Mia40 Protein Serves as an Electron Sink in the Mia40-Erv1 Import Pathway

• Published in: The Journal of biological chemistry 2015-08, Vol.290 (34), p.20804-20814
• Main Authors: Neal, Sonya E., Dabir, Deepa V., Tienson, Heather L., Horn, Darryl M., Glaeser, Kathrin, Ogozalek Loo, Rachel R., Barrientos, Antoni, Koehler, Carla M.
• Format: Article
• Language: English
• Subjects: Ascorbic Acid - pharmacology; disulfide; Disulfides - chemistry; Disulfides - metabolism; Electrons; Gene Expression Regulation, Fungal; Glutathione - pharmacology; Membrane Biology; Metallochaperones - genetics; Metallochaperones - metabolism; mitochondria; Mitochondria - drug effects; Mitochondria - metabolism; Mitochondrial Membrane Transport Proteins - genetics; Mitochondrial Membrane Transport Proteins - metabolism; Mitochondrial Proteins - genetics; Mitochondrial Proteins - metabolism; Mutation; Oxidation-Reduction; oxidation-reduction (redox); Oxidoreductases Acting on Sulfur Group Donors - genetics; Oxidoreductases Acting on Sulfur Group Donors - metabolism; Plasmids - chemistry; Plasmids - metabolism; protein import; Protein Transport; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; redox; redox regulation; Reducing Agents - pharmacology; Saccharomyces cerevisiae - drug effects; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Signal Transduction; thiol
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M115.669440

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. Background: Oxidized substrates such as Tim13 acquire two disulfide bonds simultaneously, but Mia40 has one active redox center that accepts two electrons. Results: Mia40 can acquire up to six electrons when oxidizing substrates. Conclusion: Mia40 has the flexibility to accept several electrons. Significance: Mechanistic properties of the MIA pathway are unique compared with redox pathways in the endoplasmic reticulum and bacterial periplasm.