Phosphorylation of SNAP-23 by the novel kinase SNAK regulates t-SNARE complex assembly

The docking and fusion of cargo-containing vesicles with target membranes of eukaryotic cells is mediated by the interaction of SNARE proteins present on both vesicle and target membranes. In many cases, the target membrane SNARE, or t-SNARE, exists as a complex of syntaxin with a member of the SNAP-25 family of palmitoylated proteins. We have identified a novel human kinase SNAK (SNARE kinase) that specifically phosphorylates the nonneuronal t-SNARE SNAP-23 in vivo. Interestingly, only SNAP-23 that is not assembled into t-SNARE complexes is phosphorylated by SNAK, and phosphorylated SNAP-23 resides exclusively in the cytosol. Coexpression with SNAK significantly enhances the stability of unassembled SNAP-23, and as a consequence, the assembly of newly synthesized SNAP-23 with syntaxin is augmented. These data demonstrate that phosphorylation of SNAP-23 by SNAK enhances the kinetics of t-SNARE assembly in vivo.     

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

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

Targeting of SNAP-25 to membranes is mediated by its association with the target SNARE syntaxin

The docking and fusion of synaptic vesicles with the presynaptic plasma membrane require the interaction of the vesicle-associated membrane protein VAMP with the plasma membrane proteins syntaxin and SNAP-25. Both of these proteins behave as integral membrane proteins, although they are unusual in that they insert into membranes post-translationally. Whereas VAMP and syntaxin possess hydrophobic transmembrane domains, SNAP-25 does not, and it is widely believed that SNAP-25 traffics to and inserts into membranes by post-translational palmitoylation. In pulse-chase biosynthesis studies, we now show that SNAP-25 and syntaxin rapidly bind to each other while still in the cytosol of neuroendocrine and transfected heterologous cells. Cell fractionation studies revealed that cytosolic SNAP-25.syntaxin complexes then traffic to and insert into membranes. Furthermore, the association of SNAP-25 with membranes is dramatically enhanced by syntaxin, and the transmembrane domain of syntaxin is essential for this effect. Surprisingly, despite the importance of the SNAP-25 palmitoylation domain for membrane anchoring at steady state, removal of this domain did not inhibit the initial association of newly synthesized SNAP-25 with membranes in the presence of syntaxin. These data demonstrate that the initial attachment of newly synthesized SNAP-25 to membranes is a consequence of its association with syntaxin and that it is only after syntaxin-mediated membrane tethering that SNAP-25 is palmitoylated.     

SNAP-25 is targeted to the plasma membrane through a novel membrane-binding domain.

SNAP-25, syntaxin, and synaptobrevin are SNARE proteins that mediate fusion of synaptic vesicles with the plasma membrane. Membrane attachment of syntaxin and synaptobrevin is achieved through a C-terminal hydrophobic tail, whereas SNAP-25 association with membranes appears to depend upon palmitoylation of cysteine residues located in the center of the molecule. This process requires an intact secretory pathway and is inhibited by brefeldin A. Here we show that the minimal plasma membrane-targeting domain of SNAP-25 maps to residues 85-120. This sequence is both necessary and sufficient to target a heterologous protein to the plasma membrane. Palmitoylation of this domain is sensitive to brefeldin A, suggesting that it uses the same membrane-targeting mechanism as the full-length protein. As expected, the palmitoylated cysteine cluster is present within this domain, but surprisingly, membrane anchoring requires an additional five-amino acid sequence that is highly conserved among SNAP-25 family members. Significantly, the membrane-targeting module coincides with the protease-sensitive stretch (residues 83-120) that connects the two alpha-helices that SNAP-25 contributes to the four-helix bundle of the synaptic SNARE complex. Our results demonstrate that residues 85-120 of SNAP-25 represent a protein module that is physically and functionally separable from the SNARE complex-forming domains.     

