[ { "id": "authors:vp08z-gjs20", "collection": "authors", "collection_id": "vp08z-gjs20", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:PARmbc92", "type": "article", "title": "Alternative pathways for the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases", "author": [ { "family_name": "Paravicini", "given_name": "Gerhard", "clpid": "Paravicini-G" }, { "family_name": "Horazdovsky", "given_name": "Bruce F.", "clpid": "Horazdovsky-B-F" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" } ], "abstract": "vps35 mutants of Saccharomyces cerevisiae exhibit severe defects in the localization of carboxypeptidase Y, a soluble vacuolar hydrolase. We have cloned the wild-type VPS35 gene by complementation of the vacuolar protein sorting defect exhibited by the vps35-17 mutant. Sequence analysis revealed an open reading frame predicted to encode a protein of 937 amino acids that lacks any obvious hydrophobic domains. Subcellular fractionation studies indicated that 80% of Vps35p peripherally associates with a membranous particulate cell fraction. The association of Vps35p with this fraction appears to be saturable; when overproduced, the vast majority of Vps35p remains in a soluble fraction. Disruption of the VPS35 gene demonstrated that it is not essential for yeast cell growth. However, the null allele of VPS35 results in a differential defect in the sorting of vacuolar carboxypeptidase Y (CPY), proteinase A (PrA), proteinase B (PrB), and alkaline phosphatase (ALP). proCPY was quantitatively missorted and secreted by delta vps35 cells, whereas almost all of proPrA, proPrB, and proALP were retained within the cell and converted to their mature forms, indicating delivery to the vacuole. Based on these observations, we propose that alternative pathways exist for the sorting and/or delivery of proteins to the vacuole.", "pmcid": "PMC275592", "issn": "1059-1524", "publisher": "American Society for Cell Biology", "publication": "Molecular Biology of the Cell", "publication_date": "1992-04", "series_number": "4", "volume": "3", "issue": "4", "pages": "415-427" }, { "id": "authors:9bsen-wm866", "collection": "authors", "collection_id": "9bsen-wm866", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:ROBmcb91", "type": "article", "title": "A putative zinc finger protein, Saccharomyces cerevisiae Vps18p, affects late Golgi functions required for vacuolar protein sorting and efficient alpha-factor prohormone maturation", "author": [ { "family_name": "Robinson", "given_name": "Jane S.", "clpid": "Robinson-J-S" }, { "family_name": "Graham", "given_name": "Todd R.", "clpid": "Graham-T-R" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" } ], "abstract": "Saccharomyces cerevisiae strains carrying vps18 mutations are defective in the sorting and transport of vacuolar enzymes. The precursor forms of these proteins are missorted and secreted from the mutant cells. Most vps18 mutants are temperature sensitive for growth and are defective in vacuole biogenesis; no structure resembling a normal vacuole is seen. A plasmid complementing the temperature-sensitive growth defect of strains carrying the vps18-4 allele was isolated from a centromere-based yeast genomic library. Integrative mapping experiments indicated that the 26-kb insert in this plasmid was derived from the VPS18 locus. A 4-kb minimal complementing fragment contains a single long open reading frame predicted to encode a 918-amino-acid hydrophilic protein. Comparison of the VPS18 sequence with the PEP3 sequence reported in the accompanying paper (R. A. Preston, H. F. Manolson, K. Becherer, E. Weidenhammer, D. Kirkpatrick, R. Wright, and E. W. Jones, Mol. Cell. Biol. 11:5801-5812, 1991) shows that the two genes are identical. Disruption of the VPS18/PEP3 gene (vps18 delta 1::TRP1) is not lethal but results in the same vacuolar protein sorting and growth defects exhibited by the original temperature-sensitive vps18 alleles. In addition, vps18 delta 1::TRP1 MAT alpha strains exhibit a defect in the Kex2p-dependent processing of the secreted pheromone alpha-factor. This finding suggests that vps18 mutations alter the function of a late Golgi compartment which contains Kex2p and in which vacuolar proteins are thought to be sorted from proteins destined for the cell surface. The Vps18p sequence contains a cysteine-rich, zinc finger-like motif at the COOH terminus. A mutant in which the first cysteine of this motif was changed to serine results in a temperature-conditional carboxypeptidase Y sorting defect shortly after a shift to nonpermissive conditions. We identified a similar cysteine-rich motif near the COOH terminus of another Vps protein, the Vps11/Pep5/End1 protein. Preston et al. (Mol. Cell. Biol. 11:5801-5812, 1991) present evidence that the Vps18/Pep3 protein colocalizes with the Vps11/Pep5 protein to the cytosolic face of the vacuolar membrane. Together with the similar phenotypes exhibited by both vps11 and vps18 mutants, this finding suggests that they may function at a common step during vacuolar protein sorting and that the integrity of their zinc finger motifs may be required for this function.", "issn": "0270-7306", "publisher": "Molecular and Cellular Biology", "publication": "Molecular and Cellular Biology", "publication_date": "1991-12-01", "series_number": "12", "volume": "11", "issue": "12", "pages": "5813-5824" }, { "id": "authors:ecyay-fqf92", "collection": "authors", "collection_id": "ecyay-fqf92", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:GRAjcb91", "type": "article", "title": "Compartmental organization of Golgi-specific protein modification and vacuolar protein sorting events defined in a yeast sec18 (NSF) mutant", "author": [ { "family_name": "Graham", "given_name": "Todd R.", "clpid": "Graham-T-R" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "abstract": "The sec18 and sec23 secretory mutants of Saccharomyces cerevisiae have previously been shown to exhibit temperature-conditional defects in protein transport from the ER to the Golgi complex (Novick, P., S. Ferro, and R. Schekman, 1981. Cell. 25:461-469). We have found that the Sec18 and Sec23 protein functions are rapidly inactivated upon shifting mutant cells to the nonpermissive temperature (less than 1 min). This has permitted an analysis of the potential role these SEC gene products play