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Syntaxin Is Efficiently Excluded from Sphingomyelin-enriched Domains in Supported Lipid Bilayers Containing Cholesterol

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Abstract

Formation of a trans-complex between the three SNARE proteins syntaxin, synaptobrevin and SNAP-25 drives membrane fusion. The structure of the core SNARE complex has been studied extensively. Here we have used atomic force microscopy to study the behavior of recombinant syntaxin 1A both in detergent extracts and in a lipid environment. Full-length syntaxin in detergent extracts had a marked tendency to aggregate, which was countered by addition of munc-18. In contrast, syntaxin lacking its transmembrane region was predominantly monomeric. Syntaxin could be integrated into liposomes, which formed lipid bilayers when deposited on a mica support. Supported bilayers were decorated with lipid vesicles in the presence, but not the absence, of full-length syntaxin, indicating that formation of syntaxin complexes in trans could mediate vesicle docking. Syntaxin complexes remained at the sites of docking following detergent solubilization of the lipids. Raised lipid domains could be seen in bilayers containing sphingomyelin, and these domains were devoid of syntaxin and docked vesicles in the presence, but not the absence, of cholesterol. Our results demonstrate that syntaxin is excluded from sphingomyelin-enriched domains in a cholesterol-dependent manner.

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Acknowledgements

We are grateful to Dr. J. Skepper (Multi-Imaging Laboratory, Department of Anatomy, University of Cambridge, United Kingdom) for expert assistance with the electron microscopy, and to Dr. B. Davletov (MRC Laboratory of Molecular Biology, Cambridge, United Kingdom) for many helpful discussions, and for critical reading of this manuscript. This work was supported by Grant B12816 from the Biotechnology and Biological Sciences Research Council (to R.M.H. and J.M.E.). J.C.L. was supported by an Overseas Research Scholarship and the Cambridge Commonwealth Trust.

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  1. Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom

    D. E. Saslowsky, J. C. Lawrence, R. M. Henderson & J. M. Edwardson

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  1. D. E. Saslowsky
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  2. J. C. Lawrence
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  3. R. M. Henderson
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  4. J. M. Edwardson
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Correspondence to J. M. Edwardson.

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Saslowsky, D., Lawrence, J., Henderson, R. et al. Syntaxin Is Efficiently Excluded from Sphingomyelin-enriched Domains in Supported Lipid Bilayers Containing Cholesterol . J. Membrane Biol. 194, 153–164 (2003). https://doi.org/10.1007/s00232-003-2035-7

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