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An internal GAP domain negatively regulates presynaptic dynamin in vivo: a two-step model for dynamin function.

Narayanan R, Leonard M, Song BD, Schmid SL, Ramaswami M - J. Cell Biol. (2005)

Bottom Line: We show that the ts2 mutation, which occurs in the switch 2 region of dynamin's GTPase domain, compromises GTP binding affinity.The functional rescue in vivo correlates with a reduction in both the basal and assembly-stimulated GTPase activity in vitro.These findings demonstrate that GED is indeed an internal dynamin GAP and establish that, as for other GTPase superfamily members, dynamin's function in vivo is negatively regulated by its GAP activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology and Arizona Research Laboratories Division of Neurobiology, University of Arizona, Tucson, AZ 85721, USA.

ABSTRACT
The mechanism by which the self-assembling GTPase dynamin functions in vesicle formation remains controversial. Point mutations in shibire, the Drosophila dynamin, cause temperature-sensitive (ts) defects in endocytosis. We show that the ts2 mutation, which occurs in the switch 2 region of dynamin's GTPase domain, compromises GTP binding affinity. Three second-site suppressor mutations, one in the switch 1 region of the GTPase domain and two in the GTPase effector domain (GED), dynamin's putative GAP, fully rescue the shi(ts2) defects in synaptic vesicle recycling. The functional rescue in vivo correlates with a reduction in both the basal and assembly-stimulated GTPase activity in vitro. These findings demonstrate that GED is indeed an internal dynamin GAP and establish that, as for other GTPase superfamily members, dynamin's function in vivo is negatively regulated by its GAP activity. Based on these and other observations, we propose a two-step model for dynamin during vesicle formation in which an early regulatory GTPase-like function precedes late, assembly-dependent steps during which GTP hydrolysis is required for vesicle release.

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Distinct early and late functions for dynamin: a two-step model for dynamin function in endocytosis. A model that incorporates current and previously published observations (Kosaka and Ikeda, 1983; Hinshaw and Schmid, 1995; Takei et al., 1995; Sweitzer and Hinshaw, 1998; Sever et al., 1999; Marks et al., 2001) on dynamin's role in endocytosis. The model postulates an initial regulatory GTPase function for dynamin during a rate-determining precollar step in which the steady-state levels of dynamin GTP hydrolysis and/or dynamin•GTP are critical. These parameters are positively regulated by nucleoside diphosphate kinase (NDK) and negatively regulated by dynamin's GED, an internal GAP domain. The shits2 mutation impairs GTP binding, and the defect is restored by the three intragenic Sushi mutations, which impair GTPase activity and may function to stabilize dynamin•GTP. A subsequent post-collar step in endocytosis requires dynamin self-assembly and GTP hydrolysis, both of which are mediated, in part, by GED.
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fig6: Distinct early and late functions for dynamin: a two-step model for dynamin function in endocytosis. A model that incorporates current and previously published observations (Kosaka and Ikeda, 1983; Hinshaw and Schmid, 1995; Takei et al., 1995; Sweitzer and Hinshaw, 1998; Sever et al., 1999; Marks et al., 2001) on dynamin's role in endocytosis. The model postulates an initial regulatory GTPase function for dynamin during a rate-determining precollar step in which the steady-state levels of dynamin GTP hydrolysis and/or dynamin•GTP are critical. These parameters are positively regulated by nucleoside diphosphate kinase (NDK) and negatively regulated by dynamin's GED, an internal GAP domain. The shits2 mutation impairs GTP binding, and the defect is restored by the three intragenic Sushi mutations, which impair GTPase activity and may function to stabilize dynamin•GTP. A subsequent post-collar step in endocytosis requires dynamin self-assembly and GTP hydrolysis, both of which are mediated, in part, by GED.

