[ { "id": "authors:e3mph-z5p90", "collection": "authors", "collection_id": "e3mph-z5p90", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150721-100512606", "type": "article", "title": "MitoTALENs: A general approach to reduce mutant mtDNA loads and restore oxidative phosphorylation function in mitochondrial diseases", "author": [ { "family_name": "Hashimoto", "given_name": "Masami", "clpid": "Hashimoto-Masami" }, { "family_name": "Bacman", "given_name": "Sandra R.", "clpid": "Bacman-S-R" }, { "family_name": "Peralta", "given_name": "Susana", "clpid": "Peralta-S" }, { "family_name": "Falk", "given_name": "Marni J.", "clpid": "Falk-M-J" }, { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" }, { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" }, { "family_name": "Williams", "given_name": "Sion L.", "clpid": "Williams-S-L" }, { "family_name": "Moraes", "given_name": "Carlos T.", "clpid": "Moraes-C-T" } ], "abstract": "We have designed mitochondrially targeted Transcription Activator-Like Effector Nucleases or mitoTALENs to cleave specific sequences in the mitochondrial DNA (mtDNA) with the goal of eliminating mtDNA carrying pathogenic point mutations. To test the generality of the approach we designed mitoTALENs to target two relatively common pathogenic mtDNA point mutations associated with mitochondrial diseases: the m.8344A>G tRNA^(Lys) gene mutation associated with Myoclonic Epilepsy with Ragged-Red Fibers (MERRF) and the m.13513G>A ND5 mutation associated with MELAS/Leigh Syndrome. Transmitochondrial cybrid cells harbouring the respective heteroplasmic mtDNA mutations were transfected with the respective mitoTALEN and analysed after different time periods. MitoTALENs efficiently reduced the levels of the targeted pathogenic mtDNAs in the respective cell lines. Functional assays showed that cells with heteroplasmic mutant mtDNA were able to recover respiratory capacity and oxidative phosphorylation enzymes activity after transfection with the mitoTALEN. To improve the design in the context of the low complexity of mtDNA, we designed shorter versions of the mitoTALEN specific for the MERRF m.8344A>G mutation. These shorter mitoTALENs also eliminated the mutant mtDNA. These reductions in size improve our ability to package these large sequences into viral vectors, bringing the use of these genetic tools closer to clinical trials.", "doi": "10.1038/mt.2015.126", "pmcid": "PMC4817924", "issn": "1525-0016", "publisher": "American Society of Gene & Cell Therapy", "publication": "Molecular Therapy", "publication_date": "2015-10", "series_number": "10", "volume": "23", "issue": "10", "pages": "1592-1599" }, { "id": "authors:efxc7-zn230", "collection": "authors", "collection_id": "efxc7-zn230", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20121218-075633343", "type": "article", "title": "Decreased Reactive Oxygen Species Production in Cells with Mitochondrial Haplogroups Associated with Longevity", "author": [ { "family_name": "Chen", "given_name": "Ai", "clpid": "Chen-Ai" }, { "family_name": "Raule", "given_name": "Nicola", "clpid": "Raule-N" }, { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" } ], "abstract": "Mitochondrial DNA (mtDNA) is highly polymorphic, and its variations in humans may contribute to individual differences in function. Zhang and colleagues found a strikingly higher frequency of a C150T transition in the D-loop of mtDNA from centenarians and twins of an Italian population, and also demonstrated that this base substitution causes a remodeling of the mtDNA 151 replication origin in human leukocytes and fibroblasts [1]. The C150T transition is a polymorphism associated with several haplogroups. To determine whether haplogroups that carry the C150T transition display any phenotype that may be advantageous for longevity, we analyzed cybrids carrying or not the C150T transition. These cybrids were obtained by fusing cytoplasts derived from human fibroblasts with human mtDNA-less cells (\u03c1^0 cells). We chose for cybrid construction and analysis haplogroup-matched pairs of fibroblast strains containing or not the C150T transition. In particular, we used, as one pair of mtDNA donors, a fibroblast strain of the U3a haplogroup, carrying the C150T transition and a strain of the U-K2 haplogroup, without the C150T transition, and as another pair, fibroblasts of the J2b haplogroup, carrying the C150T transition and of the J1c haplogroup, without the C150T transition. We have found no association of respiratory capacity, mtDNA level, mitochondrial gene expression level, or growth rate with the presence of the C150T transition. However, we have found that the cybrids with haplogroups that include the C150T transition have in common a lower reactive oxygen species (ROS) production rate than the haplogroup-matched cybrids without that transition. Thus, the lower ROS production rate may be a factor in the increased longevity associated with the U and the J2 haplogroups. Of further interest, we found that cybrids with the U3a haplogroup exhibited a higher respiration rate than the other cybrids examined.", "doi": "10.1371/journal.pone.0046473", "pmcid": "PMC3483264", "issn": "1932-6203", "publisher": "Public Library of Science", "publication": "PLoS ONE", "publication_date": "2012-10-29", "series_number": "10", "volume": "7", "issue": "10", "pages": "Art. No. e46473" }, { "id": "authors:jzxk7-fzs15", "collection": "authors", "collection_id": "jzxk7-fzs15", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20100512-141625107", "type": "article", "title": "Mitochondrial Fusion Is Required for mtDNA Stability in Skeletal Muscle and Tolerance of mtDNA Mutations", "author": [ { "family_name": "Chen", "given_name": "Hsiuchen", "clpid": "Chen-Hsiuchen" }, { "family_name": "Vermulst", "given_name": "Marc", "clpid": "Vermulst-M" }, { "family_name": "Wang", "given_name": "Yun E.", "clpid": "Wang-Yun-E" }, { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" }, { "family_name": "Prolla", "given_name": "Tomas A.", "clpid": "Prolla-T-A" }, { "family_name": "McCaffery", "given_name": "J. Michael", "clpid": "McCaffery-J-M" }, { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" } ], "abstract": "Mitochondria are highly mobile and dynamic organelles that continually fuse and divide. These processes allow mitochondria to exchange contents, including mitochondrial DNA (mtDNA). Here we examine the functions of mitochondrial fusion in differentiated skeletal muscle through conditional deletion of the mitofusins