Human COX20 cooperates with SCO1 and SCO2 to mature COX2 and promote the assembly of cytochrome c oxidase

Cytochrome coxidase (CIV) deficiency is one of the mostcommonrespiratory chain defects in patients presenting with mitochondrial encephalocardiomyopathies. CIV biogenesis is complicated by the dual genetic origin of its structural subunits, and assembly of a functional holoenzyme complex requires a large number of nucleusencoded assembly factors. In general, the functions of these assembly factors remain poorly understood, and mechanistic investigations of human CIV biogenesis have been limited by the availability of model cell lines. Here,wehaveusedsmall interferenceRNAandtranscription activator-like effectornucleases(TALENs)technology to create knockdown and knockout human cell lines, respectively, to study the function of the CIV assembly factor COX20 (FAM36A). These cell lines exhibit a severe, isolated CIV deficiency due to instability of COX2, a mitochondrion-encoded CIV subunit. Mitochondria lacking COX20 accumulate CIV subassemblies containing COX1 and COX4, similar to those detected in fibroblasts from patients carrying mutations in the COX2 copper chaperones SCO1 and SCO2. These results imply that in the absence of COX20, COX2 is inefficiently incorporated into earlyCIVsubassemblies. ImmunoprecipitationassaysusingastableCOX20knockout cell line expressing functional COX20-FLAG allowed us to identify an interaction betweenCOX20and newly synthesizedCOX2. Additionally, we show that SCO1 and SCO2 act on COX20-bound COX2. We propose that COX20 acts as a chaperone in the early steps ofCOX2maturation, stabilizing the newly synthesizedproteinandpresentingCOX2to its metallochaperone module, which in turn facilitates the incorporation of matureCOX2into the CIV assembly line.