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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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A, ternary SNARE complexes used in this study. Upper, schematic representation of the four ternary SNARE complexes used in this study. Lower, sequence alignment of the corresponding SNARE core domains. The heptad repeats and ionic layer are highlighted in yellow and cyan, respectively. The endogenous and mutant cysteines used for maleimide-Oregon Green 488 labeling are indicated (lowercase green c). The percentage of sequence identity between single SNARE domains is indicated on the right. B, CD of the four ternary SNARE complexes showed wavelength scans typical of α-helical structures, with characteristic minima at 208 and 220 nm and positive values below 200 nm. C, thermal melts of the four ternary SNARE complexes, monitored as loss of ellipticity at 220 nm versus temperature (25–110 °C), indicated high thermal stability. D, SEC-MALS experiments showed that the four ternary SNARE complexes are monomeric. The inset reports the predicted and measured molecular masses (in kDa) of the four ternary complexes. In the chromatogram, the lines report the molecular mass, and the curves report the light scattering (i.e. normalized Rayleigh ratio) as a function of elution volume on a WTC-100S5 column (Wyatt Technology). deg, degrees.
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Figure 1: A, ternary SNARE complexes used in this study. Upper, schematic representation of the four ternary SNARE complexes used in this study. Lower, sequence alignment of the corresponding SNARE core domains. The heptad repeats and ionic layer are highlighted in yellow and cyan, respectively. The endogenous and mutant cysteines used for maleimide-Oregon Green 488 labeling are indicated (lowercase green c). The percentage of sequence identity between single SNARE domains is indicated on the right. B, CD of the four ternary SNARE complexes showed wavelength scans typical of α-helical structures, with characteristic minima at 208 and 220 nm and positive values below 200 nm. C, thermal melts of the four ternary SNARE complexes, monitored as loss of ellipticity at 220 nm versus temperature (25–110 °C), indicated high thermal stability. D, SEC-MALS experiments showed that the four ternary SNARE complexes are monomeric. The inset reports the predicted and measured molecular masses (in kDa) of the four ternary complexes. In the chromatogram, the lines report the molecular mass, and the curves report the light scattering (i.e. normalized Rayleigh ratio) as a function of elution volume on a WTC-100S5 column (Wyatt Technology). deg, degrees.

Mentions: To compare the kinetics of NSF-driven disassembly of SNARE variants, we selected four different physiological SNARE complexes with differences in both primary sequence and domain architecture (Fig. 1A). VAMP2-syntaxin1-SNAP25 (VAMP2 is also referred to as synaptobrevin-2) is the neuronal SNARE complex involved in neurotransmitter release (2). VAMP7-syntaxin1-SNAP25 forms at the neuronal plasma membrane, where VAMP7 sustains axon outgrowth through transport of the cell adhesion molecule L1 (20, 21) and is thought to activate exocytosis of the resting pool of synaptic vesicles (22). VAMP7-syntaxin4-SNAP23 participates in synaptotagmin VII-regulated lysosomal exocytosis in fibroblasts (23). VAMP8-syntaxin4-SNAP23 is involved in cytokine/chemokine trafficking by segregating lysosomal secretory granules in mast cells (24). These four different ternary SNARE complexes allowed us to test if NSF-driven disassembly is influenced by primary sequence variation of the SNARE core domains and by differences in the N-terminal domains (i.e. VAMP2 versus VAMP7).


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)

A, ternary SNARE complexes used in this study. Upper, schematic representation of the four ternary SNARE complexes used in this study. Lower, sequence alignment of the corresponding SNARE core domains. The heptad repeats and ionic layer are highlighted in yellow and cyan, respectively. The endogenous and mutant cysteines used for maleimide-Oregon Green 488 labeling are indicated (lowercase green c). The percentage of sequence identity between single SNARE domains is indicated on the right. B, CD of the four ternary SNARE complexes showed wavelength scans typical of α-helical structures, with characteristic minima at 208 and 220 nm and positive values below 200 nm. C, thermal melts of the four ternary SNARE complexes, monitored as loss of ellipticity at 220 nm versus temperature (25–110 °C), indicated high thermal stability. D, SEC-MALS experiments showed that the four ternary SNARE complexes are monomeric. The inset reports the predicted and measured molecular masses (in kDa) of the four ternary complexes. In the chromatogram, the lines report the molecular mass, and the curves report the light scattering (i.e. normalized Rayleigh ratio) as a function of elution volume on a WTC-100S5 column (Wyatt Technology). deg, degrees.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: A, ternary SNARE complexes used in this study. Upper, schematic representation of the four ternary SNARE complexes used in this study. Lower, sequence alignment of the corresponding SNARE core domains. The heptad repeats and ionic layer are highlighted in yellow and cyan, respectively. The endogenous and mutant cysteines used for maleimide-Oregon Green 488 labeling are indicated (lowercase green c). The percentage of sequence identity between single SNARE domains is indicated on the right. B, CD of the four ternary SNARE complexes showed wavelength scans typical of α-helical structures, with characteristic minima at 208 and 220 nm and positive values below 200 nm. C, thermal melts of the four ternary SNARE complexes, monitored as loss of ellipticity at 220 nm versus temperature (25–110 °C), indicated high thermal stability. D, SEC-MALS experiments showed that the four ternary SNARE complexes are monomeric. The inset reports the predicted and measured molecular masses (in kDa) of the four ternary complexes. In the chromatogram, the lines report the molecular mass, and the curves report the light scattering (i.e. normalized Rayleigh ratio) as a function of elution volume on a WTC-100S5 column (Wyatt Technology). deg, degrees.
Mentions: To compare the kinetics of NSF-driven disassembly of SNARE variants, we selected four different physiological SNARE complexes with differences in both primary sequence and domain architecture (Fig. 1A). VAMP2-syntaxin1-SNAP25 (VAMP2 is also referred to as synaptobrevin-2) is the neuronal SNARE complex involved in neurotransmitter release (2). VAMP7-syntaxin1-SNAP25 forms at the neuronal plasma membrane, where VAMP7 sustains axon outgrowth through transport of the cell adhesion molecule L1 (20, 21) and is thought to activate exocytosis of the resting pool of synaptic vesicles (22). VAMP7-syntaxin4-SNAP23 participates in synaptotagmin VII-regulated lysosomal exocytosis in fibroblasts (23). VAMP8-syntaxin4-SNAP23 is involved in cytokine/chemokine trafficking by segregating lysosomal secretory granules in mast cells (24). These four different ternary SNARE complexes allowed us to test if NSF-driven disassembly is influenced by primary sequence variation of the SNARE core domains and by differences in the N-terminal domains (i.e. VAMP2 versus VAMP7).

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