Specific SNARE complex binding mode of the Sec1/Munc-18 protein, Sec1p

Princeton University, Princeton, New Jersey, United States
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 12/2006; 103(47):17730-5. DOI: 10.1073/pnas.0605448103
Source: PubMed


The Sec1/Munc-18 (SM) family of proteins is required for vesicle fusion in eukaryotic cells and has been linked to the membrane-fusion proteins known as soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SM proteins may activate the target-membrane SNARE, syntaxin, for assembly into the fusogenic SNARE complex. In support of an activation role, SM proteins bind directly to their cognate syntaxins. An exception is the yeast Sec1p, which does not bind the yeast plasma-membrane syntaxin, Sso1p. This exception could be explained if the SM interaction motif were blocked by the highly stable closed conformation of Sso1p. We tested the possibility of a latent binding motif using sso1 mutants in yeast and reconstituted the Sec1p binding specificity observed in vivo with purified proteins in vitro. Our results indicate there is no latent binding motif in Sso1p. Instead, Sec1p binds specifically to the ternary SNARE complex, with no detectable binding to the binary t-SNARE complex or any of the three individual SNAREs in their uncomplexed forms. We propose that vesicle fusion requires a specific interaction between the SM protein and the ternary SNARE complex.

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    • "A central challenge in developing general models for SM protein function is to reconcile the apparently divergent modes of SNARE binding reported for the four SM subfamilies. In this study, we have shown that Vps33 interacts with SNAREs and SNARE complexes in a manner closely analogous to Sec1 (Togneri et al., 2006; Morgera et al., 2011). Specifically, Vps33 interacts with individual SNARE domains and, with significantly greater affinity, quaternary SNARE complexes. "
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    ABSTRACT: 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 a SM proteins and subunit of the Vps-C complexes CORVET and HOPS, 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 in which HOPS associates with N-terminal domains of Vam3 and Vam7 through Vps33-independent interactions, 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 central conserved attribute of SM proteins.
    Preview · Article · Oct 2012 · Molecular biology of the cell
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    • "Then, Snc1/2p associates with the complex to form the SNAREpin complex, which acts as an engine to release biochemical energy to drive the vesicular and plasma membranes together. The yeast SM protein, Sec1p, regulates the SNARE complexes and the fusion rate by directly binding to the assembled SNAREpin (pattern 2) [28,29]. "
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    ABSTRACT: Network motifs, recurring subnetwork patterns, provide significant insight into the biological networks which are believed to govern cellular processes. We present a comparative network motif experimental approach, which helps to explain complex biological phenomena and increases the understanding of biological functions at the molecular level by exploring evolutionary design principles of network motifs. Using this framework to analyze the SM (Sec1/Munc18)-SNARE (N-ethylmaleimide-sensitive factor activating protein receptor) system in exocytic membrane fusion in yeast and neurons, we find that the SM-SNARE network motifs of yeast and neurons show distinct dynamical behaviors. We identify the closed binding mode of neuronal SM (Munc18-1) and SNARE (syntaxin-1) as the key factor leading to mechanistic divergence of membrane fusion systems in yeast and neurons. We also predict that it underlies the conflicting observations in SM overexpression experiments. Furthermore, hypothesis-driven lipid mixing assays validated the prediction. Therefore this study provides a new method to solve the discrepancies and to generalize the functional role of SM proteins.
    Full-text · Article · Mar 2012 · BMC Systems Biology
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    • "Note that this C-terminal Sec9 construct used in all the in vitro studies is the domain of Sec9 homologous to the mammalian SNAP-25 protein and is sufficient for Sec9 function in yeast (Brennwald et al., 1994). C-terminally V5-His 6 tagged Sec1 was expressed in Saccharomyces cerevisiae from the pYES/CT Sec1-V5-His 6 plasmid (Togneri et al., 2006). After induction with 2% galactose for 8–10 h, cells were harvested and resuspended in wash buffer ( "
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    ABSTRACT: Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function-it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6-Sec1 interaction is exclusive of Sec6-Sec9 but compatible with Sec6-exocyst assembly. In contrast, the Sec6-exocyst interaction is incompatible with Sec6-Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6-exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion.
    Full-text · Article · Nov 2011 · Molecular biology of the cell
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