Alternate interfaces may mediate homomeric and heteromeric assembly in the transmembrane domains of SNARE proteins

The fusion of a vesicle to a target membrane is mediated by temporally and spatially regulated interactions within a set of evolutionarily conserved proteins. Integral to proper fusion is the interaction between proteins originating on both vesicle and target membranes to form a protein bridge between the two membranes, known as the SNARE complex. This protein complex includes the single-pass transmembrane helix proteins: syntaxin and synaptobrevin. Experimental data and amino acid sequence analysis suggest that an interface of interaction is conserved between the transmembrane regions of the two proteins. However, conflicting reports have been presented on the role of the synaptobrevin transmembrane domain in mediating important protein-protein interactions. To address this question, a thermodynamic study was carried out to determine quantitatively the self-association propensities of the transmembrane domains of synaptobrevin and syntaxin. Our results show that the transmembrane domain of synaptobrevin has only a modest ability to self-associate, whereas the transmembrane domain of syntaxin is able to form stable homodimers. Nevertheless, by a single amino acid substitution, synaptobrevin can be driven to dimerize with the same affinity as syntaxin. Furthermore, crosslinking studies show that dimerization of synaptobrevin is promoted by oxidizing agents. Despite the presence of a conserved cysteine residue in the same location as in synaptobrevin, syntaxin dimerization is not promoted by oxidization. This analysis suggests that subtle yet distinct differences are present between the two transmembrane dimer interfaces. A syntaxin/synaptobrevin heterodimer is able to form under oxidizing conditions, and we propose that the interface of interaction for the heterodimer may resemble the homodimer interface formed by the synaptobrevin transmembrane domain. Computational analysis of the transmembrane sequences of syntaxin and synaptobrevin reveal structural models that correlate with the experimental data. These data may provide insight into the role of transmembrane segments in the mechanism of vesicle fusion.     

Peptide mimics of SNARE transmembrane segments drive membrane fusion depending on their conformational plasticity

SNARE proteins are essential for different types of intracellular membrane fusion. Whereas interaction between their cytoplasmic domains is held responsible for establishing membrane proximity, the role of the transmembrane segments in the fusion process is currently not clear. Here, we used an in vitro approach based on lipid mixing and electron microscopy to examine a potential fusogenic activity of the transmembrane segments. We show that the presence of synthetic peptides representing the transmembrane segments of the presynaptic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) synaptobrevin II (also referred to as VAMP II) or syntaxin 1A, but not of an unrelated control peptide, in liposomal membranes drives their fusion. Liposome aggregation by millimolar Ca(2+) concentrations strongly potentiated the effect of the peptides; this indicates that juxtaposition of the bilayers favours their fusion in the absence of the cytoplasmic SNARE domains. Peptide-driven fusion is reminiscent of natural membrane fusion, since it was suppressed by lysolipid and involved both bilayer leaflets. This suggests transient presence of a hemifusion intermediate followed by complete membrane merger. Structural studies of the peptides in lipid bilayers performed by Fourier transform infrared spectroscopy indicated mixtures of alpha-helical and beta-sheet conformations. In isotropic solution, circular dichroism spectroscopy showed the peptides to exist in a concentration-dependent equilibrium of alpha-helical and beta-sheet structures. Interestingly, the fusogenic activity decreased with increasing stability of the alpha-helical solution structure for a panel of variant peptides. Thus, structural plasticity of transmembrane segments may be important for SNARE protein function at a late step in membrane fusion.     

NMR analysis of the structure of synaptobrevin and of its interaction with syntaxin

Synaptobrevin is a synaptic vesicle protein that has an essential role in exocytosis and forms the SNARE complex with syntaxin and SNAP-25. We have analyzed the structure of isolated synaptobrevin and its binary interaction with syntaxin using NMR spectroscopy. Our results demonstrate that isolated synaptobrevin is largely unfolded in solution. The entire SNARE motif of synaptobrevin is capable of interacting with the isolated C-terminal SNARE motif of syntaxin but only a few residues bind to the full-length cytoplasmic region of syntaxin. This result suggests an interaction between the N- and C-terminal regions of syntaxin that competes with core complex assembly.     