Alternative splicing of SNAP-25 regulates secretion through nonconservative substitutions in the SNARE domain

The essential membrane fusion apparatus in mammalian cells, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consists of four alpha-helices formed by three proteins: SNAP-25, syntaxin 1, and synaptobrevin 2. SNAP-25 contributes two helices to the complex and is targeted to the plasma membrane by palmitoylation of four cysteines in the linker region. It is alternatively spliced into two forms, SNAP-25a and SNAP-25b, differing by nine amino acids substitutions. When expressed in chromaffin cells from SNAP-25 null mice, the isoforms support different levels of secretion. Here, we investigated the basis of that different secretory phenotype. We found that two nonconservative substitutions in the N-terminal SNARE domain and not the different localization of one palmitoylated cysteine cause the functional difference between the isoforms. Biochemical and molecular dynamic simulation experiments revealed that the two substitutions do not regulate secretion by affecting the property of SNARE complex itself, but rather make the SNAP-25b-containing SNARE complex more available for the interaction with accessory factor(s).     

SNAP-25 Palmitoylation and Plasma Membrane Targeting Require a Functional Secretory Pathway

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a palmitoylated membrane protein essential for neurotransmitter release from synaptic terminals. We used neuronal cell lines to study the biosynthesis and posttranslational processing of SNAP-25 to investigate how palmitoylation contributes to the subcellular localization of the protein. SNAP-25 was synthesized as a soluble protein that underwent palmitoylation approximately 20 min after synthesis. Palmitoylation of the protein coincided with its stable membrane association. Treatment of cells with brefeldin A or other disrupters of transport inhibited palmitoylation of newly synthesized SNAP-25 and abolished membrane association. These results demonstrate that the processing of SNAP-25 and its targeting to the plasma membrane depend on an intact transport mechanism along the exocytic pathway. The kinetics of SNAP-25 palmitoylation and membrane association and the sensitivity of these parameters to brefeldin A suggest a novel trafficking pathway for targeting proteins to the plasma membrane. In vitro, SNAP-25 stably associated with membranes was not released from the membrane after chemical deacylation. We propose that palmitoylation of SNAP-25 is required for initial membrane targeting of the protein but that other interactions can maintain membrane association in the absence of fatty acylation.     

Characterization of the Palmitoylation Domain of SNAP-25

Abstract: SNAP-25 (synaptosomal associated protein of 25 kDa) is a neural specific protein that has been implicated in the synaptic vesicle docking and fusion process. It is tightly associated with membranes, and it is one of the major palmitoylated proteins found in neurons. The functional role of palmitoylation for SNAP-25 is unclear. In this report, we show that the palmitate of SNAP-25 is rapidly turned over in PC12 cells, with a half-life of ∼3 h, and the half-life for the protein is 8 h. Mutation of Cys to Ser at positions 85, 88, 90, and 92 reduced the palmitoylation to 9, 21, 42, and 35% of the wild-type protein, respectively. Additional mutations of either Cys^85,88 or Cys^90,92 nearly abolished palmitoylation of the protein. A similar effect on membrane binding for the mutant SNAP-25 was observed, which correlated with the degree of palmitoylation. These results suggest that all four Cys residues are involved in palmitoylation and that membrane association of SNAP-25 may be regulated through dynamic palmitoylation.     

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of alpha-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the N-terminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the N-terminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins.     

Epithelial sodium channel is regulated by SNAP-23/syntaxin 1A interplay

Sodium-selective amiloride-sensitive epithelial channel (ENaC) located in the apical membrane is involved in the reabsorption of sodium in tight epithelia. The soluble N-ethylmaleimide-sensitive attachment receptors (SNAREs) mediate vesicle trafficking in a variety of cell systems. Syntaxin (a t-SNARE) has been shown to interact with and functionally regulate a number of ion channels including ENaC. In this study, we investigated the role of SNAP-23, another SNARE protein, on ENaC activity in the HT-29 colonic epithelial cell system and Xenopus oocytes. Recording of amiloride-sensitive currents in both systems suggest that SNAP-23 modulates channel function, though a much higher concentration is required to inhibit ENaC in Xenopus oocytes. The introduction of Botulinum toxin A (a neurotoxin which cleaves SNAP-23), but not Botulinum toxin B or heat-inactivated Botulinum toxin A, reversed the inhibitory effect of SNAP-23 on amiloride-sensitive currents. However, syntaxin 1A and SNAP-23 combined portray a complex scenario that suggests that this channel interacts within a quaternary complex. Synaptotagmin expression neither interacts with, nor showed any effect on amiloride-sensitive currents when co-expressed with ENaC. Pull down assays suggest mild interaction between ENaC and SNAP-23, which gets stronger in the presence of syntaxin 1A. Data further suggest that SNAP-23 possibly interacts with the N-terminal alphaENaC. These functional and biochemical approaches provide evidence for a complex relationship between ENaC and the exocytotic machinery. Our data suggest that SNARE protein interplay defines the fine regulation of sodium channel function.     

