Sly1 binds to Golgi and ER syntaxins via a conserved N-terminal peptide motif

Sec1/munc18-like proteins (SM proteins) and SNARE complexes are probably universally required for membrane fusion. However, the molecular mechanism by which they interact has only been defined for synaptic vesicle fusion where munc18 binds to syntaxin in a closed conformation that is incompatible with SNARE complex assembly. We now show that Sly1, an SM protein involved in Golgi and ER fusion, binds to a short, evolutionarily conserved N-terminal peptide of Sed5p and Ufe1p in yeast and of syntaxins 5 and 18 in vertebrates. In these syntaxins, the Sly1 binding peptide is upstream of a separate, autonomously folded N-terminal domain. These data suggest a potentially general mechanism by which SM proteins could interact with peptides in target proteins independent of core complex assembly and suggest that munc18 binding to syntaxin is an exception.     

rsly1 binding to syntaxin 5 is required for endoplasmic reticulum-to-Golgi transport but does not promote SNARE motif accessibility

Although some of the principles of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) function are well understood, remarkably little detail is known about sec1/munc18 (SM) protein function and its relationship to SNAREs. Popular models of SM protein function hold that these proteins promote or maintain an open and/or monomeric pool of syntaxin molecules available for SNARE complex formation. To address the functional relationship of the mammalian endoplasmic reticulum/Golgi SM protein rsly1 and its SNARE binding partner syntaxin 5, we produced a conformation-specific monoclonal antibody that binds only the available, but not the cis-SNARE-complexed nor intramolecularly closed form of syntaxin 5. Immunostaining experiments demonstrated that syntaxin 5 SNARE motif availability is nonuniformly distributed and focally regulated. In vitro endoplasmic reticulum-to-Golgi transport assays revealed that rsly1 was acutely required for transport, and that binding to syntaxin 5 was absolutely required for its function. Finally, manipulation of rsly1-syntaxin 5 interactions in vivo revealed that they had remarkably little impact on the pool of available syntaxin 5 SNARE motif. Our results argue that although rsly1 does not seem to regulate the availability of syntaxin 5, its function is intimately associated with syntaxin binding, perhaps promoting a later step in SNARE complex formation or function.     

Vam3p structure reveals conserved and divergent properties of syntaxins

Syntaxins and Sec1/munc18 proteins are central to intracellular membrane fusion. All syntaxins comprise a variable N-terminal region, a conserved SNARE motif that is critical for SNARE complex formation, and a transmembrane region. The N-terminal region of neuronal syntaxin 1A contains a three-helix domain that folds back onto the SNARE motif forming a 'closed' conformation; this conformation is required for munc18-1 binding. We have examined the generality of the structural properties of syntaxins by NMR analysis of Vam3p, a yeast syntaxin essential for vacuolar fusion. Surprisingly, Vam3p also has an N-terminal three-helical domain despite lacking apparent sequence homology with syntaxin 1A in this region. However, Vam3p does not form a closed conformation and its N-terminal domain is not required for binding to the Sec1/munc18 protein Vps33p, suggesting that critical distinctions exist in the mechanisms used by syntaxins to govern different types of membrane fusion.     

Dynamic conformational changes in munc18 prevent syntaxin binding

The Sec1/munc18 protein family is essential for vesicle fusion in eukaryotic cells via binding to SNARE proteins. Protein kinase C modulates these interactions by phosphorylating munc18a thereby reducing its affinity to one of the central SNARE members, syntaxin-1a. The established hypothesis is that the reduced affinity of the phosphorylated munc18a to syntaxin-1a is a result of local electrostatic repulsion between the two proteins, which interferes with their compatibility. The current study challenges this paradigm and offers a novel mechanistic explanation by revealing a syntaxin-non-binding conformation of munc18a that is induced by the phosphomimetic mutations. In the present study, using molecular dynamics simulations, we explored the dynamics of the wild-type munc18a versus phosphomimetic mutant munc18a. We focused on the structural changes that occur in the cavity between domains 3a and 1, which serves as the main syntaxin-binding site. The results of the simulations suggest that the free wild-type munc18a exhibits a dynamic equilibrium between several conformations differing in the size of its cavity (the main syntaxin-binding site). The flexibility of the cavity's size might facilitate the binding or unbinding of syntaxin. In silico insertion of phosphomimetic mutations into the munc18a structure induces the formation of a conformation where the syntaxin-binding area is rigid and blocked as a result of interactions between residues located on both sides of the cavity. Therefore, we suggest that the reduced affinity of the phosphomimetic mutant/phosphorylated munc18a is a result of the closed-cavity conformation, which makes syntaxin binding energetically and sterically unfavorable. The current study demonstrates the potential of phosphorylation, an essential biological process, to serve as a driving force for dramatic conformational changes of proteins modulating their affinity to target proteins.     