Mfn1 and Mfn2, mitochondrial GTPases essential for fusion. Loss of the mitofusins causes severe mitochondrial dysfunction, compensatory mitochondrial proliferation, and muscle atrophy. Mutant mice have severe mtDNA depletion in muscle that precedes physiological abnormalities. Moreover, the mitochondrial genomes of the mutant muscle rapidly accumulate point mutations and deletions. In a related experiment, we find that disruption of mitochondrial fusion strongly increases mitochondrial dysfunction and lethality in a mouse model with high levels of mtDNA mutations. With its dual function in safeguarding mtDNA integrity and preserving mtDNA function in the face of mutations, mitochondrial fusion is likely to be a protective factor in human disorders associated with mtDNA mutations.", "doi": "10.1016/j.cell.2010.02.026", "pmcid": "PMC2876819", "issn": "0092-8674", "publisher": "Elsevier", "publication": "Cell", "publication_date": "2010-04-16", "series_number": "2", "volume": "141", "issue": "2", "pages": "280-289" }, { "id": "authors:0kvf3-03408", "collection": "authors", "collection_id": "0kvf3-03408", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:HAJjbc07", "type": "article", "title": "Identification of a Novel Mitochondrial Complex Containing Mitofusin 2 and Stomatin-like Protein 2", "author": [ { "family_name": "H\u00e1jek", "given_name": "Petr", "clpid": "H\u00e1jek-P" }, { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" } ], "abstract": "A reverse genetics approach was utilized to discover new proteins that interact with the mitochondrial fusion mediator mitofusin 2 (Mfn2) and that may participate in mitochondrial fusion. In particular, in vivo formaldehyde cross-linking of whole HeLa cells and immunoprecipitation with purified Mfn2 antibodies of SDS cell lysates were used to detect an ~42-kDa protein. This protein was identified by liquid chromatography and tandem mass spectrometry as stomatin-like protein 2 (Stoml2), previously described as a peripheral plasma membrane protein of unknown function associated with the cytoskeleton of erythrocytes (Wang, Y., and Morrow, J. S. (2000) J. Biol. Chem. 275, 8062\u20138071). Immunoblot analysis with anti-Stoml2 antibodies showed that Stoml2 could be immunoprecipitated specifically with Mfn2 antibody either from formaldehyde-cross-linked and SDS-lysed cells or from cells lysed with digitonin. Subsequent immunocytochemistry and cell fractionation experiments fully supported the conclusion that Stoml2 is indeed a mitochondrial protein. Furthermore, demonstration of mitochondrial membrane potential-dependent import of Stoml2 accompanied by proteolytic processing, together with the results of sublocalization experiments, suggested that Stoml2 is associated with the inner mitochondrial membrane and faces the intermembrane space. Notably, formaldehyde cross-linking revealed a \"ladder\" of high molecular weight protein species, indicating the presence of high molecular weight Stoml2-Mfn2 hetero-oligomers. Knockdown of Stoml2 by the short interfering RNA approach showed a reduction of the mitochondrial membrane potential, without, however, any obvious changes in mitochondrial morphology.", "doi": "10.1074/jbc.M608168200", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "2007-02-23", "series_number": "8", "volume": "282", "issue": "8", "pages": "5670-5681" }, { "id": "authors:afwbk-8ng71", "collection": "authors", "collection_id": "afwbk-8ng71", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:DUVjbc06", "type": "article", "title": "Proteolytic Processing of OPA1 Links Mitochondrial Dysfunction to Alterations in Mitochondrial Morphology", "author": [ { "family_name": "Duvezin-Caubet", "given_name": "St\u00e9phane", "clpid": "Duvezin-Caubet-S" }, { "family_name": "Jagasia", "given_name": "Ravi", "clpid": "Jagasia-R" }, { "family_name": "Wagener", "given_name": "Johannes", "clpid": "Wagener-J" }, { "family_name": "Hofmann", "given_name": "Sabine", "clpid": "Hofmann-S" }, { "family_name": "Trifunovic", "given_name": "Aleksandra", "clpid": "Trifunovic-A" }, { "family_name": "Hansson", "given_name": "Anna", "clpid": "Hansson-A" }, { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" }, { "family_name": "Bauer", "given_name": "Matthis F.", "clpid": "Bauer-M-F" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Larsson", "given_name": "Nils-G\u00f6ran", "clpid": "Larsson-N-G" }, { "family_name": "Neupert", "given_name": "Walter", "clpid": "Neupert-W" }, { "family_name": "Reichert", "given_name": "Andrea S.", "clpid": "Reichert-A-S" } ], "abstract": "Many muscular and neurological disorders are associated with mitochondrial dysfunction and are often accompanied by changes in mitochondrial morphology. Mutations in the gene encoding OPA1, a protein required for fusion of mitochondria, are associated with hereditary autosomal dominant optic atrophy type I. Here we show that mitochondrial fragmentation correlates with processing of large isoforms of OPA1 in cybrid cells from a patient with myoclonus epilepsy and ragged-red fibers syndrome and in mouse embryonic fibroblasts harboring an error-prone mitochondrial mtDNA polymerase {gamma}. Furthermore, processed OPA1 was observed in heart tissue derived from heart-specific TFAM knock-out mice suffering from mitochondrial cardiomyopathy and in skeletal muscles from patients suffering from mitochondrial myopathies such as myopathy encephalopathy lactic acidosis and stroke-like episodes. Dissipation of the mitochondrial membrane potential leads to fast induction of proteolytic processing of OPA1 and concomitant fragmentation of mitochondria. Recovery of mitochondrial fusion depended on protein synthesis and was accompanied by resynthesis of large isoforms of OPA1. Fragmentation of mitochondria was prevented by overexpressing OPA1. Taken together, our data indicate that proteolytic processing of OPA1 has a key role in inducing fragmentation of energetically compromised mitochondria. We present the hypothesis that this pathway regulates mitochondrial morphology and serves as an early response to prevent fusion of dysfunctional mitochondria with the functional mitochondrial network.", "doi": "10.1074/jbc.M606059200", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "2006-12-08", "series_number": "49", "volume": "281", "issue": "49", "pages": "37972-37979" }, { "id": "authors:tzphd-1sv40", "collection": "authors", "collection_id": "tzphd-1sv40", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:CHEjbc05", "type": "article", "title": "Disruption