Structural basis for the Golgi membrane recruitment of Sly1p by Sed5p

Cytosolic Sec1/munc18-like proteins (SM proteins) are recruited to membrane fusion sites by interaction with syntaxin-type SNARE proteins, constituting indispensable positive regulators of intracellular membrane fusion. Here we present the crystal structure of the yeast SM protein Sly1p in complex with a short N-terminal peptide derived from the Golgi-resident syntaxin Sed5p. Sly1p folds, similarly to neuronal Sec1, into a three-domain arch-shaped assembly, and Sed5p interacts in a helical conformation predominantly with domain I of Sly1p on the opposite site of the nSec1/syntaxin-1-binding site. Sequence conservation of the major interactions suggests that homologues of Sly1p as well as the paralogous Vps45p group bind their respective syntaxins in the same way. Furthermore, we present indirect evidence that nSec1 might be able to contact syntaxin 1 in a similar fashion. The observed Sly1p-Sed5p interaction mode therefore indicates how SM proteins can stay associated with the assembling fusion machinery in order to participate in late fusion steps.     

Molecular dissection of the Munc18c/syntaxin4 interaction: implications for regulation of membrane trafficking

Sec1p/Munc18 (SM) proteins are believed to play an integral role in vesicle transport through their interaction with SNAREs. Different SM proteins have been shown to interact with SNAREs via different mechanisms, leading to the conclusion that their function has diverged. To further explore this notion, in this study, we have examined the molecular interactions between Munc18c and its cognate SNAREs as these molecules are ubiquitously expressed in mammals and likely regulate a universal plasma membrane trafficking step. Thus, Munc18c binds to monomeric syntaxin4 and the N-terminal 29 amino acids of syntaxin4 are necessary for this interaction. We identified key residues in Munc18c and syntaxin4 that determine the N-terminal interaction and that are consistent with the N-terminal binding mode of yeast proteins Sly1p and Sed5p. In addition, Munc18c binds to the syntaxin4/SNAP23/VAMP2 SNARE complex. Pre-assembly of the syntaxin4/Munc18c dimer accelerates the formation of SNARE complex compared to assembly with syntaxin4 alone. These data suggest that Munc18c interacts with its cognate SNAREs in a manner that resembles the yeast proteins Sly1p and Sed5p rather than the mammalian neuronal proteins Munc18a and syntaxin1a. The Munc18c-SNARE interactions described here imply that Munc18c could play a positive regulatory role in SNARE assembly.     

How Tlg2p/syntaxin 16 'snares' Vps45

Soluble N-ethylmaleimide sensitive factor-attachment protein receptors (SNAREs) and Sec1p/Munc18-homologs (SM proteins) play key roles in intracellular membrane fusion. The SNAREs form tight four-helix bundles (core complexes) that bring the membranes together, but it is unclear how this activity is coupled to SM protein function. Studies of the yeast trans-Golgi network (TGN)/endosomal SNARE complex, which includes the syntaxin-like SNARE Tlg2p, have suggested that its assembly requires activation by binding of the SM protein Vps45p to the cytoplasmic region of Tlg2p folded into a closed conformation. Nuclear magnetic resonance and biochemical experiments now show that Tlg2p and Pep12p, a late- endosomal syntaxin that interacts functionally but not directly with Vps45p, have a domain structure characteristic of syntaxins but do not adopt a closed conformation. Tlg2p binds tightly to Vps45p via a short N-terminal peptide motif that is absent in Pep12p. The Tlg2p/Vps45p binding mode is shared by the mammalian syntaxin 16, confirming that it is a Tlg2p homolog, and resembles the mode of interaction between the SM protein Sly1p and the syntaxins Ufe1p and Sed5p. Thus, this mechanism represents the most widespread mode of coupling between syntaxins and SM proteins.     

