title: Reconstituted syntaxin1a/SNAP25 interacts with negatively charged lipids as measured by lateral diffusion in planar supported bilayers.
abstract: According to the soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein (SNAP) receptor hypothesis (SNARE hypothesis), interactions between target SNAREs and vesicle SNAREs (t- and v-SNAREs) are required for membrane fusion in intracellular vesicle transport and exocytosis. The precise role of the SNAREs in tethering, docking, and fusion is still disputed. Biophysical measurements of SNARE interactions in planar supported membranes could potentially resolve some of the key questions regarding the mechanism of SNARE-mediated membrane fusion. As a first step toward this goal, recombinant syntaxin1A/SNAP25 (t-SNARE) was reconstituted into polymer-supported planar lipid bilayers. Reconstituted t-SNAREs in supported bilayers bound soluble green fluorescent protein/vesicle-associated membrane protein (v-SNARE), and the SNARE complexes could be specifically dissociated by NSF/alpha-SNAP in the presence of ATP. The physiological activities of SNARE complex formation were thus well reproduced in this reconstituted planar model membrane system. A large fraction (~75%) of the reconstituted t-SNARE was laterally mobile with a lateral diffusion coefficient of 7.5 x 10(-9) cm(2)/s in a phosphatidylcholine lipid background. Negatively charged lipids reduced the mobile fraction of the t-SNARE and the lipids themselves. Phosphatidylinositol-4,5-bisphosphate was more effective than phosphatidylserine in reducing the lateral mobility of the complexes. A model of how acidic lipid-SNARE interactions might alter lipid fluidity is discussed.
authors: Department of Molecular Physiology and Biological Physics and Center for Structural Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
authors: M L
authors: ML
authors: Wagner
authors: L K
authors: LK
authors: Tamm
mesh_terms: D000838:Anions / Q000378:metabolism; D000954:Antigens, Surface / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D004058:Diffusion; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism*; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008968:Molecular Conformation; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D011092:Polyethylene Glycols; D011108:Polymers / Q000737:chemistry; D011485:Protein Binding; D011791:Quartz; D050600:SNARE Proteins; D012995:Solubility; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins
chemical_list: D000838:Anions; D000954:Antigens, Surface; D002352:Carrier Proteins; D008051:Lipid Bilayers; D008081:Liposomes; D046911:Macromolecular Substances; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010713:Phosphatidylcholines; D019269:Phosphatidylinositol 4,5-Diphosphate; D010718:Phosphatidylserines; D011108:Polymers; D050600:SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D011791:Quartz; D011092:Polyethylene Glycols; C028694:1-palmitoyl-2-oleoylphosphatidylcholine