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J. Biol. Chem., Vol. 282, Issue 22, 16553-16566, June 1, 2007

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

Matthew D'Andrea-Merrins¹, Louise Chang², Alice D. Lam, Stephen A. Ernst^¶, and Edward L. Stuenkel

From the Department of Molecular and Integrative Physiology, Life Sciences Institute, and the ^¶Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109

Received for publication, November 22, 2006 , and in revised form, March 12, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 4^LE-SNAP23 Q-SNARE complexes. Fluorescent imaging of GLUT4 vesicles in 3T3L1 adipocytes revealed that syntaxin 4^LE expressed with Munc18c bypasses the requirement of insulin for GLUT4 vesicle plasma membrane docking. This effect was attenuated by reducing the Munc18c-syntaxin 4^LE 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Soluble N-ethylmaleimide-sensitive factor (NSF)³ 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). Although formation of SNARE core complexes is sufficient for membrane fusion (2, 3), 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).

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 (5-8). 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, 10). Based on overexpression studies, Munc18c was initially proposed to function as a negative regulator of GLUT4 vesicle translocation in 3T3L1 adipocytes (5, 6). 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). 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, 6, 12).

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 (13-15). 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). 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), precluding interactions with other SNAREs (16, 17). 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) (18-20). This subset of SM proteins does not hinder the kinetics of SNARE pairings (21). Comparatively, the yeast Munc18 homologue Sec1p stimulates fusion through its interaction with the ternary SNARE complex, only binding weakly to monomeric Sso1p/syntaxin (22, 23).

By analogy to the Munc18a-syntaxin 1A interaction shown in the crystal structure (14), 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), 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). Comparatively, in vitro Munc18c associates with the ternary SNARE complex (26, 27). 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). 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), whereas Munc18a deficiency was demonstrated to eliminate both evoked and spontaneous synaptic exocytosis (28). Additionally, insulin (6) as well as N-ethylmaleimide (26), 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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). 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-SNAP23^C/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) (H3/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), 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^TM accession number NP_446089 [GenBank] ) and syntaxin 4 (STX4; GenBank^TM accession number NP_112387 [GenBank] ) 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--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). 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 x 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), 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, 31). 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 x60 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, 31). The apparent efficiency of acceptor (monomeric cYFP) in complex (E_A), the apparent efficiency of donor (monomeric ECFP) in complex (E_D), and the mole fraction of acceptor to donor (RATIO) values were determined using the following equations: E_A = [((DA - ·DD)/·AA) - 1](1/E_C); E_D = [1 - (DD/((DA - ·AA - ·DD)·(/) + DD))](1/E_C); and RATIO = (/²)·[·AA/((DA - ·AA - ·DD)·(/) + DD)] (31), where images are abbreviated as follows: DD, donor excitation-donor emission; DA, donor excitation-donor emission; AA, acceptor excitation-acceptor emission; E_C, characteristic efficiency (0.37 (31)). Donor and acceptor excitations were 436 and 500 nm, respectively. Empirically determined constants were established in HEK293 cells: , 0.017; , 0.9406; , 0.0658; , 0.0147. For all measurements, E_A 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; E_D is comparable with E_A over this RATIO range.

Resolution of SNAP23^C/A-SNARE Interactions by Cytosolic Photobleach and Membrane FRAP—GFP-SNAP23^C/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-SNAP23^C/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-SNAP23^C/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, respectively. FRAP images were taken following a 1.5-min recovery period.

