N-Ethylmaleimide-sensitive Factor Acts at a Prefusion ATP-dependent Step in Ca2+-activated Exocytosis

Abstract

An ATP-dependent activity of NSF (N-ethylmaleimidesensitive factor) that rearranges soluble NSF attachment protein (SNAP) receptor (SNARE) protein complexes was proposed to be the driving force for membrane fusion. The Ca 2؉-activated fusion of secretory vesicles with the plasma membrane in permeable PC12 cells requires ATP; however, the ATP requirement is for a priming step that precedes the Ca 2؉ -triggered fusion reaction. While phosphoinositide phosphorylation is a key reaction required for priming, additional ATP-dependent reactions are also necessary. Here we report that the NSF-catalyzed rearrangement of SNARE protein complexes occurs during ATP-dependent priming. NSF with ␣-SNAP (soluble NSF attachment protein) were required for ATP-dependent priming but not Ca 2؉ -triggered fusion, indicating that NSF acts at an ATP-dependent prefusion step rather than at fusion itself. NSFcatalyzed activation of SNARE proteins may reorganize membranes to generate a vesicle-plasma membrane prefusion intermediate that is poised for conversion to full fusion by Ca 2؉ -dependent mechanisms.The regulated fusion of vesicles with the plasma membrane in neural and endocrine cells requires a core complex of proteins (synaptobrevin, syntaxin, and SNAP-25) that are specific substrates for clostridial neurotoxin proteases (1-4). This complex is proposed to function in vesicle targeting, docking or fusion. Identification of these neuronal synaptic proteins (termed SNAREs) 1 as receptors for SNAP proteins that mediate the membrane association of NSF, a protein required for constitutive membrane fusion (1), suggested that NSF may be required for Ca 2ϩ -regulated neurosecretion (5). Genetic studies in Drosophila have established an essential role for NSF in neural function (6). Stimulatory effects of ␣-SNAP on neurotransmitter secretion from chromaffin cells and squid neurons have been reported (7,8). However, the precise stage in the regulated secretory pathway at which NSF acts has not been directly established. In vitro biochemical studies demonstrated that a 20 S complex of SNAREs, NSF, and ␣/␤-SNAP was disassembled by the ATP-dependent activity of NSF, and it was suggested that NSF-catalyzed SNARE protein rearrangements drive membrane fusion (9). However, previous studies with permeable PC12 and adrenal cells had shown that MgATP was required for a priming step that precedes the final fusion steps triggered by Ca 2ϩ (10,11). In the present studies, the execution point of NSF and ␣-SNAP was established as the ATP-dependent priming step that precedes Ca 2ϩ -activated fusion. EXPERIMENTAL PROCEDURES Preparation of Permeable PC12 Cells and Secretion Assays-PC12 cells were labeled with [3 H]norepinephrine (NE; Amersham Corp.) and permeabilized with a ball homogenizer (10, 12). Two stage secretion assays were in KGlu buffer (20 mM HEPES, pH 7.2, 120 mM potassium glutamate, 20 mM potassium acetate, 2 mM EGTA) with 0.1% bovine serum albumin. Thirty-min priming incubations at 30°C contained 2 mM MgATP and 1.0 mg/ml rat brain cytosol, whic...

Assembly and disassembly of a ternary complex of synaptobrevin, syntaxin, and SNAP-25 in the membrane of synaptic vesicles

“…This view agrees well with recent experiments addressing the fusion mechanism of yeast vacuole precursor vesicles (20) and of mammalian endosomes in vitro (21). A ''priming'' role for NSF would be consistent with experiments studying exocytosis in permeabilized neuroendocrine cells that show that the final steps of exocytosis are independent of ATP (22)(23)(24). The physiological role of the vesicular complex, however, remains to be established.…”

Phospholipase C-related but Catalytically Inactive Protein (PRIP) Modulates Synaptosomal-associated Protein 25 (SNAP-25) Phosphorylation and Exocytosis

“…SNARE and synaptotagmin are believed to be "minimal membrane fusion machinery" in Ca 2ϩ -regulated exocytosis (1)(2)(3)(4)(5). In addition, numerous proteins are assumed to participate in the process in a modulatory manner; modulation is important for homeostatic adjustment according to physiological requirements (6 -13).…”

“…Exocytosis is the final stage of the secretory pathway and involves the membrane fusion of secretory vesicles with the plasma membrane. Extensive studies on the molecular mechanism of exocytosis have revealed that the basic mechanism of membrane fusion uses the proteins termed soluble N-ethylmaleimide-sensitive factor (NSF) 3 attachment protein receptors (SNAREs) (1)(2)(3)(4)(5). The SNARE complex consists of members of the synaptobrevin (also called vesicle-associated membrane protein (VAMP)) family on the vesicular membrane (v-SNARE) and syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) families on the target plasma membrane (t-SNARE).…”

SNARE Proteins Regulate H+-ATPase Redistribution to the Apical Membrane in Rat Renal Inner Medullary Collecting Duct Cells

“…1, A-C) are consistent with prior observations regarding the role of VAMP in exocytosis and now document the participation of VAMP in another exocytic process, the translocation of H ϩ -ATPase in renal epithelial cells. In neuronal cells, regulated exocytosis is thought to be mediated by the close interaction between the v-and t-SNAREs with the formation of a docking complex (20 S complex) comprised of the v-and t-SNAREs, NSF, and SNAP followed by ATP-mediated priming and Ca 2ϩ -dependent fusion (29,30). We have attempted to identify a similar 20 S-like complex in the IMCD cells.…”