Thiol Switches Controlling Mitochondrial Protein Biogenesis

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The biogenesis of mitochondria requires the import of hundreds of different protein precursors from the cytosol into the organelle. Two major import pathways were identified in the past, the preprotein import pathway leading into the matrix and the Mia40 pathway leading into the intermembrane space. During the previous funding period, we identified a critical disulfide bond in Tim17, a central subunit of the inner membrane translocase. Tim17 is the first membrane protein that was identified as a substrate of the mitochondrial oxidation machinery. The import of Tim17 into mitochondria requires the substrate-binding cleft of Mia40. Surprisingly, the formation of the disulfide bond in Tim17, however, did not depend on the oxidoreductase activity of Mia40 though still required Erv1 activity. Obviously, the Mia40 pathway controls a critical thiol switch in a core component of the matrix targeting pathway in an non-canonical reaction. The mechanistic details how the critical thiol switch in Tim17 is operated are unclear.For this project we plan to follow two goals. First, we want to study in detail how the disulfide bond in Tim17 is formed. We already generated point mutants in which the two cysteine residues are replaced by alanines. Surprisingly, the phenotype of the C77A mutant was much more severe than those of the C10A single or the C10,77A double mutants. This might point to a redox modification on C10 that in the wild-type situation is resolved in a C77-dependent reaction. We want to do mass spectrometry to identify potential modifications on the cysteine residues in Tim17. Moreover, we want to identify factors which interact with Tim17 in a cysteine-dependent manner in order to identify the oxidoreductase that oxidizes Tim17 independent of Mia40. From this part of the project we expect detailed insights into the mechanisms by with the mitochondrial disulfide relay controls protein translocation into the matrix.As a second goal we want to screen for further thiol-based redox modifications in proteins of mitochondria that impact on their ability to import proteins. Mal-functioning mitochondria are often characterized by redox changes, however, whether these influence the import of matrix proteins was not analyzed so far. We want to use redox proteomics to systematically study redox modifications on mitochondrial proteins under oxidative stress conditions and analyze the relevance of such modifications. From both parts of the project, we expect a better understanding of how redox processes in mitochondria influence the biogenesis of mitochondrial proteins. This project will considerable benefit from the scientific network of the priority program.