Quantification of Insulin-induced GLUT4 Plasma Membrane Insertion—3T3L1 adipocytes stably expressing Myc₇-GLUT4-GFP were prepared as previously described (32) using a retroviral vector pMX-GLUT4myc7-EGFP (a gift from H. Lodish) (33). Cells were electroporated as described above with pcDNA-FLAG-Munc18c and pLoxP-ECFP-syntaxin 4 or mutants of syntaxin 4 as described in the figure legends. As a control experiment to verify the functionality of N-terminally tagged syntaxin 4 (supplemental Fig. S2), adipocytes were electroporated with pcDNA-syntaxin 4^LE and pLoxP-mCerulean-Munc18c. 24 h after electroporation, adipocytes were serum-starved for 3 h in DMEM containing 0.5% FBS. Subsequently, cells were treated with 100 nM insulin for the times indicated, fixed in phosphate-buffered saline containing 4% paraformaldehyde, and quenched with 50 mM glycine for 5 min. Externalized Myc₇-GLUT4-GFP was visualized by exposing the cells to an anti-Myc monoclonal antibody 9E10 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) without permeabilization, followed by Alexa⁵⁹⁴-conjugated goat anti-mouse secondary antibody (Molecular Probes, Inc., Eugene, OR) as discussed (32). For cell counting experiments, 75 cells from each treatment possessing peripheral CFP-syntaxin 4 fluorescence were blindly examined for peripheral GFP fluorescence and/or surface Alexa⁵⁹⁴ fluorescence using an Olympus IX71 microscope equipped with a dual pass GFP/mRFP1 filter set (Chroma Technology). For quantification of GLUT4 localization, cells with equivalent CFP (syntaxin) and GFP (GLUT4) were chosen based on equivalent integrated density (cell area·intensity) from a 500-ms image taken at 436 and 480 nm, respectively. To quantify CFP and GFP localization, line scans (10-pixel width averages) drawn from the outside of the cell to the interior were normalized to fluorescence intensity at the plasma membrane region and plotted as a function of distance from the plasma membrane.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Modeling of Syntaxin 4 Structure and Generation of a Constitutively Open Syntaxin 4 Mutant—To facilitate design of syntaxin 4 mutations that would aid in characterization of the conformational states of syntaxin 4 for Munc18c binding, we constructed a homology model of syntaxin 4 based on the reported crystal structure of syntaxin 1A in complex with Munc18a (nSec1/Munc18-1) (14). The Munc18c and syntaxin 4 sequences (identity: 50%, Munc18; 46%, syntaxin) were threaded directly on the crystal structure of the Munc18a-syntaxin 1A complex (Protein Data Bank code 1DN1). No modeling predictions were made in areas with sequence gaps or insertions. Fig. 1A shows the generated Munc18c-syntaxin 4 homology model. An analysis of the model over highly conserved regions between syntaxin 1A and syntaxin 4 yielded two important observations. First, the outer surfaces of the syntaxin 4 Habc helices contain most of the nonconserved residues, whereas the regions of intramolecular interactions between the Habc domain and the H3 domain were found to be highly conserved (Fig. 1B, circled). This structural similarity suggested that the helical packing that allows syntaxin 1A to adopt a closed conformation would also allow syntaxin 4 to adopt a closed conformation. Second, the Habc-H3 linker region is stabilized against the Habc and H3 domains by a highly conserved hydrophobic pocket. A mutation in the syntaxin 1A linker helix (L165A/E166A; LE) has been reported to destabilize the N-terminal helices from interaction with the SNARE motif and result in a constitutively open syntaxin 1A conformation (15). This open conformation impairs Munc18a binding to syntaxin 1A, strongly facilitating its interaction with other SNAREs (3, 17, 29, 34). From the structural prediction (Fig. 1C), the homologous residues in syntaxin 4, Leu¹⁷³, and Glu¹⁷⁴ lie in a conserved pocket of the linker helix. The hydrophobic contacts of Leu¹⁷³ are maintained with Ile¹⁵⁶, Leu¹⁶⁰, and the semiconserved Val¹⁶⁸ (Ile¹⁴⁹, Leu¹⁵³, and Thr¹⁶⁰ in syntaxin 1A). The hydrophobic regions of the nonconserved residues, Asp¹⁷⁰ and Arg¹⁵⁷ (Ser¹⁶² and Gln¹⁵⁰ in syntaxin 1A), may form van der Waals interactions contributing to the hydrophobic pocket for Leu¹⁷³, whereas their charged portions are solvent-exposed. Glu¹⁷⁴ maintains one conserved salt bridge with Arg¹⁴⁹, although a second with Val¹⁵³ (Arg¹⁴² and Lys¹⁴⁶ in syntaxin 1A) is lost. Taken together, the model predicts that significant Leu¹⁷³ and Glu¹⁷⁴ interactions are likely to be disrupted by dual mutation to alanine (L173A/E174A), hereafter termed syntaxin 4^LE, and to confer a constitutively open conformation on the syntaxin 4 mutant.

