Gene name - Syntaxin 1A
Cytological map position - 95E1--95E2
Function - Docking and fusion protein
Keywords - vesicular docking and fusion protein, neural
Symbol - Syx1A
FlyBase ID: FBgn0013343
Genetic map position - 3-
Classification - Syntaxin
Cellular location - cell membrane
Membrane fusion is a Ca++ dependent process in which neurotransmitter-filled synaptic vesicles undergo docking and fusion with the presynaptic membrane to release neurotransmitter into the synaptic cleft. This secretion process involves the docking protein syntaxin and other synaptic protein components. Membrane fusion results from the interaction of two components of the presynaptic membrane, syntaxin and Snap 25, with two components of the vesicular membrane, Synaptotagmin and synaptobrevin/Vamp. Mediating this interaction are yet other proteins, among them a fusion protein called the N-ethylmaleimide-sensitive fusion protein (NSF) and the soluble NSF attachment proteins (SNAPs), all essential components of the intracellular membrane fusion apparatus. The four membrane components (syntaxin, Snap 25, synaptotagmin and synaptobrevin/Vamp) are termed SNAP receptors (SNAREs). The two vesicular SNAREs are known as v-SNAREs and the two target SNAREs are referred to as t-SNAREs (Söllner, 1993).
Many components of the membrane fusion apparatus have been identified in Drosophila. The t-SNARE Syntaxin 1A has been cloned as well as the v-SNARE Synaptobrevin. The known cytoplasmic components include Rop, N-ethymaleimide-sensitive fusion protein (dNSF) and the soluble NSF attachment protein (dalphaSNAP). Two proteins thus far identified are unique to the synapse: Synaptotagmin, a protein that senses Ca++ levels, and a Cysteine string protein.
All these identified components are coded for by single genes in Drosophila, with the exception of synaptobrevin, which (as in vertebrates) is present in both a neural-specifIc form and a ubiquitous form. Rop is the fly homolog of yeast Sec1p, a syntaxin interacting protein essential for late stages of secretion. dNSF is the product of comatose, a gene originally isolated on the basis of mutations that cause temperature-sensitive paralysis in the adult (Bate, 1995 and references).
Analysis of the subcellular distribution of Drosophila SYX-1A indicates both the presence of protein in presynaptic membranes and a synaptic vesicle fraction. Syntaxin 1A was originally believed to be strictly associated with the presynaptic membrane. In Drosophila, Syntaxin 1A is also associated with synaptic vesicles or possibly other small vesicles such as endosomes (Schulze, 1995).
Genetic studies with Drosophila challenge the SNARE hypothesis, at least the view that syntaxin functions solely or exclusively as a docking protein. The SNARE hypothesis proposes, in its simplest form, that vesicles dock at presynaptic active zones by the association of synaptobrevin (v-SNARE) with syntaxin (t-SNARE). Ultrastructural studies show the vesicles are targeted to the presynaptic membrane and dock normally at specialized release sites even in the absence of neural synaptobrevin or syntaxin. These vesicles are mature and functional since spontaneous vesicle fusion persists in the absence of neural synaptobrevin or syntaxin (Broadie, 1995). Studies in mammals, also indicate that the simplified SNARE hypothesis is not completely valid. The synaptic vesicle protein and likely calcium sensor synaptotagmin interacts 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).
In addition to docking, SNARE proteins are implicated as the minimal components responsible for membrane fusion. A role for fusion, as opposed to docking, would resolve much of the ambiguity concerning syntaxin function in Drosophila. Recombinant v- and t-SNARE proteins reconstituted into separate lipid bilayer vesicles assemble into SNAREpins: SNARE complexes linking two membranes. This leads to spontaneous fusion of the docked membranes at physiological temperatures. Docked and unfused intermediates can accumulate at lower temperatures and can fuse when brought to physiological temperatures. A supply of unassembled v- and t-SNAREs is needed for these intermediates to form, but not for the fusion that follows. These data imply that SNAREpins are the minimal machinery for cellular membrane fusion and do not serve exclusively as docking proteins (Weber, 1998).
