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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 has a ts defect in GTP binding. (A) The basal rates of GTP hydrolysis were determined in the presence of varying concentrations of GTP for 0.5 μM dyn1:wt (○, •) or dyn1:ts2 (□, ▪) at 20 and 39°C, as described in Materials and methods. Shown are averaged data from three independent experiments ± SEM. The kcat (B) and Km (C) values for basal GTPase activity were calculated from the data in A. Errors are SDs from the best-fit curve to the data. (D) Time course of GTP hydrolysis at 39°C for dyn1:wt (•) and dyn1:ts2 (▪) measured in the presence of physiological concentrations of GTP (100 μM). Shown are averaged data from three independent experiments ± SEM. (E–H) Same as for A–D, except that assembly-stimulated GTPase activity was measured in the presence of 0.1 mg/ml lipid nanotubules and 0.1 μM dynamin.
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fig2: Dyn1:ts2 has a ts defect in GTP binding. (A) The basal rates of GTP hydrolysis were determined in the presence of varying concentrations of GTP for 0.5 μM dyn1:wt (○, •) or dyn1:ts2 (□, ▪) at 20 and 39°C, as described in Materials and methods. Shown are averaged data from three independent experiments ± SEM. The kcat (B) and Km (C) values for basal GTPase activity were calculated from the data in A. Errors are SDs from the best-fit curve to the data. (D) Time course of GTP hydrolysis at 39°C for dyn1:wt (•) and dyn1:ts2 (▪) measured in the presence of physiological concentrations of GTP (100 μM). Shown are averaged data from three independent experiments ± SEM. (E–H) Same as for A–D, except that assembly-stimulated GTPase activity was measured in the presence of 0.1 mg/ml lipid nanotubules and 0.1 μM dynamin.

Mentions: To more precisely identify the ts biochemical defect of dyn1:ts2 responsible for the defect in endocytosis, we compared the Michaelis-Menten kinetics for basal GTPase activity of wt and mutant dynamin at both 20 and 39°C (Fig. 2, A–C). In this analysis, kcat measures the efficiency of GTP hydrolysis after binding, whereas Km, the Michaelis-Menten constant, is a composite term reflecting the rate constants for GTP association, dissociation, and catalysis. Under basal GTPase assay conditions, when hydrolysis rates are low, Km corresponds well with the affinity of dynamin for GTP (see Materials and methods). When unassembled dynamin was assayed at 20°C, both the kcat and Km were slightly increased for dyn1:ts2 relative to dyn1:wt (kcat = 0.7 ± 0.01 min−1 and 0.3 ± 0.01 min−1; and Km = 65.7 ± 3.8 μM and 35.1 ± 5.3 μM, respectively, for dyn1:ts2 and dyn1:wt) (Fig. 2, B and C). The basal GTPase activities of both wt and dyn1:ts2 increased by approximately fivefold when assayed at 39°C, and the GTPase activity of dyn1:ts2 remained approximately twofold greater than wt (kcat of 3.7 ± 0.2 min−1 vs. 2.1 ± 0.04 min−1, respectively). The most significant temperature-dependent effect observed, however, was that the Km for dyn1:ts2 assayed at 39°C was approximately sixfold increased compared with dyn1:wt (226 ± 42 μM vs. 38 ± 3 μM, respectively), suggesting a defect in GTP binding. This was confirmed using a filter assay to directly measure binding of [35S]GTPγS to dynamin (see Fig. 5 G). From these results we conclude, as predicted from earlier genetic analysis, that dyn1:ts2 exhibits a ts defect in GTP binding.


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 has a ts defect in GTP binding. (A) The basal rates of GTP hydrolysis were determined in the presence of varying concentrations of GTP for 0.5 μM dyn1:wt (○, •) or dyn1:ts2 (□, ▪) at 20 and 39°C, as described in Materials and methods. Shown are averaged data from three independent experiments ± SEM. The kcat (B) and Km (C) values for basal GTPase activity were calculated from the data in A. Errors are SDs from the best-fit curve to the data. (D) Time course of GTP hydrolysis at 39°C for dyn1:wt (•) and dyn1:ts2 (▪) measured in the presence of physiological concentrations of GTP (100 μM). Shown are averaged data from three independent experiments ± SEM. (E–H) Same as for A–D, except that assembly-stimulated GTPase activity was measured in the presence of 0.1 mg/ml lipid nanotubules and 0.1 μM dynamin.
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fig2: Dyn1:ts2 has a ts defect in GTP binding. (A) The basal rates of GTP hydrolysis were determined in the presence of varying concentrations of GTP for 0.5 μM dyn1:wt (○, •) or dyn1:ts2 (□, ▪) at 20 and 39°C, as described in Materials and methods. Shown are averaged data from three independent experiments ± SEM. The kcat (B) and Km (C) values for basal GTPase activity were calculated from the data in A. Errors are SDs from the best-fit curve to the data. (D) Time course of GTP hydrolysis at 39°C for dyn1:wt (•) and dyn1:ts2 (▪) measured in the presence of physiological concentrations of GTP (100 μM). Shown are averaged data from three independent experiments ± SEM. (E–H) Same as for A–D, except that assembly-stimulated GTPase activity was measured in the presence of 0.1 mg/ml lipid nanotubules and 0.1 μM dynamin.
Mentions: To more precisely identify the ts biochemical defect of dyn1:ts2 responsible for the defect in endocytosis, we compared the Michaelis-Menten kinetics for basal GTPase activity of wt and mutant dynamin at both 20 and 39°C (Fig. 2, A–C). In this analysis, kcat measures the efficiency of GTP hydrolysis after binding, whereas Km, the Michaelis-Menten constant, is a composite term reflecting the rate constants for GTP association, dissociation, and catalysis. Under basal GTPase assay conditions, when hydrolysis rates are low, Km corresponds well with the affinity of dynamin for GTP (see Materials and methods). When unassembled dynamin was assayed at 20°C, both the kcat and Km were slightly increased for dyn1:ts2 relative to dyn1:wt (kcat = 0.7 ± 0.01 min−1 and 0.3 ± 0.01 min−1; and Km = 65.7 ± 3.8 μM and 35.1 ± 5.3 μM, respectively, for dyn1:ts2 and dyn1:wt) (Fig. 2, B and C). The basal GTPase activities of both wt and dyn1:ts2 increased by approximately fivefold when assayed at 39°C, and the GTPase activity of dyn1:ts2 remained approximately twofold greater than wt (kcat of 3.7 ± 0.2 min−1 vs. 2.1 ± 0.04 min−1, respectively). The most significant temperature-dependent effect observed, however, was that the Km for dyn1:ts2 assayed at 39°C was approximately sixfold increased compared with dyn1:wt (226 ± 42 μM vs. 38 ± 3 μM, respectively), suggesting a defect in GTP binding. This was confirmed using a filter assay to directly measure binding of [35S]GTPγS to dynamin (see Fig. 5 G). From these results we conclude, as predicted from earlier genetic analysis, that dyn1:ts2 exhibits a ts defect in GTP binding.

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