Inserting proteins into the mitochondrial outer membrane: Deciphering the structure and mechanism of the MIM insertase

将蛋白质插入线粒体外膜：破译 MIM 插入酶的结构和机制

• 批准号: 506258237
• 负责人: Professor Dr. Doron Rapaport
• 金额: --
• 依托单位: Institute of Science and Technology Austria
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/506258237?language=en
• 关键词: Inserting; proteins; into; mitochondrial; outer

Mitochondria are involved in many cellular processes, from energy generation, ageing and cell death to key metabolic reactions. Almost all their proteins are synthesized outside the organelle and imported by sophisticated machineries composed of chaperones, receptors, motor elements, membrane translocases, and insertases. Helical proteins in the mitochondrial outer membrane (MOM) act as enzymes, components of protein import and sorting machineries, mediators of apoptosis and mitophagy, and in mediating mitochondrial fusion, fission, and motility. They are inserted into the MOM of yeast cells by an insertase called mitochondrial import (MIM). The mechanism of insertion of tens of MOM proteins is currently unknown. MIM has properties unseen in other known insertases: heterooligomeric structure composed of two small subunits, and ability to insert proteins from both sides of the membrane. A central question in molecular cell biology is how the dozens of MOM proteins integrate into the MOM in a MIM-dependent manner. Here, we will address the following questions: (i) What is the atomic structure of the MIM complex? (ii) How does MIM interact with its substrate proteins to promote their membrane integration? (iii) Does the MIM complex represent the minimal insertase machinery? We will use cryo-electron microscopy (EM) and nuclear magnetic resonance (NMR) spectroscopy and a previously developed approach that integrates both techniques to elucidate MIM’s atomic- structure. Using NMR, we will probe the flexibility of the components. In a collaborative effort, we will complement the structural data with in vivo assays in yeast cells and in organello import assays. Structure-guided mutants will be tested in vivo, and the alteration of function of these mutants will be visualized at the atomic level. Furthermore, a liposome-reconstituted MIM system will provide mechanistic insights on the structure-function relationships of the insertase. Successful completion of this project will allow us for the first time to understand the membrane insertion of MOM α-helical proteins in terms of atomic-level structure and dynamics. Due to its unique architecture, unraveling the molecular mechanisms of MIM will enlarge our general understanding of protein insertion into membranes.

线粒体参与了许多细胞过程，从能量产生、衰老、细胞死亡到关键的代谢反应。几乎所有的蛋白质都是在细胞器外合成的，并由复杂的机制输入，这些机制由伴侣、受体、运动元件、膜转位酶和插入酶组成。线粒体外膜螺旋蛋白作为酶、蛋白质输入和分选机制的组成部分、细胞凋亡和有丝分裂的中介体，以及介导线粒体的融合、分裂和运动而发挥作用。它们通过一种名为线粒体导入(MIM)的插入酶插入酵母细胞的MOM中。目前，数十种MOM蛋白的插入机制尚不清楚。MIM具有其他已知插入酶所没有的特性：由两个小亚基组成的异寡聚体结构，以及从膜两侧插入蛋白质的能力。分子细胞生物学中的一个中心问题是，数十个MoM蛋白是如何以依赖MIM的方式整合到MoM中的。在这里，我们将解决以下问题：(I)MIM络合物的原子结构是什么？(Ii)MIM如何与其底物蛋白相互作用以促进它们的膜整合？(Iii)MIM复合体是否代表最小的插入酶机制？我们将使用冷冻电子显微镜(EM)和核磁共振(NMR)光谱以及先前开发的一种集成了这两种技术的方法来阐明MIM的原子结构。使用核磁共振，我们将探索组件的灵活性。在一项合作努力中，我们将用酵母细胞的体内分析和细胞器进口的分析来补充结构数据。结构导向突变体将在体内进行测试，这些突变体的功能变化将在原子水平上可视化。此外，脂质体重组的MIM系统将提供对插入酶结构-功能关系的机械性见解。这个项目的成功完成将使我们第一次从原子级结构和动力学角度理解MoMα-螺旋蛋白质的膜插入。由于其独特的结构，揭示MIM的分子机制将扩大我们对蛋白质插入膜的一般理解。