A pair of cognate proteins, capable of pairing membranes and fusing their bilayers appears to be the simplest possible means, and one utilized by cells, to link a mechanism for specifically pairing membranes to a mechanism for fusing their bilayer. Such linking not only embodies the crux of how subcellular compartments are maintained and propagated, but also suggests a natural way that the many and diverse compartments in eukaryotic cells could have evolved in the first place. It is easy to imagine how a primitive pair of v- and t-SNARE genes could give rise to a family of such genes by duplication and mutational variation, each v-SNARE evolving in tandem with its cognate t-SNARE because they are structurally and functionally linked. Cognate v- and t-SNAREs bind each other via membrane-proximal heptad repeat regions that are predicted to form coiled-coils or closely related helical bundles. In the electron microscope, SNARE complexes are seen to be rods, 13-14 nm long and approximately 2 nm wide, as is appropriate for such a coiled-coil structure. The membrane anchors of v- and t-SNAREs emerge at the same end of the rod, implying that the rod must lie approximately in the plane of contact between the vesicles paired by a complex of a v-SNARE in one vesicle with a t-SNARE in the other vesicle, a structure termed a "SNAREpin" (Weber, 1998).
These finding cast new light on the roles of SNAPS and ATPase NSFs in membrane fusion. Following fusion, dissociation of the now thermodynamically stable SNARE complexes (which will enable later rounds of fusion), requires investment of energy and therefore additional core machinery. The need for SNAPs and the ATPase NSF as core fusion machinery can most readily be understood from this perspective, as the minimal "add-on" needed for repeated use of a bilayer-fusing helical hairpin. By separating v-SNAREs from t-SNAREs at the expense of energy from ATP, SNAPs and NSF can make them available for subsequent interbilayer-partnering. Indeed, recent evidence implies that in some cases this may be the only role for NSF and SNAPs (Weber, 1998 and references).
SYX1A may mediate membrane assembly events throughout Drosophila development. Mutations in syx1A have been isolated that reveal roles for syntaxin outside the nervous system. In the ovary, SYX1A is present in the germarium, but it is predominantly localized to nurse cell membranes. Mitotic recombination experiments in the germline show SYX1A is essential for oogenesis and may participate in membrane biogenesis in the nurse cells. Mutant ovaries are rudimentary in their appearance and indistinguishable from that of ovoD1 heterozygotes. syx1a is also required in larval imaginal discs, as certain hypomorphic mutant combinations exhibit rough eyes defects indicative of cell death. The most severly affected eyes appear to have fewer ommatidia than wild type. The ommatidia are sometimes fused and often are improperly rotated and misaligned with respect to one another. Recombinant clones that lack syx1A cause cell lethality in the developing eye. Another defect in temperature sensitive alleles is notching along the posterior margin of the wing. In mutants, incomplete compaction of the ventral nerve cord is observed. In some mutants incomplete fasciculation of the intersegmental and segmental nerves is found. The staining of the intersegmental and segmental nerve bundles in mutants is quite diffuse, suggesting that the individual axons cannot adhere to one another, perhaps due to inconsistencies in the membrane constituents. Similar defects are observed in the longitudinal tracts of the CNS when visualized with anti-fasciclin II antibody. In the wild-type CNS, three longitudinal tracts are observed on either side of the midline, and each set of axon fibers exhibits a tight arrangement. In mutants, axon bundles are irregular and slightly defasciculated (Schulze, 1996).
What might be Syntaxin's role in membrane integrity or development? Consideration of membrane formation during cellularization may provide clues to an answer. Precellular embryos exhibit ubiquitous distribution of SYX1A mRNA reflecting a maternal contribution. In young embryos during mitotic cycle 11, the plasma membrane partially envelops each dividing nucleus, forming "buds". SYX1A protein accumulates beneath these buds as they undergo cytokinesis. "Somatic buds" form around dividing somatic nuclei but undergo several rounds of formation and collapse as the nuclei beneath the buds divide. At metaphase for each mitotic division, the membrane of the bud invaginates markedly and "pseudocleavage furrows" form between the buds. SYX expression appears to follow the outline of the buds as they partially enwrap the dividing nuclei during mitotic cycles 10-14. Finally during cycle 14, the somatic nuclei become completely surrounded by the elongating membranes of the cytoplasmic buds, and cellularization is completed. SYX1A localization outlines these membranes as they form. SYX1A continues to be expressed in the membranes of each cell of the fully cellularized embryo (Schulze, 1996).
