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• W2089957341 abstract "In the process of insulin-stimulated GLUT4 vesicle exocytosis, Munc18c has been proposed to control SNARE complex formation by inactivating syntaxin 4 in a self-associated conformation. Using in vivo fluorescence resonance energy transfer in 3T3L1 adipocytes, co-immunoprecipitation, and in vitro binding assays, we provide data to indicate that Munc18c also associates with nearly equal affinity to a mutant of syntaxin 4 in a constitutively open (unfolded) state (L173A/E174A; LE). To bind to the open conformation of syntaxin 4, we found that Munc18c requires an interaction with the N terminus of syntaxin 4, which resembles Sly1 interaction with the N terminus of ER/Golgi syntaxins. However, both N and C termini of syntaxin 4 are required for Munc18c binding, since a mutation in the syntaxin 4 SNARE domain (I241A) reduces the interaction, irrespective of syntaxin 4 conformation. Using an optical reporter for syntaxin 4-SNARE pairings in vivo, we demonstrate that Munc18c blocks recruitment of SNAP23 to wild type syntaxin 4 yet associates with syntaxin 4LE-SNAP23 Q-SNARE complexes. Fluorescent imaging of GLUT4 vesicles in 3T3L1 adipocytes revealed that syntaxin 4LE expressed with Munc18c bypasses the requirement of insulin for GLUT4 vesicle plasma membrane docking. This effect was attenuated by reducing the Munc18c-syntaxin 4LE interaction with the I241A mutation, indicating that Munc18c facilitates vesicle docking. Therefore, in contradiction to previous models, our data indicates that the conformational “opening” of syntaxin 4 rather than the dissociation of Munc18c is the critical event required for GLUT4 vesicle docking. In the process of insulin-stimulated GLUT4 vesicle exocytosis, Munc18c has been proposed to control SNARE complex formation by inactivating syntaxin 4 in a self-associated conformation. Using in vivo fluorescence resonance energy transfer in 3T3L1 adipocytes, co-immunoprecipitation, and in vitro binding assays, we provide data to indicate that Munc18c also associates with nearly equal affinity to a mutant of syntaxin 4 in a constitutively open (unfolded) state (L173A/E174A; LE). To bind to the open conformation of syntaxin 4, we found that Munc18c requires an interaction with the N terminus of syntaxin 4, which resembles Sly1 interaction with the N terminus of ER/Golgi syntaxins. However, both N and C termini of syntaxin 4 are required for Munc18c binding, since a mutation in the syntaxin 4 SNARE domain (I241A) reduces the interaction, irrespective of syntaxin 4 conformation. Using an optical reporter for syntaxin 4-SNARE pairings in vivo, we demonstrate that Munc18c blocks recruitment of SNAP23 to wild type syntaxin 4 yet associates with syntaxin 4LE-SNAP23 Q-SNARE complexes. Fluorescent imaging of GLUT4 vesicles in 3T3L1 adipocytes revealed that syntaxin 4LE expressed with Munc18c bypasses the requirement of insulin for GLUT4 vesicle plasma membrane docking. This effect was attenuated by reducing the Munc18c-syntaxin 4LE interaction with the I241A mutation, indicating that Munc18c facilitates vesicle docking. Therefore, in contradiction to previous models, our data indicates that the conformational “opening” of syntaxin 4 rather than the dissociation of Munc18c is the critical event required for GLUT4 vesicle docking. Soluble N-ethylmaleimide-sensitive factor (NSF) 3The abbreviations used are: NSF, N-ethylmaleimide-sensitive factor; SNARE, soluble NSF attachment protein receptor; SM, Sec1/Munc18; FRET, fluorescence resonance energy transfer; FRAP, fluorescence recovery after photobleaching; GST, glutathione S-transferase; GFP, green fluorescent protein; YFP, yellow fluorescent protein; RFP, red fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; CFP, cyan fluorescent protein. attachment protein receptors (SNAREs) are central to vesicle fusion events in all eukaryotes. SNARE proteins anchored to both transport vesicles and their target membranes overcome the forces necessary for fusion by binding with high affinity in a ternary core complex (1.Jahn R. Scheller R.H. Nat. Rev. 2006; 7: 631-643Crossref Scopus (1942) Google Scholar). Although formation of SNARE core complexes is sufficient for membrane fusion (2.Weber T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Sollner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2021) Google Scholar, 3.Parlati F. Weber T. McNew J.A. Westermann B. Sollner T.H. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12565-12570Crossref PubMed Scopus (217) Google Scholar), essential regulatory proteins of the Sec1/Munc18 (SM) family are believed to control SNARE complex formation. Seven vertebrate SM gene family members (Munc18a/b/c, Sly1, Vps45, and Vps33a/b) have been described, which affect membrane trafficking in distinct subcellular pathways, predominantly through their association with specific syntaxin Q-SNAREs (4.Toonen R.F. Verhage M. Trends Cell Biol. 2003; 13: 177-186Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Of the varied SM-syntaxin partners, Munc18c-syntaxin 4 interactions are believed to exert a critically important role in the temporal control of insulin-stimulated GLUT4 storage vesicle exocytosis in muscle and adipose tissue (5.Tellam J.T. Macaulay S.L. McIntosh S. Hewish D.R. Ward C.W. James D.E. J. Biol. Chem. 1997; 272: 6179-6186Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 6.Thurmond D.C. Ceresa B.P. Okada S. Elmendorf J.S. Coker K. Pessin J.E. J. Biol. Chem. 1998; 273: 33876-33883Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 7.Tamori Y. Kawanishi M. Niki T. Shinoda H. Araki S. Okazawa H. Kasuga M. J. Biol. Chem. 1998; 273: 19740-19746Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 8.Dugani C.B. Klip A. EMBO Rep. 2005; 6: 1137-1142Crossref PubMed Scopus (194) Google Scholar). The delivery of GLUT4 to the cell surface occurs by distinct signaling processes: 1) GLUT4 vesicle recruitment to the plasma membrane, followed by 2) phosphatidylinositol 3-kinase-dependent vesicle docking and fusion mediated by syntaxin 4-containing SNARE complexes (9.Bose A. Robida S. Furcinitti P.S. Chawla A. Fogarty K. Corvera S. Czech M.P. Mol. Cell. Biol. 2004; 24: 5447-5458Crossref PubMed Scopus (132) Google Scholar, 10.Hodgkinson C.P. Mander A. Sale G.J. Diabetologia. 2005; 48: 1627-1636Crossref PubMed Scopus (30) Google Scholar). Based on overexpression studies, Munc18c was initially proposed to function as a negative regulator of GLUT4 vesicle translocation in 3T3L1 adipocytes (5.Tellam J.T. Macaulay S.L. McIntosh S. Hewish D.R. Ward C.W. James D.E. J. Biol. Chem. 1997; 272: 6179-6186Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 6.Thurmond D.C. Ceresa B.P. Okada S. Elmendorf J.S. Coker K. Pessin J.E. J. Biol. Chem. 1998; 273: 33876-33883Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). Furthermore, the failure of the phosphatidylinositol 3-kinase inhibitor wortmannin to suppress GLUT4 externalization in adipocytes derived from Munc18c-null mice suggests that Munc18c inhibits docking and/or fusion under the control of the phosphatidylinositol 3-kinase pathway (11.Kanda H. Tamori Y. Shinoda H. Yoshikawa M. Sakaue M. Udagawa J. Otani H. Tashiro F. Miyazaki J. Kasuga M. J. Clin. Invest. 