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

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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.