Tight coupling of the t-SNARE and calcium channel microdomains in adrenomedullary slices and not in cultured chromaffin cells

Regulated exocytosis involves calcium-dependent fusion of secretory vesicles with the plasma membrane with three SNARE proteins playing a central role: the vesicular synaptobrevin and the plasma membrane syntaxin1 and SNAP-25. Cultured bovine chromaffin cells possess defined plasma membrane microdomains that are specifically enriched in both syntaxin1 and SNAP-25. We now show that in both isolated cells and adrenal medulla slices these target SNARE (t-SNARE) patches quantitatively coincide with single vesicle secretory spots as detected by exposure of the intravesicular dopamine beta-hydroxylase onto the plasmalemma. During exocytosis, neither area nor density of the syntaxin1/SNAP-25 microdomains changes on the plasma membrane of both preparations confirming that preexisting clusters act as the sites for vesicle fusion. Our analysis reveals a high level of colocalization of L, N and P/Q type calcium channel clusters with SNAREs in adrenal slices; this close association is altered in individual cultured cells. Therefore, microdomains carrying syntaxin1/SNAP-25 and different types of calcium channels act as the sites for physiological granule fusion in "in situ" chromaffin cells. In the case of isolated cells, it is the t-SNAREs microdomains rather than calcium channels that define the sites of exocytosis.     

Plasma membrane targeting of exocytic SNARE proteins

SNARE proteins play a central role in the process of intracellular membrane fusion. Indeed, the interaction of SNAREs present on two opposing membranes is generally believed to provide the driving force to initiate membrane fusion. Eukaryotic cells express a large number of SNARE isoforms, and the function of individual SNAREs is required for specific intracellular fusion events. Exocytosis, the fusion of secretory vesicles with the plasma membrane, employs the proteins syntaxin and SNAP-25 as plasma membrane SNAREs. As a result, exocytosis is dependent upon the targeting of these proteins to the plasma membrane; however, the mechanisms that underlie trafficking of exocytic syntaxin and SNAP-25 proteins to the cell surface are poorly understood. The intracellular trafficking itinerary of these proteins is particularly intriguing as syntaxins are tail-anchored (or Type IV) membrane proteins, whereas SNAP-25 is anchored to membranes via a central palmitoylated domain-there is no common consensus for the trafficking of such proteins within the cell. In this review, we discuss the plasma membrane targeting of these essential exocytic SNARE proteins.     

Legionella pneumophila Promotes Functional Interactions between Plasma Membrane Syntaxins and Sec22b

Biogenesis of a specialized organelle that supports intracellular replication of Legionella pneumophila involves the fusion of secretory vesicles exiting the endoplasmic reticulum (ER) with phagosomes containing this bacterial pathogen. Here, we investigated host plasma membrane SNARE proteins to determine whether they play a role in trafficking of vacuoles containing L. pneumophila. Depletion of plasma membrane syntaxins by RNA interference resulted in delayed acquisition of the resident ER protein calnexin and enhanced retention of Rab1 on phagosomes containing virulent L. pneumophila, suggesting that these SNARE proteins are involved in vacuole biogenesis. Plasma membrane‐localized SNARE proteins syntaxin 2, syntaxin 3, syntaxin 4 and SNAP23 localized to vacuoles containing L. pneumophila. The ER‐localized SNARE protein Sec22b was found to interact with plasma membrane SNAREs on vacuoles containing virulent L. pneumophila, but not on vacuoles containing avirulent mutants of L. pneumophila. The addition of α‐SNAP and N‐ethylmaleimide‐sensitive factor (NSF) to the plasma membrane SNARE complexes formed by virulent L. pneumophila resulted in the dissociation of Sec22b, indicating functional pairing between these SNAREs. Thus, L. pneumophila stimulates the non‐canonical pairing of plasma membrane t‐SNAREs with the v‐SNARE Sec22b to promote fusion of the phagosome with ER‐derived vesicles. The mechanism by which L. pneumophila promotes pairing of plasma membrane syntaxins and Sec22b could provide unique insight into how the secretory vesicles could provide an additional membrane reserve subverted during phagosome maturation.     

