Dynamin : GTP controls the formation of constricted coated pits, the rate limiting step in clathrin-mediated endocytosis

The GTPase dynamin is essential for receptor-mediated endocytosis, but its function remains controversial,A domain of dynamin, termed the GTPase effec tor domain (GED), controls dynamin's high stimulated rates of GTP hydrolysi s by functioning as an assembly-dependent GAP. Dyn(K694A) and dyn(R725A) ca rry point mutations within GED resulting in reduced assembly stimulated GTP ase activity Biotinylated transferrin is more rapidly sequestered from avid in in cells transiently overexpressing either of these two activating mutan ts (Sever, S.,A.B. Muhlberg, and S.L. Schmid. 1999. Nature. 398:481-486), s uggesting that early events in receptor-mediated endocytosis are accelerate d. Using stage-specific assays and morphological analyses of stably transfo rmed cells, we have identified which events in clathrin-coated vesicle form ation are accelerated by the overexpression of dyn(K694A) and dyn(R725A). B oth mutants accelerate the formation of constricted coated pits, which we i dentify as the rate limiting step in endocytosis. Surprisingly, overexpress ion of dyn(R725A), whose primary defect is in stimulated GTP hydrolysis, bu t not dyn(K694A), whose primary defect is in self-assembly, inhibited membr ane fission leading to coated vesicle release. Together, our data support a model in which dynamin functions like a classical GTPase as a key regulato r of clathrin-mediated endocytosis.