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.     

A stable interaction between syntaxin 1a and synaptobrevin 2 mediated by their transmembrane domains

The proteins synaptobrevin (VAMP), SNAP-25 and syntaxin 1 are essential for neuronal exocytosis. They assemble into a stable ternary complex which is thought to initiate membrane fusion. In vitro, the transmembrane domains of syntaxin and synaptobrevin are not required for association. Here we report a novel interaction between synaptobrevin and syntaxin that requires the presence of the transmembrane domains. When co-reconstituted into liposomes, the proteins form a stable binary complex that cannot be disassembled by NSF and that is resistant to denaturation by SDS. Cleavage of synaptobrevin with tetanus toxin does not affect the interaction. Furthermore, the complex is formed when a truncated version of syntaxin is used that contains only 12 additional amino acid residues outside the membrane anchor. We conclude that the interaction is mediated by the transmembrane domains.     

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

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

Determinants of synaptobrevin regulation in membranes

Neuronal exocytosis is driven by the formation of SNARE complexes between synaptobrevin 2 on synaptic vesicles and SNAP-25/syntaxin 1 on the plasma membrane. It has remained controversial, however, whether SNAREs are constitutively active or whether they are down-regulated until fusion is triggered. We now show that synaptobrevin in proteoliposomes as well as in purified synaptic vesicles is constitutively active. Potential regulators such as calmodulin or synaptophysin do not affect SNARE activity. Substitution or deletion of residues in the linker connecting the SNARE motif and transmembrane region did not alter the kinetics of SNARE complex assembly or of SNARE-mediated fusion of liposomes. Remarkably, deletion of C-terminal residues of the SNARE motif strongly reduced fusion activity, although the overall stability of the complexes was not affected. We conclude that although complete zippering of the SNARE complex is essential for membrane fusion, the structure of the adjacent linker domain is less critical, suggesting that complete SNARE complex assembly not only connects membranes but also drives fusion.     

Sec22b-dependent assembly of endoplasmic reticulum Q-SNARE proteins

SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins involved in membrane fusion usually contain a conserved alpha-helix (SNARE motif) that is flanked by a C-terminal transmembrane domain. They can be classified into Q-SNARE and R-SNARE based on the structural property of their motifs. Assembly of four SNARE motifs (Qa, b, c and R) is supposed to trigger membrane fusion. We have previously shown that ER (endoplasmic reticulum)-localized syntaxin 18 (Qa) forms a complex with BNIP1 (Qb), p31/Use1 (Qc), Sec22b (R) and several peripheral membrane proteins. In the present study, we examined the interaction of syntaxin 18 with other SNAREs using pulldown assays and CD spectroscopy. We found that the association of syntaxin 18 with Sec22b induces an increase in alpha-helicity of their SNARE motifs, which results in the formation of high-affinity binding sites for BNIP1 and p31. This R-SNARE-dependent Q-SNARE assembly is quite different from the assembly mechanisms of SNAREs localized in organelles other than the ER. The implication of the mechanism of ER SNARE assembly is discussed in the context of the physiological roles of the syntaxin 18 complex.     

An activated Q‐SNARE/SM protein complex as a possible intermediate in SNARE assembly

Assembly of the SNARE proteins syntaxin1, SNAP25, and synaptobrevin into a SNARE complex is essential for exocytosis in neurons. For efficient assembly, SNAREs interact with additional proteins but neither the nature of the intermediates nor the sequence of protein assembly is known. Here, we have characterized a ternary complex between syntaxin1, SNAP25, and the SM protein Munc18‐1 as a possible acceptor complex for the R‐SNARE synaptobrevin. The ternary complex binds synaptobrevin with fast kinetics, resulting in the rapid formation of a fully zippered SNARE complex to which Munc18‐1 remains tethered by the N‐terminal domain of syntaxin1. Intriguingly, only one of the synaptobrevin truncation mutants (Syb1‐65) was able to bind to the syntaxin1:SNAP25:Munc18‐1 complex, suggesting either a cooperative zippering mechanism that proceeds bidirectionally or the progressive R‐SNARE binding via an SM template. Moreover, the complex is resistant to disassembly by NSF. Based on these findings, we consider the ternary complex as a strong candidate for a physiological intermediate in SNARE assembly.     

