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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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Sushi mutations completely suppress shits2 mutant phenotypes. (A) The three Sushi (suppressor of shits2) mutations (VS2, KVS, and Shy) do not alter levels of dynamin (top), but all completely rescue the ts paralytic behavior of shits2 mutants (bottom). (B) Synaptic vesicle endocytosis, indicated by uptake of the fluorescent endocytic tracer FM1-43 into stimulated nerve terminals, is completely blocked in shits2 but occurs efficiently in the presence of the Sushi mutations. (C) The synaptic vesicle protein synaptotagmin is trapped on the presynaptic plasma membrane and diffuses along the axon of shits2 nerve terminals stimulated at elevated temperatures (third panel), but shows normal (left two panels) synaptic vesicle distribution in the presence of the Shy, KVS, or VS2 mutations (right panels). (D) In the presence of the suppressor mutations, enhanced synaptic depression caused by use-dependent depletion of synaptic vesicles at shits2 nerve terminals stimulated at 10 Hz at 30°C is not observed (n = 5 for ts2 and VS2 and 4 for Shy and KVS). (E) Domain structure of dynamin including GTPase and GED, to which Sushi mutations VS2, KVS, and Shy are mapped. All three mutations identify residues conserved between Drosophila shibire and human dynamin-1.
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fig4: Sushi mutations completely suppress shits2 mutant phenotypes. (A) The three Sushi (suppressor of shits2) mutations (VS2, KVS, and Shy) do not alter levels of dynamin (top), but all completely rescue the ts paralytic behavior of shits2 mutants (bottom). (B) Synaptic vesicle endocytosis, indicated by uptake of the fluorescent endocytic tracer FM1-43 into stimulated nerve terminals, is completely blocked in shits2 but occurs efficiently in the presence of the Sushi mutations. (C) The synaptic vesicle protein synaptotagmin is trapped on the presynaptic plasma membrane and diffuses along the axon of shits2 nerve terminals stimulated at elevated temperatures (third panel), but shows normal (left two panels) synaptic vesicle distribution in the presence of the Shy, KVS, or VS2 mutations (right panels). (D) In the presence of the suppressor mutations, enhanced synaptic depression caused by use-dependent depletion of synaptic vesicles at shits2 nerve terminals stimulated at 10 Hz at 30°C is not observed (n = 5 for ts2 and VS2 and 4 for Shy and KVS). (E) Domain structure of dynamin including GTPase and GED, to which Sushi mutations VS2, KVS, and Shy are mapped. All three mutations identify residues conserved between Drosophila shibire and human dynamin-1.

Mentions: Strong evidence that the defect in endocytosis in shits2 is due directly to its GTP binding defect rather than indirectly to a defect in GTP hydrolysis is derived from our analysis of a new class of dynamin mutants. The so-called Suppressor of shi (Sushi) mutations were identified in an earlier genetic screen for mutations that suppressed defects in shits2 dynamin (Ramaswami et al., 1993). These mutations did not perceptibly alter protein levels (Fig. 4 A, top), but dramatically rescued the ts, paralytic phenotype (Fig. 4 A, bottom). Similarly, synaptic transmission in the visual system of Drosophila, lost in shits2 mutants at 30°C, was restored by the suppressor mutations (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200502042/DC1). Multiple lines of experimentation establish that all of the synaptic defects in shits2 mutants were rescued by the Sushi mutations. Specifically, synaptic vesicle endocytosis, which can be directly monitored as uptake of the fluorescent tracer FM1-43 into stimulated nerve terminals (Betz and Bewick, 1992; Ramaswami et al., 1994), was completely blocked in shits2, but occurred efficiently in the presence of the Sushi mutations (Fig. 4 B). The synaptic vesicle protein synaptotagmin, normally present in tight vesicle clusters localized within presynaptic varicosities, was redistributed along the axon of shits2 mutants stimulated at elevated temperatures (Estes et al., 1996); however, consistent with restored endocytosis, no such redistribution was observed in the presence of the suppressor mutations (Fig. 4 C). Finally, electrophysiological studies demonstrated that the synaptic depression that accompanies use-dependent vesicle depletion at shits2 nerve terminals was not observed in the presence of the suppressor mutations (Fig. 4 D). Thus, each of the suppressor mutations fully restores to wt both the behavioral and cellular defects of shits2 mutants.