It is estimated that more than 23 times the amount of membrane overlying the syncytium of nuclei at mitotic cycle 14 is needed to completely cellularize the 6000 blastoderm cells of the Drosophila embryo. Although the mechanism by which membrane addition occurs during cellularization is unknown, the process has been divided into two phases: a slow phase involving saltatory transport along astral microtubules and a fast phase in which apical microvilli disappear. The additions of constituents to cell membranes during the fast phase is believed to involve a flattening of the microvilli and hence may not require the synthesis and addition of new membrane. The membrane constituents have simply been stored in the microvilli, and they add to the membrane as they disappear. However, during the earlier slow phase, particles have been observed to move along the microtubules with anterograde (toward the yolk) and retrograde (toward the nucleus at the apex) speeds resembling those observed in axoplasmic flow. Hence, similar to the way that vesicles are transported to the presynaptic terminals of neurons, phospholipid vesicles may be transported further away from the periphery of the cellularizing embryo to sites of membrane addition. SYX1A may play a role in the fusion of the vesicles at these sites, as it is enriched within the cytoplasmic buds that envelop the dividing blastoderm nuclei. This localization corresponds to that of the cytoskeletal elements and products of such genes as nullo, alpha Spectrin and serendipity-alpha, genes believed to form the structural foundation for somatic membrane invagination. The approximate localization of Syx to the apical and lateral walls at cycle 14 places it where it may perform this putative function in the fusion of membranous vesicles to achieve cellularization. It is concluded that syntaxin is involved not just in release of vesicular contents during synaptic function, but is also involved in building cell membranes early in embryogenesis, and perhaps throughout embryonic and larval development (Schultze, 1996 and references).
Biochemical studies suggest that Syntaxin 1A participates in multiple protein-protein interactions in the synaptic terminal, but the in vivo significance of these interactions is poorly understood. A targeted mutagenesis approach was used to eliminate specific syntaxin binding interactions. Drosophila Syntaxin 1A has been shown to play multiple regulatory roles in neurotransmission in vivo. Syntaxin mutations that eliminate ROP/Munc-18 binding display increased neurotransmitter release, suggesting that ROP inhibits neurosecretion through its interaction with syntaxin. Syntaxin mutations that block Ca2+ channel binding also cause an increase in neurotransmitter release, suggesting that syntaxin normally functions by inhibiting Ca2+ channel opening. Additionally identified and characterized were a syntaxin Ca2+ effector domain, which may spatially organize the Ca2+ channel, a cysteine string protein, and synaptotagmin, all necessary for effective excitation-secretion coupling in the presynaptic terminal (Wu, 1999).
To dissect the putative functions of Syntaxin in vivo, partial loss-of-function mutations in syntaxin 1A (syx) were generated. Since this gene is refractory to standard mutagenesis with EMS (ethylmethylsulfonate), a site-directed mutagenesis approach was taken to interfere with specific protein-protein interactions. Syntaxin 1A has four coiled-coil domains (H1/HA, H2/HB, HC, and H3). All binding partners, with one exception (Munc-13), bind the C-terminal third of Syntaxin, including the H3 domain. Focus was thus placed on a structure-function analysis of the H3 domain (Wu, 1999).
Four mutations in the syx H3 domain were generated: two deletions (syxH3-N and syxH3-C) and two point mutations (syx2 and syx3). The syxH3-N deletion removes the majority of the N-terminal region of the H3 domain (amino acids 204-250), whereas the syxH3-C deletion removes the highly basic C-terminal 14 amino acids of the H3 domain (amino acids 253-267). For the syx2 and syx3 constructs, point mutations were made in the hydrophobic layers, which appear to be important for specific protein-protein interactions and for core complex stability. Specifically, the syx2 point mutation falls in a region thought to be required for SNAP-25 binding, and the amino acid targeted by the syx3 mutation has been shown to be important for binding to n-Sec1/Munc-18 (Wu, 1999).
To define the binding defects caused by the syx mutations, GST pulldown assays were performed. In this assay, GST alone, wild-type GST-Syx, or mutant GST-Syntaxins were immobilized on glutathione-Sepharose beads and incubated with target proteins. The binary Syb-Syx interaction is easily disrupted, because every syx mutation reveals a strong reduction if not complete absence of Syb binding. This finding is consistent with studies using vertebrate proteins and does not appear physiologically relevant, since certain mutants display robust neurotransmission. Core complex formation was tested by assaying Syb binding in the presence of SNAP-25, one of the three core complex components. The syxH3-N deletion abolishes formation of the core complex, while GST-SyxH3-C can form a core complex, although less efficiently than wild-type GST-Syx. The syx2 and syx3 point mutants are both capable of forming core complexes (Wu, 1999).
