Munc13-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion

Munc13-4 is a widely expressed member of the CAPS/Munc13 protein family proposed to function in priming secretory granules for exocytosis. Munc13-4 contains N- and C-terminal C2 domains (C2A and C2B) predicted to bind Ca(2+), but Ca(2+)-dependent regulation of Munc13-4 activity has not been described. The C2 domains bracket a predicted SNARE-binding domain, but whether Munc13-4 interacts with SNARE proteins is unknown. We report that Munc13-4 bound Ca(2+) and restored Ca(2+)-dependent granule exocytosis to permeable cells (platelets, mast, and neuroendocrine cells) dependent on putative Ca(2+)-binding residues in C2A and C2B. Munc13-4 exhibited Ca(2+)-stimulated SNARE interactions dependent on C2A and Ca(2+)-dependent membrane binding dependent on C2B. In an apparent coupling of membrane and SNARE binding, Munc13-4 stimulated SNARE-dependent liposome fusion dependent on putative Ca(2+)-binding residues in both C2A and C2B domains. Munc13-4 is the first priming factor shown to promote Ca(2+)-dependent SNARE complex formation and SNARE-mediated liposome fusion. These properties of Munc13-4 suggest its function as a Ca(2+) sensor at rate-limiting priming steps in granule exocytosis.     

Doc2 alpha and Munc13-4 regulate Ca(2+) -dependent secretory lysosome exocytosis in mast cells

The Doc2 family comprises the brain-specific Doc2alpha and the ubiquitous Doc2beta and Doc2gamma. With the exception of Doc2gamma, these proteins exhibit Ca(2+)-dependent phospholipid-binding activity in their Ca(2+)-binding C2A domain and are thought to be important for Ca(2+)-dependent regulated exocytosis. In excitatory neurons, Doc2alpha interacts with Munc13-1, a member of the Munc13 family, through its N-terminal Munc13-1-interacting domain and the Doc2alpha-Munc13-1 system is implicated in Ca(2+)-dependent synaptic vesicle exocytosis. The Munc13 family comprises the brain-specific Munc13-1, Munc13-2, and Munc13-3, and the non-neuronal Munc13-4. We previously showed that Munc13-4 is involved in Ca(2+)-dependent secretory lysosome exocytosis in mast cells, but the involvement of Doc2 in this process is not determined. In the present study, we found that Doc2alpha but not Doc2beta was endogenously expressed in the RBL-2H3 mast cell line. Doc2alpha colocalized with Munc13-4 on secretory lysosomes, and interacted with Munc13-4 through its two regions, the N terminus containing the Munc13-1-interacting domain and the C terminus containing the Ca(2+)-binding C2B domain. In RBL-2H3 cells, Ca(2+)-dependent secretory lysosome exocytosis was inhibited by expression of the Doc2alpha mutant lacking either of the Munc13-4-binding regions and the inhibition was suppressed by coexpression of Munc13-4. Knockdown of endogenous Doc2alpha also reduced Ca(2+)-dependent secretory lysosome exocytosis, which was rescued by re-expression of human Doc2alpha but not by its mutant that could not bind to Munc13-4. Moreover, Ca(2+)-dependent secretory lysosome exocytosis was severely reduced in bone marrow-derived mast cells from Doc2alpha knockout mice. These results suggest that the Doc2alpha-Muunc13-4 system regulates Ca(2+)-dependent secretory lysosome exocytosis in mast cells.     

Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon.

