title: Molecular mechanism of cholesterol- and polyphosphoinositide-mediated syntaxin clustering.
abstract: The neuronal acceptor SNARE complex that functions as the receptor for synaptic vesicle docking and fusion at the presynaptic membrane is composed of the single-span transmembrane protein syntaxin-1A and the palmitoylated soluble protein SNAP-25. Previously, we explored interactions that promote the formation of syntaxin-1A clusters in membranes. Cholesterol activates clustering in native and model membranes, and its depletion in neuroendocrine cells results in a homogeneous distribution of the protein. However, as little as 1 mol % phosphatidylinositol 4,5-bisphosphate (PI-4,5-P(2)) or 20 mol % phosphatidylserine was found to disperse syntaxin-1A clusters [Murray, D. H., and Tamm, L. K. (2009) Biochemistry 48, 4617-4625]. Strong evidence suggests that syntaxin-1A and its synaptic vesicle cognate synaptobrevin both interact directly with PI-4,5-P(2) and that this interaction activates fusion. However, the molecular details of this interaction and its relationship to the partial dispersion of syntaxin-1A clusters remain largely unexplored. Hence, we mutated the polybasic juxtamembrane motif of syntaxin-1A and found several residues that partially or fully abrogate the electrostatic interaction with PI-4,5-P(2). We further show that even in the presence of physiological concentrations of phosphatidylserine, the PI-4,5-P(2)-syntaxin interaction is sufficiently strong to disrupt syntaxin-1A clustering. The stereochemistry of PI-4,5-P(2) is not critical for this interaction as other polyphosphoinositides have similar effects. Forming an acceptor SNARE complex between syntaxin-1A and SNAP-25 weakens but does not abrogate cholesterol/PI-4,5-P(2)-controlled cluster formation. Potential consequences of these interactions with respect to synaptic vesicle fusion are discussed.
authors.affiliation: Center for Membrane Biology and Department of Molecular Physiology and Biological Physics, University of Virginia, Virginia 22908, United States.
authors.forename: David H
authors.initials: DH
authors.lastname: Murray
authors.forename: Lukas K
authors.initials: LK
authors.lastname: Tamm
mesh_terms: D000595:Amino Acid Sequence; D002784:Cholesterol / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry*; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D018129:Phosphatidylinositol Phosphates / Q000737:chemistry* / Q000235:genetics; D011510:Proteolipids / Q000737:chemistry* / Q000235:genetics; D050765:Qa-SNARE Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D050827:Syntaxin 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D008051:Lipid Bilayers; D008081:Liposomes; D018129:Phosphatidylinositol Phosphates; D011510:Proteolipids; D050765:Qa-SNARE Proteins; C494208:SNAP25 protein, human; C494212:STX1A protein, human; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; C020485:proteoliposomes; D002784:Cholesterol