org Interactive Fly, Drosophila

Syntaxin 1A


SNARE [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor] proteins are essential for membrane fusion and are conserved from yeast to humans. The SNAREs functioning in neuronal exocytosis are among the best characterized; they include the synaptic vesicle protein synaptobrevin (also referred to as VAMP) and the synaptic plasma membrane proteins SNAP-25 and syntaxin-1A. These proteins readily assemble into a stable ternary complex whose core structure has been recently solved by x-ray crystallography. Sequence alignments of the most conserved regions of SNARE proteins have been mapped onto the crystal structure of the heterotrimeric synaptic fusion complex. The association of the four alpha-helices in the synaptic fusion complex structure produces highly conserved layers of interacting amino acid side chains in the center of the four-helix bundle. Mutations in these layers reduce complex stability and cause defects in membrane traffic even in distantly related SNAREs. When syntaxin-4 is modeled into the synaptic fusion complex as a replacement of syntaxin-1A, no major steric clashes arise and the most variable amino acids localize to the outer surface of the complex. It is concluded that the main structural features of the neuronal complex are highly conserved during evolution. On the basis of these features SNARE proteins have been reclassified into Q-SNAREs and R-SNAREs, and it is proposed that fusion-competent SNARE complexes generally consist of four-helix bundles composed of three Q-SNAREs and one R-SNARE. Previously, SNARE proteins were divided into v-SNAREs (including proteins homologous to synaptobrevin) and t-SNAREs (including proteins homologous to syntaxin-1 and SNAP-25), based on their preferred localization on either the trafficking vesicle (v) or the target membrane (t), respectively. This classification scheme may not be accurate for all vesicular transport steps. Furthermore, it does not cover homotypic fusion events, that is, fusion between vesicles that are functionally and structurally equivalent. Reclassification of the SNARE proteins is proposed based on their contributions to the ionic 0 layer. The ionic layer (designated as "0" layer) at the center of the synaptic fusion complex contains the most highly conserved residues in all SNARE proteins. This layer is composed of Arg-56 of synaptobrevin-II; Gln-226 of syntaxin-1A, and Gln-53 and Gln-174 of SNAP-25B. R-SNAREs would provide an arginine (R) to this ionic layer and Q-SNAREs would provide complementary glutamines (Q). Although the R-SNAREs include most of the proteins previously classified as v-SNAREs, there are no structural reasons why the alpha-helices provided by the trafficking vesicles need to be derived only from R-SNAREs, as in the case of the synaptic fusion complex (Fasshauer, 1998).

At present, only few SNARE complexes have been characterized in which all partners have been identified and localized, such as the SNARE complex involved in yeast exocytosis. For other trafficking steps, relevant SNARE proteins are known, but it is unclear which of them interact in a particular fusion step. A well studied example is the vesicular traffic between the endoplasmic reticulum and the Golgi apparatus. Despite the assignment of several SNARE proteins it remains to be established which of them function in anterograde vs. retrograde traffic and whether intermediate fusion steps are involved. Each Q-SNARE would contribute only a single alpha-helix to the four-helix bundle of this putative complex. Although all characterized SNARE complexes involved in membrane fusion are of the 3 Q-SNARE/1 (R-SNARE) type, defined complexes have been reported that consist only of Q-SNAREs. It remains to be established whether these complexes are also four helix bundles and whether they play any role in membrane fusion events. These results support a model in which complex formation promotes membrane fusion. Indeed, the alpha-helices are closely packed in the region directly adjacent to the transmembrane domains, and this region is characterized by a conserved group of basic and aromatic residues. Furthermore, this region is sensitive to neurotoxin cleavage. Such packing may be completed only after the fusion reaction when the membrane anchors are aligned in parallel in the same membrane. Consequently, partial assembly states may occur during membrane docking in which the membrane anchors point away from one another. It remains to be established whether the free energy released by these intermediate assembly states suffices to induce lipid mixing. Perhaps this process is assisted or regulated by accessory proteins, such as synaptotagmin, which could link Ca2+-dependent exocytosis and the synaptic fusion complex (Fasshauer, 1998 and references).

The embryo of the flowering plant Arabidopsis develops by a regular pattern of cell divisions and cell shape changes. Mutations in the KNOLLE (KN) gene affect the rate and plane of cell divisions as well as cell morphology, resulting in mutant seedlings with a disturbed radial organization of tissue layers. At the cellular level, mutant embryos are characterized by incomplete cross walls and enlarged cells with polyploid nuclei. The KN gene was isolated by positional cloning. The predicted KN protein has similarity to syntaxins, a protein family involved in vesicular trafficking. During embryogenesis, KN transcripts are detected in patches of single cells or small cell groups. These results suggest a function for KN in cytokinesis (Lukowitz, 1996).

Syntaxins are thought to participate in the specific interactions between vesicles and acceptor membranes in intracellular protein trafficking. VAM3 of Saccharomyces cerevisiae encodes a 33 kDa protein (Vam3p) with a hydrophobic transmembrane segment at its C terminus. Vam3p has structural similarities to syntaxins of yeast, animal and plant cells. Deltavam3 cells accumulate spherical structures of 200-600 nm in diameter, but lack normal large vacuolar compartments. Loss of function of Vam3p results in inefficient processing of vacuolar proteins proteinase A, proteinase B and carboxypeptidase Y, and defective maturation of alkaline phosphatase. Vam3p is localized to the vacuolar membranes. Vam3p accumulates in certain regions of the vacuolar membranes. It is concluded that Vam3p is a novel member of syntaxin in vacuoles and it provides the t-SNARE function in a late step of the vacuolar assembly (Wada, 1997).

