Munc18c Interaction with Syntaxin 4 Monomers and SNARE Complex Intermediates in GLUT4 Vesicle Trafficking

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.

Section: N-terminal Region Of Syntaxin 4 Contains Basolateral Sortingmentioning

confidence: 73%

Section: Discussionmentioning

confidence: 92%

The Syntaxin 4 N Terminus Regulates Its Basolateral Targeting by Munc18c-dependent and -independent Mechanisms

Torres

Funk²,

Zegers

et al. 2011

“…Once on the cell surface, GLUT4 facilitates the uptake of excess blood glucose into the cells for disposal (20,21). Although insulin secretion and GLUT4 exocytosis are regulated by distinct trigger signals, both pathways require syntaxin-4 and SNAP-23 as the t-SNAREs, and VAMP2/synaptobrevin as the primary v-SNARE (22)(23)(24)(25)(26). In insulin secretion, the vesicle fusion reaction also involves another t-SNARE complex, syntaxin-1 and SNAP-25 (25)(26)(27).…”

mentioning

confidence: 99%

The N- and C-terminal Domains of Tomosyn Play Distinct Roles in Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Binding and Fusion Regulation

Rathore

Gulbranson

et al. 2014

Background: Tomosyn regulates vesicle fusion but its mechanism remains incompletely understood. Results: Tomosyn uses its C-terminal domain to arrest SNARE-dependent fusion reactions, whereas its N-terminal domain is required for syntaxin interaction. Conclusion:We have uncovered distinct roles of the N-and C-terminal domains of tomosyn in SNARE binding and fusion regulation. Significance: Our findings shed light upon vesicle transport in the cell.

“…However, it has been dis-puted that this N-terminal region of syntaxin 4 can independently confer binding specificity to the Munc18c isoform, as opposed to binding Munc18a or Munc18b (Munc18a and Munc18b specifically bind syntaxin isoforms 1-3). Thus, it has been suggested there are likely alternate or additional residues required to support and specify the association (37,38).…”

mentioning

confidence: 99%

The Tyrosine Phosphorylation of Munc18c Induces a Switch in Binding Specificity from Syntaxin 4 to Doc2β

Jewell

Bennett

et al. 2008

Glucose-stimulated insulin secretion is mediated by syntaxin 4-based SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein complexes and the Sec1/ Munc18 protein Munc18c. Our laboratory recently reported that Munc18c-syntaxin 4 complexes are further regulated by the competitive binding of the double C2 domain protein Doc2␤ to Munc18c, although the underlying mechanism for this is unknown. Because the Doc2␤ binding region of Munc18c contained residue Tyr-219 and this residue becomes phosphorylated in response to glucose stimulation, we hypothesized that the mechanism would involve Munc18c phosphorylation. Coimmunoprecipitation analyses using detergent lysates prepared from pervanadate-treated MIN6 beta cells revealed that the tyrosine phosphorylation of Munc18c corresponded to a 60% decrease in Glucose homeostasis requires "cross-talk" between insulin signaling in peripheral tissues (adipose and muscle) and insulin secretion from pancreatic islet beta cells. Both glucose uptake and insulin secretion are mediated by syntaxin 4-based SNARE 2 protein complexes (1, 2). The vesicle-associated SNARE protein (VAMP2) is functional in both glucose uptake and insulin granule exocytosis processes and complexes with the binary cognate receptor complex at the target membrane composed of SNAP23 and syntaxin 4 proteins (t-SNAREs) to form a SNARE heterotrimeric complex (3-11). This SNARE core complex is further regulated by the Munc18c isoform of the Sec1/Munc18 secretory protein family through its ability to directly bind to syntaxin 4 at the plasma membrane (8,(12)(13)(14)(15)(16)(17)(18)(19)(20). Although genetic models of Munc18c over-expression (12) and under-expression (13, 21) have both unequivocally substantiated Munc18c as a key and physiologically relevant regulator of glucose homeostasis by its impact upon syntaxin 4-mediated exocytosis, its precise role as a negative or positive factor in the molecular mechanism has remained unresolved. Evidence in favor of Munc18c as a negative regulator of exocytosis is principally based upon over-expression methodologies but was supported by crystallographic and NMR data showing that the neuronal Munc18 protein (nSec1/Munc18-1/ Munc18a) held its syntaxin 1 binding partner in a closed conformation in a 1:1 stoichiometric complex (22, 23) and in vitro protein-protein interaction studies stating the absence of Munc18 from the 7 S and 20 S SNARE complexes (24, 25). The role of Munc18c as a possible positive regulator in exocytosis is based upon aberrant glucose uptake and insulin secretion from Munc18c heterozygous knock-out mice and peptide interference of endogenous Munc18c-syntaxin 4 association in 3T3L1 adipocytes (13,20). In addition, recent in vitro data have detected Sec1/Munc18 proteins associated with SNARE core complexes (26 -29). Notably, these recent in vitro studies were conducted using milder detergent conditions than earlier studies that showed Sec1/Munc18 protein binding to monomeric syntaxin in cell lysates (30 -33). These differing buf...