Functionally and Spatially Distinct Modes of munc18-Syntaxin 1 Interaction
2007; Elsevier BV; Volume: 282; Issue: 16 Linguagem: Inglês
10.1074/jbc.m700227200
ISSN1083-351X
AutoresColin Rickman, Claire N. Medine, Axel Bergmann, R. R. Duncan,
Tópico(s)Pancreatic function and diabetes
ResumoEukaryotic membrane trafficking is a conserved process under tight temporal and spatial regulation in which the fusion of membranes is driven by the formation of the ternary SNARE complex. Syntaxin 1a, a core component of the exocytic SNARE complex in neurons and neuroendocrine cells, is regulated directly by munc18-1, its cognate Sec1p/munc18 (SM) protein. SM proteins show remarkable structural conservation throughout evolution, indicating a common binding mechanism and function. However, SM proteins possess disparate binding mechanisms and regulatory effects with munc18-1, the major brain isoform, classed as atypical in both its binding specificity and its mode. We now show that munc18-1 interacts with syntaxin 1a through two mechanistically distinct modes of binding, both in vitro and in living cells, in contrast to current models. Furthermore, these functionally divergent interactions occur at distinct cellular locations. These findings provide a molecular explanation for the multiple, spatially distinct roles of munc18-1. Eukaryotic membrane trafficking is a conserved process under tight temporal and spatial regulation in which the fusion of membranes is driven by the formation of the ternary SNARE complex. Syntaxin 1a, a core component of the exocytic SNARE complex in neurons and neuroendocrine cells, is regulated directly by munc18-1, its cognate Sec1p/munc18 (SM) protein. SM proteins show remarkable structural conservation throughout evolution, indicating a common binding mechanism and function. However, SM proteins possess disparate binding mechanisms and regulatory effects with munc18-1, the major brain isoform, classed as atypical in both its binding specificity and its mode. We now show that munc18-1 interacts with syntaxin 1a through two mechanistically distinct modes of binding, both in vitro and in living cells, in contrast to current models. Furthermore, these functionally divergent interactions occur at distinct cellular locations. These findings provide a molecular explanation for the multiple, spatially distinct roles of munc18-1. In neuronal and neuroendocrine cells, exocytosis is mediated by the plasma membrane proteins (t-SNAREs) 2The abbreviations used are: SNARE, soluble N-ethylmaleimide sensitive factor attachment receptor; t-SNARE, target SNARE; v-SNARE, vesicle SNARE; SM, Sec1p/Munc18; SNAP-25, synaptosome-associated protein 25 kDa; FLIM, fluorescence lifetime imaging microscopy; GST, glutathione S-transferase; N2A, Neuroblastoma 2A; TCSPC, time-correlated single photon counting; FRET, Förster resonance energy transfer; EYFP, enhanced yellow fluorescent protein; Syx, syntaxin.2The abbreviations used are: SNARE, soluble N-ethylmaleimide sensitive factor attachment receptor; t-SNARE, target SNARE; v-SNARE, vesicle SNARE; SM, Sec1p/Munc18; SNAP-25, synaptosome-associated protein 25 kDa; FLIM, fluorescence lifetime imaging microscopy; GST, glutathione S-transferase; N2A, Neuroblastoma 2A; TCSPC, time-correlated single photon counting; FRET, Förster resonance energy transfer; EYFP, enhanced yellow fluorescent protein; Syx, syntaxin. syntaxin and SNAP-25 (synaptosome-associated protein 25 kDa) and the vesicular protein synaptobrevin (v-SNARE) (1Burgoyne R.D. Morgan A. Physiol. Rev. 2003; 83: 581-632Crossref PubMed Scopus (537) Google Scholar, 2Rizo J. Sudhof T.C. Nat. Rev. Neurosci. 2002; 3: 641-653Crossref PubMed Scopus (424) Google Scholar). The cytoplasmic regions of these three proteins interact to form a trimeric, four-helical complex, the generation of which drives fusion of the two opposing bilayers (3Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1881) Google Scholar). This process is regulated by a conserved set of accessory proteins that operate throughout the trafficking pathway. The Sec1p/munc18 (SM) protein family represents one such set of modulators with SM protein mutations characterized by a severe disruption of general secretion or neurotransmitter release (4Brenner S. Genetics. 1974; 77: 71-94Crossref PubMed Google Scholar, 5Harrison S.D. Broadie K. van de Goor J. Rubin G.M. Neuron. 1994; 13: 555-566Abstract Full Text PDF PubMed Scopus (206) Google Scholar, 6Novick P. Schekman R. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1858-1862Crossref PubMed Scopus (368) Google Scholar, 7Verhage 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 (974) Google Scholar). The mammalian SM protein munc18-1 was originally isolated as a syntaxin 1-binding protein that binds to the monomeric form of syntaxin 1, rendering the t-SNARE unable to form the SDS-resistant ternary SNARE complex (8Pevsner J. Hsu S.C. Braun J.E. Calakos N. Ting A.E. Bennett M.K. Scheller R.H. Neuron. 1994; 13: 353-361Abstract Full Text PDF PubMed Scopus (522) Google Scholar, 9Hata Y. Slaughter C.A. Sudhof T.C. Nature. 1993; 366: 347-351Crossref PubMed Scopus (576) Google Scholar). Syntaxin 1 can act as a molecular switch, adopting two structurally distinct forms (10Dulubova I. Sugita S. Hill S. Hosaka M. Fernandez I. Sudhof T.C. Rizo J. EMBO J. 1999; 18: 4372-4382Crossref PubMed Scopus (541) Google Scholar). In the open form, the SNARE helix does not interact with the N-terminal three-helical regulatory domain (termed Habc) and has been shown to not interact with munc18-1 (10Dulubova I. Sugita S. Hill S. Hosaka M. Fernandez I. Sudhof T.C. Rizo J. EMBO J. 1999; 18: 4372-4382Crossref PubMed Scopus (541) Google Scholar). In contrast, the closed form of syntaxin 1, in which the N-terminal Habc domain interacts with the SNARE helix, exhibits a high affinity for munc18-1. Association of syntaxin 1 with its SNARE partners to form the ternary SNARE complex, which prevents syntaxin from adopting the closed conformation, has also been shown to preclude munc18-1 binding (11Yang B. Steegmaier M. Gonzalez Jr., L.C. Scheller R.H. J. Cell Biol. 2000; 148: 247-252Crossref PubMed Scopus (227) Google Scholar). However, a recent finding by Zilly et al. (12Zilly F.E. Sorensen J.B. Jahn R. Lang T. PLoS Biol. 2006; 4: e330Crossref PubMed Scopus (97) Google Scholar) using lysed cellular membrane sheets provided evidence that munc18-1 may interact with syntaxin 1 when in the binary SNARE complex (a heterodimer of syntaxin and SNAP-25). The interaction of munc18-1 with its cognate syntaxin is in sharp contrast to the specificity of its yeast homologue Sec1p, which binds its cognate syntaxin, Sso1p, in the ternary SNARE complex and not in the monomeric state (13Carr C.M. Grote E. Munson M. Hughson F.M. Novick P.J. J. Cell Biol. 1999; 146: 333-344Crossref PubMed Scopus (256) Google Scholar). This binding specificity has, however, since been questioned (14Scott 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 (86) Google Scholar). Another yeast SM protein, Vps45p, binds to its cognate Golgi syntaxin, Tlg2p, both in the monomeric state and in the ternary SNARE complex (15Bryant N.J. James D.E. EMBO J. 2001; 20: 3380-3388Crossref PubMed Scopus (106) Google Scholar, 16Nichols B.J. Holthuis J.C. Pelham H.R. Eur. J. Cell Biol. 1998; 77: 263-268Crossref PubMed Scopus (57) Google Scholar). This interaction can occur through an N-terminal region of Tlg2p with a similar interaction observed for its mammalian Golgi homologue, syntaxin 16 (17Dulubova I. Yamaguchi T. Gao Y. Min S.W. Huryeva I. Sudhof T.C. Rizo J. EMBO J. 2002; 21: 3620-3631Crossref PubMed Scopus (151) Google Scholar). A similar ability to bind syntaxin in both monomeric and complexed forms has been observed for the SM protein Sly1p and its cognate syntaxin Sed5p (18Grabowski R. Gallwitz D. FEBS Lett. 1997; 411: 169-172Crossref PubMed Scopus (44) Google Scholar, 19Peng R. Gallwitz D. J. Cell Biol. 2002; 157: 645-655Crossref PubMed Scopus (119) Google Scholar). This apparent discrepancy in binding mode suggests differences in binding sites and recognition motifs of SM proteins and their syntaxin partners. To date, the crystal structures of two SM proteins, munc18-1 and Sly1p (from Saccharomyces cerevisiae), in complex with their cognate SNAREs, have been solved (20Bracher A. Weissenhorn W. EMBO J. 2002; 21: 6114-6124Crossref PubMed Scopus (129) Google Scholar, 21Misura K.M. Scheller R.H. Weis W.I. Nature. 2000; 404: 355-362Crossref PubMed Scopus (599) Google Scholar). Despite exhibiting high structural homology, munc18-1 and Sly1p appear to interact with their cognate syntaxins, syntaxin 1a and Sed5p, respectively, through independent sites. Munc18-1 binds to the closed conformation of syntaxin 1a with the N-terminal Habc domain folded back on to the helix involved in SNARE complex formation (10Dulubova I. Sugita S. Hill S. Hosaka M. Fernandez I. Sudhof T.C. Rizo J. EMBO J. 1999; 18: 4372-4382Crossref PubMed Scopus (541) Google Scholar, 22Calakos N. Bennett M.K. Peterson K.E. Scheller R.H. Science. 