Synaptotagmin interaction with the syntaxin/SNAP-25 dimer is mediated by an evolutionarily conserved motif and is sensitive to inositol hexakisphosphate

Synaptotagmins are membrane proteins that possess tandem C2 domains and play an important role in regulated membrane fusion in metazoan organisms. Here we show that both synaptotagmins I and II, the two major neuronal isoforms, can interact with the syntaxin/synaptosomal-associated protein of 25 kDa (SNAP-25) dimer, the immediate precursor of the soluble NSF attachment protein receptor (SNARE) fusion complex. A stretch of basic amino acids highly conserved throughout the animal kingdom is responsible for this calcium-independent interaction. Inositol hexakisphosphate modulates synaptotagmin coupling to the syntaxin/SNAP-25 dimer, which is mirrored by changes in chromaffin cell exocytosis. Our results shed new light on the functional importance of the conserved polybasic synaptotagmin motif, suggesting that synaptotagmin interacts with the t-SNARE dimer to up-regulate the probability of SNARE-mediated membrane fusion.     

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.     

SNAP-29 is a promiscuous syntaxin-binding SNARE.

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

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.     

High affinity interaction of syntaxin and SNAP-25 on the plasma membrane is abolished by botulinum toxin E

The release of hormones and neurotransmitters requires the fusion of cargo-containing vesicles with the plasma membrane. This process of exocytosis relies on three SNARE proteins, namely syntaxin and SNAP-25 on the target plasma membrane and synaptobrevin on the vesicular membrane. In this study we examined the molecular assembly pathway that leads to formation of the fusogenic SNARE complex. We now show that the plasma membrane syntaxin and SNAP-25 interact with high affinity and equimolar stoichiometry to form a stable dimer on the pathway to the ternary SNARE complex. In bovine chromaffin cells, syntaxin and SNAP-25 colocalize in defined clusters that average 700 nm in diameter and cover 10% of the plasma membrane. Removal of the C terminus of SNAP-25 by botulinum neurotoxin E, a known neuroparalytic agent, dissociates the target SNARE dimer in vitro and disrupts the SNARE clustering in vivo. Together, our data uncover formation of stable syntaxin/SNAP-25 dimers as a central principle of the SNARE assembly pathway underlying regulated exocytosis.     

A dominant-negative variant of SNAP-23 decreases the cell surface expression of the neuronal glutamate transporter EAAC1 by slowing constitutive delivery

A family of high-affinity transporters controls the extracellular concentration of glutamate in the brain, ensuring appropriate excitatory signaling and preventing excitotoxicity. There is evidence that one of the neuronal glutamate transporters, EAAC1, is rapidly recycled on and off the plasma membrane with a half-life of no more than 5-7 min in both C6 glioma cells and cortical neurons. Syntaxin 1A has been implicated in the trafficking of several neurotransmitter transporters and in the regulation of EAAC1, but it has not been determined if this SNARE protein is required for EAAC1 trafficking. Expression of two different sets of SNARE proteins was examined in C6 glioma with Western blotting. These cells did not express syntaxin 1A, vesicle-associated membrane protein-1 (VAMP1), or synaptosomal-associated protein of 25 kDa (SNAP-25), but did express a family of SNARE proteins that has been implicated in glucose transporter trafficking, including syntaxin 4, vesicle-associated membrane protein-2 (VAMP2), and synaptosomal-associated protein of 23 kDa (SNAP-23). cDNAs encoding variants of SNAP-23 were co-transfected with Myc-tagged EAAC1 to determine if SNAP-23 function was required for maintenance of EAAC1 surface expression. Expression of a dominant-negative variant of SNAP-23 that lacks a domain required for SNARE complex assembly decreased the fraction of EAAC1 found on the cell surface and decreased total EAAC1 expression, while two control constructs had no effect. The dominant-negative variant of SNAP-23 also slowed the rate of EAAC1 delivery to the plasma membrane. These data strongly suggest that syntaxin 1A is not required for EAAC1 trafficking and provide evidence that SNAP-23 is required for constitutive recycling of EAAC1.     

