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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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The shits2 mutation (G146S) is in the highly conserved switch 2 region of the GTPase domain. (A) Alignment of Drosophila shibire, human dynamin-1, and Dictyostelium dynamin A around the ts2 mutation shows tightly conserved residues. (B) Threaded structure of human dynamin-1 GTPase domain folded on a template crystal structure of Dictyostelium dynamin A GTPase domain (Niemann et al., 2001) using Geno3D automatic comparative modeling of three-dimensional protein structure (Combet et al., 2002) and Swiss-PdbViewer (http://www.expasy.org). The four consensus GTP binding elements (green arrows) and the locations of the ts1 and ts2 mutations (yellow arrows) are indicated. (C) Temperature dependence of basal GTPase activity of dyn1:wt (○) and dyn1:ts2 (□) measured in the presence of 100 μM GTP.
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fig1: The shits2 mutation (G146S) is in the highly conserved switch 2 region of the GTPase domain. (A) Alignment of Drosophila shibire, human dynamin-1, and Dictyostelium dynamin A around the ts2 mutation shows tightly conserved residues. (B) Threaded structure of human dynamin-1 GTPase domain folded on a template crystal structure of Dictyostelium dynamin A GTPase domain (Niemann et al., 2001) using Geno3D automatic comparative modeling of three-dimensional protein structure (Combet et al., 2002) and Swiss-PdbViewer (http://www.expasy.org). The four consensus GTP binding elements (green arrows) and the locations of the ts1 and ts2 mutations (yellow arrows) are indicated. (C) Temperature dependence of basal GTPase activity of dyn1:wt (○) and dyn1:ts2 (□) measured in the presence of 100 μM GTP.

Mentions: The shits2 mutation corresponds to a Gly to Ser substitution within the switch 2 region of the GTPase domain (Fig. 1 A), known to be involved in GTP binding and hydrolysis (van der Bliek and Meyerowitz, 1991). Our finding that shits2 mutant phenotypes are substantially aggravated by a reduction in nucleoside diphosphate kinase, an enzyme that provides GTP pools accessed by dynamin (Krishnan et al., 2001; Palacios et al., 2002), suggested that this mutation might compromise GTP binding. To test this hypothesis, we generated the ts2-homologous amino acid substitution (G146S) in human dynamin-1, whose biochemical properties in vitro have been thoroughly studied (for review see Song and Schmid, 2003). The validity of this experimental design is supported by (1) the high conservation of GTPase domain sequences (Fig. 1 A) between human and Drosophila dynamins (Chen et al., 1991; van der Bliek and Meyerowitz, 1991); (2) the fact that the entire three-dimensional structures of both GTPase domains are easily folded on a template crystal structure of the Dictyostelium dynamin A GTPase domain (Niemann et al., 2001) (Fig. 1 B); and (3) our finding that HeLa cells exhibit a ts defect in endocytosis similar to shits1 flies when overexpressing dyn1:G273D, the human homologue of shits1 (Damke et al., 1995).


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)

The shits2 mutation (G146S) is in the highly conserved switch 2 region of the GTPase domain. (A) Alignment of Drosophila shibire, human dynamin-1, and Dictyostelium dynamin A around the ts2 mutation shows tightly conserved residues. (B) Threaded structure of human dynamin-1 GTPase domain folded on a template crystal structure of Dictyostelium dynamin A GTPase domain (Niemann et al., 2001) using Geno3D automatic comparative modeling of three-dimensional protein structure (Combet et al., 2002) and Swiss-PdbViewer (http://www.expasy.org). The four consensus GTP binding elements (green arrows) and the locations of the ts1 and ts2 mutations (yellow arrows) are indicated. (C) Temperature dependence of basal GTPase activity of dyn1:wt (○) and dyn1:ts2 (□) measured in the presence of 100 μM GTP.
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Related In: Results  -  Collection

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

fig1: The shits2 mutation (G146S) is in the highly conserved switch 2 region of the GTPase domain. (A) Alignment of Drosophila shibire, human dynamin-1, and Dictyostelium dynamin A around the ts2 mutation shows tightly conserved residues. (B) Threaded structure of human dynamin-1 GTPase domain folded on a template crystal structure of Dictyostelium dynamin A GTPase domain (Niemann et al., 2001) using Geno3D automatic comparative modeling of three-dimensional protein structure (Combet et al., 2002) and Swiss-PdbViewer (http://www.expasy.org). The four consensus GTP binding elements (green arrows) and the locations of the ts1 and ts2 mutations (yellow arrows) are indicated. (C) Temperature dependence of basal GTPase activity of dyn1:wt (○) and dyn1:ts2 (□) measured in the presence of 100 μM GTP.
Mentions: The shits2 mutation corresponds to a Gly to Ser substitution within the switch 2 region of the GTPase domain (Fig. 1 A), known to be involved in GTP binding and hydrolysis (van der Bliek and Meyerowitz, 1991). Our finding that shits2 mutant phenotypes are substantially aggravated by a reduction in nucleoside diphosphate kinase, an enzyme that provides GTP pools accessed by dynamin (Krishnan et al., 2001; Palacios et al., 2002), suggested that this mutation might compromise GTP binding. To test this hypothesis, we generated the ts2-homologous amino acid substitution (G146S) in human dynamin-1, whose biochemical properties in vitro have been thoroughly studied (for review see Song and Schmid, 2003). The validity of this experimental design is supported by (1) the high conservation of GTPase domain sequences (Fig. 1 A) between human and Drosophila dynamins (Chen et al., 1991; van der Bliek and Meyerowitz, 1991); (2) the fact that the entire three-dimensional structures of both GTPase domains are easily folded on a template crystal structure of the Dictyostelium dynamin A GTPase domain (Niemann et al., 2001) (Fig. 1 B); and (3) our finding that HeLa cells exhibit a ts defect in endocytosis similar to shits1 flies when overexpressing dyn1:G273D, the human homologue of shits1 (Damke et al., 1995).

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