Structural and functional studies of import and intermembrane space transfer of mitochondrial membrane proteins

线粒体是铁-S簇生物发生所必需的，是细胞凋亡的关键，参与多种代谢途径，并以其在三磷酸腺苷合成中的作用而闻名。所有这些过程都依赖于外膜的β-桶通道和内膜的α-螺旋代谢物载体。与绝大多数线粒体蛋白类似，所有这些通道和转运蛋白都在细胞核中编码，并在胞质核糖体上翻译。经典的线粒体前体蛋白含有一个N-末端的前序列，这是靶向和输入所必需的，并且足够的，然后它被切割以产生成熟的蛋白质。该项目的目标是全面描述具有内部靶向信号的线粒体膜蛋白前体的进口。在靶向外膜转位酶(TOM)和转位后，这些疏水前体通过水膜间空间进行分选。随后，在分选和组装机械(SAM)的帮助下，通过载体转位酶(TIM22)进入内膜，β-桶前体被插入到外层和α-螺旋代谢物载体中。主要的开放问题是三种TOM受体对具有内部靶向信号的膜蛋白的特异性靶向机制，TIM伴侣系统在膜间隙中蛋白质伴侣的结构基础以及膜插入酶复合体的转移。我们将通过综合结构生物学和生化的方法，分析不同冗余的TOM受体对靶向β-桶蛋白和代谢产物载体的贡献。此外，我们还将确定TIM伴侣系统通过水膜间隙将膜蛋白前体从TOM复合体转移到SAM和TIM22复合体的机制。我们将结合核磁共振、小角X射线散射等生物物理方法分析膜蛋白前体与受体结构域和伴侣形成的复合体的结构和动力学，确定相对结合亲和力，并在结构水平上研究伴侣蛋白向TIM22复合体的Sam50-Potra结构域和Tim54受体的转移。这些生物物理和结构方法的补充将是产生受体和伴侣蛋白的定点突变体，并通过生长试验和对分离的线粒体进行导入实验进行体内和体外分析。综上所述，我们将通过深入描述线粒体两种最丰富的膜蛋白类别的导入来补充经典线粒体前体蛋白的导入知识，这也将揭示叶绿体和革兰氏阴性细菌的重要原理。

Mitochondria are essential for Fe-S cluster biogenesis, crucial for apoptosis, involved in numerous metabolic pathways and well known for their role in ATP synthesis. All these processes depend on beta-barrel channels in the outer membrane and alpha-helical metabolite carriers in the inner membrane. Similar to the vast majority of mitochondrial proteins, all these channels and transporters are encoded in the nucleus and translated on cytosolic ribosomes. Classical mitochondrial precursor proteins contain an N-terminal presequence which is required and sufficient for targeting and import and afterwards it is cleaved off to produce the mature protein. The goal of this project is a comprehensive description of the import of mitochondrial membrane protein precursors with internal targeting signals. After targeting to the translocase of the outer membrane (TOM) and translocation these hydrophobic precursors are sorted through the aqueous intermembrane space. Subsequently, beta-barrel precursors are inserted with the help of the sorting and assembly machinery (SAM) into the outer and alpha-helical metabolite carriers by the carrier translocase (TIM22) into the inner membrane. Major open questions are the specific targeting mechanisms of the three Tom receptors for membrane proteins with internal targeting signals, the structural basis of protein chaperoning in the intermembrane space by the TIM chaperone system and the transfer to the membrane insertase complexes.By an integrated structural biology and biochemical approach, we will analyze the contribution of the different redundant Tom receptors for the targeting of beta-barrel proteins and metabolite carriers. Moreover, we will determine the mechanism of membrane protein precursor transfer by the TIM chaperone system from the TOM complex through the aqueous intermembrane space to the SAM and TIM22 complexes. We will analyze the structure and dynamics of the complexes formed by the membrane protein precursors and the receptor domains and chaperones by combining NMR, SAXS and other biophysical approaches, determine relative binding affinities, and study the preprotein transfer from chaperones to the Sam50-POTRA domain and to the Tim54 receptor of the TIM22 complex at the structural level. These biophysical and structural approaches will be complemented by generation of site-specific point mutants of the receptor and chaperone proteins and their in vivo and in vitro analysis by growth assays and import experiments into isolated mitochondria. Taken together, we will complement the knowledge for the import of the classical mitochondrial precursor proteins by an in-depth description of the import of the two most abundant membrane protein classes of mitochondria and this will reveal important principles also for chloroplasts and Gram negative bacteria.