View larger version (26K): [in this window] [in a new window]   FIGURE 1. Homology modeling of the Munc18c-syntaxin 4 complex based on the Munc18a-syntaxin 1A crystal structure. A, Munc18c and syntaxin 4 were threaded directly into the Munc18a-syntaxin 1A crystal structure based on a ClustalW alignment. Munc18c is colored gray, and the syntaxin 4 helical domains are colored as follows: Ha in pale blue, H bin gold, H cin cyan, H 3 in green, and the Habc-H3 linker region in orange. The syntaxin 4 point mutants used in this study (red) are mapped onto the Munc18c-syntaxin 4 structural model: L173A/E174A, LE; I217A; I241A. B, structural map of nonconserved syntaxin 4 residues viewed down the axis of the syntaxin 4 H3 domain (green) as it packs against the Habc helices. Side chains of nonconserved residues are shown. A region of highly conserved packing between the SNARE (H3) domain and the N-terminal helices is circled (rust, Ha; blue, Hb; magenta, Hc; red, H3; indigo, Habc-H3 linker). C, all contacts proposed to interact with the syntaxin 4 linker residues Leu173 and Glu174 are colored as conserved (yellow) and nonconserved (magenta); differing syntaxin 1A residues are shown after a slash. Residue numbers correspond to the rat syntaxin 4 sequence (GenBankTM, NP_112387).

View larger version (44K): [in this window] [in a new window]   FIGURE 2. Comparison of trypsin proteolysis between wild type and mutant syntaxin 4LE. A, immunoblot comparing the effect of trypsin digest (60 nm, 3 min) on GST-syntaxin 4-(1-275) wild type and GST-syntaxin 4-(1-275)LE (L173A/E174A). Cleavage products were fractionated on SDS-PAGE and visualized by Western blot using a syntaxin 4 polyclonal antibody. Equivalent amounts of each undigested protein (control) are also shown. B, immunoblot as in A, comparing the products of trypsin digest of wild type or mutant GST-syntaxin 1A-(1-267)LE (L165A/E166A), visualized using an anti-syntaxin 1A monoclonal antibody. Representative blots were chosen from four independent experiments.

View larger version (37K): [in this window] [in a new window]   FIGURE 3. FRET measurements of direct Munc18c and syntaxin 4 interaction in 3T3L1 adipocytes. A, FRET measured by sensitized emission in living 3T3L1 adipocytes transfected with cYFP-Munc18c and CFP-syntaxin 4 or mutants as indicated. EA and ED are the apparent FRET efficiencies for the acceptor (cYFP-Munc18c) and donor (CFP-syntaxin 4) in complex, respectively. RATIO is the mole fraction cYFP/CFP. The pseudocolored EA, ED, and RATIO images represent a spatial map of FRET efficiency; the color bar indicates scaling. B, averaged EA values for each co-transfection condition. Cells with a similar RATIO (averaging 0.9-1.1) were chosen for comparison. The numbers above columns indicate the number of cells imaged. Significant differences are from the wild type Munc18c-syntaxin 4 treatment: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Our FRET experiments in adipocytes described above demonstrated a significant interaction of Munc18c with syntaxin 4^LE, which contrasts with the strongly reduced interaction reported for the LE mutant of syntaxin 1A with Munc18a in vitro and in vivo (17, 29). This indicates that divergence occurs among vertebrate SM-syntaxin protein interactions. To directly compare the interaction of Munc18c and Munc18a with their cognate syntaxins in vivo, we measured FRET between the protein pairs when expressed in a common reporter cell line, HEK293 cells. Fig. 4A indicates that the apparent efficiency (E_A) of cYFP-Munc18c in complex with CFP-syntaxin 4^LE was mildly reduced from that measured with CFP-syntaxin 4. By contrast, the apparent efficiency of energy transfer between Munc18a and syntaxin 1A^LE was 50% lower than that measured for syntaxin 1A, a result consistent with the strong reduction in affinity conferred by the syntaxin 1A^LE mutation reported in other studies (17, 29). To further gauge the competence of Munc18c to interact with syntaxin 4^LE in live cells, we examined the ability of RFP-syntaxin 4^LE to compete with wild type CFP-syntaxin 4 for the binding of cYFP-Munc18c (Fig. 4B). The use of an RFP probe facilitated the setting of roughly equivalent levels of the RFP-syntaxins between treatments. We found that RFP-syntaxin 4^LE expression impaired cYFP-Munc18c energy transfer to CFP-syntaxin 4by 70%. When the RFP and CFP probes were exchanged to measure FRET between cYFP-Munc18c and CFP-syntaxin 4^LE in the presence of RFP-syntaxin 4, energy transfer was similarly reduced. Taken together, these data demonstrate that Munc18c binds efficiently to syntaxin 4 in either a closed or open conformation.

