title: Mitochondrial thiol oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles.
abstract: Erv1 (essential for respiration and viability 1), is an essential component of the MIA (mitochondrial import and assembly) pathway, playing an important role in the oxidative folding of mitochondrial intermembrane space proteins. In the MIA pathway, Mia40, a thiol oxidoreductase with a CPC motif at its active site, oxidizes newly imported substrate proteins. Erv1 a FAD-dependent thiol oxidase, in turn reoxidizes Mia40 via its N-terminal Cys30-Cys33 shuttle disulfide. However, it is unclear how the two shuttle cysteine residues of Erv1 relay electrons from the Mia40 CPC motif to the Erv1 active-site Cys130-Cys133 disulfide. In the present study, using yeast genetic approaches we showed that both shuttle cysteine residues of Erv1 are required for cell growth. In organelle and in vitro studies confirmed that both shuttle cysteine residues were indeed required for import of MIA pathway substrates and Erv1 enzyme function to oxidize Mia40. Furthermore, our results revealed that the two shuttle cysteine residues of Erv1 are functionally distinct. Although Cys33 is essential for forming the intermediate disulfide Cys33-Cys130' and transferring electrons to the redox active-site directly, Cys30 plays two important roles: (i) dominantly interacts and receives electrons from the Mia40 CPC motif; and (ii) resolves the Erv1 Cys33-Cys130 intermediate disulfide. Taken together, we conclude that both shuttle cysteine residues are required for Erv1 function, and play complementary, but distinct, roles to ensure rapid turnover of active Erv1.
authors.affiliation: *Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K.
authors.forename: Swee Kim
authors.initials: SK
authors.lastname: Ang
authors.affiliation: *Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K.
authors.forename: Mengqi
authors.initials: M
authors.lastname: Zhang
authors.affiliation: †Department of Life Sciences, University of Parma, I-43100 Parma, Italy.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Lodi
authors.affiliation: *Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K.
authors.forename: Hui
authors.initials: H
authors.lastname: Lu
mesh_terms: D000595:Amino Acid Sequence; D003545:Cysteine / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism*; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004220:Disulfides; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors; C073790:ERV1 protein, S cerevisiae; D003545:Cysteine