Regulated exocytosis involves the Ca(2+)-triggered fusion of secretory vesicles with the plasma membrane, by activation of vesicle membrane Ca(2+)-binding proteins [1]. The Ca(2+)-binding sites of these proteins are likely to lie within 30 nm of the vesicle surface, a domain in which changes in Ca2+ concentration cannot be resolved by conventional fluorescence microscopy. A fluorescent indicator for Ca2+ called a yellow 'cameleon' (Ycam2) - comprising a fusion between a cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13 and an enhanced yellow-emitting GFP - which is targetable to specific intracellular locations, has been described [2]. Here, we generated a fusion between phogrin, a protein that is localised to secretory granule membranes [3], and Ycam2 (phogrin-Ycam2) to monitor changes in Ca2+ concentration ([Ca2+]) at the secretory vesicle surface ([Ca2+]gd) through alterations in fluorescence resonance energy transfer (FRET) between the linked cyan and yellow fluorescent proteins (CFP and YFP, respectively) in Ycam2. In both neuroendocrine PC12 and MIN6 pancreatic beta cells, apparent resting values of cytosolic [Ca2+] and [Ca2+](gd) were similar throughout the cell. In MIN6 cells following the activation of Ca2+ influx, the minority of vesicles that were within approximately 1 microm of the plasma membrane underwent increases in [Ca2+](gd) that were significantly greater than those experienced by deeper vesicles, and greater than the apparent cytosolic [Ca2+] change. The ability to image both global and compartmentalised [Ca2+] changes with recombinant targeted cameleons should extend the usefulness of these new Ca2+ probes.     

DOC2B,C2 Domains,and Calcium: A Tale of Intricate Interactions

Ca⁺²-dependent exocytosis involves vesicle docking, priming, fusion, and recycling. This process is performed and regulated by a vast number of synaptic proteins and depends on proper protein–protein and protein–lipid interactions. Double C2 domain (DOC2) is a protein family of three isoforms found while screening DNA libraries with a C2 probe. DOC2 has three domains: the Munc13-interacting domain and tandem C2s (designated C2A and C2B) connected by a short polar linker. The C2 domain binds phospholipids in a Ca²⁺-dependent manner. This review focuses on the ubiquitously expressed isoform DOC2B. Sequence alignment of the tandem C2 protein family in mouse revealed high homology (81%) between rabphilin-3A and DOC2B proteins. We created a structural model of DOC2B's C2A based on the crystal structure of rabphilin-3A with and without calcium and found that the calcium-binding loops of DOC2B move upon calcium binding, enabling efficient plasma membrane penetration of its C2A. Here, we discuss the potential relation between the DOC2B bioinformatical model and its function and suggest a possible working model for its interaction with other proteins of the exocytotic machinery, including Munc13, Munc18, and syntaxin.     

CAPS and Munc13 utilize distinct PIP2-linked mechanisms to promote vesicle exocytosis

Phosphoinositides provide compartment-specific signals for membrane trafficking. Plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP₂) is required for Ca²⁺-triggered vesicle exocytosis, but whether vesicles fuse into PIP₂-rich membrane domains in live cells and whether PIP₂ is metabolized during Ca²⁺-triggered fusion were unknown. Ca²⁺-dependent activator protein in secretion 1 (CAPS-1; CADPS/UNC31) and ubMunc13-2 (UNC13B) are PIP₂-binding proteins required for Ca²⁺-triggered vesicle exocytosis in neuroendocrine PC12 cells. These proteins are likely effectors for PIP₂, but their localization during exocytosis had not been determined. Using total internal reflection fluorescence microscopy in live cells, we identify PIP₂-rich membrane domains at sites of vesicle fusion. CAPS is found to reside on vesicles but depends on plasma membrane PIP₂ for its activity. Munc13 is cytoplasmic, but Ca²⁺-dependent translocation to PIP₂-rich plasma membrane domains is required for its activity. The results reveal that vesicle fusion into PIP₂-rich membrane domains is facilitated by sequential PIP₂-dependent activation of CAPS and PIP₂-dependent recruitment of Munc13. PIP₂ hydrolysis only occurs under strong Ca²⁺ influx conditions sufficient to activate phospholipase Cη2 (PLCη2). Such conditions reduce CAPS activity and enhance Munc13 activity, establishing PLCη2 as a Ca²⁺-dependent modulator of exocytosis. These studies provide a direct view of the spatial distribution of PIP₂ linked to vesicle exocytosis via regulation of lipid-dependent protein effectors CAPS and Munc13.     

