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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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Model of SNARE complex disassembly. α-SNAP forms an initial 1:1 complex with the ternary SNARE complex (probably via the t-SNARE components of the complex) with an affinity of 1.5 μm (see Fig. 3). NSF binds to this initial α-SNAP-SNARE complex and then disassembles the ternary SNARE complex through hydrolysis of ATP. Additional α-SNAP molecules may be recruited at this stage or during the disassembly process.
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Figure 5: Model of SNARE complex disassembly. α-SNAP forms an initial 1:1 complex with the ternary SNARE complex (probably via the t-SNARE components of the complex) with an affinity of 1.5 μm (see Fig. 3). NSF binds to this initial α-SNAP-SNARE complex and then disassembles the ternary SNARE complex through hydrolysis of ATP. Additional α-SNAP molecules may be recruited at this stage or during the disassembly process.

Mentions: Our data suggest a model (Fig. 5) in which one α-SNAP molecule binds the t-SNARE components of the ternary SNARE complex with medium affinity (KD = 0.3–1.5 μm). NSF binds the α-SNAP-SNARE complex with high affinity in an arrangement that is not influenced by the VAMP and syntaxin N-terminal domains. The processive mechanism of ATP-driven disassembly by NSF is then initiated (19). At this stage or during the processive disassembly, additional α-SNAP molecules may be recruited. It is possible that two of three putative α-SNAP molecules in the 20S complex do not concurrently contact the SNARE complex. The low resolution of the cryo-EM reconstruction of the 20S complex would allow this possibility (18). If this were the case, subsequent binding and unbinding events of α-SNAP molecules with the SNARE complex could be an important part of the processive disassembly mechanism by NSF.


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)

Model of SNARE complex disassembly. α-SNAP forms an initial 1:1 complex with the ternary SNARE complex (probably via the t-SNARE components of the complex) with an affinity of 1.5 μm (see Fig. 3). NSF binds to this initial α-SNAP-SNARE complex and then disassembles the ternary SNARE complex through hydrolysis of ATP. Additional α-SNAP molecules may be recruited at this stage or during the disassembly process.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Model of SNARE complex disassembly. α-SNAP forms an initial 1:1 complex with the ternary SNARE complex (probably via the t-SNARE components of the complex) with an affinity of 1.5 μm (see Fig. 3). NSF binds to this initial α-SNAP-SNARE complex and then disassembles the ternary SNARE complex through hydrolysis of ATP. Additional α-SNAP molecules may be recruited at this stage or during the disassembly process.
Mentions: Our data suggest a model (Fig. 5) in which one α-SNAP molecule binds the t-SNARE components of the ternary SNARE complex with medium affinity (KD = 0.3–1.5 μm). NSF binds the α-SNAP-SNARE complex with high affinity in an arrangement that is not influenced by the VAMP and syntaxin N-terminal domains. The processive mechanism of ATP-driven disassembly by NSF is then initiated (19). At this stage or during the processive disassembly, additional α-SNAP molecules may be recruited. It is possible that two of three putative α-SNAP molecules in the 20S complex do not concurrently contact the SNARE complex. The low resolution of the cryo-EM reconstruction of the 20S complex would allow this possibility (18). If this were the case, subsequent binding and unbinding events of α-SNAP molecules with the SNARE complex could be an important part of the processive disassembly mechanism by NSF.

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