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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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Comparison of ATPase stimulation and disassembly kinetics for ternary and binary SNARE complexes. All bar diagrams show means ± S.D. from three replicates. Statistical comparisons were by one-way analysis of variance with a Bonferroni post hoc test. **, p < 0.01; ***, p < 0.001. A, stimulation of ATPase activity by ternary SNARE complexes in the presence of α-SNAP. All four ternary SNARE complexes similarly stimulated the ATPase activity of NSF. B, steady-state kinetics of NSF-driven disassembly of the four ternary SNARE complexes. The means ± S.D. of kcat and Km values were obtained from fitting the Michaelis-Menten equation (y = kcat·x/(Km + x)) to the observed initial disassembly rates as a function of SNARE complex concentration for three independent experiments (see also supplemental Fig. S1A). Km values are statistically similar for the four ternary complexes, although there was a small difference between the kcat obtained for the complexes containing syntaxin1-SNAP25 and those containing syntaxin4-SNAP23. C, steady-state kinetics of NSF-driven disassembly for the four ternary SNARE complexes and the neuronal t-SNARE (syntaxin1-SNAP25) complex as a function of α-SNAP concentration. The means ± S.D. of Km and kcat values were obtained by fitting the Michaelis-Menten equation to the observed initial disassembly rates as a function of α-SNAP concentration for three independent experiments (see also supplemental Fig. S1B). Similar to B, a small difference was observed between the kcat of the complexes containing syntaxin1-SNAP25 and the kcat of those containing syntaxin4-SNAP23. The slight variation of kcat values in B and C is likely due to batch-to-batch variability of different NSF preparations and temperature variations across different instruments and experiments.
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Figure 2: Comparison of ATPase stimulation and disassembly kinetics for ternary and binary SNARE complexes. All bar diagrams show means ± S.D. from three replicates. Statistical comparisons were by one-way analysis of variance with a Bonferroni post hoc test. **, p < 0.01; ***, p < 0.001. A, stimulation of ATPase activity by ternary SNARE complexes in the presence of α-SNAP. All four ternary SNARE complexes similarly stimulated the ATPase activity of NSF. B, steady-state kinetics of NSF-driven disassembly of the four ternary SNARE complexes. The means ± S.D. of kcat and Km values were obtained from fitting the Michaelis-Menten equation (y = kcat·x/(Km + x)) to the observed initial disassembly rates as a function of SNARE complex concentration for three independent experiments (see also supplemental Fig. S1A). Km values are statistically similar for the four ternary complexes, although there was a small difference between the kcat obtained for the complexes containing syntaxin1-SNAP25 and those containing syntaxin4-SNAP23. C, steady-state kinetics of NSF-driven disassembly for the four ternary SNARE complexes and the neuronal t-SNARE (syntaxin1-SNAP25) complex as a function of α-SNAP concentration. The means ± S.D. of Km and kcat values were obtained by fitting the Michaelis-Menten equation to the observed initial disassembly rates as a function of α-SNAP concentration for three independent experiments (see also supplemental Fig. S1B). Similar to B, a small difference was observed between the kcat of the complexes containing syntaxin1-SNAP25 and the kcat of those containing syntaxin4-SNAP23. The slight variation of kcat values in B and C is likely due to batch-to-batch variability of different NSF preparations and temperature variations across different instruments and experiments.

Mentions: We measured the stimulation of NSF ATPase activity by the four different ternary SNARE complexes. All four complexes similarly stimulated the steady-state rate of NSF-catalyzed ATP hydrolysis activity (Fig. 2A), suggesting that the stimulation of ATPase activity is independent of SNARE primary sequence variation and N-terminal domain architecture.


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)

Comparison of ATPase stimulation and disassembly kinetics for ternary and binary SNARE complexes. All bar diagrams show means ± S.D. from three replicates. Statistical comparisons were by one-way analysis of variance with a Bonferroni post hoc test. **, p < 0.01; ***, p < 0.001. A, stimulation of ATPase activity by ternary SNARE complexes in the presence of α-SNAP. All four ternary SNARE complexes similarly stimulated the ATPase activity of NSF. B, steady-state kinetics of NSF-driven disassembly of the four ternary SNARE complexes. The means ± S.D. of kcat and Km values were obtained from fitting the Michaelis-Menten equation (y = kcat·x/(Km + x)) to the observed initial disassembly rates as a function of SNARE complex concentration for three independent experiments (see also supplemental Fig. S1A). Km values are statistically similar for the four ternary complexes, although there was a small difference between the kcat obtained for the complexes containing syntaxin1-SNAP25 and those containing syntaxin4-SNAP23. C, steady-state kinetics of NSF-driven disassembly for the four ternary SNARE complexes and the neuronal t-SNARE (syntaxin1-SNAP25) complex as a function of α-SNAP concentration. The means ± S.D. of Km and kcat values were obtained by fitting the Michaelis-Menten equation to the observed initial disassembly rates as a function of α-SNAP concentration for three independent experiments (see also supplemental Fig. S1B). Similar to B, a small difference was observed between the kcat of the complexes containing syntaxin1-SNAP25 and the kcat of those containing syntaxin4-SNAP23. The slight variation of kcat values in B and C is likely due to batch-to-batch variability of different NSF preparations and temperature variations across different instruments and experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750193&req=5

Figure 2: Comparison of ATPase stimulation and disassembly kinetics for ternary and binary SNARE complexes. All bar diagrams show means ± S.D. from three replicates. Statistical comparisons were by one-way analysis of variance with a Bonferroni post hoc test. **, p < 0.01; ***, p < 0.001. A, stimulation of ATPase activity by ternary SNARE complexes in the presence of α-SNAP. All four ternary SNARE complexes similarly stimulated the ATPase activity of NSF. B, steady-state kinetics of NSF-driven disassembly of the four ternary SNARE complexes. The means ± S.D. of kcat and Km values were obtained from fitting the Michaelis-Menten equation (y = kcat·x/(Km + x)) to the observed initial disassembly rates as a function of SNARE complex concentration for three independent experiments (see also supplemental Fig. S1A). Km values are statistically similar for the four ternary complexes, although there was a small difference between the kcat obtained for the complexes containing syntaxin1-SNAP25 and those containing syntaxin4-SNAP23. C, steady-state kinetics of NSF-driven disassembly for the four ternary SNARE complexes and the neuronal t-SNARE (syntaxin1-SNAP25) complex as a function of α-SNAP concentration. The means ± S.D. of Km and kcat values were obtained by fitting the Michaelis-Menten equation to the observed initial disassembly rates as a function of α-SNAP concentration for three independent experiments (see also supplemental Fig. S1B). Similar to B, a small difference was observed between the kcat of the complexes containing syntaxin1-SNAP25 and the kcat of those containing syntaxin4-SNAP23. The slight variation of kcat values in B and C is likely due to batch-to-batch variability of different NSF preparations and temperature variations across different instruments and experiments.
Mentions: We measured the stimulation of NSF ATPase activity by the four different ternary SNARE complexes. All four complexes similarly stimulated the steady-state rate of NSF-catalyzed ATP hydrolysis activity (Fig. 2A), suggesting that the stimulation of ATPase activity is independent of SNARE primary sequence variation and N-terminal domain architecture.

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