Endosomal Fusion upon SNARE Knockdown is Maintained by Residual SNARE Activity and Enhanced Docking

SNARE proteins mediate membrane fusion in the secretory pathway of eukaryotic cells. Genetic deletion and siRNA-based knockdown have been instrumental in assigning given SNAREs to defined intracellular transport steps. However, SNARE depletion occasionally results in barely detectable phenotypes. To understand how cells cope with SNARE loss, we have knocked down several SNAREs functioning in early endosome fusion. Surprisingly, knockdown of syntaxin 13, syntaxin 6 and vti1a, alone or in combinations, did not result in measurable changes of endosomal trafficking or fusion. We found that the residual SNARE levels (typically ∼10%) were sufficient for a substantial amount of SNARE–SNARE interactions. Conversely, in wild-type cells, most SNARE molecules were concentrated in clusters, constituting a spare pool not readily available for interactions. Additionally, the knockdown organelles exhibited enhanced docking. We conclude that SNAREs are expressed at much higher levels than needed for maintenance of organelle fusion, and that loss of SNAREs is compensated for by the co-regulation of the docking machinery.     

SNAP-29 is a promiscuous syntaxin-binding SNARE.

SNARE proteins are key regulators of membrane fusion and are proposed to dictate the specificity with which particular vesicles fuse with particular target organelles. On intracellular organelles that serve as targets for transport vesicles, organelle-specific syntaxins form heterodimers with either SNAP-23 or its recently described homolog SNAP-29. We have performed a variety of in vitro and in vivo binding assays in an attempt to determine whether SNAP-23 and SNAP-29 differ in their ability to form binary SNARE complexes with different intracellular syntaxins. While SNAP-23 preferentially binds to plasma membrane-localized syntaxins, SNAP-29 binds to both plasma membrane and intracellular syntaxins equally well. Furthermore, binding to SNAP-29 augments the ability of syntaxin to bind to vesicle-associated SNAREs and the presence of vesicle SNAREs dramatically increases SNAP-29 binding to syntaxin. These data suggest that SNAP-23 preferentially regulates plasma membrane-vesicle fusion events while SNAP-29 plays a role in the maintenance of various intracellular protein trafficking pathways.     

The t-SNAREs syntaxin4 and SNAP23 but not v-SNARE VAMP2 are indispensable to tether GLUT4 vesicles at the plasma membrane in adipocyte

SNARE proteins (VAMP2, syntaxin4, and SNAP23) have been thought to play a key role in GLUT4 trafficking by mediating the tethering, docking and subsequent fusion of GLUT4-containing vesicles with the plasma membrane. The precise functions of these proteins have remained elusive, however. We have now shown that depletion of the vesicle SNARE (v-SNARE) VAMP2 by RNA interference in 3T3-L1 adipocytes inhibited the fusion of GLUT4 vesicles with the plasma membrane but did not affect tethering of the vesicles to the membrane. In contrast, depletion of the target SNAREs (t-SNAREs) syntaxin4 or SNAP23 resulted in impairment of GLUT4 vesicle tethering to the plasma membrane. Our results indicate that the t-SNAREs syntaxin4 and SNAP23 are indispensable for the tethering of GLUT4 vesicles to the plasma membrane, whereas the v-SNARE VAMP2 is not required for this step but is essential for the subsequent fusion event.     

Sly1 protein bound to Golgi syntaxin Sed5p allows assembly and contributes to specificity of SNARE fusion complexes

Fusion of transport vesicles with their target organelles involves specific membrane proteins, SNAREs, which form tight complexes bridging the membranes to be fused. Evidence from yeast and mammals indicates that Sec1 family proteins act as regulators of membrane fusion by binding to the target membrane SNAREs. In experiments with purified proteins, we now made the observation that the ER to Golgi core SNARE fusion complex could be assembled on syntaxin Sed5p tightly bound to the Sec1-related Sly1p. Sly1p also bound to preassembled SNARE complexes in vitro and was found to be part of a vesicular/target membrane SNARE complex immunoprecipitated from yeast cell lysates. This is in marked contrast to the exocytic SNARE assembly in neuronal cells where high affinity binding of N-Sec1/Munc-18 to syntaxin 1A precluded core SNARE fusion complex formation. We also found that the kinetics of SNARE complex formation in vitro with either Sly1p-bound or free Sed5p was not significantly different. Importantly, several presumably nonphysiological SNARE complexes easily generated with Sed5p did not form when the syntaxin was first bound to Sly1p. This indicates for the first time that a Sec1 family member contributes to the specificity of SNARE complex assembly.     