in transport events distal to the ER. The sec-dependent transport of alpha-factor (alpha f) and carboxypeptidase Y (CPY) biosynthetic intermediates present throughout the secretory pathway was monitored in temperature shift experiments. We found that Sec18p/NSF function was required sequentially for protein transport from the ER to the Golgi complex, through multiple Golgi compartments and from the Golgi complex to the cell surface. In contrast, Sec23p function was required in the Golgi complex, but only for transport of alpha f out of an early compartment. Together, these studies define at least three functionally distinct Golgi compartments in yeast. From cis to trans these compartments contain: (a) An alpha 1\u21926 mannosyltransferase; (b) an alpha 1\u21923 mannosyltransferase; and (c) the Kex2 endopeptidase. \n\nSurprisingly, we also found that a pool of Golgi-modified CPY (p2 CPY) located in a compartment distal to the alpha 1\u21923 mannosyltransferase does not require Sec18p function for final delivery to the vacuole. This compartment appears to be equivalent to the Kex2 compartment as we show that a novel vacuolar CPY-alpha f-invertase fusion protein undergoes efficient Kex2-dependent cleavage resulting in the secretion of invertase. We propose that this Kex2 compartment is the site in which vacuolar proteins are sorted from proteins destined to be secreted.", "doi": "10.1083/jcb.114.2.207", "pmcid": "PMC2289075", "issn": "0021-9525", "publisher": "Rockefeller University Press", "publication": "Journal of Cell Biology", "publication_date": "1991-07-15", "series_number": "2", "volume": "114", "issue": "2", "pages": "207-218" }, { "id": "authors:0nwfq-ndq66", "collection": "authors", "collection_id": "0nwfq-ndq66", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:HERmcb90", "type": "article", "title": "Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae", "author": [ { "family_name": "Herman", "given_name": "Paul K.", "clpid": "Herman-P-K" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" } ], "abstract": "VPS34 gene function is required for the efficient localization of a variety of vacuolar proteins. We have cloned and sequenced the wild-type VPS34 gene in order to gain a better understanding of the role of its protein product in this intracellular sorting pathway. Interestingly, disruption of the VPS34 locus resulted in a temperature-sensitive growth defect, indicating that the VPS34 gene is essential for vegetative growth only at elevated growth temperatures. As with the original vps34 alleles, vps34 null mutants exhibited severe vacuolar protein sorting defects and possessed a morphologically normal vacuolar structure. The VPS34 gene DNA sequence identifies an open reading frame that could encode a hydrophilic protein of 875 amino acids. The predicted protein sequence lacks any apparent signal sequence or membrane-spanning domains, suggesting that Vps34p does not enter the secretory pathway. Results from immunoprecipitation experiments with antiserum prepared against a TrpE-Vps34 fusion protein were consistent with this prediction: a rare, unglycosylated protein of approximately 95,000 Da was detected in extracts of wild-type Saccharomyces cerevisiae cells. Cell fractionation studies indicated that a significant portion of the Vps34p is found associated with a particulate fraction of yeast cells. This particulate Vps34p was readily solubilized by treatment with 2 M urea but not with Triton X-100, suggesting that the presence of Vps34p in this pelletable structure is mediated by protein-protein interactions. vp34 mutant cells also exhibited a defect in the normal partitioning of the vacuolar compartment between mother and daughter cells during cell division. In more than 80% of the delta vps34 dividing cells examined, no vacuolar structures were observed in the newly emerging bud, whereas in wild-type dividing cells, more than 95% of the buds had a detectable vacuolar compartment. Our results suggest that the Vps34p may act as a component of a relatively large intracellular structure that functions to facilitate specific steps of the vacuolar protein delivery and inheritance pathways.", "issn": "0270-7306", "publisher": "Molecular and Cellular Biology", "publication": "Molecular and Cellular Biology", "publication_date": "1990-12-01", "series_number": "12", "volume": "10", "issue": "12", "pages": "6742-6754" }, { "id": "authors:tz32n-h3v51", "collection": "authors", "collection_id": "tz32n-h3v51", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:VIDjcb90", "type": "article", "title": "In vitro reconstitution of intercompartmental protein transport to the yeast vacuole", "author": [ { "family_name": "Vida", "given_name": "Thomas A.", "clpid": "Vida-T-A" }, { "family_name": "Graham", "given_name": "Todd R.", "clpid": "Graham-T-R" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "abstract": "Toward a detailed understanding of protein sorting in the late secretory pathway, we have reconstituted intercompartmental transfer and proteolytic maturation of a yeast vacuolar protease, carboxypeptidase Y (CPY). This in vitro reconstitution uses permeabilized yeast spheroplasts that are first radiolabeled in vivo under conditions that kinetically trap ER and Golgi apparatus-modified precursor forms of CPY (p1 and p2, respectively). After incubation at 25 degrees C, up to 45% of the p2CPY that is retained in the perforated cells can be proteolytically converted to mature CPY (mCPY). This maturation is specific for p2CPY, requires exogenously added ATP, an ATP regeneration system, and is stimulated by cytosolic protein extracts. The p2CPY processing shows a 5-min lag period and is then linear for 15-60 min, with a sharp temperature optimum of 25-30 degrees C. After hypotonic extraction, the compartments that contain p2 and mCPY show different osmotic stability characteristics as p2 and mCPY can be separated with centrifugation into a pellet and supernatant, respectively. Like CPY maturation in vivo, the observed in vitro reaction is dependent on the PEP4 gene product, proteinase A, which is the principle processing enzyme. After incubation with ATP and cytosol, mCPY was recovered in a vacuole-enriched fraction from perforated spheroplasts using Ficoll step-gradient centrifugation. The p2CPY precursor was not recovered in