A Novel Tetanus Neurotoxin-insensitive Vesicle-associated Membrane Protein in SNARE Complexes of the Apical Plasma Membrane of Epithelial Cells

The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been demonstrated that clostridial neurotoxins (NTs) proteolyze the vesicle SNAREs (v-SNAREs) vesicle-associated membrane protein (VAMP)/brevins and their partners, the target SNAREs (t-SNAREs) syntaxin 1 and SNAP25. Yet, several exocytotic events, including apical exocytosis in epithelial cells, are insensitive to numerous clostridial NTs, suggesting the presence of SNARE-independent mechanisms of exocytosis. In this study we found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs. In epithelial cells, TI-VAMP–containing vesicles were concentrated in the apical domain, and the protein was detected at the apical plasma membrane by immunogold labeling on ultrathin cryosections. Syntaxin 3 and SNAP23 were codistributed at the apical plasma membrane where they formed NEM-dependent SNARE complexes with TI-VAMP and cellubrevin. We suggest that TI-VAMP, SNAP23, and syntaxin 3 can participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specific exocytosis in clostridial NT-resistant pathways.     

Syntaxin and VAMP association with lipid rafts depends on cholesterol depletion in capacitating sperm cells

Sperm cells represent a special exocytotic system since mature sperm cells contain only one large secretory vesicle, the acrosome, which fuses with the overlying plasma membrane during the fertilization process. Acrosomal exocytosis is believed to be regulated by activation of SNARE proteins. In this paper, we identified specific members of the SNARE protein family, i.e., the t-SNAREs syntaxin1 and 2, and the v-SNARE VAMP, present in boar sperm cells. Both syntaxins were predominantly found in the plasma membrane whereas v-SNAREs are mainly located in the outer acrosomal membrane of these cells. Under non-capacitating conditions both syntaxins and VAMP are scattered in well-defined punctate structures over the entire sperm head. Bicarbonate-induced in vitro activation in the presence of BSA causes a relocalization of these SNAREs to a more homogeneous distribution restricted to the apical ridge area of the sperm head, exactly matching the site of sperm zona binding and subsequent induced acrosomal exocytosis. This redistribution of syntaxin and VAMP depends on cholesterol depletion and closely resembles the previously reported redistribution of lipid raft marker proteins. Detergent-resistant membrane isolation and subsequent analysis shows that a significant proportion of syntaxin emerges in the detergent-resistant membrane (raft) fraction under such conditions, which is not the case under those conditions where cholesterol depletion is blocked. The v-SNARE VAMP displays a similar cholesterol depletion-dependent lateral and raft redistribution. Taken together, our results indicate that redistribution of syntaxin and VAMP during capacitation depends on association of these SNAREs with lipid rafts and that such a SNARE-raft association may be essential for spatial control of exocytosis and/or regulation of SNARE functioning.     

Exocytotic mechanism studied by truncated and zero layer mutants of the C-terminus of SNAP-25

The highly conserved SNARE proteins, SNAP-25, syntaxin and synaptobrevin, form a tight ternary complex, which is essential for exocytosis. Crystallization of this complex revealed a four-helix bundle with an unusual hydrophilic layer (zero layer) in its center. In order to evaluate the role of this layer in different kinetic components of secretion, we used the Semliki Forest virus (SFV) system to infect adrenal chromaffin cells with SNAP-25 Q174L, a point mutant in the zero layer. Using combined flash photolysis of caged calcium and membrane capacitance measurements, we investigated its effect on the exocytotic burst and sustained phase of exocytosis with high time resolution. Cells expressing SNAP-25 Q174L displayed a selective reduction in the sustained phase, while the two components of the exocytotic burst remained unaffected. Furthermore, the exocytotic response to the second flash was significantly reduced, indicating a decrease in refilling kinetics. We therefore conclude that the zero layer is critical for the formation of SNARE complexes, but that it plays no role in the dynamic equilibrium between the two exocytosis-competent vesicle pools.     

