "0305-1048", "publisher": "Oxford University Press", "publication": "Nucleic Acids Research", "publication_date": "1976-02", "series_number": "2", "volume": "3", "issue": "2", "pages": "477-492" }, { "id": "authors:phxf8-70h38", "collection": "authors", "collection_id": "phxf8-70h38", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210317-083952421", "type": "article", "title": "Studies on chromatin. V. A model for the structure of chromatin subunit", "author": [ { "family_name": "Varshavsky", "given_name": "A. J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Georgiev", "given_name": "G. P.", "clpid": "Georgiev-Georgiy-P" } ], "abstract": "A new model for the fine structure of the chromatin subunit (or 'nucleosome') is proposed. The model is based on previous experimental findings [1\u201314] and on two new suggestions, namely:\n\n(1)\nEight histones form a toroidal-shaped core of the nucleosome and are arranged in the following circular sequense: .\n\n(2)\nDNA is 'kinked' around a toroidal-shaped histone core in a 'solenoid-like' mode, each kink occurring every 10 base pairs along DNA.\n\nThe electron microscopic evidence for a toroidal shape of the nucleosome is described in the preceding paper [13]. The possibility of the existence of kinks in the DNA double helix was considered recently by Crick and Klug [14]. The proposed model of the nucleosome, being more detailed than earlier models permits us to explain in direct structural terms the yet unordered set of data bearing on the pattern of histone-histone interactions in chromatin, the results of a mild deoxyribonuclease digestion of DNA within the nucleosomal particle and also the quantitative data on the unwinding of the DNA duplex upon formation of the nucleosome.", "doi": "10.1007/bf00356996", "issn": "0301-4851", "publisher": "Springer", "publication": "Molecular Biology Reports", "publication_date": "1975-10", "series_number": "3", "volume": "2", "issue": "3", "pages": "255-262" }, { "id": "authors:vpzfn-h1115", "collection": "authors", "collection_id": "vpzfn-h1115", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210317-085746135", "type": "article", "title": "Studies on chromatin. III. v-Bodies and Free DNA in Chromatin Lacking Histone H1", "author": [ { "family_name": "Varshavsky", "given_name": "A. J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Bakayev", "given_name": "V. V.", "clpid": "Bakayev-V-V" } ], "abstract": "Chromatin lacking histone H1 was found by electron microscopy to contain 'beaded' deoxyribonucleoprotein fibers. Adjacent beads are connected with each other by threads having a DNA-like appearance. At least some of threads are shown to be free DNA stretches. Average length and the content of free DNA stretches in histone H1-depleted chromatin depends on the ionic conditions of the medium. The appearance of individual beads is similar to that of chromatin subunits or v-bodies [1] in the original chromatin.\n\nThus, in agreement with the X-ray data [2], histone H1 apparently is not required for maintenance of a compact state of DNA in chromatin subunits.", "doi": "10.1007/bf00356990", "issn": "0301-4851", "publisher": "Springer", "publication": "Molecular Biology Reports", "publication_date": "1975-10", "series_number": "3", "volume": "2", "issue": "3", "pages": "209-217" }, { "id": "authors:762py-mx864", "collection": "authors", "collection_id": "762py-mx864", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210317-090059214", "type": "article", "title": "Studies on chromatin. IV. Evidence for a toroidal shape of chromatin subunits", "author": [ { "family_name": "Varshavsky", "given_name": "A. J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Bakayev", "given_name": "V. V.", "clpid": "Bakayev-V-V" } ], "abstract": "Electron microscopy of purified chromatin subunits (\u03bd-bodies [17] or nucleosomes [2] revealed a hole or at least a deep indentation in the globular nucleosome. A hole in the nucleosome was visualized using rotatory shadowing with platinum-palladium or more directly, by negative staining with sodium phosphotungstate. The diameter of the hole as measured from negatively stained samples is 10\u201325\u00c5. The external diameter of the negatively stained nucleosome equals 75\u00b115\u00c5.\n\nAlthough most of the data are formally compatible with either a hole or a deep indentation in the nucleosome, some views of the particles in the negatively stained samples suggest a hole rather than an indentation.