A Genetic Dissection of Mitochondrial Respiratory Chain Biogenesis

Mitochondria house the mitochondrial respiratory chain (MRC), the main site for cellular respiration and energy production. The MRC consists of five large protein complexes (I-V) in the inner mitochondrial membrane. Despite the fundamental role of the MRC in cellular energy generation and its involvement in several human diseases, we still do not know all the proteins required for its formation. To address this gap in our knowledge, we have developed an integrative approach based on clues from evolutionary history and protein localization to shortlist 56 uncharacterized proteins that are physically localized to the mitochondria of yeast and humans. A recent study on mitochondrial disease patients identified potential pathogenic mutations in one of our prioritized candidate genes, C1orf31, suggesting its involvement in MRC biogenesis. Due to the lack of an assigned function to C1orf31, it was not possible to prove the pathogenicity of the patient mutations. Therefore, our study focused on determining the role of C1orf31 in MRC biogenesis. We identified a yeast, Saccharomyces cerevisiae, ortholog of C1orf31, named Coa6, by BLAST analysis, allowing us to use this genetically tractable model system to quickly decipher the protein’s function. Using coa6Δ yeast cells, we show that Coa6 is required for respiratory growth, cellular respiration, and MRC complex IV biogenesis. A sequence analysis of Coa6 identified a conserved, non-canonical, putative copper-binding motif, suggesting its role in copper delivery to MRC complex IV, the only copper-containing MRC complex. Indeed, copper supplementation rescues the respiratory defect of coa6Δ yeast cells, while copper starvation exacerbates the respiratory growth phenotype. Furthermore, we show that conserved residues in the putative copper-binding motif, including the residues mutated in the human mitochondrial disease patient, are essential for Coa6 function, thus confirming the pathogenicity of the patient mutations. Based on these results, we hypothesize that Coa6 is a mitochondrial copper metallochaperone required for delivery of copper to MRC complex IV. In support of this hypothesis, we show that the coa6Δ phenotype is exacerbated when the gene for another known copper metallochaperone, Sco2, is deleted, indicating that these two proteins have an overlapping function in delivering copper to MRC complex IV