Thiol Switches Controlling Mitochondrial Protein Biogenesis

线粒体的生物发生需要将数百种不同的蛋白质前体从细胞质导入细胞器。过去鉴定了两种主要的输入途径，前蛋白输入途径导致基质和Mia40途径导致膜间隙。在上一个资助期间，我们在内膜转位酶的中心亚基Tim17中确定了一个关键的二硫键。Tim17是第一个被鉴定为线粒体氧化机制底物的膜蛋白。Tim17导入线粒体需要Mia40的底物结合裂缝。然而，令人惊讶的是，Tim17中二硫键的形成不依赖于Mia40的氧化还原酶活性，尽管仍然需要Erv1活性。显然，Mia40途径在非典型反应中控制基质靶向途径的核心组分中的关键巯基开关。Tim17中关键的巯基开关是如何运作的机制细节尚不清楚。首先，我们想详细研究Tim17中的二硫键是如何形成的。我们已经产生了点突变体，其中两个半胱氨酸残基被丙氨酸取代。令人惊讶的是，C77A突变体的表型比C10A单突变体或C10，77A双突变体的表型严重得多。这可能指向在野生型情况下在C77依赖性反应中解决的C10上的氧化还原修饰。我们想用质谱法来鉴定Tim17中半胱氨酸残基的潜在修饰。此外，我们想确定的因素，以半胱氨酸依赖的方式与Tim17相互作用，以确定氧化还原酶，氧化Tim17独立的Mia40。从这个项目的这一部分，我们希望详细了解线粒体二硫键中继控制蛋白质易位到matrix.As第二个目标，我们希望筛选进一步的巯基为基础的线粒体蛋白质的氧化还原修饰，影响其输入蛋白质的能力的机制。功能障碍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到目前为止还没有分析。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质的氧化还原修饰，并分析这些修饰的相关性。从该项目的两个部分，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质的生物合成。该项目将大大受益于优先计划的科学网络。

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.