this fraction indicating that intercompartmental transport to the vacuole takes place. In addition, intracompartmental processing of p2CPY with autoactivated, prevacuolar zymogen pools of proteinase A cannot account for this reconstitution. Stimulation of in vitro processing with energy and cytosol took place efficiently when the expression of PEP4, under control of the GAL1 promoter, was induced then completely repressed before radiolabeling spheroplasts. Finally, reconstitution of p2CPY maturation was not possible with vps mutant perforated cells suggesting that VPS gene product function is necessary for intercompartmental transport to the vacuole in vitro.", "doi": "10.1083/jcb.111.6.2871", "pmcid": "PMC2116412", "issn": "0021-9525", "publisher": "Rockefeller University Press", "publication": "Journal of Cell Biology", "publication_date": "1990-12", "series_number": "6, Pt2", "volume": "111", "issue": "6, Pt2", "pages": "2871-2884" }, { "id": "authors:f8wzk-n4m83", "collection": "authors", "collection_id": "f8wzk-n4m83", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:BANmcb90", "type": "article", "title": "Characterization of yeast Vps33p, a protein required for vacuolar protein sorting and vacuole biogenesis", "author": [ { "family_name": "Banta", "given_name": "Lois M.", "clpid": "Banta-L-M" }, { "family_name": "Vida", "given_name": "Thomas A.", "clpid": "Vida-T-A" }, { "family_name": "Herman", "given_name": "Paul K.", "clpid": "Herman-P-K" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" } ], "abstract": "vps33 mutants missort and secrete multiple vacuolar hydrolases and exhibit extreme defects in vacuolar morphology. Toward a molecular understanding of the role of the VPS33 gene in vacuole biogenesis, we have cloned this gene from a yeast genomic library by complementation of a temperature-sensitive vps33 mutation. Gene disruption demonstrated that VPS33 was not essential but was required for growth at high temperatures. At the permissive temperature, vps33 null mutants exhibited defects in vacuolar protein localization and vacuole morphology similar to those seen in most of the original mutant alleles. Sequence analysis revealed a putative open reading frame sufficient to encode a protein of 691 amino acids. Hydropathy analysis indicated that the deduced product of the VPS33 gene is generally hydrophilic, contains no obvious signal sequence or transmembrane domains, and is therefore unlikely to enter the secretory pathway. Polyclonal antisera raised against TrpE-Vps33 fusion proteins recognized a protein in yeast cells of the expected molecular weight, approximately 75,000. In cell fractionation studies, Vps33p behaved as a cytosolic protein. The predicted VPS33 gene product possessed sequence similarity with a number of ATPases and ATP-binding proteins specifically in their ATP-binding domains. One vps33 temperature-sensitive mutant contained a missense mutation near this region of sequence similarity; the mutation resulted in a Leu-646----Pro substitution in Vps33p. This temperature-sensitive mutant strain contained normal vacuoles at the permissive temperature but lacked vacuoles specifically in the bud at the nonpermissive temperature. Our data suggest that Vps33p acts in the cytoplasm to facilitate Golgi-to-vacuole protein delivery. We propose that as a consequence of the vps33 protein-sorting defects, abnormalities in vacuolar morphology and vacuole assembly result.", "issn": "0270-7306", "publisher": "Molecular and Cellular Biology", "publication": "Molecular and Cellular Biology", "publication_date": "1990-09-01", "series_number": "9", "volume": "10", "issue": "9", "pages": "4638-4649" }, { "id": "authors:etr55-pcj39", "collection": "authors", "collection_id": "etr55-pcj39", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:WEIpnas90", "type": "article", "title": "Mutants of Saccharomyces cerevisiae that Block Intervacuole Vesicular Traffic and Vacuole Division and Segregation", "author": [ { "family_name": "Weisman", "given_name": "Lois S.", "clpid": "Weisman-L-S" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" }, { "family_name": "Wickner", "given_name": "William T.", "clpid": "Wickner-W-T" } ], "abstract": "Intervacuole vesicular exchange and the segregation of parental vacuole material into the bud are strikingly impaired in a temperature-sensitive yeast mutant, vac1-1. At the nonpermissive temperature, haploid vac1-1 cells show a pronounced delay in separation of mature buds from the mother cell and accumulate cells with multiple buds. At both the permissive and restrictive temperatures, daughter cells are produced that lack a detectable vacuole or contain a very small vacuole. In zygotes, vacuoles from a vac1-1 strain are defective as donors, or recipients, of the vesicles of intervacuole vesicular traffic. These defects are specific for the vacuole in that the segregation of nuclear DNA and of mitochondria into the bud appears normal. The isolation of the vac1-1 mutation is a first step in the genetic characterization of vacuole division and segregation.", "doi": "10.1073/pnas.87.3.1076", "pmcid": "PMC53413", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1990-02-01", "series_number": "3", "volume": "87", "issue": "3", "pages": "1076-1080" }, { "id": "authors:jve45-wy585", "collection": "authors", "collection_id": "jve45-wy585", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120510-114856892", "type": "article", "title": "The Saccharomyces cerevisiae SEC14 Gene Encodes a Cytosolic Factor That Is Required for Transport of Secretory Proteins from the Yeast Golgi Complex", "author": [ { "family_name": "Bankaitis", "given_name": "Vytas A.", "clpid": "Bankaitis-V-A" }, { "family_name": "Malehorn", "given_name": "David E.", "clpid": "Malehorn-D-E" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" }, { "family_name": "Greene", "given_name": "Robert", "clpid": "Greene-R" } ], "abstract": "We have obtained and characterized a genomic clone of SEC14, a Saccharomyces cerevisiae gene whose product is required for export of yeast secretory proteins from the Golgi complex. Gene disruption experiments indicated that SEC14 is an essential gene for yeast vegetative growth. Nucleotide sequence analysis revealed the presence of an intron within the SEC14 structural gene, and predicted the synthesis