Crystal structure of a SNARE complex

The evolutionarily conserved SNARE proteins and their complexes are involved in the fusion of vesicles with their target membranes; however, the overall organization and structural details of these complexes are unknown. The X-ray crystal structure at 2.4 A resolution of a core synaptic fusion complex containing syntaxin-1 A, synaptobrevin-II and SNAP-25B is reported. One of the identifying characteristics of SNARE proteins is their ability to form tight, SDS-resistant, ternary complexes. The SNARE complex is a parallel four-helix bundle with one helix contributed by each of Syntaxin and VAMP and two contributed by SNAP-25. The formation of a trans-membrane complex, with VAMP on the transport vesicle and Syntaxin and SNAP-25 on the target membrane, is thought to lead to the fusion of the two membranes, resulting in a cis-membrane complex. Conserved leucine-zipper-like layers are found at the center of the synaptic fusion complex. Embedded within these leucine-zipper layers is an ionic layer consisting of an arginine and three glutamine residues contributed from each of the four alpha-helices. These residues are highly conserved across the entire SNARE family. The regions flanking the leucine-zipper-like layers contain a hydrophobic core similar to that of more general four-helix-bundle proteins. The surface of the synaptic fusion complex is highly grooved and possesses distinct hydrophilic, hydrophobic and charged regions. These characteristics may be important for membrane fusion and for the binding of regulatory factors affecting neurotransmission (Sutton, 1998).

Conformational switch of syntaxin-1 controls synaptic vesicle fusion

During synaptic vesicle fusion, the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) protein syntaxin-1 exhibits two conformations that both bind to Munc18-1: a 'closed' conformation outside the SNARE complex and an 'open' conformation in the SNARE complex. Although SNARE complexes containing open syntaxin-1 and Munc18-1 are essential for exocytosis, the function of closed syntaxin-1 is unknown. Knockin/knockout mice were generated that expressed only open syntaxin-1B. Syntaxin-1BOpen mice were viable but succumbed to generalized seizures at 2 to 3 months of age. Binding of Munc18-1 to syntaxin-1 was impaired in syntaxin-1BOpen synapses, and the size of the readily releasable vesicle pool was decreased; however, the rate of synaptic vesicle fusion was dramatically enhanced. Thus, the closed conformation of syntaxin-1 gates the initiation of the synaptic vesicle fusion reaction, which is then mediated by SNARE-complex/Munc18-1 assemblies (Gerber, 2006).

Opening syntaxin-1 facilitates the fusion of synaptic vesicles on the background of a smaller readily-releasable vesicle pool (RRP) without changing the recruitment of vesicles into the RRP. Consistent with this conclusion, it was found that the syntaxin-1BOpen mutation accelerates sucrose-induced release and significantly boosts the relative amount and fractional release rate induced at lower sucrose concentrations. Moreover, the syntaxin-1BOpen mutation increases the apparent Ca2+ sensitivity of neurotransmitter release and occludes the phorbol-ester-induced potentiation of release. Overall, these results establish that although the RRP is smaller in syntaxin-1BOpen synapses, their resident RRP vesicles are more fusogenic (Gerber, 2008).

The closed conformation of syntaxin-1 performs three functions upstream of the canonical role of syntaxin-1 as a SNARE protein in membrane fusion: (1) Closed syntaxin-1, but not the SNARE complex, mediates vesicle docking in chromaffin cells but not in synapses. The same differential phenotype is observed upon deletion of Munc18-1, suggesting that the Munc18-1-syntaxin-1 complex docks chromaffin but not synaptic vesicles. (2) The closed syntaxin-1 conformation stabilizes syntaxin-1 and Munc18-1, whereas opening syntaxin-1 decreases syntaxin-1 and Munc18-1 levels and thereby lowers the RRP size. (3) Opening syntaxin-1 accelerates the rate of synaptic vesicle fusion, accounting for the fulminant epilepsy observed in synaxin-1BOpen mutant mice (Gerber, 2008).

Ca2+ and sucrose trigger fusion of primed synaptic vesicles. Primed vesicles are thought to be suspended in a metastable state in which SNARE complexes are assembled but the bilayers have not yet fused. It is proposed that primed vesicles are associated with a variable number of assembled SNARE complexes and that this number dictates the sucrose- and Ca2+-sensitivity of a given vesicle. To account for the synaptic phenotype of syntaxin-1BOpen mutant mice, it is hypothesized that the syntaxin-1BOpen mutation increases the average number of assembled SNARE complexes per vesicle and thereby enhances their Ca2+ and sucrose sensitivity. In contrast, the destabilization of syntaxin-1 and Munc18-1 by the syntaxin-1BOpen mutation decreases the total number of primed vesicles and thus the RRP, even though the primed vesicles are more fusogenic. The decrease in RRP is not due to the increased spontaneous release rate because its spontaneous fusion rate is still over 100-fold less than the vesicle repriming rate and because much higher spontaneous fusion rates in synaptotagmin-mutant mice do not decrease the RRP size. An alternative hypothesis would be that primed vesicles lack assembled SNARE complexes and Ca2+ or hypertonic sucrose trigger fusion of primed vesicles by inducing the opening of syntaxin-1 and assembly of SNARE complexes. The simplicity of this second model is attractive, but it cannot account for the speed of Ca2+-triggered fusion or for its dependence on complexin, which binds to assembled SNARE complexes. Independent of which model is correct, the results demonstrate that syntaxin-1 performs multiple functions in exocytosis that go beyond its role as a SNARE protein to include the control of vesicle docking and the regulation of the vesicle fusion rate (Gerber, 2008).

