Clathrin-mediated endocytosis is one of the processes by which cells internalize macromolecules. Ultrastructural and biochemical analyses have established that the formation of clathrin-coated vesicles proceeds through several distinct steps (Schmid 1997
). Clathrin coat assembly is initiated by the binding of coat components to a docking site at the plasma membrane. Through the addition or rearrangement of coat constituents, the initially flat pit gains curvature, becoming progressively more invaginated, until the neck is constricted. Finally, membrane fission releases the coated vesicle, carrying its cargo into the cell interior (Gaidarov et al. 1999
bearing a temperature-sensitive allele of the dynamin homologue shibire
, electron microscopy of the nerve terminals reveals the accumulation of endocytic pits, most of which are encircled at their necks by a single or double electron-dense band of similar dimensions to dynamin rings (Koenig and Ikeda 1989
). In mammalian cells, overexpression of dominant negative dynamin mutants inhibits endocytosis and leads to the accumulation of invaginated coated pits (Damke et al. 1994
). Notably, dynamin collars have not been detected in nonneuronal cells, even in the shibire
flies. In vitro studies have shown that when dynamin is assembled around lipid templates, GTP hydrolysis induces a conformational change which, depending on the composition of the lipid, causes either constriction of the dynamin spirals, resulting in vesiculation (Sweitzer and Hinshaw 1998
), or an increase in the spacing between the rungs of the assembled dynamin helix (Stowell et al. 1999
). Together, these and other observations have led to various models (Hinshaw and Schmid 1995
; Warnock and Schmid 1996
; McNiven 1998
; Stowell et al. 1999
) proposing that dynamin functions as a mechanochemical enzyme whose stimulated rate of GTP hydrolysis is required for pinching off vesicles from the plasma membrane (for review see Sever et al. 2000
Dynamin mutants that are specifically defective in assembly stimulated GTPase activity (Sever et al. 1999
) were designed to test its function in endocytosis. Dynamin's stimulated GTPase activity is controlled by an intramolecular GAP, encoded within dynamin's GTPase effector domain (GED, amino acids 658–750) that becomes activated upon self-assembly (Muhlberg et al. 1997
; Sever et al. 1999
). A lysine residue, K694, is involved in intermolecular GED–GED interactions that occur upon self-assembly and are required for GAP activation; whereas an arginine residue, R725, participates more directly in catalysis (Sever et al. 1999
). Substitution of either one of these residues with alanine specifically impairs the GAP-dependent, assembly stimulated rate of GTP hydrolysis, without affecting dynamin's basal GTPase rate. In the case of the dyn(R725A) mutant, the GAP defect is due to impaired catalytic activity, whereas in the case of the dyn(K694A) mutant, the defect is due to impaired self-assembly.
Unexpectedly, transient overexpression of either of these dynamin mutants accelerated the rate of receptor-mediated endocytosis as measured by the sequestration of biotinylated transferrin (B-Tfn) from avidin (Sever et al. 1999
). These results challenged the view that dynamin acts as a mechanochemical enzyme because neither the maximal stimulated rate of GTP hydrolysis nor self-assembly appeared to be prerequisites for efficient endocytosis. Instead, because both of these activating mutants of dynamin are predicted to prolong dynamin in its GTP-bound form, these results suggested that, like other members of the GTPase superfamily, dynamin:GTP controls a rate-limiting step in endocytosis by recruiting a downstream partner.
Here, we report a detailed biochemical and morphological analysis of the functional consequences of overexpression of dyn(K694A) and dyn(R725A) in stably transformed cells aimed at addressing two unresolved questions. First, which event(s) in the formation of clathrin-coated vesicles are regulated by the GTP-bound form of dynamin? Second, given that dynamin:GTP is the active form, what is the role of rapid, assembly stimulated GTP hydrolysis? By using assays that distinguish between different steps in the formation of clathrin-coated vesicles, we show that both of the activating mutants accelerate the formation of constricted coated pits and more rapid sequestration of B-Tfn. However, overexpression of dyn (R725A), which is predicted to be defective in disassembly, decreased the rate of vesicle release. Together, our results suggest that dynamin:GTP controls the formation of constricted coated pits, and that the role of the stimulated rate of GTP-hydrolysis may be to switch dynamin off and release it from the membrane so that it does not impede membrane fission.