Syntaphilin: a syntaxin-1 clamp that controls SNARE assembly

Syntaxin-1 is a key component of the synaptic vesicle docking/fusion machinery that forms the SNARE complex with VAMP/synaptobrevin and SNAP-25. Identifying proteins that modulate SNARE complex formation is critical for understanding the molecular mechanisms underlying neurotransmitter release and its modulation. We have cloned and characterized a protein called syntaphilin that is selectively expressed in brain. Syntaphilin competes with SNAP-25 for binding to syntaxin-1 and inhibits SNARE complex formation by absorbing free syntaxin-1. Transient overexpression of syntaphilin in cultured hippocampal neurons significantly reduces neurotransmitter release. Furthermore, introduction of syntaphilin into presynaptic superior cervical ganglion neurons in culture inhibits synaptic transmission. These findings suggest that syntaphilin may function as a molecular clamp that controls free syntaxin-1 availability for the assembly of the SNARE complex, and thereby regulates synaptic vesicle exocytosis.     

Opposing functions of two sub‐domains of the SNARE‐complex in neurotransmission

The SNARE‐complex consisting of synaptobrevin‐2/VAMP‐2, SNAP‐25 and syntaxin‐1 is essential for evoked neurotransmission and also involved in spontaneous release. Here, we used cultured autaptic hippocampal neurons from Snap‐25 null mice rescued with mutants challenging the C‐terminal, N‐terminal and middle domains of the SNARE‐bundle to dissect out the involvement of these domains in neurotransmission. We report that the stabilities of two different sub‐domains of the SNARE‐bundle have opposing functions in setting the probability for both spontaneous and evoked neurotransmission. Destabilizing the C‐terminal end of the SNARE‐bundle abolishes spontaneous neurotransmitter release and reduces evoked release probability, indicating that the C‐terminal end promotes both modes of release. In contrast, destabilizing the middle or deleting the N‐terminal end of the SNARE‐bundle increases both spontaneous and evoked release probabilities. In both cases, spontaneous release was affected more than evoked neurotransmission. In addition, the N‐terminal deletion delays vesicle priming after a high‐frequency train. We propose that the stability of N‐terminal two‐thirds of the SNARE‐bundle has a function for vesicle priming and limiting spontaneous release.     

Three-dimensional structure of the complexin/SNARE complex

During neurotransmitter release, the neuronal SNARE proteins synaptobrevin/VAMP, syntaxin, and SNAP-25 form a four-helix bundle, the SNARE complex, that pulls the synaptic vesicle and plasma membranes together possibly causing membrane fusion. Complexin binds tightly to the SNARE complex and is essential for efficient Ca(2+)-evoked neurotransmitter release. A combined X-ray and TROSY-based NMR study now reveals the atomic structure of the complexin/SNARE complex. Complexin binds in an antiparallel alpha-helical conformation to the groove between the synaptobrevin and syntaxin helices. This interaction stabilizes the interface between these two helices, which bears the repulsive forces between the apposed membranes. These results suggest that complexin stabilizes the fully assembled SNARE complex as a key step that enables the exquisitely high speed of Ca(2+)-evoked neurotransmitter release.     

An isolated pool of vesicles recycles at rest and drives spontaneous neurotransmission

Spontaneous synaptic vesicle fusion is a common property of all synapses. To trace the origin of spontaneously fused vesicles in hippocampal synapses, we tagged vesicles with fluorescent styryl dyes, antibodies against synaptotagmin-1, or horseradish peroxidase. We could show that synaptic vesicles recycle at rest, and after spontaneous exo-endocytosis, they populate a reluctantly releasable pool of limited size. Interestingly, vesicles in this spontaneously labeled pool were more likely to re-fuse spontaneously compared to vesicles labeled with activity. We found that blocking vesicle refilling at rest selectively depleted neurotransmitter from spontaneously fusing vesicles without significantly altering evoked transmission. Furthermore, in the absence of the vesicle SNARE protein synaptobrevin (VAMP), activity-dependent and spontaneously recycling vesicles could mix, suggesting a role for synaptobrevin in the separation of the two pools. Taken together these results suggest that spontaneously recycling vesicles and activity-dependent recycling vesicles originate from distinct pools with limited cross-talk with each other.     