\n\nThe possible significance of a toroidal structure of the chromatin subunit is discussed in the accompanying paper [3].", "doi": "10.1007/bf00356995", "issn": "0301-4851", "publisher": "Springer", "publication": "Molecular Biology Reports", "publication_date": "1975-10", "series_number": "3", "volume": "2", "issue": "3", "pages": "247-254" }, { "id": "authors:pg31j-yjt45", "collection": "authors", "collection_id": "pg31j-yjt45", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201216-124002260", "type": "article", "title": "Very long stretches of free DNA in chromatin", "author": [ { "family_name": "Varshavsky", "given_name": "A. J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Ilyin", "given_name": "Yu. V.", "clpid": "Ilyin-Yu-V" }, { "family_name": "Georgiev", "given_name": "G. P.", "clpid": "Georgiev-G-P" } ], "abstract": "The arrangement of histones along the DNA in chromosomes has been studied using digestion of the chromatin with DNase and titration of the chromatin with polylysine or certain dyes as probes. The precision of these probes is limited by the extent to which DNA partially covered with histones will react with the enzymes or titration agents used. Furthermore, it has been noted that histones are redistributed along or between DNA fibres under conditions arising during the normal course of isolation and handling of the chromatin. This phenomenon may have strongly influenced the results obtained previously.", "doi": "10.1038/250602a0", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "1974-08-16", "series_number": "5467", "volume": "250", "issue": "5467", "pages": "602-606" }, { "id": "authors:x9vzn-chm79", "collection": "authors", "collection_id": "x9vzn-chm79", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210317-085445588", "type": "article", "title": "Histone-histone proximity in chromatin as seen by imidoester cross-linking", "author": [ { "family_name": "Ilyin", "given_name": "Yu. V.", "clpid": "Ilyin-Yu-V" }, { "family_name": "Bayev", "given_name": "A. A., Jr.", "clpid": "Bayev-A-A-Jr" }, { "family_name": "Zhuze", "given_name": "A. L.", "clpid": "Zhuze-A-L" }, { "family_name": "Varshavsky", "given_name": "A. J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" } ], "abstract": "After treatment of purified chromatin with dimethyl adipimidate (a reversible crosslinking reagent) a number of histone oligomers could be isolated in the soluble form from the chromatin. It was shown that all five histones take part in the dimethyl adipimidate-induced oligomer formation.\n\nOnly a few kinds of histone oligomers (with unknown composition) could be isolated in the soluble form after treatment of chromatic with formaldehyde.", "doi": "10.1007/bf00309568", "issn": "0301-4851", "publisher": "Springer", "publication": "Molecular Biology Reports", "publication_date": "1974-03", "series_number": "6", "volume": "1", "issue": "6", "pages": "343-348" }, { "id": "authors:sjjcz-sfw80", "collection": "authors", "collection_id": "sjjcz-sfw80", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201216-124001838", "type": "article", "title": "On the Structural Organization of the Transcriptional Unit in Animal Chromosomes", "author": [ { "family_name": "Georgiev", "given_name": "Georgiy P.", "clpid": "Georgiev-Georgiy-P" }, { "family_name": "Varshavsky", "given_name": "Alexandr Ja.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Ryskov", "given_name": "Akexej P.", "clpid": "Ryskov-Alexej-P" }, { "family_name": "Church", "given_name": "Robert B.", "clpid": "Church-Robert-B" } ], "abstract": "Three levels of genome organization in eukaryotes are considered in this paper: (1) the structure of the transcriptional unit (or transcripton); (2) the arrangement of proteins along unfolded chromosomal DNA in chromatin; and (3) the mode of DNA packing in the chromatin.", "doi": "10.1101/sqb.1974.038.01.089", "issn": "0091-7451", "publisher": "Cold Spring Harbor Laboratory", "publication": "Cold Spring Harbor Symposia on Quantitative Biology", "publication_date": "1974-01-01", "volume": "38", "pages": "869-884" }, { "id": "authors:kg689-ev673", "collection": "authors", "collection_id": "kg689-ev673", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210317-085119478", "type": "article", "title": "Long molecules of free DNA in the sheared chromatin preparation", "author": [ { "family_name": "Varshavsky", "given_name": "A. J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Ilyin", "given_name": "Yu. V.", "clpid": "Ilyin-Yu-V" }, { "family_name": "Georgiev", "given_name": "G. P.", "clpid": "Georgiev-Georgiy-P" } ], "abstract": "Hydrodynamic shearing of chromatin in the presence of Mg\u00b2\u207a ions produces two discrete types of particles: (1) molecules of completely free DNA which comprise 20\u201323% of the total DNA and (2) histone-covered DNA molecules which contain all five histone fractions. The average length of free DNA molecules depends on the intensity of shearing and can be as high as 1000 base pairs or more. Shearing of chromatin in the absence of Mg\u00b2\u207a produces a heterogeneous population of DNP particles; no free DNA is liberated. However, the addition of Mg\u00b2\u207a to this preparation results in appearance of free DNA molecules and in a complete restoration of the above 'bimodal' distribution.