2005; 115: 291-301Crossref PubMed Scopus (105) Google Scholar). Although the exact mechanism for this inhibition is not fully understood, Munc18c has been proposed to preclude the binding of syntaxin 4 to its cognate SNAREs, VAMP2 (vesicle-associated membrane protein 2) and SNAP23 (synaptosome-associated protein of 23 kDa), preventing GLUT4 externalization in the absence of insulin (5.Tellam J.T. Macaulay S.L. McIntosh S. Hewish D.R. Ward C.W. James D.E. J. Biol. Chem. 1997; 272: 6179-6186Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 6.Thurmond D.C. Ceresa B.P. Okada S. Elmendorf J.S. Coker K. Pessin J.E. J. Biol. Chem. 1998; 273: 33876-33883Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 12.Araki S. Tamori Y. Kawanishi M. Shinoda H. Masugi J. Mori H. Niki T. Okazawa H. Kubota T. Kasuga M. Biochem. Biophys. Res. Commun. 1997; 234: 257-262Crossref PubMed Scopus (85) Google Scholar). In the absence of fine structural information, a model of Munc18c control over syntaxin 4-SNARE complex formation is not easily predicted by other SM-syntaxin interactions due to the diversity in their modes of action. For example, the Munc18a-syntaxin 1A complex participates in regulated neuroexocytosis and has high sequence homology to Munc18c-syntaxin 4. Analysis of the syntaxin 1A structure showed that an autonomously folded N-terminal three-helix bundle, termed the Habc domain, reversibly folds over a helical H3 domain containing the SNARE motif (13.Margittai M. Widengren J. Schweinberger E. Schroder G.F. Felekyan S. Haustein E. Konig M. Fasshauer D. Grubmuller H. Jahn R. Seidel C.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15516-15521Crossref PubMed Scopus (231) Google Scholar, 14.Misura K.M. Scheller R.H. Weis W.I. Nature. 2000; 404: 355-362Crossref PubMed Scopus (617) Google Scholar, 15.Dulubova I. Sugita S. Hill S. Hosaka M. Fernandez I. Sudhof T.C. Rizo J. EMBO J. 1999; 18: 4372-4382Crossref PubMed Scopus (553) Google Scholar). These two helical domains are separated by a short structured linker proposed to allow syntaxin 1A to rapidly fluctuate between a folded (closed) conformation and an unfolded (open) conformation (13.Margittai M. Widengren J. Schweinberger E. Schroder G.F. Felekyan S. Haustein E. Konig M. Fasshauer D. Grubmuller H. Jahn R. Seidel C.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15516-15521Crossref PubMed Scopus (231) Google Scholar). The open conformation facilitates SNARE-SNARE pairing resulting from accessibility of the SNARE motif. The closed (SNARE pairing inactive) conformation of syntaxin 1A is stabilized by interaction with Munc18a (15.Dulubova I. Sugita S. Hill S. Hosaka M. Fernandez I. Sudhof T.C. Rizo J. EMBO J. 1999; 18: 4372-4382Crossref PubMed Scopus (553) Google Scholar), precluding interactions with other SNAREs (16.Calakos N. Bennett M.K. Peterson K.E. Scheller R.H. Science. 1994; 263: 1146-1149Crossref PubMed Scopus (368) Google Scholar, 17.Liu J. Ernst S.A. Gladycheva S.E. Lee Y.Y. Lentz S.I. Ho C.S. Li Q. Stuenkel E.L. J. Biol. Chem. 2004; 279: 55924-55936Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Thus, the association of Munc18 suggests a mechanism for the regulation of the syntaxin SNARE motif through the control of syntaxin conformation. However, there is a lack of general correlation between SM function and a particular conformation of syntaxin. The situation is further complicated, because not all syntaxins assume folded conformations. For instance, the SM proteins Sly1 and Vps45, which regulate trafficking of intracellular transport vesicles, bind to a conserved N-terminal domain found upstream of the Habc domain in their cognate syntaxins (Sly1 to syntaxins 5 and 18, and Vps45 to syntaxin 16) (18.Bryant N.J. James D.E. J. Cell Biol. 2003; 161: 691-696Crossref PubMed Scopus (32) Google Scholar, 19.Dulubova I. Yamaguchi T. Gao Y. Min S.W. Huryeva I. Sudhof T.C. Rizo J. EMBO J. 2002; 21: 3620-3631Crossref PubMed Scopus (152) Google Scholar, 20.Yamaguchi T. Dulubova