Next to be examined were the abilities of the GST-Syx mutant proteins to bind ROP, Synaptotagmin (Syt), and the synprint site of the N-type Ca2+ channel. GST-SyxH3-N shows an abolishment of ROP binding, but binds Synaptotagmin and synprint. Conversely, the GST-SyxH3-C deletion can bind ROP, but shows a severe reduction in Synaptotagmin and synprint binding, relative to wild-type GST-Syx. Therefore, these findings suggest that the N-terminal region of the H3 domain is essential for core complex formation and ROP binding, while the C-terminal 14 amino acids of the H3 domain are required for efficient binding to Synaptotagmin and synprint (Wu, 1999).
Binding analysis of the point mutations show that the syx2 and syx3 mutations selectively disrupt binding to different Syntaxin partners. The syx3 mutation specifically abolishes ROP binding to Syntaxin. Surprisingly, the most striking binding defect caused by the syx2 point mutation is an abolishment of binding to synprint. This effect is specific for syx2, as GST-Syx3 and GST-SyxH3-N are capable of binding synprint. Curiously, syx2 lies within a domain not required for synprint binding, as demonstrated by GST-SyxH3-N, suggesting that the point mutation induces a conformational change in the C-terminal region of the H3 domain. Although unexpected, structural studies have shown that amino acid substitutions can cause long-range effects on the binding properties of a protein. In contrast, the point mutations do not significantly affect Synaptotagmin binding, when compared to wild-type GST-Syx. These data confirm that, similar to vertebrate syntaxin 1A, the H3 domain of Drosophila Syntaxin is important for core complex formation and binding to ROP, Synaptotagmin, and synprint. Further, in vitro findings show that specific Syntaxin protein interactions can be selectively blocked by point mutations in the H3 domain (Wu, 1999).
To address the in vivo effects of the syxH3-N, syxH3-C, syx2, and syx3 mutations, these mutations were introduced into flies. Since a 13.5 kb genomic fragment containing syx (syxwt) can rescue a null allele (syx229) to adult viability, each mutation was introduced in the 13.5 kb fragment, sequenced, and used to generate transgenic flies. The wild-type genomic rescue construct was used as control (syxwt). Transgenic flies bearing the mutant and wild-type constructs were then crossed into syxP1697 (hypomorph) and syx229 (null) backgrounds. To control for position effects, several independent transgenic lines were established for each mutant construct. Single embryo Westerns showed that SyxH3-N, Syx2, and Syx3 mutant proteins are produced at levels similar to Syxwt controls. However, SyxH3-C mutant protein is expressed relatively poorly, compared to other mutants. Different transgenic lines for the same mutation expressed similar amounts of mutant Syntaxin protein, indicating that position effects do not significantly affect protein levels. Immunocytochemical stainings indicate that the mutant Syntaxins are expressed in a spatial and temporal pattern indistinguishable from wild-type (Wu, 1999).
Mutants and wild-type controls were first assessed for their ability to rescue the lethality of syxP1697 and syx229. The deletion mutations are the most severe, since syxH3-N and syxH3-C mutants are embryonic lethal in both the null and partial loss-of-function background. In contrast, the point mutations cause milder phenotypes, allowing animals to live to adulthood in the hypomorphic background, but not in the null background (Wu, 1999).
Wild-type Drosophila embryos undergo peristaltic muscular contractions prior to hatching. syxH3-N and syxH3-C mutants do not undergo spontaneous muscle contractions, suggesting that neurotransmission is severely affected. However, in response to tactile stimulation, syxH3-C mutants are capable of weak, local contractions, whereas syxH3-N mutants fail to respond. In contrast, syx2 and syx3 mutant animals are capable of spontaneous peristaltic contractions and robust touch response. These data suggest that synaptic transmission is more severely affected in syxH3-N and syxH3-C mutants than in syx2 or syx3 mutants (Wu, 1999).
In wild-type Drosophila embryos, epidermal cells secrete a cuticle apically. The presence of cuticle can easily be visualized by the presence of cuticular denticle belts, which is a convenient assay for nonneuronal secretion. syx null mutants (syx229) fail to secrete cuticle and completely lack denticles, indicating an essential role for Syntaxin in nonneuronal secretion. The ability of these different syx mutants to carry out nonneuronal secretion was examined. All mutants except syxH3-N are capable of secreting cuticle normally, suggesting that the inability to form core complexes blocks nonneuronal secretion. Moreover, perturbations of specific syntaxin protein interactions, most importantly the Syntaxin-ROP interaction, does not appear to affect nonneuronal secretion (Wu, 1999).