of fusion results in mitochondrial heterogeneity and dysfunction", "author": [ { "family_name": "Chen", "given_name": "Hsiuchen", "clpid": "Chen-Hsiuchen" }, { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" }, { "family_name": "Chan", "given_name": "David C.", "orcid": "0000-0002-0191-2154", "clpid": "Chan-D-C" } ], "abstract": "Mitochondria undergo continual cycles of fusion and fission, and the balance of these opposing processes regulates mitochondrial morphology. Paradoxically, cells invest many resources to maintain tubular mitochondrial morphology, when reducing both fusion and fission simultaneously achieves the same end. This observation suggests a requirement for mitochondrial fusion, beyond maintenance of organelle morphology. Here, we show that cells with targeted null mutations in Mfn1 or Mfn2 retained low levels of mitochondrial fusion and escaped major cellular dysfunction. Analysis of these mutant cells showed that both homotypic and heterotypic interactions of Mfns are capable of fusion. In contrast, cells lacking both Mfn1 and Mfn2 completely lacked mitochondrial fusion and showed severe cellular defects, including poor cell growth, widespread heterogeneity of mitochondrial membrane potential, and decreased cellular respiration. Disruption of OPA1 by RNAi also blocked all mitochondrial fusion and resulted in similar cellular defects. These defects in Mfn-null or OPA1-RNAi mammalian cells were corrected upon restoration of mitochondrial fusion, unlike the irreversible defects found in fzo yeast. In contrast, fragmentation of mitochondria, without severe loss of fusion, did not result in such cellular defects. Our results showed that key cellular functions decline as mitochondrial fusion is progressively abrogated.", "doi": "10.1074/jbc.M503062200", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "2005-07-15", "series_number": "28", "volume": "280", "issue": "28", "pages": "26185-26192" }, { "id": "authors:94k6m-spm04", "collection": "authors", "collection_id": "94k6m-spm04", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:DUAjbc03", "type": "article", "title": "Mitochondrial Outer Membrane Permeability Change and Hypersensitivity to Digitonin Early in Staurosporine-induced Apoptosis", "author": [ { "family_name": "Duan", "given_name": "Shili", "clpid": "Duan-Shili" }, { "family_name": "H\u00e1jek", "given_name": "Petr", "clpid": "H\u00e1jek-P" }, { "family_name": "Lin", "given_name": "Catherine", "clpid": "Lin-Catherine-P" }, { "family_name": "Shin", "given_name": "Soo Kyung", "clpid": "Shin-Soo-Kyung" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" } ], "abstract": "We have shown here that the apoptosis inducer staurosporine causes an early decrease in the endogenous respiration rate in intact 143B.TK- cells. On the other hand, the activity of cytochrome c oxidase is unchanged for the first 8 h after staurosporine treatment, as determined by oxygen consumption measurements in intact cells. The decrease in the endogenous respiration rate precedes the release of cytochrome c from mitochondria. Moreover, we have ruled out caspases, permeability transition, and protein kinase C inhibition as being responsible for the decrease in respiration rate. Furthermore, overexpression of the gene for Bcl-2 does not prevent the decrease in respiration rate. The last finding suggests that Bcl-2 acts downstream of the perturbation in respiration. The evidence of normal enzymatic activities of complex I and complex III in staurosporine-treated 143B.TK- osteosarcoma cells indicates that the cause of the respiration decrease is probably an alteration in the permeability of the outer mitochondrial membrane. Presumably, the voltage-dependent anion channel closes, thereby preventing ADP and oxidizable substrates from being taken up into mitochondria. This interpretation was confirmed by another surprising finding, namely that, in staurosporine-treated 143B.TK- cells permeabilized with digitonin at a concentration not affecting the mitochondrial membranes in naive cells, the outer mitochondrial membrane loses its integrity; this leads to a reversal of its impermeability to exogenous substrates. The loss of outer membrane integrity leads also to a massive premature release of cytochrome c from mitochondria. Most significantly, Bcl-2 overexpression prevents the staurosporine-induced hypersensitivity of the outer membrane to digitonin. Our experiments have thus revealed early changes in the outer mitochondrial membrane, which take place long before cytochrome c is released from mitochondria in intact cells.", "doi": "10.1074/jbc.M209269200", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "2003-01-10", "series_number": "2", "volume": "278", "issue": "2", "pages": "1346-1353" }, { "id": "authors:v0hvn-tp750", "collection": "authors", "collection_id": "v0hvn-tp750", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:BAIjbc01", "type": "article", "title": "Lack of complex I activity in human cells carrying a mutation in MtDNA-encoded ND4 subunit is corrected by the Saccharomyces cerevisiae NADH-quinone oxidoreductase (NDI1) gene", "author": [ { "family_name": "Bai", "given_name": "Yidong", "clpid": "Bai-Yidong" }, { "family_name": "H\u00e1jek", "given_name": "Petr", "clpid": "H\u00e1jek-Petr" }, { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" }, { "family_name": "Chan", "given_name": "Elisa", "clpid": "Chan-Elisa" }, { "family_name": "Seo", "given_name": "Byoung Boo", "clpid": "Seo-Byoung-Boo" }, { "family_name": "Matsuno-Yagi", "given_name": "Akemi", "clpid": "Matsuno-Yagi-Akemi" }, { "family_name": "Yagi", "given_name": "Takao", "clpid": "Yagi-Takao" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" } ], "abstract": "The gene for the single subunit, rotenone-insensitive, and flavone-sensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1) can completely restore the NADH dehydrogenase activity in mutant human cells that lack the essential mitochondrial DNA (mtDNA)-encoded subunit ND4. In particular, the NDI1 gene was introduced into the nuclear genome of the human 143B.TK cell line derivative C4T, which carries a homoplasmic frameshift mutation in the ND4 gene. Two transformants with a low or high level of expression of the exogenous gene were chosen for a detailed analysis. In these cells the corresponding protein is localized in mitochondria, its NADH-binding site faces the matrix compartment as in yeast mitochondria, and in perfect correlation with its abundance