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

线粒体参与许多细胞过程，从能量产生，衰老和细胞死亡到关键代谢反应。几乎所有的蛋白质都是在细胞器外合成的，并由分子伴侣、受体、运动元件、膜转位酶和插入酶组成的复杂机制输入。螺旋蛋白在线粒体外膜（mtDNA）作为酶，蛋白质输入和分选机制的组成部分，介导细胞凋亡和线粒体自噬，并在介导线粒体融合，分裂和运动。它们通过一种称为线粒体输入（MIM）的插入酶插入到酵母细胞的线粒体中。目前，数十种蛋白质的插入机制尚不清楚。MIM具有在其他已知插入酶中看不到的特性：由两个小亚基组成的异源寡聚结构，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种β-淀粉样蛋白如何以依赖于MIM的方式整合到β-淀粉样蛋白中。在这里，我们将解决以下问题：（i）MIM复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其膜整合？(iii)MIM复合体是否代表最小的插入酶机制？我们将使用低温电子显微镜（EM）和核磁共振（NMR）光谱和先前开发的方法，集成了这两种技术来阐明MIM的原子结构。使用NMR，我们将探测组件的灵活性。在一个合作的努力，我们将补充结构数据与酵母细胞中的体内测定和organello进口测定。结构导向突变体将在体内进行测试，这些突变体的功能改变将在原子水平上可视化。此外，脂质体重构的MIM系统将提供插入酶的结构-功能关系的机制见解。该项目的成功完成将使我们第一次从原子水平的结构和动力学方面了解α-螺旋蛋白质的膜插入。由于其独特的结构，解开MIM的分子机制将扩大我们对蛋白质插入膜的一般理解。

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.