The N-terminal domains of syntaxin 7 and vti1b form three-helix bundles that differ in their ability to regulate SNARE complex assembly

The SNAREs syntaxin 7, syntaxin 8, vti1b, and endobrevin/VAMP8 function in the fusion of late endosomes. Although the core complex formed by these SNAREs is very similar to the neuronal SNARE complex, it differs from the neuronal complex in that three of the four SNAREs contain extended N-terminal regions of unknown structure and function. Here we show that the N-terminal regions of syntaxin 7, syntaxin 8, and vti1b contain well folded alpha-helical domains. Multidimensional NMR spectroscopy revealed that in syntaxin 7 and vti1b, the domains form three-helix bundles resembling those of syntaxin 1, Sso1p, and Vam3p. The three-helix bundle domain of vti1b is the first of its kind identified in a SNARE outside the syntaxin family. Only syntaxin 7 adopts a closed conformation, whereas in vti1b and syntaxin 8, the N-terminal domains do not interact with the adjacent SNARE motifs. Accordingly, the rate of SNARE complex assembly is retarded about 7-fold when syntaxin 7 contains its N-terminal domain, whereas the N-terminal domains of vti1b and syntaxin 8 have no influence on assembly kinetics. We conclude that three-helix bundles represent a common fold for SNARE N-terminal domains, not restricted to the syntaxin family. However, they differ in their ability to adopt closed conformations and thus to regulate the assembly of SNARE complexes.     

The SNARE protein vti1a functions in dense‐core vesicle biogenesis

The SNARE protein vti1a is proposed to drive fusion of intracellular organelles, but recent data also implicated vti1a in exocytosis. Here we show that vti1a is absent from mature secretory vesicles in adrenal chromaffin cells, but localizes to a compartment near the trans‐Golgi network, partially overlapping with syntaxin‐6. Exocytosis is impaired in vti1a null cells, partly due to fewer Ca²⁺‐channels at the plasma membrane, partly due to fewer vesicles of reduced size and synaptobrevin‐2 content. In contrast, release kinetics and Ca²⁺‐sensitivity remain unchanged, indicating that the final fusion reaction leading to transmitter release is unperturbed. Additional deletion of the closest related SNARE, vti1b, does not exacerbate the vti1a phenotype, and vti1b null cells show no secretion defects, indicating that vti1b does not participate in exocytosis. Long‐term re‐expression of vti1a (days) was necessary for restoration of secretory capacity, whereas strong short‐term expression (hours) was ineffective, consistent with vti1a involvement in an upstream step related to vesicle generation, rather than in fusion. We conclude that vti1a functions in vesicle generation and Ca²⁺‐channel trafficking, but is dispensable for transmitter release.     

Endosomal SNARE proteins regulate CFTR activity and trafficking in epithelial cells

The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein is a chloride channel localized at the apical plasma membrane of epithelial cells. We previously described that syntaxin 8, an endosomal SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) protein, interacts with CFTR and regulates its trafficking to the plasma membrane and hence its channel activity. Syntaxin 8 belongs to the endosomal SNARE complex which also contains syntaxin 7, vti1b and VAMP8. Here, we report that these four endosomal SNARE proteins physically and functionally interact with CFTR. In LLC-PK1 cells transfected with CFTR and in Caco-2 cells endogenously expressing CFTR, we demonstrated that endosomal SNARE protein overexpression inhibits CFTR activity but not swelling- or calcium-activated iodide efflux, indicating a specific effect upon CFTR activity. Moreover, co-immunoprecipitation experiments in LLC-PK1-CFTR cells showed that CFTR and SNARE proteins belong to a same complex and pull-down assays showed that VAMP8 and vti1b preferentially interact with CFTR N-terminus tail. By cell surface biotinylation and immunofluorescence experiments, we evidenced that endosomal SNARE overexpression disturbs CFTR apical targeting. Finally, we found a colocalization of CFTR and endosomal SNARE proteins in Rab11-positive recycling endosomes, suggesting a new role for endosomal SNARE proteins in CFTR trafficking in epithelial cells.     