Lipid Dynamics in Exocytosis

Regulated exocytosis of neurotransmitter- and hormone-containing vesicles underpins neuronal and hormonal communication and relies on a well-orchestrated series of molecular interactions. This in part involves the upstream formation of a complex of SNAREs and associated proteins leading to the eventual fusion of the vesicle membrane with the plasma membrane, a process that enables content release. Although the role of lipids in exocytosis is intuitive, it has long been overlooked at least compared to the extensive work on SNAREs. Here, we will present the latest advances in this rapidly developing field revealing that lipids actually play an active role in exocytosis by focusing on cholesterol, 3′-phosphorylated phosphoinositides and phosphatidic acid.     

Structure-function study of mammalian Munc18-1 and C. elegans UNC-18 implicates domain 3b in the regulation of exocytosis

Munc18-1 is an essential synaptic protein functioning during multiple stages of the exocytotic process including vesicle recruitment, docking and fusion. These functions require a number of distinct syntaxin-dependent interactions; however, Munc18-1 also regulates vesicle fusion via syntaxin-independent interactions with other exocytotic proteins. Although the structural regions of the Munc18-1 protein involved in closed-conformation syntaxin binding have been thoroughly examined, regions of the protein involved in other interactions are poorly characterised. To investigate this we performed a random transposon mutagenesis, identifying domain 3b of Munc18-1 as a functionally important region of the protein. Transposon insertion in an exposed loop within this domain specifically disrupted Mint1 binding despite leaving affinity for closed conformation syntaxin and binding to the SNARE complex unaffected. The insertion mutation significantly reduced total amounts of exocytosis as measured by carbon fiber amperometry in chromaffin cells. Introduction of the equivalent mutation in UNC-18 in Caenorhabditis elegans also reduced neurotransmitter release as assessed by aldicarb sensitivity. Correlation between the two experimental methods for recording changes in the number of exocytotic events was verified using a previously identified gain of function Munc18-1 mutation E466K (increased exocytosis in chromaffin cells and aldicarb hypersensitivity of C. elegans). These data implicate a novel role for an exposed loop in domain 3b of Munc18-1 in transducing regulation of vesicle fusion independent of closed-conformation syntaxin binding.     

Insight in the exocytotic process in chromaffin cells: regulation by trimeric and monomeric G proteins

Catecholamine secretion from chromaffin cells has been used for a long time as a general model to study exocytosis of large dense core secretory granules. Permeabilization and microinjection techniques have brought the possibility to dissect at the molecular level the multi-protein machinery involved in this complex physiological process. Regulated exocytosis comprises distinct and sequential steps including the priming of secretory granules, the formation of a docking complex between granules and the plasma membrane and the subsequent fusion of the granule with the plasma membrane. Key proteins involved in the exocytotic machinery have been identified. For instance, SNAREs which participate in the docking events in most intracellular transport steps along the secretory pathway, play a role in exocytosis in both neuronal and endocrine cells. However, in contrast to intracellular transport processes for which the highest fusion efficiency is required after correct targeting of the vesicles, the number of exocytotic events in activated secretory cells needs to be tightly controlled. We describe here the multistep control exerted by heterotrimeric and monomeric G proteins on the progression of secretory granules from docking to fusion and the molecular nature of some of their downstream effectors in neuroendocrine chromaffin cells.     

Exocytosis in neuroendocrine cells: new tasks for actin

Most secretory cells undergoing calcium-regulated exocytosis in response to cell surface receptor stimulation display a dense subplasmalemmal actin network, which is remodeled during the exocytotic process. This review summarizes new insights into the role of the cortical actin cytoskeleton in exocytosis. Many earlier findings support the actin-physical-barrier model whereby transient depolymerization of cortical actin filaments permits vesicles to gain access to their appropriate docking and fusion sites at the plasma membrane. On the other hand, data from our laboratory and others now indicate that actin polymerization also plays a positive role in the exocytotic process. Here, we discuss the potential functions attributed to the actin cytoskeleton at each major step of the exocytotic process, including recruitment, docking and fusion of secretory granules with the plasma membrane. Moreover, we present actin-binding proteins, which are likely to link actin organization to calcium signals along the exocytotic pathway. The results cited in this review are derived primarily from investigations of the adrenal medullary chromaffin cell, a cell model that is since many years a source of information concerning the molecular machinery underlying exocytosis.     