The C-terminal transmembrane region of synaptobrevin binds synaptophysin from adult synaptic vesicles

Synaptophysin and synaptobrevin are abundant membrane proteins of neuronal small synaptic vesicles. In mature, differentiated neurons they form the synaptophysin/synaptobrevin (Syp/Syb) complex. Synaptobrevin also interacts with the plasma membrane-associated proteins syntaxin and SNAP25, thereby forming the SNARE complex necessary for exocytotic membrane fusion. The two complexes are mutually exclusive. Synaptobrevin is a C-terminally membrane-anchored protein with one transmembrane domain. While its interaction with its SNARE partners is mediated exclusively by its N-terminal cytosolic region it has been unclear so far how binding to synaptophysin is accomplished. Here, we show that synaptobrevin can be cleaved in its synaptophysin-bound form by tetanus toxin and botulinum neurotoxin B, or by botulinum neurotoxin D, leaving shorter or longer C-terminal peptide chains bound to synaptophysin, respectively. A recombinant, C-terminally His-tagged synaptobrevin fragment bound to nickel beads specifically bound synaptophysin, syntaxin and SNAP25 from vesicular detergent extracts. After cleavage by tetanus toxin or botulinum toxin D light chain, the remaining C-terminal fragment no longer interacted with syntaxin or SNAP 25. In contrast, synaptophysin was still able to bind to the residual C-terminal synaptobrevin cleavage product. In addition, the His-tagged C-terminal synaptobrevin peptide 68-116 was also able to bind synaptophysin in detergent extracts from adult brain membranes. These data suggest that synaptophysin interacts with the C-terminal transmembrane part of synaptobrevin, thereby allowing the N-terminal cytosolic chain to interact freely with the plasma membrane-associated SNARE proteins. Thus, by binding synaptobrevin, synaptophysin may positively modulate neurotransmission.     

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.     

Action of complexin on SNARE complex

Calcium-dependent synaptic vesicle exocytosis requires three SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins: synaptobrevin/vesicle-associated membrane protein in the vesicular membrane and syntaxin and SNAP-25 in the presynaptic membrane. The SNAREs form a thermodynamically stable complex that is believed to drive fusion of vesicular and presynaptic membranes. Complexin, also known as synaphin, is a neuronal cytosolic protein that acts as a positive regulator of synaptic vesicle exocytosis. Complexin binds selectively to the neuronal SNARE complex, but how this promotes exocytosis remains unknown. Here we used purified full-length and truncated SNARE proteins and a gel shift assay to show that the action of complexin on SNARE complex depends strictly on the transmembrane regions of syntaxin and synaptobrevin. By means of a preparative immunoaffinity procedure to achieve total extraction of SNARE complex from brain, we demonstrated that complexin is the only neuronal protein that tightly associates with it. Our data indicated that, in the presence of complexin, the neuronal SNARE proteins assemble directly into a complex in which the transmembrane regions interact. We propose that complexin facilitates neuronal exocytosis by promoting interaction between the complementary syntaxin and synaptobrevin transmembrane regions that reside in opposing membranes prior to fusion.     

Evidence that late-endosomal SNARE multimerization complex is promoted by transmembrane segments

Assembly of SNARE proteins into quaternary complexes is a critical step in membrane docking and fusion. Here, we have studied the influence of the transmembrane segments on formation of the late endosomal SNARE complex. The complex was assembled in vitro from full-length recombinant SNAREs and from mutants, where the transmembrane segments were either deleted or replaced by oligo-alanine sequences. We show that endobrevin, syntaxin 7, syntaxin 8, and vti1b readily form a complex. This complex forms a dimer as well as multimeric structures. Interestingly, the natural transmembrane segments accelerate the conversion of the quaternary complex to the dimeric form and are essential for multimerization. These in vitro results suggest that the transmembrane segments are responsible for supramolecular assembly of the endosomal SNARE complex.     