Mentions: Based on these considerations and our new findings, we suggest a two-step model for dynamin function (Fig. 6) in which an initial regulatory GTPase-like mechanism operates during early, rate-limiting steps in endocytosis, whereas self-assembly and assembly-stimulated GTP hydrolysis are essential for later stages in vesicle formation. In this model, dynamin might function early in endocytosis as a scaffolding molecule, interacting through its COOH-terminal proline/arginine-rich domain with SH3 domain–containing endocytic accessory factors that control coated pit assembly and maturation. It is possible that dynamin's interactions with SH3 domain–containing effectors are influenced by its GTP-bound state and/or by GTP turnover at steady state, although this has not been shown. Nonetheless, we suggest that dynamin's interactions with these effector molecules enables it to function as a kinetic timer monitoring molecular events during early stages of coated pit formation and maturation. At later stages, dynamin self-assembles at the necks where assembly-stimulated GTP hydrolysis may function directly in membrane fission. In this model, dynamin assembly controls the transition to late events in vesicle formation, and may be triggered by accessory proteins, for example amphiphysin or SNX9 (Yoshida et al., 2004; Soulet et al., 2005) and/or by the accumulation of dynamin•GTP, as it is known that dynamin self-assembly, under physiological conditions, is strongly enhanced in the presence of nonhydrolyzable analogues of GTP (Takei et al., 1995; Carr and Hinshaw, 1997). Finally, there is now considerable evidence that GTP hydrolysis by assembled dynamin drives concerted conformational changes that can generate a constrictive force on liposomes and LTs, which would facilitate vesicle release. (Sweitzer and Hinshaw, 1998; Stowell et al., 1999; Takei et al., 1999; Marks et al., 2001; Danino et al., 2004) GTP hydrolysis also triggers rapid disassembly of the dynamin complex (Warnock et al., 1996), which might influence interactions with accessory proteins required for membrane fission and/or recycling. Although we provide compelling evidence that presynaptic dynamin functions mechanistically like a regulatory GTPase whose activity is negatively regulated by an internal GAP, further experiments are required to completely establish the details of both early and post-collar events in endocytic vesicle formation.


An internal GAP domain negatively regulates presynaptic dynamin in vivo: a two-step model for dynamin function.

Narayanan R, Leonard M, Song BD, Schmid SL, Ramaswami M - J. Cell Biol. (2005)

Distinct early and late functions for dynamin: a two-step model for dynamin function in endocytosis. A model that incorporates current and previously published observations (Kosaka and Ikeda, 1983; Hinshaw and Schmid, 1995; Takei et al., 1995; Sweitzer and Hinshaw, 1998; Sever et al., 1999; Marks et al., 2001) on dynamin's role in endocytosis. The model postulates an initial regulatory GTPase function for dynamin during a rate-determining precollar step in which the steady-state levels of dynamin GTP hydrolysis and/or dynamin•GTP are critical. These parameters are positively regulated by nucleoside diphosphate kinase (NDK) and negatively regulated by dynamin's GED, an internal GAP domain. The shits2 mutation impairs GTP binding, and the defect is restored by the three intragenic Sushi mutations, which impair GTPase activity and may function to stabilize dynamin•GTP. A subsequent post-collar step in endocytosis requires dynamin self-assembly and GTP hydrolysis, both of which are mediated, in part, by GED.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171915&req=5

