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A more recent version of this article appeared on May 1, 2004
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Submitted on January 9, 2004
Revised on February 13, 2004
Accepted on February 14, 2004
1 Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037
* Corresponding author. E-mail address: slschmid{at}scripps.edu.
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, I690, K694 and I697 suggests that interactions with an amphipathic helix in GED are required for self-assembly. In particular, Dyn1-I690K 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-I690K 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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