restores partially or fully NADH-dependent respiration that is rotenone-insensitive, flavone-sensitive, and antimycin A-sensitive. Thus the yeast enzyme has become coupled to the downstream portion of the human respiratory chain. Furthermore, the P:O ratio with malate/glutamate-dependent respiration in the transformants is approximately two-thirds of that of the wild-type 143B.TK cells, as expected from the lack of proton pumping activity in the yeast enzyme. Finally, whereas the original mutant cell line C4T fails to grow in medium containing galactose instead of glucose, the high NDI1-expressing transformant has a fully restored capacity to grow in galactose medium. The present observations substantially expand the potential of the yeast NDI1 gene for the therapy of mitochondrial diseases involving complex I deficiency.", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "2001-10-19", "series_number": "42", "volume": "276", "issue": "42", "pages": "38808-38813" }, { "id": "authors:fa4bb-qqt25", "collection": "authors", "collection_id": "fa4bb-qqt25", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:CHOjbc00", "type": "article", "title": "The Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, and Stroke-like Episode Syndrome-associated Human Mitochondrial tRNALeu(UUR) Mutation Causes Aminoacylation Deficiency and Concomitant Reduced Association of mRNA with Ribosomes", "author": [ { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" }, { "family_name": "Enr\u00edquez", "given_name": "Jos\u00e9 Antonio", "clpid": "Enr\u00edquez-J-A" }, { "family_name": "Micol", "given_name": "Vicente", "clpid": "Micol-V" }, { "family_name": "Fernandez-Silva", "given_name": "Patricio", "clpid": "Fern\u00e1dez-Silva-P" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" } ], "abstract": "The pathogenetic mechanism of the mitochondrial tRNALeu(UUR) A3243G transition associated with the mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome has been investigated in transmitochondrial cell lines constructed by transfer of mutant mitochondrial DNA (mtDNA)-carrying mitochondria from three genetically unrelated MELAS patients or of isogenic wild-type mtDNA-carrying organelles into human mtDNA-less cells. An in vivo footprinting analysis of the mtDNA segment within the tRNALeu(UUR) gene that binds the transcription termination factor failed to reveal any difference in occupancy of sites or qualitative interaction with the protein between mutant and wild-type mtDNAs. Cell lines nearly homoplasmic for the mutation exhibited a strong (70-75%) reduction in the level of aminoacylated tRNALeu(UUR) and a decrease in mitochondrial protein synthesis rate. The latter, however, did not show any significant correlation between synthesis defect of the individual polypeptides and number or proportion of UUR codons in their mRNAs, suggesting that another step, other than elongation, may be affected. Sedimentation analysis in sucrose gradient showed a reduction in size of the mitochondrial polysomes, while the distribution of the two rRNA components and of the mRNAs revealed decreased association of mRNA with ribosomes and, in the most affected cell line, pronounced degradation of the mRNA associated with slowly sedimenting structures. Therefore, several lines of evidence indicate that the protein synthesis defect in A3243G MELAS mutation-carrying cells is mainly due to a reduced association of mRNA with ribosomes, possibly as a consequence of the tRNALeu(UUR) aminoacylation defect.", "doi": "10.1074/jbc.M908734199", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "2000-06-25", "series_number": "25", "volume": "275", "issue": "25", "pages": "19198-19209" }, { "id": "authors:h7yjv-r7406", "collection": "authors", "collection_id": "h7yjv-r7406", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:HELnar99", "type": "article", "title": "Search for differences in post-transcriptional modification patterns of mitochondrial DNA-encoded wild-type and mutant human tRNALys and tRNALeu(UUR)", "author": [ { "family_name": "Helm", "given_name": "Mark", "clpid": "Helm-M" }, { "family_name": "Florentz", "given_name": "Catherine", "clpid": "Florentz-C" }, { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" } ], "abstract": "Post-transcriptional modifications are characteristic features of tRNAs and have been shown in a number of cases to influence both their structural and functional properties, including structure stabilization, amino-acylation and codon recognition. We have developed an approach which allows the investigation of the post-transcriptional modification patterns of human mitochondrial wild-type and mutant tRNAs at both the qualitative and the quantitative levels. Specific tRNA species are long-term labeled in vivo with [P-32]orthophosphate, isolated in a highly selective way, enzymatically digested to mononucleotides and then subjected to two-dimensional thin layer chromatographic analysis. The wild-type tRNA(LyS) and the corresponding tRNALyS carrying the A8344G mutation associated with the MERRF (Myoclonic Epilepsy with Ragged Red Fibers) syndrome exhibit the same modified nucleotides at the same molar concentrations. By contrast, a quantitatively different modification pattern was observed between the wild-type tRNA(LeU(UUR)) and its counterpart carrying the A3243G mutation associated with the MELAS (Mitochondrial Myopathy, Encephalopathy with Lactic Acidosis and Stroke-like episodes) syndrome, the latter exhibiting a 50% decrease in m(2)G content. Complementary sequencing of tRNA(Leu(UUR)) has allowed the localization of this modification at position 10 within the D-stem of the tRNA. The decreased lever of this modification may have important implications for understanding the molecular mechanism underlying the MELAS-associated mitochondrial dysfunction.", "doi": "10.1093/nar/27.3.756", "issn": "0305-1048", "publisher": "Nucleic Acids Research", "publication": "Nucleic Acids Research", "publication_date": "1999-02-01", "series_number": "3", "volume": "27", "issue": "3", "pages": "756-763" }, { "id": "authors:02rea-vhf04", "collection": "authors", "collection_id": "02rea-vhf04", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20111222-100052561", "type": "article", "title": "The Myoclonic Epilepsy and Ragged-Red Fiber Mutation Provides New Insights into Human Mitochondrial Function and Genetics", "author": [ { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" } ], "abstract": "Of the ~50 mtDNA point mutations, reported so far,\nthat cause disease in humans, \u2265 35 occur in tRNA genes\n(Schon et al. 1997). Of these, perhaps the best-studied\nis the A \u2192 G transition at position 8344 in the tRNA^(Lys)\ngene, the mutation that causes myoclonic epilepsy and\nragged-red fiber syndrome (MERRF; Shoffner et al.