The mitochondrial respiratory chain (MRC) consists of four multi-protein complexes in the inner membrane of mitochondria and is the main site of cellular respiration. MRC biogenesis is facilitated by several assembly factors, many of which remain unidentified and uncharacterized. Using an integrative genomic approach, we recently identified an evolutionarily conserved role of a previously uncharacterized mitochondrial protein, Coa6, in the assembly of MRC complex IV, also known as cytochrome c oxidase (CcO). Follow-up experiments showed that the CcO assembly defect could be rescued by copper supplementation, implicating Coa6 in mitochondrial copper metabolism. However, the precise molecular function of Coa6 in CcO assembly remained unknown. To understand the function and regulation of Coa6, we have purified Coa6 to homogeneity and generated a polyclonal antiCoa6 antibody, which was used to study Coa6 regulation under different nutrient, chemical, and genetic perturbations. By measuring Coa6 protein levels under these perturbations, we found that like Cox2, a copper containing subunit of CcO, Coa6 is regulated by copper abundance, further supporting its role in copper delivery to CcO. In order to place Coa6 in the CcO copper delivery pathway, we performed a genetic epistasis analysis and found synthetic lethal interactions between Coa6 and Sco2, a well-known mitochondrial copper metallochaperone, and Coa6 and Cox12, a known subunit of CcO. These results suggest overlapping but non-redundant roles of Coa6, Cox12 and Sco2 in copper delivery to Cox2.