Multiple SNARE interactions of an SM protein: Sed5p/Sly1p binding is dispensable for transport

Sec1/Munc18 (SM) proteins are central to intracellular transport and neurotransmitter release but their exact role is still elusive. Several SM proteins, like the neuronal N-Sec1 and the yeast Sly1 protein, bind their cognate t-SNAREs with high affinity. This has been thought to be critical for their function. Here, we show that various mutant forms of Sly1p and the Golgi-localized syntaxin Sed5p, which abolish their high-affinity interaction, are fully functional in vivo, indicating that the tight interaction of the two molecules per se is not relevant for proper function. Mutant Sly1p unable to bind Sed5p is excluded from core SNARE complexes, also demonstrating that Sly1p function is not directly coupled to assembled SNARE complexes thought to execute membrane fusion. We also find that wild-type Sly1p and mutant Sly1p unable to bind Sed5p directly interact with selected ER-to-Golgi and intra-Golgi nonsyntaxin SNAREs. The newly identified, direct interactions of the SM protein with nonsytaxin SNAREs might provide a molecular mechanism by which SNAREs can be activated to engage in pairing and assemble into fusogenic SNARE complexes.     

Sly1 protein bound to Golgi syntaxin Sed5p allows assembly and contributes to specificity of SNARE fusion complexes

Fusion of transport vesicles with their target organelles involves specific membrane proteins, SNAREs, which form tight complexes bridging the membranes to be fused. Evidence from yeast and mammals indicates that Sec1 family proteins act as regulators of membrane fusion by binding to the target membrane SNAREs. In experiments with purified proteins, we now made the observation that the ER to Golgi core SNARE fusion complex could be assembled on syntaxin Sed5p tightly bound to the Sec1-related Sly1p. Sly1p also bound to preassembled SNARE complexes in vitro and was found to be part of a vesicular/target membrane SNARE complex immunoprecipitated from yeast cell lysates. This is in marked contrast to the exocytic SNARE assembly in neuronal cells where high affinity binding of N-Sec1/Munc-18 to syntaxin 1A precluded core SNARE fusion complex formation. We also found that the kinetics of SNARE complex formation in vitro with either Sly1p-bound or free Sed5p was not significantly different. Importantly, several presumably nonphysiological SNARE complexes easily generated with Sed5p did not form when the syntaxin was first bound to Sly1p. This indicates for the first time that a Sec1 family member contributes to the specificity of SNARE complex assembly.     

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.     

A Targeted siRNA Screen to Identify SNAREs Required for Constitutive Secretion in Mammalian Cells

The role of SNAREs in mammalian constitutive secretion remains poorly defined. To address this, we have developed a novel flow cytometry‐based assay for measuring constitutive secretion and have performed a targeted SNARE and Sec1/Munc18 (SM) protein‐specific siRNA screen (38 SNAREs, 4 SNARE‐like proteins and 7 SM proteins). We have identified the endoplasmic reticulum (ER)/Golgi SNAREs syntaxin 5, syntaxin 17, syntaxin 18, GS27, SLT1, Sec20, Sec22b, Ykt6 and the SM protein Sly1, along with the post‐Golgi SNAREs SNAP‐29 and syntaxin 19, as being required for constitutive secretion. Depletion of SNAP‐29 or syntaxin 19 causes a decrease in the number of fusion events at the cell surface and in SNAP‐29‐depleted cells causes an increase in the number of docked vesicles at the plasma membrane as determined by total internal reflection fluorescence (TIRF) microscopy. Analysis of syntaxin 19‐interacting partners by mass spectrometry indicates that syntaxin 19 can form SNARE complexes with SNAP‐23, SNAP‐25, SNAP‐29, VAMP3 and VAMP8, supporting its role in Golgi to plasma membrane transport or fusion. Surprisingly, we have failed to detect any requirement for a post‐Golgi‐specific R‐SNARE in this process.     

The Sec1/Munc18 Protein Vps45 Regulates Cellular Levels of Its SNARE Binding Partners Tlg2 and Snc2 in Saccharomyces cerevisiae

