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Disassembly of all SNARE complexes by N-ethylmaleimide-sensitive factor (NSF) is initiated by a conserved 1:1 interaction between α-soluble NSF attachment protein (SNAP) and SNARE complex.

Vivona S, Cipriano DJ, O'Leary S, Li YH, Fenn TD, Brunger AT - J. Biol. Chem. (2013)

Bottom Line: By measuring SNARE-stimulated ATP hydrolysis rates, Michaelis-Menten constants for disassembly, and SNAP-SNARE binding constants for four different ternary SNARE complexes and one binary complex, we found a conserved mechanism, not influenced by N-terminal SNARE domains. α-SNAP and the ternary SNARE complex form a 1:1 complex as revealed by multiangle light scattering.We propose a model of NSF-mediated disassembly in which the reaction is initiated by a 1:1 interaction between α-SNAP and the ternary SNARE complex, followed by NSF binding.Subsequent additional α-SNAP binding events may occur as part of a processive disassembly mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University Medical School, Stanford, California 94305, USA.

ABSTRACT
Vesicle trafficking in eukaryotic cells is facilitated by SNARE-mediated membrane fusion. The ATPase NSF (N-ethylmaleimide-sensitive factor) and the adaptor protein α-SNAP (soluble NSF attachment protein) disassemble all SNARE complexes formed throughout different pathways, but the effect of SNARE sequence and domain variation on the poorly understood disassembly mechanism is unknown. By measuring SNARE-stimulated ATP hydrolysis rates, Michaelis-Menten constants for disassembly, and SNAP-SNARE binding constants for four different ternary SNARE complexes and one binary complex, we found a conserved mechanism, not influenced by N-terminal SNARE domains. α-SNAP and the ternary SNARE complex form a 1:1 complex as revealed by multiangle light scattering. We propose a model of NSF-mediated disassembly in which the reaction is initiated by a 1:1 interaction between α-SNAP and the ternary SNARE complex, followed by NSF binding. Subsequent additional α-SNAP binding events may occur as part of a processive disassembly mechanism.

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α-SNAP-SNARE interaction and stoichiometry.A, binding of α-SNAP to the four ternary SNARE complexes. The means ± S.D. of the equilibrium dissociation constant (KD) were obtained from fitting the equation y = Fmax·x/(KD + x) to the increase in basal fluorescence of the labeled SNARE complexes as a function of α-SNAP concentration for three independent experiments (see also supplemental Fig. S2). No significant difference (p < 0.01) was observed for the four SNARE complexes by one-way analysis of variance with a Bonferroni post hoc test. B, biolayer interferometry of α-SNAP interacting with biotinylated VAMP2-syntaxin1-SNAP25 loaded onto streptavidin sensors. Three replicates of association and dissociation phases are shown. One global fit was used to fit all of the data. C, upper, SEC-MALS of the ΔNVAMP7-ΔNsyntaxin1-SNAP25 core complex (referred to as the ΔNSNARE complex), α-SNAP, 1:1 (i.e. 30 μm) and 1:3 mixtures of ΔNSNARE and α-SNAP, and a 1:3 mixture of VAMP2-syntaxin1-SNAP25 (referred to as the SNARE complex) and α-SNAP. Molecular mass and normalized differential refractive index chromatograms of the four samples are overlaid and reported as a function of elution volume using a WTC-030S5 column (Wyatt Technology). Lower, the predicted and measured molecular masses (in kDa) are shown.
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Figure 3: α-SNAP-SNARE interaction and stoichiometry.A, binding of α-SNAP to the four ternary SNARE complexes. The means ± S.D. of the equilibrium dissociation constant (KD) were obtained from fitting the equation y = Fmax·x/(KD + x) to the increase in basal fluorescence of the labeled SNARE complexes as a function of α-SNAP concentration for three independent experiments (see also supplemental Fig. S2). No significant difference (p < 0.01) was observed for the four SNARE complexes by one-way analysis of variance with a Bonferroni post hoc test. B, biolayer interferometry of α-SNAP interacting with biotinylated VAMP2-syntaxin1-SNAP25 loaded onto streptavidin sensors. Three replicates of association and dissociation phases are shown. One global fit was used to fit all of the data. C, upper, SEC-MALS of the ΔNVAMP7-ΔNsyntaxin1-SNAP25 core complex (referred to as the ΔNSNARE complex), α-SNAP, 1:1 (i.e. 30 μm) and 1:3 mixtures of ΔNSNARE and α-SNAP, and a 1:3 mixture of VAMP2-syntaxin1-SNAP25 (referred to as the SNARE complex) and α-SNAP. Molecular mass and normalized differential refractive index chromatograms of the four samples are overlaid and reported as a function of elution volume using a WTC-030S5 column (Wyatt Technology). Lower, the predicted and measured molecular masses (in kDa) are shown.