1994; 263: 1146-1149Crossref PubMed Scopus (361) Google Scholar), whereas Sly1p binds the N terminus of Sed5p through an independent binding site. The disparate modes of interaction between SM proteins and their cognate syntaxins, observed in different species and for different membrane compartments, are at odds with the conserved role for SM proteins in membrane fusion indicated by genetic experiments (2Rizo J. Sudhof T.C. Nat. Rev. Neurosci. 2002; 3: 641-653Crossref PubMed Scopus (424) Google Scholar, 23Toonen R.F. Verhage M. Trends Cell Biol. 2003; 13: 177-186Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Importantly, effects exerted by munc18-1 on different stages in the syntaxin lifecycle, from trafficking (24Rowe J. Corradi N. Malosio M.L. Taverna E. Halban P. Meldolesi J. Rosa P. J. Cell Sci. 1999; 112: 1865-1877Crossref PubMed Google Scholar), through vesicle docking (25Voets T. Toonen R.F. Brian E.C. de Wit H. Moser T. Rettig J. Sudhof T.C. Neher E. Verhage M. Neuron. 2001; 31: 581-591Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar), to the final fusion event itself (26Fisher R.J. Pevsner J. Burgoyne R.D. Science. 2001; 291: 875-878Crossref PubMed Scopus (172) Google Scholar), cannot be explained using the current model of the munc18-1-syntaxin 1a interaction. In this study, we analyzed in detail the syntaxin 1-munc18-1 interaction using mutations shown to lock syntaxin 1 in the open conformation (10Dulubova I. Sugita S. Hill S. Hosaka M. Fernandez I. Sudhof T.C. Rizo J. EMBO J. 1999; 18: 4372-4382Crossref PubMed Scopus (541) Google Scholar). Here we show that munc18-1 binds to an evolutionarily conserved motif at the N terminus of syntaxin 1 in addition to the closed form binding mode. Using quantitative colocalization and fluorescence lifetime imaging microscopy (FLIM), we show that these two binding modes of syntaxin1 and munc18-1 are spatially segregated in living cells. Furthermore, although the closed form binding mode prevents progress through the SNARE assembly pathway, syntaxin 1 in the open form can progress to the ternary SNARE complex with munc18-1 remaining associated. This is the first demonstration that the major neuronal and neuroendocrine SNARE, syntaxin 1, and its cognate SM protein, munc18-1, can interact through dual modes of binding. Vectors and Cell Culture—Plasmids encoding glutathione S-transferase (GST) fusion proteins with syntaxin 1a (amino acids 1–261, cytoplasmic domain), SNAP-25 (amino acids 1–206), and synaptobrevin (amino acids 1–96) were described previously (27Hu K. Carroll J. Fedorovich S. Rickman C. Sukhodub A. Davletov B. Nature. 2002; 415: 646-650Crossref PubMed Scopus (156) Google Scholar, 28Kweon D.H. Chen Y. Zhang F. Poirier M. Kim C.S. Shin Y.K. Biochemistry. 2002; 41: 5449-5452Crossref PubMed Scopus (21) Google Scholar). A plasmid encoding a polyhistidine-tagged munc18-1 (amino acids 1–594) was as described previously (29Graham M.E. Sudlow A.W. Burgoyne R.D. J. Neurochem. 1997; 69: 2369-2377Crossref PubMed Scopus (49) Google Scholar). Generation of N-terminal truncations of syntaxin 1a was performed by PCR and subsequent ligation into HindIII/KpnI and HindIII/XbaI sites of pmCerulean-C1 and PGEX-KG, respectively, or into pTargeT (Promega). Distance between the fusion tag and syntaxin 1a was maintained in all constructs. The (L165A,E166A) mutation was generated by site-directed mutagenesis using a QuikChange II XL kit (Stratagene). An EYFP-munc18-1 fusion was generated using similar standard techniques. Neuroblastoma 2A (N2A) cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 10 mm l-glutamine, 50 units of penicillin, 50 μg/ml streptomycin and maintained at 37 °C in 5% (v/v) CO2, 95% (v/v) air. All transfections were performed using ExGen 500 (Fermentas). Protein Biochemistry—Recombinant GST fusion proteins were expressed and purified as described previously (30Rickman C. Davletov B. Chem. Biol. 2005; 12: 545-553Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). For in vitro binding reactions, 2 μg of GST-syntaxin 1a or SNAP-25 was immobilized on glutathione-Sepharose beads (GE Healthcare) and incubated in a total volume of 100 μl of 20 mm HEPES, pH 7.4, 100 mm NaCl, 1 mm EDTA, 0.1% Triton X-100 (Buffer A) with 3 μg of protein to be tested for binding for 1 h at 4 °C. Beads were washed by low speed centrifugation, and bound protein was eluted in SDS-containing sample buffer followed by SDS-PAGE and Coomassie Blue staining. For binding reactions involving munc18-1, GST-fused syntaxin 1a was incubated either with freshly prepared detergent rat