Rcf2 revealed in cryo-EM structures of hypoxic isoforms of mature mitochondrial III-IV supercomplexes

Abstract

The organization of the mitochondrial electron transport chain proteins into supercomplexes (SCs) is now undisputed; however, their assembly process, or the role of differential expression isoforms, remain to be determined. In Saccharomyces cerevisiae, cytochrome c oxidase (CIV) forms SCs of varying stoichiometry with cytochrome bc1 (CIII). Recent studies have revealed, in normoxic growth conditions, an interface made exclusively by Cox5A, the only yeast respiratory protein that exists as one of two isoforms depending on oxygen levels. Here we present the cryo-EM structures of the III2-IV1 and III2-IV2 SCs containing the hypoxic isoform Cox5B solved at 3.4 and 2.8 Å, respectively. We show that the change of isoform does not affect SC formation or activity, and that SC stoichiometry is dictated by the level of CIII/CIV biosynthesis. Comparison of the CIV5B- and CIV5A-containing SC structures highlighted few differences, found mainly in the region of Cox5. Additional density was revealed in all SCs, independent of the CIV isoform, in a pocket formed by Cox1, Cox3, Cox12, and Cox13, away from the CIII–CIV interface. In the CIV5B-containing hypoxic SCs, this could be confidently assigned to the hypoxia-induced gene 1 (Hig1) type 2 protein Rcf2. With conserved residues in mammalian Hig1 proteins and Cox3/Cox12/Cox13 orthologs, we propose that Hig1 type 2 proteins are stoichiometric subunits of CIV, at least when within a III-IV SC.

Composition and stage dynamics of mitochondrial complexes inPlasmodium falciparum

“…Supplementary Figure 5. CIV from S.cerevisiae (95). Subunits that were lost in the evolution to P.falciparum, or whose homology to P. falciparum proteins is barely detectable, are in yellow.…”

HIGD-Driven Regulation of Cytochrome c Oxidase Biogenesis and Function

“…The C-terminal portion of Rcf2, comprising two transmembrane α-helixes and a final C-terminal α-helix extending in the intermembrane space (IMS), has been identified in hypoxic respiratory SCs reconstructed by cryo-electron microscopy (cryo-EM) [34] (Figure 2b). The transmembrane portion of Rcf2, including the HIG1 domain [36,51], forms extensive interactions with the transmembrane domains of Cox3 and Cox13, whereas the IMS α-helix, a unique feature of yeast HIGD proteins, interacts with Cox12 [34].…”

“…The C-terminal portion of Rcf2, comprising two transmembrane α-helixes and a final C-terminal α-helix extending in the intermembrane space (IMS), has been identified in hypoxic respiratory SCs reconstructed by cryo-electron microscopy (cryo-EM) [34] (Figure 2b). The transmembrane portion of Rcf2, including the HIG1 domain [36,51], forms extensive interactions with the transmembrane domains of Cox3 and Cox13, whereas the IMS α-helix, a unique feature of yeast HIGD proteins, interacts with Cox12 [34]. The relevance of the conserved HIG1 domain for Rcf2 protein function is highlighted by the observations that the Rcf2 HIG1 domain alone can interact with SCs in isolated mitochondria [61], and the expression of a truncated Rcf1 protein containing just the HIG1 domain is sufficient to support respiratory growth [36].…”

“…Indeed, the incorporation of Cox13 into CIV occurs before the binding of Rcf2 [20], and a small fraction of Rcf2 was detected co-sedimenting with Cox12-Cox13-containing CIV species upon extraction with the mild detergent digitonin [36,61], but not n-Dodecyl β-D-Maltoside (DDM) [19], suggesting a transient or labile interaction. The stable presence of Rcf2 in hypoxic SCs suggests that, at least under certain conditions, this protein may be a stable SC component required to modulate cytochrome c-mediated electron transfer of CIV enzymatic activity in response to the cellular energetic requirements [34].…”

“…The QRRQ motifs in Rcf1 and Rcf2 are marked in red. The lower panel shows the overlapping of the NMR structure of Rcf2 (PDB 6LUL) (in gold) and the partial cryo-EM structure for Rcf2 as bound to hypoxic respiratory supercomplex (SC) CIII 2 +CIV (PDB 6T15) [34] (in red). The Rcf2 transmembrane helices resolved by NMR that overlap with the Rcf2 cryo-electron microscopy (cryo-EM) structure are presented in yellow.…”