FIS1 and DNM1L Cooperate in Mitochondrial Fission: Convergence of Evolution and Intelligent Design

Mitochondrial fission helps to maintain proper mitochondrial homeostasis in a poorly understood manner despite its association with human disease. Several proteins have been identified in this process, but only two -- FIS1 and DNM1L -- are found in every species that contain mitochondria. We asked whether these proteins might cooperate together in this process. We found that FIS1 has the ability to directly recruit DNM1L, but is auto-inhibited by an N-terminal arm. A point mutant was rationally designed to relieve auto-inhibition and found to increase DNM1L binding and impair mitochondrial fission. This point mutant also increased the population of a latent dimeric state of FIS1 highlighting the complex nature of protein-protein interactions in fission. To address this, we devised an unbiased and general method to rapidly identify residues critical to protein interfaces and applied this technology to yeast Fis1 interactions. Of the >3000 Fis1 alleles screened, ∼9% selectively disrupted interactions with one of the three protein partners including DNM1L. To test the functional consequences, each allele was parsed into its corresponding point mutation and tested for mitochondrial fission. Of 211 yeast Fis1 mutants tested to date, 97 resulted in nonfunctional fission indicating that our method identifies residues essential for mitochondrial fission. Orthologous mutations were introduced into human Fis1 and also found to impair interactions with DNM1L and mitochondrial morphology. Analysis of these data supports a new model for the assembly of the mitochondrial fission machinery.