Intracellular membrane trafficking pathways must be tightly regulated to ensure proper functioning of all eukaryotic cells. Central to membrane trafficking is the formation of specific SNARE (soluble N-ethylmeleimide-sensitive factor attachment protein receptor) complexes between proteins on opposing lipid bilayers. The Sec1/Munc18 (SM) family of proteins play an essential role in SNARE-mediated membrane fusion, and like the SNAREs are conserved through evolution from yeast to humans. The SM protein Vps45 is required for the formation of yeast endosomal SNARE complexes and is thus essential for traffic through the endosomal system. Here we report that, in addition to its role in regulating SNARE complex assembly, Vps45 regulates cellular levels of its SNARE binding partners: the syntaxin Tlg2 and the v-SNARE Snc2: Cells lacking Vps45 have reduced cellular levels of Tlg2 and Snc2; and elevation of Vps45 levels results in concomitant increases in the levels of both Tlg2 and Snc2. As well as regulating traffic through the endosomal system, the Snc v-SNAREs are also required for exocytosis. Unlike most vps mutants, cells lacking Vps45 display multiple growth phenotypes. Here we report that these can be reversed by selectively restoring Snc2 levels in vps45 mutant cells. Our data indicate that as well as functioning as part of the machinery that controls SNARE complex assembly, Vps45 also plays a key role in determining the levels of its cognate SNARE proteins; another key factor in regulation of membrane traffic.     

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.     

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.     

Sec1/Munc18 protein Vps33 binds to SNARE domains and the quaternary SNARE complex

Soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) proteins catalyze membrane fusion events in the secretory and endolysosomal systems, and all SNARE-mediated fusion processes require cofactors of the Sec1/Munc18 (SM) family. Vps33 is an SM protein and subunit of the Vps-C complexes HOPS (homotypic fusion and protein sorting) and CORVET (class C core vacuole/endosome tethering), which are central regulators of endocytic traffic. Here we present biochemical studies of interactions between Saccharomyces cerevisiae vacuolar SNAREs and the HOPS holocomplex or Vps33 alone. HOPS binds the N-terminal H_abc domain of the Qa-family SNARE Vam3, but Vps33 is not required for this interaction. Instead, Vps33 binds the SNARE domains of Vam3, Vam7, and Nyv1. Vps33 directly binds vacuolar quaternary SNARE complexes, and the affinity of Vps33 for SNARE complexes is greater than for individual SNAREs. Through targeted mutational analyses, we identify missense mutations of Vps33 that produce a novel set of defects, including cargo missorting and the loss of Vps33-HOPS association. Together these data suggest a working model for membrane docking: HOPS associates with N-terminal domains of Vam3 and Vam7 through Vps33-independent interactions, which are followed by binding of Vps33, the HOPS SM protein, to SNARE domains and finally to the quaternary SNARE complex. Our results also strengthen the hypothesis that SNARE complex binding is a core attribute of SM protein function.     

Crystal Structures of the Sec1/Munc18 (SM) Protein Vps33, Alone and Bound to the Homotypic Fusion and Vacuolar Protein Sorting (HOPS) Subunit Vps16*

Intracellular membrane fusion requires the regulated assembly of SNARE (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor) proteins anchored in the apposed membranes. To exert the force required to drive fusion between lipid bilayers, juxtamembrane SNARE motifs zipper into four-helix bundles. Importantly, SNARE function is regulated by additional factors, none more extensively studied than the SM (Sec1/Munc18-like) proteins. SM proteins interact with both individual SNAREs and SNARE complexes, likely chaperoning SNARE complex formation and protecting assembly intermediates from premature disassembly by NSF. Four families of SM proteins have been identified, and representative members of two of these families (Sec1/Munc18 and Sly1) have been structurally characterized. We report here the 2.6 Å resolution crystal structure of an SM protein from the third family, Vps33. Although Vps33 shares with the first two families the same basic three-domain architecture, domain 1 is displaced by 15 Å, accompanied by a 40° rotation. A unique feature of the Vps33 family of SM proteins is that its members function as stable subunits within a multi-subunit tethering complex called HOPS (homotypic fusion and vacuolar protein sorting). Integration into the HOPS complex depends on the interaction between Vps33 and a second HOPS subunit, Vps16. The crystal structure of Vps33 bound to a C-terminal portion of Vps16, also at 2.6 Å resolution, reveals the structural basis for this interaction. Despite the extensive interface between the two HOPS subunits, the conformation of Vps33 is only subtly affected by binding to Vps16.     