I. Min S.W. Chen X. Rizo J. Sudhof T.C. Dev. Cell. 2002; 2: 295-305Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). This subset of SM proteins does not hinder the kinetics of SNARE pairings (21.Peng R. Gallwitz D. J. Cell Biol. 2002; 157: 645-655Crossref PubMed Scopus (121) Google Scholar). Comparatively, the yeast Munc18 homologue Sec1p stimulates fusion through its interaction with the ternary SNARE complex, only binding weakly to monomeric Sso1p/syntaxin (22.Scott B.L. Van Komen J.S. Irshad H. Liu S. Wilson K.A. McNew J.A. J. Cell Biol. 2004; 167: 75-85Crossref PubMed Scopus (88) Google Scholar, 23.Carr C.M. Grote E. Munson M. Hughson F.M. Novick P.J. J. Cell Biol. 1999; 146: 333-344Crossref PubMed Scopus (260) Google Scholar). By analogy to the Munc18a-syntaxin 1A interaction shown in the crystal structure (14.Misura K.M. Scheller R.H. Weis W.I. Nature. 2000; 404: 355-362Crossref PubMed Scopus (617) Google Scholar), the most prevalent model for Munc18c regulation of SNARE complex formation equates the association of Munc18c with a closed syntaxin 4 conformation to the inhibition of GLUT4 vesicle externalization. However, recent evidence suggests that exocytotic SM-syntaxin interactions may possess multiple binding and functional states. For example, Munc18a has been shown to bind to SNARE complexes and facilitate SNARE-mediated fusion in vitro (24.Shen J. Tareste D.C. Paumet F. Rothman J.E. Melia T.J. Cell. 2007; 128: 183-195Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar), the basis of which is probably a binding mode for the SM protein utilizing both the N terminus of syntaxin 1A and interactions with the SNARE bundle (25.Dulubova I. Khvotchev M. Liu S. Huryeva I. Sudhof T.C. Rizo J. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 2697-2702Crossref PubMed Scopus (256) Google Scholar). Comparatively, in vitro Munc18c associates with the ternary SNARE complex (26.Widberg C.H. Bryant N.J. Girotti M. Rea S. James D.E. J. Biol. Chem. 2003; 278: 35093-35101Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 27.Latham C.F. Lopez J.A. Hu S.H. Gee C.L. Westbury E. Blair D.H. Armishaw C.J. Alewood P.F. Bryant N.J. James D.E. Martin J.L. Traffic. 2006; 7: 1408-1419Crossref PubMed Scopus (100) Google Scholar). In addition, Munc18c possesses an interaction site for the N-terminal amino acids of syntaxin 4 resembling the syntaxin binding sites on Sly1 and Vps45 (27.Latham C.F. Lopez J.A. Hu S.H. Gee C.L. Westbury E. Blair D.H. Armishaw C.J. Alewood P.F. Bryant N.J. James D.E. Martin J.L. Traffic. 2006; 7: 1408-1419Crossref PubMed Scopus (100) Google Scholar). However, genetic studies suggest that the mechanism of Munc18c action may be different from Munc18a. For example, the insulin-induced fusion of GLUT4 vesicles was enhanced in adipocytes prepared from Munc18c-null mice (11.Kanda H. Tamori Y. Shinoda H. Yoshikawa M. Sakaue M. Udagawa J. Otani H. Tashiro F. Miyazaki J. Kasuga M. J. Clin. Invest. 2005; 115: 291-301Crossref PubMed Scopus (105) Google Scholar), whereas Munc18a deficiency was demonstrated to eliminate both evoked and spontaneous synaptic exocytosis (28.Verhage M. Maia A.S. Plomp J.J. Brussaard A.B. Heeroma J.H. Vermeer H. Toonen R.F. Hammer R.E. van den Berg T.K. Missler M. Geuze H.J. Sudhof T.C. Science. 