Human mitochondrial cytochrome c oxidase assembly factor COX18 acts transiently as a membrane insertase within the subunit 2 maturation module

Abstract

Edited by John M. Denu Defects in mitochondrial cytochrome c oxidase or respiratory chain complex IV (CIV) assembly are a frequent cause of human mitochondrial disorders. Specifically, mutations in four conserved assembly factors impinging the biogenesis of the mitochondrion-encoded catalytic core subunit 2 (COX2) result in myopathies. These factors afford stability of newly synthesized COX2 (the dystonia-ataxia syndrome protein COX20), a protein with two transmembrane domains, and maturation of its copper center, Cu A (cardiomyopathy proteins SCO1, SCO2, and COA6). COX18 is an additional COX2 assembly factor that belongs to the Oxa1 family of membrane protein insertases. Here, we used a gene-editing approach to generate a human COX18 knock-out HEK293T cell line that displays isolated complete CIV deficiency. We demonstrate that COX20 stabilizes COX2 during insertion of its N-proximal transmembrane domain, and subsequently, COX18 transiently interacts with COX2 to promote translocation across the inner membrane of the COX2 C-tail that contains the apo-Cu A site. The release of COX18 from this complex coincides with the binding of the SCO1-SCO2-COA6 copper metallation module to COX2-COX20 to finalize COX2 biogenesis. Therefore, COX18 is a new candidate when screening for mitochondrial disorders associated with isolated CIV deficiency.In nucleated human cells, mitochondria are central organelles that harbor the enzymes required for cellular respiration and aerobic energy production through oxidative phosphorylation (OXPHOS).2 These multimeric complexes are formed by subunits encoded in the nuclear and mitochondrial genomes that are assembled into functional units with the assistance of a myriad of nucleus-encoded ancillary proteins, generally called assembly factors. Mutations in OXPHOS subunits or assembly factors result in failure to fulfill the energy demands of most tissues with a major incidence in those with high energy

Building the CuA site of cytochrome c oxidase: A complicated, redox-dependent process driven by a surprisingly large complement of accessory proteins

“…However, OXA1L knockdown in HEK293 cells leads to a diminution in the abundance of Complexes I and V rather than COX (44). This has led to the suggestion that in mammals COX20 may be sufficient for insertion of the N-terminal transmembrane domain of COX2, given that its hydrophilic N-terminus is shorter than that of yeast Cox2 and does not require proteolytic processing upon membrane insertion (45). Further studies of OXA1L are required to discriminate between these distinct mechanistic possibilities, but are challenged by its broad function as an insertase of subunits of multiple complexes of oxidative phosphorylation.…”

“…In yeast, deletion of COX18 stalls Cox2 maturation and leads to the accumulation of a species in which the N-terminal transmembrane domain is inserted into the membrane and the leader sequence has been cleaved (50). Deletion of COX18 in human cells similarly leads to the accumulation of a stalled COX2-COX20 complex (45). Although COX18 belongs to the OXA1 protein family (50), COX2 is its only known substrate in both yeast and mammals (45).…”

“…Deletion of COX18 in human cells similarly leads to the accumulation of a stalled COX2-COX20 complex (45). Although COX18 belongs to the OXA1 protein family (50), COX2 is its only known substrate in both yeast and mammals (45). Yeast experiments in which Oxa1 was overexpressed in a COX18 null strain failed to restore growth on a nonfermentable carbon source even though some export of the C-terminal tail of Cox2 was observed, emphasizing that Cox18 translocates the tail across the membrane and delivers it in a state competent for metallation of its Cu A site (51).…”

“…These results led Shoubridge and coworkers to propose that SCO2 acts upstream of SCO1 to stabilize newly synthesized COX2, and that the subsequent maturation of COX2 is contingent upon the formation of a ternary complex containing both SCO proteins and COX2 (31). However, it is now clear that another small, soluble twin Cx 9 C motif-containing protein, COA6, is also an essential part of the metallochaperone module that associates with the COX20-COX2 complex to catalyze copper insertion into the Cu A site of COX2 (23,45,69,74,78) (Figure 3).…”

“…Following the export of its Cterminal tail into the IMS, COX2 remains bound to COX20 and is competent for Cu A site metallation (29,45). Physical interaction studies indicate that dissociation of COX18 from the COX20-COX2 complex coincides with the recruitment of a metallochaperone module composed of SCO1, SCO2 and COA6 (45).…”