Calcium concentration threshold and translocation kinetics of EGFP-DOC2B expressed in cultured Aplysia neurons

The double C2 domain protein family (DOC2) is characterized by two calcium-binding domains (C2). Upon binding to calcium, the affinity of the protein to phospholipids is significantly increased, leading to translocation of the protein from the cytosol to the plasma membrane. These properties, and the binding domain of DOC2B to Munc13, suggested that DOC2B could play a role in augmentation and potentiation of synaptic release. Nevertheless, the level of the free intracellular calcium concentration ([Ca(2+)](i)) which triggers its translocation under in vivo conditions, is not known. Using cultured Aplysia neurons that express rat EGFP-DOC2B, we found that the [Ca(2+)](i) increment necessary to induce EGFP-DOC2B translocation is approximately 200 nM in the bulk of the cytoplasm. The rate of EGFP-DOC2B recruitment to the plasma membrane is slower than the [Ca(2+)](i) elevation rate, while the detachment of EGFP-DOC2B from it is faster than the calcium removal. The extent of EGFP-DOC2B translocation to the plasma membrane reflects local submembrane [Ca(2+)](i). Our observations are consistent with the view that DOC2B can participate in the regulation of neurotransmitter release. It should be noted that EGFP-DOC2B could be used as a tool to map sub-membrane calcium dynamics under physiological conditions.     

Synaptotagmin C2A loop 2 mediates Ca2+-dependent SNARE interactions essential for Ca2+-triggered vesicle exocytosis

Synaptotagmins contain tandem C2 domains and function as Ca(2+) sensors for vesicle exocytosis but the mechanism for coupling Ca(2+) rises to membrane fusion remains undefined. Synaptotagmins bind SNAREs, essential components of the membrane fusion machinery, but the role of these interactions in Ca(2+)-triggered vesicle exocytosis has not been directly assessed. We identified sites on synaptotagmin-1 that mediate Ca(2+)-dependent SNAP25 binding by zero-length cross-linking. Mutation of these sites in C2A and C2B eliminated Ca(2+)-dependent synaptotagmin-1 binding to SNAREs without affecting Ca(2+)-dependent membrane binding. The mutants failed to confer Ca(2+) regulation on SNARE-dependent liposome fusion and failed to restore Ca(2+)-triggered vesicle exocytosis in synaptotagmin-deficient PC12 cells. The results provide direct evidence that Ca(2+)-dependent SNARE binding by synaptotagmin is essential for Ca(2+)-triggered vesicle exocytosis and that Ca(2+)-dependent membrane binding by itself is insufficient to trigger fusion. A structure-based model of the SNARE-binding surface of C2A provided a new view of how Ca(2+)-dependent SNARE and membrane binding occur simultaneously.     

Cyclin-dependent kinase 5 associated with p39 promotes Munc18-1 phosphorylation and Ca(2+)-dependent exocytosis

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine protein kinase that requires association with a regulatory protein, p35 or p39, to form an active enzyme. Munc18-1 plays an essential role in membrane fusion, and its function is regulated by phosphorylation. We report here that both p35 and p39 were expressed in insulin-secreting beta-cells, where they exhibited individual subcellular distributions and associated with membranous organelles of different densities. Overexpression of Cdk5, p35, or p39 showed that Cdk5 and p39 augmented Ca(2+)-induced insulin exocytosis. Suppression of p39 and Cdk5, but not of p35, by antisense oligonucleotides selectively inhibited insulin exocytosis. Transient transfection of primary beta-cells with Munc18-1 templates mutated in potential Cdk5 or PKC phosphorylation sites, in combination with Cdk5 and the different Cdk5 activators, suggested that Cdk5/p39-promoted Ca(2+)-dependent insulin secretion from primary beta-cells by phosphorylating Munc18-1 at a biochemical step immediately prior to vesicle fusion.     