Synaptobrevins (v-SNAREs)

Synaptobrevins are vesicle-associated proteins implicated in neurotransmitter release by both biochemical studies and perturbation experiments that use botulinum toxins. To test these models in vivo, the first synaptobrevin mutants in metazoans were isolated and characterized. Neurotransmission is severely disrupted in mutant animals. Mutants lacking snb-1 die just after completing embryogenesis. The dying animals retain some capability for movement, although they are extremely uncoordinated and incapable of feeding. Several hypomorphic snb-1 mutants were isolated and characterized. Although fully viable, these mutants exhibit a variety of behavioral abnormalities that are consistent with a general defect in the efficacy of synaptic transmission. The viable mutants are resistant to an acetylcholinesterase inhibitor, indicating that cholinergic transmission is impaired. Extracellular recordings from pharyngeal muscle also demonstrate severe defects in synaptic transmission in the mutants. The molecular lesions in the hypomorphic alleles reside on the hydrophobic face of a proposed amphipathic-helical region implicated biochemically in interacting with the t-SNAREs syntaxin and SNAP-25. Double mutants lacking both the v-SNAREs synaptotagmin and snb-1 are phenotypically similar to snb-1 mutants and less severe than syntaxin mutants. This work demonstrates that synaptobrevin is essential for viability and is required for functional synaptic transmission. However, the analysis also suggests that transmitter release is not completely eliminated by removal of either one or both v-SNAREs (Nonet, 1998).

Syntaxin is a cytoplasmically oriented plasma membrane protein and VAMP (vesicle-associated membrane protein; synaptobrevin) is a protein associated with the secretory vesicle membrane. These two proteins form part of a complex that is thought to mediate the fusion of plasma and vesicle membranes during exocytosis. This paper reports the identification of syntaxin and VAMP homologs in sea urchin sperm. During fertilization, sea urchin sperm release the contents of a single vesicle, the acrosomal vesicle, exposing the membrane that is destined to fuse with the egg. During acrosomal exocytosis, the plasma membrane over the acrosomal vesicle fuses at multiple points with the acrosomal membrane (vesiculation) and syntaxin and VAMP are shed with the resulting membrane vesicles. Sea urchin sperm syntaxin and VAMP are associated in a complex that has been detected by immunoprecipitation. Following acrosomal exocytosis, syntaxin and VAMP cosediment to denser fractions on sucrose gradients; this shows that they have undergone associative changes during or after the acrosome reaction. Syntaxin and VAMP localization and loss during acrosomal exocytosis support a role for these proteins in regulating the acrosome reaction (Schulz, 1997).

Exocytosis of synaptic vesicles requires the formation of a fusion complex consisting of the synaptic vesicle protein synaptobrevin (vesicle-associated membrane protein, or VAMP) and the plasma membrane proteins syntaxin and soluble synaptosomal-associated protein of 25 kDa (or SNAP 25). In search of mechanisms that regulate the assembly of the fusion complex, it was found that synaptobrevin also binds to the vesicle protein synaptophysin and that synaptophysin-bound synaptobrevin cannot enter the fusion complex. Using a combination of immunoprecipitation, cross-linking, and in vitro interaction experiments, the synaptophysin-synaptobrevin complex has been shown to be upregulated during neuronal development. In embryonic rat brain, the complex is not detectable, although synaptophysin and synaptobrevin are expressed and are localized to the same nerve terminals and to the same pool of vesicles. In contrast, the ability of synaptobrevin to participate in the fusion complex is detectable as early as embryonic day 14. The binding of synaptoporin (a closely related homolog of synaptophysin) to synaptobrevin changes in a similar manner during development. Recombinant synaptobrevin binds to synaptophysin derived from adult brain extracts but not to that derived from embryonic brain extracts. Furthermore, the soluble cytosol fraction of adult, but not of embryonic, synaptosomes contains a protein that induces synaptophysin-synaptobrevin complex formation in embryonic vesicle fractions. It is concluded that complex formation is regulated during development and is mediated by a posttranslational modification of synaptophysin. Furthermore, it is proposed that the synaptophysin-synaptobrevin complex is not essential for exocytosis but rather provides a reserve pool of synaptobrevin for exocytosis that can be readily recruited during periods of high synaptic activity (Becher, 1999).

Neurotransmitter release involves the assembly of a heterotrimeric SNARE complex composed of the vesicle protein synaptobrevin (VAMP 2) and two plasma membrane partners, syntaxin 1 and SNAP-25. Calcium influx is thought to control this process via Ca2+-binding proteins that associate with components of the SNARE complex. Ca2+/calmodulin or phospholipids bind in a mutually exclusive fashion to a C-terminal domain of VAMP (VAMP77-90), and residues involved were identified by plasmon resonance spectroscopy. Microinjection of wild-type VAMP77-90, but not mutant peptides, inhibits catecholamine release from chromaffin cells monitored by carbon fiber amperometry. Pre-incubation of PC12 pheochromocytoma cells with the irreversible calmodulin antagonist ophiobolin A inhibits Ca2+-dependent human growth hormone release in a permeabilized cell assay. Treatment of permeabilized cells with tetanus toxin light chain (TeNT) also suppresses secretion. In the presence of TeNT, exocytosis is restored by transfection of TeNT-resistant (Q76V, F77W) VAMP, but additional targeted mutations in VAMP77-90 abolishes its ability to rescue release. The calmodulin- and phospholipid-binding domain of VAMP 2 is thus required for Ca2+-dependent exocytosis, possibly to regulate SNARE complex assembly (Quetglas, 2002).

