PURIFICATION, CHARACTERIZATION, AND LOCALIZATION OF YEAST COX17P, A MITOCHONDRIAL COPPER SHUTTLE

Cox17p was previously shown to be essential for the expression of cyto chrome oxidase in Saccharomyces cerevisiae. In the present study COX17 has been placed under the control of the GAL10 promoter in an autonom ously replicating plasmid. A yeast transformant harboring the high cop y construct was used to purify Cox17p to homogeneity. Purified Cox17p contains 0.2-0.3 mol of copper per mol of protein. The molar copper co ntent is increased to 1.8 after incubation of Cox17p in the presence o f a 6-fold molar excess of cuprous chloride under reduced conditions. An antibody against Cox17p was obtained by immunization of rabbits wit h a carboxyl-terminal peptide coupled to bovine serum albumin. The ant iserum detects Cox17p in both the mitochondrial and soluble protein fr actions of wild type yeast and of the transformant overexpressing Cox1 7p. Exposure of intact mitochondria to hypotonic conditions causes mos t of Cox17p to be released as a soluble protein indicating that the mi tochondrial fraction of Cox17p is localized in the intermembrane space . These results are consistent with the previously proposed function o f Cox17p, namely in providing cytoplasmic copper for mitochondrial uti lization.