Disassembly of the reconstituted synaptic vesicle membrane fusion complex in vitro

EMBO J. 1995 May 15;14(10):2317-25.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Toxins / pharmacology
  • Base Sequence
  • Carrier Proteins / metabolism
  • Macromolecular Substances
  • Membrane Fusion / physiology*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Models, Biological
  • Molecular Sequence Data
  • Munc18 Proteins
  • Mutation
  • Nerve Tissue Proteins / metabolism*
  • Neurotoxins / pharmacology
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Secondary
  • Qa-SNARE Proteins
  • R-SNARE Proteins
  • Recombinant Proteins / metabolism
  • Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins
  • Structure-Activity Relationship
  • Synaptic Vesicles / metabolism*
  • Synaptosomal-Associated Protein 25
  • Vesicular Transport Proteins*

Substances

  • Bacterial Toxins
  • Carrier Proteins
  • Macromolecular Substances
  • Membrane Proteins
  • Munc18 Proteins
  • Nerve Tissue Proteins
  • Neurotoxins
  • Qa-SNARE Proteins
  • R-SNARE Proteins
  • Recombinant Proteins
  • Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins
  • Synaptosomal-Associated Protein 25
  • Vesicular Transport Proteins