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...

Sec1p/Munc18 (SM) proteins are essential for SNARE-mediated membrane trafficking. The formulation of unifying hypotheses for the function of the SM protein family has been hampered by the observation that two of its members bind their cognate syntaxins (Sxs) in strikingly different ways. The SM protein Vps45p binds its Sx Tlg2p in a manner analogous to that captured by the Sly1p–Sed5p crystal structure, whereby the NH2-terminal peptide of the Sx inserts into a hydrophobic pocket on the outer face of domain I of the SM protein. In this study, we report that although this mode of interaction is critical for the binding of Vps45p to Tlg2p, the SM protein also binds Tlg2p-containing SNARE complexes via a second mode that involves neither the NH2 terminus of Tlg2p nor the region of Vps45p that facilitates this interaction. Our findings point to the possibility that SM proteins interact with their cognate SNARE proteins through distinct mechanisms at different stages in the SNARE assembly/disassembly cycle.

Signal recognition particle (SRP) targets proteins for co-translational insertion through or into the endoplasmic reticulum membrane. Mammalian SRP slows nascent chain elongation by the ribosome during targeting in vitro. This`elongation arrest' activity requires the SRP9/14 subunit of the particle and interactions of the C-terminus of SRP14. We have puri®ed SRP from Saccharomyces cerevisiae and demonstrated that it too has elongation arrest activity. A yeast SRP containing Srp14p truncated at its C-terminus (DC29) did not maintain elongation arrest, was substantially de®cient in promoting translocation and interfered with targeting by wild-type SRP. In vivo, this mutation conferred a constitutive defect in the coupling of protein translation and translocation and temperature-sensitive growth, but only a slight defect in protein translocation. In combination, these data indicate that the primary defect in SRP DC29 is in elongation arrest, and that this is a physiologically important and conserved function of eukaryotic SRP.

Sec1/Munc18 (SM) proteins are involved in various intracellular membrane trafficking steps. Many SM proteins bind to appropriate syntaxin homologues involved in these steps, suggesting that SM proteins function as syntaxin chaperones. Organisms with mutations in SM genes, however, exhibit defects in either early (docking) or late (fusion) stages of exocytosis, implying that SM proteins may have multiple functions. To gain insight into the role of SM proteins, we introduced mutations modeled on those identified in Caenorhabditis elegans, Drosophila melanogaster, and Saccharomyces cerevisiae into mammalian Munc18-1. As expected, several mutants exhibited reduced binding to syntaxin1A. However, three mutants displayed wild-type syntaxin binding affinities, indicating syntaxin-independent defects. Expression of these mutants in chromaffin cells either increased the rate and extent of exocytosis or altered the kinetics of individual release events. This latter effect was associated with a reduced Mint binding affinity in one mutant, implying a potential mechanism for the observed alteration in release kinetics. Furthermore, this phenotype persisted when the mutation was combined with a second mutation that greatly reduced syntaxin binding affinity. These results clarify the data on the function of SM proteins in mutant organisms and indicate that Munc18-1 controls multiple stages of exocytosis via both syntaxin-dependent and -independent protein interactions.