Regulated exocytosis at the synaptic terminal was assayed in syntaxin mutants. The syxH3-N deletion abolishes core complex formation. Synaptic transmission was examined in syxH3-N mutants using whole-cell patch-clamp analysis at the embryonic neuromuscular junction (NMJ). Three different wild-type transgenic controls (syxwt-1, syxwt-2, syxwt-3) showed robust evoked neurotransmission. However, evoked and spontaneous neurotransmission is completely abolished in syxH3-N animals, showing that the syxH3-N mutant phenotype is identical to that of syntaxin null mutants. As shown for the syx null mutant, direct glutamate pressure ejection at the synapse of syxH3-N mutants reveals that the postsynaptic muscles respond normally to neurotransmitter, indicating that the block in neurotransmission is presynaptic in nature. The complete absence of neurotransmitter release in syxH3-N mutants, taken together with the absence of nonneuronal secretion in these mutants, suggests that the core complex is essential for both neuronal and nonneuronal vesicular fusion (Wu, 1999).
To determine the function of the Ca2+ effector domain in neurotransmission, the electrophysiological phenotype of syxH3-C deletion mutants was analyzed. In the majority of mutant animals, evoked transmission is observed, but EJC amplitude is strongly reduced, compared to syxwt controls. In the remaining mutants, evoked secretion is abolished. In contrast to the complete absence of neurotransmission in syxH3-N deletion mutants, syxH3-C deletion mutants reveal strongly reduced evoked responses but an increase in the number of spontaneous quantal events. Both features have been previously associated with synaptotagmin mutants (Wu, 1999).
In addition to a severe reduction of EJC amplitude in syxH3-C mutants, the mutants also exhibit decreased reliability of excitation-secretion coupling. Specifically, evoked neurotransmission in syxH3-C mutants is characterized by asynchronous release, low fidelity of release, and a high failure rate. Normally, synchronized fusion of synaptic vesicles is tightly coupled to the Ca2+ influx resulting from a single nerve stimulation, and the amount of neurotransmitter released in response to a single stimulation is relatively consistent at the NMJ. However, a single stimulation at syxH3-C synapses can elicit multiple, nonsynchronized EJCs at variable latencies. Furthermore, the variability of release in syxH3-C mutants is significantly increased compared to syxwt controls. Finally, whereas the NMJs of syxwt animals always release neurotransmitter in response to a nerve stimulation in 1.8 mM extracellular Ca2+, syxH3-C mutants fail to respond approximately 25% of the time. Collectively, these phenotypes suggest that loss of the Ca2+ effector domain causes defects in the ability of the fusion machinery to properly regulate synchronized release of synaptic vesicles in response to a Ca2+ influx. These features are hallmarks of synaptotagmin null mutations, suggesting that syxH3-C mutants reveal defects in either the sensing of or response to Ca2+ in synaptic vesicle fusion (Wu, 1999).
One intriguing interaction is the binding of syntaxin 1A to members of the Munc-18/n-Sec1/ROP family. Genetic studies of Sec1 homologs have shown that these proteins perform a positive function in exocytosis, because mutants show severe defects in both neuronal and nonneuronal secretion. Therefore, it has been suggested that the syntaxin-Munc-18/n-Sec1 interaction positively regulates secretion. However, more recent studies of ROP, the Drosophila Sec1 homolog, have demonstrated that this protein also performs an inhibitory role in exocytosis in vivo. Hence, it is unclear whether the positive or inhibitory function of ROP/n-sec1 is mediated by its interaction with Syntaxin or by interaction with other proteins (Wu, 1999).
To define the function of the Syntaxin-ROP complex, a phenotypic analysis was carried out of the syx3 mutant, since the syx3 mutation selectively disrupts the Syntaxin-ROP interaction. Since the syx3 mutation affects a hydrophobic residue that may be important for core complex stability, the heat lability of SDS-resistant core complexes containing GST-Syx3 was tested. The syx3 mutation does not significantly alter core complex stability relative to wild-type control, although the levels of SDS-resistant core complexes are somewhat reduced at 54°C for GST-Syx3, compared to wild-type GST-Syx. Thus, syx3 mutants are capable of forming stable core complexes but specifically fail to bind to ROP (Wu, 1999).
