title: Dynamin-catalyzed membrane fission requires coordinated GTP hydrolysis.
abstract: Dynamin is the most-studied membrane fission machinery and has served as a paradigm for studies of other fission GTPases; however, several critical questions regarding its function remain unresolved. In particular, because most dynamin GTPase domain mutants studied to date equally impair both basal and assembly-stimulated GTPase activities, it has been difficult to distinguish their respective roles in clathrin-mediated endocytosis (CME) or in dynamin catalyzed membrane fission. Here we compared a new dynamin mutant, Q40E, which is selectively impaired in assembly-stimulated GTPase activity with S45N, a GTP-binding mutant equally defective in both basal and assembly-stimulated GTPase activities. Both mutants potently inhibit CME and effectively recruit other endocytic accessory proteins to stalled coated pits. However, the Q40E mutant blocks at a later step than S45N, providing additional evidence that GTP binding and/or basal GTPase activities of dynamin are required throughout clathrin coated pit maturation. Importantly, using in vitro assays for assembly-stimulated GTPase activity and membrane fission, we find that the latter is much more potently inhibited by both dominant-negative mutants than the former. These studies establish that efficient fission from supported bilayers with excess membrane reservoir (SUPER) templates requires coordinated GTP hydrolysis across two rungs of an assembled dynamin collar.
authors: Department of Cell Biology, The Scripps Research Institute, La Jolla, California, United States of America.
authors: Ya-Wen
authors: YW
authors: Liu
authors: Juha-Pekka
authors: JP
authors: Mattila
authors: Sandra L
authors: SL
authors: Schmid
mesh_terms: D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism / Q000648:ultrastructure; D034281:Dynamins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D009154:Mutation; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization
chemical_list: D002966:Clathrin; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D034281:Dynamins
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