SNAREpin Assembly by Munc18-1 Requires Previous Vesicle Docking by Synaptotagmin 1

Regulated exocytosis requires the general membrane fusion machinery-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins. Using reconstituted giant unilamellar vesicles containing preassembled t-SNARE proteins (syntaxin 1·SNAP-25), we determined how Munc18-1 controls the docking, priming, and fusion of small unilamellar vesicles containing the v-SNARE VAMP2 and the Ca(2+) sensor synaptotagmin 1. In vitro assays allowed us to position Munc18-1 in the center of a sequential reaction cascade; vesicle docking by synaptotagmin 1 is a prerequisite for Munc18-1 to accelerate trans-SNARE complex (SNAREpin) assembly and membrane fusion. Complexin II stalls SNAREpin zippering at a late stage and, hence, contributes to synchronize membrane fusion in a Ca(2+)- and synaptotagmin 1-dependent manner. Thus, at the neuronal synapse, the priming factor Munc18-1 may accelerate the conversion of docked synaptic vesicles into a readily releasable pool by activating SNAREs for efficient membrane fusion.     

The C2B domain of rabphilin directly interacts with SNAP-25 and regulates the docking step of dense core vesicle exocytosis in PC12 cells

Rabphilin is a membrane trafficking protein on secretory vesicles that consists of an N-terminal Rab-binding domain and C-terminal tandem C2 domains. The N-terminal part of rabphilin has recently been shown to function as an effector domain for both Rab27A and Rab3A in PC12 cells (Fukuda, M., Kanno, E., and Yamamoto, A. (2004) J. Biol. Chem. 279, 13065-13075), but the function of the C2 domains of rabphilin during secretory vesicle exocytosis is largely unknown. In this study we investigated the interaction between rabphilin and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors, VAMP-2/synaptobrevin-2, syntaxin IA, and SNAP-25) and SNARE-associated proteins (Munc18-1 and Munc13-1) and found that the C2B domain of rabphilin, but not of other Rab27A-binding proteins with tandem C2 domains (i.e. Slp1-5), directly interacts with a plasma membrane protein, SNAP-25. The interaction between rabphilin and SNAP-25 occurs even in the absence of Ca(2+) (EC(50) = 0.817 microm SNAP-25), but 0.5 mm Ca(2+) increases the affinity for SNAP-25 2-fold (EC(50) = 0.405 microm SNAP-25) without changing the B(max) value (1.06 mol of SNAP-25/mol of rabphilin). Furthermore, vesicle dynamics were imaged by total internal reflection fluorescence microscopy in a single PC12 cell expressing a lumen-targeted pH-insensitive yellow fluorescent protein (Venus), neuropeptide Y-Venus. Expression of the wild-type rabphilin in PC12 cells significantly increased the number of docked vesicles to the plasma membrane without altering the kinetics of individual secretory events, whereas expression of the mutant rabphilin lacking the C2B domain, rabphilin-DeltaC2B, decreased the number of docked vesicle or fusing at the plasma membrane. These findings suggest that rabphilin is involved in the docking step of regulated exocytosis in PC12 cells, possibly through interaction between the C2B domain and SNAP-25.     

Munc13 homology domain-1 in CAPS/UNC31 mediates SNARE binding required for priming vesicle exocytosis

Neuropeptide and peptide hormone secretion from neural and endocrine cells occurs by Ca(2+)-triggered dense-core vesicle exocytosis. The membrane fusion machinery consisting of vesicle and plasma membrane SNARE proteins needs to be assembled for Ca(2+)-triggered vesicle exocytosis. The related Munc13 and CAPS/UNC31 proteins that prime vesicle exocytosis are proposed to promote SNARE complex assembly. CAPS binds SNARE proteins and stimulates SNARE complex formation on liposomes, but the relevance of SNARE binding to CAPS function in cells had not been determined. Here we identify a core SNARE-binding domain in CAPS as corresponding to Munc13 homology domain-1 (MHD1). CAPS lacking a single helix in MHD1 was unable to bind SNARE proteins or to support the Ca(2+)-triggered exocytosis of either docked or newly arrived dense-core vesicles. The results show that MHD1 is a SNARE-binding domain and that SNARE protein binding is essential for CAPS function in dense-core vesicle exocytosis.     