Mentions: We hypothesized that our results could be explained by a conserved interaction between the adaptor protein α-SNAP and the SNARE complex. To test this hypothesis, we took advantage of the observation that the fluorescence quantum yield of complexes labeled at their C termini with Oregon Green is sensitive to α-SNAP binding (supplemental Fig. S2), likely due to the change of chemical environment sensed by the fluorophores upon interaction with α-SNAP. Measuring the change in Oregon Green fluorescence intensity as a function of α-SNAP concentration and fitting the resulting titration curves with a simple first-order binding model (y = Fmax·x/(KD + x)) yielded no significant differences among dissociation constants (KD) (Fig. 3A and supplemental Fig. S2), supporting an overall common mode of binding of α-SNAP to all SNARE complexes, in agreement with the similar steady-state kinetics of the disassembly reaction (Fig. 2). This effect is α-SNAP-specific, as no change is seen with BSA (supplemental Fig. S2). It is also in agreement with previous reports suggesting that α-SNAP binds to the C terminus of the SNARE complex (17, 26, 32).


Disassembly of all SNARE complexes by N-ethylmaleimide-sensitive factor (NSF) is initiated by a conserved 1:1 interaction between α-soluble NSF attachment protein (SNAP) and SNARE complex.

Vivona S, Cipriano DJ, O'Leary S, Li YH, Fenn TD, Brunger AT - J. Biol. Chem. (2013)

