The mitochondrial respiratory chain enzymes are organized as individual complexes and supercomplexes, whose biogenesis remains to be fully understood. To disclose the role of the human Hypoxia Inducible Gene Domain family proteins HIGD1A and HIGD2A in these processes, we generate and characterize HIGD-knockout (KO) cell lines. We show that HIGD2A controls and coordinates the modular assembly of isolated and supercomplexed complex IV (CIV) by acting on the COX3 assembly module. In contrast, HIGD1A regulates CIII and CIII-containing supercomplex biogenesis by supporting the incorporation of UQCRFS1. HIGD1A also clusters with COX4-1 and COX5A CIV subunits and, when overexpressed, suppresses the CIV biogenesis defect of HIGD2A-KO cells. We conclude that HIGD1A and HIGD2A have both independent and overlapping functions in the biogenesis of respiratory complexes and supercomplexes. Our data illuminate the existence of multiple pathways to assemble these structures by dynamic HIGD-mediated CIV biogenesis, potentially to adapt to changing environmental and nutritional conditions.

The mitochondrial respiratory chain is organized in a dynamic set of supercomplexes (SCs). The COX7A2L protein is essential for mammalian SC III2+IV assembly. However, its function in respirasome (SCs I+III2+IVn) biogenesis remains controversial. To unambiguously determine the COX7A2L role, we generated COX7A2L-knockout (COX7A2L-KO) HEK293T and U87 cells. COX7A2L-KO cells lack SC III2+IV but have enhanced complex III steady-state levels, activity, and assembly rate, normal de novo complex IV biogenesis, and delayed respirasome formation. Nonetheless, the KOs have normal respirasome steady-state levels, and only larger structures (SCs I1-2+III2+IV2-n or megacomplexes) were undetected. Functional substrate-driven competition assays showed normal mitochondrial respiration in COX7A2L-KO cells in standard and nutritional-, environmental-, and oxidative-stress-challenging conditions. We conclude that COX7A2L establishes a regulatory checkpoint for the biogenesis of CIII2 and specific SCs, but the COX7A2L-dependent MRC remodeling is essential neither to maintain mitochondrial bioenergetics nor to cope with acute cellular stresses.

By using negatively charged Coomassie brilliant blue G-250 dye to induce a charge shift on proteins, blue native polyacrylamide gel electrophoresis (BN-PAGE) allows resolution of enzymatically active multiprotein complexes extracted from cellular or subcellular lysates while retaining their native conformation. BN-PAGE was first developed to analyze the size, composition, and relative abundance of the complexes and supercomplexes that form the mitochondrial respiratory chain and OXPHOS system. Here, we present a detailed protocol of BN-PAGE to obtain robust and reproducible results.

Mpc proteins are highly conserved from yeast to humans and are necessary for the uptake of pyruvate at the inner mitochondrial membrane, which is used for leucine and valine biosynthesis and as a fuel for respiration. Our analysis of the yeast MPC gene family suggests that amino acid biosynthesis, respiration rate and oxidative stress tolerance are regulated by changes in the Mpc protein composition of the mitochondria. Mpc2 and Mpc3 are highly similar but functionally different: Mpc2 is most abundant under fermentative non stress conditions and important for amino acid biosynthesis, while Mpc3 is the most abundant family member upon salt stress or when high respiration rates are required. Accordingly, expression of the MPC3 gene is highly activated upon NaCl stress or during the transition from fermentation to respiration, both types of regulation depend on the Hog1 MAP kinase. Overexpression experiments show that gain of Mpc2 function leads to a severe respiration defect and ROS accumulation, while Mpc3 stimulates respiration and enhances tolerance to oxidative stress. Our results identify the regulated mitochondrial pyruvate uptake as an important determinant of respiration rate and stress resistance.