To address the effects of disrupting the Syntaxin-ROP interaction on synaptic transmission, whole-cell patch-clamp analysis at the NMJ was performed. syx3 mutants demonstrate a very significantly enhanced evoked junctional current (EJC) amplitude when compared to syxwt controls. Two independent transgenic lines show similarly increased evoked responses. To rule out the possibility that the enhancement of evoked response results from a postsynaptic change, the amplitude of spontaneous miniature excitatory junctional currents (mEJCs) was measured. These data show that quantal size is not significantly altered in syx3 mutants, indicating that the increase in evoked response results from an increase in the number of quanta released. Hence, these data demonstrate that the ROP-Syntaxin complex is not essential for neurotransmission and likely inhibits it (Wu, 1999).
One particularly interesting regulatory interaction examined in this study was the binding of syntaxin 1A to synaptic N-, P-, and Q-type calcium channels, at the cytoplasmic loop between domains II and III, called the synprint site. A functional correlate of this binding was proposed on the basis of synprint peptide competition experiments, which suggested that inhibiting the syntaxin-Ca2+ channel interaction caused a reduction in evoked synaptic transmission, with an increase in asynchronous release. Thus, this interaction was proposed to tether the core complex at Ca2+ channels in order to localize the fusion machinery near the site of Ca2+ influx and potentiate neurotransmission. However, studies in which syntaxin 1A was expressed in Xenopus oocytes have shown that the protein functions to inhibit Ca2+ channels. In this context, syntaxin may function to reduce random or unregulated Ca2+ channel openings, and loss of this function would be postulated to lead to enhanced neurotransmitter release. Therefore, a syntaxin-Ca2+ channel interaction has been suggested to play at least two roles in neurotransmitter release: a potentiating tethering function and an inhibitory regulatory function (Wu, 1999 and references).
A phenotypic analysis of syx2, which specifically impairs synprint binding was conducted to study the function of the Syntaxin-Ca2+ channel complex in vivo. To quantitate the reduction of synprint binding to GST-Syx2, bound synprint was detected using 125I-labeled secondary antibodies and phosphorimaging. Binding of the synprint peptide to wild-type GST-Syx is dose dependent and saturable with half-maximal binding at approximately 0.4 µM under these conditions. GST-Syx2 binding to synprint shows an approximately 67% reduction in binding at 1.5 µM synprint, compared to wild-type GST-Syx. To rule out the possibility that the syx2 mutation impairs core complex stability, the heat lability of SDS-resistant core complexes containing GST-Syx2 was tested: results indicate that in this assay the syx2 mutation does not affect core complex stability (Wu, 1999).
syx2 mutants reveal very significantly increased EJC amplitude compared to syxwt controls. Two independent lines, syx2-1 and syx2-2, have similarly increased EJC amplitudes. To ensure that this increase in evoked response represents a presynaptic phenomenon, mEJC amplitude was measured. mEJC amplitude is not significantly altered in syx2-1 or syx2-2 mutants, compared to syxwt. Therefore, syx2 mutants, in which synprint binding is specifically impaired, reveal a dramatic increase of evoked neurotransmitter release (Wu, 1999).
In addition to an increase in evoked response, the mEJC frequency is also greatly increased in both syx2-1 and syx2-2 lines. At the Drosophila embryonic NMJ, a population of mEJC events is Ca2+ dependent, and these Ca2+-dependent spontaneous fusions are thought to be triggered by random openings of Ca2+ channels. Thus, the increase in mEJC frequency in syx2 mutants may represent an inability of these mutant Syntaxins to prevent random openings of Ca2+ channels. If additional quantal events observed in syx2 mutants are due to increased openings of Ca2+ channels, then, in the absence of Ca2+, the mEJC frequency of syx2 mutants should be reduced. mEJC frequency in syx2 mutants is significantly reduced in the absence of Ca2+. In addition, even in zero Ca2+, syx2 mutants show significantly more quantal events than syxwt, suggesting that the Syntaxin-Ca2+ channel interaction also physically inhibits spontaneous fusion in a manner independent of Ca2+. These data suggest that the Syntaxin-Ca2+ channel interaction inhibits evoked and spontaneous neurotransmission in vivo (Wu, 1999).
