Docked secretory vesicles undergo Ca2+-activated exocytosis in a cell-free system.
The Ca2+-activated fusion of secretory vesicles with the plasma membrane responsible for regulated neurotransmitter and hormone secretion has previously been studied in permeable neuroendocrine cells, where requirements for ATP and cytosolic proteins were identified. As reported here, Ca2+-activated fusion mechanisms are also preserved following cell homogenization. The release of norepinephrine (NE) and other vesicle constituents from a PC12 cell membrane fraction was activated by micromolar Ca2+ (EC50 approximately 3 microM) and exhibited a dependence upon MgATP and cytosol. Ca2+-dependent NE release was inhibited by botulinum neurotoxins and by CAPS (Ca2+-dependent activator protein for secretion) antibody implying that syntaxin, synaptobrevin, SNAP-25 (synaptosomal-associated protein of 25 kDa), and CAPS are required for regulated exocytosis in this system. The exocytosis-competent membrane fraction 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 docked vesicles were competent for exocytosis under the reconstitution conditions used. A cell-free system for Ca2+-activated fusion will facilitate studies on the roles of essential proteins such as syntaxin, synaptobrevin, SNAP-25, and CAPS that act at post-docking steps in the regulated exocytotic pathway.
Martin
T F
TF
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA. tfmartin@facstaff.wisc.edu
Kowalchyk
J A
JA
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000648:ultrastructure*; D002474:Cell-Free System; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000648:ultrastructure*; D005089:Exocytosis / Q000187:drug effects*; D016716:PC12 Cells; D051381:Rats
D002118:Calcium