2000; 287: 864-869Crossref PubMed Scopus (1008) Google Scholar). Additionally, insulin (6.Thurmond D.C. Ceresa B.P. Okada S. Elmendorf J.S. Coker K. Pessin J.E. J. Biol. Chem. 1998; 273: 33876-33883Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar) as well as N-ethylmaleimide (26.Widberg C.H. Bryant N.J. Girotti M. Rea S. James D.E. J. Biol. Chem. 2003; 278: 35093-35101Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), which inhibits NSF-mediated SNARE core complex disassembly, have been reported to dissociate Munc18c from syntaxin 4. Thus, it is not yet evident how the Munc18c-syntaxin 4 interaction fits with other models for SM-syntaxin interactions. The purpose of this study was to define the molecular properties of the Munc18c interaction with syntaxin 4, specifically how the Munc18c-syntaxin 4 complex influences SNARE core complex formation in the pathway of GLUT4 cycling. Our investigations utilized quantitative optical techniques, including fluorescence resonance energy transfer (FRET) and biochemical approaches, to directly report the state of the Munc18c-syntaxin 4 interaction in vivo. Using mutational analyses, we address the mode of Munc18c binding to syntaxin 4, including where and when the interaction occurs and which structural motifs are important for function. Our findings indicate that syntaxin 4 exists in a closed conformation that is stabilized by Munc18c, thus rendering the syntaxin 4 SNARE domain unavailable for SNARE complex formation. Using the system of insulin-stimulated GLUT4 exocytosis, we furthermore demonstrate that the binding of Munc18c to an open conformation of syntaxin 4 facilitates GLUT4 vesicle docking. Therefore, the change in the Munc18c-syntaxin 4 binding state precipitated by conformational opening of syntaxin is probably the critical regulatory point in the temporal sequence for the initiation of vesicle docking and fusion events. Chemicals and Expression Constructs−The following antibodies were used at a 1:1000 dilution for Western blotting: anti-syntaxin 4 rabbit polyclonal (Sigma), anti-syntaxin 1A mouse monoclonal HPC-1 (Sigma), anti-SNAP23 polyclonal (Synaptic Systems), anti-FLAG polyclonal (Sigma), and anti-c-Myc mouse monoclonal 9B11 (Cell Signaling). To generate N-terminal fluoroprotein-labeled constructs, the following cDNAs were subcloned into the SalI-XbaI sites of pDNR-dual for use with the Cre recombinase-mediated Creator System (Clontech): rat Munc18a (pGex-KG-Munc18a), rat syntaxin 1A (pGEX-syntaxin 1A11), rat Munc18c (pcDNA3-FLAG-Munc18c), human syntaxin 4 (pcDNA4/TO/syntaxin 4-Myc2-His), and human SNAP23 (pcDNA3-SNAP23) (gifts from J. Pevsner, R. Scheller, J. Pessin, and T. Weimbs (syntaxin 4 and SNAP23), respectively). The recipient vectors pLoxP-ECFP-C1 and pLoxP-EcYFP-C1 (Q39M mutant of pEYFP-C1; citrine) were generated and mutated to their monomeric forms (A206K) from pLoxP-EGFP-C1 (Clontech) as described previously (17.Liu J. Ernst S.A. Gladycheva S.E. Lee Y.Y. Lentz S.I. Ho C.S. Li Q. Stuenkel E.L. J. Biol. Chem. 2004; 279: 55924-55936Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). To prepare pLoxP-mRFP1-C1, the cYFP sequence from pLoxP-EcYFP-C1 was replaced with mRFP1 (monomeric form) from pRSETB-mRFP1 (a gift from R. Tsien) by PCR cloning. cDNAs in pDNR-dual were subcloned into the indicated recipient vectors using Cre recombinase according to the manufacturer’s instructions. To prepare recombinant fusion proteins in Escherichia coli, syntaxin 4-(1-275) and Munc18c were PCR-amplified and cloned downstream of glutathione S-transferase (GST) cDNA contained in the pGEX-KG plasmid; pGEX-syntaxin 1A11 (syntaxin 1A-(1-267)) was a gift from R. Scheller. pGex-KG-FLAG-loxP-Munc18c was created using a pGex-KG construct modified to include a loxP site suitable for use with the Clontech Creator system and an N-terminal FLAG epitope. The PCR-based QuikChange site-directed mutagenesis kit (Stratagene) was used to construct the following mutants: EGFP-SNAP23C/A, a cytosolic mutant lacking cysteine palmitoylation sites at 80, 83, 85, and 87, which were mutated to alanine; Munc18c (R240L); syntaxin 1A (L165A/E166A, LE; I209A; and I233A), and the homologous mutations in syntaxin 4 (L173A/E174A, LE; I217A; and