Mutations in the COX17 gene of Saccharomyces cerevisiae cause a respiratory deficiency due to a block in the production of a functional cytochrome oxidase complex. Because cox17 mutants are able to express both the mitochondrially and nuclearly encoded subunits of cytochrome oxidase, the Cox17p most likely affects some late posttranslational step of the assembly pathway. A fragment of yeast nuclear DNA capable of complementing the mutation has been cloned by transformation of the cox17 mutant with a library of genomic DNA. Subcloning and sequencing of the COX17 gene revealed that it codes for a cysteine-rich protein with a molecular weight of 8,057. Unlike other previously described accessory factors involved in cytochrome oxidase assembly, all of which are components of mitochondria, Cox17p is a cytoplasmic protein. The cytoplasmic location of Cox17p suggested that it might have a function in delivery of a prosthetic group to the holoenzyme. A requirement of Cox17p in providing the copper prosthetic group of cytochrome oxidase is supported by the finding that a cox17 null mutant is rescued by the addition of copper to the growth medium. Evidence is presented indicating that Cox17p is not involved in general copper metabolism in yeast but rather has a more specific function in the delivery of copper to mitochondria.

C129/U1 is a respiratory defective mutant of Saccharomyces cerevisiae arrested in cytochrome oxidase assembly due to a mutation in COX17, a nuclear gene encoding a low molecular weight cytoplasmic protein proposed to function in mitochondrial copper recruitment. In the present study we show that the respiratory defect of C129/U1 is rescuable by two multicopy suppressors, SCO1 and SCO2. SCO1 was earlier reported to code for a mitochondrial inner membrane protein with an essential function in cytochrome oxidase assembly (Buchwald, P., Krummeck, G., and Rodel, G. (1991) Mol. Gen. Genet. 229, 413-420). SCO2 is a homologue of SCO1, whose product is also localized in the mitochondrial membrane but is not required for respiration.SCO1 also suppresses a cox17 null mutant, indicating that overexpression of Sco1p can compensate for the absence of Cox17p. In contrast, neither copper, COX17 on a multicopy plasmid, or a combination of the two is able to restore respiration in sco1 mutants. Rescue of cox17 mutants by Sco1p suggests that this mitochondrial protein plays a role either in mitochondrial copper transport or insertion of copper into the active site of cytochrome oxidase. Although SCO2 can also partially restore respiratory growth in the cox17 null mutant, rescue in this case requires addition of copper to the growth medium. SCO2 does not suppress a sco1 null mutant, although it is able to partially rescue a sco1 point mutant. We interpret the ability of SCO2 to restore respiration in cox17, but not in sco1 mutants, to indicate that Sco1p and Sco2p have overlapping but not identical functions.

Ubiquinone (coenzyme Q or Q) is a lipophilic metabolite that functions in the electron transport chain in the plasma membrane of prokaryotes and in the inner mitochondrial membrane of eukaryotes. Q-deficient mutants of Saccharomyces cerevisiae fall into eight complementation groups (coq1-coq8). Yeast mutants from the coq5 complementation group lack Q and as a result are respiration-defective and fail to grow on nonfermentable carbon sources. A nuclear gene, designated COQ5 was isolated from a yeast genomic library based on its ability to restore growth of a representative coq5 mutant on media containing glycerol as the sole carbon source. The DNA segment responsible for the complementation contained an open reading frame (GenBank TM accession number Z49210) with 44% sequence identity over 262 amino acids to UbiE, which is required for a C-methyltransferase step in the Q and menaquinone biosynthetic pathways in Escherichia coli. Both the ubiE and COQ5 coding sequences contain sequence motifs common to a wide variety of S-adenosyl-L-methionine-dependent methyltransferases. A gene fusion expressing a biotinylated form of Coq5p retains function, as assayed by the complementation of the coq5 mutant. This Coq5-biotinylated fusion protein is located in mitochondria.The synthesis of two farnesylated analogs of intermediates in the ubiquinone biosynthetic pathway is reported. These reagents have been used to develop in vitro C-methylation assays with isolated yeast mitochondria. These studies show that Coq5p is required for the C-methyltransferase step that converts 2-methoxy-6-polyprenyl-1,4-benzoquinone to 2-methoxy-5-methyl-6-polyprenyl-1,4-benzoquinone.

Three complementation groups of a pet mutant collection have been found to be composed of respiratory-deficient deficient mutants with lesions in mitochondrial protein synthesis. Recombinant plasmids capable of restoring respiration were cloned by transformation of representatives of each complementation group with a yeast genomic library. The plasmids were used to characterize the complementing genes and to institute disruption of the chromosomal copies of each gene in respiratory-proficient yeast. The sequences of the cloned genes indicate that they code for isoleucyl-, arginyl- and glutamyl-tRNA synthetases. The properties of the mutants used to obtain the genes and of strains with the disrupted genes indicate that all three aminoacyl-tRNA synthetases function exclusively in mitochondrial proteins synthesis. The ISM1 gene for mitochondrial isoleucyl-tRNA synthetase has been localized to chromosome XVI next to UME5. The MSR1 gene for the arginyl-tRNA synthetase was previously located on yeast chromosome VIII. The third gene MSE1 for the mitochondrial glutamyl-tRNA synthetase has not been localized. The identification of three new genes coding for mitochondrial-specific aminoacyl-tRNA synthetases indicates that in Saccharomyces cerevisiae at least 11 members of this protein family are encoded by genes distinct from those coding for the homologous cytoplasmic enzymes.

Three complementation groups of a pet mutant collection have been found to be composed of respiratory‐deficient mutants with lesions in mitochondrial protein synthesis. Recombinant plasmids capable of restoring respiration were cloned by transformation of representatives of each complementation group with a yeast genomic library. The plasmids were used to characterize the complementing genes and to institute disruption of the chromosomal copies of each gene in respiratory‐proficient yeast. The sequences of the cloned genes indicate that they code for isoleucyl‐, arginyl‐ and glutamyl‐tRNA synthetases. The properties of the mutants used to obtain the genes and of strains with the disrupted genes indicate that all three aminoacyl‐tRNA synthetases function exclusively in mitochondrial proteins synthesis. The ISMI gene for mitochondrial isoleucyl‐tRNA synthetase has been localized to chromosome XVI next to UME5. The MSR1 gene for the arginyl‐tRNA synthetase was previously located on yeast chromosome VIII. The third gene MSE1 for the mitochondrial glutamyl‐tRNA synthetase has not been localized. The identification of three new genes coding for mitochondrial‐specific aminoacyl‐tRNA synthetases indicates that in Saccharomyces cerevisiae at least 11 members of this protein family are encoded by genes distinct from those coding for the homologous cytoplasmic enzymes.