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 a nd the synaptosomal SNAP receptors (SNAREs) synaptobrevin, syntaxin an d SNAP-25 is essential for synaptic vesicle exocytosis, Formation of t his 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 neu rotoxins and a synaptotagmin peptide, affect the properties of the 20S complex. Cleavage of synaptobrevin and SNAP-25 by the neurotoxic clos tridial proteases tetanus toxin and botulinum toxin A had no effect on assembly and disassembly of the 20S complex; however, the stability o f its SDS-resistant SNARE core was compromised, This SDS-resistant low energy conformation of the SNAREs constitutes the physiological targe t of NSF, as indicated by its ATP-dependent disassembly in the presenc e of SNAP and NSF, Synaptotagmin peptides caused inhibition of in vitr o binding of this protein to the SNAREs, a result that is inconsistent with synaptotagmin's proposed role as a regulator of SNAP binding, Ou r data can be reconciled by the idea that NSF and SNAP generate synapt otagmin-containing intermediates in synaptic vesicle fusion, which cat alyse neurotransmitter release.