Journal
MOLECULAR BIOLOGY OF THE CELL
Volume 15, Issue 5, Pages 2243-2252Publisher
AMER SOC CELL BIOLOGY
DOI: 10.1091/mbc.E04-01-0015
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Funding
- NHLBI NIH HHS [5T32 HL 07695, T32 HL007695] Funding Source: Medline
- NIGMS NIH HHS [R01 GM042455, GM 42455] Funding Source: Medline
- NIMH NIH HHS [MH 61345, R37 MH061345, R01 MH061345] Funding Source: Medline
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Dynamin GTPase activity is required for its biological function in clathrin-mediated endocytosis; however, the role of self-assembly has not been unambiguously established. Indeed, overexpression of a dynamin mutant, Dyn1-K694A, with impaired ability to self-assemble has been shown to stimulate endocytosis in HeLa cells (Sever et al., Nature 1999, 398, 481). To identify new, assembly-incompetent mutants of dynamin 1, we made point mutations in the GTPase effector/ assembly domain (GED) and tested for their effects on self-assembly and clathrin-mediated endocytosis. Mutation of three residues, 1690, K694, and 1697, suggests that interactions with an amphipathic helix in GED are required for self-assembly. In particular, Dyn1-1690K failed to exhibit detectable assembly-stimulated GTPase activity under all assay conditions. Overexpression of this assembly-incompetent mutant inhibited transferrin endocytosis as potently as the GTPase-defective dominant-negative mutant, Dyn1-K44A. However, worm-like endocytic intermediates accumulated in cells expressing Dyn1-1690K that were structurally distinct from long tubules that accumulated in cells expressing Dyn1-K44A. Together these results provide new structural insight into the role of GED in self-assembly and assembly-stimulated GTPase activity and establish that dynamin self-assembly is essential for clathrin-mediated endocytosis.
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