brain extract, as described previously (30Rickman C. Davletov B. Chem. Biol. 2005; 12: 545-553Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), or with freshly prepared detergent bacterial extract containing expressed recombinant His6-munc18-1. Circular dichroism experiments were performed as described previously (31Rickman C. Jimenez J.L. Graham M.E. Archer D.A. Soloviev M. Burgoyne R.D. Davletov B. Mol. Biol. Cell. 2006; 17: 283-294Crossref PubMed Scopus (60) Google Scholar). For the measurement of syntaxin 1a affinity for munc18-1, 0.2 pmol of GST-fused syntaxin 1a was immobilized on glutathione-Sepharose beads and incubated with increasing concentrations of munc18-1 in detergent bacterial extract in a reaction volume of 0.5 ml at 21 °C. The beads were washed three times in buffer A, and bound protein was analyzed by Western immunoblotting using a monoclonal anti-munc18-1 antibody (BD Biosciences) and West Dura enhanced chemiluminescence kit (Pierce). The chemiluminescent signal was imaged using a cooled 16-bit CCD and quantified using ImageJ. The concentration of munc18-1 in the detergent bacterial extract was measured by purification of munc18-1 and in-gel quantification using Sypro Red (Invitrogen). The calculated intensity volumes were fitted with a variable slope dose-response relationship using Prism (GraphPad). For binding assays using a purified syntaxin 1a-munc18-1 complex, bacterial detergent extracts containing expressed GST-Syx1–261 (or variant) and His6-munc18-1 were mixed. The resulting protein complex was purified sequentially on glutathione-Sepharose resin and Profinity IMAC resin (Bio-Rad). Purity was assessed by Sypro Red in-gel staining to confirm an equimolar stoichiometric ratio (30Rickman C. Davletov B. Chem. Biol. 2005; 12: 545-553Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Confocal Laser Scanning Microscopy and Image Analysis— All imaging experiments were performed using a Zeiss LSM 510 Axiovert confocal laser scanning microscope, equipped with a pulsed excitation source (MIRA 900 Ti:Sapphire femto-second pulsed laser with a coupled VERDI 10-watt pump laser (Coherent). Data were acquired using a 1024 × 1024 pixel image size, using a Zeiss Plan NeoFLUAR 1.4 NA ×63 oil immersion lens or a Zeiss C-Apochromat 1.2 NA ×63 water-corrected immersion objective lens. All imaging was performed using living cells, maintained at 37 °C in 5% (v/v) CO2, 95% (v/v) air. Image data, acquired at Nyquist sampling rates, were deconvolved using Huygens software (Scientific Volume Imaging), and the resulting three-dimensional models were analyzed using NIH ImageJ software. Residual maps were generated by calculating the residual of each voxel from the linear regression fit to the intensities of each channel within each voxel. The resulting residuals are displayed on a color scale from -1 to 1 (with zero residual colored cyan) with brightness corresponding to the combined intensity of the two channels. Cell peripheries were determined using transmitted light imaging combined with confocal data. TCSPC-FLIM Acquisition and Analysis—Time-correlated single photon counting (TCSPC) measurements were made under 800–820 nm two-photon excitation, which efficiently excited cerulean without any detectable direct excitation or emission from EYFP, using a non-descanned detector (R3809U-50) multichannel plate-photomultiplier tube or a fast photomultiplier tube (H7422; both Hamamatsu Photonics UK) coupled directly to the rear port of the Axiovert microscope. TCSPC recordings were acquired for between 10 and 60 s, and mean photon counts were between 105 and 106 cps. Images were recorded at 256 × 256 pixels from a 1024 × 1024 image scan with 256 time bins over a 12-ns period (32Duncan R.R. Bergmann A. Cousin M.A. Apps D.K. Shipston M.J. J. Microsc. (Oxf.). 2004; 215: 1-12Crossref PubMed Scopus (116) Google Scholar). Off-line FLIM data analysis used pixel-based fitting software (SPCImage, Becker & Hickl). The fluorescence was assumed to follow a multiexponential decay. In addition, an adaptive offset correction was performed. A constant offset takes into consideration the time-independent baseline due to dark noise of the detector and the background caused by room light, calculated from the average number of photons per channel preceding the rising part of the fluorescence trace. To fit the parameters of the multiexponential decay to the fluorescence decay trace measured by the system, a convolution with the instrumental response function was carried out. The optimization of the fit