eprintid: 10095020 rev_number: 24 eprint_status: archive userid: 608 dir: disk0/10/09/50/20 datestamp: 2020-04-27 07:50:02 lastmod: 2021-10-06 22:54:57 status_changed: 2020-04-27 07:50:02 type: article metadata_visibility: show creators_name: Hartley, AM creators_name: Meunier, B creators_name: Pinotsis, N creators_name: Maréchal, A title: Rcf2 revealed in cryo-EM structures of hypoxic isoforms of mature mitochondrial III-IV supercomplexes ispublished: inpress divisions: UCL divisions: B02 divisions: C08 divisions: D09 divisions: G03 keywords: Hig1 proteins, bioenergetics, cytochrome c oxidase, electron transport chain, respiratory supercomplexes note: © 2020 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND). 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. date: 2020-04-14 date_type: published official_url: https://doi.org/10.1073/pnas.1920612117 oa_status: green full_text_type: pub language: eng primo: open primo_central: open_green verified: verified_manual elements_id: 1776723 doi: 10.1073/pnas.1920612117 pii: 1920612117 lyricists_name: MARECHAL, Amandine lyricists_id: AMARE34 actors_name: Flynn, Bernadette actors_id: BFFLY94 actors_role: owner full_text_status: public publication: Proceedings of the National Academy of Sciences event_location: United States issn: 1091-6490 citation: Hartley, AM; Meunier, B; Pinotsis, N; Maréchal, A; (2020) Rcf2 revealed in cryo-EM structures of hypoxic isoforms of mature mitochondrial III-IV supercomplexes. Proceedings of the National Academy of Sciences 10.1073/pnas.1920612117 <https://doi.org/10.1073/pnas.1920612117>. (In press). Green open access document_url: https://discovery.ucl.ac.uk/id/eprint/10095020/7/1920612117.full.pdf