Ivor Muroff

The respiratory defect of Saccharomyces cerevisiae mutants assigned to complementation group G4 of apet strain collection stems from their failure to synthesize cytochrome oxidase. The mutations do not affect expression of either the mitochondrially or nuclearly encoded subunits of the enzyme. The cytochrome oxidase deficiency also does not appear to be related to mitochondrial copper metabolism or heme abiosynthesis. These data suggest that the mutants are likely to be impaired in assembly of the enzyme. A gene designated COX15has been cloned by transformation of mutants from complementation group G4. This gene is identical to reading frame YER141w on chromosome 5. To facilitate further studies, Cox15p has been expressed as a biotinylated protein. Biotinylated Cox15p fully restores cytochrome oxidase incox15 mutants, indicating that the carboxyl-terminal sequence with biotin does not affect its function. Cox15p is a constituent of the mitochondrial inner membrane and, because of its resistance to proteolysis, probably is largely embedded in the phospholipid bilayer of the membrane. The present studies further emphasize the complexity of cytochrome oxidase assembly and report a new constituent of mitochondria involved in this process. The existence of COX15 homologs in Schizosaccharomyces pombeand Caenorhabditis elegans suggests that it may be widely distributed in eucaryotic organisms. The respiratory defect of Saccharomyces cerevisiae mutants assigned to complementation group G4 of apet strain collection stems from their failure to synthesize cytochrome oxidase. The mutations do not affect expression of either the mitochondrially or nuclearly encoded subunits of the enzyme. The cytochrome oxidase deficiency also does not appear to be related to mitochondrial copper metabolism or heme abiosynthesis. These data suggest that the mutants are likely to be impaired in assembly of the enzyme. A gene designated COX15has been cloned by transformation of mutants from complementation group G4. This gene is identical to reading frame YER141w on chromosome 5. To facilitate further studies, Cox15p has been expressed as a biotinylated protein. Biotinylated Cox15p fully restores cytochrome oxidase incox15 mutants, indicating that the carboxyl-terminal sequence with biotin does not affect its function. Cox15p is a constituent of the mitochondrial inner membrane and, because of its resistance to proteolysis, probably is largely embedded in the phospholipid bilayer of the membrane. The present studies further emphasize the complexity of cytochrome oxidase assembly and report a new constituent of mitochondria involved in this process. The existence of COX15 homologs in Schizosaccharomyces pombeand Caenorhabditis elegans suggests that it may be widely distributed in eucaryotic organisms. The completion of the Saccharomyces cerevisiae genome sequence (1Goffeau A. Barrell B.G. Bussey H. Davis R.W. Dujon B. Feldmann H. Galibert F. Hoheisel J.D. Jacq C. Johnson M. Louis E.J. Mewes H.W. Murakami Y. Philippsen P. Tettelin H. Oliver S.G. Science. 1996; 274: 546-567Crossref PubMed Scopus (3208) Google Scholar) has spawned new large scale projects designed to unravel the functions of the numerous unknown reading frames. A substantial fraction of the total information in yeast chromosomal DNA is comprised of PET genes that are essential for the biogenesis of respiratory competent mitochondria. Mutations in these genes were shown in the early 1950s (2Sherman F. Genetics. 1963; 48: 375-385Crossref PubMed Google Scholar, 3Sherman F. Slonimski P.P. Biochim. Biophys. Acta. 1964; 90: 1-15Crossref PubMed Scopus (164) Google Scholar) to affect the ability of yeast to respire. Renewed efforts to mutationally saturate for this class of genes (4Tzagoloff A. Dieckmann C.L. Microbiol. Rev. 1990; 54: 211-225Crossref PubMed Google Scholar, 5Burkl G. Demmer W. Holzner H. Schweizer E. Bandlow W. Schweyen R.J. Thomas D.Y. Wolf K. Kaudewitz E.F. Genetics Biogenesis and Bioenergetics of Mitochondria. Walter de Gruyter, Berlin1976: 39-48Crossref Google Scholar) have helped to expand our knowledge of the extent and nature of the contribution made by the nucleus toward maintenance of respiring mitochondria.Biochemical studies of petmutants 1The abbreviations used are: petmutant, nuclear respiratory deficient mutant of yeast; ρomutant, cytoplasmic petite mutant lacking mitochondrial DNA; PAGE, polyacrylamide gel electrophoresis; kb, kilobase pair. 1The abbreviations used are: petmutant, nuclear respiratory deficient mutant of yeast; ρomutant, cytoplasmic petite mutant lacking mitochondrial DNA; PAGE, polyacrylamide gel electrophoresis; kb, kilobase pair. have revealed that the assembly of respiratory chain enzymes is governed by an unexpectedly large number of genes. For example, some three dozen complementation groups have been reported to consist of mutants displaying a selective deficiency in cytochrome oxidase (4Tzagoloff A. Dieckmann C.L. Microbiol. Rev. 1990; 54: 211-225Crossref PubMed Google Scholar, 6McEwen J.E. Cameron V.L. Poyton R.O. J. Bacteriol. 