Characterization of the Munc13-calmodulin interaction by photoaffinity labeling

Sensing of and response to transient increases in the residual presynaptic Ca2+ levels are important adaptive mechanisms that define the short-term plasticity characteristics of neurons. Due to their essential function in synaptic vesicle priming and in the modulation of synaptic strength, Munc13 proteins have emerged as key regulators of these adaptive mechanisms. Indeed, Munc13-1 and ubMunc13-2 contain a conserved calmodulin (CaM) binding site and the Ca2+ -dependent interaction of these Munc13 isoforms with CaM constitutes a molecular mechanism that transduces residual Ca2+ signaling to the synaptic exocytotic machinery. Here, we used Munc13-derived model peptides in photoaffinity labeling (PAL) experiments to demonstrate the stoichiometric and Ca2+ -dependent CaM binding of the other members of the Munc13 family, bMunc13-2 and Munc13-3, via structurally distinct non-conserved binding sites. A PAL-based Ca2+ titration assay revealed that all Munc13 isoforms can form a complex with CaM already at low Ca2+ concentrations just above resting levels, underscoring the Ca2+ sensor/effector function of this interaction in short-term synaptic plasticity phenomena.     

Differential control of vesicle priming and short-term plasticity by Munc13 isoforms

Presynaptic short-term plasticity is an important adaptive mechanism regulating synaptic transmitter release at varying action potential frequencies. However, the underlying molecular mechanisms are unknown. We examined genetically defined and functionally unique axonal subpopulations of synapses in excitatory hippocampal neurons that utilize either Munc13-1 or Munc13-2 as synaptic vesicle priming factor. In contrast to Munc13-1-dependent synapses, Munc13-2-driven synapses show pronounced and transient augmentation of synaptic amplitudes following high-frequency stimulation. This augmentation is caused by a Ca(2+)-dependent increase in release probability and releasable vesicle pool size, and requires phospholipase C activity. Thus, differential expression of Munc13 isoforms at individual synapses represents a general mechanism that controls short-term plasticity and contributes to the heterogeneity of synaptic information coding.     

Functional interaction of the active zone proteins Munc13-1 and RIM1 in synaptic vesicle priming

Synaptic neurotransmitter release is restricted to active zones, where the processes of synaptic vesicle tethering, priming to fusion competence, and Ca2+-triggered fusion are taking place in a highly coordinated manner. We show that the active zone components Munc13-1, an essential vesicle priming protein, and RIM1, a Rab3 effector with a putative role in vesicle tethering, interact functionally. Disruption of this interaction causes a loss of fusion-competent synaptic vesicles, creating a phenocopy of Munc13-1-deficient neurons. RIM1 binding and vesicle priming are mediated by two distinct structural modules of Munc13-1. The Munc13-1/RIM1 interaction may create a functional link between synaptic vesicle tethering and priming, or it may regulate the priming reaction itself, thereby determining the number of fusion-competent vesicles.     

Calcium-induced calcium release contributes to somatic secretion of serotonin in leech Retzius neurons

