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Vesicular transport of cellular cargo requires targeted membrane fusion and formation of a SNARE protein complex that draws the two apposing fusing membranes together. Insulin-regulated delivery and fusion of glucose transporter-4 storage vesicles at the cell surface is dependent on two key proteins: the SNARE integral membrane protein Syntaxin4 (Sx4) and the soluble regulatory protein Munc18c. Many reported in vitro studies of Munc18c:Sx4 interactions and of SNARE complex formation have used soluble Sx4 constructs lacking the native transmembrane domain. As a consequence, the importance of the Sx4 C-terminal anchor remains poorly understood. Here we show that soluble C-terminally truncated Sx4 dissociates more rapidly from Munc18c than Sx4 where the C-terminal transmembrane domain is replaced with a T4-lysozyme fusion. We also show that Munc18c appears to inhibit SNARE complex formation when soluble C-terminally truncated Sx4 is used but does not inhibit SNARE complex formation when Sx4 is C-terminally anchored (by a C-terminal His-tag bound to resin, by a C-terminal T4L fusion or by the native C-terminal transmembrane domain in detergent micelles). We conclude that the C-terminus of Sx4 is critical for its interaction with Munc18c, and that the reported inhibitory role of Munc18c may be an artifact of experimental design. These results support the notion that a primary role of Munc18c is to support SNARE complex formation and membrane fusion. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

When nerve cells communicate, vesicles from one neuron fuse with the presynaptic membrane releasing chemicals that signal to the next. Similarly, when insulin binds its receptor on adipocytes or muscle, glucose transporter-4 vesicles fuse with the cell membrane, allowing glucose to be imported. These essential processes require the interaction of SNARE proteins on vesicle and cell membranes, as well as the enigmatic protein Mund 8 that binds the SNARE protein Syntaxin. Here, we show that in solution the neuronal protein Syntaxinla interacts with Mund 8-1 whether or not the Syntaxinla N-peptide is present. Conversely, the adipocyte protein Syntaxin4 does not bind its partner Munc18c unless the N-peptide is present. Solution-scattering data for the Munc18-1:Syntaxin1a complex in the absence of the N-peptide indicates that this complex adopts the inhibitory closed binding mode, exemplified by a crystal structure of the complex. However, when the N-peptide is present, the solution-scattering data indicate both Syntaxinla and Syntaxin4 adopt extended conformations in complexes with their respective Mund 8 partners. The low-resolution solution structure of the open Munc18:Syntaxin binding mode was modeled using data from cross-linking/mass spectrometry, small-angle X-ray scattering, and small-angle neutron scattering with contrast variation, indicating significant differences in MundRSyntaxin interactions compared with the closed binding mode. Overall, our results indicate that the neuronal Mund8-1:Syntaxin1a proteins can adopt two alternate and functionally distinct binding modes, closed and open, depending on the presence of the N-peptide, whereas Munc18c:Syntaxin4 adopts only the open binding mode. [ABSTRACT FROM AUTHOR]