Phosphorylation of SNAP-23 in activated human platelets

Phosphorylation of SNARE proteins may provide a critical link between cell activation and secretory processes. Platelets contain all three members of the SNAP-23/25/29 gene family, but by comparison to brain tissue, SNAP-23 is the most highly enriched of these proteins in platelets. SNAP-23 function is required for exocytosis from platelet alpha, dense, and lysosomal granules. SNAP-23 was phosphorylated largely on serine residues in platelets activated with thrombin. Phosphorylation kinetics paralleled or preceded granule secretion. Inhibition studies suggested that SNAP-23 phosphorylation proceeds largely through a protein kinase C (PKC) mechanism and purified PKC directly phosphorylated recombinant (r-) SNAP-23 (up to 0.3 mol of phosphate/mol of protein). Five major tryptic phosphopeptides were identified in cellular SNAP-23 isolated from activated platelets; three phosphopeptides co-migrated with those identified in PKC-phosphorylated r-SNAP-23. In contrast, only one major phosphopeptide was identified when SNAP-23, engaged in a ternary SNARE complex, was phosphorylated by PKC. Ion trap mass spectrometry revealed that platelet SNAP-23 was phosphorylated at Ser23/Thr24 and Ser161, after cell activation by thrombin; these sites were also identified in PKC-phosphorylated r-SNAP-23. SNAP-23 mutants that mimic phosphorylation at Ser23/Thr24 inhibited syntaxin 4 interactions, whereas a phosphorylation mutant of Ser161 had only minor effects. Taken together these studies show that SNAP-23 is phosphorylated in platelets during cell activation through a PKC-related mechanism at two or more sites with kinetics that parallel or precede granule secretion. Because mutants that mimic SNAP-23 phosphorylation affect syntaxin 4 interactions, we hypothesize that SNAP-23 phosphorylation may be important for modulating SNARE-complex interactions during membrane trafficking and fusion.     

Palmitoylated peptides from the cysteine-rich domain of SNAP-23 cause membrane fusion depending on peptide length,position of cysteines,and extent of palmitoylation

Synaptosome-associated proteins SNAP-23/25, members of a family of proteins essential for exocytosis, have a highly conserved central cysteine-rich domain that plays an important role in membrane targeting. More than one cysteine in this domain is modified by palmitic acid through a thioester linkage. In an effort to address the biological significance of acylation of this domain, we have generated synthetic peptides corresponding to the cysteine-rich region of SNAP-23 and covalently modified the cysteines with palmitic acid. The interaction of acylated and nonacylated peptides with lipid vesicles and natural membranes has been investigated. Our results indicate that palmitoylation is essential for membrane association. The palmitoylated peptides were able to fuse both model and natural membranes. The extent of fusion depended on the length of the peptides and the number and positions of covalently linked palmitic acids. Peptide-mediated fusion was suppressed by lysolipid and involved both outer and inner leaflets of the lipid bilayer, which is characteristic of natural membrane fusion. Our results suggest an important role for the cysteine-rich palmitoylated domain of SNAP-23 in promoting membrane fusion in cells.     

Botulinum neurotoxin E-insensitive mutants of SNAP-25 fail to bind VAMP but support exocytosis

Neurotransmitter release from synaptic vesicles is mediated by complex machinery, which includes the v- and t-SNAP receptors (SNAREs), vesicle-associated membrane protein (VAMP), synaptotagmin, syntaxin, and synaptosome-associated protein of 25 kDa (SNAP-25). They are essential for neurotransmitter exocytosis because they are the proteolytic substrates of the clostridial neurotoxins tetanus neurotoxin and botulinum neurotoxins (BoNTs), which cause tetanus and botulism, respectively. Specifically, SNAP-25 is cleaved by both BoNT/A and E at separate sites within the COOH-terminus. We now demonstrate, using toxin-insensitive mutants of SNAP-25, that these two toxins differ in their specificity for the cleavage site. Following modification within the COOH-terminus, the mutants completely resistant to BoNT/E do not bind VAMP but were still able to form a sodium dodecyl sulfate-resistant complex with VAMP and syntaxin. Furthermore, these mutants retain function in vivo, conferring BoNT/E-resistant exocytosis to transfected PC12 cells. These data provide information on structural requirements within the C-terminal domain of SNAP-25 for its function in exocytosis and raise doubts about the significance of in vitro binary interactions for the in vivo functions of synaptic protein complexes.     

The t-SNARE Complex: A Close Up

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

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

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