fig6: Distinct early and late functions for dynamin: a two-step model for dynamin function in endocytosis. A model that incorporates current and previously published observations (Kosaka and Ikeda, 1983; Hinshaw and Schmid, 1995; Takei et al., 1995; Sweitzer and Hinshaw, 1998; Sever et al., 1999; Marks et al., 2001) on dynamin's role in endocytosis. The model postulates an initial regulatory GTPase function for dynamin during a rate-determining precollar step in which the steady-state levels of dynamin GTP hydrolysis and/or dynamin•GTP are critical. These parameters are positively regulated by nucleoside diphosphate kinase (NDK) and negatively regulated by dynamin's GED, an internal GAP domain. The shits2 mutation impairs GTP binding, and the defect is restored by the three intragenic Sushi mutations, which impair GTPase activity and may function to stabilize dynamin•GTP. A subsequent post-collar step in endocytosis requires dynamin self-assembly and GTP hydrolysis, both of which are mediated, in part, by GED.
Mentions: Based on these considerations and our new findings, we suggest a two-step model for dynamin function (Fig. 6) in which an initial regulatory GTPase-like mechanism operates during early, rate-limiting steps in endocytosis, whereas self-assembly and assembly-stimulated GTP hydrolysis are essential for later stages in vesicle formation. In this model, dynamin might function early in endocytosis as a scaffolding molecule, interacting through its COOH-terminal proline/arginine-rich domain with SH3 domain–containing endocytic accessory factors that control coated pit assembly and maturation. It is possible that dynamin's interactions with SH3 domain–containing effectors are influenced by its GTP-bound state and/or by GTP turnover at steady state, although this has not been shown. Nonetheless, we suggest that dynamin's interactions with these effector molecules enables it to function as a kinetic timer monitoring molecular events during early stages of coated pit formation and maturation. At later stages, dynamin self-assembles at the necks where assembly-stimulated GTP hydrolysis may function directly in membrane fission. In this model, dynamin assembly controls the transition to late events in vesicle formation, and may be triggered by accessory proteins, for example amphiphysin or SNX9 (Yoshida et al., 2004; Soulet et al., 2005) and/or by the accumulation of dynamin•GTP, as it is known that dynamin self-assembly, under physiological conditions, is strongly enhanced in the presence of nonhydrolyzable analogues of GTP (Takei et al., 1995; Carr and Hinshaw, 1997). Finally, there is now considerable evidence that GTP hydrolysis by assembled dynamin drives concerted conformational changes that can generate a constrictive force on liposomes and LTs, which would facilitate vesicle release. (Sweitzer and Hinshaw, 1998; Stowell et al., 1999; Takei et al., 1999; Marks et al., 2001; Danino et al., 2004) GTP hydrolysis also triggers rapid disassembly of the dynamin complex (Warnock et al., 1996), which might influence interactions with accessory proteins required for membrane fission and/or recycling. Although we provide compelling evidence that presynaptic dynamin functions mechanistically like a regulatory GTPase whose activity is negatively regulated by an internal GAP, further experiments are required to completely establish the details of both early and post-collar events in endocytic vesicle formation.

Bottom Line: We show that the ts2 mutation, which occurs in the switch 2 region of dynamin's GTPase domain, compromises GTP binding affinity.The functional rescue in vivo correlates with a reduction in both the basal and assembly-stimulated GTPase activity in vitro.These findings demonstrate that GED is indeed an internal dynamin GAP and establish that, as for other GTPase superfamily members, dynamin's function in vivo is negatively regulated by its GAP activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology and Arizona Research Laboratories Division of Neurobiology, University of Arizona, Tucson, AZ 85721, USA.

ABSTRACT
The mechanism by which the self-assembling GTPase dynamin functions in vesicle formation remains controversial. Point mutations in shibire, the Drosophila dynamin, cause temperature-sensitive (ts) defects in endocytosis. We show that the ts2 mutation, which occurs in the switch 2 region of dynamin's GTPase domain, compromises GTP binding affinity. Three second-site suppressor mutations, one in the switch 1 region of the GTPase domain and two in the GTPase effector domain (GED), dynamin's putative GAP, fully rescue the shi(ts2) defects in synaptic vesicle recycling. The functional rescue in vivo correlates with a reduction in both the basal and assembly-stimulated GTPase activity in vitro. These findings demonstrate that GED is indeed an internal dynamin GAP and establish that, as for other GTPase superfamily members, dynamin's function in vivo is negatively regulated by its GAP activity. Based on these and other observations, we propose a two-step model for dynamin during vesicle formation in which an early regulatory GTPase-like function precedes late, assembly-dependent steps during which GTP hydrolysis is required for vesicle release.

Show MeSH
Related in: MedlinePlus