Mechanism of calcium-independent synaptotagmin binding to target SNAREs

Synaptic vesicle exocytosis requires three SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins: syntaxin and SNAP-25 on the plasma membrane (t-SNAREs) and synaptobrevin/VAMP on the synaptic vesicles (v-SNARE). Vesicular synaptotagmin 1 is essential for fast synchronous SNARE-mediated exocytosis and interacts with the SNAREs in brain material. To uncover the step at which synaptotagmin becomes linked to the three SNAREs, we purified all four proteins from brain membranes and analyzed their interactions. Our study reveals that, in the absence of calcium, native synaptotagmin 1 binds the t-SNARE heterodimer, formed from syntaxin and SNAP-25. This interaction is both stoichiometric and of high affinity. Synaptotagmin contains two divergent but conserved C2 domains that can act independently in calcium-triggered phospholipid binding. We now show that both C2 domains are strictly required for the calcium-independent interaction with the t-SNARE heterodimer, indicating that the double C2 domain structure of synaptotagmin may have evolved to acquire a function beyond calcium/phospholipid binding.     

Differential Phosphorylation of Syntaxin and Synaptosome-Associated Protein of 25 kDa (SNAP-25) Isoforms

Abstract : The synaptic plasma membrane proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) are central participants in synaptic vesicle trafficking and neurotransmitter release. Together with the synaptic vesicle protein synaptobrevin/vesicle-associated membrane protein (VAMP), they serve as receptors for the general membrane trafficking factors N -ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (α-SNAP). Consequently, syntaxin, SNAP-25, and VAMP (and their isoforms in other membrane trafficking pathways) have been termed SNAP receptors (SNAREs). Because protein phosphorylation is a common and important mechanism for regulating a variety of cellular processes, including synaptic transmission, we have investigated the ability of syntaxin and SNAP-25 isoforms to serve as substrates for a variety of serine/threonine protein kinases. Syntaxins 1A and 4 were phosphorylated by casein kinase II, whereas syntaxin 3 and SNAP-25 were phosphorylated by Ca²⁺ - and calmodulin-dependent protein kinase II and cyclic AMP-dependent protein kinase, respectively. The biochemical consequences of SNARE protein phosphorylation included a reduced interaction between SNAP-25 and phosphorylated syntaxin 4 and an enhanced interaction between phosphorylated syntaxin 1A and the synaptic vesicle protein synaptotagmin I, a potential Ca²⁺ sensor in triggering synaptic vesicle exocytosis. No other effects on the formation of SNARE complexes (comprised of syntaxin, SNAP-25, and VAMP) or interactions involving n-Sec1 or α-SNAP were observed. These findings suggest that although phosphorylation does not directly regulate the assembly of the synaptic SNARE complex, it may serve to modulate SNARE complex function through other proteins, including synaptotagmin I.     

The t-SNAREs syntaxin 1 and SNAP-25 are present on organelles that participate in synaptic vesicle recycling

Syntaxin 1 and synaptosome-associated protein of 25 kD (SNAP-25) are neuronal plasmalemma proteins that appear to be essential for exocytosis of synaptic vesicles (SVs). Both proteins form a complex with synaptobrevin, an intrinsic membrane protein of SVs. This binding is thought to be responsible for vesicle docking and apparently precedes membrane fusion. According to the current concept, syntaxin 1 and SNAP-25 are members of larger protein families, collectively designated as target-SNAP receptors (t-SNAREs), whose specific localization to subcellular membranes define where transport vesicles bind and fuse. Here we demonstrate that major pools of syntaxin 1 and SNAP-25 recycle with SVs. Both proteins cofractionate with SVs and clathrin-coated vesicles upon subcellular fractionation. Using recombinant proteins as standards for quantitation, we found that syntaxin 1 and SNAP-25 each comprise approximately 3% of the total protein in highly purified SVs. Thus, both proteins are significant components of SVs although less abundant than synaptobrevin (8.7% of the total protein). Immunoisolation of vesicles using synaptophysin and syntaxin specific antibodies revealed that most SVs contain syntaxin 1. The widespread distribution of both syntaxin 1 and SNAP-25 on SVs was further confirmed by immunogold electron microscopy. Botulinum neurotoxin C1, a toxin that blocks exocytosis by proteolyzing syntaxin 1, preferentially cleaves vesicular syntaxin 1. We conclude that t- SNAREs participate in SV recycling in what may be functionally distinct forms.     