A conserved membrane-spanning amino acid motif drives homomeric and supports heteromeric assembly of presynaptic SNARE proteins

Assembly of the SNARE proteins synaptobrevin/VAMP, syntaxin, and SNAP-25 to binary and ternary complexes is important for docking and/or fusion of presynaptic vesicles to the neuronal plasma membrane prior to regulated neurotransmitter release. Despite the well characterized structure of their cytoplasmic assembly domains, little is known about the role of the transmembrane segments in SNARE protein assembly and function. Here, we identified conserved amino acid motifs within the transmembrane segments that are required for homodimerization of synaptobrevin II and syntaxin 1A. Minimal motifs of 6-8 residues grafted onto an otherwise monomeric oligoalanine host sequence were sufficient for self-interaction of both transmembrane segments in detergent solution or membranes. These motifs constitute contiguous areas of interfacial residues assuming alpha-helical secondary structures. Since the motifs are conserved, they also contributed to heterodimerization of synaptobrevin II and syntaxin 1A and therefore appear to constitute interaction domains independent of the cytoplasmic coiled coil regions. Interactions between the transmembrane segments may stabilize the SNARE complex, cause its multimerization to previously observed multimeric superstructures, and/or be required for the fusogenic activity of SNARE proteins.     

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.     

Ca2+-dependent phosphorylation of syntaxin-1A by the death-associated protein (DAP) kinase regulates its interaction with Munc18

Syntaxin-1 is a key component of the synaptic vesicle docking/fusion machinery that binds with VAMP/synaptobrevin and SNAP-25 to form the SNARE complex. Modulation of syntaxin binding properties by protein kinases could be critical to control of neurotransmitter release. Using yeast two-hybrid selection with syntaxin-1A as bait, we have isolated a cDNA encoding the C-terminal domain of death-associated protein (DAP) kinase, a calcium/calmodulin-dependent serine/threonine protein kinase. Expression of DAP kinase in adult rat brain is restricted to particular neuronal subpopulations, including the hippocampus and cerebral cortex. Biochemical studies demonstrate that DAP kinase binds to and phosphorylates syntaxin-1 at serine 188. This phosphorylation event occurs both in vitro and in vivo in a Ca2+-dependent manner. Syntaxin-1A phosphorylation by DAP kinase or its S188D mutant, which mimics a state of complete phosphorylation, significantly decreases syntaxin binding to Munc18-1, a syntaxin-binding protein that regulates SNARE complex formation and is required for synaptic vesicle docking. Our results suggest that syntaxin is a DAP kinase substrate and provide a novel signal transduction pathway by which syntaxin function could be regulated in response to intracellular [Ca2+] and synaptic activity.     

Calmodulin and lipid binding to synaptobrevin regulates calcium-dependent exocytosis

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 Ca(2+)-binding proteins that associate with components of the SNARE complex. Ca(2+)/calmodulin or phospholipids bind in a mutually exclusive fashion to a C-terminal domain of VAMP (VAMP(77-90)), and residues involved were identified by plasmon resonance spectroscopy. Microinjection of wild-type VAMP(77-90), but not mutant peptides, inhibited catecholamine release from chromaffin cells monitored by carbon fibre amperometry. Pre-incubation of PC12 pheochromocytoma cells with the irreversible calmodulin antagonist ophiobolin A inhibited Ca(2+)-dependent human growth hormone release in a permeabilized cell assay. Treatment of permeabilized cells with tetanus toxin light chain (TeNT) also suppressed secretion. In the presence of TeNT, exocytosis was restored by transfection of TeNT-resistant (Q(76)V, F(77)W) VAMP, but additional targeted mutations in VAMP(77-90) abolished its ability to rescue release. The calmodulin- and phospholipid-binding domain of VAMP 2 is thus required for Ca(2+)-dependent exocytosis, possibly to regulate SNARE complex assembly.     