The specificity of SNARE pairing in biological membranes is mediated by both proof-reading and spatial segregation

Soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins mediate organelle fusion in the secretory pathway. Different fusion steps are catalyzed by specific sets of SNARE proteins. Here we have used the SNAREs mediating the fusion of early endosomes and exocytosis, respectively, to investigate how pairing specificity is achieved. Although both sets of SNAREs promiscuously assemble in vitro, there is no functional crosstalk. We now show that they not only colocalize to overlapping microdomains in the membrane of early endosomes of neuroendocrine cells, but also form cis-complexes promiscuously, with the proportion of the different complexes being primarily dependent on mass action. Addition of soluble SNARE molecules onto native membranes revealed preference for cognate SNAREs. Furthermore, we found that SNAREs are laterally segregated at endosome contact sites, with the exocytotic synaptobrevin being depleted. We conclude that specificity in endosome fusion is mediated by the following two synergistically operating mechanisms: (i) preference for the cognate SNARE in 'trans' interactions and (ii) lateral segregation of SNAREs, leading to relative enrichment of the cognate ones at the prospective fusion sites.     

Cholesterol and regulated exocytosis: A requirement for unitary exocytotic events

Since the 1970s, much effort was been expended researching mechanisms of regulated exocytosis. Early work focused mainly on the role of proteins. Most notably the discovery of SNARE proteins in the 1980s and the zippering hypothesis brought us much closer to understanding the complex interactions in membrane fusion between vesicle and plasma membranes, a pivotal component of regulated exocytosis. However, most likely due to the predictions of the Singer-Nicholson fluid mosaic membrane model, the lipid components of the exocytotic machinery remained largely overlooked. Lipids were considered passive constituents of cellular membranes, not contributing much, if anything, to the process of exocytosis and membrane fusion. Since the 1990s, this so-called proteocentric view has been gradually giving way to the new perspective best described with the term proteolipidic. Many lipids were found to be of great importance in the regulation of exocytosis. Here we highlight the role of cholesterol. Furthermore, by using high-resolution cell-attached membrane capacitance measurements, we have monitored unitary exocytotic events in cholesterol-depleted membranes. We show that the frequency of these events is attenuated, providing evidence at the single vesicle level that cholesterol directly influences the merger of the vesicle and the plasma membranes.     

Selective expression of a sec1/munc18 member in sea urchin eggs and embryos

Regulated secretion is mediated by SNAREs (soluble NSF attachment receptors) and their regulators and effectors, which include the SM (sec1/munc18) family of proteins. Homologs of the SNAREs have been identified in sea urchins, associated with cortical granule exocytosis at fertilization, with membranes of the cleavage furrow, and in secretory cells later in development. To contribute to the understanding of regulated secretion in sea urchins we have cloned the single SM protein homolog from two species of sea urchin, Lytechinus variegatus and Strongylocentrotus purpuratus. In oocytes and eggs, we find that it localizes to the plasma membrane and the cortical region of the egg, consistent with a role in one of the steps leading to cortical granule exocytosis. The protein is also expressed throughout development, enriched in membranes of the cleavage furrow in early embryos, and in cells of the gut in advanced embryos. Furthermore, we find that sec1/munc18 co-localizes with its cognate binding partner syntaxin. Finally, our biochemical analysis shows that the protein associates with rab3 in high molecular weight complexes, suggesting that the exocytotic machinery functions as a multi-protein subunit to mediate regulated secretion in sea urchins. These results will be instrumental in the future to functionally test the SNARE regulators associated with multiple membrane fusion events.     