of a hydrophilic polypeptide of 35 kD in molecular mass. In confirmation, immunoprecipitation experiments demonstrated SEC14p to be an unglycosylated polypeptide, with an apparent molecular mass of some 37 kD, that behaved predominantly as a cytosolic protein in subcellular fractionation experiments. These data were consistent with the notion that SEC14p is a cytosolic factor that promotes protein export from yeast Golgi. Additional radiolabeling experiments also revealed the presence of SEC14p-related polypeptides in extracts prepared from the yeasts Kluyveromyces lactis and Schizosaccharomyces pombe. Furthermore, the K. lactis SEC14p was able to functionally complement S. cerevisiae sec14ts defects. These data suggested a degree of conservation of SEC14p structure and function in these yeasts species.", "doi": "10.1083/jcb.108.4.1271", "pmcid": "PMC2115512", "issn": "0021-9525", "publisher": "Rockefeller University Press", "publication": "Journal of Cell Biology", "publication_date": "1989-04", "series_number": "4", "volume": "108", "issue": "4", "pages": "1271-1281" }, { "id": "authors:0z0gf-cd955", "collection": "authors", "collection_id": "0z0gf-cd955", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:BEDmcb89", "type": "article", "title": "Sequence and structural requirements of a mitochondrial protein import signal defined by saturation cassette mutagenesis", "author": [ { "family_name": "Bedwell", "given_name": "David M.", "clpid": "Bedwell-D-M" }, { "family_name": "Strobel", "given_name": "Scott A.", "clpid": "Strobel-S-A" }, { "family_name": "Yun", "given_name": "Kyuson", "clpid": "Yun-Kyuson" }, { "family_name": "Jongeward", "given_name": "Gregg D.", "clpid": "Jongeward-G-D" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" } ], "abstract": "The Saccharomyces cerevisiae F1-ATPase beta subunit precursor contains redundant mitochondrial protein import information at its NH2 terminus (D. M. Bedwell, D. J. Klionsky, and S. D. Emr, Mol. Cell. Biol. 7:4038-4047, 1987). To define the critical sequence and structural features contained within this topogenic signal, one of the redundant regions (representing a minimal targeting sequence) was subjected to saturation cassette mutagenesis. Each of 97 different mutant oligonucleotide isolates containing single (32 isolates), double (45 isolates), or triple (20 isolates) point mutations was inserted in front of a beta-subunit gene lacking the coding sequence for its normal import signal (codons 1 through 34 were deleted). The phenotypic and biochemical consequences of these mutations were then evaluated in a yeast strain deleted for its normal beta-subunit gene (delta atp2). Consistent with the lack of an obvious consensus sequence for mitochondrial protein import signals, many mutations occurring throughout the minimal targeting sequence did not significantly affect its import competence. However, some mutations did result in severe import defects. In these mutants, beta-subunit precursor accumulated in the cytoplasm, and the yeast cells exhibited a respiration defective phenotype. Although point mutations have previously been identified that block mitochondrial protein import in vitro, a subset of the mutations reported here represents the first single missense mutations that have been demonstrated to significantly block mitochondrial protein import in vivo. The previous lack of such mutations in the beta-subunit precursor apparently relates to the presence of redundant import information in this import signal. Together, our mutants define a set of constraints that appear to be critical for normal activity of this (and possibly other) import signals. These include the following: (i) mutant signals that exhibit a hydrophobic moment greater than 5.5 for the predicted amphiphilic alpha-helical conformation of this sequence direct near normal levels of beta-subunit import (ii) at least two basic residues are necessary for efficient signal function, (iii) acidic amino acids actively interfere with import competence, and (iv) helix-destabilizing residues also interfere with signal function. These experimental observations provide support for mitochondrial protein import models in which both the structure and charge of the import signal play a critical role in directing mitochondrial protein targeting and import.", "pmcid": "PMC362691", "issn": "0270-7306", "publisher": "American Society for Microbiology", "publication": "Molecular and Cellular Biology", "publication_date": "1989-03", "series_number": "3", "volume": "9", "issue": "3", "pages": "1014-1025" }, { "id": "authors:7mkt8-61754", "collection": "authors", "collection_id": "7mkt8-61754", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:ROBmcb88", "type": "article", "title": "Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases", "author": [ { "family_name": "Robinson", "given_name": "Jane S.", "clpid": "Robinson-J-S" }, { "family_name": "Klionsky", "given_name": "Daniel J.", "clpid": "Klionsky-D-J" }, { "family_name": "Banta", "given_name": "Lois M.", "clpid": "Banta-L-M" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" } ], "abstract": "Using a selection for spontaneous mutants that mislocalize a vacuolar carboxypeptidase Y (CPY)-invertase fusion protein to the cell surface, we identified vacuolar protein targeting (vpt) mutants in 25 new vpt complementation groups. Additional alleles in each of the eight previously identified vpt complementation groups (vpt1 through vpt8) were also obtained. Representative alleles from each of the 33 vpt complementation groups (vpt1 through vpt33) were shown to exhibit defects in the sorting and processing of several native vacuolar proteins, including the soluble hydrolases CPY, proteinase A, and proteinase B. Of the 33 complementation groups, 19 were found to contain mutant alleles that led to extreme defects. In these mutants, CPY accumulated in its Golgi complex-modified precursor form which was secreted by the mutant cells. Normal protein secretion appeared to be unaffected in the vpt mutants. The lack of significant leakage of cytosolic markers from the vpt mutant cells indicated that the vacuolar protein-sorting defects associated with these mutants do not result from cell lysis. In addition, the observation that the precursor rather than the mature forms of CPY, proteinase