Determining the mechanism of mitochondrial outer membrane fusion

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Project Summary This proposal examines the mitofusin proteins, which catalyze both mitochondrial tethering and outer membrane fusion. Mitochondrial shape is central to mitochondrial function and is closely linked to changes in cellular physiology during development, stress, and aging. Mitochondrial fusion increases metabolic production, protects against excessive degradation of mitochondria, reduces sensitivity of the cell to apoptotic stimuli and allows functional complementation of damaged organelles. Compromised mitochondrial function and decreased organelle connectivity are associated with neurodegeneration and other age-related diseases. Furthermore, mutations in the genes encoding components of the mitochondrial fusion machine cause peripheral neuropathy, optic atrophy, myopathy and ataxia. Both mitochondrial division and mitochondrial fusion are mediated by conserved dynamin superfamily proteins. This diverse family of mechanochemical GTPases couple self-assembly and conformational changes to membrane remodeling events throughout the cell. Mitofusin is the mitochondrial outer membrane fusion machine and must assemble both in cis within a single membrane, and in trans across two mitochondria. The molecular details, including of the composition of these assemblies, the regulation of their formation and how they contribute to membrane tethering and fusion are not known. We address these gaps in knowledge utilizing biochemical analyses of mitofusin function. In Aim 1, we will elucidate mechanisms of allosteric regulation that control assembly of mitofusin into fusion- competent complexes. Our collection of functional variants and novel approaches will provide a powerful tool in dissecting the contribution of each mitofusin functional domain to cis oligomerization and trans complex formation. Our work characterizing CMT2A-associated variants of mitofusin revealed that molecular defects of mitofusin can be compensated for by cytosolic factors in cells. In Aim 2, we use a reductionist approach that combines cell biology, biochemistry, and reconstituted assays developed in my lab to determine how the cytosolic mitofusin effectors Bax and MSTO1 alter mitofusin assembly to regulate mitochondrial fusion. This research program draws on our strengths in cellular and biochemical analysis of mitochondrial biology and will yield fundamental insights not only for mitochondrial dynamics but for the mechanisms through which dynamin superfamily proteins in general operate.