Ca(2+)-synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion

In nerve terminals, exocytosis is mediated by SNARE proteins and regulated by Ca(2+) and synaptotagmin-1 (syt). Ca(2+) promotes the interaction of syt with anionic phospholipids and the target membrane SNAREs (t-SNAREs) SNAP-25 and syntaxin. Here, we have used a defined reconstituted fusion assay to determine directly whether syt-t-SNARE interactions couple Ca(2+) to membrane fusion by comparing the effects of Ca(2+)-syt on neuronal (SNAP-25, syntaxin and synaptobrevin) and yeast (Sso1p, Sec9c and Snc2p) SNAREs. Ca(2+)-syt aggregated neuronal and yeast SNARE liposomes to similar extents via interactions with anionic phospholipids. However, Ca(2+)-syt was able to bind and stimulate fusion mediated by only neuronal SNAREs and had no effect on yeast SNAREs. Thus, Ca(2+)-syt regulates fusion through direct interactions with t-SNAREs and not solely through aggregation of vesicles. Ca(2+)-syt drove assembly of SNAP-25 onto membrane-embedded syntaxin, providing direct evidence that Ca(2+)-syt alters t-SNARE structure.     

Secretory Vesicles Are Preferentially Targeted to Areas of Low Molecular SNARE Density

Intercellular communication is commonly mediated by the regulated fusion, or exocytosis, of vesicles with the cell surface. SNARE (soluble N-ethymaleimide sensitive factor attachment protein receptor) proteins are the catalytic core of the secretory machinery, driving vesicle and plasma membrane merger. Plasma membrane SNAREs (tSNAREs) are proposed to reside in dense clusters containing many molecules, thus providing a concentrated reservoir to promote membrane fusion. However, biophysical experiments suggest that a small number of SNAREs are sufficient to drive a single fusion event. Here we show, using molecular imaging, that the majority of tSNARE molecules are spatially separated from secretory vesicles. Furthermore, the motilities of the individual tSNAREs are constrained in membrane micro-domains, maintaining a non-random molecular distribution and limiting the maximum number of molecules encountered by secretory vesicles. Together our results provide a new model for the molecular mechanism of regulated exocytosis and demonstrate the exquisite organization of the plasma membrane at the level of individual molecular machines.     

Ternary SNARE complexes are enriched in lipid rafts during mast cell exocytosis

Lipid rafts are membrane microdomains rich in cholesterol and glycosphingolipids that have been implicated in the regulation of intracellular protein trafficking. During exocytosis, a class of proteins termed SNAREs mediate secretory granule-plasma membrane fusion. To investigate the role of lipid rafts in secretory granule exocytosis, we examined the raft association of SNARE proteins and SNARE complexes in rat basophilic leukemia (RBL) mast cells. The SNARE protein SNAP-23 co-localized with a lipid raft marker and was present in detergent-insoluble lipid raft microdomains in RBL cells. By contrast, only small amounts (<20%) of the plasma membrane SNARE syntaxin 4 or the granule-associated SNARE vesicle-associated membrane protein (VAMP)-2 were present in these microdomains. Despite this, essentially all syntaxin 4 and most of VAMP-2 in these rafts were present in SNARE complexes containing SNAP-23, while essentially none of these complexes were present in nonraft membranes. Whereas SNAP-23 is membrane anchored by palmitoylation, the association of the transmembrane protein syntaxin 4 with lipid rafts was because of its binding to SNAP-23. After stimulating mast cells exocytosis, the amount of syntaxin 4 and VAMP-2 present in rafts increased twofold, and these proteins were now present in raft-associated phospho-SNAP-23/syntaxin 4/VAMP-2 complexes, revealing differential association of SNARE fusion complexes during the process of regulated exocytosis.     

