1. Mia40 Protein Serves as an Electron Sink in the Mia40-Erv1 Import Pathway*
- Author
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Neal, Sonya E, Dabir, Deepa V, Tienson, Heather L, Horn, Darryl M, Glaeser, Kathrin, Ogozalek Loo, Rachel R, Barrientos, Antoni, and Koehler, Carla M
- Subjects
Biochemistry and Cell Biology ,Biological Sciences ,Ascorbic Acid ,Disulfides ,Electrons ,Gene Expression Regulation ,Fungal ,Glutathione ,Metallochaperones ,Mitochondria ,Mitochondrial Membrane Transport Proteins ,Mitochondrial Precursor Protein Import Complex Proteins ,Mitochondrial Proteins ,Mutation ,Oxidation-Reduction ,Oxidoreductases Acting on Sulfur Group Donors ,Plasmids ,Protein Transport ,Recombinant Proteins ,Reducing Agents ,Saccharomyces cerevisiae ,Saccharomyces cerevisiae Proteins ,Signal Transduction ,disulfide ,mitochondria ,oxidation-reduction ,protein import ,redox ,redox regulation ,thiol ,Chemical Sciences ,Medical and Health Sciences ,Biochemistry & Molecular Biology ,Biological sciences ,Biomedical and clinical sciences ,Chemical sciences - 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.
- Published
- 2015