α-SNAP-SNARE interaction and stoichiometry.A, binding of α-SNAP to the four ternary SNARE complexes. The means ± S.D. of the equilibrium dissociation constant (KD) were obtained from fitting the equation y = Fmax·x/(KD + x) to the increase in basal fluorescence of the labeled SNARE complexes as a function of α-SNAP concentration for three independent experiments (see also supplemental Fig. S2). No significant difference (p < 0.01) was observed for the four SNARE complexes by one-way analysis of variance with a Bonferroni post hoc test. B, biolayer interferometry of α-SNAP interacting with biotinylated VAMP2-syntaxin1-SNAP25 loaded onto streptavidin sensors. Three replicates of association and dissociation phases are shown. One global fit was used to fit all of the data. C, upper, SEC-MALS of the ΔNVAMP7-ΔNsyntaxin1-SNAP25 core complex (referred to as the ΔNSNARE complex), α-SNAP, 1:1 (i.e. 30 μm) and 1:3 mixtures of ΔNSNARE and α-SNAP, and a 1:3 mixture of VAMP2-syntaxin1-SNAP25 (referred to as the SNARE complex) and α-SNAP. Molecular mass and normalized differential refractive index chromatograms of the four samples are overlaid and reported as a function of elution volume using a WTC-030S5 column (Wyatt Technology). Lower, the predicted and measured molecular masses (in kDa) are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: α-SNAP-SNARE interaction and stoichiometry.A, binding of α-SNAP to the four ternary SNARE complexes. The means ± S.D. of the equilibrium dissociation constant (KD) were obtained from fitting the equation y = Fmax·x/(KD + x) to the increase in basal fluorescence of the labeled SNARE complexes as a function of α-SNAP concentration for three independent experiments (see also supplemental Fig. S2). No significant difference (p < 0.01) was observed for the four SNARE complexes by one-way analysis of variance with a Bonferroni post hoc test. B, biolayer interferometry of α-SNAP interacting with biotinylated VAMP2-syntaxin1-SNAP25 loaded onto streptavidin sensors. Three replicates of association and dissociation phases are shown. One global fit was used to fit all of the data. C, upper, SEC-MALS of the ΔNVAMP7-ΔNsyntaxin1-SNAP25 core complex (referred to as the ΔNSNARE complex), α-SNAP, 1:1 (i.e. 30 μm) and 1:3 mixtures of ΔNSNARE and α-SNAP, and a 1:3 mixture of VAMP2-syntaxin1-SNAP25 (referred to as the SNARE complex) and α-SNAP. Molecular mass and normalized differential refractive index chromatograms of the four samples are overlaid and reported as a function of elution volume using a WTC-030S5 column (Wyatt Technology). Lower, the predicted and measured molecular masses (in kDa) are shown.
Mentions: We hypothesized that our results could be explained by a conserved interaction between the adaptor protein α-SNAP and the SNARE complex. To test this hypothesis, we took advantage of the observation that the fluorescence quantum yield of complexes labeled at their C termini with Oregon Green is sensitive to α-SNAP binding (supplemental Fig. S2), likely due to the change of chemical environment sensed by the fluorophores upon interaction with α-SNAP. Measuring the change in Oregon Green fluorescence intensity as a function of α-SNAP concentration and fitting the resulting titration curves with a simple first-order binding model (y = Fmax·x/(KD + x)) yielded no significant differences among dissociation constants (KD) (Fig. 3A and supplemental Fig. S2), supporting an overall common mode of binding of α-SNAP to all SNARE complexes, in agreement with the similar steady-state kinetics of the disassembly reaction (Fig. 2). This effect is α-SNAP-specific, as no change is seen with BSA (supplemental Fig. S2). It is also in agreement with previous reports suggesting that α-SNAP binds to the C terminus of the SNARE complex (17, 26, 32).

Bottom Line: By measuring SNARE-stimulated ATP hydrolysis rates, Michaelis-Menten constants for disassembly, and SNAP-SNARE binding constants for four different ternary SNARE complexes and one binary complex, we found a conserved mechanism, not influenced by N-terminal SNARE domains. α-SNAP and the ternary SNARE complex form a 1:1 complex as revealed by multiangle light scattering.We propose a model of NSF-mediated disassembly in which the reaction is initiated by a 1:1 interaction between α-SNAP and the ternary SNARE complex, followed by NSF binding.Subsequent additional α-SNAP binding events may occur as part of a processive disassembly mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Physiology, Stanford University Medical School, Stanford, California 94305, USA.

ABSTRACT
Vesicle trafficking in eukaryotic cells is facilitated by SNARE-mediated membrane fusion. The ATPase NSF (N-ethylmaleimide-sensitive factor) and the adaptor protein α-SNAP (soluble NSF attachment protein) disassemble all SNARE complexes formed throughout different pathways, but the effect of SNARE sequence and domain variation on the poorly understood disassembly mechanism is unknown. By measuring SNARE-stimulated ATP hydrolysis rates, Michaelis-Menten constants for disassembly, and SNAP-SNARE binding constants for four different ternary SNARE complexes and one binary complex, we found a conserved mechanism, not influenced by N-terminal SNARE domains. α-SNAP and the ternary SNARE complex form a 1:1 complex as revealed by multiangle light scattering. We propose a model of NSF-mediated disassembly in which the reaction is initiated by a 1:1 interaction between α-SNAP and the ternary SNARE complex, followed by NSF binding. Subsequent additional α-SNAP binding events may occur as part of a processive disassembly mechanism.

Show MeSH
Related in: MedlinePlus