Munc18-1 stabilizes syntaxin 1, but is not essential for syntaxin 1 targeting and SNARE complex formation

Munc18-1, a member of the Sec1/Munc18 (SM) protein family, is essential for synaptic vesicle exocytosis. Munc18-1 binds tightly to the SNARE protein syntaxin 1, but the physiological significance and functional role of this interaction remain unclear. Here we show that syntaxin 1 levels are reduced by 70% in munc18-1 knockout mice. Pulse-chase analysis in transfected HEK293 cells revealed that Munc18-1 directly promotes the stability of syntaxin 1, consistent with a chaperone function. However, the residual syntaxin 1 in munc18-1 knockout mice is still correctly targeted to synapses and efficiently forms SDS-resistant SNARE complexes, demonstrating that Munc18-1 is not required for syntaxin 1 function as such. These data demonstrate that the Munc18-1 interaction with syntaxin 1 is physiologically important, but does not represent a classical chaperone-substrate relationship. Instead, the presence of SNARE complexes in the absence of membrane fusion in munc18-1 knockout mice indicates that Munc18-1 either controls the spatially correct assembly of core complexes for SNARE-dependent fusion, or acts as a direct component of the fusion machinery itself.     

Munc18a controls SNARE assembly through its interaction with the syntaxin N-peptide

Sec1/Munc18-like (SM) proteins functionally interact with SNARE proteins in vesicular fusion. Despite their high sequence conservation, structurally disparate binding modes for SM proteins with syntaxins have been observed. Several SM proteins appear to bind only to a short peptide present at the N terminus of syntaxin, designated the N-peptide, while Munc18a binds to a 'closed' conformation formed by the remaining portion of syntaxin 1a. Here, we show that the syntaxin 16 N-peptide binds to the SM protein Vps45, but the remainder of syntaxin 16 strongly enhances the affinity of the interaction. Likewise, the N-peptide of syntaxin 1a serves as a second binding site in the Munc18a/syntaxin 1a complex. When the syntaxin 1a N-peptide is bound to Munc18a, SNARE complex formation is blocked. Removal of the N-peptide enables binding of syntaxin 1a to its partner SNARE SNAP-25, while still bound to Munc18a. This suggests that Munc18a controls the accessibility of syntaxin 1a to its partners, a role that might be common to all SM proteins.     

The syntaxin 4 N terminus regulates its basolateral targeting by munc18c-dependent and -independent mechanisms

To generate and maintain epithelial cell polarity, specific sorting of proteins into vesicles destined for the apical and basolateral domain is required. Syntaxin 3 and 4 are apical and basolateral SNARE proteins important for the specificity of vesicle fusion at the apical and basolateral plasma membrane domains, respectively, but how these proteins are specifically targeted to these domains themselves is unclear. Munc18/SM proteins are potential regulators of this process. Like syntaxins, they are crucial for exocytosis and vesicle fusion. However, how munc18c and syntaxin 4 regulate the function of each other is unclear. Here, we investigated the requirement of syntaxin 4 in the delivery of basolateral membrane and secretory proteins, the basolateral targeting of syntaxin 4, and the role of munc18c in this targeting. Depletion of syntaxin 4 resulted in significant reduction of basolateral targeting, suggesting no compensation by other syntaxin forms. Mutational analysis identified amino acids Leu-25 and to a lesser extent Val-26 as essential for correct localization of syntaxin 4. Recently, it was shown that the N-terminal peptide of syntaxin 4 is involved in binding to munc18c. A mutation in this region that affects munc18c binding shows that munc18c binding is required for stabilization of syntaxin 4 at the plasma membrane but not for its correct targeting. We conclude that the N terminus serves two functions in membrane targeting. First, it harbors the sorting motif, which targets syntaxin 4 basolaterally in a munc18c-independent manner and second, it allows for munc18c binding, which stabilizes the protein in a munc18c-dependent manner.     

