Molecular Mechanisms Behind Mitochondrial Fission

Abstract

Mitochondria undergo a remarkable sequence of fission and fusion events during its lifecycle yet the molecular basis of these events is just emerging. Fis1 appears essential to mitochondrial fission by directly or indirectly recruiting Drp1, a dynamin-related mechanoenzyme to sites of scission. A genetic screen in yeast identified non-functional alleles of Fis1 alleles but the root of this misfunction is not known. We have taken biased and unbiased approaches to define Fis1 mechanism, which have converged on a new model. Specifically, we determined that these non-functional alleles unexpectedly 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. Surprisingly, either obligate monomers or dimers are impaired in fission, indicating that either the failure to dimerize or excessive dimerization interfere with Fis1 function. To dissect this further, we have developed a novel method to rapidly identify 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 this analysis, we parsed each hit sequence 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 these residues are essential for mitochondrial fission. An analysis of these mutations supports a new model for the assembly of the mitochondrial fission machinery. We anticipate that this method will be useful in defining critical residues in other macromolecular machines and “signaling hub” proteins with multiple binding partners.