Biogenesis of alpha-helical mitochondrial outer membrane proteins in higher eukaryotes

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Project Summary/Abstract Mitochondria are essential organelles of endosymbiotic origin which have evolved to play critical roles in eukaryotic physiology. Mitochondrial activity is dependent on proteins embedded in the outer mitochondrial membrane (OMM), which mediate mitochondrial-cytoplasmic communication, and critical aspects of cellular function such as apoptosis and the innate immune response. a-helical proteins are an important subset of OMM proteins. However, how they get inserted into the lipid bilayer of the OMM in mammalian cells has until recently been unclear. Work by myself, in collaboration with Rebecca Voorhees and Jonathan Weissman’s labs, has identified the OMM resident protein MTCH2, a defining member of a novel family of insertases, as both necessary and sufficient for the insertion of a-helical proteins into the OMM. MTCH2 is a diverged member of the solute carrier family 25 (SLC25), and has evolved to exploit the canonical transporter fold for insertion. Bioinformatic analysis reveals that MTCH2 has a homolog in peroxisomes, and orthologs across holozoa, suggesting a common mechanism for a-helical protein insertion across membranes and eukaryotes. Building on this finding, this proposal aims to develop a comprehensive understanding of how a-helical proteins are correctly targeted to the OMM and inserted into the lipid bilayer across eukaryotes. This work will address a fundamental question in cell biology, as the OMM proteome has evolved to support increasingly more complex functions in higher eukaryotes. a-helical proteins are defined by the presence of one or more transmembrane domains (TAs), though their TMs can vary significantly in number and a range of biophysical characteristics such as hydrophobicity. This proposal aims to first develop a deeper understanding of MTCH2 function. First, in vivo and biochemical techniques will be combined to map the route a substrate TM takes through MTCH2 into the lipid bilayer, directly testing whether MTCH2’s conserved hydrophilic groove has a direct role in this process. Second, this work will establish whether structural homologs of MTCH2 have retained their insertase ability across eukaryotes. The biochemical skills and knowledge acquired by defining the molecular basis for MTCH2 will be combined with systematic functional genomics to establish how a-helical proteins get targeted to the OMM, and whether other factors besides MTCH2 are required to support the biogenesis of this diverse class. Cumulatively, this proposal will provide important insights into the mechanisms that have evolved to support eukaryotic life. Further, intimate knowledge of the machinery that governs a-helical OMM protein insertion will be critical for developing new treatments associated with outer mitochondrial membrane protein dysregulation, including neurodegenerative diseases such as Parkinson’s and Alzheimer’s.