Neuronal SNAREs do not trigger fusion between synthetic membranes but do promote PEG-mediated membrane fusion

At low surface concentrations that permit formation of impermeable membranes, neuronal soluble N-ethyl maleimide sensitive factor attachment protein receptor (SNARE) proteins form a stable, parallel, trans complex when vesicles are brought into contact by a low concentration of poly(ethylene glycol) (PEG). Surprisingly, formation of a stable SNARE complex does not trigger fusion under these conditions. However, neuronal SNAREs do promote fusion at low protein/lipid ratios when triggered by higher concentrations of PEG. Promotion of PEG-triggered fusion required phosphatidylserine and depended only on the surface concentration of SNAREs and not on the formation of a trans SNARE complex. These results were obtained at protein surface concentrations reported for synaptobrevin in synaptic vesicles and with an optimally fusogenic lipid composition. At a much higher protein/lipid ratio, vesicles joined by SNARE complex slowly mixed lipids at 37 degrees C in the absence of PEG, in agreement with earlier reports. However, vesicles containing syntaxin at a high protein/lipid ratio (>or=1:250) lost membrane integrity. We conclude that the neuronal SNARE complex promotes fusion by joining membranes and that the individual proteins syntaxin and synaptobrevin disrupt membranes so as to favor formation of a stalk complex and to promote conversion of the stalk to a fusion pore. These effects are similar to the effects of viral fusion peptides and transmembrane domains, but they are not sufficient by themselves to produce fusion in our in vitro system at surface concentrations documented to occur in synaptic vesicles. Thus, it is likely that proteins or factors other than the SNARE complex must trigger fusion in vivo.     

A Targeted siRNA Screen to Identify SNAREs Required for Constitutive Secretion in Mammalian Cells

The role of SNAREs in mammalian constitutive secretion remains poorly defined. To address this, we have developed a novel flow cytometry‐based assay for measuring constitutive secretion and have performed a targeted SNARE and Sec1/Munc18 (SM) protein‐specific siRNA screen (38 SNAREs, 4 SNARE‐like proteins and 7 SM proteins). We have identified the endoplasmic reticulum (ER)/Golgi SNAREs syntaxin 5, syntaxin 17, syntaxin 18, GS27, SLT1, Sec20, Sec22b, Ykt6 and the SM protein Sly1, along with the post‐Golgi SNAREs SNAP‐29 and syntaxin 19, as being required for constitutive secretion. Depletion of SNAP‐29 or syntaxin 19 causes a decrease in the number of fusion events at the cell surface and in SNAP‐29‐depleted cells causes an increase in the number of docked vesicles at the plasma membrane as determined by total internal reflection fluorescence (TIRF) microscopy. Analysis of syntaxin 19‐interacting partners by mass spectrometry indicates that syntaxin 19 can form SNARE complexes with SNAP‐23, SNAP‐25, SNAP‐29, VAMP3 and VAMP8, supporting its role in Golgi to plasma membrane transport or fusion. Surprisingly, we have failed to detect any requirement for a post‐Golgi‐specific R‐SNARE in this process.     

SNAREs in mammalian sperm: possible implications for fertilization

Soluble N-ethylmalameide-sensitive factor attachment protein receptor (SNARE) proteins are present in mammalian sperm and could be involved in critical membrane fusion events during fertilization, namely the acrosome reaction. Vesicle-associated membrane protein/synaptobrevin, a SNARE on the membrane of a vesicular carrier, and syntaxin 1, a SNARE on the target membrane, as well as the calcium sensor synaptotagmin I, are present in the acrosome of mammalian sperm (human, rhesus monkey, bull, hamster, mouse). Sperm SNAREs are sloughed off during the acrosome reaction, paralleling the release of sperm membrane vesicles and acrosomal contents, and SNARE antibodies inhibit both the acrosome reaction and fertilization, without inhibiting sperm-egg binding. In addition, sperm SNAREs may be responsible, together with other sperm components, for the asynchronous male DNA decondensation that occurs following intracytoplasmic sperm injection, an assisted reproduction technique that bypasses normal sperm-egg surface interactions. The results suggest the participation of sperm SNAREs during membrane fusion events at fertilization in mammals.     

