The N-ethylmaleimide-sensitive fusion protein and alpha-SNAP induce a conformational change in syntaxin.
The N-ethylmaleimide-sensitive fusion protein (NSF) plays an essential role in intracellular membrane fusion events and has been implicated in the exocytosis of synaptic vesicles. NSF binds through soluble NSF attachment proteins (SNAPs) to a complex of neuronal membrane proteins comprised of synaptobrevin, syntaxin, and SNAP-25. Disassembly of this complex by NSF is thought to be a critical step in the molecular events which lead to vesicle fusion with the plasma membrane. Here we have studied the interaction of alpha-SNAP and NSF with individual components of this complex and have identified syntaxin as a primary substrate for NSF/alpha-SNAP. We find that alpha-SNAP binds directly to syntaxin 1A as well as weakly to SNAP-25, while it does not bind to synaptobrevin II. NSF binds to syntaxin through alpha-SNAP and in the presence of ATP catalyzes a conformational rearrangement which abolishes binding of itself and alpha-SNAP. This reaction leads to the previously described disassembly of the fusion complex, since synaptobrevin binding to syntaxin is also reduced. alpha-SNAP binds to a carboxyl-terminal syntaxin fragment (residues 194-288) that also binds synaptobrevin and SNAP-25. However, NSF action on this syntaxin fragment has no effect on the binding of alpha-SNAP or synaptobrevin. This suggests that the conformational change normally induced by NSF in syntaxin depends on an interaction between carboxyl- and amino-terminal domains of syntaxin.
Hanson
P I
PI
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Otto
H
H
Barton
N
N
Jahn
R
R
D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism / Q000494:pharmacology*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D008969:Molecular Sequence Data; D052066:N-Ethylmaleimide-Sensitive Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism; D011487:Protein Conformation / Q000187:drug effects; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D033921:Vesicular Transport Proteins; D052066:N-Ethylmaleimide-Sensitive Proteins