A, proteinase B were secreted from the vpt mutants was consistent with the fact that mislocalization occurred at a stage after Golgi complex-specific modification, but before final vacuolar sorting of these enzymes. Vacuolar membrane protein sorting appeared to be unaffected in the majority of the vpt mutants. However, a subset of the vpt mutants (vpt11, vpt16, vpt18, and vpt33) was found to exhibit defects in the sorting of a vacuolar membrane marker enzyme, alpha-mannosidase. Up to 50% of the alpha-mannosidase enzyme activity was found to be mislocalized to the cell surface in these vpt mutants. Seven of the vpt complementation groups (vpt3, vpt11, vpt15, vpt16, vpt18, vpt29, and vpt33) contained alleles that led to a conditional lethal phenotype; the mutants were temperature sensitive for vegetative cell growth. This temperature-sensitive phenotype has been shown to be recessive and to cosegregate with the vacuolar protein-sorting defect in each case. Tetrad analysis showed that vpt3 mapped to the right arm of chromosome XV and that vpt15 mapped to the right arm of chromosome II. Intercrosses with other mutants that exhibited defects in vacuolar protein sorting or function (vpl, sec, pep, and end mutants) revealed several overlaps among these different sets of genes. Together, these data indicate that more than 50 gene products are involved, directly or indirectly, in the process of vacuolar protein sorting.", "issn": "0270-7306", "publisher": "Molecular and Cellular Biology", "publication": "Molecular and Cellular Biology", "publication_date": "1988-11-01", "series_number": "11", "volume": "8", "issue": "11", "pages": "4936-4948" }, { "id": "authors:br7vh-tme48", "collection": "authors", "collection_id": "br7vh-tme48", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:EAKmcb88", "type": "article", "title": "Characterization of a component of the yeast secretion machinery: identification of the SEC18 gene product", "author": [ { "family_name": "Eakle", "given_name": "Kurt A.", "clpid": "Eakle-K-A" }, { "family_name": "Bernstein", "given_name": "Mitchell", "clpid": "Bernstein-M" }, { "family_name": "Emr", "given_name": "S.D.", "clpid": "Emr-S-D" } ], "abstract": "SEC18 gene function is required for secretory protein transport between the endoplasmic reticulum (ER) and the Golgi complex. We cloned the SEC18 gene by complementation of the sec18-1 mutation. Gene disruption has shown that SEC18 is essential for yeast cell growth. Sequence analysis of the gene revealed a 2,271-base-pair open reading frame which could code for a protein of 83.9 kilodaltons. The predicted protein sequence showed no significant similarity to other known protein sequences. In vitro transcription and translation of SEC18 led to the synthesis of two proteins of approximately 84 and 82 kilodaltons. Antisera raised against a Sec18-beta-galactosidase fusion protein also detected two proteins (collectively referred to as Sec18p) in extracts of 35S-labeled yeast cells identical in size to those seen by in vitro translation. Mapping of the 5' end of the SEC18 mRNA revealed only one major start site for transcription, which indicates that the multiple forms of Sec18p do not arise from mRNAs with different 5' ends. Results of pulse-chase experiments indicated that the two forms of Sec18p are not the result of posttranslational processing. We suggest that translation initiating at different in-frame AUG start codons is likely to account for the presence of two forms of Sec18p. Hydrophobicity analysis indicated that the proteins were hydrophilic in nature and lacked any region that would be predicted to serve as a signal sequence or transmembrane anchor. Although potential sites for N-linked glycosylation were present in the Sec18p sequence, the sizes of the in vivo SEC18 gene products were unaffected by the drug tunicamycin, indicating that Sec18p does not enter the secretory pathway. These results suggest that Sec18p resides in the cell cytoplasm. While preliminary cell fractionation studies showed that Sec18p is not associated with the ER or Golgi complex, association with a 100,000 x g pellet fraction was observed. This suggests that Sec18p may bind transiently to small vesicles such as those presumed to participate in secretory protein transport between ER and the Golgi complex.", "issn": "0270-7306", "publisher": "Molecular and Cellular Biology", "publication": "Molecular and Cellular Biology", "publication_date": "1988-10-01", "series_number": "10", "volume": "8", "issue": "10", "pages": "4098-4109" }, { "id": "authors:b4db3-1bf08", "collection": "authors", "collection_id": "b4db3-1bf08", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120531-141222457", "type": "article", "title": "Organelle Assembly in Yeast: Characterization of Yeast Mutants Defective in Vacuolar Biogenesis and Protein Sorting", "author": [ { "family_name": "Banta", "given_name": "Lois M.", "clpid": "Banta-L-M" }, { "family_name": "Robinson", "given_name": "Jane S.", "clpid": "Robinson-J-S" }, { "family_name": "Klionsky", "given_name": "Daniel J.", "clpid": "Klionsky-D-J" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" } ], "abstract": "Yeast vacuole protein targeting (vpt) mutants exhibit defects in the sorting and processing of multiple vacuolar hydrolases. To evaluate the impact these vpt mutations have on the biogenesis and functioning of the lysosome-like vacuole, we have used light and electron microscopic techniques to analyze the vacuolar morphology in the mutants. These observations have permitted us to assign the vpt mutants to three distinct classes. The class A vpt mutants (26 complementation groups) contain 1-3 large vacuoles that are morphologically indistinguishable from those in the parental strain, suggesting that only a subset of the proteins destined for delivery to this compartment is mislocalized. One class A mutant (vpt13) is very sensitive to low pH and exhibits a defect in vacuole acidification. Consistent with a potential role for vacuolar pH in protein sorting, we found that bafilomycin A1, a specific inhibitor of the vacuolar ATPase, as well as the weak base ammonium acetate and the proton ionophore carbonyl cyanide m-chlorophenylhydrazone, collapse the pH gradient across the vacuolar membrane and cause the missorting and secretion of two vacuolar hydrolases