Cloning and characterization of COX18, a Saccharomyces cerevisiae PET generequired for the assembly of cytochrome oxidase

Nuclear mutants of Saccharomyces cerevisiae assigned to complementation gro up G34 are respiratory-deficient and lack cytochrome oxidase activity and t he characteristic spectral peaks of cytochromes a and a,. The corresponding gene was cloned by complementation, sequenced, and identified as reading f rame YGR062C on chromosome VII. This gene was named COX18, The COX18 gene p roduct is a polypeptide of 316 amino acids with a putative amino-terminal m itochondrial targeting sequence and predicted transmembrane domains. Respir atory chain carriers other than cytochromes a and a, and the ATPase complex are present at near wild-type levels in cox18 mutants, indicating that the mutations specifically affect cytochrome oxidase. The synthesis of Cox1p a nd Cox3p in mutant mitochondria is normal whereas Cox2p is barely detected among labeled mitochondrial polypeptides. Transcription of COX2 does not re quire COX18 function, and a chimeric COX3-COX2 mRNA did not suppress the re spiratory defect in the null mutant, indicating that the mutation does not impair transcription or translation of the mRNA. Western analysis of cytoch rome oxidase subunits shows that inactivation of the COX18 gene greatly red uces the steady state amounts of subunit 2 and results in variable decrease s in other subunits of cytochrome oxidase. A gene fusion expressing a bioti nylated form of Cox18p complements cox18 mutants. Biotinylated Cox18p is a mitochondrial integral membrane protein. These results indicate Cox18p to b e a new member of a group of mitochondrial proteins that function at a late stage of the cytochrome oxidase assembly pathway.