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

Abstract

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.

Mitochondrial dynamics and division in budding yeast

“…Overproduction of a dynamin GED mutation predicted to prolong the GTP-bound state was shown to enhance the rate of endocytosis in vivo, consistent with the idea that dynamin-GTP regulates a rate-limiting step in endocytosis [36]. A later study established that this rate-limiting step was the formation of constricted coated pits [35]. The effect of Dnm1p's AH/GED on the rate of GTP hydrolysis by assembled Dnm1p has not yet been measured.…”

“…Evidence that the GTPase cycle of Dnm1p might regulate this rate-limiting step in mitochondrial fission comes from mutational studies of the Dnm1p AH domain [28]. In previous in vitro studies of mammalian dynamin, the AH domain was shown to stimulate GTP hydrolysis by dynamin after the protein assembled to form higher-order structures [35,36]. Based on these findings, dynamin's AH domain was renamed the GED [36].…”

The GTPase Effector Domain Sequence of the Dnm1p GTPase Regulates Self-Assembly and Controls a Rate-limiting Step in Mitochondrial Fission

“…When overexpressed in mammalian cells, both of these mutant forms of dynamin increase the rate of formation of constricted coated pits, the rate-limiting step in endocytosis. However, membrane fission and coated vesicle release is blocked by dyn R725A but occurs normally in dyn K694A (Sever et al, 1999(Sever et al, , 2000. Sever et al (1999Sever et al ( , 2000 suggested that these findings argue against a mechanochemical role for dynamin (Kelly, 1999;van der Bliek, 1999b;Yang and Cerione, 1999).…”

“…K694 is involved in dynamin tetramer assembly via GED-GED interactions, whereas R725 appears to play a direct role in catalysis. Surprisingly, in vivo analysis indicated that overexpression of both the K694A and R725A GED mutant dynamin proteins increased the rate of formation of constricted coated pits in mammalian cells (Sever et al, 1999(Sever et al, , 2000.…”

“…Immunogold labeling studies reveal that these Dnm1p complexes cluster at sites where mitochondrial tubules are constricted (Bleazard et al, 1999). Based on the dynamin GED analysis published by Sever et al (1999Sever et al ( , 2000, we favor a model in which Dnm1p:GTP controls the formation of constricted mitochondrial tubules and the recruitment/activity of downstream partners required for fission. GTP hydrolysis by Dnm1p might then lead to the actual fission event.…”