in wild-type cells. Mutants in the three class B vpt complementation groups exhibit a fragmented vacuole morphology. In these mutants, no large normal vacuoles are observed. Instead, many (20-40) smaller vacuole-like organelles accumulate. The class C vpt mutants, which constitute four complementation groups, exhibit extreme defects in vacuole biogenesis. The mutants lack any organelle resembling a normal vacuole but accumulate other organelles including vesicles, multilamellar membrane structures, and Golgi-related structures. Heterozygous class C zygotes reassemble normal vacuoles rapidly, indicating that some of the accumulated aberrant structures may be intermediates in vacuole formation. These class C mutants also exhibit sensitivity to osmotic stress, suggesting an osmoregulatory role for the vacuole. The vpt mutants should provide insights into the normal physiological role of the vacuole, as well as allowing identification of components required for vacuole protein sorting and/or vacuole assembly.", "doi": "10.1083/jcb.107.4.1369", "pmcid": "PMC2115260", "issn": "0021-9525", "publisher": "Rockefeller University Press", "publication": "Journal of Cell Biology", "publication_date": "1988-10-01", "series_number": "4", "volume": "107", "issue": "4", "pages": "1369-1383" }, { "id": "authors:na3t7-09223", "collection": "authors", "collection_id": "na3t7-09223", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:KLImcb88", "type": "article", "title": "Intracellular sorting and processing of a yeast vacuolar hydrolase: proteinase A propeptide contains vacuolar targeting information", "author": [ { "family_name": "Klionsky", "given_name": "Daniel J.", "clpid": "Klionsky-D-J" }, { "family_name": "Banta", "given_name": "Lois M.", "clpid": "Banta-L-M" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" } ], "abstract": "An inactive precursor form of proteinase A (PrA) transits through the early secretory pathway before final vacuolar delivery. We used gene fusions between the gene coding for PrA (PEP4) and the gene coding for the secretory enzyme invertase (SUC2) to identify vacuolar protein-sorting information in the PrA precursor. We found that the 76-amino-acid preprosegment of PrA contains at least two sorting signals: an amino-terminal signal peptide that is cleaved from the protein at the level of the endoplasmic reticulum followed by the prosegment which functions as a vacuolar protein-sorting signal. PrA-invertase hybrid proteins that carried this sequence information were accurately sorted to the yeast vacuole as determined by cell fractionation and immunolocalization studies. Hybrid proteins lacking all or a portion of the PrA prosegment were secreted from the cell. Our gene fusion data together with an analysis of the wild-type PrA protein indicated that N-linked carbohydrate modifications are not required for vacuolar sorting of this protein. Furthermore, results obtained with a set of deletion mutations constructed in the PrA prosegment indicated that this sequence also contributes to proper folding of this polypeptide into a stable transit-competent molecule.", "issn": "0270-7306", "publisher": "Molecular and Cellular Biology", "publication": "Molecular and Cellular Biology", "publication_date": "1988-05-01", "series_number": "5", "volume": "8", "issue": "5", "pages": "2105-2116" }, { "id": "authors:8ryqf-9gg79", "collection": "authors", "collection_id": "8ryqf-9gg79", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:BEDmcb87", "type": "article", "title": "The yeast F1-ATPase beta subunit precursor contains functionally redundant mitochondrial protein import information", "author": [ { "family_name": "Bedwell", "given_name": "David M.", "clpid": "Bedwell-D-M" }, { "family_name": "Klionsky", "given_name": "Daniel J.", "clpid": "Klionsky-D-J" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" } ], "abstract": "The NH2 terminus of the yeast F1-ATPase beta subunit precursor directs the import of this protein into mitochondria. To define the functionally important components of this import signal, oligonucleotide-directed mutagenesis was used to introduce a series of deletion and missense mutations into the gene encoding the F1-beta subunit precursor. Among these mutations were three nonoverlapping deletions, two within the 19-amino-acid presequence (delta 5-12 and delta 16-19) and one within the mature protein (delta 28-34). Characterization of the mitochondrial import properties of various mutant F1-beta subunit proteins containing different combinations of these deletions showed that import was blocked only when all three deletions were combined. Mutant proteins containing all possible single and pairwise combinations of these deletions were found to retain the ability to direct mitochondrial import of the F1-beta subunit. These data suggest that the F1-beta subunit contains redundant import information at its NH2 terminus. In fact, we found that deletion of the entire F1-beta subunit presequence did not prevent import, indicating that a functional mitochondrial import signal is present near the NH2 terminus of the mature protein. Furthermore, by analyzing mitochondrial import of the various mutant proteins in [rho-] yeast, we obtained evidence that different segments of the F1-beta subunit import signal may act in an additive or cooperative manner to optimize the import properties of this protein.", "issn": "0270-7306", "publisher": "Molecular and Cellular Biology", "publication": "Molecular and Cellular Biology", "publication_date": "1987-11-01", "series_number": "11", "volume": "7", "issue": "11", "pages": "4038-4047" }, { "id": "authors:8d0h1-w3886", "collection": "authors", "collection_id": "8d0h1-w3886", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150127-091612638", "type": "article", "title": "Isolation of yeast mutants defective in protein targeting to the vacuole", "author": [ { "family_name": "Bankaitis", "given_name": "Vytas A.", "orcid": "0000-0002-1654-6759", "clpid": "Bankaitis-Vytas-A" }, { "family_name": "Johnson", "given_name": "Lianna M.", "orcid": "0000-0002-0396-8426", "clpid": "Johnson-Lianna-M" }, { "family_name": "Emr", "given_name": "Scott D.", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" } ], "abstract": "We have constructed a PRC1-SUC2 gene fusion that directs the synthesis