Taken together, these observations suggest that Syntaxin binding to Ca2+ channels reduces both spontaneous and evoked openings of the channels. The findings do not support an essential function for Syntaxin in tethering the fusion machinery near Ca2+ channels, since loss of this function should lead to a reduction in synchronized evoked release. An attractive alternative candidate for such a tethering function is SNAP-25, which has also been shown to bind the synprint site. How are these data reconciled with the observation that injection of synprint peptides into cultured neurons inhibits neurotransmission and causes asynchronous release? In addition to syntaxin, synaptotagmin and CSP have been shown to bind synprint peptides, and the data suggest that the synprint peptide binds to the Ca2+ effector domain of Syntaxin. Therefore, one interpretation of these results is that injection of synprint peptides may inhibit the function of these proteins or their interaction with the syntaxin Ca2+ effector domain, resulting in a phenotype similar to that observed in syxH3-C mutants. The syx3 and syx2 mutations do not appear to alter the stability of their core complexes, suggesting that the structure of the core complex bundle is intact. However, the possibility that these mutations cause additional defects, such as affecting the conformation or function of the N-terminal domain of Syntaxin, which has been suggested to regulate core complex assembly, cannot be excluded (Wu, 1999).
To investigate whether CSP could regulate the Syntaxin-Ca2+ channel interaction, an examination was performed to see if CSP can be found in a complex with Syntaxin. Syntaxin complexes were immunoprecipitated from Drosophila head extracts with anti-Syntaxin. As shown for vertebrate proteins, Drosophila Synaptotagmin coimmunoprecipitates with Syntaxin. In addition, CSP also coimmunoprecipitates with Syntaxin. Since coimmunoprecipitation does not demonstrate direct binding, an examination was carried out of whether CSP interacts with Syx in a GST pulldown assay. CSP binds to wild-type GST-Syx, but not to GST alone. These data show that CSP can bind directly to Syntaxin in vitro and is present in a complex with Syntaxin in vivo (Wu, 1999).
The region deleted in GST-SyxH3-N is essential for core complex formation and ROP binding, but not Synaptotagmin or synprint binding. Conversely, the region deleted in GST-SyxH3-C is required for efficient binding to Synaptotagmin and to synprint, but not ROP. To determine if these regions are also important for CSP binding, an examination was made of the ability of CSP to interact with GST-SyxH3-N and GST-SyxH3-C, as well as GST-Syx2 and GST-Syx3. While CSP can interact with GST-SyxH3-N, CSP binding to GST-SyxH3-C is reduced. In addition, CSP binds to GST-Syx2 and GST-Syx3. To quantitate the amount of CSP, Synaptotagmin, and synprint binding to wild-type GST-Syx and GST-SyxH3-C, binding experiments were performed. Binding of CSP to Syntaxin is dose dependent and saturable. The apparent stoichiometry of this interaction is approximately 0.3:1 (CSP:GST-Syx), when Syntaxin is immobilized on beads. Maximal binding of CSP to GST-SyxH3-C is reduced approximately 80%. Maximal Synaptotagmin binding to GST-SyxH3-C is severely reduced using these conditions, compared to wild-type GST-Syx. In addition, synprint binding to GST-SyxH3-C is also reduced, relative to wild-type GST-Syx. The syxH3-C deletion causes approximately 85% reduction in maximal synprint binding. Hence, these binding data suggest that the region deleted in syxH3-C is required for binding of three proteins implicated in Ca2+ signaling: CSP, synprint, and Synaptotagmin. It is proposed that the N-terminal domain of Syntaxin acts as a core domain, while the highly basic C-terminal region of the H3 domain functions as a Ca2+ effector domain, spatially localizing proteins required for the production, regulation, and sensing of the Ca2+ signal (Wu, 1999).
A possible model to explain CSP function and its in vitro binding to Syntaxin is that the role of CSP is to alleviate the Syntaxin-mediated inhibition of Ca2+ channels, thereby signaling the presence of synaptic vesicles and preparing the machinery for fusion. This model makes a simple prediction, namely that CSP should be able to compete with Syntaxin for the binding to synprint. To test this possibility, immobilized wild-type GST-Syntaxin protein was incubated with synprint peptide in the presence of increasing amounts of CSP or Synaptotagmin. CSP effectively competes the Syntaxin-synprint interaction in a dose-dependent manner. Synaptotagmin can also compete the Syntaxin-synprint interaction, but somewhat less effectively. Therefore, while both CSP and Synaptotagmin can interact with the Ca2+ effector domain of Syntaxin, CSP appears to be a more effective competitor of the Syntaxin-synprint interaction than Synaptotagmin. These data further suggest that CSP, Synaptotagmin, and synprint bind to the Ca2+ effector domain of Syntaxin. Additionally, these data support the hypothesis that these proteins participate in competitive interactions to regulate Ca2+ entry and are consistent with the idea that CSP may disassemble Syntaxin-Ca2+ channel complexes by interacting with Syntaxin, the synprint site, or both (Wu, 1999).