A discontinuous SNAP-25 C-terminal coil supports exocytosis

Membrane fusion requires the formation of four-helical bundles comprised of the SNARE proteins syntaxin, vesicle-associated membrane protein (VAMP), and the synaptosomal-associated protein of 25 kDa (SNAP-25). Botulinum neurotoxin E cleaves the C-terminal coil of SNAP-25, inhibiting exocytosis of norepinephrine from permeabilized PC12 cells. Addition of a 26-mer peptide comprising the C terminus of SNAP-25 that is cleaved by the toxin restores exocytosis, demonstrating that continuity of the SNAP-25 C-terminal helix is not critical for its function. By contrast, vesicle-associated membrane protein peptides could not rescue botulinum neurotoxin D-treated cells, suggesting that helix continuity is critical for VAMP function. Much higher concentrations of the SNAP-25 C-terminal peptide are required for rescuing exocytosis (K(assembly) = approximately 460 microm) than for binding to other SNAREs in vitro (Kd < 5 microm). Each residue of the peptide was mutated to alanine to assess its functional importance. Whereas most mutants rescue exocytosis with lower efficiency than the wild type peptide, D186A rescues with higher efficiency, and kinetic analysis suggests this is because of higher affinity for the cellular binding site. This is consistent with Asp-186 contributing to negative regulation of the fusion process.     

Syntaxin 6: the promiscuous behaviour of a SNARE protein

Vesicle flow within the cell is responsible for the dynamic maintenance of and communication between intracellular compartments. In addition, vesicular transport is crucial for communication between the cell and its surrounding environment. The ability of a vesicle to recognise and fuse with an appropriate compartment or vesicle is determined by its protein and lipid composition as well as by proteins in the cytosol. SNARE proteins present on both vesicle as well as target organelle membranes provide one component necessary for the process of membrane fusion. While in mammalian cells the main focus of interest about SNARE function has centred on those involved in exocytosis, recent data on SNAREs involved in intracellular membrane-trafficking steps have provided a deeper insight into the properties of these proteins. We take, as an example, the promiscuous SNARE syntaxin 6, a SNARE involved in multiple membrane fusion events. The properties of syntaxin 6 reveal similarities but also differences in the behaviour of intracellular SNAREs and the highly specialised exocytotic SNARE molecules.     

Mast cell degranulation requires N-ethylmaleimide-sensitive factor-mediated SNARE disassembly

Mast cells possess specialized granules that, upon stimulation of surface FcR with IgE, fuse with the plasma membrane, thereby releasing inflammatory mediators. A family of membrane fusion proteins called SNAREs, which are present on both the granule and the plasma membrane, plays a role in the fusion of these granules with the plasma membrane of mast cells. In addition to the SNAREs themselves, it is likely that the SNARE accessory protein, N-ethylmaleimide-sensitive factor (NSF), affects the composition and structure of the SNARE complex. NSF is a cytoplasmic ATPase that disassembles the SNARE complexes. To investigate the role of NSF in mast cell degranulation, we developed an assay to measure secretion from transiently transfected RBL (rat basophilic leukemia)-2H3 mast cells (a tumor analog of mucosal mast cells). RBL-2H3 cells were cotransfected with a plasmid encoding a human growth hormone secretion reporter along with either wild-type NSF or an NSF mutant that lacks ATPase activity. Human growth hormone was targeted to and released from secretory granules in RBL-2H3 cells, and coexpression with mutant NSF dramatically inhibited regulated exocytosis from the transfected cells. Biochemical analysis of SNARE complexes in these cells revealed that overexpression of the NSF mutant decreased disassembly and resulted in an accumulation of SNARE complexes. These data reveal a role for NSF in mast cell exocytosis and highlight the importance of SNARE disassembly, or priming, in regulated exocytosis from mast cells.     

Revisiting the role of SNAREs in exocytosis and membrane fusion

For over a decade SNARE hypotheses have been proposed to explain the mechanism of membrane fusion, yet the field still lacks sufficient evidence to conclusively identify the minimal components of native fusion. Consequently, debate concerning the postulated role(s) of SNAREs in membrane fusion continues. The focus of this review is to revisit original literature with a current perspective. Our analysis begins with the earliest studies of clostridial toxins, leading to various cellular and molecular approaches that have been used to test for the roles of SNAREs in exocytosis. We place much emphasis on distinguishing between specific effects on membrane fusion and effects on other critical steps in exocytosis. Although many systems can be used to study exocytosis, few permit selective access to specific steps in the pathway, such as membrane fusion. Thus, while SNARE proteins are essential to the physiology of exocytosis, assay limitations often prevent definitive conclusions concerning the molecular mechanism of membrane fusion. In all, the SNAREs are more likely to function upstream as modulators or priming factors of fusion.     