I241A). Syntaxin 4-(1-188) (ΔH3/TM) and syntaxin 1A-(1-181) were created by inserting an XbaI site with the TAG stop codon in frame at Leu189 and Ile182, respectively. The same XbaI site along with a SalI site upstream of the start codon was used to facilitate construction of the following syntaxin chimeras: syntaxin 41 (syntaxin 4-(1-188) + syntaxin 1A-(182-288)) and syntaxin 14 (syntaxin 1A-(1-181) + syntaxin 4-(189-297)). Before use, the XbaI restriction sites were back-mutated in the syntaxin 14 and syntaxin 41 plasmids to eliminate the stop codon and restore the Leu189 in the syntaxin 4 fragments and the Ile182 in the syntaxin 1A fragments. The sequence fidelity of all expression constructs was confirmed by DNA sequencing (University of Michigan DNA Sequencing Core). Cell Culture and Transfection−3T3L1 fibroblasts (American Type Culture Collection, Manassas, VA) were maintained in DMEM containing 10% bovine serum (Invitrogen). Following 2 days of confluence, differentiation was induced by the addition of DMEM with 10% fetal bovine serum (FBS) containing 167 nm insulin, 0.25 μm dexamethasone, and 0.25 mm isobutylmethylxanthine for 3 days, followed by DMEM/FBS containing insulin for 2 days and subsequent removal of insulin for 2 days. Adipocytes were transfected by electroporation (0.16 kV, 950 micro-farads) in phosphate-buffered saline, pH 7.4 (Invitrogen), with 50-200 μg of each plasmid. After electroporation, cells were allowed to adhere to coverglass for 18-24 h in DMEM/FBS prior to imaging. In selected experiments, cells were exposed to 100 nm insulin after 3 h of serum starvation in DMEM low glucose (Invitrogen) containing 0.5% FBS. HEK293-S3 cells, which contain a stably transfected N-type calcium channel (17.Liu J. Ernst S.A. Gladycheva S.E. Lee Y.Y. Lentz S.I. Ho C.S. Li Q. Stuenkel E.L. J. Biol. Chem. 2004; 279: 55924-55936Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), were cultured in RPMI 1640 (Invitrogen) supplemented with 10% FBS (Invitrogen), 0.4 mg/ml hygromycin (Invitrogen), 0.4 mg/ml Geneticin (Invitrogen), and 1% penicillin/streptomycin (Invitrogen). Cells were plated on coverglass affixed to the bottom of 35-mm plates 12-24 h prior to transfection using Lipofectamine2000 (Invitrogen). Before live cell imaging, cells were transferred to physiologic saline solution containing 140 mm NaCl, 5 mm KCl, 5 mm glucose, 1 mm MgCl2, 2.2 mm CaCl2, 5 mm NaHCO3, and 10 mm HEPES (pH 7.4). Homology Modeling of Munc18c-Syntaxin 4 Complex−Rat Munc18c (Stxbp3; GenBank™ accession number NP_446089) and syntaxin 4 (STX4; GenBank™ accession number NP_112387) sequences were threaded into the Munc18a-syntaxin 1A structure (Protein Data Bank code 1DN1) using O, based on a ClustalW alignment. Helical register, assessed using PredictProtein, was comparable; sequence gaps and insertions were not modeled. Figures were made with MOL-SCRIPT, RASTER 3D version 2.1.2, and GIMP. GST Fusion Proteins−Chemically competent BL21DE3(RIPL) E. coli (Invitrogen) containing pGex-KG-syntaxin 4-(1-275), pGex-KG-syntaxin 4-(1-275)LE, or pGEX-KG-Munc18c were cultured and induced with 100 nm isopropyl 1-thio-β-d-galactopyranoside for 5-6 h at 27 °C. Bacteria were lysed by French press (15,000 p.s.i. pressure differential), and the eluent was solubilized in phosphate-buffered saline containing 1% Triton X-100 for 1 h on ice. Proteins were subsequently purified using glutathione-Sepharose (Amersham Biosciences). Cleavage of the GST moiety from the fusion protein was accomplished by treatment with 1.4 NIH units of human thrombin (Amersham Biosciences) for 16 h at 4 °C. Purity of all isolated fusion proteins was confirmed by SDS-PAGE fractionation and subsequent visualization with Coomassie Blue or Western blotting. Protein