1985; 161: 831-835Crossref PubMed Google Scholar). In addition to mutations in the structural genes, these strains are also affected in: 1) processing of the mitochondrial cytochrome oxidase pre-mRNAs (7McEwen J.E. Ko C. Kloeckener-Gruissem B. Poyton R.O. J. Biol. Chem. 1986; 261: 11872-11879Abstract Full Text PDF PubMed Google Scholar, 8Pel H.J. Tzagoloff A. Grivell L.A. Curr. Genet. 1992; 21: 139-146Crossref PubMed Scopus (34) Google Scholar, 9Seraphin B. Simon M. Faye G. EMBO J. 1988; 7: 1455-1464Crossref PubMed Scopus (50) Google Scholar), 2) translation of the resultant mRNAs (10Costanzo M.C. Seaver E.C. Fox T.D. EMBO J. 1986; 5: 3637-3641Crossref PubMed Scopus (59) Google Scholar, 11Poutre C.G. Fox T.D. Genetics. 1987; 115: 637-647Crossref PubMed Google Scholar), 3) heme abiosynthesis (12Tzagoloff A. Nobrega M. Gorman N. Sinclair P. Biochem. Mol. Biol. Int. 1993; 31: 593-598PubMed Google Scholar), 4) copper import and transfer to the apoenzyme (13Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 14504-14509Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar,14Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 20531-20535Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar), and 5) as yet poorly understood events in the pathway leading to the functional enzyme (15McEwen J.E. Hong K.H. Park S. Preciado G.T. Curr. Genet. 1993; 23: 9-14Crossref PubMed Scopus (57) Google Scholar, 16Bonnefoy N. Chalvet F. Hamel P. Slonimski P.P. Dujardin G. J. Mol. Biol. 1994; 239: 201-212Crossref PubMed Scopus (179) Google Scholar, 17Glerum D.M. Koerner T.J. Tzagoloff A. J. Biol. Chem. 1995; 270: 15585-15590Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). To learn more about the assembly of this heteroligomeric membrane complex, we have continued to screen for, and to analyze pet mutants with lesions in cytochrome oxidase. In this article, we report the properties of mutants from complementation group G4 whose cytochrome oxidase deficiency results from mutations in COX15 (identical to reading frame YER141w on chromosome 5). We show that mutations in this gene have no effect on the expression of the mitochondrial or nuclear cytochrome oxidase genes. Since cox15 mutants also do not appear to be impaired in heme a synthesis and copper metabolism, the product of this gene (Cox15p) is most likely involved in some aspect of the assembly process itself. We also present evidence that Cox15p is imported into mitochondria and is a constituent of the inner membrane. The completion of the Saccharomyces cerevisiae genome sequence (1Goffeau A. Barrell B.G. Bussey H. Davis R.W. Dujon B. Feldmann H. Galibert F. Hoheisel J.D. Jacq C. Johnson M. Louis E.J. Mewes H.W. Murakami Y. Philippsen P. Tettelin H. Oliver S.G. Science. 1996; 274: 546-567Crossref PubMed Scopus (3208) Google Scholar) has spawned new large scale projects designed to unravel the functions of the numerous unknown reading frames. A substantial fraction of the total information in yeast chromosomal DNA is comprised of PET genes that are essential for the biogenesis of respiratory competent mitochondria. Mutations in these genes were shown in the early 1950s (2Sherman F. Genetics. 1963; 48: 375-385Crossref PubMed Google Scholar, 3Sherman F. Slonimski P.P. Biochim. Biophys. Acta. 1964; 90: 1-15Crossref PubMed Scopus (164) Google Scholar) to affect the ability of yeast to respire. Renewed efforts to mutationally saturate for this class of genes (4Tzagoloff A. Dieckmann C.L. Microbiol. Rev. 1990; 54: 211-225Crossref PubMed Google Scholar, 5Burkl G. Demmer W. Holzner H. Schweizer E. Bandlow W. Schweyen R.J. Thomas D.Y. Wolf K. Kaudewitz E.F. Genetics Biogenesis and Bioenergetics of Mitochondria. Walter de Gruyter, Berlin1976: 39-48Crossref Google Scholar) have helped to expand our knowledge of the extent and nature of the contribution made by the nucleus toward maintenance of respiring mitochondria. Biochemical studies of petmutants 1The abbreviations used are: petmutant, nuclear respiratory deficient mutant of yeast; ρomutant, cytoplasmic petite mutant lacking mitochondrial DNA; PAGE, polyacrylamide gel electrophoresis; kb, kilobase pair. 1The abbreviations used are: petmutant, nuclear respiratory deficient mutant of yeast; ρomutant, cytoplasmic petite mutant lacking mitochondrial DNA; PAGE, polyacrylamide gel electrophoresis; kb, kilobase pair. have revealed that the assembly of respiratory chain enzymes is governed by an unexpectedly large number of genes. For example, some three dozen complementation groups have been reported to consist of mutants displaying a selective deficiency in cytochrome oxidase (4Tzagoloff A. Dieckmann C.L. Microbiol. Rev. 1990; 54: 211-225Crossref PubMed Google Scholar, 6McEwen J.E. Cameron V.L. Poyton R.O. J. Bacteriol. 1985; 161: 831-835Crossref PubMed Google Scholar). In addition to mutations in the structural genes, these strains are also affected in: 1) processing of the mitochondrial cytochrome oxidase pre-mRNAs (7McEwen J.E. Ko C. Kloeckener-Gruissem B. Poyton R.O. J. Biol. Chem. 1986; 261: 11872-11879Abstract Full Text PDF PubMed Google Scholar, 8Pel H.J. Tzagoloff A. Grivell L.A. Curr. Genet. 