in Saccharomyces cerevisiae of a hybrid polypeptide consisting of a 433-residue amino-terminal domain derived from the yeast vacuolar protease carboxypeptidase Y (CPY; EC 3.4.16.1) and a 511-residue carboxyl-terminal domain derived from the secreted yeast enzyme invertase (EC 3.2.1.26). Fractionation data indicated that this amount of CPY primary sequence is sufficient to quantitatively divert invertase to the yeast vacuole. The phenotypic consequence of localizing active invertase to the vacuole has enabled us to select for mutants that \"mislocalize\" the hybrid protein to the cell surface. The corresponding mutations that lead to this effect are all trans-acting and recessive, and they define at least eight complementation groups. These vacuolar protein targeting (vpt) mutants also exhibit hybrid protein independent defects in wild-type CPY delivery to the yeast vacuole. Precursor forms of CPY accumulate in the mutants and are secreted into the yeast periplasm and extracellular medium. The vpt mutants should provide useful information pertaining to the mechanisms by which yeast cells regulate vacuolar protein traffic.", "doi": "10.1073/pnas.83.23.9075", "pmcid": "PMC387077", "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": "9075-9079" }, { "id": "authors:vrx7y-16804", "collection": "authors", "collection_id": "vrx7y-16804", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120627-080630340", "type": "article", "title": "The Amino Terminus of the Yeast F_1-ATPase \u03b2-Subunit\n Precursor Functions as a Mitochondrial Import Signal", "author": [ { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" }, { "family_name": "Vassarotti", "given_name": "Alessio", "clpid": "Vassarotti-A" }, { "family_name": "Garrett", "given_name": "Jinnie", "clpid": "Garrett-J" }, { "family_name": "Geller", "given_name": "Bruce L.", "clpid": "Geller-B-L" }, { "family_name": "Takeda", "given_name": "Masaharu", "clpid": "Takeda-M" }, { "family_name": "Douglas", "given_name": "Michael G.", "clpid": "Douglas-M-G" } ], "abstract": "The ATP2 gene of Saccharomyces cerevisiae codes for the cytoplasmically synthesized beta-subunit protein of the mitochondrial F1-ATPase. To define the amino acid sequence determinants necessary for the in vivo targeting and import of this protein into mitochondria, we have constructed gene fusions between the ATP2 gene and either the Escherichia coli lacZ gene or the S. cerevisiae SUC2 gene (which codes for invertase). The ATP2-lacZ and ATP2-SUC2 gene fusions code for hybrid proteins that are efficiently targeted to yeast mitochondria in vivo. The mitochondrially associated hybrid proteins fractionate with the inner mitochondrial membrane and are resistant to proteinase digestion in the isolated organelle. Results obtained with the gene fusions and with targeting-defective ATP2 deletion mutants provide evidence that the amino-terminal 27 amino acids of the beta-subunit protein precursor are sufficient to direct both specific sorting of this protein to yeast mitochondria and its import into the organelle. Also, we have observed that certain of the mitochondrially associated Atp2-LacZ and Atp2-Suc2 hybrid proteins confer a novel respiration-defective phenotype to yeast cells.", "doi": "10.1083/jcb.102.2.523", "pmcid": "PMC2114072", "issn": "0021-9525", "publisher": "Rockefeller University Press", "publication": "Journal of Cell Biology", "publication_date": "1986-02-01", "series_number": "2", "volume": "102", "issue": "2", "pages": "523-533" }, { "id": "authors:j3wds-j3h73", "collection": "authors", "collection_id": "j3wds-j3h73", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:SCHAjcb85", "type": "article", "title": "Invertase signal and mature sequence substitutions that delay intercompartmental transport of active enzyme", "author": [ { "family_name": "Schauer", "given_name": "Irene", "clpid": "Schauer-I" }, { "family_name": "Emr", "given_name": "Scott", "orcid": "0000-0002-5408-6781", "clpid": "Emr-S-D" }, { "family_name": "Gross", "given_name": "Coleman", "clpid": "Gross-C" }, { "family_name": "Schekman", "given_name": "Randy", "clpid": "Schekman-R" } ], "abstract": "The role of structural signals in intercompartmental transport has been addressed by the isolation of yeast invertase (SUC2) mutations that cause intracellular accumulation of active enzyme. Two mutations that delay transport of core-glycosylated invertase, but not acid phosphatase, have been mapped in the 5' coding region of SUC2. Both mutations reduce specifically the transport of invertase to a compartment, presumably in the Golgi body, where outer chain carbohydrate is added. Subsequent transport to the cell surface is not similarly delayed. One mutation (SUC2-s1) converts an ala codon to val at position -1 in the signal peptide; the other (SUC2-s2) changes a thr to an ile at position +64 in the mature protein. Mutation s1 results in about a 50-fold reduced rate of invertase transport to the Golgi body which is attributable to defective signal peptide cleavage. While peptide cleavage normally occurs at an ala-ser bond, the s1 mutant form is processed slowly at the adjacent ser-met position giving rise to mature invertase with an N-terminal met residue. s2 mutant invertase is transported about sevenfold more slowly than normal, with no delay in signal peptide cleavage, and no detectable abnormal physical property of the enzyme. This substitution may interfere with the interaction of invertase and a receptor that facilitates transport to the Golgi body.", "doi": "10.1083/jcb.100.5.1664", "pmcid": "PMC2113855", "issn": "0021-9525", "publisher": "Rockefeller University Press", "publication": "Journal of Cell Biology", "publication_date": "1985-05", "series_number": "5", "volume": "100", "issue": "5", "pages": "1664-1675" }, { "id": "authors:bagqa-3dr21", "collection": "authors", "collection_id": "bagqa-3dr21", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:DOUpnas84", "type": "article", "title": "Intracellular Targeting and Import of an F1-ATPase \u03b2-subunit-\u03b2-galactosidase Hybrid Protein into Yeast Mitochondria", "author": [ { "family_name": "Douglas", "given_name": "Michael G.", "clpid": "Douglas-M-G" }, { "family_name": "Geller", "given_name": "Bruce L.", "clpid": "Geller-B-L" }, { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" } ], "abstract": "The