Negative regulation of syntaxin4/SNAP-23/VAMP2-mediated membrane fusion by Munc18c <i>In Vitro</i>

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

Skeletal muscle munc18c and syntaxin 4 in human obesity

<p>Abstract</p> <p>Background</p> <p>Animal and cell culture data suggest a critical role for Munc18c and Syntaxin 4 proteins in insulin mediated glucose transport in skeletal muscle, but no studies have been published in humans.</p> <p>Methods</p> <p>We investigated the effect of a 12 vs. 48 hr fast on insulin action and skeletal muscle Munc18c and Syntaxin 4 protein in lean and obese subjects. Healthy lean (n = 14; age = 28.0 +/- 1.4 yr; BMI = 22.8 +/- 0.42 kg/m²) and obese subjects (n = 11; age = 34.6 +/- 2.3 yr; BMI = 36.1 +/- 1.5 kg/m²) were studied twice following a 12 and 48 hr fast. Skeletal muscle biopsies were obtained before a 3 hr 40 mU/m²/min hyperinsulinemic-euglycemic clamp with [6,6-²H₂]glucose infusion.</p> <p>Results</p> <p>Glucose rate of disappearance (Rd) during the clamp was lower in obese vs. lean subjects after the 12 hr fast (obese: 6.25 +/- 0.67 vs. lean: 9.42 +/- 1.1 mg/kgFFM/min, p = 0.007), and decreased significantly in both groups after the 48 hr fast (obese 3.49 +/- 0.31 vs. lean: 3.91 +/- 0.42 mg/kgFFM/min, p = 0.002). Munc18c content was not significantly different between lean and obese subjects after the 12 hour fast, and decreased after the 48 hr fast in both groups (p = 0.013). Syntaxin 4 content was not altered by obesity or fasting duration. There was a strong positive relationship between plasma glucose concentration and Munc18c content in lean and obese subjects during both 12 and 48 hr fasts (R²= 0.447, p = 0.0015). Significant negative relationships were also found between Munc18c and FFA (p = 0.041), beta-hydroxybutyrate (p = 0.039), and skeletal muscle AKT content (p = 0.035) in lean and obese subjects.</p> <p>Conclusion</p> <p>These data indicate Munc18c and Syntaxin 4 are present in human skeletal muscle. Munc18c content was not significantly different between lean and obese subjects, and is therefore unlikely to explain obesity-induced insulin resistance. Munc18c content decreased after prolonged fasting in lean and obese subjects concurrently with reduced insulin action. These data suggest changes in Munc18c content in skeletal muscle are associated with short-term changes in insulin action in humans.</p

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

Milligram quantities of homogeneous recombinant full-length mouse Munc18c from Escherichia coli cultures

Vesicle fusion is an indispensable cellular process required for eukaryotic cargo delivery. The Sec/Munc18 protein Munc18c is essential for insulin-regulated trafficking of glucose transporter4 (GLUT4) vesicles to the cell surface in muscle and adipose tissue. Previously, our biophysical and structural studies have used Munc18c expressed in SF9 insect cells. However to maximize efficiency, minimize cost and negate any possible effects of post-translational modifications of Munc18c, we investigated the use of Escherichia coli as an expression host for Munc18c. We were encouraged by previous reports describing Munc18c production in E. coli cultures for use in in vitro fusion assay, pulldown assays and immunoprecipitations. Our approach differs from the previously reported method in that it uses a codon-optimized gene, lower temperature expression and autoinduction media. Three N-terminal His-tagged constructs were engineered, two with a tobacco etch virus (TEV) or thrombin protease cleavage site to enable removal of the fusion tag. The optimized protocol generated 1-2 mg of purified Munc18c per L of culture at much reduced cost compared to Munc18c generated using insect cell culture. The purified recombinant Munc18c protein expressed in bacteria was monodisperse, monomeric, and functional. In summary, we developed methods that decrease the cost and time required to generate functional Munc18c compared with previous insect cell protocols, and which generates sufficient purified protein for structural and biophysical studies

Munc18a Scaffolds SNARE Assembly to Promote Membrane Fusion

Munc18a is an SM protein required for SNARE-mediated fusion. The molecular details of how Munc18a acts to enhance neurosecretion have remained elusive. Here, we use in vitro fusion assays to characterize how specific interactions between Munc18a and the neuronal SNAREs enhance the rate and extent of fusion. We show that Munc18a interacts directly and functionally with the preassembled t-SNARE complex. Analysis of Munc18a point mutations indicates that Munc18a interacts with helix C of the Syntaxin1a NRD in the t-SNARE complex. Replacement of the t-SNARE SNAP25b with yeast Sec9c had little effect, suggesting that Munc18a has minimal contact with SNAP25b within the t-SNARE complex. A chimeric Syntaxin built of the Syntaxin1a NRD and the H3 domain of yeast Sso1p and paired with Sec9c eliminated stimulation of fusion, suggesting that Munc18a/Syntaxin1a H3 domain contacts are important. Additionally, a Syntaxin1A mutant lacking a flexible linker region that allows NRD movement abolished stimulation of fusion. These experiments suggest that Munc18a binds to the Syntaxin1a NRD and H3 domain within the assembled t-SNARE complex, positioning them for productive VAMP2 binding. In this capacity, Munc18a serves as a platform for trans-SNARE complex assembly that facilitates efficient SNARE-mediated membrane fusion