The trafficking of two endogenous axonal membrane proteins, VAMP2 (Drosophila homolog: Synaptobrevin-62A) and NgCAM, has been examined in order to elucidate the cellular events that underlie their polarization. VAMP2 is delivered to the surface of both axons and dendrites, but preferentially endocytosed from the dendritic membrane. A mutation in the cytoplasmic domain of VAMP2 that inhibits endocytosis abolished its axonal polarization. In contrast, the targeting of NgCAM depends on sequences in its ectodomain, which mediate its sorting into carriers that preferentially deliver their cargo proteins to the axonal membrane. These observations show that neurons use two distinct mechanisms to polarize proteins to the axonal domain: selective retention in the case of VAMP2; selective delivery in the case of NgCAM (Sampo, 2003).

Nearly all neurons are polarized into two structurally and functionally distinct domains, the axon and the dendrites. Consistent with the different physiological properties of axons and dendrites and their different roles in cell signaling, many cell surface proteins are preferentially distributed either to the axonal or somatodendritic domain. In a general sense, the trafficking pathways involved in the biosynthesis of integral membrane proteins are well understood. These proteins are synthesized in the rough endoplasmic reticulum, pass through the Golgi complex, and are packaged into carrier vesicles, which are transported into the axons and dendrites where they deliver their contents to the plasma membrane by exocytic fusion. With respect to the trafficking of proteins destined for different destinations within the cell, however, many fundamental questions remain unanswered. Where along these pathways does the trafficking of axonal and dendritic proteins diverge? What underlying mechanisms lead to the selective localization of such proteins on the cell surface? With regard to the second question, there are two general mechanisms that could account for the selective localization of polarized proteins on the cell surface. Selectivity along the trafficking pathways en route to the plasma membrane could ensure that proteins destined for different domains are segregated from one another into different carriers and only delivered to the plasma membrane of the appropriate domain. Alternatively, proteins could be delivered equally to the plasma membrane of both domains but retained on the cell surface only in the appropriate domain (Sampo, 2003).

Both mechanisms, selective delivery and selective retention, contribute to the maintenance of polarity in epithelial cells. Many apical proteins and basolateral proteins are sorted into distinct transport carriers, either as they exit the Golgi complex or within an endosomal compartment in the cell periphery, and these carriers deliver their cargoes exclusively to the apical or basolateral surface. Other proteins, such as Na,K-ATPase and ß1 integrin, are delivered in equal amounts to both domains. The proteins that reach the inappropriate domain are rapidly removed by endocytosis, whereas those that reach the appropriate domain interact with submembranous cytoskeletal proteins, which stabilize them in the membrane and prevent their endocytosis (Sampo, 2003 and references therein).

Studies of neuronal protein targeting have revealed motifs that are required for their appropriate localization, but it is unclear if these motifs mediate selective sorting and delivery or selective retention. Both mechanisms could plausibly contribute to the maintenance of polarity in nerve cells, and there have been few experimental tests to distinguish between them. In the case of dendritic targeting, the available evidence favors the selective sorting and delivery model. The same motifs that govern the targeting of some dendritic proteins in neurons have been shown to mediate selective delivery to the basolateral surface in epithelial cells. Moreover, live-cell imaging studies have shown that carriers labeled by expression of GFP-tagged transferrin receptor, a dendritic protein, are transported into dendrites but excluded from axons, implying that such carriers deliver their protein contents only to the somatodendritic membrane. Similar results have been observed for several other GFP-tagged dendritic proteins, including the acidic amino acid transporter EAAT3, the EGF receptor, and the metabotropic glutamate receptor mGluR1a , indicating that selective sorting and delivery is likely to contribute to the polarization of many dendritic proteins (Sampo, 2003 and references therein).

The mechanisms that underlie the polarization of axonal proteins are less well understood, but several lines of evidence raise the possibility that selective retention rather than selective delivery may play a particularly important role. (1) In contrast to the situation for dendritic proteins, transport carriers labeled following expression of the GFP-tagged axonal protein NgCAM are transported into dendrites as well as axons. It is not known if the NgCAM-containing carriers that enter dendrites fuse with and deliver their contents to the dendritic membrane or simply are returned to the cell body and eventually reach the axon. (2) Axons and dendrites differ in the molecular composition of their submembranous cytoskeleton and in the components of their endocytic machinery, which could allow for the selective retention of proteins in the axon (Sampo, 2003 and references therein).