The Habc domain and the SNARE core complex are connected by a highly flexible linker

Syntaxin 1a is a member of the SNARE superfamily of small, mostly membrane-bound proteins that mediate membrane fusion in all eukaryotic cells. Upon membrane fusion, syntaxin 1 forms a stable complex with its partner SNAREs. Syntaxin contains a C-terminal transmembrane domain, an adjacent SNARE motif that interacts with its partner SNAREs, and an N-terminal Habc domain. The Habc domain reversibly folds back upon the SNARE motif, resulting in a "closed" conformation that is stabilized by binding to the protein munc18. The SNARE motif and the Habc domain are separated by a linker region of about 40 amino acids. When syntaxin is complexed with munc18, the linker is structured and consists of a mix of turns and small alpha-helices. When syntaxin is complexed with its partner SNAREs, the Habc domain is dissociated, but the structure of the linker region is not known. Here we used site-directed spin labeling and EPR spectroscopy to determine the structure of the linker region of syntaxin in the SNARE complex. We found that the entire linker region of syntaxin is unstructured except for three residues at the N-terminal and six residues at the C-terminal boundary whereas the structures of the flanking regions in the Habc domain and the SNARE motif correspond to the high-resolution structures of the isolated fragments. We conclude that the linker region exhibits a high degree of conformational flexibility.     

Cellular levels of the syntaxin Tlg2p are regulated by a single mode of binding to Vps45p

Sec1p/Munc18 (SM) proteins play a key role in the regulation of soluble N-ethylmaleimide-sensitive fusion (NSF)-attachment protein receptor (SNARE)-mediated intracellular membrane trafficking events in all eukaryotic cells. Understanding the molecular mechanisms by which SM proteins function has not been straight forward as SM proteins bind to their cognate SNARE proteins by at least two distinct mechanisms, suggesting that they provide more than one function. We have previously characterised two binding modes used by the yeast SM protein Vps45p to interact with its SNARE proteins. In one of these modes, the N terminus of the syntaxin Tlg2p inserts into a hydrophobic pocket in the SM protein. We now report that disruption of this high-affinity binding between Vps45p and Tlg2p leads to downregulation of Tlg2p, and propose that this pocket-mode of binding of SM proteins to their cognate syntaxins serves to regulate cellular levels of the syntaxin.     

Munc18-1 and Syntaxin1: Unraveling the Interactions Between the Dynamic Duo

All neurotransmitter and hormone regulated secretory events involve the action of three soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, syntaxin, SNAP-25, and synaptobrevin. The SNARE proteins interact to form a four alpha-helical complex, involving syntaxin and SNAP-25 on the plasma membrane and synaptobrevin on the vesicular membrane, bringing the opposing membranes together, promoting bilayer merger and membrane fusion. The process of regulated secretion is an adaptation of the membrane fusion events which occur at multiple steps throughout the intracellular trafficking pathway, in each case catalyzed by SNARE protein isoforms. At all of these locations, the SNAREs are joined by a member of the Sec1p/Munc18 (SM) protein family which selectively bind to syntaxin isoforms. From their initial identification, the SM proteins were known to be essential for membrane fusion, however, over the intervening decades, deciphering the precise mechanism of action of the SM proteins has proved problematic. Recent studies, investigating the interactions of munc18-1 and syntaxin1, provide an explanation for previous, apparently conflicting, observations yielding a new understanding of their cellular functions.     

Vesicle Fusion Probability Is Determined by the Specific Interactions of Munc18

Mammalian-regulated secretion is absolutely dependent on four evolutionarily conserved proteins: three SNARE proteins and munc18. Dissecting the functional outcomes of the spatially organized protein interactions between these factors has been difficult because of the close interrelationship between different binding modes. Here, we investigated the spatial distribution of single munc18 molecules at the plasma membrane of cells and the underlying interactions between syntaxin and munc18. Disruption of munc18 binding to the N-terminal peptide motif of syntaxin did not alter munc18 localization on the plasma membrane but had a pronounced influence on the behavior of secretory vesicles and their likelihood to undergo fusion. We therefore conclude that interaction with the syntaxin N-peptide can confer differential release probabilities to secretory vesicles and may contribute to the delineation of secretory vesicle pools.     