SNAP-25 traffics to the plasma membrane by a syntaxin-independent mechanism

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are essential for vesicle docking and fusion. SNAP-25, syntaxin 1A, and synaptobrevin/vesicle-associated membrane protein (VAMP) are SNARE proteins that mediate fusion of synaptic vesicles with the plasma membrane. It has been proposed that interactions of SNAP-25 with syntaxin 1A are required for initial membrane attachment of SNAP-25 (Vogel, K., Cabaniols, J.-P., and Roche, P. (2000) J. Biol. Chem. 275, 2959-2965). However, we have shown previously that residues 85-120 of the SNAP-25 interhelical domain, which do not interact with syntaxin, are necessary and sufficient for palmitoylation and plasma membrane localization of a green fluorescent protein reporter molecule (Gonzalo, S., Greentree, W. K., and Linder, M. E. (1999) J. Biol. Chem. 274, 21313-21318). To clarify the role of syntaxin in membrane targeting of SNAP-25, we studied a SNAP-25 point mutant (G43D) that does not interact with syntaxin. SNAP-25 G43D/green fluorescent protein was palmitoylated and localized at the plasma membrane. Newly synthesized SNAP-25 G43D had the same kinetics of membrane association as the wild-type protein. Furthermore, expression of a cytosolic mutant syntaxin 1A did not interfere with SNAP-25 membrane interactions or palmitoylation in the neuronal cell line NG108-15. Exogenously expressed SNAP-25 targets efficiently to the plasma membrane in cells of neuronal origin but only partially in HeLa cells, a neurosecretion-incompetent line. This phenotype was not rescued when syntaxin 1A was co-expressed with SNAP-25. Our data support a syntaxin-independent mechanism of membrane targeting for SNAP-25.     

Clostridial neurotoxins compromise the stability of a low energy SNARE complex mediating NSF activation of synaptic vesicle fusion.

A 20S complex composed of the cytosolic fusion proteins NSF and SNAP and the synaptosomal SNAP receptors (SNAREs) synaptobrevin, syntaxin and SNAP-25 is essential for synaptic vesicle exocytosis. Formation of this complex is thought to be regulated by synaptotagmin, the putative calcium sensor of neurotransmitter release. Here we have examined how different inhibitors of neurotransmitter release, e.g. clostridial neurotoxins and a synaptotagmin peptide, affect the properties of the 20S complex. Cleavage of synaptobrevin and SNAP-25 by the neurotoxic clostridial proteases tetanus toxin and botulinum toxin A had no effect on assembly and disassembly of the 20S complex; however, the stability of its SDS-resistant SNARE core was compromised. This SDS-resistant low energy conformation of the SNAREs constitutes the physiological target of NSF, as indicated by its ATP-dependent disassembly in the presence of SNAP and NSF. Synaptotagmin peptides caused inhibition of in vitro binding of this protein to the SNAREs, a result that is inconsistent with synaptotagmin's proposed role as a regulator of SNAP binding. Our data can be reconciled by the idea that NSF and SNAP generate synaptotagmin-containing intermediates in synaptic vesicle fusion, which catalyse neurotransmitter release.     

