Regulated neurotransmitter secretion is a specialized version of a general membrane fusion mechanism in which exocytotic fusion is strictly Ca 2ϩ -regulated. Studies of this process have yielded insights into universal mechanisms for intracellular membrane fusion and the identity of core components of the fusion machinery (1-3). VAMP, 1 syntaxin, and SNAP25 are the neural protein substrates for clostridial neurotoxins, a family of highly selective proteases that potently inhibits neurosecretion (4, 5). These proteins are soluble N-ethylmaleimidesensitive factor attachment protein receptors (SNAREs) that mediate the membrane association of N-ethylmaleimide-sensitive factor, a protein required for membrane fusion (6). The SNARE proteins are capable of assembling into extremely stable heterotrimeric complexes (7-9) that consist of a four-helix bundle in parallel alignment (10 -13). A current hypothesis suggests that the formation of SNARE complexes in trans across apposing membranes promotes intimate bilayer interactions and provides the energy to drive membrane fusion (10,(13)(14)(15)(16) (1,18,22,23). The precise role of synaptotagmin and its Ca 2ϩ -dependent interactions with phospholipids or syntaxin in regulated neurosecretion remains to be determined.Studies of regulated exocytosis in membrane preparations from neuroendocrine cells demonstrated that SNAREs are required for a late Ca 2ϩ -dependent step that occurs after vesicle docking and ATP-dependent priming and immediately before fusion (28,29). Tetanus toxin and botulinum neurotoxin (BoNT) B, C1, and E completely inhibited the Ca 2ϩ -dependent triggering of exocytosis, which implied that VAMP, syntaxin, and SNAP25 participate in steps close to or at fusion. Paradoxically BoNT A, which like BoNT E cleaves SNAP25, was only partially inhibitory for triggered fusion despite efficient proteolysis of SNAP25 (28). Because these toxins cleave SNAP25 at distinct C-terminal sites (Arg 180 -Ile 181 for BoNT E and Gln 197 -Arg 198 for BoNT A; see Refs. 30 and 31), it was inferred that the domain within the C terminus of SNAP25 between the toxin cleavage sites plays a distinct role in Ca 2ϩ -dependent membrane fusion events (28). In the present study, this domain of SNAP25 is revealed to be essential for Ca 2ϩ -dependent interactions with synaptotagmin. This finding clarifies classical observations that BoNT A inhibition of neurosecretion is partially reversed by elevating neuronal Ca 2ϩ levels (32). Moreover, the results suggest that an important role for the C terminus of SNAP25 in regulated exocytosis is to mediate Ca 2ϩ -dependent interactions between synaptotagmin and SNARE protein complexes.
EXPERIMENTAL PROCEDURES
Assays for Ca2ϩ -activated Exocytosis-PC12 cells grown in Dulbecco's modified culture medium supplemented with 5% horse serum and * This work was supported in part by National Institutes of Health Research Grants DK25861 and DK40428 (to T. F. J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This ar...