Clostridial neurotoxins compromise the stability of a low energy SNARE complex mediating NSF activation of synaptic vesicle fusion.
A 20S complex composed of the cytosolic fusion proteins NSF and SNAP and the synaptosomal SNAP receptors (SNAREs) synaptobrevin, syntaxin and SNAP-25 is essential for synaptic vesicle exocytosis. Formation of this complex is thought to be regulated by synaptotagmin, the putative calcium sensor of neurotransmitter release. Here we have examined how different inhibitors of neurotransmitter release, e.g. clostridial neurotoxins and a synaptotagmin peptide, affect the properties of the 20S complex. Cleavage of synaptobrevin and SNAP-25 by the neurotoxic clostridial proteases tetanus toxin and botulinum toxin A had no effect on assembly and disassembly of the 20S complex; however, the stability of its SDS-resistant SNARE core was compromised. This SDS-resistant low energy conformation of the SNAREs constitutes the physiological target of NSF, as indicated by its ATP-dependent disassembly in the presence of SNAP and NSF. Synaptotagmin peptides caused inhibition of in vitro binding of this protein to the SNAREs, a result that is inconsistent with synaptotagmin's proposed role as a regulator of SNAP binding. Our data can be reconciled by the idea that NSF and SNAP generate synaptotagmin-containing intermediates in synaptic vesicle fusion, which catalyse neurotransmitter release.
Pellegrini
L L
LL
Abteilung Neurochemie, Max-Planck Institut für Hirnforschung, Frankfurt am Main, Germany.
O'Connor
V
V
Lottspeich
F
F
Betz
H
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001905:Botulinum Toxins / Q000494:pharmacology; D001921:Brain / Q000378:metabolism; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D005089:Exocytosis / Q000187:drug effects; D006868:Hydrolysis; D046911:Macromolecular Substances; D008561:Membrane Fusion / Q000187:drug effects*; D008562:Membrane Glycoproteins / Q000494:pharmacology; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism*; D052066:N-Ethylmaleimide-Sensitive Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism / Q000494:pharmacology; D018377:Neurotransmitter Agents / Q000037:antagonists & inhibitors; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D051381:Rats; D050600:SNARE Proteins; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D013572:Synaptic Vesicles / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25; D050857:Synaptotagmins; D013744:Tetanus Toxin / Q000494:pharmacology*; D033921:Vesicular Transport Proteins
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D050825:Synaptosomal-Associated Protein 25; D013744:Tetanus Toxin; D033921:Vesicular Transport Proteins; D050857:Synaptotagmins; D012967:Sodium Dodecyl Sulfate; D000255:Adenosine Triphosphate; D001905:Botulinum Toxins; D052066:N-Ethylmaleimide-Sensitive Proteins; C498737:Nsf protein, rat