DOCKED SECRETORY VESICLES UNDERGO CA2-ACTIVATED EXOCYTOSIS IN A CELL-FREE SYSTEM()

The Ca2+-activated fusion of secretory vesicles with the plasma membra ne responsible for regulated neurotransmitter and hormone secretion ha s previously been studied in permeable neuroendocrine cells, where req uirements for ATP and cytosolic proteins were identified, As reported here, Ca2+-activated fusion mechanisms are also preserved following ce ll homogenization. The release of norepinephrine (NE) and other vesicl e constituents from a PC12 cell membrane fraction was activated by mic romolar Ca2+ (EC50 similar to 3 mu M) and exhibited a dependence upon MgATP and cytosol, Ca2+-dependent NE release was inhibited by botulinu m neurotoxins and by CAPS (Ca2+-dependent activator protein for secret ion) antibody implying that syntaxin, synaptobrevin, SNAP-25 (synaptos omal-associated protein of 25 kDa), and CAPS are required for regulate d exocytosis in this system, The exocytosis-competent membrane fractio n consisted of rapidly sedimenting dense core vesicles associated with plasma membrane fragments. Free vesicles did not release NE either in the absence or presence of plasma membranes, indicating that only doc ked vesicles were competent for exocytosis under the reconstitution co nditions used, A cell-free system for Ca2+-activated fusion will facil itate studies on the roles of essential proteins such as syntaxin, syn aptobrevin, SNAP-25, and CAPS that act at post-docking steps in the re gulated exocytotic pathway.