This study analyzes the trafficking of two endogenously expressed axonal proteins, NgCAM and VAMP2, in order to determine which mechanism, selective retention or selective delivery, accounts for their polarity. L1 and its chick homolog NgCAM are members of the Ig superfamily of neural cell adhesion molecules, which are thought to play a role in axonal pathfinding and fasciculation. When NgCAM is expressed in cultured hippocampal neurons, it is highly polarized to the axonal surface. VAMP2 is a synaptic vesicle v-SNARE that is required for calcium-dependent exocytosis at presynaptic specializations. Although VAMP2 is a component of synaptic vesicles, a significant fraction of VAMP2 is also present on the axonal surface. GFP-tagged VAMP2 is highly polarized to the axonal surface when expressed in cultured hippocampal neurons, and carriers containing GFP-tagged VAMP2 are transported into both dendrites and axons, like those containing NgCAM. Thus, existing data concerning the trafficking of both NgCAM and VAMP2 are equally compatible with the selective retention and selective delivery models. In order to determine which mechanism accounts for the polarization of these proteins, this study has assessed whether these proteins are preferentially endocytosed from the dendritic surface. Regions within these proteins have been identified that are required for their polarization to the axonal surface and whether these regions are likely to mediate selective retention or selective sorting has been examined. The findings indicate that the polarization of these two axonal proteins depends on distinct mechanisms: selective retention in the case of VAMP2, selective delivery in the case of NgCAM (Sampo, 2003).

The axonal polarization of NgCAM depends on information contained in the FnIII repeats in its extracellular domain. By analogy to results on apical targeting in epithelial cells, deletions within the ectodomain of NgCAM could well disrupt its sorting, allowing it to enter an inappropriate population of carriers, which deliver cargoes to the dendritic membrane. Mutations in the ectodomain are unlikely to interfere with endocytosis. Indeed, NgCAM mutants (lacking the FnIII domains) inappropriately reach the dendritic surface, and their endocytosis from the dendritic surface is readily detectable by antibody uptake. These observations strongly imply that wild-type NgCAM is preferentially delivered to the axonal membrane. Additional methods that allow direct visualization of the fusion of NgCAM carriers with the plasma membrane, such as total internal reflection fluorescence microscopy (TIR-FM), will be required to quantify differences in rates of exocytosis of these carriers in axons and dendrites. If NgCAM is selectively delivered to the axonal plasma membrane while VAMP2 is delivered equally to both axonal and dendritic domains, these two proteins must reach the membrane via different carriers. In addition, the preferential fusion of NgCAM- but not VAMP2-containing carriers with the axonal membrane implies that the former contain unique v-SNAREs (or other proteins that govern interaction with the fusion machinery) and that these proteins interact with axon-specific t-SNAREs or other tethering proteins. If this model is correct, it remains to be determined how VAMP2 and NgCAM become segregated into different carriers and where in the cell their trafficking diverges (Sampo, 2003).

Proteins that interact with syntaxin: SNAP25 (a v-SNARE)

During the process of docking and fusion of synaptic vesicles to the presynaptic membrane, several presynaptic proteins bind sequentially to a core complex associating two proteins of the presynaptic membrane, syntaxin and SNAP 25 (both v-SNAREs), and a protein of synaptic vesicles, VAMP/synaptobrevin (a t-SNARE). This core complex was immunoprecipitated after solubilization of pure cholinergic synaptosomes of Torpedo electric organ, using anti-syntaxin or anti-VAMP immunobeads. Syntaxin and VAMP, which are transported by the rapid axonal flow to the nerve endings accumulate at the proximal end of an electric nerve ligature and are already engaged in complexes, as in synaptosomes. In unligated nerves as well, significant amounts of VAMP associate with syntaxin. Hence, syntaxin is already associated with SNAP 25 and VAMP during axonal transport, before reaching nerve endings (Shiff, 1997).

The highly conserved proteins syntaxin and SNAP-25 are part of a protein complex that is thought to play a key role in exocytosis of synaptic vesicles. Previous work has demonstrated that syntaxin and SNAP-25 bind to one another with high affinity and that their binding regions are predicted to form coiled coils. Circular dichroism spectroscopy was used to study the alpha-helicity of the individual proteins and to gain insight into structural changes associated with complex formation. Syntaxin displays approximately 43% alpha-helical content. In contrast, the alpha-helical content of SNAP-25 is low under physiological conditions. Formation of the SNAP-25-syntaxin complex is associated with a dramatic increase in alpha-helicity. Interaction of a 90-residue NH2-terminal fragment of SNAP-25 comprising the minimal syntaxin binding domain leads to a similar but less pronounced increase in alpha-helicity. Single amino acid replacements in the putative hydrophobic core of this fragment with hydrophilic amino acids abolishes the induced structural change and disrupts the interaction monitored by binding assays. Replacements with hydrophobic residues has no effect. These findings are consistent with induced coiled coil formation upon binding of syntaxin and SNAP-25 (Fasshauer, 1997).

Neurotransmitter release requires the specific docking of synaptic vesicles to the presynaptic plasma membrane followed by a calcium-triggered fusion event. This study describes the previously unsuspected interaction of the synaptic vesicle protein and likely calcium sensor synaptotagmin with the plasma membrane t-SNARE SNAP-25. This interaction appears to resolve the apparent paradox that synaptic vesicles are capable of docking even when VAMP (vesicle-associated membrane protein) or syntaxin is cleaved or deleted and suggests that two species of v-SNAREs (VAMP and synaptotagmin) and two species of t-SNAREs (SNAP-25 and syntaxin) interact to functionally dock synaptic vesicles (Schiavo, 1997).

The membrane protein syntaxin participates in several protein-protein interactions that have been implicated in neurotransmitter release. To probe the physiological importance of these interactions, Botulinum toxin C1 (which cleaves syntaxin) and the H3 domain of syntaxin (which mediates binding to other proteins) were microinjected into the squid giant presynaptic terminal. Both reagents inhibit synaptic transmission yet did not affect the number or distribution of synaptic vesicles at the presynaptic active zone. The recombinant H3 domain inhibits the interactions between syntaxin and SNAP-25 that underlie the formation of stable SNARE complexes in vitro. These data support the notion that syntaxin-mediated SNARE complexes are necessary for docked synaptic vesicles to fuse (O'Connor, 1997).

