Martin T F, Kowalchyk J A
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
J Biol Chem. 1997 May 30;272(22):14447-53. doi: 10.1074/jbc.272.22.14447.
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.
分泌小泡与质膜的钙离子激活融合负责调节神经递质和激素分泌，此前已在可渗透的神经内分泌细胞中进行过研究，在这些细胞中确定了对ATP和胞质蛋白的需求。如本文所报道，细胞匀浆后钙离子激活的融合机制也得以保留。去甲肾上腺素（NE）和其他小泡成分从PC12细胞膜组分中的释放被微摩尔浓度的钙离子激活（EC50约为3微摩尔），并且表现出对MgATP和胞质溶胶的依赖性。钙离子依赖性的NE释放受到肉毒杆菌神经毒素和CAPS（钙离子依赖性分泌激活蛋白）抗体的抑制，这意味着 syntaxin、突触小泡蛋白、SNAP-25（25 kDa的突触体相关蛋白）和CAPS是该系统中调节性胞吐作用所必需的。具有胞吐能力的膜组分由与质膜片段相关的快速沉降的致密核心小泡组成。游离小泡在有无质膜的情况下均不释放NE，这表明在所用的重构条件下，只有对接的小泡才有胞吐能力。用于钙离子激活融合的无细胞系统将有助于研究在调节性胞吐途径的对接后步骤中起作用的必需蛋白质，如syntaxin、突触小泡蛋白、SNAP-25和CAPS的作用。
