The dynamin-related protein Mgm1p assembles into oligomers and hydrolyzes GTP to function in mitochondrial membrane fusion.
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.
Meglei
Gabriela
G
Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
McQuibban
G Angus
GA
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
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