Mitochondrial disease genesCOA6,COX6BandSCO2have overlapping roles in COX2 biogenesis

Abstract

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.

The mitochondrion: a central architect of copper homeostasis

“…55 In retrospect, these findings are not that surprising given the relative severity of the copper deficiency in various SCO1 cell types, 45 and the fact that SCO1 catalyzes the transfer of copper to the Cu A site of COX2 in a reaction that is facilitated by SCO2 and COA6. 54, 56, 58, 87 These observations in fact collectively support the idea that SCO1 sits at the head of a mitochondrial pathway, and that its functional status directly impinges upon COX assembly and the generation of a signal that regulates copper homeostasis (Figure 2). 23, 24 The exact identity of this mitochondrial signal and how it is transduced to copper handling machinery localized elsewhere in the cell is not yet known.…”

“…28 Its copper is coordinated via a Cx 3 C motif and a conserved histidine residue, 51–53 and is transferred directly to the Cu A site of COX2 54 in a reaction that also requires the COX assembly factors SCO2 and COA6 (Figure 1). 55–58 Interestingly, a multitude of other IMS-localized proteins containing canonical twin Cx 9 C motifs have been implicated in COX assembly in yeast, 59 many of which have evolutionarily conserved mammalian homologues with undefined functions. We fully expect that future studies will identify members of this protein family that fulfill unique roles in delivering copper to COX, prioritizing copper utilization within the IMS, and regulating the generation and transduction of a copper-dependent, mitochondrial signal.…”

Cox2p of yeast cytochrome oxidase assembles as a stand-alone subunit with the Cox1p and Cox3p modules

“…The presence of Cox20p in B4 and of Cox18p in both B3 and B4 suggests that the interaction of Cox18p with the Cox2p precursor precedes that of Cox20p. Yeast Sco1p and Coa6p, like their human counterparts, form complexes with Cox2p intermediates (39,40). In both cases the interactions appear to be transient as only a small fraction of the newly translated Cox2p co-immunopurified with tagged Sco1p and Coa6p.…”

Biochemical pathway for the biosynthesis of the CuA center in bacterial cytochrome c oxidase

“…Work by Morgada et al . showed that Sco2 functions as a reductase for both, Sco1 and Cox2 (CoxB‐like), in a process that is possibly assisted by yet another assembly factor, COA6 . Finally, investigating Cu A synthesis in vitro with a bacterial sytem, Abriata et al .…”

“…More specifically, the intermembrane space protein Cox17 was described not only as a metallochaperone transferring Cu 1+ to Sco1 (ScoI-like) [47], but also as a possible reductase for the copperbinding site of Sco1 [48]. Work by Morgada et al [39] showed that Sco2 functions as a reductase for both, Sco1 and Cox2 (CoxB-like), in a process that is possibly assisted by yet another assembly factor, COA6 [49]. Finally, investigating Cu A synthesis in vitro with a bacterial sytem, Abriata et al [34] reported that a ScoI-like protein was fully sufficient to reduce the oxidized Cu A -binding site in ba 3 -type Cox subunit II of T. thermophilus.…”