The SM protein Sly1 accelerates assembly of the ER-Golgi SNARE complex.
ABSTRACT: Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) and Sec1/Munc18 (SM) proteins constitute the core of an ancient vesicle fusion machine that diversified into distinct sets that now function in different trafficking steps in eukaryotic cells. Deciphering their precise mode of action has proved challenging. SM proteins are thought to act primarily through one type of SNARE protein, the syntaxins. Despite high structural similarity, however, contrasting binding modes have been found for different SM proteins and syntaxins. Whereas the secretory SM protein Munc18 binds to the ?closed conformation" of syntaxin 1, the ER-Golgi SM protein Sly1 interacts only with the N-peptide of Sed5. Recent findings, however, indicate that SM proteins might interact simultaneously with both syntaxin regions. In search for a common mechanism, we now reinvestigated the Sly1/Sed5 interaction. We found that individual Sed5 adopts a tight closed conformation. Sly1 binds to both the closed conformation and the N-peptide of Sed5, suggesting that this is the original binding mode of SM proteins and syntaxins. In contrast to Munc18, however, Sly1 facilitates SNARE complex formation by loosening the closed conformation of Sed5.
Project description:Sec1Munc18-like (SM) proteins functionally interact with soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) in membrane fusion, but the mechanisms of these interactions differ. In vertebrates, SM proteins that mediate exocytosis (Munc18-1, 18-2, and 18c) bind to the closed conformation of syntaxins 1-4, which requires the N-terminal H(abc) domains and SNARE motifs of these syntaxins. In contrast, SM proteins that mediate Golgi and endoplasmic reticulum fusion (Sly1 and Vps45) bind only to short N-terminal sequences of syntaxins 5, 16, or 18, independently of their H(abc) domains and SNARE motifs. We now show that Munc18-1, Sly1, and Vps45 interact with cognate syntaxins via similar, autonomously folded N-terminal domains, but the syntaxin 5-binding surface of the Sly1 N-terminal domain is opposite to the syntaxin 1-binding surface of the Munc18-1 N-terminal domain. In transfected cells, the N-terminal domain of Sly1 specifically disrupts the structure of the Golgi complex, supporting the notion that the interaction of Sly1 with syntaxin 5 is essential for fusion. These data, together with previous results, suggest that a relatively small N-terminal domain of SM proteins is dedicated to mechanistically distinct interactions with SNAREs, leaving the remaining large parts of SM proteins free to execute their as yet unknown function as effector domains.
Project description: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.
Project description: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.
Project description:The membrane fusion necessary for vesicle trafficking is driven by the assembly of heterologous SNARE proteins orchestrated by the binding of Sec1/Munc18 (SM) proteins to specific syntaxin SNARE proteins. However, the precise mode of interaction between SM proteins and SNAREs is debated, as contrasting binding modes have been found for different members of the SM protein family, including the three vertebrate Munc18 isoforms. While different binding modes could be necessary, given their roles in different secretory processes in different tissues, the structural similarity of the three isoforms makes this divergence perplexing. Although the neuronal isoform Munc18a is well-established to bind tightly to both the closed conformation and the N-peptide of syntaxin 1a, thereby inhibiting SNARE complex formation, Munc18b and -c, which have a more widespread distribution, are reported to mainly interact with the N-peptide of their partnering syntaxins and are thought to instead promote SNARE complex formation. We have reinvestigated the interaction between Munc18c and syntaxin 4 (Syx4). Using isothermal titration calorimetry, we found that Munc18c, like Munc18a, binds to both the closed conformation and the N-peptide of Syx4. Furthermore, using a novel kinetic approach, we found that Munc18c, like Munc18a, slows down SNARE complex formation through high-affinity binding to syntaxin. This strongly suggests that secretory Munc18s in general control the accessibility of the bound syntaxin, probably preparing it for SNARE complex assembly.
Project description: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.
