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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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Dyn1:ts2 and the Sushi mutants exhibit wt self-assembly activities. (A) Wt and mutant dynamin proteins (2 μM) were incubated in 20 mM Hepes, pH 7.5, 2 mM MgCl2, and 10 mM KCl for 20 min at 39°C. Mixtures were sedimented at 14,000 rpm for 10 min at 4°C; pellets (P) and supernatants (S) were collected and subjected to SDS-PAGE. Proteins were detected by Coomassie blue staining. (B) Wt and mutant dynamin proteins were incubated as above except in buffer containing 150 mM KCl and with LTs. Pellets and supernatants after sedimentation are shown. (C) Negative-stained electron micrographs of wt and mutant dynamins assembled onto PI-4,5-P2–containing LTs. Bar, 50 nm.
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fig3: Dyn1:ts2 and the Sushi mutants exhibit wt self-assembly activities. (A) Wt and mutant dynamin proteins (2 μM) were incubated in 20 mM Hepes, pH 7.5, 2 mM MgCl2, and 10 mM KCl for 20 min at 39°C. Mixtures were sedimented at 14,000 rpm for 10 min at 4°C; pellets (P) and supernatants (S) were collected and subjected to SDS-PAGE. Proteins were detected by Coomassie blue staining. (B) Wt and mutant dynamin proteins were incubated as above except in buffer containing 150 mM KCl and with LTs. Pellets and supernatants after sedimentation are shown. (C) Negative-stained electron micrographs of wt and mutant dynamins assembled onto PI-4,5-P2–containing LTs. Bar, 50 nm.

Mentions: Results consistent with this conclusion were also obtained when dynamin's assembly-stimulated GTP hydrolysis was measured using lipid tubules (LTs) as a template for dynamin self-assembly (Fig. 2, E–G). The GTPase activity of both proteins was increased 50- to 100-fold upon assembly onto LT templates, and the maximum rate of LT-stimulated GTPase activity measured at 39°C was similar for dyn1:ts2 (93.6 ± 4.4 min−1) and dyn1:wt (99.1 ± 3.5 min−1) (Fig. 2 F). In contrast, dyn1:ts2 exhibited an approximately sixfold increase in Km for GTP compared with dyn1:wt (208 ± 29 vs. 34 ± 6, respectively) (Fig. 2 G). As the kcat values for these two proteins are comparable, this suggests that dyn1:ts2 exhibits a sixfold decrease in binding affinity relative to dyn1:wt. Importantly, the proteins were found to be indistinguishable in their ability to self-assemble into sedimentable structures at low salt concentration (Fig. 3 A), or into sedimentable helical arrays on LTs at physiological salt concentration (Fig. 3, B and C). From these data we conclude that the major ts defect of dyn1:ts2 is its reduced ability to bind GTP.


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)

Dyn1:ts2 and the Sushi mutants exhibit wt self-assembly activities. (A) Wt and mutant dynamin proteins (2 μM) were incubated in 20 mM Hepes, pH 7.5, 2 mM MgCl2, and 10 mM KCl for 20 min at 39°C. Mixtures were sedimented at 14,000 rpm for 10 min at 4°C; pellets (P) and supernatants (S) were collected and subjected to SDS-PAGE. Proteins were detected by Coomassie blue staining. (B) Wt and mutant dynamin proteins were incubated as above except in buffer containing 150 mM KCl and with LTs. Pellets and supernatants after sedimentation are shown. (C) Negative-stained electron micrographs of wt and mutant dynamins assembled onto PI-4,5-P2–containing LTs. Bar, 50 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171915&req=5

fig3: Dyn1:ts2 and the Sushi mutants exhibit wt self-assembly activities. (A) Wt and mutant dynamin proteins (2 μM) were incubated in 20 mM Hepes, pH 7.5, 2 mM MgCl2, and 10 mM KCl for 20 min at 39°C. Mixtures were sedimented at 14,000 rpm for 10 min at 4°C; pellets (P) and supernatants (S) were collected and subjected to SDS-PAGE. Proteins were detected by Coomassie blue staining. (B) Wt and mutant dynamin proteins were incubated as above except in buffer containing 150 mM KCl and with LTs. Pellets and supernatants after sedimentation are shown. (C) Negative-stained electron micrographs of wt and mutant dynamins assembled onto PI-4,5-P2–containing LTs. Bar, 50 nm.
Mentions: Results consistent with this conclusion were also obtained when dynamin's assembly-stimulated GTP hydrolysis was measured using lipid tubules (LTs) as a template for dynamin self-assembly (Fig. 2, E–G). The GTPase activity of both proteins was increased 50- to 100-fold upon assembly onto LT templates, and the maximum rate of LT-stimulated GTPase activity measured at 39°C was similar for dyn1:ts2 (93.6 ± 4.4 min−1) and dyn1:wt (99.1 ± 3.5 min−1) (Fig. 2 F). In contrast, dyn1:ts2 exhibited an approximately sixfold increase in Km for GTP compared with dyn1:wt (208 ± 29 vs. 34 ± 6, respectively) (Fig. 2 G). As the kcat values for these two proteins are comparable, this suggests that dyn1:ts2 exhibits a sixfold decrease in binding affinity relative to dyn1:wt. Importantly, the proteins were found to be indistinguishable in their ability to self-assemble into sedimentable structures at low salt concentration (Fig. 3 A), or into sedimentable helical arrays on LTs at physiological salt concentration (Fig. 3, B and C). From these data we conclude that the major ts defect of dyn1:ts2 is its reduced ability to bind GTP.

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