Synaptic vesicle docking and fusion are mediated by the assembly of a stable SNARE core complex of proteins, which includes the synaptic vesicle membrane protein VAMP/synaptobrevin and the plasmalemmal proteins syntaxin and SNAP-25. Another SNAP-25-binding protein, called Snapin, has been identified. Snapin is enriched in neurons and exclusively located on synaptic vesicle membranes. It is associated with the SNARE complex through direct interaction with SNAP-25. Binding of recombinant Snapin-CT to SNAP-25 blocks the association of the SNARE complex with synaptotagmin. Introduction of Snapin-CT and peptides containing the SNAP-25 binding sequence into presynaptic superior cervical ganglion neurons in culture reversibly inhibits synaptic transmission. These results suggest that Snapin is an important component of the neurotransmitter release process through its modulation of the sequential interactions between the SNAREs and synaptotagmin (Ilardi, 1999).

SNARE proteins are known to play a role in regulating intracellular protein transport between donor and target membranes. This docking and fusion process involves the interaction of specific vesicle-SNAREs (e.g. VAMP) with specific cognate target-SNAREs (e.g. syntaxin and SNAP-23). Using human SNAP-23 as the bait in a yeast two-hybrid screen of a human B-lymphocyte cDNA library, the 287-amino-acid SNARE protein syntaxin 11 was identified. Like other syntaxin family members, syntaxin 11 binds to the SNARE proteins VAMP and SNAP-23 in vitro and also exists in a complex with SNAP-23 in transfected HeLa cells and in native human B lymphocytes. Unlike other syntaxin family members, no obvious transmembrane domain is present in syntaxin 11. Nevertheless, syntaxin 11 is predominantly membrane-associated and colocalizes with the mannose 6-phosphate receptor on late endosomes and the trans-Golgi network. These data suggest that syntaxin 11 is a SNARE that acts to regulate protein transport between late endosomes and the trans-Golgi network in mammalian cells (Valdez, 1999).

cAMP-dependent protein kinase A (PKA) can modulate synaptic transmission by acting directly on unknown targets in the neurotransmitter secretory machinery. Snapin, a protein of relative molecular mass 15,000 has been identified that is implicated in neurotransmission by binding to SNAP-25, as a possible target. Deletion mutation and site-directed mutagenetic experiments pinpoint the phosphorylation site to serine 50. PKA-phosphorylation of Snapin significantly increases its binding to synaptosomal-associated protein-25 (SNAP-25). Mutation of Snapin serine 50 to aspartic acid (S50D) mimics this effect of PKA phosphorylation and enhances the association of synaptotagmin with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Furthermore, treatment of rat hippocampal slices with nonhydrolyzable cAMP analog induces in vivo phosphorylation of Snapin and enhances the interaction of both Snapin and synaptotagmin with the SNARE complex. In adrenal chromaffin cells, overexpression of the Snapin S50D mutant leads to an increase in the number of release-competent vesicles. These results indicate that Snapin may be a PKA target for modulating transmitter release through the cAMP-dependent signal-transduction pathway (Chheda, 2001).

The results presented here indicate that Snapin can serve as a target for PKA at the synapse to modulate transmitter release and neuronal plasticity through a second-messenger pathway. Phosphorylation of Snapin at serine 50, both in vitro and in vivo, leads to an increase in binding of Snapin to SNAP-25. Furthermore, this phosphorylation apparently strengthens the association of synaptotagmin, a proposed Ca2+ sensor for exocytosis, with the assembled SNARE complex. In physiological experiments, overexpression of constitutively phosphorylated Snapin (S50D Snapin) leads to an increase in the number of release-ready vesicles in adrenal chromaffin cells, whereas overexpression of the unphosphorylated form (S50A Snapin) reduces this number. In the context of the current model of exocytosis in chromaffin cells, these findings lead to the following conclusions: (1) both the S50D and S50A mutations increase the magnitude of the sustained component of exocytosis, indicating that Snapin acts as a positive modulator of the priming step by increasing the forward rate constant of priming; (2) S50D Snapin increases the size of the exocytotic burst, wheresa S50A Snapin reduces it. Thus, phosphorylation of Snapin leads to stabilization of release-ready vesicles, most probably by reducing the backward rate constant for the priming-unpriming reaction (Chheda, 2001).

Synaptotagmin is a proposed Ca2+ sensor on the vesicle for regulated exocytosis and exhibits Ca2+-dependent binding to phospholipids, syntaxin, and SNAP-25 in vitro, but any understanding of the mechanism by which Ca2+ triggers membrane fusion is uncertain. SNAP-25 plays a role in the Ca2+ regulation of secretion. Synaptotagmins I and IX associate with SNAP-25 during Ca2+-dependent exocytosis in PC12 cells, and C-terminal amino acids in SNAP-25 (Asp179, Asp186, Asp193) have been identified that are required for Ca2+-dependent synaptotagmin binding. Replacement of SNAP-25 in PC12 cells with SNAP-25 containing C-terminal Asp mutations leads to a loss-of-function in regulated exocytosis at the Ca2+-dependent fusion step. These results indicate that the Ca2+-dependent interaction of synaptotagmin with SNAP-25 is essential for the Ca2+-dependent triggering of membrane fusion (Zhang, 2002).

