Mitochondrial Precursor Proteins in the Cytosol: Identification and Characterization of Signals and Factors that Coordinate Early Steps in Mitochondrial Protein Biogenesis

线粒体是真核细胞的重要细胞器。它们由800到1500个蛋白质组成，其中大部分是在细胞质中作为前体蛋白质合成的。细胞质中前体蛋白被维持在未折叠的，进口的形式，靶向线粒体，被表面受体识别并被分类到特定的线粒体亚室的反应，我们对这些反应知之甚少。这些线粒体蛋白靶向的早期过程是在蛋白酶体系统的监视下进行的，以避免细胞质中非进口前体蛋白的积累，但细节在很大程度上是难以捉摸的。在这个项目中，我们想要解决两个相关的问题：首先，我们想要了解含有序列的前体蛋白的成熟区域的哪些特征支持它们通过细胞质靶向，哪些特征在将前体蛋白引导到线粒体内的特定位置中发挥作用。最近，我们发现了与线粒体表面受体Tom70结合的内部基质靶向信号样序列（iMTS-Ls）。在该项目的第一部分，我们将更详细地研究这些iMTS-Ls的相关性，分析它们与线粒体表面前体靶向的相关性，以及它们在内膜蛋白拓扑形成中的作用。大多数内膜蛋白在其跨膜结构域前或后含有由iMTS-Ls组成的特征性模式，但这些模式的具体功能尚不清楚。该项目的第二个目标是了解细胞对线粒体前体蛋白胞质积累的反应。初步实验表明，细胞质中前体水平的增加导致强烈的细胞应激反应，导致细胞质中伴侣蛋白和蛋白水解系统成分的上调。与此同时，细胞减少线粒体前体蛋白的表达，可能是为了减轻它们在细胞质中的毒性作用。我们设计了识别和表征允许细胞引发这些反应的信号级联的策略，并已经确定了关键的转录因子。此外，我们打算鉴定调节蛋白酶体降解前体蛋白的成分，以清除细胞质中容易聚集的未折叠前蛋白，并研究它们在这一过程中的功能。从我们的研究中，我们期望详细了解调节线粒体蛋白生物发生早期步骤的反应，要么通过刺激前体蛋白进入线粒体的有效进口（第1部分），要么通过细胞质质量控制系统去除积累的前体蛋白（第2部分）。

Mitochondria are essential organelles of eukaryotic cells. They consist of 800 to 1500 proteins, most of which are synthesized in the cytosol as precursor proteins. Little is known about the reactions in the cytosol by which precursor proteins are maintained in an unfolded, import-competent form, targeted to the mitochondria, recognized by surface receptors and sorted into the specific mitochondrial subcompartments. These early processes of mitochondrial protein targeting are under the surveillance of the proteasome system in order to avoid the accumulation of non-imported precursor proteins in the cytosol, but details are largely elusive.In this project, we want to address two related questions: First, we want to understand which features of the mature regions of presequence-containing precursor proteins support their targeting through the cytosol and which features play a role to direct precursor proteins to their specific locations within mitochondria. Recently, we identified internal matrix targeting signal-like sequences (iMTS-Ls) which bind to the mitochondrial surface receptor Tom70. In the first part of this project we will study the relevance of these iMTS-Ls in more detail, analysing their relevance for precursor targeting to the mitochondrial surface, as well as their role in the topogenesis of inner membrane proteins. Most inner membrane proteins contain characteristic patterns consisting of iMTS-Ls in front of, or following, their transmembrane domains, but the specific functions of these patterns are not clear.The second goal of this project is to understand the cellular response to the cytosolic accumulation of mitochondrial precursor proteins. Preliminary experiments show that increased levels of precursors in the cytosol leads to a strong cellular stress response causing the upregulation of chaperones and of components of the proteolytic system of the cytosol. In parallel, cells reduce the expression of mitochondrial precursor proteins, presumably in order to attenuate their toxic effects in the cytosol. We designed strategies to identify and characterize the signaling cascades that allow the cell to elicit these reactions and have already identified critical transcription factors. Moreover, we intend to identify the components that regulate the degradation of precursor proteins by the proteasome, in order to clear the cytosol from aggregation-prone unfolded preproteins and to study their function in this process. From our studies we expect detailed insights into the reactions that regulate the early steps in mitochondrial protein biogenesis, either by stimulating the efficient import of precursor proteins into mitochondria (part 1) or by removing accumulating precursor proteins by the cytosolic quality control system (part 2).