Evidence that late-endosomal SNARE multimerization complex is promoted by transmembrane segments

Assembly of SNARE proteins into quaternary complexes is a critical step in membrane docking and fusion. Here, we have studied the influence of the transmembrane segments on formation of the late endosomal SNARE complex. The complex was assembled in vitro from full-length recombinant SNAREs and from mutants, where the transmembrane segments were either deleted or replaced by oligo-alanine sequences. We show that endobrevin, syntaxin 7, syntaxin 8, and vti1b readily form a complex. This complex forms a dimer as well as multimeric structures. Interestingly, the natural transmembrane segments accelerate the conversion of the quaternary complex to the dimeric form and are essential for multimerization. These in vitro results suggest that the transmembrane segments are responsible for supramolecular assembly of the endosomal SNARE complex.     

Neuronal SNAREs do not trigger fusion between synthetic membranes but do promote PEG-mediated membrane fusion

At low surface concentrations that permit formation of impermeable membranes, neuronal soluble N-ethyl maleimide sensitive factor attachment protein receptor (SNARE) proteins form a stable, parallel, trans complex when vesicles are brought into contact by a low concentration of poly(ethylene glycol) (PEG). Surprisingly, formation of a stable SNARE complex does not trigger fusion under these conditions. However, neuronal SNAREs do promote fusion at low protein/lipid ratios when triggered by higher concentrations of PEG. Promotion of PEG-triggered fusion required phosphatidylserine and depended only on the surface concentration of SNAREs and not on the formation of a trans SNARE complex. These results were obtained at protein surface concentrations reported for synaptobrevin in synaptic vesicles and with an optimally fusogenic lipid composition. At a much higher protein/lipid ratio, vesicles joined by SNARE complex slowly mixed lipids at 37 degrees C in the absence of PEG, in agreement with earlier reports. However, vesicles containing syntaxin at a high protein/lipid ratio (>or=1:250) lost membrane integrity. We conclude that the neuronal SNARE complex promotes fusion by joining membranes and that the individual proteins syntaxin and synaptobrevin disrupt membranes so as to favor formation of a stalk complex and to promote conversion of the stalk to a fusion pore. These effects are similar to the effects of viral fusion peptides and transmembrane domains, but they are not sufficient by themselves to produce fusion in our in vitro system at surface concentrations documented to occur in synaptic vesicles. Thus, it is likely that proteins or factors other than the SNARE complex must trigger fusion in vivo.     

Homo- and heterooligomeric SNARE complexes studied by site-directed spin labeling

SNARE (soluble NSF acceptor protein receptor) proteins are thought to mediate membrane fusion by assembling into heterooligomeric complexes that connect the fusing membranes and initiate the fusion reaction. Here we used site-directed spin labeling to map conformational changes that occur upon homo- and heterooligomeric complex formation of neuronal SNARE proteins. We found that the soluble domains of synaptobrevin, SNAP-25, and syntaxin 1 are unstructured. At higher concentrations, the SNARE motif of syntaxin 1 forms homooligomeric helical bundles with at least some of the alpha-helices aligned in parallel. In the assembled SNARE complex, mapping of thirty side chain positions yielded spectra which are in good agreement with the recently published crystal structure. The loop region of SNAP-25 that connects the two SNARE motifs is largely unstructured. C-terminal truncation of synaptobrevin resulted in complexes that are completely folded N-terminal of the truncation but become unstructured at the C-terminal end. The binary complex of syntaxin and SNAP-25 consists of a parallel four helix-bundle with properties resembling that of the ternary complex.     