parameters was performed by using the Levenberg-Marquardt algorithm, minimizing the weighted χ-square quantity. This approach can be used to separate the interacting from the non-interacting donor fraction in our Förster resonance energy transfer (FRET) systems. The long lifetime component τ2 was determined by control assays with cerulean alone or expressed with (non-interacting) EYFP as described above. This value was subsequently used as a fixed τ2 lifetime for all other experiments. As controls for nonspecific FRET or FRET between green fluorescent proteins that may form dimers spontaneously when overexpressed in cells, we determined the fluorescence lifetimes of cerulean-Syx1–288 alone, cerulean alone, or cerulean-Syx1–288 co-transfected with EYFP (not shown). No FRET was detected in any of these experiments. Likewise, experiments using ceruleanA206K-fused Syx1–288 revealed no self-dimerization between fluorescent proteins. We analyzed the conservation of amino acids in syntaxin 1 homologues from evolutionarily distant species along with Sed5p from S. cerevisiae (Fig. 1A). There is a high degree of similarity between the syntaxin homologues throughout the sequence corresponding to the SNARE helix and head domain of syntaxin. Of note is the additional spike in similarity in the first 15 amino acids. Analysis of the sequence contained in this region demonstrated a high degree of conservation of amino acids with a lysine-aspartate-arginine (KDR) motif absolutely conserved in both Sed5p and the syntaxin homologues. A structural alignment of munc18-1 and Sly1p demonstrates the highly conserved topology of these two proteins despite an overall sequence identity of 18% (Fig. 1B). Highlighted on the bound Sed5p peptide (Fig. 1B) are the residues absolutely conserved in the sequence alignment in Fig. 1A. The conservation of these residues suggested that the N-terminal of syntaxin 1a may be capable of interacting with munc18-1 in a similar manner and that an open mutation of syntaxin 1a (10Dulubova I. Sugita S. Hill S. Hosaka M. Fernandez I. Sudhof T.C. Rizo J. EMBO J. 1999; 18: 4372-4382Crossref PubMed Scopus (541) Google Scholar) could still support an interaction with munc18-1. We therefore determined the ability of syntaxin 1a (Syx1–261) and an open mutant of syntaxin (L165A,E166A) (10Dulubova I. Sugita S. Hill S. Hosaka M. Fernandez I. Sudhof T.C. Rizo J. EMBO J. 1999; 18: 4372-4382Crossref PubMed Scopus (541) Google Scholar) (Syx 1–261 (Open)) to bind to munc18-1 in rat brain detergent extracts. Both forms of syntaxin readily bound munc18-1 (Fig. 1C). As the crystal structure of the syntaxin 1a-munc18-1 complex (notably excluding the first 26 amino acids of syntaxin) indicates that only closed syntaxin can bind to munc18-1 (21Misura K.M. Scheller R.H. Weis W.I. Nature. 2000; 404: 355-362Crossref PubMed Scopus (599) Google Scholar), these findings suggest that an additional mechanism and site of interaction exists. To determine whether the conserved amino acids found at the syntaxin N terminus are involved in a direct interaction with munc18-1, we generated a series of truncations of syntaxin in both the wild-type and the open mutant forms. These purified proteins were incubated with a bacterial detergent extract containing recombinant His6-munc18-1 (Fig. 1D). Syx1–261 and Syx 1–261 (Open) both readily bound recombinant munc18-1 from bacterial detergent extracts, replicating the observation using detergent brain extracts. The N-terminal truncations of syntaxin 1a had no affect on their ability to bind munc18-1, but importantly, the combination of N-terminal truncation and the open mutations severely diminished munc18-1 interaction. As truncation of the first six amino acids produced a decrease in binding of munc18-1 to open mutants of syntaxin 1a (Fig. 1D), similar to the longer deletions, we concentrated on this short truncation in further experiments. Circular dichroism analysis confirmed that the mutations and truncations incorporated into syntaxin 1a resulted in no major changes to the secondary structure of the proteins (Fig. 1E). Because binding reactions, as used in Fig. 1D, are strongly dependent on the relative concentrations of the interacting partners used, the affinity of munc18-1 for syntaxin and for the mutant forms was measured. Syx1–261, Syx1–261 (Open), and Syx7–261 (Δ6) all had a similar affinity for munc18-1. Only when the open mutation was combined with truncation of the N terminus (Syx7–261 (Open) (Δ6)) was there a significant reduction in the affinity of