Fis1 and Drp1 cooperate in mitochondrial homeostasis (756.8)

Abstract

Defects in mitochondrial fission and fusion cause numerous human disorders yet how these defects affect mitochondrial function and contribute to disease is unknown. Mitochondrial fission requires the dynamin‐related GTPase Drp1 and a lethal mutation (A395D) was reported in a neonate with microcephaly, abnormal brain development, optic atrophy, and lactic acidemia (Waterham et al., 2007, N. Engl. J. Med. 356, 1736‐1741). Another Drp1 mutation (C446F) was reported to cause severe cardiomyopathy in mice (Ashrafian et al., 2010, PLoS Genet 6, e1001000). We are determining how these mutants affect mechanoenzyme activity and function. For A395D, we found impaired localization, recruitment, assembly, and GTP hydrolysis. For C446F, we found impaired GTP hydrolysis and assembly. Thus, these mutants impair higher order assembly of Drp1 at mitochondria, leading to decreased fission, elongated mitochondria, and altered cellular distribution of mitochondria. Curiously, these mutants show enhanced in vitro interactions with the mitochondrial protein Fis1, a possible recruiter of Drp1. To determine the consequences of disrupting these interactions, 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 yeast Drp1. 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 Drp1 and mitochondrial morphology consistent with a role for Fis1 in mitochondrial homeostasis.Grant Funding Source: RO1‐GM067180