SNAP-25 IS REQUIRED FOR A LATE POSTDOCKING STEP IN CA2-DEPENDENT EXOCYTOSIS()

The Ca2+-activated fusion of large dense core vesicles (LDCVs) with th e plasma membrane is reconstituted in mechanically permeabilized PC12 cells by provision of millimolar MgATP and cytosolic proteins, Ca2+-ac tivated LDCV exocytosis was inhibited completely by the type E but not the type A botulinum neurotoxin (BoNT) even though both BoNTs were eq ually effective in proteolytically cleaving the synaptosome-associated protein of 25 kDa (SNAP-25). The greater inhibition of exocytosis by BoNT E correlated with a greater destabilization of detergent-extracte d complexes consisting of SNAP-25, synaptobrevin, and syntaxin. LDCVs in permeable PC12 cells can be poised at a late postdocking, prefusion state by MgATP dependent priming processes catalyzed by N-ethylmaleim ide sensitive factor and priming in exocytosis proteins. BoNT E comple tely blocked Ca2+-activated LDCV exocytosis in ATP-primed cells, where as BoNT A was only slightly inhibitory, implying that the C-terminal r egion of SNAP-25 (Ile(181)-Gln(197)) between the cleavage sites for Bo NT E and BoNT A is essential for late postdocking steps. A required ro le for SNAP-25 at this stage was also indicated by inhibition of Ca2+- activated LDCV fusion in ATP-primed cells by a C-terminal peptide anti body. We conclude that plasma membrane SNAP-25, particularly residues 181-197, is required for Ca2+-regulated membrane fusion at a step beyo nd LDCV docking and ATP utilization.