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

线粒体膜蛋白输入和膜间空间转移的结构和功能研究

• 批准号: 406757425
• 负责人: Professor Dr. Nils Wiedemann
• 金额: --
• 依托单位: Institut für Biochemie und Molekularbiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/406757425?language=en
• 关键词: Structural; functional; studies; import; intermembrane

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.

线粒体对Fe-S簇的生物合成至关重要，对细胞凋亡至关重要，参与许多代谢途径，并且众所周知其在ATP合成中的作用。所有这些过程都取决于外膜中的β桶通道和内膜中的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，并在细胞溶质核糖体上翻译。经典的线粒体前体蛋白含有N-末端前序列，其对于靶向和输入是必需的且足够的，并且之后其被切割以产生成熟蛋白。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输入。在靶向外膜的易位酶（TOM）和易位之后，这些疏水前体通过水性膜间空间被分选。随后，β-桶前体在分选和组装机械（SAM）的帮助下插入到外部和α-螺旋代谢物载体中，通过载体移位酶（TIM 22）插入到内膜中。主要的开放性问题是三种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分子伴侣系统在膜间隙中的蛋白质伴侣作用的结构基础以及转移到膜插入酶复合物。我们将分析不同的冗余Tom受体对β-桶蛋白和代谢物载体靶向的贡献。此外，我们将确定膜蛋白前体转移的TIM分子伴侣系统从TOM复合物通过水膜间隙的SAM和TIM 22复合物的机制。我们将结合NMR、SAXS和其他生物物理方法，分析膜蛋白前体与受体结构域和伴侣蛋白形成的复合物的结构和动力学，确定相对结合亲和力，并研究前蛋白从伴侣蛋白转移到Sam 50-POTRA结构域和TIM 22复合物的Tim 54受体的结构水平。这些生物物理和结构的方法将通过产生受体和伴侣蛋白的位点特异性点突变体及其通过生长测定和导入分离的线粒体的实验进行的体内和体外分析来补充。两者合计，我们将通过深入描述线粒体的两种最丰富的膜蛋白类的进口来补充经典线粒体前体蛋白的进口知识，这将揭示叶绿体和革兰氏阴性细菌的重要原则。