Syntaxin-1蛋白的多重功能(英文)

Syntaxin-1是特异性地分布在神经细胞突触前质膜上的蛋白。它早期被作为分子量为35 kD的synaptotagmin-1结合蛋白,但很快就被认识到是细胞质膜融合的关键蛋白。Syntaxin-1通过与SNAP25和Synaptobrevin/VAMP蛋白聚合,进而形成被认为是神经突触囊泡融合必要因子的SNARE核心复合体。作为一个多结构域的蛋白,syntaxin-1与多个突触蛋白相互作用,其作用远超出了仅作为SNARE核心复合体中一个蛋白质成员的作用。本文着重介绍了有关syntaxin-1与其它SNARE组份蛋白、munc18蛋白和钙离子通道的相互作用及其功能的最新研究进展。全面揭示syntaxin-1作为SNARE核心复合体成员的功能以及超越这一功能的作用,还有待于对其结构以及与其它突触蛋白相互作用特性的进一步深刻理解。     

The identification of a novel endoplasmic reticulum to Golgi SNARE complex used by the prechylomicron transport vesicle

Dietary long chain fatty acids are absorbed in the intestine, esterified to triacylglycerol, and packaged in the unique lipoprotein of the intestine, the chylomicron. The rate-limiting step in the transit of chylomicrons through the enterocyte is the exit of chylomicrons from the endoplasmic reticulum in prechylomicron transport vesicles (PCTV) that transport chylomicrons to the cis-Golgi. Because chylomicrons are 250 nm in average diameter and lipid absorption is intermittent, we postulated that a unique SNARE pairing would be utilized to fuse PCTV with their target membrane, cis-Golgi. PCTV loaded with [(3)H]triacylglycerol were incubated with cis-Golgi and were separated from the Golgi by a sucrose step gradient. PCTV-chylomicrons acquire apolipoprotein-AI (apoAI) only after fusion with the Golgi. PCTV became isodense with Golgi upon incubation and were considered fused when their cargo chylomicrons acquired apoAI but docked when they did not. PCTV, docked with cis-Golgi, were solubilized in 2% Triton X-100, and proteins were immunoprecipitated using VAMP7 or rBet1 antibodies. In both cases, a 112-kDa complex was identified in nonboiled samples that dissociated upon boiling. The constituents of the complex were VAMP7, syntaxin 5, vti1a, and rBet1. Antibodies to each SNARE component significantly inhibited fusion of PCTV with cis-Golgi. Membrin, Sec22b, and Ykt6 were not found in the 112-kDa complex. We conclude that the PCTV-cis-Golgi SNARE complex is composed of VAMP7, syntaxin 5, Bet1, and vti1a.     

The neuronal t-SNARE complex is a parallel four-helix bundle

Assembly of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex is an essential step for neurotransmitter release in synapses. The presynaptic plasma membrane associated proteins (t-SNAREs), SNAP-25 (synaptosome-associated protein of 25,000 Da) and syntaxin 1A may form an intermediate complex that later binds to vesicle-associated membrane protein 2 (VAMP2). Using spin labeling electron paramagnetic resonance (EPR), we found that the two t-SNARE proteins assemble into a parallel four-helix bundle that consists of two identical syntaxin 1A components and the N-terminal and C-terminal domains of SNAP-25. Although the structure is generally similar to that of the final SNARE complex, the middle region of the helical bundle appears more flexible in the t-SNARE complex. Such flexibility might facilitate interactions between VAMP2 and the t-SNARE complex.     

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.     

Synaptotagmin-12, a synaptic vesicle phosphoprotein that modulates spontaneous neurotransmitter release

Central synapses exhibit spontaneous neurotransmitter release that is selectively regulated by cAMP-dependent protein kinase A (PKA). We now show that synaptic vesicles contain synaptotagmin-12, a synaptotagmin isoform that differs from classical synaptotagmins in that it does not bind Ca(2+). In synaptic vesicles, synaptotagmin-12 forms a complex with synaptotagmin-1 that prevents synaptotagmin-1 from interacting with SNARE complexes. We demonstrate that synaptotagmin-12 is phosphorylated by cAMP-dependent PKA on serine(97), and show that expression of synaptotagmin-12 in neurons increases spontaneous neurotransmitter release by approximately threefold, but has no effect on evoked release. Replacing serine(97) by alanine abolishes synaptotagmin-12 phosphorylation and blocks its effect on spontaneous release. Our data suggest that spontaneous synaptic-vesicle exocytosis is selectively modulated by a Ca(2+)-independent synaptotagmin isoform, synaptotagmin-12, which is controlled by cAMP-dependent phosphorylation.     