Munc18-1 mutations that strongly impair SNARE-complex binding support normal synaptic transmission

Synaptic transmission depends critically on the Sec1p/Munc18 protein Munc18-1, but it is unclear whether Munc18-1 primarily operates as a integral part of the fusion machinery or has a more upstream role in fusion complex assembly. Here, we show that point mutations in Munc18-1 that interfere with binding to the free Syntaxin1a N-terminus and strongly impair binding to assembled SNARE complexes all support normal docking, priming and fusion of synaptic vesicles, and normal synaptic plasticity in munc18-1 null mutant neurons. These data support a prevailing role of Munc18-1 before/during SNARE-complex assembly, while its continued association to assembled SNARE complexes is dispensable for synaptic transmission.     

An InCytes from MBC Selection: Yeast Sec1p Functions before and after Vesicle Docking

Sec1/Munc18 (SM) proteins bind cognate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and stimulate vesicle membrane fusion. Before fusion, vesicles are docked to specific target membranes. Regulation of vesicle docking is attributed to some but not all SM proteins, suggesting specialization of this earlier function. Yeast Sec1p seems to function only after vesicles are docked and SNARE complexes are assembled. Here, we show that yeast Sec1p is required before and after SNARE complex assembly, in support of general requirements for SM proteins in both vesicle docking and fusion. Two classes of sec1 mutants were isolated. Class A mutants are tightly blocked in cell growth and secretion at a step before SNARE complex assembly. Class B mutants have a SNARE complex binding defect, with a range in severity of cell growth and secretion defects. Mapping the mutations onto an SM protein structure implicates a peripheral bundle of helices for the early, docking function and a deep groove, opposite the syntaxin-binding cleft on nSec1/Munc-18, for the interaction between Sec1p and the exocytic SNARE complex.     

Selective expression of a sec1/munc18 member in sea urchin eggs and embryos

Regulated secretion is mediated by SNAREs (soluble NSF attachment receptors) and their regulators and effectors, which include the SM (sec1/munc18) family of proteins. Homologs of the SNAREs have been identified in sea urchins, associated with cortical granule exocytosis at fertilization, with membranes of the cleavage furrow, and in secretory cells later in development. To contribute to the understanding of regulated secretion in sea urchins we have cloned the single SM protein homolog from two species of sea urchin, Lytechinus variegatus and Strongylocentrotus purpuratus. In oocytes and eggs, we find that it localizes to the plasma membrane and the cortical region of the egg, consistent with a role in one of the steps leading to cortical granule exocytosis. The protein is also expressed throughout development, enriched in membranes of the cleavage furrow in early embryos, and in cells of the gut in advanced embryos. Furthermore, we find that sec1/munc18 co-localizes with its cognate binding partner syntaxin. Finally, our biochemical analysis shows that the protein associates with rab3 in high molecular weight complexes, suggesting that the exocytotic machinery functions as a multi-protein subunit to mediate regulated secretion in sea urchins. These results will be instrumental in the future to functionally test the SNARE regulators associated with multiple membrane fusion events.     

A conformational switch in syntaxin during exocytosis: role of munc18.

Syntaxin 1, an essential protein in synaptic membrane fusion, contains a helical autonomously folded N-terminal domain, a C-terminal SNARE motif and a transmembrane region. The SNARE motif binds to synaptobrevin and SNAP-25 to assemble the core complex, whereas almost the entire cytoplasmic sequence participates in a complex with munc18-1, a neuronal Sec1 homolog. We now demonstrate by NMR spectroscopy that, in isolation, syntaxin adopts a 'closed' conformation. This default conformation of syntaxin is incompatible with core complex assembly which requires an 'open' syntaxin conformation. Using site-directed mutagenesis, we find that disruption of the closed conformation abolishes the ability of syntaxin to bind to munc18-1 and to inhibit secretion in PC12 cells. These results indicate that syntaxin binds to munc18-1 in a closed conformation and suggest that this conformation represents an essential intermediate in exocytosis. Our data suggest a model whereby, during exocytosis, syntaxin undergoes a large conformational switch that mediates the transition between the syntaxin-munc18-1 complex and the core complex.     