\nThese findings support a previously proposed 'asymmetric hairpin' model of DNA packing in the chromatin [1\u20133].", "doi": "10.1007/bf00357642", "issn": "0301-4851", "publisher": "Springer", "publication": "Molecular Biology Reports", "publication_date": "1973-12", "series_number": "4", "volume": "1", "issue": "4", "pages": "201-207" }, { "id": "authors:wm6jb-de263", "collection": "authors", "collection_id": "wm6jb-de263", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210317-084722770", "type": "article", "title": "Redistribution of histones during unfolding of chromosomal DNA", "author": [ { "family_name": "Varshavsky", "given_name": "Alexander J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Georgiev", "given_name": "Georgii P.", "clpid": "Georgiev-Georgii-P" } ], "abstract": "Just in the course of unfolding of chromosomal deoxyribonucleoproteins performed in the absence of magnesium ions all DNA-bound histones may be redistributed. This type of redistribution is completely blocked in already 'unfolded' DNP preparations. If the unfolding-induced histone redistribution is allowed it leads to a significant decrease of the average length of free DNA segments in the unfolded, histone F1-depleted DNP. The implications of these data on the mode of DNA packing in chromatin are discussed.", "doi": "10.1007/bf00357154", "issn": "0301-4851", "publisher": "Springer", "publication": "Molecular Biology Reports", "publication_date": "1973-10", "series_number": "3", "volume": "1", "issue": "3", "pages": "143-148" }, { "id": "authors:tmmyv-t0555", "collection": "authors", "collection_id": "tmmyv-t0555", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210317-084350197", "type": "article", "title": "Arrangement of histones along DNA in chromosomal deoxyribonucleoprotein lacking histone F1", "author": [ { "family_name": "Varshavsky", "given_name": "A. J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Georgiev", "given_name": "G. P.", "clpid": "Georgiev-Georgiy-P" } ], "abstract": "In deoxyribonucleoprotein from which histone F1 and most nonhistone proteins were removed by treatment with tRNA in the presence of Mg\u00b2\u207a, one can find very long stretches of completely free DNA (average length of about 4\u00d710\u00b3 base pairs). They alternate with stretches of DNA (\u223c16\u00d710\u00b3 base pairs) which are nearly uniformly covered with the other four histones.", "doi": "10.1007/bf00357586", "issn": "0301-4851", "publisher": "Springer", "publication": "Molecular Biology Reports", "publication_date": "1973-08", "series_number": "2", "volume": "1", "issue": "2", "pages": "87-92" }, { "id": "authors:8cf3q-dfw23", "collection": "authors", "collection_id": "8cf3q-dfw23", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201202-154043213", "type": "article", "title": "Clustered arrangement of histones F2a1 and F3 along DNA in chromosomal deoxyribonucleoproteins", "author": [ { "family_name": "Varshavsky", "given_name": "A. J.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Georgiev", "given_name": "G. P.", "clpid": "Georgiev-G-P" } ], "abstract": "Molecules of the arginine-rich histones (F2a1 and F3) are arranged on DNA in clusters separated from each other by stretches of DNA free of either F2a1 or F3 with average length of about 900 base pairs. Such histone F3, F2a1-free stretches of DNA constitute at least 35% (and probably considerably more) of the total mouse nuclear DNA.", "doi": "10.1016/0005-2787(72)90166-9", "issn": "0005-2787", "publisher": "Elsevier", "publication": "Biochimica et Biophysica Acta", "publication_date": "1972-11-09", "series_number": "4", "volume": "281", "issue": "4", "pages": "669-674" }, { "id": "authors:3z6b3-31232", "collection": "authors", "collection_id": "3z6b3-31232", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20201202-154043608", "type": "article", "title": "Collapse of extended deoxyribonucleoprotein molecules upon increase of the ionic strength of solution", "author": [ { "family_name": "Varshavsky", "given_name": "A. Ya.", "orcid": "0000-0002-4011-258X", "clpid": "Varshavsky-A" }, { "family_name": "Ilyin", "given_name": "Yu. V.", "clpid": "Ilyin-Yu-V" }, { "family_name": "Kadyckov", "given_name": "V. I.", "clpid": "Kadyckov-V-I" }, { "family_name": "Senchenkov", "given_name": "E. P.", "clpid": "Senchenkov-E-P" } ], "abstract": "The increase of the ionic strength of extremely dilute deoxyribonucleo protein (DNP) solution from 0.001 to 0.1 results in an intramolecular structural transition, which is reflected by a 2.5-fold increase of the sedimentation coefficient of the DNP and by the electron microscopic appearance of DNP molecules. The rate of this structural transition is considerably higher at 37\u00b0 than at 0\u00b0. Both sedimentation and electron microscopic data allow one to suggest the existence of a supercoiled DNA in collapsed DNP molecules. \n\nRemoval of the very lysine-rich histone (F1) and of some nonhistone proteins from the DNP results in the loss of its ability to undergo the above structural transition.", "doi": "10.1016/0005-2787(71)90797-0", "issn": "0005-2787", "publisher": "Elsevier", "publication": "Biochimica et Biophysica Acta", "publication_date": "1971-09-24", "series_number": "3", "volume": "246", "issue": "3", "pages": "583-588" } ]