syntaxin 1a for munc18-1 (Fig. 1F). These in vitro data suggest that syntaxin 1a can interact with munc18-1 through two distinct binding mechanisms. It is unclear, however, whether these binding modes are employed in a cellular context. To address this question, fluorescent fusions of munc18-1 and syntaxin 1a were visualized in living N2A cells. A quantitative approach was used to investigate colocalization of syntaxin 1a and munc18-1. In addition to the merger of the two emission channels, to display areas of coincidence, the intensities of both channels within each voxel were displayed as a frequency histogram and fit by linear regression. From this fit, residuals for each voxel were calculated and displayed as a residual map to highlight areas of covariance (expected if two proteins interact with a defined stoichiometry) within an image. Syntaxin 1a and munc18-1 exhibited a high degree of coincidence and of intensity covariance on the plasma membrane and in intracellular membranes (Fig. 2A). Similarly, Syx7–288 (Δ6) and Syx1–288 (Open) demonstrated a high degree of colocalization with munc18-1. Importantly, however, munc18-1 no longer colocalized with syntaxin when both the N-terminal truncation and the open syntaxin mutations were combined (Syx7–288 (Δ6) (Open)). Munc18-1 is a soluble protein with no localization signals, and as such, its membrane association relies on its interaction with syntaxin 1a (33Schutz D. Zilly F. Lang T. Jahn R. Bruns D. Eur. J. Neurosci. 2005; 21: 2419-2432Crossref PubMed Scopus (46) Google Scholar); when co-expressed with Syx7–288 (Open) (Δ6), munc18-1 adopted a cytoplasmic localization. As a negative control for random colocalization, unfused munc18-1 and EYFP were co-expressed with cerulean-syntaxin 1a. Pearson's correlation coefficient analysis demonstrated no significant difference between cerulean-Syx1–288, -Syx1–288 (Open), and -Syx7–288 (Δ6) covariance with EYFP-munc18-1 (Fig. 2B). However, the combined N-terminal deletion and open mutation of syntaxin 1a (Syx7–288 (Open) (Δ6)) resulted in a large decrease in covariance to a level not significantly different from the negative control. This quantitative analysis of colocalization between mutant proteins in living cells provided evidence for a role of the N terminus of syntaxin 1a in binding to munc18-1. However, colocalization data are limited by the resolution of the microscope (maximally 200 nm) and do not directly indicate interaction. To increase our understanding of each mode of syntaxin 1a binding to munc18-1, we employed FLIM. FLIM quantifies the fluorescence lifetime of a fluorophore, the duration of which is sensitive to the microenvironment it inhabits (32Duncan R.R. Bergmann A. Cousin M.A. Apps D.K. Shipston M.J. J. Microsc. (Oxf.). 2004; 215: 1-12Crossref PubMed Scopus (116) Google Scholar). Thus, FRET, to an interacting acceptor molecule, dramatically shortens the donor fluorescence lifetime; this effect can be quantified directly in each pixel of an image. Furthermore, the low light levels required for FLIM allowed us to select cells expressing very low levels of exogenous proteins. Using this technique, the fraction of non-interacting and interacting donor fluorescence lifetimes in each pixel can be resolved (Fig. 3, A–D) (32Duncan R.R. Bergmann A. Cousin M.A. Apps D.K. Shipston M.J. J. Microsc. (Oxf.). 2004; 215: 1-12Crossref PubMed Scopus (116) Google Scholar). This approach revealed that fewer Syx1–288 and munc18-1 molecules interact on the plasma membrane when compared with intracellular locations (Mann-Whitney, p < 0.007, n = 18) and confirmed that Syx1–288 (Open) and munc18-1 interact in cells (Fig. 3F). Surprisingly, FRET (i.e. donor fluorescence lifetime and amplitude ratios) between cerulean-Syx1–288 (Open) and EYFP-munc18-1 was substantially decreased in intracellular membranes when compared with Syx1–288 and munc18-1 (p < 0.001, n = 8; Fig. 3F) with cell surface interactions maintained in evenly distributed puncta. In contrast, cerulean-Syx7–288 (Δ6) showed no decrease in interaction in intracellular membranes but a large reduction in FRET on the cell surface (p < 0.001, n = 8). These effects were additive; cerulean-Syx7–288 (Open) (Δ6) exhibited decreased FRET both intracellularly and on the cell surface (Fig. 3F). Thus, the two binding modes are spatially distinct with syntaxin 1a N-terminal deletion affecting plasma membrane interactions and abolition of syntaxin closed conformation binding affecting predominantly intracellular complexes. Why does deletion of the syntaxin