Proteins that interact with syntaxin: NSF

N-ethylmaleimide-sensitive fusion protein (NSF) and alpha-SNAP play key roles in vesicular traffic through the secretory pathway. NSF is able to associate with Golgi membranes in an ATP-dependent fashion. This association is dependent on three peripheral membrane proteins, termed soluble NSF attachment proteins (SNAPs). alpha-SNAP and NSF are associated in a 20S complex with three membrane proteins: syntaxin, SNAP-25 (synaptosomal associated protein of 25 kD) and vesicle-associate membrane protein (VAMP), collectively termed SNAP receptors (SNAREs). In this study, NH2- and COOH-terminal truncation mutants of alpha-SNAP were assayed for the ability to bind NSF and stimulate its ATPase activity. Deletion of up to 160 NH2-terminal amino acids has little effect on the ability of alpha-SNAP to stimulate the ATPase activity of NSF. However, deletion of as few as 10 COOH-terminal amino acids results in a marked decrease. Both NH2-terminal (1-160) and COOH-terminal (160-295) fragments of alpha-SNAP are able to bind to NSF, suggesting that alpha-SNAP contains distinct NH2- and COOH-terminal binding sites for NSF. Sequence alignment of known SNAPs reveals only leucine 294 to be conserved in the final 10 amino acids of alpha-SNAP. Mutation of leucine 294 to alanine [alpha-SNAP(L294A)] results in a decrease in the ability to stimulate NSF ATPase activity but has no effect on the ability of this mutant to bind NSF. alpha-SNAP (1-285) and alpha-SNAP (L294A) are unable to stimulate Ca2+-dependent exocytosis in permeabilized chromaffin cells. In addition, alpha-SNAP (1-285) and alpha-SNAP (L294A) are able to inhibit the stimulation of exocytosis by exogenous alpha-SNAP. alpha-SNAP, alpha-SNAP (1-285), and alpha-SNAP (L294A) are all able to become incorporated into a 20S complex and recruit NSF. In the presence of MgATP, alpha-SNAP (1-285) and alpha-SNAP (L294A) are unable to fully disassemble the 20S complex and do not allow vesicle-associated membrane protein dissociation to any greater level than seen in control incubations. These findings imply that alpha-SNAP stimulation of NSF ATPase activity may be required for 20S complex disassembly and for the alpha-SNAP stimulation of exocytosis (Barnard, 1997).

The membrane proteins synaptobrevin, syntaxin, and SNAP-25 form the core of a ubiquitous fusion machine that interacts with the soluble proteins NSF and alpha-SNAP. During regulated exocytosis, membrane fusion is usually strictly controlled by Ca2+ ions. However, the mechanism by which Ca2+ regulates exocytosis is still unclear. The membranes of exocrine secretory granules contain an 18-kDa protein, syncollin, that binds to syntaxin at low Ca2+ concentrations and dissociates at concentrations known to stimulate exocytosis. Syncollin binds to the cytoplasmic domain of syntaxin and also to the C-terminal region (albeit less well), but fails to bind the N-terminal region. alpha-SNAP displaces syncollin from immobilized syntaxin in a concentration dependent manner. Syncollin has a single hydrophobic domain at its N-terminus and shows no significant homology with any known protein. Recombinant syncollin inhibits fusion in vitro between zymogen granules and pancreatic plasma membranes; its potency falls as Ca2+ concentration rises. It has been suggested that syncollin acts as a Ca2(+)-sensitive regulator of exocytosis in exocrine tissues (Edwardson, 1997).

Specific interaction has been demonstrated between the GluR2 (AMPA) receptor subunit C-terminal peptide (see Drosophila Glutamate receptor IIA and Glutamate receptor IIB), an ATPase N-ethylmaleimide-sensitive fusion protein (NSF), and alpha- and beta-soluble NSF attachment proteins (SNAPs), as well as the dendritic colocalization of these proteins. The assembly of the GluR2-NSF-SNAP complex is ATP hydrolysis reversible and resembles the binding of NSF and SNAP with the SNAP receptor (SNARE) membrane fusion apparatus. This paper provides evidence that the molar ratio of NSF to SNAP in the GluR2-NSF-SNAP complex is similar to that of the t-SNARE syntaxin-NSF-SNAP complex. NSF is known to disassemble the SNARE protein complex in a chaperone-like interaction driven by ATP hydrolysis. A model is proposed in which NSF functions as a chaperone in the molecular processing of the AMPA receptor (Osten, 1998).

The findings that NSF and alpha- and beta-SNAPs interact with GluR2 in a complex, which in several respects resembles the interaction of NSF and SNAP at the SNARE, can be interpreted to support a functional model of the GluR2-NSF-SNAP binding. In this model, the NSF-SNAP complex is required in chaperone-like priming of the AMPA receptors during a continuous process required for receptor function. This process could involve receptor recycling between the postsynaptic membrane and a cytoplasmic pool. As has been proposed for NSF function at the SNARE complexes, the interaction of NSF and SNAP with the AMPA receptor could involve the disruption of multiprotein complexes, such as those formed between the membrane-inserted receptor and the proteins of the postsynaptic density (such as GRIP). NSF-driven disassembly of these complexes could be required for the proper sorting of these proteins at specific times during development, as for example, prior to a new cycle of insertion and anchoring, or in the processing of newly synthesized receptors (Osten, 1998 and references).

