Disassembly of the reconstituted synaptic vesicle membrane fusion complex in vitro.
The interaction of the presynaptic membrane proteins SNAP-25 and syntaxin with the synaptic vesicle protein synaptobrevin (VAMP) plays a key role in the regulated exocytosis of neurotransmitters. Clostridial neurotoxins, which proteolyze these polypeptides, are potent inhibitors of neurotransmission. The cytoplasmic domains of the three membrane proteins join into a tight SDS-resistant complex (Hayashi et al., 1994). Here, we show that this reconstituted complex, as well as heterodimers composed of syntaxin and SNAP-25, can be disassembled by the concerted action of the N-ethylmaleimide-sensitive factor, NSF, and the soluble NSF attachment protein, alpha-SNAP. alpha-SNAP binds to predicted alpha-helical coiled-coil regions of syntaxin and SNAP-25, shown previously to be engaged in their direct interaction. Synaptobrevin, although incapable of binding alpha-SNAP individually, induced a third alpha-SNAP binding site when associated with syntaxin and SNAP-25 into heterotrimers. NSF released prebound alpha-SNAP from full-length syntaxin but not from a syntaxin derivative truncated at the N-terminus. Disassembly of complexes containing this syntaxin mutant was impaired, indicating a critical role for the N-terminal domain in the alpha-SNAP/NSF-mediated dissociation process. Complexes containing C-terminally deleted SNAP-25 derivatives, as generated by botulinal toxins type A and E, were dissociated more efficiently. In contrast, the N-terminal fragment generated from synaptobrevin by botulinal toxin type F produced an SDS-sensitive complex that was poorly dissociated.
Hayashi
T
T
Department of Microbiology, Federal Research Center for Virus Diseases of Animals, Tübingen, Germany.
Yamasaki
S
S
Nauenburg
S
S
Binz
T
T
Niemann
H
H
D001427:Bacterial Toxins / Q000494:pharmacology; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism; D046911:Macromolecular Substances; D008561:Membrane Fusion / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D051938:Munc18 Proteins; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009498:Neurotoxins / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D013329:Structure-Activity Relationship; D013572:Synaptic Vesicles / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins
D001427:Bacterial Toxins; D002352:Carrier Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; D009498:Neurotoxins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D011994:Recombinant Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins