Botulinum neurotoxin E-insensitive mutants of SNAP-25 fail to bind VAMP but support exocytosis

Neurotransmitter release from synaptic vesicles is mediated by complex mach inery, which includes the v- and t-SNAP receptors (SNAREs), vesicle-associa ted membrane protein (VAMP), synaptotagmin, syntaxin, and synaptosome-assoc iated protein of 25 kDa (SNAP;25). They are essential for neurotransmitter exocytosis because they are the proteolytic substrates of the clostridial n eurotoxins tetanus neurotoxin and botulinum neurotoxins (BoNTs), which caus e tetanus and botulism, respectively. Specifically, SNAP-25 is cleaved by b oth BoNT/A and E at separate sites within the COOH-terminus. We now demonst rate, using toxin-insensitive mutants of SNAP-25, that these two toxins dif fer in their specificity for the cleavage site. Following modification with in the COOH-terminus, the mutants completely resistant to BoNT/E do not bin d VAMP but were still able to form a sodium dodecyl sulfate-resistant compl ex with VAMP and syntaxin. Furthermore, these mutants retain function in vi vo, conferring BoNT/E-resistant exocytosis to transfected PC12 cells. These data provide information on structural requirements within the C-terminal domain of SNAP-25 for its function in exocytosis and mise doubts about the significance of in vitro binary interactions for the in vivo functions of s ynaptic protein complexes.