CLONING AND CHARACTERIZATION OF COX14, WHOSE PRODUCT IS REQUIRED FOR ASSEMBLY OF YEAST CYTOCHROME-OXIDASE

Nuclear respiration-deficient mutants of Saccharomyces cerevisiae previously assigned to complementation group G93 lack cytochromes a and a(3) and detectable cytochrome oxidase activity, Other respiratory chain carriers and the ATPase complex are present at near wild-type levels, indicating that the mutations specifically affect cytochrome oxidase, Since synthesis of the mitochondrially derived subunits 1, 2, and 3 of cytochrome oxidase is normal, the defect cannot be related to transcription of the endogenous genes or processing and translation of the corresponding RNAs, The results of Western analysis of the cytochrome oxidase subunits encoded in nuclear DNA also argues against an effect of the mutations on expression of these constituents. The G93 mutants are complemented by a nuclear gene, designated COX14, The product of this gene is a low molecular mass protein of 7,960 Da, A gene fusion expressing a biotinylated form of Cox14p complements cox14 mutants, indicating partial functional equivalence. The biotinylated derivative has been helpful in localizing Cox14p to the mitochondrial membrane and demonstrating that it is not a hitherto unrecognized subunit of cytochrome oxidase, although it does appear to be associated with a high molecular weight complex, This evidence, combined with the assembly-arrested phenotype of cox14 mutants, indicates that Cox14p, like several other recently described mitochondrial constituents, provides an important function at some late stage of the cytochrome oxidase assembly pathway.