Determinants of synaptobrevin regulation in membranes

Neuronal exocytosis is driven by the formation of SNARE complexes between synaptobrevin 2 on synaptic vesicles and SNAP-25/syntaxin 1 on the plasma membrane. It has remained controversial, however, whether SNAREs are constitutively active or whether they are down-regulated until fusion is triggered. We now show that synaptobrevin in proteoliposomes as well as in purified synaptic vesicles is constitutively active. Potential regulators such as calmodulin or synaptophysin do not affect SNARE activity. Substitution or deletion of residues in the linker connecting the SNARE motif and transmembrane region did not alter the kinetics of SNARE complex assembly or of SNARE-mediated fusion of liposomes. Remarkably, deletion of C-terminal residues of the SNARE motif strongly reduced fusion activity, although the overall stability of the complexes was not affected. We conclude that although complete zippering of the SNARE complex is essential for membrane fusion, the structure of the adjacent linker domain is less critical, suggesting that complete SNARE complex assembly not only connects membranes but also drives fusion.     

Mixed and non-cognate SNARE complexes. Characterization of assembly and biophysical properties.

Assembly of soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) proteins between two opposing membranes is thought to be the key event that initiates membrane fusion. Many new SNARE proteins have recently been localized to distinct intracellular compartments, supporting the view that sets of specific SNAREs are specialized for distinct trafficking steps. We have now investigated whether other SNAREs can form complexes with components of the synaptic SNARE complex including synaptobrevin/VAMP 2, SNAP-25, and syntaxin 1. When the Q-SNAREs syntaxin 2, 3, and 4, and the R-SNARE endobrevin/VAMP 8 were used in various combinations, heat-resistant complexes were formed. Limited proteolysis revealed that these complexes contained a protease-resistant core similar to that of the synaptic complex. All complexes were disassembled by the ATPase N-ethylmaleimide-sensitive fusion protein and its cofactor alpha-SNAP. Circular dichroism spectroscopy showed that major conformational changes occur during assembly, which are associated with induction of structure from unstructured monomers. Furthermore, no preference for synaptobrevin was observed during the assembly of the synaptic complex when endobrevin/VAMP 8 was present in equal concentrations. We conclude that cognate and non-cognate SNARE complexes are very similar with respect to biophysical properties, assembly, and disassembly, suggesting that specificity of membrane fusion in intracellular membrane traffic is not due to intrinsic specificity of SNARE pairing.     

Munc18-1 and Syntaxin1: Unraveling the Interactions Between the Dynamic Duo

All neurotransmitter and hormone regulated secretory events involve the action of three soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, syntaxin, SNAP-25, and synaptobrevin. The SNARE proteins interact to form a four alpha-helical complex, involving syntaxin and SNAP-25 on the plasma membrane and synaptobrevin on the vesicular membrane, bringing the opposing membranes together, promoting bilayer merger and membrane fusion. The process of regulated secretion is an adaptation of the membrane fusion events which occur at multiple steps throughout the intracellular trafficking pathway, in each case catalyzed by SNARE protein isoforms. At all of these locations, the SNAREs are joined by a member of the Sec1p/Munc18 (SM) protein family which selectively bind to syntaxin isoforms. From their initial identification, the SM proteins were known to be essential for membrane fusion, however, over the intervening decades, deciphering the precise mechanism of action of the SM proteins has proved problematic. Recent studies, investigating the interactions of munc18-1 and syntaxin1, provide an explanation for previous, apparently conflicting, observations yielding a new understanding of their cellular functions.     

SNARE membrane trafficking dynamics in vivo

The ER/Golgi soluble NSF attachment protein receptor (SNARE) membrin, rsec22b, and rbet1 are enriched in approximately 1-micrometer cytoplasmic structures that lie very close to the ER. These appear to be ER exit sites since secretory cargo concentrates in and exits from these structures. rsec22b and rbet1 fused to fluorescent proteins are enriched at approximately 1-micrometer ER exit sites that remained more or less stationary, but periodically emitted streaks of fluorescence that traveled generally in the direction of the Golgi complex. These exit sites were reused and subsequent tubules or streams of vesicles followed similar trajectories. Fluorescent membrin- enriched approximately 1-micrometer peripheral structures were more mobile and appeared to translocate through the cytoplasm back and forth, between the periphery and the Golgi area. These mobile structures could serve to collect secretory cargo by fusing with ER-derived vesicles and ferrying the cargo to the Golgi. The post-Golgi SNAREs, syntaxin 6 and syntaxin 13, when fused to fluorescent proteins each displayed characteristic patterns of movement. However, syntaxin 13 was the only SNARE whose life cycle appeared to involve interactions with the plasma membrane. These studies reveal the in vivo spatiotemporal dynamics of SNARE proteins and provide new insight into their roles in membrane trafficking.     

