Regulated exocytosis of secretory granules or dense-core granules has been examined in many well-characterized cell types including neurons, neuroendocrine, endocrine, exocrine, and hemopoietic cells and also in other less well-studied cell types. Secretory granule exocytosis occurs through mechanisms with many aspects in common with synaptic vesicle exocytosis and most likely uses the same basic protein components. Despite the widespread expression and conservation of a core exocytotic machinery, many variations occur in the control of secretory granule exocytosis that are related to the specialized physiological role of particular cell types. In this review we describe the wide range of cell types in which regulated secretory granule exocytosis occurs and assess the evidence for the expression of the conserved fusion machinery in these cells. The signals that trigger and regulate exocytosis are reviewed. Aspects of the control of exocytosis that are specific for secretory granules compared with synaptic vesicles or for particular cell types are described and compared to define the range of accessory control mechanisms that exert their effects on the core exocytotic machinery.

Protein phosphorylation by protein kinase C (PKC) has been implicated in the control of neurotransmitter release and various forms of synaptic plasticity. The PKC substrates responsible for phosphorylation-dependent changes in regulated exocytosis in vivo have not been identified. Munc18a is essential for neurotransmitter release by exocytosis and can be phosphorylated by PKC in vitro on Ser-306 and Ser-313. We demonstrate that it is phosphorylated on Ser-313 in response to phorbol ester treatment in adrenal chromaffin cells. Mutation of both phosphorylation sites to glutamate reduces its affinity for syntaxin and so acts as a phosphomimetic mutation. Unlike phorbol ester treatment, expression of Munc18 with this phosphomimetic mutation in PKC phosphorylation sites did not affect the number of exocytotic events. The mutant did, however, produce changes in single vesicle release kinetics, assayed by amperometry, which were identical to those caused by phorbol ester treatment. Furthermore, the effects of phorbol ester treatment on release kinetics were occluded in cells expressing phosphomimetic Munc18. These results suggest that the dynamics of vesicle release events during exocytosis are controlled by PKC directly through phosphorylation of Munc18 on Ser-313. Phosphorylation of Munc18 by PKC may provide a mechanism for the control of exocytosis and thereby synaptic plasticity.Protein phosphorylation has been long known as an important mechanism for the regulation of exocytosis although, with only a few exceptions such as the synapsins (1), the targets for regulation by phosphorylation in vivo are unknown. Treatment with phorbol esters modifies regulated exocytosis in many different neuronal and non-neuronal (2, 3) cell types leading to increased vesicle recruitment into the ready releasable pool (4 -6), acceleration of fusion pore expansion (7), or changes in the kinetics of exocytosis (8,9). PKC also has a key role in synaptic plasticity (10). The effects of phorbol ester were originally attributed to activation of PKC 1 although the PKC substrates responsible had not been identified, and it is not known if the same target regulates all of the parameters modified by phorbol esters. The SNARE proteins, syntaxin 1, SNAP-25, and VAMP play key roles in exocytosis (11-13), and formation of the SNARE complex has been suggested to be a driving force for membrane fusion (14). The syntaxin-binding protein Munc18a (15) (29) and synaptotagmin I (25). In no case has the functional consequences of these phosphorylation events for exocytosis been established. Indeed, the phosphorylation of SNAP-25 by PKC in PC12 cells lagged well behind the effects of phorbol ester on the extent of exocytosis (29). In that study, it was also shown that the phorbol ester effects had both a PKC-dependent and a PKC-independent component. The synaptic protein Munc13 has been identified as an alternative phorbol ester-binding protein (30, 31), and recently it has been suggested that the effects of phorbol ester on synaptic transmission are mediat...

N-ethylmaleimide–sensitive fusion protein (NSF) and α-SNAP play key roles in vesicular traffic through the secretory pathway. In this study, NH2- and COOH-terminal truncation mutants of α-SNAP were assayed for ability to bind NSF and stimulate its ATPase activity. Deletion of up to 160 NH2-terminal amino acids had little effect on the ability of α-SNAP to stimulate the ATPase activity of NSF. However, deletion of as few as 10 COOH-terminal amino acids resulted in a marked decrease. Both NH2-terminal (1–160) and COOH-terminal (160–295) fragments of α-SNAP were able to bind to NSF, suggesting that α-SNAP contains distinct NH2- and COOH-terminal binding sites for NSF. Sequence alignment of known SNAPs revealed only leucine 294 to be conserved in the final 10 amino acids of α-SNAP. Mutation of leucine 294 to alanine (α-SNAP(L294A)) resulted in a decrease in the ability to stimulate NSF ATPase activity but had no effect on the ability of this mutant to bind NSF. α-SNAP (1–285) and α-SNAP (L294A) were unable to stimulate Ca2+-dependent exocytosis in permeabilized chromaffin cells. In addition, α-SNAP (1–285), and α-SNAP (L294A) were able to inhibit the stimulation of exocytosis by exogenous α-SNAP. α-SNAP, α-SNAP (1–285), and α-SNAP (L294A) were all able to become incorporated into a 20S complex and recruit NSF. In the presence of MgATP, α-SNAP (1–285) and α-SNAP (L294A) were unable to fully disassemble the 20S complex and did not allow vesicle-associated membrane protein dissociation to any greater level than seen in control incubations. These findings imply that α-SNAP stimulation of NSF ATPase activity may be required for 20S complex disassembly and for the α-SNAP stimulation of exocytosis.

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