title: Mia40 Protein Serves as an Electron Sink in the Mia40-Erv1 Import Pathway.
abstract: A redox-regulated import pathway consisting of Mia40 and Erv1 mediates the import of cysteine-rich proteins into the mitochondrial intermembrane space. Mia40 is the oxidoreductase that inserts two disulfide bonds into the substrate simultaneously. However, Mia40 has one redox-active cysteine pair, resulting in ambiguity about how Mia40 accepts numerous electrons during substrate oxidation. In this study, we have addressed the oxidation of Tim13 in vitro and in organello. Reductants such as glutathione and ascorbate inhibited both the oxidation of the substrate Tim13 in vitro and the import of Tim13 and Cmc1 into isolated mitochondria. In addition, a ternary complex consisting of Erv1, Mia40, and substrate, linked by disulfide bonds, was not detected in vitro. Instead, Mia40 accepted six electrons from substrates, and this fully reduced Mia40 was sensitive to protease, indicative of conformational changes in the structure. Mia40 in mitochondria from the erv1-101 mutant was also trapped in a completely reduced state, demonstrating that Mia40 can accept up to six electrons as substrates are imported. Therefore, these studies support that Mia40 functions as an electron sink to facilitate the insertion of two disulfide bonds into substrates.
authors.affiliation: Department of Chemistry and Biochemistry and the Molecular Biology Institute, UCLA, Los Angeles, California 90095.
authors.forename: Sonya E
authors.initials: SE
authors.lastname: Neal
authors.affiliation: Department of Biology, Loyola Marymount University, Los Angeles, California 90045.
authors.forename: Deepa V
authors.initials: DV
authors.lastname: Dabir
authors.affiliation: Department of Chemistry and Biochemistry and the Molecular Biology Institute, UCLA, Los Angeles, California 90095.
authors.forename: Heather L
authors.initials: HL
authors.lastname: Tienson
authors.affiliation: Department of Neurology and Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, Florida 33136.
authors.forename: Darryl M
authors.initials: DM
authors.lastname: Horn
authors.affiliation: Department of Chemistry and Biochemistry and the Molecular Biology Institute, UCLA, Los Angeles, California 90095.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Glaeser
authors.affiliation: Department of Chemistry and Biochemistry and the Molecular Biology Institute, UCLA, Los Angeles, California 90095.
authors.forename: Rachel R
authors.initials: RR
authors.lastname: Ogozalek Loo
authors.affiliation: Department of Neurology and Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, Florida 33136.
authors.forename: Antoni
authors.initials: A
authors.lastname: Barrientos
authors.affiliation: Department of Chemistry and Biochemistry and the Molecular Biology Institute, UCLA, Los Angeles, California 90095. Electronic address: koehler@chem.ucla.edu.
authors.forename: Carla M
authors.initials: CM
authors.lastname: Koehler
mesh_terms: D001205:Ascorbic Acid / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004583:Electrons; D015966:Gene Expression Regulation, Fungal; D005978:Glutathione / Q000494:pharmacology; D056511:Metallochaperones / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010084:Oxidation-Reduction; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019163:Reducing Agents / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: C529981:CMC1 protein, S cerevisiae; D004220:Disulfides; C495320:MIA40 protein, S cerevisiae; D056511:Metallochaperones; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D019163:Reducing Agents; D029701:Saccharomyces cerevisiae Proteins; C403093:TIM13 protein, S cerevisiae; D050862:Oxidoreductases Acting on Sulfur Group Donors; C073790:ERV1 protein, S cerevisiae; D005978:Glutathione; D001205:Ascorbic Acid
keywords: disulfide; mitochondria; oxidation-reduction (redox); protein import; redox; redox regulation; thiol