We analyzed the contribution of calcium (Ca2+)-induced Ca2+ release to somatic secretion in serotonergic Retzius neurons of the leech. Somatic secretion was studied by the incorporation of fluorescent dye FM1-43 upon electrical stimulation with trains of 10 impulses and by electron microscopy. Quantification of secretion with FM1-43 was made in cultured neurons to improve optical resolution. Stimulation in the presence of FM1-43 produced a frequency-dependent number of fluorescent spots. While a 1-Hz train produced 19.5+/-5.0 spots/soma, a 10-Hz train produced 146.7+/-20.2 spots/soma. Incubation with caffeine (10 mM) to induce Ca2+ release from intracellular stores without electrical stimulation and external Ca2+, produced 168+/-21.7 spots/soma. This staining was reduced by 49% if neurons were preincubated with the Ca2+- ATPase inhibitor thapsigargin (200 nM). Moreover, in neurons stimulated at 10 Hz in the presence of ryanodine (100 microM) to block Ca2+-induced Ca2+ release, FM1-43 staining was reduced by 42%. In electron micrographs of neurons at rest or stimulated at 1 Hz in the ganglion, endoplasmic reticulum lay between clusters of dense core vesicles and the plasma membrane. In contrast, in neurons stimulated at 20 Hz, the vesicle clusters were apposed to the plasma membrane and flanked by the endoplasmic reticulum. These results suggest that Ca2+-induced Ca2+ release produces vesicle mobilization and fusion in the soma of Retzius neurons, and supports the idea that neuronal somatic secretion shares common mechanisms with secretion by excitable endocrine cells.     

Protein kinase calpha targeting is regulated by temporal and spatial changes in intracellular free calcium concentration [Ca(2+)](i).

Protein kinase C (PKC) isoforms exert specific intracellular functions, but the different isoforms display little substrate specificity in vitro. Selective PKC isoform targeting may be a mechanism to achieve specificity. We used a green fluorescent fusion protein (GFP) to test the hypothesis that local changes in [Ca(2+)](i) regulate translocation of PKCalpha and that different modes of Ca(2+) and Ca(2+) release play a role in PKCalpha targeting. We constructed deletion mutants of PKCalpha to analyze the Ca(2+)-sensitive domains and their role in targeting. Confocal microscopy was used and [Ca(2+)](i) was measured by fluo-3. The fusion protein PKCalpha-GFP was expressed in vascular smooth muscle cells and showed a cytosolic distribution similar to the wild-type PKCalpha protein. The Ca(2+) ionophore ionomycin induced a speckled cytosolic PKCalpha-GFP distribution, followed by membrane translocation, while depolarization by KCl induced primarily membrane translocation. Selective voltage-operated Ca(2+) channel opening led to a localized accumulation of PKCalpha-GFP near the plasma membrane. Opening Ca(2+) stores with InsP(3), thapsigargin, or ryanodine induced a specific PKCalpha-GFP targeting to distinct intracellular areas. The G-protein-coupled receptor agonist thrombin induced a rapid translocation of the fusion protein to focal domains. The tyrosine kinase receptor agonist PDGF induced Ca(2+) influx and led to a linear PKCalpha-GFP membrane association. PKCalpha-GFP deletion mutants demonstrated that the C2 domain, but not the catalytic subunit, is necessary for Ca(2+)-induced PKCalpha targeting. Targeting was also abolished when the ATP binding site was deleted. We conclude that PKCalpha can rapidly be translocated to distinct intracellular or membrane domains by local increases in [Ca(2+)](i). The targeting mechanism is dependent on the C2 and ATP binding site of the enzyme. Localized [Ca(2+)](i) changes determine the spatial and temporal targeting of PKCalpha.     

Calcium sensors in regulated exocytosis

Neurotransmitter release, hormone secretion and a variety of other secretory process are tightly regulated with exocytotic fusion of secretory vesicles being triggered by a rise in cytosolic Ca2+ concentration. A series of proteins that act as part of a conserved core machinery for vesicle docking and fusion throughout the cell have been identified. In regulated exocytosis this core machinery must be controlled by Ca(2+)-sensor proteins that allow rapid activation of the fusion process following elevation of cytosolic Ca2+ concentration. The properties of such Ca2+ sensors are known from physiological studies but their molecular identity remains to be unequivocally established. The multiple Ca(2+)-dependent steps in the exocytotic pathway suggest the likely involvement of several Ca(2+)-binding proteins with distinct properties. Functional evidence for the role of various Ca(2+)-binding proteins and their possible sites of action is accumulating but a definitive identification of the major Ca(2+)-sensor in the final step of Ca(2+)-triggered membrane fusion in different cell types awaits further analysis.     

