SILAC-based complexome profiling dissects the structural organization of the human respiratory supercomplexes in SCAFIKO cells

The study of the mitochondrial respiratory chain (MRC) function in relation with its structural organization is of great interest due to the central role of this system in eukaryotic cell metabolism. The complexome profiling technique has provided invaluable information for our understanding of the...

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Published in:Biochimica et biophysica acta. Bioenergetics 2021-07, Vol.1862 (7), p.148414-148414, Article 148414
Main Authors: Fernández-Vizarra, Erika, López-Calcerrada, Sandra, Formosa, Luke E., Pérez-Pérez, Rafael, Ding, Shujing, Fearnley, Ian M., Arenas, Joaquín, Martín, Miguel A., Zeviani, Massimo, Ryan, Michael T., Ugalde, Cristina
Format: Article
Language:English
Subjects:
COX7A2
COX7A2L/SCAFI
Mitochondria
oxidative phosphorylation
respiratory chain
respiratory supercomplexes
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Summary:The study of the mitochondrial respiratory chain (MRC) function in relation with its structural organization is of great interest due to the central role of this system in eukaryotic cell metabolism. The complexome profiling technique has provided invaluable information for our understanding of the composition and assembly of the individual MRC complexes, and also of their association into larger supercomplexes (SCs) and respirasomes. The formation of the SCs has been highly debated, and their assembly and regulation mechanisms are still unclear. Previous studies demonstrated a prominent role for COX7A2L (SCAFI) as a structural protein bridging the association of individual MRC complexes III and IV in the minor SC III2 + IV, although its relevance for respirasome formation and function remains controversial. In this work, we have used SILAC-based complexome profiling to dissect the structural organization of the human MRC in HEK293T cells depleted of SCAFI (SCAFIKO) by CRISPR-Cas9 genome editing. SCAFI ablation led to a preferential loss of SC III2 + IV and of a minor subset of respirasomes without affecting OXPHOS function. Our data suggest that the loss of SCAFI-dependent respirasomes in SCAFIKO cells is mainly due to alterations on early stages of CI assembly, without impacting the biogenesis of complexes III and IV. Contrary to the idea of SCAFI being the main player in respirasome formation, SILAC-complexome profiling showed that, in wild-type cells, the majority of respirasomes (ca. 70%) contained COX7A2 and that these species were present at roughly the same levels when SCAFI was knocked-out. We thus demonstrate the co-existence of structurally distinct respirasomes defined by the preferential binding of complex IV via COX7A2, rather than SCAFI, in human cultured cells. •SILAC-CP allows a comprehensive view of the human MRC in SCAFIKO HEK293T cells.•SCAFI loss does not trigger major metabolic adaptations in human HEK293T cells.•SCAFI loss induces subtle early CI assembly defects without affecting CIII and CIV.•In human cultured cells most respirasomes contain COX7A2 instead of SCAFI.
ISSN:0005-2728
1879-2650
DOI:10.1016/j.bbabio.2021.148414