\n1990). This disorder is an encephalomyopathy characterized\nby myoclonic epilepsy, ataxia, and mitochondrial\nmyopathy, with additional dysfunction, in some individuals,\nin tissues other than brain and skeletal muscle\n(Schon et al. 1997). Histological analysis of transverse\nsections of a MERRF patient's skeletal muscle, stained\nwith Gomori's modified trichrome stain, reveals the presence\nof \"ragged red fibers,\" muscle fibers that exhibit\nperipheral blotchy, red patches that represent subsarcolemmal accumulations of mitochondria.", "doi": "10.1086/301813", "issn": "0002-9297", "publisher": "Elsevier", "publication": "American Journal of Human Genetics", "publication_date": "1998-04", "series_number": "4", "volume": "62", "issue": "4", "pages": "745-751" }, { "id": "authors:kgn7p-84880", "collection": "authors", "collection_id": "kgn7p-84880", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20191030-134818569", "type": "article", "title": "Myoclonic Epilepsy and Ragged Red Fibers (MERRF) Syndrome: Selective Vulnerability of CNS Neurons Does Not Correlate with the Level of Mitochondrial tRNA^(lys) Mutation in Individual Neuronal Isolates", "author": [ { "family_name": "Zhou", "given_name": "Li", "clpid": "Zhou-Li" }, { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Miller", "given_name": "Carol A.", "clpid": "Miller-C-A" } ], "abstract": "Selective vulnerability of subpopulations of neurons is a striking feature of neurodegeneration. Mitochondrially transmitted diseases are no exception. In this study CNS tissues from a patient with myoclonus epilepsy and ragged red fibers (MERRF) syndrome, which results from an A to G transition of nucleotide (nt) 8344 in the mitochondrial tRNA^(Lys) gene, were examined for the proportion of mutant mtDNA. Either individual neuronal somas or the adjacent neuropil and glia were microdissected from cryostat tissue sections of histologically severely affected brain regions, including dentate nuclei, Purkinje cells, and inferior olivary nuclei, and from a presumably less affected neuronal subpopulation, the anterior horn cells of the spinal cord. Mutant and normal mtDNA were quantified after PCR amplification with a mismatched primer and restriction enzyme digestion. Neurons and the surrounding neuropil and glia from all CNS regions that were analyzed exhibited high proportions of mutant mtDNA, ranging from 97.6 \u00b1 0.7% in Purkinje cells to 80.6 \u00b1 2.8% in the anterior horn cells. Within each neuronal group that was analyzed, neuronal soma values were similar to those in the surrounding neuropil and glia or in the regional tissue homogenate. Surprisingly, as compared with controls, neuronal loss ranged from 7% of the Purkinje cells to 46% of the neurons of the dentate nucleus in MERRF cerebellum. Thus, factors other than the high proportion of mutant mtDNA, in particular nuclear-controlled neuronal differences among various regions of the CNS, seem to contribute to the mitochondrial dysfunction and ultimate cell death.", "doi": "10.1523/jneurosci.17-20-07746.1997", "pmcid": "PMC6793887", "issn": "0270-6474", "publisher": "Society for Neuroscience", "publication": "Journal of Neuroscience", "publication_date": "1997-10-15", "series_number": "20", "volume": "17", "issue": "20", "pages": "7746-7753" }, { "id": "authors:aq1nm-46a30", "collection": "authors", "collection_id": "aq1nm-46a30", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:HOFjbc96", "type": "article", "title": "Respiration and Growth Defects in Transmitochondrial Cell Lines Carrying the 11778 Mutation Associated with Leber's Hereditary Optic Neuropathy", "author": [ { "family_name": "Hofhaus", "given_name": "G\u00f6tz", "clpid": "Hofhaus-G" }, { "family_name": "Johns", "given_name": "Donald R.", "clpid": "Johns-D-R" }, { "family_name": "Hurko", "given_name": "Orest", "clpid": "Hurko-O" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" } ], "abstract": "Mitochondrial DNA from two genetically unrelated patients carrying the mutation at position 11778 that causes Leber's hereditary optic neuropathy has been transferred with mitochondria into human mtDNA-less 0206 cells. As analyzed in several transmitochondrial cell lines thus obtained, the mutation, which is in the gene encoding subunit ND4 of the respiratory chain NADH dehydrogenase (ND), did not affect the synthesis, size, or stability of ND4, nor its incorporation into the enzyme complex. However, NADH dehydrogenase-dependent respiration, as measured in digitonin-permeabilized cells, was specifically decreased by approximately 40% in cells carrying the mutation. This decrease, which was significant at the 99.99% confidence level, was correlated with a significantly reduced ability of the mutant cells to grow in a medium containing galactose instead of glucose, indicating a clear impairment in their oxidative phosphorylation capacity. On the contrary, no decrease in rotenone-sensitive NADH dehydrogenase activity, using a water-soluble ubiquinone analogue as electron acceptor, was detected in disrupted mitochondrial membranes. This is the first cellular model exhibiting in a foreign nuclear background mitochondrial DNA-linked biochemical defects underlying the optic neuropathy phenotype.", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "1996-05-31", "series_number": "22", "volume": "271", "issue": "22", "pages": "13155-13161" }, { "id": "authors:cxvzx-tnb98", "collection": "authors", "collection_id": "cxvzx-tnb98", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150624-150439462", "type": "article", "title": "MtDNA mutation in MERRF syndrome causes defective aminoacylation of tRNA^(Lys) and premature translation termination", "author": [ { "family_name": "Enriquez", "given_name": "Jos\u00e9 Antonio", "clpid": "Enr\u00edquez-J-A" }, { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" } ], "abstract": "We have investigated the pathogenetic mechanism of the mitochondrial tRNA^(Lys) gene mutation (position 8344) associated with MERRF encephalomyopathy in several mitochondrial DMA (mtDNA)\u2212less cell transformants carrying the mutation and in control cells. A decrease of 50\u221260% in the specific tRNA^(Lys) aminoacylation capacity per cell was found in mutant