A transient N-terminal interaction of SNAP-25 and syntaxin nucleates SNARE assembly

The SNARE proteins syntaxin, SNAP-25, and synaptobrevin play a central role during Ca(2+)-dependent exocytosis at the nerve terminal. Whereas syntaxin and SNAP-25 are located in the plasma membrane, synaptobrevin resides in the membrane of synaptic vesicles. It is thought that gradual assembly of these proteins into a membrane-bridging ternary SNARE complex ultimately leads to membrane fusion. According to this model, syntaxin and SNAP-25 constitute an acceptor complex for synaptobrevin. In vitro, however, syntaxin and SNAP-25 form a stable complex that contains two syntaxin molecules, one of which is occupying and possibly obstructing the binding site of synaptobrevin. To elucidate the assembly pathway of the synaptic SNAREs, we have now applied a combination of fluorescence and CD spectroscopy. We found that SNARE assembly begins with the slow and rate-limiting interaction of syntaxin and SNAP-25. Their interaction was prevented by N-terminal but not by C-terminal truncations, suggesting that for productive assembly all three participating helices must come together simultaneously. This suggests a complicated nucleation process that might be the reason for the observed slow assembly rate. N-terminal truncations of SNAP-25 and syntaxin also prevented the formation of the ternary complex, whereas neither N- nor C-terminal shortened synaptobrevin helices lost their ability to interact. This suggests that binding of synaptobrevin occurs after the establishment of the syntaxin-SNAP-25 interaction. Moreover, binding of synaptobrevin was inhibited by an excess of syntaxin, suggesting that a 1:1 interaction of syntaxin and SNAP-25 serves as the on-pathway SNARE assembly intermediate.     

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.     

Inhibition of neurotransmitter release in the lamprey reticulospinal synapse by antibody-mediated disruption of SNAP-25 function

Exocytosis - syntaxin - synaptobrevin - SNARE synaptic vesicle The lamprey giant reticulospinal synapse can be used to manipulate the molecular machinery of synaptic vesicle exocytosis by presynaptic microinjection. Here we test the effect of disrupting the function of the SNARE protein SNAP-25. Polyclonal SNAP-25 antibodies were shown in an in vitro assay to inhibit the binding between syntaxin and SNAP-25. When microinjected presynaptically, these antibodies produced a potent inhibition of the synaptic response. Ba2+ spikes recorded in the presynaptic axon were not altered, indicating that the effect was not due to a reduced presynaptic Ca2+ entry. Electron microscopic analysis showed that synaptic vesicle clusters had a similar organization in synapses of antibody-injected axons as in control axons, and the number of synaptic vesicles in apparent contact with the presynaptic plasma membrane was also similar. Clathrin-coated pits, which normally occur at the plasma membrane around stimulated synapses, were not detected after injection of SNAP-25 antibodies, consistent with a blockade of vesicle cycling. Thus, SNAP-25 antibodies, which disrupt the interaction with syntaxin, inhibit neurotransmitter release without affecting the number of synaptic vesicles at the plasma membrane. These results provide further support to the view that the formation of SNARE complexes is critical for membrane fusion, but not for the targeting of synaptic vesicles to the presynaptic membrane.     

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.     

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.     

The relation of protein binding to function: what is the significance of munc18 and synaptotagmin binding to syntaxin 1, and where are the corresponding binding sites?

The Q-SNARE syntaxin 1 is a central component of the synaptic membrane fusion machinery. Syntaxin probably interacts with multiple proteins during synaptic vesicle exocytosis. In vitro, the tightest binding partners for syntaxin 1 are other SNAREs (synaptobrevin/VAMP and SNAP-25) and munc18-1 (also known as rbsec1/nsec1). Recent studies on Drosophila syntaxin led to the surprising finding that a syntaxin mutant which does not bind the munc18-homolog Rop nevertheless functionally substitutes for wild-type syntaxin in membrane fusion (Wu et al., Neuron 23, 593-605, 1999). This observation suggested that syntaxin 1 binding to munc18-1 is not essential for fusion, a puzzling conclusion in view of the tight binding of munc18 and syntaxin homologs in all organisms. To address this issue, we have now reinvestigated the interaction of syntaxin with munc18 and Rop. We find that the syntaxin sequence that was mutated in the Drosophila studies is not essential for munc18/Rop binding, and that the mutant is in fact able to bind to munc18/Rop. Thus the fact that the mutant syntaxin rescues release cannot be used as an argument that munc18 binding is not essential. In addition to munc18 and SNAREs, several other proteins have been suggested to interact with various domains of syntaxin 1, notably the calcium-sensor synaptotagmin and the vesicle protein CSP. Our results confirm that the SNARE motif in syntaxin binds to synaptotagmin, but this interaction does not require the very C-terminus of the motif. Interestingly, binding of synaptotagmin appears to be decreased in the closed conformation of syntaxin. In contrast, no interaction of CSP with syntaxin was detected even under low-stringency conditions. Our data suggest 1., that assays measuring protein/protein interactions that involve syntaxin may be more difficult to evaluate than is often assumed because of the sticky nature of the proteins involved, and 2., that it is currently not possible to draw conclusions about the importance of the various interactions with the available data from Drosophila or vertebrates.     