View larger version (37K): [in this window] [in a new window]   FIGURE 4. Comparison of syntaxin 4 and syntaxin 1A LE mutants on SM protein binding. A, averaged apparent energy transfer (EA) between the indicated isoforms of cYFP-Munc18 and CFP-syntaxin or CFP-syntaxinLE co-expressed in HEK293-S3 cells. RATIO values between 0.9 and 1.1 were selected. B, competition by RFP-syntaxin 4LE/CFP-syntaxin 4 or RFP-syntaxin 4/CFP-syntaxin 4LE pairs for binding to cYFP-Munc18c as measured by the energy transfer between the CFP and cYFP probes in living HEK293-S3 cells. Cells selected for analysis exhibited similar average RATIO (2.0 ± 0.10 for both treatments). The number of observations is indicated above each column. *, p < 0.05; **, p < 0.01; ***, p < 0.001. C, EA at each image pixel was plotted against its corresponding inverse RATIO value for FRET between cYFP-Munc18c and CFP-syntaxin 4 (black, n = 61) or CFP-syntaxin 4LE (gray, n = 67). Data were fit with a single exponential; the 95% confidence interval is displayed. Vertical dashed lines indicate the RATIO range over which EA values were averaged for FRET experiments shown in A.

View larger version (43K): [in this window] [in a new window]   FIGURE 5. In vitro binding relationship between Munc18c and syntaxin 4WT or syntaxin 4LE. Immunoblots of the binding reaction between bacterially expressed and purified 500 nM syntaxin 4-(1-275) (black squares, solid line) or syntaxin 4-(1-275)LE (open circles, dashed line) and given quantities of Munc18c spotted on nitrocellulose. The syntaxin 4 immunoreactive signal was quantified as a percentage of maximal binding (n = 4 spots/condition); averaged results (right) are from three independent experiments. Syntaxin 4-(1-275) and syntaxin 4-(1-275)LE used for binding were spotted on nitrocellulose to verify that equivalent amounts were used for binding and demonstrate equivalent immunoreactivity (below).

The Syntaxin 4 Binding Sites for Munc18c—The interaction of Munc18c with multiple conformations of syntaxin 4 is in direct contrast to the belief that vertebrate exocytotic SM proteins bind solely to the closed form of syntaxin. As a possible explanation, Munc18c-syntaxin 4 interactions may resemble a subset of SM proteins that function in the ER, Golgi, TGN, and endosomal networks, Sly1 and Vps45, which are known to require only short N-terminal sequences of their cognate syntaxins (18-20). Since syntaxin 4 shares some sequence homology with the motif that allows rSly1 to bind the N terminus of syntaxins 5 and 18 (Fig. 6A), it remained possible that the N terminus of syntaxin 4 upstream of the Habc domain could be important for binding to Munc18c, as first suggested by in vitro studies (27). Yamaguchi et al. (20) previously demonstrated that an arginine substitution at Asp⁵ in Sed5p/syntaxin 5, a residue common to syntaxins 1-4 as Asp³, resulted in complete loss of Sly1p binding when mutated to arginine. Shown in Fig. 6B, CFP-syntaxin 4^D3R results in a 40% reduction in FRET with cYFP-Munc18c when compared with the wild type proteins expressed in HEK293-S3 cells. Moreover, the D3R mutant completely eliminated Munc18c binding to the constitutively open mutant of syntaxin 4. However, the N terminus of syntaxin 4 is insufficient to bind Munc18c, since a mutant of CFP-syntaxin 4 lacking its H3 and transmembrane domains (H3/TM) exhibited negligible energy transfer with cYFP-Munc18c. The N terminus of syntaxin 4 could therefore stabilize the association of Munc18c with both the closed and open syntaxin 4 conformations. Furthermore, these results point to a mode of Munc18c-syntaxin 4 binding that shares the characteristics of SM binding to both ER/Golgi syntaxins and the exocytotic syntaxins, which require both the N and C termini of syntaxin for SM binding.

