Center for Membrane Biology and Department of Molecular Physiology and Biological Physics, University of Virginia, Virginia 22908, United States.
Biochemistry. 2011 Oct 25;50(42):9014-22. doi: 10.1021/bi201307u. Epub 2011 Sep 27.
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.
作为突触小泡停泊和融合的受体，在突触前膜起作用的神经元接受体 SNARE 复合物由单跨膜蛋白 syntaxin-1A 和棕榈酰化可溶性蛋白 SNAP-25 组成。先前，我们探索了促进 syntaxin-1A 簇在膜中形成的相互作用。胆固醇激活天然和模型膜中的聚类，而神经内分泌细胞中胆固醇的耗竭导致蛋白质的均匀分布。然而，仅发现 1 mol%的磷脂酰肌醇 4,5-二磷酸（PI-4,5-P(2)）或 20 mol%的磷脂酰丝氨酸可以分散 syntaxin-1A 簇[Murray, D. H., and Tamm, L. K. (2009) Biochemistry 48, 4617-4625]。有强有力的证据表明，syntaxin-1A 及其突触小泡同源 synaptobrevin 都直接与 PI-4,5-P(2)相互作用，并且这种相互作用激活融合。然而，这种相互作用的分子细节及其与 syntaxin-1A 簇部分分散的关系在很大程度上仍未得到探索。因此，我们突变了 syntaxin-1A 的多碱性近膜基序，并发现了几个残基，这些残基部分或完全消除了与 PI-4,5-P(2)的静电相互作用。我们进一步表明，即使存在生理浓度的磷脂酰丝氨酸，PI-4,5-P(2)-syntaxin 相互作用也足够强，可以破坏 syntaxin-1A 聚类。PI-4,5-P(2)的立体化学对于这种相互作用并不关键，因为其他多磷酸肌醇具有类似的作用。在 syntaxin-1A 和 SNAP-25 之间形成接受体 SNARE 复合物会削弱但不会消除胆固醇/PI-4,5-P(2)控制的聚类形成。讨论了这些相互作用对突触小泡融合的潜在影响。