1992; 21: 139-146Crossref PubMed Scopus (34) Google Scholar, 9Seraphin B. Simon M. Faye G. EMBO J. 1988; 7: 1455-1464Crossref PubMed Scopus (50) Google Scholar), 2) translation of the resultant mRNAs (10Costanzo M.C. Seaver E.C. Fox T.D. EMBO J. 1986; 5: 3637-3641Crossref PubMed Scopus (59) Google Scholar, 11Poutre C.G. Fox T.D. Genetics. 1987; 115: 637-647Crossref PubMed Google Scholar), 3) heme abiosynthesis (12Tzagoloff A. Nobrega M. Gorman N. Sinclair P. Biochem. Mol. Biol. Int. 1993; 31: 593-598PubMed Google Scholar), 4) copper import and transfer to the apoenzyme (13Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 14504-14509Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar,14Glerum D.M. Shtanko A. Tzagoloff A. J. Biol. Chem. 1996; 271: 20531-20535Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar), and 5) as yet poorly understood events in the pathway leading to the functional enzyme (15McEwen J.E. Hong K.H. Park S. Preciado G.T. Curr. Genet. 1993; 23: 9-14Crossref PubMed Scopus (57) Google Scholar, 16Bonnefoy N. Chalvet F. Hamel P. Slonimski P.P. Dujardin G. J. Mol. Biol. 1994; 239: 201-212Crossref PubMed Scopus (179) Google Scholar, 17Glerum D.M. Koerner T.J. Tzagoloff A. J. Biol. Chem. 1995; 270: 15585-15590Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). To learn more about the assembly of this heteroligomeric membrane complex, we have continued to screen for, and to analyze pet mutants with lesions in cytochrome oxidase. In this article, we report the properties of mutants from complementation group G4 whose cytochrome oxidase deficiency results from mutations in COX15 (identical to reading frame YER141w on chromosome 5). We show that mutations in this gene have no effect on the expression of the mitochondrial or nuclear cytochrome oxidase genes. Since cox15 mutants also do not appear to be impaired in heme a synthesis and copper metabolism, the product of this gene (Cox15p) is most likely involved in some aspect of the assembly process itself. We also present evidence that Cox15p is imported into mitochondria and is a constituent of the inner membrane.

The yeast HO gene is transcribed transiently during G1 as cells undergo START. START-specific HO activation requires two proteins, SWI4 and SWI6, which act via a motif (CACGA4) repeated up to 10 times within the URS2 region of the HO promoter. We identified a DNA-binding activity containing SWI4 and SWI6 that recognizes the CACGA4 sequences within URS2. Two forms of SWI4,6-DNA complexes called L and U can be distinguished by their electrophoretic mobility. L complexes can be detected at all stages of the cell cycle, but U complexes are only detected in cells that have undergone START. The formation of U complexes may be the trigger of HO activation. The SWI6 protein is concentrated in the nucleus throughout G1, but at some point in S or G2 significant amounts accumulate in the cytoplasm. This change in cellular location of the SWI6 protein might contribute to the turnoff of HO transcription after cells have undergone START.

The synthesis of cytochrome b in yeast depends on the expression of both mitochondrial and nuclear gene products that act at the level of processing of the pre-mRNA, translation of the mRNA, and maturation of the apoprotein during its assembly with the nuclear-encoded subunits of coenzyme QH2-cytochrome c reductase. Previous studies indicated one of the nuclear genes (CBP2) to code for a protein that is needed for the excision of the terminal intervening sequence from the pre-mRNA. We show here that the intervening sequence can promote its own excision in the presence of high concentrations of magnesium ion (50 mM), but that at physiological concentrations of the divalent cation (5 mM), the splicing reaction requires the presence of the CBP2-encoded product. These results provide strong evidence for a direct participation of the protein in splicing, most likely in stabilizing a splicing competent structure in the RNA. The conversion of apocytochrome b to the functional cytochrome has been examined in mutants lacking one or multiple structural subunits of the coenzyme QH2-cytochrome c reductase complex. Based on the phenotypes of the different mutants studied, the following have been concluded. (i) The assembly of catalytically active enzyme requires the synthesis of all except the 17 kDa subunit. (ii) Membrane insertion of the individual subunits is not contingent on protein-protein interactions. (iii) Assembly of the subunits occurs in the lipid bilayer following their insertion. (iv) The attachment of haem to apocytochrome b is a late event in assembly after an intermediate complex of the structural subunits has been formed. This complex minimally is composed of apocytochrome b, the non haem iron protein and all the non-catalytic subunits except for the 17 kDa core 3 subunit.