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Redox Characterization of Proteins Involved in the Mitochondrial Intermembrane Space Pathway

  • Author(s): Neal, Sonya Elina
  • Advisor(s): Koehler, Carla M
  • et al.
Abstract

A redox-regulated import pathway consisting of Mia40 and Erv1 was identified to mediate the import of cysteine-rich proteins into the mitochondrial intermembrane space. Mia40 mediates the import and oxidative folding of substrates and Erv1 is a putative oxidant for Mia40. The mechanism involved in the oxidation of substrates and subsequent release from Mia40 is not clear. In this study, we have reconstituted the disulfide exchange reaction in vitro using Tim13 as the substrate. By reconstituting the Mia40-Erv1 import pathway with cysteine mutants of Tim13, we were able to determine that C57, C61 and C73 are required for oxidative folding by Mia40. Moreover, we show that Tim13 is able to induce a conformational change and reduce 6 cysteines in Mia40. Finally, in organello studies confirmed that in yeast strains deficient in Erv1, Mia40 remains in a reduced state. In all, these experiments suggest that Mia40 is able to act as an electron sink in which it accumulates electrons in the presence of Tim13.

Subsequently, the Mia40-Erv1 import pathway has electron acceptors to continue the process of disulfide bond formation. The aerobic electron acceptors include oxygen and cytochrome c, but an anaerobic acceptor has not been identified. Here we show that the fumarate reductase Osm1, which transfers electrons from fumarate to generate succinate, fills this gap as a new electron acceptor. In addition to microsomes, Osm1 localizes to the IMS and assembles with Erv1 in a complex. In reconstitution studies, Erv1 transfers electrons to Osm1 in the oxidation of Tim13. Comparative assays support that Osm1 accepts electrons with similar efficiency as cytochrome c and that the cell has strategies to coordinate expression of the terminal electron acceptors. Thus, Osm1 is a new terminal electron acceptor in the IMS that may also have a similar function in the endoplasmic reticulum.

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