Involvement of complexin 2 in docking, locking and unlocking of different SNARE complexes during sperm capacitation and induced acrosomal exocytosis

Acrosomal exocytosis (AE) is an intracellular multipoint fusion reaction of the sperm plasma membrane (PM) with the outer acrosomal membrane (OAM). This unique exocytotic event enables the penetration of the sperm through the zona pellucida of the oocyte. We previously observed a stable docking of OAM to the PM brought about by the formation of the trans-SNARE complex (syntaxin 1B, SNAP 23 and VAMP 3). By using electron microscopy, immunochemistry and immunofluorescence techniques in combination with functional studies and proteomic approaches, we here demonstrate that calcium ionophore-induced AE results in the formation of unilamellar hybrid membrane vesicles containing a mixture of components originating from the two fused membranes. These mixed vesicles (MV) do not contain the earlier reported trimeric SNARE complex but instead possess a novel trimeric SNARE complex that contained syntaxin 3, SNAP 23 and VAMP 2, with an additional SNARE interacting protein, complexin 2. Our data indicate that the earlier reported raft and capacitation-dependent docking phenomenon between the PM and OAM allows a specific rearrangement of molecules between the two docked membranes and is involved in (1) recruiting SNAREs and complexin 2 in the newly formed lipid-ordered microdomains, (2) the assembly of a fusion-driving SNARE complex which executes Ca(2+)-dependent AE, (3) the disassembly of the earlier reported docking SNARE complex, (4) the recruitment of secondary zona binding proteins at the zona interacting sperm surface. The possibility to study separate and dynamic interactions between SNARE proteins, complexin and Ca(2+) which are all involved in AE make sperm an ideal model for studying exocytosis.     

The R-SNARE motif of tomosyn forms SNARE core complexes with syntaxin 1 and SNAP-25 and down-regulates exocytosis

Tomosyn is a 130-kDa syntaxin-binding protein that contains a large N-terminal domain with WD40 repeats and a C-terminal domain homologous to R-SNAREs. Here we show that tomosyn forms genuine SNARE core complexes with the SNAREs syntaxin 1 and SNAP-25. In vitro studies with recombinant proteins revealed that complex formation proceeds from unstructured monomers to a stable four-helical bundle. The assembled complex displayed features typical for SNARE core complexes, including a profound hysteresis upon unfolding-refolding transitions. No stable complexes were formed between the SNARE motif of tomosyn and either syntaxin or SNAP-25 alone. Furthermore, both native tomosyn and its isolated C-terminal domain competed with synaptobrevin for binding to endogenous syntaxin and SNAP-25 on inside-out sheets of plasma membranes. Tomosyn-SNARE complexes were effectively disassembled by the ATPase N-ethylmaleimide-sensitive factor together with its cofactor alpha-SNAP. Moreover, the C-terminal domain of tomosyn was as effective as the cytoplasmic portion of synaptobrevin in inhibiting evoked exocytosis in a cell-free preparation derived from PC12 cells. Similarly, overexpression of tomosyn in PC12 cells resulted in a massive reduction of exocytosis, but the release parameters of individual exocytotic events remained unchanged. We conclude that tomosyn is a soluble SNARE that directly competes with synaptobrevin in the formation of SNARE complexes and thus may function in down-regulating exocytosis.     

Regulation of insulin exocytosis by calcium-dependent protein kinase C in beta cells

The control of insulin release from pancreatic beta cells helps ensure proper blood glucose level, which is critical for human health. Protein kinase C has been shown to be one key control mechanism for this process. After glucose stimulation, calcium influx into beta cells triggers exocytosis of insulin-containing dense-core granules and activates protein kinase C via calcium-dependent phospholipase C-mediated generation of diacylglycerol. Activated protein kinase C potentiates insulin release by enhancing the calcium sensitivity of exocytosis, likely by affecting two main pathways that could be linked: (1) the reorganization of the cortical actin network, and (2) the direct phosphorylation of critical exocytotic proteins such as munc18, SNAP25, and synaptotagmin. Here, we review what is currently known about the molecular mechanisms of protein kinase C action on each of these pathways and how these effects relate to the control of insulin release by exocytosis. We identify remaining challenges in the field and suggest how these challenges might be addressed to advance our understanding of the regulation of insulin release in health and disease.     