View larger version (28K): [in this window] [in a new window]   FIGURE 6. Identification of the syntaxin 4 domains required for Munc18c binding. A, diagram illustrating the relative position of syntaxin 4 mutants used to identify interacting sites with Munc18c. Alignments of the syntaxin N termini are shown for the four vertebrate exocytotic isoforms as well as syntaxins 5 and 18 (Sed5p and Ufe1p in yeast, respectively). The aspartic acid residue mutated in syntaxin 4D3R is highlighted in gray. The SM binding partners for each syntaxin isoform are listed, although not all combinations exhibit equal affinity (20). In syntaxin 4 chimeric mutants, termed 14 and 41 (not pictured), the syntaxin 4 Habc or H3 domains were replaced with syntaxin 1A sequence upstream or downstream of Leu189 as detailed under "Experimental Procedures." Syntaxin 4H3/TM contains a stop codon in place of Leu189. B and C, averaged apparent energy transfer (EA) between cYFP-Munc18c and the indicated mutants of CFP-syntaxin 4. Averaged EA values determined for FRET RATIO values of 0.9-1.1 were selected. Significant differences are relative to syntaxin 4WT or syntaxin 4LE (white bars; controls redisplayed from Fig. 4A). Numbers of observations are indicated above bars.*, p < 0.05; **, p < 0.01; ***, p < 0.001.

Effects of Syntaxin 4 Conformation and Munc18c Binding on Q-SNARE Complex Formation—The nucleation of syntaxin 4-mediated SNARE complex formation is believed to be negatively regulated by Munc18c (5-7, 11, 12, 36). Since Munc18c binds to both closed and open conformations of isolated syntaxin 4 (Figs. 3, 4, 5 and 6) as well as to the syntaxin 4-SNAP23-VAMP2 complex in vitro (26, 27), we next investigated whether the opening of syntaxin 4 is sufficient to relieve the Munc18c inhibition of SNARE complex nucleation in vivo. To address this question, we developed an optical reporter system to assess how Munc18c affects the interaction of closed or constitutively open syntaxin 4 with SNAP23 (Fig. 7A). We expressed limiting quantities of syntaxin 4 in living HEK293-S3 cells with an excess of GFP-SNAP23^C/A, a cytosolic mutant of SNAP23 lacking its four-cysteine palmitoylation sequence (39). After a 1-min photobleach of a delimited region within the cytosol, GFP-SNAP23^C/A was resolved at the plasma membrane, consistent with the formation of a dimeric SNAP23-syntaxin 4 complex. The additional expression of mRFP-Munc18c resulted in the complete loss of GFP-SNAP23^C/A signal after photobleach, signifying that GFP-SNAP23^C/A was freely diffusible and not tethered at the plasma membrane. This result intimates that Munc18c binding to syntaxin 4 occludes SNAP23 binding to syntaxin 4. Indeed, the presence of membrane-associated mRFP-Munc18c was observed after the photobleach, indicative of its binding to syntaxin 4. The membrane association of Munc18c was not due to binding of endogenous proteins in the HEK293 cells, since expressed Munc18c was cytosolic in the absence of expressed syntaxin 4 (data not shown). In summary, the data are consistent with Munc18c occluding syntaxin 4 interactions with SNAP23 in vitro (12). Of specific importance, we next investigated if the association of Munc18c with the open form of syntaxin 4 inhibits SNAP23 binding. For this analysis, we coexpressed syntaxin 4^LE with GFP-SNAP23^C/A in the absence or presence of mRFP-Munc18c. After photobleach, GFP-SNAP23^C/A remained bound to syntaxin 4^LE despite the association of RFP-Munc18c at the membrane, signifying that the conformational transition of syntaxin 4 is sufficient to relieve the inhibition of Munc18c on SNAP23 binding. The localization of GFP-SNAP23^C/A in both experiments was quantified using line scans drawn from the outside of the cell to its center (Fig. 7B). The sharp "ear" in the line scan is consistent with membrane localization of GFP-SNAP23^C/A, whereas the GFP intensity plateaus in cells where photobleach of diffusible GFP-SNAP23^C/A has occurred upon the addition of mRFP1-Munc18c. To confirm that a Munc18c-syntaxin 4^LE-SNAP23^C/A complex is formed on the plasma membrane, we performed FRAP experiments on the plasma membrane following the cytosolic photobleach. As anticipated, in the syntaxin 4^WT/RFP-Munc18c/GFP-SNAP23^C/A coexpression condition, no further bleaching of GFP was observed on the membrane, although membrane-associated RFP-Munc18c signal recovered after membrane photobleach, consistent with the Munc18c inhibition of Q-SNARE complex formation (supplemental Fig. S1). However, that both RFP-Munc18c and GFP-SNAP23^C/A signals recover after a subsequent plasma membrane photobleach when coexpressed with syntaxin 4^LE supports the interpretation that a Munc18c-syntaxin 4^LE-SNAP23^C/A complex is formed specifically at the plasma membrane.