N-terminal amino acids reduce interaction with munc18-1 at the plasma membrane? We have shown that N-terminal binding to munc18-1 is utilized when syntaxin is opened. This being the case, open syntaxin might be able to bind both munc18-1 and the other SNARE(s) simultaneously at the cell surface; loss of N-terminal interaction would thus dissociate munc18-1 in the presence of SNAP-25. To explore this hypothesis, we used highly purified complexes of syntaxin 1a bound to munc18-1. It was thus possible to probe the effect of N-terminal truncation and the open mutation on progression of syntaxin through the SNARE assembly pathway (Fig. 4A). Incubation of Syx1–261/munc18-1 with immobilized SNAP-25 demonstrated that in this form, syntaxin is unable to engage into either the binary SNARE complex or the ternary SNARE complex (Fig. 4A, left panel). The N-terminal truncation of syntaxin did not allow interaction of the Syx7–261 (Δ6)/munc18-1 complex with no SNAP-25 binding detected (Fig. 4A, center panel). However, incorporation of the open mutation into syntaxin allowed Syx1–261(Open)/munc18-1 to interact with SNAP-25 forming both the binary SNARE complex (t-SNARE heterodimer) and the ternary SNARE complex (Fig. 4A, right panel). As a control, munc18-1 alone was incubated with immobilized SNAP-25 (Fig. 4B). As shown previously (11Yang B. Steegmaier M. Gonzalez Jr., L.C. Scheller R.H. J. Cell Biol. 2000; 148: 247-252Crossref PubMed Scopus (227) Google Scholar), munc18-1 was unable to interact directly with SNAP-25. Importantly, munc18-1 remained bound to syntaxin 1a throughout the SNARE assembly pathway. This SNARE complex-munc18-1 interaction cannot include closed form syntaxin, and we conclude therefore that the N-terminal mode of interaction is essential at the cell surface for this reason. Our data demonstrate that munc18-1 can interact with syntaxin 1a through two discrete modes of binding that are spatially distinct within the cell. The closed form of syntaxin-munc18-1 binding is a structurally constrained state with properties comparable with those previously described for this complex (8Pevsner J. Hsu S.C. Braun J.E. Calakos N. Ting A.E. Bennett M.K. Scheller R.H. Neuron. 1994; 13: 353-361Abstract Full Text PDF PubMed Scopus (522) Google Scholar, 21Misura K.M. Scheller R.H. Weis W.I. Nature. 2000; 404: 355-362Crossref PubMed Scopus (599) Google Scholar). This mode of interaction is of high affinity and occurs primarily, but not exclusively, on intracellular membranes (Figs. 1 and 3). We speculate that this conformation of syntaxin 1a is clamped by munc18-1 to allow the passage of syntaxin through the endoplasmic reticulum and Golgi apparatus, preventing ectopic intracellular SNARE complex formation. In addition to this form of binding, we now show that syntaxin 1a and munc18-1 can interact through an evolutionarily conserved motif at the N terminus of syntaxin. It is of note that an early clone of syntaxin 1a (22Calakos N. Bennett M.K. Peterson K.E. Scheller R.H. Science. 1994; 263: 1146-1149Crossref PubMed Scopus (361) Google Scholar) lacks the conserved N-terminal amino acids identified in Fig. 1 and has since been widely used to examine syntaxin 1a protein-protein interactions. In contrast to a previous report (10Dulubova I. Sugita S. Hill S. Hosaka M. Fernandez I. Sudhof T.C. Rizo J. EMBO J. 1999; 18: 4372-4382Crossref PubMed Scopus (541) Google Scholar) using rat brain extracts (as in Fig. 1D), munc18-1 readily binds to a mutation that prevents syntaxin 1a from adopting a closed conformation, exhibiting no reduction in affinity for this interaction (Fig. 1). We are unable to explain this discrepancy, although it should be noted that the mutants used in the NMR studies had a 26-amino acid N-terminal truncation as this gave better spectra (10Dulubova I. Sugita S. Hill S. Hosaka M. Fernandez I. Sudhof T.C. Rizo J. EMBO J. 1999; 18: 4372-4382Crossref PubMed Scopus (541) Google Scholar). Importantly, the full-length form of this mutant syntaxin rescues the Caenorhabditis elegans unc13 phenotype (34Richmond J.E. Weimer R.M. Jorgensen E.M. Nature. 