Identification of a high-affinity network of secretagogin-binding proteins involved in vesicle secretion

Secretagogin is a hexa EF-hand Ca(2+)-binding protein expressed in neuroendocrine, pancreatic endocrine and retinal cells. The protein has been noted for its expression in specific neuronal subtypes in the support of hierarchical organizing principles in the mammalian brain. Secretagogin has previously been found to interact with SNAP25 involved in Ca(2+)-induced exocytosis. Here, the cellular interaction network of secretagogin has been expanded with nine proteins: SNAP-23, DOC2alpha, ARFGAP2, rootletin, KIF5B, β-tubulin, DDAH-2, ATP-synthase and myeloid leukemia factor 2, based on screening of a high content protein array and validation and quantification of binding with surface plasmon resonance and GST pulldown assays. All targets have association rate constants in the range 10(4)-10(6) M(-1) s(-1), dissociation rate constants in the range 10(-3)-10(-5) s(-1) and equilibrium dissociation constants in the 100 pM to 10 nM range. The novel target SNAP23 is an essential component of the high affinity receptor for the general membrane fusion machinery and an important regulator of transport vesicle docking and fusion. Complementary roles in vesicle trafficking are known for ARFGAP2 and DOC2alpha in regulating fusion of vesicles to membranes, kinesin 5B and tubulin for transport of vesicles in the cell, while rootletin builds up the rootlet believed to function as a scaffold for vesicles. The identification of a discrete network of interacting proteins that mediate secretion and vesicle trafficking suggests a regulatory role for secretagogin in these processes.     

Transient, phorbol ester-induced DOC2-Munc13 interactions in vivo.

Munc13-1 and DOC2 have been implicated in the regulation of exocytosis. Here we demonstrate in vivo that these two proteins undergo a transient phorbol ester-mediated and protein kinase C-independent interaction, resulting in the translocation of DOC2 from a vesicular localization to the plasma membrane. The translocation of DOC2 is dependent upon the DOC2 Munc interacting domain that binds specifically to Munc13-1, whereas the association of DOC2 with intracellular membranes is dependent on its C2 domains. This is the first direct in vivo demonstration of a protein-protein interaction between two presynaptic proteins and may represent a molecular basis for phorbol ester-dependent enhancement of exocytosis.     

Tomosyn inhibits synaptotagmin-1-mediated step of Ca2+-dependent neurotransmitter release through its N-terminal WD40 repeats

Neurotransmitter release is triggered by Ca(2+) binding to a low affinity Ca(2+) sensor, mostly synaptotagmin-1, which catalyzes SNARE-mediated synaptic vesicle fusion. Tomosyn negatively regulates Ca(2+)-dependent neurotransmitter release by sequestering target SNAREs through the C-terminal VAMP-like domain. In addition to the C terminus, the N-terminal WD40 repeats of tomosyn also have potent inhibitory activity toward Ca(2+)-dependent neurotransmitter release, although the molecular mechanism underlying this effect remains elusive. Here, we show that through its N-terminal WD40 repeats tomosyn directly binds to synaptotagmin-1 in a Ca(2+)-dependent manner. The N-terminal WD40 repeats impaired the activities of synaptotagmin-1 to promote SNARE complex-mediated membrane fusion and to bend the lipid bilayers. Decreased acetylcholine release from N-terminal WD40 repeat-microinjected superior cervical ganglion neurons was relieved by microinjection of the cytoplasmic domain of synaptotagmin-1. These results indicate that, upon direct binding, the N-terminal WD40 repeats negatively regulate the synaptotagmin-1-mediated step of Ca(2+)-dependent neurotransmitter release. Furthermore, we show that synaptotagmin-1 binding enhances the target SNARE-sequestering activity of tomosyn. These results suggest that the interplay between tomosyn and synaptotagmin-1 underlies inhibitory control of Ca(2+)-dependent neurotransmitter release.