An Assembly-incompetent Mutant Establishes a Requirement for Dynamin Self-assembly in Clathrin-mediated Endocytosis in Vivo

Byeong Doo Song, Defne Yarar, Sandra L. Schmid

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)2243-2252
Number of pages10
JournalMolecular Biology of the Cell
Volume15
Issue number5
DOIs
StatePublished - May 2004

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Dynamins
Clathrin
GTP Phosphohydrolases
Endocytosis
Dynamin I
Transferrin
HeLa Cells
Point Mutation
Mutation

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

An Assembly-incompetent Mutant Establishes a Requirement for Dynamin Self-assembly in Clathrin-mediated Endocytosis in Vivo. / Song, Byeong Doo; Yarar, Defne; Schmid, Sandra L.

In: Molecular Biology of the Cell, Vol. 15, No. 5, 05.2004, p. 2243-2252.

Research output: Contribution to journalArticle

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