cells. Furthermore, several lines of evidence reveal that the severe protein synthesis impairment in MERRF mutation\u2212carrying cells is due to premature termination of translation at each or near each lysine codon, with the deficiency of aminoacylated tRNA^(Lys) being the most likely cause of this phenomenon.", "doi": "10.1038/ng0595-47", "issn": "1061-4036", "publisher": "Nature Publishing Group", "publication": "Nature Genetics", "publication_date": "1995-05", "series_number": "1", "volume": "10", "issue": "1", "pages": "47-55" }, { "id": "authors:ppabv-1ys50", "collection": "authors", "collection_id": "ppabv-1ys50", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:YONpnas92", "type": "article", "title": "Marked replicative advantage of human mtDNA carrying a point mutation that causes the MELAS encephalomyopathy", "author": [ { "family_name": "Yoneda", "given_name": "Makoto", "clpid": "Yoneda-Makoto" }, { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" }, { "family_name": "Martinuzzi", "given_name": "Andrea", "clpid": "Martinuzzi-A" }, { "family_name": "Hurko", "given_name": "Orest", "clpid": "Hurko-O" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" } ], "abstract": "The segregation of mutant and wild-type mtDNA was investigated in transformants constructed by transferring human mitochondria from individuals belonging to four pedigrees with the MELAS encephalomyopathy-associated mtDNA mutation (MELAS is mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) into human mtDNA-less (rho-degrees) cells. Five of 13 clonal cell lines containing mixtures of wild-type and mutant mtDNAs were found to undergo a rapid shift of their genotype toward the pure mutant type. The other 8 cell lines, which included 6 exhibiting nearly homoplasmic mutant mtDNA, on the contrary, maintained a stable genotype. Subcloning experiments and growth rate measurements clearly indicated that an intracellular replicative advantage of mutant mtDNA was mainly responsible for the dramatic shift toward the mutant genotype observed in the unstable cell lines.", "doi": "10.1073/pnas.89.23.11164", "pmcid": "PMC50510", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1992-12-01", "series_number": "23", "volume": "89", "issue": "23", "pages": "11164-11168" }, { "id": "authors:jgvey-30d16", "collection": "authors", "collection_id": "jgvey-30d16", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120309-160913724", "type": "article", "title": "MELAS Mutation in mtDNA Binding Site for Transcription Termination Factor Causes Defects in Protein Synthesis and in Respiration but no Change in Levels of Upstream and Downstream Mature Transcripts", "author": [ { "family_name": "Chomyn", "given_name": "A.", "clpid": "Chomyn-A" }, { "family_name": "Martinuzzi", "given_name": "A.", "clpid": "Martinuzzi-A" }, { "family_name": "Yoneda", "given_name": "M.", "clpid": "Yoneda-M" }, { "family_name": "Daga", "given_name": "A.", "clpid": "Daga-A" }, { "family_name": "Hurko", "given_name": "O.", "clpid": "Hurko-O" }, { "family_name": "Johns", "given_name": "D.", "clpid": "Johns-D" }, { "family_name": "Lai", "given_name": "S. T.", "clpid": "Lai-Susan-T" }, { "family_name": "Nonaka", "given_name": "I.", "clpid": "Nonaka-I" }, { "family_name": "Angelini", "given_name": "C.", "clpid": "Angelini-C" }, { "family_name": "Attardi", "given_name": "G.", "clpid": "Attardi-G" } ], "abstract": "The pathogenetic mechanism of the mitochondrial tRNA^(Leu)_(UUR) gene mutation responsible for the MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) syndrome was investigated in transformants obtained by transfer of mitochondria from three genetically unrelated MELAS patients into human mitochondrial DNA (mtDNA)-less (\u03c1\u00b0) cells. Marked defects in mitochondrial protein synthesis and respiratory activity were observed in transformants containing virtually pure mutant mtDNA, as compared to the parent of the \u03c1\u00b0 cells (the 143B cell line) or to transformants containing exclusively wild-type mtDNA, derived from one of the patients or a maternally related asymptomatic individual. A striking protective effect against the mutation was exerted in the transformants by levels of residual wild-type mtDNA above 6%. The MELAS mutation occurs within the mtDNA binding site for a protein factor (mTERF) that promotes termination of transcription at the 16S rRNA/tRNA^(Leu)_(UUR) gene boundary. A marked decrease in affinity of purified mTERF for the mutant target sequence was observed in in vitro assays. By contrast, RNA transfer hybridization experiments failed to show any significant change in the steady-state amounts of the two rRNA species, encoded upstream of the termination site, and of the mRNAs encoded downstream, in the transformants carrying the MELAS mutation.", "doi": "10.1073/pnas.89.10.4221", "pmcid": "PMC49053", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1992-05-15", "series_number": "10", "volume": "89", "issue": "10", "pages": "4221-4225" }, { "id": "authors:fvvph-c1w69", "collection": "authors", "collection_id": "fvvph-c1w69", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20120418-153850946", "type": "article", "title": "In Vitro Genetic Transfer of Protein Synthesis and Respiration Defects to Mitochondrial DNA-Less Cells with Myopathy-Patient Mitochondria", "author": [ { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" }, { "family_name": "Meola", "given_name": "Giovanni", "clpid": "Meola-G" }, { "family_name": "Bresolin", "given_name": "Nereo", "clpid": "Bresolin-N" }, { "family_name": "Lai", "given_name": "Susan T.", "clpid": "Lai-Susan-T" }, { "family_name": "Scarlato", "given_name": "Guglielmo", "clpid": "Scarlato-G" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" } ], "abstract": "A severe mitochondrial protein synthesis defect in myoblasts from a patient with mitochondrial myopathy was transferred with myoblast mitochondria into two genetically unrelated mitochondrial DNA (mtDNA)-less human cell lines, pointing to an mtDNA alteration as being responsible and sufficient for causing the disease. The transfer of the defect correlated with marked deficiencies in respiration and cytochrome c oxidase activity of the transformants and the presence in their mitochondria of mtDNA carrying a tRNA^(Lys) mutation. Furthermore, apparently complete segregation of the defective genotype and phenotype was observed in the transformants derived from the heterogeneous proband myoblast population, suggesting that the mtDNA heteroplasmy in