View larger version (44K): [in this window] [in a new window]   FIGURE 7. Munc18c effects on Q-SNARE complex nucleation. A, confocal images of HEK293-S3 cells expressing GFP-SNAP23C/A and syntaxin 4 in the absence or presence of RFP-Munc18c are shown after a 1-min photobleach (488 nm) of a region in the cytosol. The localization of diffusible GFP-SNAP23C/A after cytosolic photobleach was used as an optical reporter for SNARE pairings with syntaxin 4. B, averaged GFP-SNAP23C/A fluorescence intensity remaining after photobleach was quantified by plotting the average GFP pixel intensity across the plasma membrane region for cells co-expressing syntaxin 4 (top) or syntaxin 4LE (bottom) in the absence (black squares) or presence (gray circles) of RFP-Munc18c. Line scans are normalized to peak GFP intensity (n = 8-12 cells analyzed per experiment; three independent experiments). C, immunoblots showing co-immunoprecipitation of endogenous SNAP23 from HEK293-S3 cell lysates containing expressed syntaxin 4WT-Myc2 or syntaxin 4LE-Myc2 in the presence or absence of expressed FLAG-Munc18c. Lysates were incubated with anti-Myc monoclonal antibody (9B11), pulled down with protein G-Sepharose, and subject to 12% SDS-PAGE fractionation and Western blot analysis. Blots were cut and separately exposed to rabbit polyclonal antibodies against syntaxin 4, SNAP23, and FLAG. Cell lysates (Lys) and supernatants (Sup) are shown for reference. No SNAP23 or FLAG-Munc18c pull down was observed when syntaxin 4-Myc2 was absent (right) (n = 4).

View larger version (24K): [in this window] [in a new window]   FIGURE 8. Munc18c interaction with syntaxin 4LE-SNAP23 Q-SNARE complexes. HEK293-S3 cell lysates expressing either syntaxin 4WT or syntaxin 4LE with FLAG-Munc18c and Myc-SNAP23 were subject to immunoprecipitation (as detailed in Fig. 7C) using Myc antibodies (left and middle). Expression conditions for each co-immunoprecipitation are indicated below immunoblots. Syntaxin 4 was omitted in a control experiment (right). Blots were probed for SNAP23 and VAMP2 in addition to the overexpressed proteins syntaxin 4 and FLAG-Munc18c (n = 3).

