Phosphatidylinositol 4,5-bisphosphate (PI 4,5-P2) on the plasma membrane is essential for vesicle exocytosis but its role in membrane fusion has not been determined. Here, we quantify the concentration of PI 4,5-P2 as ∼6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles. At this concentration of PI 4,5-P2 soluble NSF attachment protein receptor (SNARE)–dependent liposome fusion is inhibited. Inhibition by PI 4,5-P2 likely results from its intrinsic positive curvature–promoting properties that inhibit formation of high negative curvature membrane fusion intermediates. Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P2, suggesting that syntaxin sequesters PI 4,5-P2 to alleviate inhibition. To define an essential rather than inhibitory role for PI 4,5-P2, we test a PI 4,5-P2–binding priming factor required for vesicle exocytosis. Ca2+-dependent activator protein for secretion promotes increased rates of SNARE-dependent fusion that are PI 4,5-P2 dependent. These results indicate that PI 4,5-P2 regulates fusion both as a fusion restraint that syntaxin-1 alleviates and as an essential cofactor that recruits protein priming factors to facilitate SNARE-dependent fusion.

Synaptotagmin is a proposed Ca2+ sensor on the vesicle for regulated exocytosis and exhibits Ca2+-dependent binding to phospholipids, syntaxin, and SNAP-25 in vitro, but the mechanism by which Ca2+ triggers membrane fusion is uncertain. Previous studies suggested that SNAP-25 plays a role in the Ca2+ regulation of secretion. We found that synaptotagmins I and IX associate with SNAP-25 during Ca2+-dependent exocytosis in PC12 cells, and we identified C-terminal amino acids in SNAP-25 (Asp179, Asp186, Asp193) that are required for Ca2+-dependent synaptotagmin binding. Replacement of SNAP-25 in PC12 cells with SNAP-25 containing C-terminal Asp mutations led to a loss-of-function in regulated exocytosis at the Ca2+-dependent fusion step. These results indicate that the Ca2+-dependent interaction of synaptotagmin with SNAP-25 is essential for the Ca2+-dependent triggering of membrane fusion.

Regulated neurotransmitter secretion is a specialized version of a general membrane fusion mechanism in which exocytotic fusion is strictly Ca 2ϩ -regulated. Studies of this process have yielded insights into universal mechanisms for intracellular membrane fusion and the identity of core components of the fusion machinery (1-3). VAMP, 1 syntaxin, and SNAP25 are the neural protein substrates for clostridial neurotoxins, a family of highly selective proteases that potently inhibits neurosecretion (4, 5). These proteins are soluble N-ethylmaleimidesensitive factor attachment protein receptors (SNAREs) that mediate the membrane association of N-ethylmaleimide-sensitive factor, a protein required for membrane fusion (6). The SNARE proteins are capable of assembling into extremely stable heterotrimeric complexes (7-9) that consist of a four-helix bundle in parallel alignment (10 -13). A current hypothesis suggests that the formation of SNARE complexes in trans across apposing membranes promotes intimate bilayer interactions and provides the energy to drive membrane fusion (10,(13)(14)(15)(16) (1,18,22,23). The precise role of synaptotagmin and its Ca 2ϩ -dependent interactions with phospholipids or syntaxin in regulated neurosecretion remains to be determined.Studies of regulated exocytosis in membrane preparations from neuroendocrine cells demonstrated that SNAREs are required for a late Ca 2ϩ -dependent step that occurs after vesicle docking and ATP-dependent priming and immediately before fusion (28,29). Tetanus toxin and botulinum neurotoxin (BoNT) B, C1, and E completely inhibited the Ca 2ϩ -dependent triggering of exocytosis, which implied that VAMP, syntaxin, and SNAP25 participate in steps close to or at fusion. Paradoxically BoNT A, which like BoNT E cleaves SNAP25, was only partially inhibitory for triggered fusion despite efficient proteolysis of SNAP25 (28). Because these toxins cleave SNAP25 at distinct C-terminal sites (Arg 180 -Ile 181 for BoNT E and Gln 197 -Arg 198 for BoNT A; see Refs. 30 and 31), it was inferred that the domain within the C terminus of SNAP25 between the toxin cleavage sites plays a distinct role in Ca 2ϩ -dependent membrane fusion events (28). In the present study, this domain of SNAP25 is revealed to be essential for Ca 2ϩ -dependent interactions with synaptotagmin. This finding clarifies classical observations that BoNT A inhibition of neurosecretion is partially reversed by elevating neuronal Ca 2ϩ levels (32). Moreover, the results suggest that an important role for the C terminus of SNAP25 in regulated exocytosis is to mediate Ca 2ϩ -dependent interactions between synaptotagmin and SNARE protein complexes. EXPERIMENTAL PROCEDURES Assays for Ca2ϩ -activated Exocytosis-PC12 cells grown in Dulbecco's modified culture medium supplemented with 5% horse serum and * This work was supported in part by National Institutes of Health Research Grants DK25861 and DK40428 (to T. F. J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This ar...