Selective interaction of complexin with the neuronal SNARE complex. Determination of the binding regions

Complexins are evolutionarily conserved proteins that specifically bind to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and thus may regulate SNARE function. Using purified proteins, we have performed a detailed analysis of the structure of complexin and of its interaction with SNARE proteins. NMR spectroscopy revealed that isolated complexins have no tertiary structure but contain an unusual alpha-helical middle domain of approximately 58 amino acids that overlaps with the most highly conserved region of the molecules. Complexins form a stable stoichiometric complex with the central domain of the ternary SNARE complex, whereas no binding was observed to monomeric SNAREs. Using a combination of limited proteolysis, deletion mutagenesis, and NMR spectroscopy, we found that the helical middle region of complexin is responsible for binding to the SNARE complex. Binding was highly sensitive to substitution of syntaxin 1 or synaptobrevin 2 with other SNARE homologs but less sensitive to substitution of SNAP-25. In addition, a stretch of 12 amino acids in the middle of the SNARE motif of syntaxin 1A was able to confer binding activity to the non-binding relative syntaxin 4. Furthermore, disassembly of ternary complexes is not affected by complexins. We conclude that complexins are specific ligands of the neuronal core complex that bind with a central alpha-helical domain, probably to the middle of the surface groove formed by synaptobrevin and syntaxin. Complexins may regulate the function of ternary complexes and control membrane fusion through this interaction.     

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

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

The interaction between syntaxin 1A and cystic fibrosis transmembrane conductance regulator Cl- channels is mechanistically distinct from syntaxin 1A-SNARE interactions

Syntaxin 1A binds to and inhibits epithelial cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels and synaptic Ca(2+) channels in addition to participating in SNARE complex assembly and membrane fusion. We exploited the isoform-specific nature of the interaction between syntaxin 1A and CFTR to identify residues in the H3 domain of this SNARE (SNARE motif) that influence CFTR binding and regulation. Mutating isoform-specific residues that map to the surface of syntaxin 1A in the SNARE complex led to the identification of two sets of hydrophilic residues that are important for binding to and regulating CFTR channels or for binding to the syntaxin regulatory protein Munc-18a. None of these mutations affected syntaxin 1A binding to other SNAREs or the assembly and stability of SNARE complexes in vitro. Conversely, the syntaxin 1A-CFTR interaction was unaffected by mutating hydrophobic residues in the H3 domain that influence SNARE complex stability and Ca(2+) channel regulation. Thus, CFTR channel regulation by syntaxin 1A involves hydrophilic interactions that are mechanistically distinct from the hydrophobic interactions that mediate SNARE complex formation and Ca(2+) channel regulation by this t-SNARE.     

Intracellular interaction between syntaxin and Munc 18-1 revealed by fluorescence resonance energy transfer

Neurosecretion is catalyzed by assembly of a soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)-complex composed of SNAP-25, synaptobrevin and syntaxin. Munc 18-1 is known to bind to syntaxin in vitro. This interaction prevents assembly of the SNARE-complex, but might also affect intracellular targeting of the proteins. We have fused syntaxin and Munc 18 to the yellow- (YFP) or cyan-fluorescence-protein (CFP) and expressed the constructs in CHO- and MDCK-cells. We have studied their localization with confocal microscopy and a possible protein-protein interaction with fluorescence-resonance energy transfer (FRET). YFP-syntaxin localizes to intracellular membranes. CFP-Munc 18 is present in the cytoplasm as expected for a protein lacking membrane targeting domains. However, Munc 18 is redirected to internal membranes when syntaxin is coexpressed, but only limited transport of the proteins to the plasma membrane was observed. An interaction between Munc 18 and syntaxin could be demonstrated by FRET using two methods, sensitized acceptor fluorescence and acceptor photobleaching. A mutation in syntaxin (L165A, E166A), which is known to inhibit binding to Munc 18 in vitro, prevents colocalization of the proteins and also the FRET signal. Thus, a protein-protein interaction between Munc 18 and syntaxin occurs on intracellular membranes, which is required but not sufficient for quantitative transport of both proteins to the plasma membrane.