Project description:Both SM proteins (for Sec1/Munc18-like proteins) and SNARE proteins (for soluble NSF-attachment protein receptors) are essential for intracellular membrane fusion, but the general mechanism of coupling between their functions is unclear, in part because diverse SM protein/SNARE binding modes have been described. During synaptic vesicle exocytosis, the SM protein Munc18-1 is known to bind tightly to the SNARE protein syntaxin-1, but only when syntaxin-1 is in a closed conformation that is incompatible with SNARE complex formation. We now show that Munc18-1 also binds tightly to assembled SNARE complexes containing syntaxin-1. The newly discovered Munc18-1/SNARE complex interaction involves contacts of Munc18-1 with the N-terminal H(abc) domain of syntaxin-1 and the four-helical bundle of the assembled SNARE complex. Together with earlier studies, our results suggest that binding of Munc18-1 to closed syntaxin-1 is a specialization that evolved to meet the strict regulatory requirements of neuronal exocytosis, whereas binding of Munc18-1 to assembled SNARE complexes reflects a general function of SM proteins involved in executing membrane fusion.
Project description:The Sec1/Munc18 (SM) protein Munc18-1 and the SNAREs syntaxin-1, SNAP-25 and synaptobrevin form the core of the membrane fusion machinery that triggers neurotransmitter release. Munc18-1 binds to syntaxin-1 folded into a closed conformation and to the SNARE complex formed by the three SNAREs, which involves an open syntaxin-1 conformation. The former interaction is likely specialized for neurotransmitter release, whereas SM protein/SNARE complex interactions are likely key for all types of intracellular membrane fusion. It is currently unclear whether the closed conformation is highly or only marginally populated in isolated syntaxin-1, and whether Munc18-1 stabilizes the close conformation or helps to open it to facilitate SNARE complex formation. A detailed NMR analysis now suggests that the closed conformation is almost quantitatively populated in isolated syntaxin-1 in the absence of oligomerization, and indicates that its structure is very similar to that observed previously in the crystal structure of the Munc18-1/syntaxin-1 complex. Moreover, we demonstrate that Munc18-1 binding prevents opening of the syntaxin-1 closed conformation. These results support a model whereby the closed conformation constitutes a key intrinsic property of isolated syntaxin-1 and Munc18-1 binding stabilizes this conformation; in this model, Munc18-1 plays in addition an active role in downstream events after another factor(s) helps to open the syntaxin-1 conformation.
Project description:The Sec1/Munc18 (SM) protein family regulates intracellular trafficking through interactions with individual SNARE proteins and assembled SNARE complexes. Revealing a common mechanism of this regulation has been challenging, largely because of the multiple modes of interaction observed between SM proteins and their cognate syntaxin-type SNAREs. These modes include binding of the SM to a closed conformation of syntaxin, binding to the N-terminal peptide of syntaxin, binding to assembled SNARE complexes, and/or binding to nonsyntaxin SNAREs. The SM protein Vps45p, which regulates endosomal trafficking in yeast, binds the conserved N-terminal peptide of the syntaxin Tlg2p. We used size exclusion chromatography and a quantitative fluorescent gel mobility shift assay to reveal an additional binding site that does not require the Tlg2p N-peptide. Characterization of Tlg2p mutants and truncations indicate that this binding site corresponds to a closed conformation of Tlg2p. Furthermore, the Tlg2p N-peptide competes with the closed conformation for binding, suggesting a fundamental regulatory mechanism for SM-syntaxin interactions in SNARE assembly and membrane fusion.
Project description:Fusion of intracellular trafficking vesicles is mediated by the assembly of SNARE proteins into membrane-bridging complexes. SNARE-mediated membrane fusion requires Sec1/Munc18-family (SM) proteins, SNARE chaperones that can function as templates to catalyze SNARE complex assembly. Paradoxically, the SM protein Munc18-1 traps the Qa-SNARE protein syntaxin-1 in an autoinhibited closed conformation. Here we present the structure of a second SM-Qa-SNARE complex, Vps45-Tlg2. Strikingly, Vps45 holds Tlg2 in an open conformation, with its SNARE motif disengaged from its Habc domain and its linker region unfolded. The domain 3a helical hairpin of Vps45 is unfurled, exposing the presumptive R-SNARE binding site to allow template complex formation. Although Tlg2 has a pronounced tendency to form homo-tetramers, Vps45 can rescue Tlg2 tetramers into stoichiometric Vps45-Tlg2 complexes. Our findings demonstrate that SM proteins can engage Qa-SNAREs using at least two different modes, one in which the SNARE is closed and one in which it is open.
Project description: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.