Impact of COX7A2L in the biogenesis of human mitochondrial respiratory chain supercomplexes

Resumo

Mitochondrial respiratory chain (MRC) complexes I, III and IV are associated in large supramolecular structures termed supercomplexes (SCs) and respirasomes, whose biogenesis and functional and pathophysiological relevance remain unclear. Such superstructures originate interdependences between the individual OXPHOS complexes, with major biomedical implications for the diagnosis of mitochondrial disorders because combined deficiencies of these complexes can be attributed to a genetic defect of a single MRC complex. Furthermore, the functional implication of the mitochondrial protein COX7A2L / COX7RP / SCAFI in the biogenesis of SCs is a subject of intense and controversial debate. To elucidate the role of COX7A2L in the structural organization of human MRC, blue native electrophoresis, proteomic analyses and COX7A2L downregulation assays were performed in control and mutant transmitochondrial cybrids lacking one of each MRC complexes. Our results showed that human COX7A2L binds primarily to the SC-unbound complex III dimer (CIII2) and to a minor extent to free complex IV, to specifically promote the stabilization of the SC III2+IV without affecting the respirasomes formation. Further analyses in TALEN-mediated COX7A2L knockout (COX7A2L-KO) HEK293T cells demonstrated that the lack of COX7A2L prevents SC III2+IV formation without affecting de novo complex IV biogenesis, enhances the biogenetic rates of CIII2, and delays the formation of the complex III-containing respirasomes that, nonetheless, accumulate to control steady-state levels. Functional substrate competition assays revealed that, upon the induction of complex I deficiency, COX7A2L-KO cells significantly favour electron flux through complex II. Altogether, our data suggest that COX7A2L establishes a regulatory checkpoint for the biogenesis of CIII2 and complex III-containing SCs that limits succinate oxidation when complex I activity is compromised, and that independent regulatory mechanisms co-exist for the biogenesis of SC III2+IV and the respirasomes. The primary role of COX7A2L in the biogenesis of complex III-related MRC structures was confirmed through comparative analyses of the assembly and composition of human SCs between control cells and two mutant cybrids lacking complex IV due to pathogenic nonsense mutations in the COX1 and COX2 subunits, respectively. In the absence of complex IV, three different bands corresponding to fully-assembled SC I+III2 were identified, which also contained COX7A2L. Surprisingly, two of these SC I+III2 bands showed the presence of specific complex IV subunits that regularly take part in late steps of complex IV assembly. Reversible inhibition of mitochondrial translation demonstrated that the biogenesis of COX7A2L-containing SC I+III2 is completed prior to the stepwise association of complex IV subunits. Radioactive pulse-chase analyses additionally showed that the insertion of COX subunits is essential to stabilize the COX7A2L-containing SC I+III2. Our results thus reveal the existence of alternative COX assembly lines for the synthesis of free- versus SC-associated complex IV.