View larger version (53K): [in this window] [in a new window]   FIGURE 9. Effects of syntaxin 4 conformation and Munc18c complexation on GLUT4 vesicle docking in 3T3L1 adipocytes. A, confocal images of differentiated 3T3L1 adipocytes stably expressing Myc7-GLUT4-GFP, transfected with Munc18c and CFP-syntaxin 4WT, CFP-syntaxin 4LE, CFP-syntaxin 4I241A or CFP-syntaxin 4LE + I241A as indicated. Cells were fixed before or after treatment with 100 nM insulin (30 min at 37C) and stained without permeabilization against the Myc epitope located in the first exofacial loop of GLUT4. B, relative expression levels of CFP-syntaxin 4 (or mutants as indicated) and Myc7-GLUT4-GFP were determined in adipocytes based on fluorescence intensity. Munc18c was coexpressed for each condition, and results were quantified from unstimulated cells. The numbers above columns indicate the number of cells imaged. No significant differences in CFP or GFP expression were found between treatments. C, relative plasma membrane localization of CFP-syntaxin 4 (436 nm) and Myc7-GLUT4-GFP (480 nm) for cells in B. Fluorescence intensity along a 10-pixel width line scan was determined for each cell. The plasma membrane is set at pixel 0. Plots show averaged data ± S.E. A representative cell expressing Munc18c, CFP-syntaxin 4LE, and Myc7-GLUT4-GFP is shown for clarity with a typical region selected for line scan (left).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
SM proteins exert functional effects at different stages of SNARE complex assembly primarily through specific, nonconserved, interactions with cognate syntaxins. In this study, we have focused on identifying and characterizing the molecular underpinnings of the interactions between the ubiquitous SM protein Munc18c and its Q-SNARE cognate, syntaxin 4, an interaction of critical importance in the regulation of insulin-stimulated GLUT4 vesicle exocytosis in adipose tissue and muscle (8). Using a combination of optical techniques and biochemical approaches, we have demonstrated that Munc18c is capable of multiple binding interactions with syntaxin 4 in vivo, which probably occur during the stepwise assembly of SNARE complexes. These interactions include Munc18c binding to a closed conformation of syntaxin 4 that is inhibitory to SNARE complex nucleation as well as to an open conformation, which permits Munc18c binding to heteromeric Q-SNARE complexes. Point mutants were used to identify the syntaxin 4 SNARE domain as essential for mediating Munc18c interaction with both syntaxin 4 conformations, although the N terminus of syntaxin 4 was strictly required, but insufficient, for Munc18c binding to the open conformation. Last, we provide evidence that these multiple Munc18c binding states with Q-SNAREs are critical determinants of distinct functional effects on Glut4 vesicle translocation in differentiated 3T3L1 adipocytes.

Recent reports have indicated that Munc18c possesses an ability to directly interact with syntaxin 4 while it is bound to other SNARE proteins (26, 27). Our findings in living 3T3L1 adipocytes extend the analysis of direct Munc18c interactions to an in vivo situation, with a specific focus on its interactions with syntaxin 4. We report that Munc18c exhibits nearly equal FRET efficiency with either wild type syntaxin 4 or a mutant that maintains a constitutively open conformation (L173A/E174A; LE). Therefore, the affinity of Munc18c for syntaxin 4 appears to be largely unaffected by the conformational state of syntaxin 4. Our direct in vitro binding studies using bacterially expressed GST fusion proteins as well as co-immunoprecipitation results from cell lysates confirmed the in vivo findings. We and others have previously reported that Munc18a also exhibits limited binding to the constitutively open LE mutant of syntaxin 1A in vivo, (15, 17), a finding that has been recently extended to include Munc18a interaction with syntaxin 1A in heteromeric Q-SNARE complexes (40) and fully assembled SNARE complexes (24, 25). Comparatively, the yeast exocytotic SM protein Sec1p interacts weakly with a closed conformation of Sso1p, favoring binding to Sso1p-Snc2p Q-SNARE complexes (22, 23). Therefore, the interaction of exocytotic SM proteins with their cognate syntaxins appears to be converging on a general model where exocytotic Sec/Munc18 proteins demonstrate multiple binding states.