this population was to a large extent intercellular. The present work thus establishes a direct link between mtDNA alteration and a biochemical defect.", "doi": "10.1128/MCB.11.4.2236", "pmcid": "PMC359920", "issn": "0270-7306", "publisher": "American Society for Microbiology", "publication": "Molecular and Cellular Biology", "publication_date": "1991-04", "series_number": "4", "volume": "11", "issue": "4", "pages": "2236-2244" }, { "id": "authors:5301w-7gh97", "collection": "authors", "collection_id": "5301w-7gh97", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:CHOjbc88", "type": "article", "title": "The site of synthesis of the iron-sulfur subunits of the flavoprotein and iron-protein fractions of human NADH dehydrogenase", "author": [ { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" }, { "family_name": "Patel", "given_name": "Salil D.", "clpid": "Patel-S-D" }, { "family_name": "Cleeter", "given_name": "Michael W. J.", "clpid": "Cleeter-M-W-J" }, { "family_name": "Ragan", "given_name": "C. Ian", "clpid": "Ragan-C-I" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" } ], "abstract": "The site of synthesis of the iron-sulfur subunits of the flavoprotein and iron-protein fractions of the human respiratory chain NADH dehydrogenase has been investigated to test the possibility that any of them is synthesized in mitochondria. For this purpose, antibodies specific for individual subunits of the bovine enzyme, which cross- reacted with the homologous human subunits in immunoblot assays, were tested against HeLa cell mitochondrial proteins labeled in vivo with [35S]methionine in the absence or presence of inhibitors of mitochondrial or cytoplasmic protein synthesis. The results clearly indicated that all the iron-sulfur subunits of the flavoprotein and iron-protein fractions of human complex I are synthesized in the cytosol and are, therefore, encoded in nuclear genes.", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "1988-11-05", "series_number": "31", "volume": "263", "issue": "31", "pages": "16395-16400" }, { "id": "authors:mj50h-zba24", "collection": "authors", "collection_id": "mj50h-zba24", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20150130-124807876", "type": "article", "title": "URF6, last unidentified reading frame of human mtDNA, codes for an NADH dehydrogenase subunit", "author": [ { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" }, { "family_name": "Cleeter", "given_name": "Michael W. J.", "clpid": "Cleeter-M-W-J" }, { "family_name": "Ragan", "given_name": "C. Ian", "clpid": "Ragan-C-I" }, { "family_name": "Riley", "given_name": "Marcia", "clpid": "Riley-M" }, { "family_name": "Doolittle", "given_name": "Russell F.", "clpid": "Doolittle-R-F" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" } ], "abstract": "The polypeptide encoded in URF6, the last unassigned reading frame of human mitochondrial DNA, has been identified with antibodies to peptides predicted from the DNA sequence. Antibodies prepared against highly purified respiratory chain NADH dehydrogenase from beef heart or against the cytoplasmically synthesized 49-kilodalton iron-sulfur subunit isolated from this enzyme complex, when added to a deoxycholate or a Triton X-100 mitochondrial lysate of HeLa cells, specifically precipitated the URF6 product together with the six other URF products previously identified as subunits of NADH dehydrogenase. These results strongly point to the URF6 product as being another subunit of this enzyme complex. Thus, almost 60% of the protein coding capacity of mammalian mitochondrial DNA is utilized for the assembly of the first enzyme complex of the respiratory chain. The absence of such information in yeast mitochondrial DNA dramatizes the variability in gene content of different mitochondrial genomes.", "doi": "10.1126/science.3764430", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "1986-10-31", "series_number": "4776", "volume": "234", "issue": "4776", "pages": "614-618" }, { "id": "authors:60ead-b4579", "collection": "authors", "collection_id": "60ead-b4579", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:MARpnas86b", "type": "article", "title": "Identification of the polypeptides encoded in the unassigned reading frames 2, 4, 4L, and 5 of human mitochondrial DNA", "author": [ { "family_name": "Mariottini", "given_name": "Paolo", "clpid": "Mariottini-P" }, { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" }, { "family_name": "Riley", "given_name": "Marcia", "clpid": "Riley-M" }, { "family_name": "Cottrell", "given_name": "Barbara", "clpid": "Cottrell-B" }, { "family_name": "Doolittle", "given_name": "Russell F.", "clpid": "Doolittle-R-F" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" } ], "abstract": "In previous work, antibodies prepared against chemically synthesized peptides predicted from the DNA sequence were used to identify the polypeptides encoded in three of the eight unassigned reading frames (URFs) of human mitochondrial DNA (mtDNA). In the present study, this approach has been extended to other human mtDNA URFs. In particular, antibodies directed against the NH2-terminal octapeptide of the putative URF2 product specifically precipitated component 11 of the HeLa cell mitochondrial translation products, the reaction being inhibited by the specific peptide. Similarly, antibodies directed against the COOH-terminal nonapeptide of the putative URF4 product reacted specifically with components 4 and 5, and antibodies against a COOH-terminal heptapeptide of the presumptive URF4L product reacted specifically with component 26. Antibodies against the NH2-terminal heptapeptide of the putative product of URF5 reacted with component 1, but only to a marginal extent; however, the results of a trypsin fingerprinting analysis of component 1 point strongly to this component as being the authentic product of URF5. The polypeptide assignments to the mtDNA URFs analyzed here are supported by the relative electrophoretic mobilities of proteins 11, 4-5, 26, and 1, which are those expected for the molecular weights predicted from the DNA sequence for the products of URF2, URF4, URF4L, and URF5, respectively. With the present assignment, seven of the eight human mtDNA URFs have been shown to be expressed in HeLa cells.", "doi": "10.1073/pnas.83.6.1563", "pmcid": "PMC323123", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1986-03-15", "series_number": "6", "volume": "83", "issue": "6", "pages": "1563-1567" }, { "id": "authors:bm8t2-c2y17", "collection": "authors", "collection_id": "bm8t2-c2y17", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:MARjbc86", "type": "article", "title": "Antibodies