CAPS-1 is required for Ca2+-triggered fusion of dense-core vesicles with the plasma membrane, but its site of action and mechanism are unknown. We analyzed the kinetics of Ca2+-triggered exocytosis reconstituted in permeable PC12 cells. CAPS-1 increased the initial rate of Ca2+-triggered vesicle exocytosis by acting at a rate-limiting, Ca2+-dependent prefusion step. CAPS-1 activity depended upon prior ATP-dependent priming during which PIP2 synthesis occurs. CAPS-1 activity and binding to the plasma membrane depended upon PIP2. Ca2+ was ineffective in triggering vesicle fusion in the absence of CAPS-1 but instead promoted desensitization to CAPS-1 resulting from decreased plasma membrane PIP2. We conclude that CAPS-1 functions following ATP-dependent priming as a PIP2 binding protein to enhance Ca2+-dependent DCV exocytosis. Essential prefusion steps in dense-core vesicle exocytosis involve sequential ATP-dependent synthesis of PIP2 and the subsequent PIP2-dependent action of CAPS-1. Regulation of PIP2 levels and CAPS-1 activity would control the secretion of neuropeptides and monoaminergic transmitters.

The activation of G protein-coupled receptors (GPCRs) can result in an inhibition of Ca(2+)-dependent hormone and neurotransmitter secretion. This has been attributed in part to G protein inhibition of Ca(2+) influx. However, a frequently dominant inhibitory effect, of unknown mechanism, also occurs distal to Ca(2+) entry. Here we characterize direct inhibitory actions of G protein betagamma (Gbetagamma) on Ca(2+)-triggered vesicle exocytosis in permeable PC12 cells. Gbetagamma inhibition was rapid (<1 s) and was attenuated by cleavage of synaptosome-associated protein of 25 kD (SNAP25). Gbetagamma bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, and binding was reduced to SNARE complexes containing cleaved SNAP25 or by Ca(2+)-dependent synaptotagmin binding. Here we show inhibitory coupling between GPCRs and vesicle exocytosis mediated directly by Gbetagamma interactions with the Ca(2+)-dependent fusion machinery.

Exocytotic secretion in neuroendocrine cells is actiNeurotransmitter and peptide hormone secretion are mediated by the fusion of secretory vesicles with the plasma membrane, an exocytotic process that requires ATP and is strongly dependent upon and activated by Ca 2ϩ . Insights into the molecular basis of regulated membrane fusion have been provided by the identification of several required synaptic proteins such as vesicle-associated membrane protein/synaptobrevin, syntaxin and SNAP-25, which are substrates for clostridial neurotoxin proteases (1) and receptors for N-ethylmaleimide-sensitive factor/SNAP proteins (2), and synaptotagmin, a vesicle Ca 2ϩ

Neurotransmitter and peptide hormone secretion require fusion between secretory vesicles and the plasma membrane, an exocytotic process activated by cytoplasmic Ca 2ϩ elevation. Understanding regulated secretion requires identification of molecular components that mediate docking and fusion reactions and delineation of rate-limiting steps that are Ca 2ϩ -regulated (1). The synaptic vesicle protein synaptobrevin and the presynaptic membrane proteins syntaxin and SNAP-25 1 are required components of the exocytotic apparatus as indicated by the inhibitory action of BoNTs and Tetx, which involves the specific endoproteolytic cleavage of these protein substrates (2, 3). The three toxin substrates were independently identified as receptors for SNAPs, proteins required for the membrane binding of NSF, a late acting component in constitutive membrane fusion reactions (4). A characterized ternary complex containing synaptobrevin, syntaxin, and SNAP-25 was suggested to represent a docking complex that mediates the targeting, docking, or fusion of secretory vesicles (5, 6). Identification of an ATP-dependent catalytic activity of NSF/SNAP that promotes the disassembly of ternary complexes in vitro led to the suggestion that a similar reaction in vivo was responsible for late steps in membrane fusion (7). Ca 2ϩ -triggered secretion in permeable neuroendocrine cells requires ATP; however, the requirement for ATP precedes that for Ca 2ϩ (8, 9). ATP hydrolysis is required for prefusion events that prime the exocytotic apparatus, whereas Ca 2ϩ -activated fusion proceeds in the absence of ATP (8 -11).2 In priming, ATP serves as a substrate for polyphosphoinositide synthesis (10, 11) and as a substrate for the SNAP-dependent ATPase activity of NSF that catalyzes rearrangement of docking protein complexes.2 These studies experimentally identify a late step in the exocytotic pathway beyond LDCV docking and ATP utilization that is proximal to Ca 2ϩ -dependent fusion reactions. To characterize events that lead to or are directly involved in membrane fusion, it is important to identify molecular components that act at this late Ca were permeabilized by passage through a ball homogenizer (12). Secretion assays were conducted either as single stage or as two-stage assays (9,14). For the former, permeable cells were incubated for 15 min at 30°C in KGlu-BSA buffer supplemented with CaCl 2 (to achieve 10 M Ca 2ϩ f ), 0.002 M MgATP, and 0.5 mg/ml rat brain cytosol. Two-stage assays were conducted as separate priming incubations (30 min at 30°C in KGlu-BSA buffer supplemented with 0.002 M MgATP plus 1 mg/ml rat brain cytosol) followed by triggering incubations (1-3 min at 30°C in KGlu-BSA buffer supplemented with 10 M Ca 2ϩ plus 0.5 mg/ml rat brain cytosol) with extensive washing between incubations. [ 3 H]NE release was determined by centrifugation of permeable cells at 800 ϫ g for 30 min and scintillation counting of 3 H in supernatants and in cell pellets to express NE release as percent of the total 3 H. The SNAP-25 antibody used to inhi...

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