concentration was measured using the DC protein assay (Bio-Rad) against a bovine serum albumin standard (Sigma). Trypsin Proteolysis Assay−Trypsin digest of recombinant GST-syntaxin 4-(1-275) and GST-syntaxin 4-(1-275)LE was modeled after Graham et al. (29.Graham M.E. Barclay J.W. Burgoyne R.D. J. Biol. Chem. 2004; 279: 32751-32760Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Proteins were incubated at 5 μm concentration in a total volume of 50 μl containing 60 nm trypsin (Type IX-S; Sigma) in phosphate-buffered saline (pH 7.4) for the times indicated. During trypsin incubation, GST fusion proteins remained bound to glutathione-Sepharose beads. The reactions were terminated by the addition of SDS sample buffer and immediate boiling. After separation by 12% SDS-PAGE, the samples were transferred to nitrocellulose and probed for syntaxin 1A or syntaxin 4. Proteins were visualized with a horseradish peroxidase-conjugated secondary antibody and ECL substrate (Amersham Biosciences). Co-immunoprecipitation−For each treatment, a 6-well plate of confluent HEK293-S3 cells was transiently transfected with the indicated constructs. After a 2-day expression period, cells were rinsed twice in physiologic saline solution and lysed in a Dounce homogenizer in a buffer containing 2% sucrose, 1 mm EDTA, and 20 mm Tris (pH 7.5). After the homogenate was centrifuged (300 × g for 3 min) to pellet the nuclei, the supernatant was diluted 1:1 in immunoprecipitation buffer (150 Tris, pH 7.4, 1 mm MgCl2, 0.1 mm EGTA, 2% Triton X-100). Samples were normalized for lysate volume and concentration (2-3 μg/μl) and incubated with 10 μg of anti-Myc 9B11 monoclonal antibody (Cell Signaling) for 4 h at 4 °C. The samples were then incubated with Protein G-Sepharose beads (Pierce) for 1 h, and washed in immunoprecipitation buffer. Finally the pellet was resuspended in SDS sample buffer and subject to fractionation by SDS-PAGE and Western blotting. In Vitro Binding Assay−For all binding reactions in vitro, the GST moiety was cleaved from purified GST-syntaxin 4-(1-275), GST-syntaxin 4-(1-275)LE, and GST-Munc18c. Reactions used 0.03-30 pmol of Munc18c in a dilution series spotted in quadruplicate on nitrocellulose (BA-83; Schleicher and Schuell). After blocking in 2% milk, the blots were incubated with 500 nm syntaxin 4-(1-275) or syntaxin 4-(1-275)LE for ≥12 h at 4 °C. Syntaxin 4 proteins were visualized using an anti-syntaxin 4 polyclonal antibody and horseradish peroxidase-conjugated secondary antibody as described above. Integrated intensity (area·intensity) was quantified using Metamorph (version 6.3r5; Universal Imaging, Inc., Malvern, PA). Measurement of FRET Stoichiometry by Sensitized Emission−Live cell imaging of FRET was performed on transfected 3T3L1 adipocytes and HEK293 cells 24 h after transfection. Since false positive FRET signals have been observed when cytoplasmic donors come into contact with membrane-compartmentalized acceptors (30.Vogel S.S. Thaler C. Koushik S.V. Sci. STKE. 2006; 2006: re2PubMed Google Scholar), syntaxin 4 was purposely tagged with CFP. However, performing the experiments using CFP-Munc18c and cYFP-syntaxin 4 resulted in similar FRET efficiency values (data not shown). Measurement of sensitized emission FRET was carried out as previously described (17.Liu J. Ernst S.A. Gladycheva S.E. Lee Y.Y. Lentz S.I. Ho C.S. Li Q. Stuenkel E.L. J. Biol. Chem. 2004; 279: 55924-55936Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 31.Hoppe A. Christensen K. Swanson J.A. Biophys. J. 2002; 83: 3652-3664Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). The methodology employed an inverted fluorescence microscope (Olympus, IX71) equipped with the following components: a TILL-Photonics Polychrome