Defects in the mitochondrial lifecycle from impaired fission and fusion cause numerous human disorders yet how these defects affect mitochondrial function and contribute to disease is unknown. For mitochondrial fission at least two proteins are required: the outer membrane protein Fis1 and the dynamin-related mechanoenzyme, Drp1. We are determining the molecular basis of two disease-causing mutations of Drp1, one of which was lethal. For this lethal mutation, A395D, we found impaired higher order assembly of Drp1 at mitochondria, leading to decreased fission and altered cellular distribution of elongated mitochondria. Analysis of this and other mutants led us to propose a new model in which the Fis1 protein mediates Drp1 assembly. We are testing this model using different approaches from structural to genetics. Structurally, we have found that three non-functional Fis1 alleles form elevated amounts of dimer in vitro compared to wild type, suggesting that enhanced dimerization of Fis1 interferes with mitochondrial fission. We rationally designed a single point mutation that disrupts in vitro dimerization of Fis1 and the three dimer-promoting alleles, suggesting a common structural basis for dimerization of the wild type and mutant proteins. Surprisingly, either obligate monomers or dimers are impaired in fission, indicating that either the failure to dimerize or excessive dimerization interfere with Fis1 function. These findings highlight the complex nature of protein-protein interactions in fission and accordingly we have developed a novel screen to rapidly identify the critical, or hotspot, residues at protein interfaces. We call this method hotspot and used it to define essential Fis1 residues. We randomly generated a library of Fis1 variants and simultaneously screened for disruption of
Fis1-mediated yeast two-hybrid interactions with each binding partner. Of the >3000 colonies screened, ~9% selectively disrupted interactions with one of the three protein partners. To test the functional consequence of our hotspot analysis, we parsed each hit sequence, which contained multiple amino acid changes, into its corresponding single point mutations and tested viability in a growth assay that directly reports on mitochondrial fission. Of 97 Fis1 mutants tested to date, 40 resulted in nonfunctional fission indicating that residues identified by hotspot are essential for mitochondrial fission. Combining these results with our structure-based design data is converging on a model in which a Fis1 checkpoint acts to regulate Drp1
assembly in fission.
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