SNARE bundle and syntaxin N-peptide constitute a minimal complement for Munc18-1 activation of membrane fusion

Sec1/Munc18 (SM) proteins activate intracellular membrane fusion through binding to cognate SNAP receptor (SNARE) complexes. The synaptic target membrane SNARE syntaxin 1 contains a highly conserved H_abc domain, which connects an N-peptide motif to the SNARE core domain and is thought to participate in the binding of Munc18-1 (the neuronal SM protein) to the SNARE complex. Unexpectedly, we found that mutation or complete removal of the H_abc domain had no effect on Munc18-1 stimulation of fusion. The central cavity region of Munc18-1 is required to stimulate fusion but not through its binding to the syntaxin H_abc domain. SNAP-25, another synaptic SNARE subunit, contains a flexible linker and exhibits an atypical conjoined Q_bc configuration. We found that neither the linker nor the Q_bc configuration is necessary for Munc18-1 promotion of fusion. As a result, Munc18-1 activates a SNARE complex with the typical configuration, in which each of the SNARE core domains is individually rooted in the membrane bilayer. Thus, the SNARE four-helix bundle and syntaxin N-peptide constitute a minimal complement for Munc18-1 activation of fusion.     

The relation of protein binding to function: what is the significance of munc18 and synaptotagmin binding to syntaxin 1, and where are the corresponding binding sites?

The Q-SNARE syntaxin 1 is a central component of the synaptic membrane fusion machinery. Syntaxin probably interacts with multiple proteins during synaptic vesicle exocytosis. In vitro, the tightest binding partners for syntaxin 1 are other SNAREs (synaptobrevin/VAMP and SNAP-25) and munc18-1 (also known as rbsec1/nsec1). Recent studies on Drosophila syntaxin led to the surprising finding that a syntaxin mutant which does not bind the munc18-homolog Rop nevertheless functionally substitutes for wild-type syntaxin in membrane fusion (Wu et al., Neuron 23, 593-605, 1999). This observation suggested that syntaxin 1 binding to munc18-1 is not essential for fusion, a puzzling conclusion in view of the tight binding of munc18 and syntaxin homologs in all organisms. To address this issue, we have now reinvestigated the interaction of syntaxin with munc18 and Rop. We find that the syntaxin sequence that was mutated in the Drosophila studies is not essential for munc18/Rop binding, and that the mutant is in fact able to bind to munc18/Rop. Thus the fact that the mutant syntaxin rescues release cannot be used as an argument that munc18 binding is not essential. In addition to munc18 and SNAREs, several other proteins have been suggested to interact with various domains of syntaxin 1, notably the calcium-sensor synaptotagmin and the vesicle protein CSP. Our results confirm that the SNARE motif in syntaxin binds to synaptotagmin, but this interaction does not require the very C-terminus of the motif. Interestingly, binding of synaptotagmin appears to be decreased in the closed conformation of syntaxin. In contrast, no interaction of CSP with syntaxin was detected even under low-stringency conditions. Our data suggest 1., that assays measuring protein/protein interactions that involve syntaxin may be more difficult to evaluate than is often assumed because of the sticky nature of the proteins involved, and 2., that it is currently not possible to draw conclusions about the importance of the various interactions with the available data from Drosophila or vertebrates.     