IV xenon lamp-based monochrometer (TILL-Photonics, Grafelfing, Germany), a polychroic mirror that allowed excitation of multiple fluorophores (436-500 nm; Chroma Technology Corp., Brattleboro, VT), a Planapo ×60 water immersion objective (1.2 numerical aperture), a multispec microimager (Optical Insights, Santa Fe, NM) containing dichroic splitter (505dcxr) and emission filters (D465/30 and HQ535/30) to allow simultaneous two-channel monitoring of emission fluorescence, and a cooled digital CCD camera (TILL IMAGO QE). The multispec microimager hardware was calibrated to allow pixel-by-pixel alignment of images, and offline adjustments were made using the TILL-Vision software. All analyses of the acquired images were performed using Metamorph image-processing software as previously described (17.Liu J. Ernst S.A. Gladycheva S.E. Lee Y.Y. Lentz S.I. Ho C.S. Li Q. Stuenkel E.L. J. Biol. Chem. 2004; 279: 55924-55936Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 31.Hoppe A. Christensen K. Swanson J.A. Biophys. J. 2002; 83: 3652-3664Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). The apparent efficiency of acceptor (monomeric cYFP) in complex (EA), the apparent efficiency of donor (monomeric ECFP) in complex (ED), and the mole fraction of acceptor to donor (RATIO) values were determined using the following equations: EA = γ[((DA - β·DD)/α·AA) - 1](1/EC); ED = [1 - (DD/((DA - α·AA - β·DD)·(ξ/γ) + DD))](1/EC); and RATIO = (ξ/γ2)·[α·AA/((DA - α·AA - β·DD)·(ξ/γ) + DD)] (31.Hoppe A. Christensen K. Swanson J.A. Biophys. J. 2002; 83: 3652-3664Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar), where images are abbreviated as follows: DD, donor excitation-donor emission; DA, donor excitation-donor emission; AA, acceptor excitation-acceptor emission; EC, characteristic efficiency (0.37 (31.Hoppe A. Christensen K. Swanson J.A. Biophys. J. 2002; 83: 3652-3664Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar)). Donor and acceptor excitations were 436 and 500 nm, respectively. Empirically determined constants were established in HEK293 cells: α, 0.017; β, 0.9406; γ, 0.0658; ξ, 0.0147. For all measurements, EA values were determined in regions of the cell where the mole fraction between cYFP-Munc18c and CFP-syntaxin 4 (RATIO) was between 0.9 and 1.1; ED is comparable with EA over this RATIO range. Resolution of SNAP23C/A-SNARE Interactions by Cytosolic Photobleach and Membrane FRAP−GFP-SNAP23C/A bound to syntaxin 4 at the plasma membrane was observed by expression of the proteins when viewed with the FV500 Olympus FluoView laser-scanning confocal microscope. In order to resolve the relative amount of plasmalemmal GFP-SNAP23C/A, images were taken before and after 1 min of cytosolic photobleach (>90% bleach) using the 488-nm laser line of the argon laser. The localization of GFP-SNAP23C/A fluorescence intensity remaining after photobleach was quantified using linescans averaged over a 10-pixel width (∼1/8 cell diameter) normalized to peak intensity for each cell. For FRAP experiments, cytosolic photobleach was followed by a 10-s photobleach (>50%) of the cell membrane using simultaneous excitation with the 488-nm line of the Argon laser and 543-nm line of the helium neon laser to facilitate simultaneous bleach of GFP and RFP, respe" @default.
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• W2089957341 date "2007-06-01" @default.
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• W2089957341 title "Munc18c Interaction with Syntaxin 4 Monomers and SNARE Complex Intermediates in GLUT4 Vesicle Trafficking" @default.
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• W2089957341 doi "https://doi.org/10.1074/jbc.m610818200" @default.
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