The GTPase effector domain sequence of the Dnm1p GTPase regulates self-assembly and controls a rate-limiting step in mitochondrial fission.
Dnm1p belongs to a family of dynamin-related GTPases required to remodel different cellular membranes. In budding yeast, Dnm1p-containing complexes assemble on the cytoplasmic surface of the outer mitochondrial membrane at sites where mitochondrial tubules divide. Our previous genetic studies suggested that Dnm1p's GTPase activity was required for mitochondrial fission and that Dnm1p interacted with itself. In this study, we show that bacterially expressed Dnm1p can bind and hydrolyze GTP in vitro. Coimmunoprecipitation studies and yeast two-hybrid analysis suggest that Dnm1p oligomerizes in vivo. With the use of the yeast two-hybrid system, we show that this Dnm1p oligomerization is mediated, in part, by a C-terminal sequence related to the GTPase effector domain (GED) in dynamin. The Dnm1p interactions characterized here are similar to those reported for dynamin and dynamin-related proteins that form higher order structures in vivo, suggesting that Dnm1p assembles to form rings or collars that surround mitochondrial tubules. Based on previous findings, a K705A mutation in the Dnm1p GED is predicted to interfere with GTP hydrolysis, stabilize active Dnm1p-GTP, and stimulate a rate-limiting step in fission. Here we show that expression of the Dnm1 K705A protein in yeast enhances mitochondrial fission. Our results provide evidence that the GED region of a dynamin-related protein modulates a rate-limiting step in membrane fission.
Fukushima
N H
NH
Department of Biology, University of Utah, Salt Lake City, UT 84112, USA.
Brisch
E
E
Keegan
B R
BR
Bleazard
W
W
Shaw
J M
JM
D000595:Amino Acid Sequence; D034284:Dynamin I; D034281:Dynamins; D004926:Escherichia coli; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000737:chemistry; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008561:Membrane Fusion; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D005656:Fungal Proteins; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; D006160:Guanosine Triphosphate; D034284:Dynamin I; D020558:GTP Phosphohydrolases; C094774:DNM1 protein, S cerevisiae; D034281:Dynamins