against the COOH-terminal undecapeptide of subunit II, but not those against the NH2-terminal decapeptide, immunoprecipitate the whole human cytochrome c oxidase complex", "author": [ { "family_name": "Mariottini", "given_name": "Paolo", "clpid": "Mariottini-P" }, { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" }, { "family_name": "Doolittle", "given_name": "Russell F.", "clpid": "Doolittle-R-F" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" } ], "abstract": "Antibodies against synthetic peptides derived from the DNA sequence of human cytochrome c oxidase subunit II (COII) have been tested for their capacity to immunoprecipitate the whole enzyme complex. Antibodies against the COOH-terminal undecapeptide of COII (anti-COII-C), when incubated with a Triton X-100 mitochondrial lysate from HeLa cells pulse-labeled with [35S]methionine under conditions selective for mitochondrial protein synthesis and chased for 18 h in unlabeled medium, precipitated the pulse-labeled three largest subunits (mitochondrially synthesized) of cytochrome c oxidase in proportions close to equimolarity. Antibodies against the NH2-terminal decapeptide of COII (anti-COII-N), although equally reactive as the anti-COII-C antibodies with the sodium dodecyl sulfate-solubilized COII, did not precipitate any of the three labeled subunits from the Triton X-100 mitochondrial lysate. In other experiments, all the 13 subunits which have been identified in the mammalian cytochrome c oxidase were immunoprecipitated from a Triton X-100 mitochondrial lysate of cells long-term labeled with [35S]methionine by anti-COII-C antibodies, but not by anti-COII-N antibodies. By contrast, in immunoblots of total mitochondrial proteins dissociated with sodium dodecyl sulfate, the anti-COII-C antibodies reacted specifically only with COII. These results strongly suggest that, in the native cytochrome c oxidase complex, the epitope recognized by the anti-COII-C antibodies is in the COII subunit and that, therefore, in such complex, the COOH-terminal peptide of COII is exposed to antibodies, whereas the NH2-terminal peptide is not accessible.", "issn": "0021-9258", "publisher": "American Society for Biochemistry and Molecular Biology", "publication": "Journal of Biological Chemistry", "publication_date": "1986-03-05", "series_number": "7", "volume": "261", "issue": "7", "pages": "3355-3362" }, { "id": "authors:ghz70-b0034", "collection": "authors", "collection_id": "ghz70-b0034", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:CHOpnas83", "type": "article", "title": "Identification of the polypeptides encoded in the ATPase 6 gene and in the unassigned reading frames 1 and 3 of human mtDNA", "author": [ { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" }, { "family_name": "Mariottini", "given_name": "Paolo", "clpid": "Mariottini-P" }, { "family_name": "Gonzalez-Cadavid", "given_name": "Nestor", "clpid": "Gonzalez-Cadavid-N" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" }, { "family_name": "Strong", "given_name": "Donna D.", "clpid": "Strong-D-D" }, { "family_name": "Trovato", "given_name": "Dennis", "clpid": "Trovato-D" }, { "family_name": "Riley", "given_name": "Marcia", "clpid": "Riley-M" }, { "family_name": "Doolittle", "given_name": "Russell F.", "clpid": "Doolittle-R-F" } ], "abstract": "Antibodies prepared against chemically synthesized peptides predicted from the DNA sequence have been used to identify the polypeptides encoded in the ATPase 6 gene and in unidentified reading frames (URFs) 1 and 3 of human mtDNA. In particular, antibodies directed against the COOH-terminal nonapeptide of the putative polypeptide encoded in the ATPase 6 reading frame immunoprecipitated specifically component 17 of the HeLa cell mitochondrial translation products, the reaction being inhibited by the specific peptide. Similarly, antibodies directed against the COOH-terminal undecapeptide of the putative URF1 product or against the COOH-terminal heptapeptide of the presumptive URF3 product were effective in immunoprecipitating specifically component 12 or, respectively, component 24 of the mitochondrial translation products. The sizes of proteins 17, 12, and 24, as estimated from their electrophoretic mobilities, are compatible with their being the products of the ATPase 6 gene, URF1, and URF3, respectively.", "doi": "10.1073/pnas.80.18.5535", "pmcid": "PMC384292", "issn": "0027-8424", "publisher": "National Academy of Sciences", "publication": "Proceedings of the National Academy of Sciences of the United States of America", "publication_date": "1983-09-15", "series_number": "18", "volume": "80", "issue": "18", "pages": "5535-5539" }, { "id": "authors:dq1v1-df680", "collection": "authors", "collection_id": "dq1v1-df680", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:CHOnar81", "type": "article", "title": "Alignment of the amino terminal amino acid sequence of human cytochrome c oxidase subunits I and II with the sequence of their putative mRNAs", "author": [ { "family_name": "Chomyn", "given_name": "Anne", "clpid": "Chomyn-A" }, { "family_name": "Hunkapiller", "given_name": "Michael W.", "clpid": "Hunkapiller-M-W" }, { "family_name": "Attardi", "given_name": "Giuseppe", "clpid": "Attardi-G" } ], "abstract": "Thirteen of the first fifteen amino acids from the NH2-terminus of the primary sequence of human cytochrome c oxidase subunit I and eleven of the first twelve amino acids of subunit II have been identified by microsequencing procedures. These sequences have been compared with the recently determined 5'-end proximal sequences of the HeLa cell mitochondrial mRNAS and unambiguously aligned with two of them. This alignment has allowed the identification of the putative mRNA for subunit I, and has shown that the initiator codon for this subunit is only three nucleotides away from the 5'-end of its mRNA; furthermore, the results have substantiated the idea that the translation of human cytochrome c oxidase subunit II starts directly at the 5'-end of its putative mRNA, as had been previously inferred on the basis of the sequence homology of human mitochondrial DNA with the primary sequence of the bovine subunit.", "issn": "0305-1048", "publisher": "Nucleic Acids Research", "publication": "Nucleic Acids Research", "publication_date": "1981-02-25", "series_number": "4", "volume": "9", "issue": "4", "pages": "867-877" } ]