Calcium-independent stimulation of membrane fusion and SNAREpin formation by synaptotagmin I

Neurotransmitter release requires the direct coupling of the calcium sensor with the machinery for membrane fusion. SNARE proteins comprise the minimal fusion machinery, and synaptotagmin I, a synaptic vesicle protein, is the primary candidate for the main neuronal calcium sensor. To test the effect of synaptotagmin I on membrane fusion, we incorporated it into a SNARE-mediated liposome fusion assay. Synaptotagmin I dramatically stimulated membrane fusion by facilitating SNAREpin zippering. This stimulatory effect was topologically restricted to v-SNARE vesicles (containing VAMP 2) and only occurred in trans to t-SNARE vesicles (containing syntaxin 1A and SNAP-25). Interestingly, calcium did not affect the overall fusion reaction. These results indicate that synaptotagmin I can directly accelerate SNARE-mediated membrane fusion and raise the possibility that additional components might be required to ensure tight calcium coupling.     

The transmembrane domain of syntaxin 1A is critical for cytoplasmic domain protein-protein interactions

Assembly of the plasma membrane proteins syntaxin 1A and SNAP-25 with the vesicle protein synaptobrevin is a critical step in neuronal exocytosis. Syntaxin is anchored to the inner face of presynaptic plasma membrane via a single C-terminal membrane-spanning domain. Here we report that this transmembrane domain plays a critical role in a wide range of syntaxin protein-protein interactions. Truncations or deletions of the membrane-spanning domain reduce synaptotagmin, alpha/beta-SNAP, and synaptobrevin binding. In contrast, deletion of the transmembrane domain potentiates SNAP-25 and rbSec1A/nsec-1/munc18 binding. Normal partner protein binding activity of the isolated cytoplasmic domain could be "rescued" by fusion to the transmembrane segments of synaptobrevin and to a lesser extent, synaptotagmin. However, efficient rescue was not achieved by replacing deleted transmembrane segments with corresponding lengths of other hydrophobic amino acids. Mutations reported to diminish the dimerization of the transmembrane domain of syntaxin did not impair the interaction of full-length syntaxin with other proteins. Finally, we observed that membrane insertion and wild-type interactions with interacting proteins are not correlated. We conclude that the transmembrane domain, via a length-dependent and sequence-specific mechanism, affects the ability of the cytoplasmic domain to engage other proteins.     

Functional and physical coupling of voltage-sensitive calcium channels with exocytotic proteins: ramifications for the secretion mechanism

The secretion of neurotransmitters is a rapid Ca(2+)-regulated process that brings about vesicle fusion with the plasma membrane. This rapid process (< 100 microseconds) involves multiple proteins located at the plasma and vesicular membranes. Because of their homology to proteins participating in constitutive secretion and protein trafficking, they have been characterized extensively. The sequential events that lead these proteins to vesicle docking and fusion are still unclear. We will review recent studies that demonstrate the operative role played by voltage-sensitive Ca(2+) channels and discuss the relevance for the process of evoked transmitter release. The regulation of Ca(2+) influx by syntaxin, synaptosome-associated protein of 25 kDa (SNAP-25) and synaptotagmin, and the reciprocity of these proteins in controlling the kinetic properties of the channel will be discussed. Calcium channel and synaptic proteins expressed in Xenopus oocytes demonstrate a strong functional interaction, which could be pertinent to the mechanism of secretion. First, the voltage-sensitive Ca(2+) channels are negatively modulated by syntaxin: this inhibition is reversed by synaptotagmin. Second, the modulation of N-type Ca(2+) channel activation kinetics strongly suggests that the vesicle could be docked at the plasma membrane through direct interaction with synaptotagmin. Finally, these interactions provide evidence for the assembly of the voltage-sensitive Ca(2+) channel with syntaxin 1A, SNAP-25 and synaptotagmin into an excitosome complex: a putative fusion complex with a potential role in the final stages of secretion. Studies suggest that cross-talk between the synaptic proteins and the channel in a tightly organized complex may enable a rapid secretory response to an incoming signal such as membrane depolarization.     