Three SYX-1A transcripts, 3.5- 4.2- and 8.0-kb are abundant in young embryos. At 3-9 hours of development the 3.5-kb transcript slowly disappears while 4.2-, 8.0- and 9.0-kb messages predominant until the end of embryogenesis. Between 9-15 hours, two additional transcripts (7.0 and 12 kb) appear. During first instar larval life, five messages are present, whereas second and third instars utilize three and two messages respectively. During pupariation, all five transcripts reappear, and the adult expression pattern resembles that of pupae. Three of the adult transcripts are the same as those found in young embryos, suggesting some or all of these messages represent the maternal contribution to the embryo. Since the cDNA is contained within a single exon, it is likely that alternative splicing, various promoters, and different polyadenylation signals are used to generate this heterogeneity (Schulze, 1996).
The protein exhibits 70% amino acid identity with rat syntaxin-1A over its full length and 25% identity with yeast Sso1p. Identity with Rat syntaxin-5 is only 22% (Schulze, 1995).
A monoclonal antibody, mAb 44D5, has been used to identify and clone Drosophila syntaxin 1 (Dsynt1), a homolog of rat syntaxin 1. The deduced amino acid sequence of the Dsynt1 cDNA cloned is highly homologous to rat syntaxin 1A. Dsynt1 contains 291 amino acid residues and like other members of the syntaxin family is an integral membrane protein, with a transmembrane region at its carboxy-terminus and several regions of the molecule predicted to be in a coiled-coil conformation (Cerezo, 1995).
Conserved domains have been analyzed in t-SNAREs (soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein [SNAP] receptors in the target membrane). These proteins are believed to be involved in the fusion of transport vesicles with their target membrane. A new homology domain has been identified that is common in the two protein families ( the syntaxin and SNAP-25 [synaptosome-associated protein of 25 kDa] families) previously identified to act as t-SNAREs, and which therefore constitute a new superfamily. This homology domain of approximately 60 amino acids is predicted to form a coiled-coil structure. The significance of this homology domain could be demonstrated by a partial suppression of the coiled-coil properties of the domain profile. In proteins belonging to the syntaxin family, a single homology domain is located near the transmembrane domain, whereas the members of the SNAP-25 family possess two homology domains. This domain has also been identified in several proteins that have been implicated in vesicular transport but do not belong to any of the t-SNARE protein families. Several new yeast, nematode, and mammalian proteins have been identified that belong to the new superfamily. The evolutionary conservation of the SNARE coiled-coil homology domain suggests that this domain has a similar function in different membrane fusion proteins (Weimbs, 1997).
The interaction between the proteins syntaxin 1A and SNAP-25 is a key step in synaptic vesicle docking and fusion. To define the SNAP-25 binding domain on syntaxin, peptides were prepared that span the syntaxin H3 domain (residues 191-266), the region previously shown to be important for binding to SNAP-25. The affinities of these peptides for binding to SNAP-25 were then determined. A minimal binding domain was identified within a region of 32 amino acids (residues 189-220). Its affinity for SNAP-25 is substantially enhanced by C-terminal extension (residues 221-266). Circular dichroism reveals the presence of substantial alpha-helicity in the H3 domain and in the 32-mer minimal binding domain, but not in H3 peptides that do not bind to SNAP-25. At temperatures that denature the alpha-helix of the minimal binding domain peptide, SNAP-25 binding is lost. Selected mutations in evolutionarily conserved residues of the amphiphilic alpha-helix within the minimal binding domain (e.g., residues 205 and 209) greatly reduce the affinity for SNAP-25 but have no major effect on secondary structure, suggesting that these residues may interact directly with SNAP-25. The H3 domain peptide and the minimal binding domain peptide inhibit norepinephrine release from PC12 cells. These results suggest that specific amino acid residues in the H3 domain, positioned by the underlying alpha-helical structure, are important for its binding to SNAP-25 and support the notion that this interaction is important for presynaptic vesicular exocytosis (Zhong, 1997).
date revised: 5 MAY 98
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