神经递质释放过程中的可溶性NSF附着蛋白受体(SNARE)和相关蛋白

近年来,对突触小泡释放神经递质分子机制的研究迅速发展,发现了大量位于神经末梢的蛋白质.它们之间的相互作用与突触小泡释放神经递质相关,特别是位于突触小泡膜上的突触小泡蛋白/突触小泡相关膜蛋白(synaptobrevin/VAMP),位于突触前膜上的syntaxin和突触小体相关蛋白(synaptosome-associated protein of 25 ku),三者聚合形成的可溶性NSF附着蛋白受体(SNARE)核心复合体在突触小泡的胞裂外排、释放递质过程中有重要作用.而一些已知及未知的与SNARE蛋白有相互作用的蛋白质,可通过调节SNARE核心复合体的形成与解离来影响突触小泡的胞裂外排,从而可以调节突触信号传递的效率及强度,在突触可塑性的形成中起重要作用.     

Are neuronal SNARE proteins Ca2+ sensors?

The neuronal SNARE complex formed by synaptobrevin, syntaxin and SNAP-25 plays a central role in Ca2+-triggered neurotransmitter release. The SNARE complex contains several potential Ca2+-binding sites on the surface, suggesting that the SNAREs may be involved directly in Ca2+-binding during release. Indeed, overexpression of SNAP-25 bearing mutations in two putative Ca2+ ligands (E170A/Q177A) causes a decrease in the Ca2+-cooperativity of exocytosis in chromaffin cells. To test whether the SNARE complex might function in Ca2+-sensing, we analyzed its Ca2+-binding properties using transverse relaxation optimized spectroscopy (TROSY)-based NMR methods. Several Ca2+-binding sites are found on the surface of the SNARE complex, but most of them are not specific for Ca2+ and all have very low affinity. Moreover, we find that the E170A/Q177A SNAP-25 mutation does not alter interactions between the SNAREs and the Ca2+ sensor synaptotagmin 1, but severely impairs SNARE complex assembly. These results suggest that the SNAREs do not act directly as Ca2+ receptors but SNARE complex assembly is coupled tightly to Ca2+-sensing during neurotransmitter release.     

Dual inhibition of SNARE complex formation by tomosyn ensures controlled neurotransmitter release

Neurotransmitter release from presynaptic nerve terminals is regulated by soluble NSF attachment protein receptor (SNARE) complex–mediated synaptic vesicle fusion. Tomosyn inhibits SNARE complex formation and neurotransmitter release by sequestering syntaxin-1 through its C-terminal vesicle-associated membrane protein (VAMP)–like domain (VLD). However, in tomosyn-deficient mice, the SNARE complex formation is unexpectedly decreased. In this study, we demonstrate that the N-terminal WD-40 repeat domain of tomosyn catalyzes the oligomerization of the SNARE complex. Microinjection of the tomosyn N-terminal WD-40 repeat domain into neurons prevented stimulated acetylcholine release. Thus, tomosyn inhibits neurotransmitter release by catalyzing oligomerization of the SNARE complex through the N-terminal WD-40 repeat domain in addition to the inhibitory activity of the C-terminal VLD.     

SNAREs in neurons--beyond synaptic vesicle exocytosis (Review)

The paradigm for soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) function in mammalian cells has been built on advancements in our understanding of structural and biochemical aspects of synaptic vesicle exocytosis, involving specifically synaptobrevin, syntaxin 1 and SNAP25. Interestingly, a good number of SNAREs which are not directly involved in neurotransmitter exocytosis, are either brain-enriched or have distinct neuron-specific functions. Syntaxins 12/13 regulates glutamate receptor recycling via its interaction with neuron-enriched endosomal protein of 21 kDa (NEEP21). TI-VAMP/VAMP7 is essential for neuronal morphogenesis and mediates the vesicular transport processes underlying neurite outgrowth. Ykt6 is highly enriched in the cerebral cortex and hippocampus and is targeted to a novel compartment in neurons. Syntaxin 16 has a moderate expression level in many tissues, but is rather enriched in the brain. Here, we review and discuss the neuron-specific physiology and possible pathology of these and other (such as SNAP-29 and Vti1a-beta) members of the SNARE family.