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.     

The t-SNARE Complex: A Close Up

The SNARE proteins, syntaxin, SNAP-25, and synaptobrevin have long been known to provide the driving force for vesicle fusion in the process of regulated exocytosis. Of particular interest is the initial interaction between SNAP-25 and syntaxin to form the t-SNARE heterodimer, an acceptor for subsequent synaptobrevin engagement. In vitro studies have revealed at least two different dynamic conformations of t-SNARE heterodimer defined by the degree of association of the C-terminal SNARE motif of SNAP-25 with syntaxin. At the plasma membrane, these proteins are organized into dense clusters of 50–60 nm in diameter. More recently, the t-SNARE interaction within these clusters was investigated in live cells at the molecular level, estimating each cluster to contain 35–70 t-SNARE molecules. This work reported the presence of both partially and fully zippered t-SNARE complex at the plasma membrane in agreement with the earlier in vitro findings. It also revealed a spatial segregation into distinct clusters containing predominantly one conformation apparently patterned by the surrounding lipid environment. The reason for this dynamic t-SNARE complex in exocytosis is uncertain; however, it does take us one step closer to understand the complex sequence of events leading to vesicle fusion, emphasizing the role of both membrane proteins and lipids.     

Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly.

Clostridial neurotoxins inhibit neurotransmitter release by selective and specific intracellular proteolysis of synaptobrevin/VAMP, synaptosomal-associated protein of 25 kDa (SNAP-25) or syntaxin. Here we show that in binary reactions synaptobrevin binds weakly to both SNAP-25 and syntaxin, and SNAP-25 binds to syntaxin. In the presence of all three components, a dramatic increase in the interaction strengths occurs and a stable sodium dodecyl sulfate-resistant complex forms. Mapping of the interacting sequences reveals that complex formation correlates with the presence of predicted alpha-helical structures, suggesting that membrane fusion involves intermolecular interactions via coiled-coil structures. Most toxins only attack the free, and not the complexed, proteins, and proteolysis of the proteins by different clostridial neurotoxins has distinct inhibitory effects on the formation of synaptobrevin-syntaxin-SNAP-25 complexes. Our data suggest that synaptobrevin, syntaxin and SNAP-25 associate into a unique stable complex that functions in synaptic vesicle exocytosis.     

Identification of SNAP-47, a novel Qbc-SNARE with ubiquitous expression

The SNARE proteins are essential components of the intracellular fusion machinery. It is thought that they form a tight four-helix complex between membranes, in effect initiating fusion. Most SNAREs contain a single coiled-coil region, referred to as the SNARE motif, directly adjacent to a single transmembrane domain. The neuronal SNARE SNAP-25 defines a subfamily of SNARE proteins with two SNARE helices connected by a longer linker, comprising also the proteins SNAP-23 and SNAP-29. We now report the initial characterization of a novel vertebrate homologue termed SNAP-47. Northern blot and immunoblot analysis revealed ubiquitous tissue distribution, with particularly high levels in nervous tissue. In neurons, SNAP-47 shows a widespread distribution on intracellular membranes and is also enriched in synaptic vesicle fractions. In vitro, SNAP-47 substituted for SNAP-25 in SNARE complex formation with the neuronal SNAREs syntaxin 1a and synaptobrevin 2, and it also substituted for SNAP-25 in proteoliposome fusion. However, neither complex assembly nor fusion was as efficient as with SNAP-25.     

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.     

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.