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)

Sushi mutations completely suppress shits2 mutant phenotypes. (A) The three Sushi (suppressor of shits2) mutations (VS2, KVS, and Shy) do not alter levels of dynamin (top), but all completely rescue the ts paralytic behavior of shits2 mutants (bottom). (B) Synaptic vesicle endocytosis, indicated by uptake of the fluorescent endocytic tracer FM1-43 into stimulated nerve terminals, is completely blocked in shits2 but occurs efficiently in the presence of the Sushi mutations. (C) The synaptic vesicle protein synaptotagmin is trapped on the presynaptic plasma membrane and diffuses along the axon of shits2 nerve terminals stimulated at elevated temperatures (third panel), but shows normal (left two panels) synaptic vesicle distribution in the presence of the Shy, KVS, or VS2 mutations (right panels). (D) In the presence of the suppressor mutations, enhanced synaptic depression caused by use-dependent depletion of synaptic vesicles at shits2 nerve terminals stimulated at 10 Hz at 30°C is not observed (n = 5 for ts2 and VS2 and 4 for Shy and KVS). (E) Domain structure of dynamin including GTPase and GED, to which Sushi mutations VS2, KVS, and Shy are mapped. All three mutations identify residues conserved between Drosophila shibire and human dynamin-1.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Sushi mutations completely suppress shits2 mutant phenotypes. (A) The three Sushi (suppressor of shits2) mutations (VS2, KVS, and Shy) do not alter levels of dynamin (top), but all completely rescue the ts paralytic behavior of shits2 mutants (bottom). (B) Synaptic vesicle endocytosis, indicated by uptake of the fluorescent endocytic tracer FM1-43 into stimulated nerve terminals, is completely blocked in shits2 but occurs efficiently in the presence of the Sushi mutations. (C) The synaptic vesicle protein synaptotagmin is trapped on the presynaptic plasma membrane and diffuses along the axon of shits2 nerve terminals stimulated at elevated temperatures (third panel), but shows normal (left two panels) synaptic vesicle distribution in the presence of the Shy, KVS, or VS2 mutations (right panels). (D) In the presence of the suppressor mutations, enhanced synaptic depression caused by use-dependent depletion of synaptic vesicles at shits2 nerve terminals stimulated at 10 Hz at 30°C is not observed (n = 5 for ts2 and VS2 and 4 for Shy and KVS). (E) Domain structure of dynamin including GTPase and GED, to which Sushi mutations VS2, KVS, and Shy are mapped. All three mutations identify residues conserved between Drosophila shibire and human dynamin-1.
Mentions: Strong evidence that the defect in endocytosis in shits2 is due directly to its GTP binding defect rather than indirectly to a defect in GTP hydrolysis is derived from our analysis of a new class of dynamin mutants. The so-called Suppressor of shi (Sushi) mutations were identified in an earlier genetic screen for mutations that suppressed defects in shits2 dynamin (Ramaswami et al., 1993). These mutations did not perceptibly alter protein levels (Fig. 4 A, top), but dramatically rescued the ts, paralytic phenotype (Fig. 4 A, bottom). Similarly, synaptic transmission in the visual system of Drosophila, lost in shits2 mutants at 30°C, was restored by the suppressor mutations (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200502042/DC1). Multiple lines of experimentation establish that all of the synaptic defects in shits2 mutants were rescued by the Sushi mutations. Specifically, synaptic vesicle endocytosis, which can be directly monitored as uptake of the fluorescent tracer FM1-43 into stimulated nerve terminals (Betz and Bewick, 1992; Ramaswami et al., 1994), was completely blocked in shits2, but occurred efficiently in the presence of the Sushi mutations (Fig. 4 B). The synaptic vesicle protein synaptotagmin, normally present in tight vesicle clusters localized within presynaptic varicosities, was redistributed along the axon of shits2 mutants stimulated at elevated temperatures (Estes et al., 1996); however, consistent with restored endocytosis, no such redistribution was observed in the presence of the suppressor mutations (Fig. 4 C). Finally, electrophysiological studies demonstrated that the synaptic depression that accompanies use-dependent vesicle depletion at shits2 nerve terminals was not observed in the presence of the suppressor mutations (Fig. 4 D). Thus, each of the suppressor mutations fully restores to wt both the behavioral and cellular defects of shits2 mutants.

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