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.
Highlights
NSF and ␣-SNAP disassemble all SNARE complexes
VAMP2-syntaxin1-SNAP25 (VAMP2 is referred to as synaptobrevin-2) is the neuronal SNARE complex involved in neurotransmitter release [2]
VAMP7-syntaxin4-SNAP23 participates in synaptotagmin VII-regulated lysosomal exocytosis in fibroblasts [23]
Summary
NSF and ␣-SNAP disassemble all SNARE complexes. Results: The disassembly kinetics is conserved for different ternary and binary SNARE complexes. ␣-SNAP and the ternary SNARE complex form a 1:1 complex. The ATPase NSF (N-ethylmaleimide-sensitive factor) and the adaptor protein ␣-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. We propose a model of NSF-mediated disassembly in which the reaction is initiated by a 1:1 interaction between ␣-SNAP and the ternary SNARE complex, followed by NSF binding. We performed comparative studies of binding constants and disassembly kinetics of four different physiological ternary SNARE complexes and one binary (t-SNARE) complex with NSF and ␣-SNAP. We propose a conserved SNARE complex disassembly mechanism in which one ␣-SNAP binds the SNARE complex at any given time, with subsequent ␣-SNAP binding events possibly occurring during the disassembly process
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