title: The dynamin-related protein Mgm1p assembles into oligomers and hydrolyzes GTP to function in mitochondrial membrane fusion.
abstract: Mitochondrial dynamics resulting from competing membrane fusion and fission reactions are required for normal cellular function in eukaryotes. Mgm1p, a dynamin-related protein, is a key component in yeast mitochondrial fusion and is evolutionarily conserved. Previous studies suggest that Mgm1p mediates mitochondrial inner membrane fusion in a manner similar to that of other dynamin proteins that use GTP hydrolysis and oligomerization to induce structural changes in lipid bilayers; however, a direct demonstration of these activities has yet to be presented. Here we show that purified Mgm1p forms low-order oligomers that are dependent on protein concentration, suggesting a dynamic and reversible interaction. We further demonstrate that Mgm1p has GTPase activity and kinetic properties consistent with a mechanoenzyme and with a role in inner membrane mitochondrial fusion. Mutations of key residues in conserved motifs of the GTPase domain show markedly reduced or diminished GTPase activity. A mutation in the GTPase effector domain, involved in assembly and assembly-stimulated GTP hydrolysis, has basal GTPase activity similar to that of wild-type Mgm1p but has a weaker propensity to form oligomers. Finally, our data indicate that Mgm1p interacts specifically with negatively charged phospholipids found in mitochondrial membranes, and point mutations in the predicted lipid-binding domain abrogate these interactions. These findings suggest the presence of a putative lipid-binding domain, providing insight into how this protein mediates inner membrane fusion. Together, these data indicate that Mgm1p mediates fusion through oligomerization, GTP hydrolysis, and lipid binding in a manner similar to those of other dynamin mechanoenzymes.
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Meglei
authors.forename: G Angus
authors.initials: GA
authors.lastname: McQuibban
mesh_terms: D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D004229:Dithiothreitol / Q000494:pharmacology; D034281:Dynamins / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D050356:Lipid Metabolism / Q000187:drug effects; D008561:Membrane Fusion / Q000187:drug effects; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology
chemical_list: C082883:MGM1 protein, S cerevisiae; D024101:Mitochondrial Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D012965:Sodium Chloride; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D034281:Dynamins; D004229:Dithiothreitol