Our data establishing that Munc18c is able to interact with an open conformation of syntaxin 4 in vivo prompted experiments to map the primary structural sites that mediate the interaction. To date, the interacting sites between these proteins have been postulated to resemble the crystal structure of Munc18a in complex with syntaxin 1A (14). Indeed, our initial threading of the Munc18c and syntaxin 4 sequences directly into the Munc18a-syntaxin 1A crystal structure predicted similar interactions between Munc18c and the C terminus of syntaxin 4 when it is in the closed conformation. In confirmation of this prediction, we show that homologous I241A and I233A SNARE domain mutations in syntaxin 4 and syntaxin 1A, respectively, both dramatically reduced binding to their SM partners (17, 29). Furthermore, the entire SNARE domain of syntaxin 1A substituted for the syntaxin 4 SNARE domain exhibited no consequence for Munc18c binding. Notably, the complementary substitution (i.e. replacement of syntaxin 1A SNARE motif with syntaxin 4 sequence) resulted in a complete loss of Munc18a binding to the syntaxin 1A chimera (data not shown). These results correlate well with the binding of Munc18c to both syntaxin 4 and, with lesser affinity, syntaxin 1A, whereas Munc18a shows specificity for syntaxin 1A (41).

By comparison with the relatively well defined interactions of Munc18c with a closed state of syntaxin 4, the specific structural determinants that allow interaction of Munc18c with an open conformation of syntaxin 4 have been largely unstudied. In the current study, we show that the N terminus of syntaxin 4 is required for binding of Munc18c to the open mutant of syntaxin 4, but not to wild type syntaxin 4, via examination of an arginine substitution at syntaxin 4 Asp³ in FRET binding assays. Notably, this residue is conserved in both exocytotic and ER/Golgi syntaxins (syntaxins 1-4, 5, and 18), suggesting that SM protein interaction with the N terminus of syntaxin is a common mechanism for association with the open conformation in intracellular SNARE complexes. This is consistent with the N-terminal motif of Sed5p/syntaxin 5 and Ufe1p/syntaxin 18 providing a platform for their SM partner, Sly1p, to bind both monomeric syntaxin proteins and SNARE complexes (20, 21). Our findings complement a recent report that used an in vitro binding assay to elucidate the Munc18c binding pocket predicted to hold the N terminus of syntaxin 4 (27). However, a notable discrepancy between these collective findings is whether the syntaxin 4 N terminus is sufficient for Munc18c binding. Using an in vitro binding assay, Latham et al. (27) reported significant association between Munc18c and a mutant syntaxin 4 that lacked the SNARE motif (H3 domain) and transmembrane domain (syntaxin 4^H3/TM). However, our in vivo analysis using a FRET-based assay displayed a loss Munc18c interaction with syntaxin 4^H3/TM, thereby suggesting that the N-terminal interaction is insufficient to mediate the Munc18c-syntaxin 4 interaction, in parallel to the in vitro findings of Ter Beest et al. (42). The necessity of the syntaxin 4 C terminus was additionally confirmed using the I241A mutation of syntaxin 4, a residue predicted to bind to the central cavity of Munc18c. The syntaxin 4^I241A mutant eliminated Munc18c binding to constitutively open syntaxin 4 and severely reduced interaction with wild type syntaxin 4. Therefore, our data favor a model in which Munc18c interaction with either the open or closed conformation of syntaxin 4 depends upon SNARE motif interactions, with an additional requirement for an N-terminal interaction with syntaxin 4 in the open conformation. A similar requirement has been demonstrated for Munc18a binding to the open conformation of syntaxin 1A in SNARE complexes (25).

The SNARE assembly cycle begins with syntaxin nucleation of Qb- and Qc-SNARE complexes followed by the final SNARE motif contributed by vesicular R-SNAREs (43). It has remained unclear which steps are the relevant sites of action for the exocytotic SM proteins. In the present study, we demonstrate that Munc18c can interact with a SNARE pairing-competent conformation of syntaxin 4 in vivo. These findings extend in vitro binding studies, which showed Munc18c associated with preformed syntaxin 4-SNAP23 Q-SNARE complexes as well as ternary SNARE complexes composed of syntaxin 4, SNAP23, and VAMP2 (26, 27). It is difficult to directly demonstrate that Munc18c-containing SNARE complexes form in vivo within the regulated secretory pathway. However, as support that these complexes form, we show that constitutively open syntaxin 4^LE recruits both Munc18c and diffusible GFP-SNAP23^C/A to the plasma membrane, as resolved by photobleach/FR