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| The EMBO Journal
(1999) 18, 4372–4382, doi:10.1093/emboj/18.16.4372 |
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| A conformational switch in syntaxin during exocytosis: role of munc18 |
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| Irina Dulubova1, 2, Shuzo Sugita3, 4, 5, Sandra Hill1, 2, Masahiro Hosaka3, 4, 5, Imma Fernandez1, 2, Thomas C. Südhof3, 4, 5 and Josep Rizo1, 2 |
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1 Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, USA 2 Department of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, USA 3 Department of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, USA 4 Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, USA 5 Center for Basic Neuroscience, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235, USA To whom correspondence should be addressed Thomas C. Südhof, Tsudho@MEDNET.swmed.edu Josep Rizo, jose@arnie.swmed.edu Received 4 May 1999; Revised 29 June 1999; Accepted 29 June 1999. |
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| Abstract |
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| Syntaxin 1, an essential protein in synaptic membrane fusion, contains a helical autonomously folded N-terminal domain, a C-terminal SNARE motif and a transmembrane region. The SNARE motif binds to synaptobrevin and SNAP-25 to assemble the core complex, whereas almost the entire cytoplasmic sequence participates in a complex with munc18-1, a neuronal Sec1 homolog. We now demonstrate by NMR spectroscopy that, in isolation, syntaxin adopts a 'closed' conformation. This default conformation of syntaxin is incompatible with core complex assembly which requires an 'open' syntaxin conformation. Using site-directed mutagenesis, we find that disruption of the closed conformation abolishes the ability of syntaxin to bind to munc18-1 and to inhibit secretion in PC12 cells. These results indicate that syntaxin binds to munc18-1 in a closed conformation and suggest that this conformation represents an essential intermediate in exocytosis. Our data suggest a model whereby, during exocytosis, syntaxin undergoes a large conformational switch that mediates the transition between the syntaxin–munc18-1 complex and the core complex. |
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| Keywords: exocytosis, munc18, protein NMR, SNARE, syntaxin |
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