An assembly-incompetent mutant establishes a requirement for dynamin self-assembly in clathrin-mediated endocytosis in vivo.
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.
Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Byeong Doo
BD
Song
Defne
D
Yarar
Sandra L
SL
Schmid
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000032:analysis / Q000378:metabolism*; D034281:Dynamins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D016415:Sequence Alignment; D041701:Swiss 3T3 Cells; D013752:Tetracycline / Q000494:pharmacology; D014168:Transferrin / Q000032:analysis / Q000378:metabolism
D002966:Clathrin; D008081:Liposomes; D014168:Transferrin; D034281:Dynamins; D013752:Tetracycline