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Published online 4 September 2000. doi:10.1083/jcb.150.5.1137
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© The Rockefeller University Press, 0021-9525/2000//1137 $5.00
The Journal of Cell Biology, Volume 150, Number 5, , 2000 1137-1148

Original Article

Dynamin:Gtp Controls the Formation of Constricted Coated Pits, the Rate Limiting Step in Clathrin-Mediated Endocytosis



Sanja Severa, Hanna Damkea, and Sandra L. Schmida

a Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037
Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037.(858) 784-9126(858) 784-2311

slschmid{at}scripps.edu

The GTPase dynamin is essential for receptor-mediated endocytosis, but its function remains controversial. A domain of dynamin, termed the GTPase effector domain (GED), controls dynamin's high stimulated rates of GTP hydrolysis by functioning as an assembly-dependent GAP. Dyn(K694A) and dyn(R725A) carry point mutations within GED resulting in reduced assembly stimulated GTPase activity. Biotinylated transferrin is more rapidly sequestered from avidin in cells transiently overexpressing either of these two activating mutants (Sever, S., A.B. Muhlberg, and S.L. Schmid. 1999. Nature. 398:481–486), suggesting that early events in receptor-mediated endocytosis are accelerated. Using stage-specific assays and morphological analyses of stably transformed cells, we have identified which events in clathrin-coated vesicle formation are accelerated by the overexpression of dyn(K694A) and dyn(R725A). Both mutants accelerate the formation of constricted coated pits, which we identify as the rate limiting step in endocytosis. Surprisingly, overexpression of dyn(R725A), whose primary defect is in stimulated GTP hydrolysis, but not dyn(K694A), whose primary defect is in self-assembly, inhibited membrane fission leading to coated vesicle release. Together, our data support a model in which dynamin functions like a classical GTPase as a key regulator of clathrin-mediated endocytosis.

Key Words: dynamin • endocytosis • clathrin-coated vesicles • GTPase

© 2000 The Rockefeller University Press

The present address of S. Sever is Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Dana Farber Cancer Institute, Boston, MA 02115.

Abbreviations used in this paper: BSS-Tfn, biotinylated transferrin; dyn, dynamin; GED, GTPase effector domain; MesNa, 2-mercaptoethanesulfonic acid; Tfn, transferrin; tet, tetracycline.


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