Thiol Switches Controlling Mitochondrial Protein Biogenesis

线粒体的生物发生需要从细胞质中输入数百种不同的蛋白质前体到细胞器中。过去确定了两种主要的进口途径，即进入基质的前蛋白进口途径和进入膜间空间的Mia40途径。在之前的资助期间，我们在Tim17中发现了一个关键的二硫键，Tim17是内膜转位酶的一个中心亚基。Tim17是第一个被鉴定为线粒体氧化机制底物的膜蛋白。Tim17进入线粒体需要Mia40的底物结合间隙。然而，令人惊讶的是，Tim17中二硫键的形成并不依赖于Mia40的氧化还原酶活性，尽管仍然需要Erv1活性。显然，在非典型反应中，Mia40通路控制着基质靶向通路核心组分中的一个关键硫醇开关。Tim17中的关键硫醇开关如何操作的机制细节尚不清楚。对于这个项目，我们计划遵循两个目标。首先，我们想详细研究Tim17中的二硫键是如何形成的。我们已经产生了两个半胱氨酸残基被丙氨酸取代的点突变体。令人惊讶的是，C77A突变体的表型比C10A单突变体或c1077a双突变体的表型严重得多。这可能表明C10的氧化还原修饰在野生型情况下在c77依赖性反应中得到解决。我们想用质谱法鉴定Tim17中半胱氨酸残基的潜在修饰。此外，我们希望确定以半胱氨酸依赖方式与Tim17相互作用的因子，以确定独立于Mia40氧化Tim17的氧化还原酶。从项目的这一部分，我们期望通过线粒体二硫化物接力控制蛋白质转运到基质中的机制的详细见解。作为第二个目标，我们希望在线粒体蛋白质中筛选进一步的基于硫醇的氧化还原修饰，这些修饰会影响它们进口蛋白质的能力。功能异常的线粒体通常以氧化还原变化为特征，然而，这些变化是否影响基质蛋白的输入迄今尚未分析。我们希望利用氧化还原蛋白质组学系统地研究氧化应激条件下线粒体蛋白的氧化还原修饰，并分析这些修饰的相关性。从项目的两个部分，我们期望更好地了解线粒体中的氧化还原过程如何影响线粒体蛋白的生物发生。该项目将从科学网络优先规划中获得可观的收益。

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.