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

线粒体是Fe-S簇生物发生所必需的，对细胞凋亡至关重要，参与许多代谢途径，并以其在ATP合成中的作用而闻名。所有这些过程都依赖于外膜的β -桶状通道和内膜的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细胞质核糖体上翻译。经典的线粒体前体蛋白含有一个n端前体序列，它是靶向和进口所必需的，然后被切割掉以产生成熟蛋白。该项目的目标是全面描述线粒体膜蛋白前体与内部靶向信号的进口。在靶向外膜转位酶（TOM）并进行易位后，这些疏水前体通过水膜间隙进行分类。随后，β -桶前体在分选和组装机制（SAM）的帮助下，通过载体转位酶（TIM22）插入到内膜的外螺旋代谢载体和α -螺旋代谢载体中。主要的开放问题是三种Tom受体对膜蛋白具有内部靶向信号的特异性靶向机制，TIM伴侣系统在膜间空间中陪伴蛋白质的结构基础以及向膜插入酶复合物的转移。通过综合结构生物学和生化方法，我们将分析不同的冗余Tom受体对靶向β -桶蛋白和代谢物载体的贡献。此外，我们将确定膜蛋白前体通过TIM伴侣系统从TOM复合物通过水膜间空间转移到SAM和TIM22复合物的机制。我们将结合NMR、SAXS等生物物理方法，分析膜蛋白前体与受体结构域和伴侣蛋白形成复合物的结构和动力学，确定相对结合亲和力，并在结构水平上研究伴侣蛋白向Sam50-POTRA结构域和Tim54受体的前体蛋白转移。这些生物物理和结构方法将被受体和伴侣蛋白的位点特异性点突变体的产生以及它们的体内和体外生长分析和分离线粒体的导入实验所补充。综上所述，我们将通过深入描述线粒体中两种最丰富的膜蛋白的进口来补充经典线粒体前体蛋白的进口知识，这也将揭示叶绿体和革兰氏阴性菌的重要原理。

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.