Assembly of nuclear DNA‐encoded subunits into mitochondrial complex IV, and their preferential integration into supercomplex forms in patient mitochondria

Abstract

Complex IV is the terminal enzyme of the mitochondrial respiratory chain. In humans, biogenesis of complex IV involves the coordinated assembly of 13 subunits encoded by both mitochondrial and nuclear genomes. The early stages of complex IV assembly involving mitochondrial DNA‐encoded subunits CO1 and CO2 have been well studied. However, the latter stages, during which many of the nuclear DNA‐encoded subunits are incorporated, are less well understood. Using in vitro import and assembly assays, we found that subunits Cox6a, Cox6b and Cox7a assembled into pre‐existing complex IV, while Cox4‐1 and Cox6c subunits assembled into subcomplexes that may represent rate‐limiting intermediates. We also found that Cox6a and Cox7a are incorporated into a novel intermediate complex of approximately 250 kDa, and that transition of subunits from this complex to the mature holoenzyme had stalled in the mitochondria of patients with isolated complex IV deficiency. A number of complex IV subunits were also found to integrate into supercomplexes containing combinations of complex I, dimeric complex III and complex IV. Subunit assembly into these supercomplexes was also observed in mitochondria of patients in whom monomeric complex IV was selectively reduced. We conclude that newly imported nuclear DNA‐encoded subunits can integrate into the complex IV holoenzyme and supercomplex forms by associating with pre‐existing subunits and intermediate assembly complexes.

Lethal mitochondrial cardiomyopathy in a hypomorphic Med30 mouse mutant is ameliorated by ketogenic diet

“…Mitochondrial cardiomyopathy has been associated with numerous mutations in both mitochondrial and nuclear DNA-encoded genes (14,(35)(36)(37)(38). Our data indicate that genetic lesions affecting Mediator complex subunits should also be considered as a possible cause of primary cardiomyopathies in humans, particularly in pediatric cases given the importance of MED30 to early cardiac function during the period of weaning in mice.…”

A stabilizing factor for mitochondrial respiratory supercomplex assembly regulates energy metabolism in muscle

“…COX7A has been considered as a late-assembling subunit similar to COX6A ref. 29, and in vitro translated COX7A was first incorporated into complex IV and supercomplex III þ IV based on in vitro import and assembly assays 30 . Thus, it is likely that COX7A has a role in the late steps of holo-complex IV formation, as well as in the assembly of III and IV, and is located in the periphery of complex IV.…”

Mitochondrial respiratory chain dysfunction: Implications in neurodegeneration

“…Pulse-chase experiments that analyzed the time-course incorporation of the thirteen mitochondrial-encoded proteins into RC complexes and supercomplexes suggested that supercomplexes originated by the direct association of single fully-assembled complexes (Acin-Perez et al, 2008). However, the observation that, in the absence of monomeric complex IV, newly-imported nuclear subunits from this complex were preferentially integrated into supercomplexes, suggested that the respirasomes formation could also be achieved through the association of partiallyassembled complexes and free subunits (Lazarou et al, 2009). In a recent study, our group addressed the biosynthetic pathway of mitochondrial RC supercomplexes by partially depleting control cell lines of OXPHOS complexes with doxycycline, a reversible inhibitor of mitochondrial translation.…”