Crystal structure of the endosomal SNARE complex reveals common structural principles of all SNAREs.

SNARE proteins are crucial for intracellular membrane fusion in all eukaryotes. These proteins assemble into tight complexes that connect membranes and may induce fusion. The crystal structure of the neuronal core complex is represented by an unusually long bundle of four alpha-helices connected by 16 layers of mostly hydrophobic amino acids. Here we report the 1.9 A resolution crystal structure of an endosomal SNARE core complex containing four SNAREs: syntaxin 7, syntaxin 8, vti1b and endobrevin/VAMP-8. Despite limited sequence homology, the helix alignment and the layer structure of the endosomal complex are remarkably similar to those of the neuronal complex. However, subtle variations are evident that characterize different SNARE subfamilies. We conclude that the structure of the SNARE core complex is an evolutionarily conserved hallmark of all SNARE complexes and is intimately associated with the general role of SNAREs in membrane fusion.     

Synaptic vesicles are constitutively active fusion machines that function independently of Ca2+

BACKGROUND: In neurons, release of neurotransmitter occurs through the fusion of synaptic vesicles with the plasma membrane. Many proteins required for this process have been identified, with the SNAREs syntaxin 1, SNAP-25, and synaptobrevin thought to constitute the core fusion machinery. However, there is still a large gap between our understanding of individual protein-protein interactions and the functions of these proteins revealed by perturbations in intact synaptic preparations. To bridge this gap, we have used purified synaptic vesicles, together with artificial membranes containing core-constituted SNAREs as reaction partners, in fusion assays. RESULTS: By using complementary experimental approaches, we show that synaptic vesicles fuse constitutively, and with high efficiency, with proteoliposomes containing the plasma membrane proteins syntaxin 1 and SNAP-25. Fusion is inhibited by clostridial neurotoxins and involves the formation of SNARE complexes. Despite the presence of endogenous synaptotagmin, Ca(2+) does not enhance fusion, even if phosphatidylinositol 4,5-bisphosphate is present in the liposome membrane. Rather, fusion kinetics are dominated by the availability of free syntaxin 1/SNAP-25 acceptor sites for synaptobrevin. CONCLUSIONS: Synaptic vesicles are constitutively active fusion machines, needing only synaptobrevin for activity. Apparently, the final step in fusion does not involve the regulatory activities of other vesicle constituents, although these may be involved in regulating earlier processes. This is particularly relevant for the calcium-dependent regulation of exocytosis, which, in addition to synaptotagmin, requires other factors not present in the vesicle membrane. The in vitro system described here provides an ideal starting point for unraveling of the molecular details of such regulatory events.     

Docking of liposomes to planar surfaces mediated by trans-SNARE complexes

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a key role in membrane fusion in the secretory pathway. In vitro, SNAREs spontaneously assemble into helical SNARE complexes with the transmembrane domains at the C-terminal end. During fusion, SNAREs are thought to bridge the two membranes and assemble in a zipper-like fashion, pulling the membranes together and initiating fusion. However, it is not clear to what extent SNARE assembly contributes to membrane attachment and membrane fusion. Using the neuronal SNAREs synaptobrevin (VAMP), SNAP-25, and syntaxin as examples, we show here that liposomes containing synaptobrevin firmly attach to planar surfaces containing immobilized syntaxin. Attachment requires the formation of SNARE complexes because it is dependent on the presence of SNAP-25. Binding is competed for by soluble SNARE fragments, with noncognate SNAREs such as endobrevin (VAMP8), VAMP4, and VAMP7 (Ti-VAMP) being effective but less potent in some cases. Furthermore, although SNAP-23 is unable to substitute for SNAP-25 in the attachment assay, it forms complexes of comparable stability and is capable of substituting in liposome fusion assays. Vesicle attachment is initiated by SNARE assembly at the N-terminal end of the helix bundle. We conclude that SNAREs can indeed form stable trans-complexes that result in vesicle attachment if progression to fusion is prevented, further supporting the zipper model of SNARE function.