Mitochondrial disease genes COA6, COX6B and SCO2 have overlapping roles in COX2 biogenesis.

Biogenesis of cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain, is a complex process facilitated by several assembly factors. Pathogenic mutations were recently reported in one such assembly factor, COA6, and our previous work linked Coa6 function to mitochondrial copper metabolism and expression of Cox2, a copper-containing subunit of CcO. However, the precise role of Coa6 in Cox2 biogenesis remained unknown. Here we show that yeast Coa6 is an orthologue of human COA6, and like Cox2, is regulated by copper availability, further implicating it in copper delivery to Cox2. In order to place Coa6 in the Cox2 copper delivery pathway, we performed a comprehensive genetic epistasis analysis in the yeast Saccharomyces cerevisiae and found that simultaneous deletion of Coa6 and Sco2, a mitochondrial copper metallochaperone, or Coa6 and Cox12/COX6B, a structural subunit of CcO, completely abrogates Cox2 biogenesis. Unlike Coa6 deficient cells, copper supplementation fails to rescue Cox2 levels of these double mutants. Overexpression of Cox12 or Sco proteins partially rescues the coa6Δ phenotype, suggesting their overlapping but non-redundant roles in copper delivery to Cox2. These genetic data are strongly corroborated by biochemical studies demonstrating physical interactions between Coa6, Cox2, Cox12 and Sco proteins. Furthermore, we show that patient mutations in Coa6 disrupt Coa6-Cox2 interaction, providing the biochemical basis for disease pathogenesis. Taken together, these results place COA6 in the copper delivery pathway to CcO and, surprisingly, link it to a previously unidentified function of CcO subunit Cox12 in Cox2 biogenesis.