Disruption of N-ethylmaleimide-sensitive fusion protein- (NSF-) GluR2 interaction by infusion into cultured hippocampal neurons of a blocking peptide (pep2m) causes a rapid decrease in the frequency but no change in the amplitude of AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs). NMDA receptor-mediated mEPSCs were not changed. Viral expression of pep2m reduces the surface expression of AMPA receptors, whereas NMDA receptor surface expression in the same living cells is unchanged. In permeabilized neurons, the total amount of GluR2 immunoreactivity is unchanged, and a punctate distribution of GluR2 is observed throughout the dendritic tree. These data suggest that the NSF-GluR2 interaction is required for the surface expression of GluR2-containing AMPA receptors and that disruption of the interaction leads to the functional elimination of AMPA receptors at synapses. Based on these findings and the known properties of NSF, a model is favored in which the interaction between NSF and GluR2 is involved in the part of the cycling process that is necessary for the insertion and/or stabilization of AMPA receptors at the postsynaptic membrane. By analogy with its known presynaptic functions, NSF could act at the AMPA receptor complex by stripping the receptors of associated proteins. Candidate proteins interacting with GluR2 include the PDZ-containing proteins GRIP, ABP, and PICK1. Removal of associated proteins could prime or "reset" the AMPA receptor complex to a naive state, thereby allowing insertion into the postsynaptic membrane. If the action of NSF is prevented, for example, by peptide block, the receptors cannot be appropriately processed, and insertion/reinsertion of the reconfigured receptors into the postsynaptic membrane cannot occur (Noel, 1999).

The precise biochemical role of N-ethylmaleimide-sensitive factor (NSF) in membrane fusion mediated by SNARE proteins is unclear. To provide further insight into the function of NSF, a mutation was introduced into mammalian NSF that, in Drosophila NSF-1, leads to temperature-sensitive neuroparalysis. This mutation is like the comatose mutation and renders the mammalian NSF temperature sensitive for fusion of postmitotic Golgi vesicles and tubules into intact cisternae. Unexpectedly, at the temperature that is permissive for membrane fusion, this mutant NSF binds to, but cannot disassemble, SNARE complexes and exhibits almost no ATPase activity. A well-charaterized NSF mutant containing an inactivating point mutation in the catalytic site of its ATPase domain is equally active in the Golgi-reassembly assay. These data indicate that the need for NSF during postmitotic Golgi membrane fusion may be distinct from its ATPase-dependent ability to break up SNARE pairs (Muller, 1999).

Results obtained with the comatose-like mutant were substantiated by using a distinct mutant, NSF(E329Q), that is defective in ATPase activity and SNARE disassembly. This mutation results in a ~75% reduction in ATPase activity, which completely abolishes its ability to stimulate fusion in an intra-Golgi transport assay. These results indicate a positive correlation between the membrane-fusion-promoting function of NSF and its ATPase activity. In contrast, the ATPase-defective NSF (E329Q), like NSF(G274E), is capable of promoting cisternal regrowth to ~80% of the level of wild-type NSF. Together, data obtained using the mutant NSF proteins indicate that NSF's ATPase activity may not be directly linked to postmitotic Golgi membrane fusion (Muller, 1999).

As NSF(G274E) and NSF(E329Q) both lack the ability to break up SNARE complexes, NSF-dependent SNARE disassembly seems to be uncoupled from membrane fusion of postmitotic Golgi fragments. The break-up of SNARE complexes is thought to be essential for the recycling of these proteins for further rounds of fusion; thus, these data indicate that recycling may not be needed for Golgi reassembly in the cell-free assay. On the basis of current models, it is therefore predicted that there is an abundant source of disassembled SNAREs on mitotic Golgi fragments before reassembly. If true, this could help to explain the discrepancy in the requirement for NSF's ATPase activity in other published membrane-fusion assays but not during mitotic Golgi reassembly (Muller, 1999).

However, this leaves open the nature of the distinct role for NSF during the membrane-fusion process. Interestingly, assembly of synaptic 20S complexes is temperature sensitive in the presence of the NSF mutant, which indicates that the presence of NSF in a SNARE complex might be critical. One possibility is that NSF is needed to prime (for example, by folding or assisting in accessory-factor recruitment) the SNAREs on MGFs in preparation for fusion. Although priming of vacuole and, perhaps, Golgi SNAREs correlates with the presence of Mg-ATP, it is not clear that ATP hydrolysis is needed for this process. Another possibility is that a checkpoint exists to ensure that NSF is recruited to the fusion site in preparation for its later function in breaking up SNARE complexes. This would certainly explain why NSF has been found on synaptic and clathrin-coated vesicles that still have to dock and fuse. Such recruitment would target NSF to the site at which its action will be needed. A final possibility is that NSF takes part in the actual fusion process itself. It is, therefore, interesting that NSF-SNAPs can directly fuse liposomes together in an ATP-dependent manner. Furthermore, this happens most efficiently when the NSF-SNAP complex has the lowest ATPase activity, the key feature of the NSF mutant (Muller, 1999).

In conclusion, these studies of the Drosophila comatose analog in mammalian NSF provide clear evidence that NSF has a role in membrane fusion that is divorced from its ability to break up SNARE complexes. The likelihood is that NSF has multiple roles and further structure/function studies should provide the means for their dissection (Muller, 1999).

Proteins that interact with syntaxin: Synaptotagmin

Continued: see Syntaxin 1A Evolutionary Homologs part 2/3 | part 3/3

Syntaxin 1A: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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