Identification of the Neuroblastoma-amplified Gene Product as a Component of the Syntaxin 18 Complex Implicated in Golgi-to-Endoplasmic Reticulum Retrograde Transport

Syntaxin 18, a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) protein implicated in endoplasmic reticulum (ER) membrane fusion, forms a complex with other SNAREs (BNIP1, p31, and Sec22b) and several peripheral membrane components (Sly1, ZW10, and RINT-1). In the present study, we showed that a peripheral membrane protein encoded by the neuroblastoma-amplified gene (NAG) is a subunit of the syntaxin 18 complex. NAG encodes a protein of 2371 amino acids, which exhibits weak similarity to yeast Dsl3p/Sec39p, an 82-kDa component of the complex containing the yeast syntaxin 18 orthologue Ufe1p. Under conditions favoring SNARE complex disassembly, NAG was released from syntaxin 18 but remained in a p31-ZW10-RINT-1 subcomplex. Binding studies showed that the extreme N-terminal region of p31 is responsible for the interaction with NAG and that the N- and the C-terminal regions of NAG interact with p31 and ZW10-RINT-1, respectively. Knockdown of NAG resulted in a reduction in the expression of p31, confirming their intimate relationship. NAG depletion did not substantially affect Golgi morphology and protein export from the ER, but it caused redistribution of Golgi recycling proteins accompanied by a defect in protein glycosylation. These results together suggest that NAG links between p31 and ZW10-RINT-1 and is involved in Golgi-to-ER transport.     

The Aspergillus nidulans syntaxin PepAPep12 is regulated by two Sec1/Munc‐18 proteins to mediate fusion events at early endosomes,late endosomes and vacuoles

Syntaxins are target‐SNAREs that crucially contribute to determine membrane compartment identity. Three syntaxins, Tlg2p, Pep12p and Vam3p, organize the yeast endovacuolar system. Remarkably, filamentous fungi lack the equivalent of the yeast vacuolar syntaxin Vam3p, making unclear how these organisms regulate vacuole fusion. We show that the nearly essential Aspergillus nidulans syntaxin PepA^Pep12, present in all endocytic compartments between early endosomes and vacuoles, shares features of Vam3p and Pep12p, and is capable of forming compositional equivalents of all known yeast endovacuolar SNARE bundles including that formed by yeast Vam3p for vacuolar fusion. Our data further indicate that regulation by two Sec1/Munc‐18 proteins, Vps45 in early endosomes and Vps33 in early and late endosomes/vacuoles contributes to the wide domain of PepA^Pep12 action. The syntaxin TlgB^Tlg2 localizing to the TGN appears to mediate retrograde traffic connecting post‐Golgi (sorting) endosomes with the TGN. TlgB^Tlg2 is dispensable for growth but becomes essential if the early Golgi syntaxin SedV^Sed5 is compromised, showing that the Golgi can function with a single syntaxin, SedV^Sed5. Remarkably, its pattern of associations with endosomal SNAREs is consistent with SedV^Sed5 playing roles in retrograde pathway(s) connecting endocytic compartments downstream of the post‐Golgi endosome with the Golgi, besides more conventional intra‐Golgi roles.     

SM-protein-controlled ER-associated degradation discriminates between different SNAREs

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a specialized activity of the ubiquitin-proteasome system that is involved in clearing the ER of aberrant proteins and regulating the levels of specific ER-resident proteins. Here we show that the yeast ER-SNARE Ufe1, a syntaxin (Qa-SNARE) involved in ER membrane fusion and retrograde transport from the Golgi to the ER, is prone to degradation by an ERAD-like mechanism. Notably, Ufe1 is protected against degradation through binding to Sly1, a known SNARE regulator of the Sec1-Munc18 (SM) protein family. This mechanism is specific for Ufe1, as the stability of another Sly1 partner, the Golgi Qa-SNARE Sed5, is not influenced by Sly1 interaction. Thus, our findings identify Sly1 as a discriminating regulator of SNARE levels and indicate that Sly1-controlled ERAD might regulate the balance between different Qa-SNARE proteins.     