2001; 412: 338-341Crossref PubMed Scopus (314) Google Scholar), interpreted as circumventing the requirement for Unc13 to dissociate Unc18 and open syntaxin. However, we now show that this mutation permits syntaxin to progress through the SNARE assembly pathway, all the while still associated with munc18-1 (Fig. 4). Little or no information is available to describe the intracellular locations of SM-syntaxin interactions. A recent report found that Vps45p, an SM homologue in S. cerevisiae, can interact in vitro with its cognate syntaxin through an undefined second mode of binding in addition to its well characterized N-terminal interaction (35Carpp L.N. Ciufo L.F. Shanks S.G. Boyd A. Bryant N.J. J. Cell Biol. 2006; 173: 927-936Crossref PubMed Scopus (84) Google Scholar). Additionally, the mammalian SM protein munc18-3 binds its partner syntaxin 4 principally through an N-terminal interaction in vitro (36Latham 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 (98) Google Scholar). However, the authors of this report (36Latham 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 (98) Google Scholar) were unable to exclude that a second mode of binding exists. It is therefore credible that a dual mode of interaction with cognate syntaxins is a conserved feature of the SM protein family. Why have SM proteins evolved multiple functions and binding modes, and how do they control vesicle fusion? We propose that syntaxin 1a is transported to the plasma membrane in a closed conformation with the Habc regulatory head domain interacting with the SNARE helix, bound by munc18-1 (Fig. 4C). This is in agreement with a previously suggested chaper-one role for munc18-1 (24Rowe J. Corradi N. Malosio M.L. Taverna E. Halban P. Meldolesi J. Rosa P. J. Cell Sci. 1999; 112: 1865-1877Crossref PubMed Google Scholar) and would most closely resemble the binding mode observed in the crystal structure of the syntaxin1a/munc18-1 complex (21Misura K.M. Scheller R.H. Weis W.I. Nature. 2000; 404: 355-362Crossref PubMed Scopus (599) Google Scholar), although it may also include the additional interaction of munc18-1 with the N terminus of syntaxin 1a. This mode of binding would preclude any aberrant interaction with intracellular or trafficking plasma membrane SNAREs until delivery to the plasma membrane. At the plasma membrane, the syntaxin 1a would change conformation from a closed to an open state under the regulation of an as yet undefined plasma membrane factor (30Rickman C. Davletov B. Chem. Biol. 2005; 12: 545-553Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 34Richmond J.E. Weimer R.M. Jorgensen E.M. Nature. 2001; 412: 338-341Crossref PubMed Scopus (314) Google Scholar, 37Barclay J.W. Craig T.J. Fisher R.J. Ciufo L.F. Evans G.J. Morgan A. Burgoyne R.D. J. Biol. Chem. 2003; 278: 10538-10545Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). In this form, munc18-1 binding would occur through the N terminus of syntaxin 1a through a similar interaction as observed in the Sed5p/Sly1p crystal structure (20Bracher A. Weissenhorn W. EMBO J. 2002; 21: 6114-6124Crossref PubMed Scopus (129) Google Scholar). This conformational change of syntaxin 1a would permit the subsequent interaction of syntaxin with SNAP-25 to form the binary t-SNARE heterodimer while retaining the interaction with munc18 through the N-terminal mode of binding. This complex may form a scaffold for vesicle docking, a proposed function for munc18-1 (25Voets T. Toonen R.F. Brian E.C. de Wit H. Moser T. Rettig J. Sudhof T.C. Neher E. Verhage M. Neuron. 2001; 31: 581-591Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 38Toonen R.F. Kochubey O. de Wit H. Gulyas-Kovacs A. Konijnenburg B. Sorensen J.B. Klingauf J. Verhage M. EMBO J. 2006; 25: 3725-3737Crossref PubMed Scopus (137) Google Scholar) that cannot be explained adequately using current models. Subsequent triggering of exocytosis would then drive engagement of synaptobrevin with the t-SNARE heterodimer to form the four-helical ternary SNARE complex formation driving membrane fusion. The association of munc18-1 with the ternary SNARE complex through the N terminus of syntaxin 1a can explain the observed effect that munc18-1 can exert on the final fusion step (26Fisher R.J. Pevsner J. Burgoyne R.D. Science. 2001; 291: 875-878Crossref PubMed Scopus (172) Google Scholar). It is reasonable to expect that the function of munc18-1 here parallels that of sec1p, which apparently stabilizes the yeast exocytic SNARE complex and is required for vesicle fusion (13Carr C.M. Grote E. Munson M. Hughson F.M. Novick P.J. J. Cell Biol. 1999; 146: 333-344Crossref PubMed Scopus (256) Google Scholar). The interaction of munc18-1 with different conformational forms of syntaxin, at spatially distinct sites, can explain the multiple, and somewhat controversial, roles that munc18-1 has been proposed to play in the cell and so help unify thinking on SM protein structure and function.
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