Trafficking of green fluorescent protein-tagged SNARE proteins in HSY cells

SNARE proteins are widely accepted to be involved in the docking and fusion process of intracellular vesicle trafficking. VAMP-2, syntaxin-4, and SNAP-23 are plausible candidate SNARE proteins for non-neuronal exocytosis. Thus, we examined the localization, protein-protein interaction, and intracellular trafficking of these proteins by expressing them as green fluorescent protein (GFP)- and FLAG-tagged fusion proteins in various cells, including HSY cells, a human parotid epithelial cell line. GFP-VAMP-2 was ex-pressed strongly in the Golgi area and weakly on the plasma membrane. Although GFP-SNAP-23 seemed to be expressed universally in the cytosol, the GFP signal was clearly seen on the plasma membrane, when soluble GFP-SNAP-23 was removed by treatment with saponin. GFP-syntaxin-4 was undetectable on the plasma membrane but was strongly expressed on unidentified unusually large vesicles. GFP-syntaxin-4 without its transmembrane domain was still incompletely soluble and observed as aggregates. When syntaxin-4 and munc18c were coexpressed, syntaxin-4 was translocated at least in part to the plasma membrane. The protein-protein interaction between syntaxin-4 and VAMP-2 with their transmembrane domains was markedly inhibited on coexpression of munc18c. These results suggest that munc18c plays an important role in the trafficking of syntaxin-4 to its proper destination by preventing premature interactions with other proteins, including SNARE proteins.     

Negative Regulation of Syntaxin4/SNAP-23/VAMP2-Mediated Membrane Fusion by Munc18c In Vitro

Background

Translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the plasma membrane is responsible for the increased rate of glucose transport into fat and muscle cells in response to insulin. This represents a specialised form of regulated membrane trafficking. Intracellular membrane traffic is subject to multiple levels of regulation by conserved families of proteins in all eukaryotic cells. Notably, all intracellular fusion events require SNARE proteins and Sec1p/Munc18 family members. Fusion of GLUT4-containing vesicles with the plasma membrane of insulin-sensitive cells involves the SM protein Munc18c, and is regulated by the formation of syntaxin 4/SNAP23/VAMP2 SNARE complexes.

Methodology/Principal Findings

Here we have used biochemical approaches to characterise the interaction(s) of Munc18c with its cognate SNARE proteins and to examine the role of Munc18c in regulating liposome fusion catalysed by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. We demonstrate that Munc18c makes contacts with both t- and v-SNARE proteins of this complex, and directly inhibits bilayer fusion mediated by the syntaxin 4/SNAP23/VAMP2 SNARE complex.

Conclusion/Significance

Our reductionist approach has enabled us to ascertain a direct inhibitory role for Munc18c in regulating membrane fusion mediated by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. It is important to note that two different SM proteins have recently been shown to stimulate liposome fusion mediated by their cognate SNARE complexes. Given the structural similarities between SM proteins, it seems unlikely that different members of this family perform opposing regulatory functions. Hence, our findings indicate that Munc18c requires a further level of regulation in order to stimulate SNARE-mediated membrane fusion.     

Characteristics of SNARE proteins are defined by distinctive properties of SNARE motifs

BackgroundSyntaxin-1A and Sso1 are syntaxin family SNARE proteins engaged in synaptic vesicle fusion and yeast exocytosis. The syntaxin-1A SNARE motif can form a fusogenic SNARE complex with Sso1 partners. However, a chimera in which the SNARE motif in syntaxin-1A is introduced into Sso1 was not functional in yeast because the chimera is retained in the ER. Through the analysis of the transport defect of Sso1/syntaxin-1A chimeric SNAREs, we found that their SNARE motifs have distinctive properties.MethodsSso1, syntaxin-1A, and Sso1/syntaxin-1A chimeric SNAREs were expressed in yeast cells and their localization and interaction with other SNAREs are analyzed.ResultsSNARE proteins containing the syntaxin-1A SNARE motif exhibit a transport defect because they form a cis-SNARE complex in the ER. Ectopic SNARE complex formation can be prevented in syntaxin-1A by binding to a Sec1/Munc-18-like (SM) protein. In contrast, the SNARE motif of Sso1 does not form an ectopic SNARE complex. Additionally, we found that the SNARE motif in syntaxin-1A, but not that in Sso1, self-interacts, even when it is in the inactive form and bound to the SM protein.ConclusionsThe SNARE motif in syntaxin-1A, but not in Sso1, likely forms ectopic SNARE complex. Because of this property, the SM protein is necessary for syntaxin-1A to prevent its promiscuous assembly and to promote its export from the ER.General significanceProperties of SNARE motifs affect characteristics of SNARE proteins. The regulatory mechanisms of SNARE proteins are, in part, designed to handle such properties.