The Sec1p/Munc18 protein Vps45p binds its cognate SNARE proteins via two distinct modes

Sec1p/Munc18 (SM) proteins are essential for SNARE-mediated membrane trafficking. The formulation of unifying hypotheses for the function of the SM protein family has been hampered by the observation that two of its members bind their cognate syntaxins (Sxs) in strikingly different ways. The SM protein Vps45p binds its Sx Tlg2p in a manner analogous to that captured by the Sly1p-Sed5p crystal structure, whereby the NH2-terminal peptide of the Sx inserts into a hydrophobic pocket on the outer face of domain I of the SM protein. In this study, we report that although this mode of interaction is critical for the binding of Vps45p to Tlg2p, the SM protein also binds Tlg2p-containing SNARE complexes via a second mode that involves neither the NH2 terminus of Tlg2p nor the region of Vps45p that facilitates this interaction. Our findings point to the possibility that SM proteins interact with their cognate SNARE proteins through distinct mechanisms at different stages in the SNARE assembly/disassembly cycle.     

Structure-based functional analysis reveals a role for the SM protein Sly1p in retrograde transport to the endoplasmic reticulum

Sec1p/Munc18 (SM) proteins are essential for membrane fusion events in eukaryotic cells. Here we describe a systematic, structure-based mutational analysis of the yeast SM protein Sly1p, which was previously shown to function in anterograde endoplasmic reticulum (ER)-to-Golgi and intra-Golgi protein transport. Five new temperature-sensitive (ts) mutants, each carrying a single amino acid substitution in Sly1p, were identified. Unexpectedly, not all of the ts mutants exhibited striking anterograde ER-to-Golgi transport defects. For example, in cells of the novel sly1-5 mutant, transport of newly synthesized lysosomal and secreted proteins was still efficient, but the ER-resident Kar2p/BiP was missorted to the outside of the cell, and two proteins, Sed5p and Rer1p, which normally shuttle between the Golgi and the ER, failed to relocate to the ER. We also discovered that in vivo, Sly1p was associated with a SNARE complex formed on the ER, and that in vitro, the SM protein directly interacted with the ER-localized nonsyntaxin SNAREs Use1p/Slt1p and Sec20p. Furthermore, several conditional mutants defective in Golgi-to-ER transport were synthetically lethal with sly1-5. Together, these results indicate a previously unrecognized function of Sly1p in retrograde transport to the endoplasmic reticulum.     

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.     

Direct interaction between the COG complex and the SM protein,Sly1, is required for Golgi SNARE pairing

The crucial roles of Sec1/Munc18 (SM)‐like proteins in membrane fusion have been evidenced in genetic and biochemical studies. SM proteins interact directly with SNAREs and contribute to SNARE pairing by a yet unclear mechanism. Here, we show that the SM protein, Sly1, interacts directly with the conserved oligomeric Golgi (COG) tethering complex. The Sly1–COG interaction is mediated by the Cog4 subunit, which also interacts with Syntaxin 5 through a different binding site. We provide evidence that disruption of Cog4–Sly1 interaction impairs pairing of SNAREs involved in intra‐Golgi transport thereby markedly attenuating Golgi‐to‐ER retrograde transport. These results highlight the mechanism by which SM proteins link tethering to SNAREpin assembly.     

Interaction of SNAREs with ArfGAPs precedes recruitment of Sec18p/NSF

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are key components of the fusion machinery in vesicular transport and in homotypic membrane fusion. We previously found that ADP-ribosylation factor GTPase activating proteins (ArfGAPs) promoted a conformational change on SNAREs that allowed recruitment of the small GTPase Arf1p in stoichiometric amounts. Here, we show that the ArfGAP Gcs1p accelerates vesicle (v)-target membrane (t)-SNARE complex formation in vitro, indicating that ArfGAPs may act as folding chaperones. These SNARE complexes were resolved in the presence of ATP by the yeast homologues of alpha-soluble N-ethylmaleimide-sensitive factor attachment protein and N-ethylmaleimide-sensitive factor, Sec17p and Sec18p, respectively. In addition, Sec18p and Sec17p also recognized the "activated" SNAREs even when they were not engaged in v-t-SNARE complexes. Here again, the induction of a conformational change by ArfGAPs was essential. Surprisingly, recruitment of Sec18p to SNAREs did not require Sec17p or ATP hydrolysis. Moreover, Sec18p displaced prebound Arf1p from SNAREs, indicating that Sec18p may have more than one function: first, to ensure that all vesicle coat proteins are removed from the SNAREs before the engagement in a trans-SNARE complex; and second, to resolve cis-SNARE complexes after fusion has occurred.