SNARE complex regulation by phosphorylation

SNAREs (soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptors) are ubiquitous proteins that direct vesicular trafficking and exocytosis. In neurons, SNAREs act to mediate release of neurotransmitters, which is a carefully regulated process. Calcium influx has long been shown to be the key trigger of release. However, calcium alone cannot regulate the degree of vesicle content release. For example, only a limited number of docked vesicles releases neurotransmitters when calcium entry occurs; this suggests that exocytosis is regulated by other factors besides calcium influx. Regulation of the degree of release is best explained by looking at the many enzymatic proteins that interact with the SNARE complex. These proteins have been hypothesized to regulate the formation, stability, or disassembly of the SNARE complex and therefore may regulate neurotransmitter release. One group of enzymatic regulators is the protein kinases. These proteins phosphorylate sites on both SNARE proteins and proteins that interact with SNARE proteins. Recent research has identified some of the specific effects that phosphorylation (or dephosphorylation) at these sites can produce. Additionally, palmitoylation of SNAP-25, regulates the localization, and hence activity of this key SNARE protein. This review focuses on the location and effects of phosphorylation on SNARE regulation.     

Genetic evidence for an inhibitory role of tomosyn in insulin‐stimulated GLUT4 exocytosis

Exocytosis is a vesicle fusion process driven by soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs). A classic exocytic pathway is insulin‐stimulated translocation of the glucose transporter type 4 (GLUT4) from intracellular vesicles to the plasma membrane in adipocytes and skeletal muscles. The GLUT4 exocytic pathway plays a central role in maintaining blood glucose homeostasis and is compromised in insulin resistance and type 2 diabetes. A candidate regulator of GLUT4 exocytosis is tomosyn, a soluble protein expressed in adipocytes. Tomosyn directly binds to GLUT4 exocytic SNAREs in vitro but its role in GLUT4 exocytosis was unknown. In this work, we used CRISPR‐Cas9 genome editing to delete the two tomosyn‐encoding genes in adipocytes. We observed that both basal and insulin‐stimulated GLUT4 exocytosis was markedly elevated in the double knockout (DKO) cells. By contrast, adipocyte differentiation and insulin signaling remained intact in the DKO adipocytes. In a reconstituted liposome fusion assay, tomosyn inhibited all the SNARE complexes underlying GLUT4 exocytosis. The inhibitory activity of tomosyn was relieved by NSF and α‐SNAP, which act in concert to remove tomosyn from GLUT4 exocytic SNAREs. Together, these studies revealed an inhibitory role for tomosyn in insulin‐stimulated GLUT4 exocytosis in adipocytes. We suggest that tomosyn‐arrested SNAREs represent a reservoir of fusion capacity that could be harnessed to treat patients with insulin resistance and type 2 diabetes.     

Structure and function of SNARE and SNARE-interacting proteins

This review focuses on the so-called SNARE (soluble N-ethyl maleimide sensitive factor attachment protein receptor) proteins that are involved in exocytosis at the pre-synpatic plasma membrane. SNAREs play a role in docking and fusion of synaptic vesicles to the active zone, as well as in the Ca2+-triggering step itself, most likely in combination with the Ca2+ sensor synaptotagmin. Different SNARE domains are involved in different processes, such as regulation, docking, and fusion. SNAREs exhibit multiple configurational, conformational, and oliogomeric states. These different states allow SNAREs to interact with their matching SNARE partners, auxiliary proteins, or with other SNARE domains, often in a mutually exclusive fashion. SNARE core domains undergo progressive disorder to order transitions upon interactions with other proteins, culminating with the fully folded post-fusion (cis) SNARE complex. Physiological concentrations of neuronal SNAREs can juxtapose membranes, and promote fusion in vitro under certain conditions. However, significantly more work will be required to reconstitute an in vitro system that faithfully mimics the Ca2+-triggered fusion of a synaptic vesicle at the active zone.