gene coding for the yeast mitochondrial F1-ATPase \u03b2 subunit (ATP2) has been fused to the Escherichia coli lacZ gene. The chimeric ATP2-lacZ gene codes for a hybrid protein consisting of some 350 amino acids of the F1-ATPase \u03b2 subunit at its amino terminus and a large enzymatically active portion of the lacZ gene product, \u03b2-galactosidase (\u03b2-D-galactoside galactohydrolase, EC 3.2.1.23), at its carboxyl terminus. The \u03b2-subunit-\u03b2 -galactosidase hybrid protein is expressed in both E. coli and yeast. In yeast, this hybrid molecule is targeted to the mitochondrion and is protected in isolated mitochondria from added protease under conditions in which an outer membrane enzymatic marker is digested. Yeast cells carrying the ATP2-lacZ gene fusion on plasmid p\u03b2 Z1 are unable to grow on a nonfermentable carbon source. Upon loss of the p\u03b2 Z1 plasmid, growth of the cured host strain on the nonfermentable substrate is restored. In the presence of the \u03b2-subunit-\u03b2-galactosidase hybrid protein, the energy-transducing capacity of the mitochondrial membrane as measured by the 32Pi-ATP exchange reaction is only 9% of that measured in the absence of the gene fusion product. The results indicate that it is the presence of the \u03b2-subunit-\u03b2-galactosidase hybrid protein within mitochondria that interferes with function(s) essential for respiratory growth. These observations open up the prospect of genetic characterization of the signals and cellular machinery responsible for mitochondrial protein delivery.", "doi": "10.1073/pnas.81.13.3983", "pmcid": "PMC345352", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1984-07-01", "series_number": "13", "volume": "81", "issue": "13", "pages": "3983-3987" }, { "id": "authors:mb91s-0hn39", "collection": "authors", "collection_id": "mb91s-0hn39", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120709-104828314", "type": "article", "title": "An MF \u03b11-SUC2 (\u03b1-factor-invertase) gene fusion for study of protein localization and gene expression in yeast", "author": [ { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" }, { "family_name": "Schekman", "given_name": "Randy", "clpid": "Schekman-R" }, { "family_name": "Flessel", "given_name": "Monica C.", "clpid": "Flessel-M-C" }, { "family_name": "Thorner", "given_name": "Jeremy", "clpid": "Thorner-J" } ], "abstract": "The peptide mating pheromone alpha-factor and the hydrolytic enzyme invertase (beta-D-fructofuranoside fructohydrolase, EC 3.2.1.26) are processed from larger precursor proteins during their secretion from yeast cells (Saccharomyces cerevisiae). An in-frame fusion of the structural genes for these two proteins was constructed by connecting the 5'-flanking region and prepro-leader portion of the coding sequence of the alpha-factor gene (MF alpha 1) to a large fragment of the invertase gene (SUC2) lacking its 5'-flanking region and the coding information for the first four amino acids of its signal sequence. Sites that have been implicated in normal proteolytic processing of the alpha-factor precursor have been retained in this construction. The chimeric gene directs synthesis of a high level of active invertase that is secreted efficiently into the periplasmic space, permitting cell growth on sucrose-containing media. This extracellular invertase appears to contain no prepro-alpha-factor sequences. The initial intracellular product is, however, a hybrid protein that can be detected either by treatment of the cells with the drug tunicamycin or by blockage of secretion in a temperature-conditional secretion-defective mutant (sec18). Therefore, prior to its efficient proteolytic removal, the alpha-factor portion of the hybrid protein apparently provides the necessary information for efficient export of the substantially larger protein invertase. Similar to MF alpha 1, the MF alpha 1-SUC2 fusion is expressed in alpha haploids at levels 65-75 times higher than in a haploids or in a/alpha diploids; also, high-level expression is eliminated in mat alpha 1 mutants but not in mat alpha 2 mutants. Unlike expression of SUC2, expression of the fusion is not affected by glucose concentration. Hence, the 5'-flanking region present in the fusion (about 950 base pairs) is sufficient to confer alpha cell-specific expression to the hybrid gene.", "doi": "10.1073/pnas.80.23.7080", "pmcid": "PMC389996", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1983-12-01", "series_number": "23", "volume": "80", "issue": "23", "pages": "7080-7084" }, { "id": "authors:fz8bw-yeb21", "collection": "authors", "collection_id": "fz8bw-yeb21", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:EMRpnas83a", "type": "article", "title": "Importance of secondary structure in the signal sequence for protein secretion", "author": [ { "family_name": "Emr", "given_name": "Scott D.", "clpid": "Emr-S-D" }, { "family_name": "Silhavy", "given_name": "Thomas J.", "clpid": "Silhavy-T-J" } ], "abstract": "Mutant Escherichia coli strains in which export of the LamB protein (coded for by the lamB gene) to the outer membrane of the cell is prevented have been described previously. One of these mutant strains contains a small (12-base pair) deletion mutation within the region of the lamB gene that codes for the NH2-terminal signal sequence. In this mutant strain, export but not synthesis of the LamB protein is blocked. We have isolated pseudorevertants that restore export of functional LamB protein to the outer membrane. DNA sequence analysis showed that two of the revertants contain a point mutation in addition to the original deletion. These point mutations lead to amino acid substitutions within the signal sequence. Our results indicate that these secondary mutations efficiently suppress the export defect caused by the deletion mutation. Analysis of the secondary structure of the wild-type, mutant, and pseudorevertant LamB signal sequences suggests that the secondary mutations restore export by allowing the formation of a stable \u03b1-helical conformation in the central, hydrophobic region of the signal sequence.", "doi": "10.1073/pnas.80.15.4599", "pmcid": "PMC384091", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1983-08-01", "series_number": "15", "volume": "80", "issue": "15", "pages": "4599-4603" } ]