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.
Highlights
Oxidized substrates such as Tim13 acquire two disulfide bonds simultaneously, but Mia40 has one active redox center that accepts two electrons
A redox-regulated import pathway consisting of Mia40 and Erv1 mediates the import of cysteine-rich proteins into the mitochondrial intermembrane space
Reductants Inhibit Substrate Oxidation and Import—Previous studies showed that Tim13 has a single midpoint potential and that two disulfide bonds are acquired simultaneously [19]; a partially oxidized intermediate has not been detected
Summary
Oxidized substrates such as Tim acquire two disulfide bonds simultaneously, but Mia has one active redox center that accepts two electrons. Mia in mitochondria from the erv101 mutant was trapped in a completely reduced state, demonstrating that Mia can accept up to six electrons as substrates are imported These studies support that Mia functions as an electron sink to facilitate the insertion of two disulfide bonds into substrates. The Mia40/Erv import pathway in the mitochondrial intermembrane space mediates the translocation of proteins that contain disulfide bonds [2,3,4]. The N terminus contains a redox-active CPC motif that is typically oxidized and forms a transient disulfide bond with the substrate as the substrate enters the intermembrane space [16, 17]. We report that Mia can be fully reduced both in vitro and in mitochondria, indicating that Mia has the capacity to serve as an electron sink to collect electrons during the oxidation of imported substrates
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