RECONSTITUTION OF SNARE-MEDIATED FUSION WITH NATIVE AND ARTIFICIAL MEMBRANES

SNARE蛋白在膜融合中起关键作用。介导神经元胞吐的SNAREs是最具代表性的，它们包括囊泡蛋白synaptobrevin/VAMP和质膜驻留蛋白syntaxin 1和SNAP-25。SNAREs经历了一个与主要构象变化相关的组装-拆卸循环。在膜之间组装snare被认为是催化双分子层的融合。然而，用SNAREs重组的脂质体的融合过程动力学缓慢，到目前为止，还没有一个已知的控制胞外分泌或其他细胞内融合反应的调节步骤从纯化的成分中重组出来。在这里，我们建议详细描述蛋白质脂质体与神经元SNARE蛋白的融合，无论是与含有相应SNAREs的脂质体还是与含有相应SNAREs的生物膜作为内源性蛋白。已经开发出敏感的测定方法，允许测量囊泡对接和融合。初步结果表明，含有SNAREs的脂质体的融合速率在很大程度上取决于膜中SNAREs的构象状态。首先，我们将定义决定含有synaptobrevin的脂质体与含有syntaxin 1和SNAP-25的脂质体融合动力学的条件，包括构象和寡聚状态以及调节蛋白（如synaptotagmin 1， complexins和Munc-18）的影响。其次，我们将深入研究syntaxin 1 /SNAP-25脂质体与突触囊泡的融合，重点是确定在初步实验中观察到的增强融合动力学的因素。第三，我们将研究含synaptobrevin脂质体与血浆倒草坪的结合和融合

SNARE proteins play a key role in membrane fusion. The SNAREs mediating neuronal exocytosis are among the best characterized, they include the vesicle protein synaptobrevin/VAMP and the plasma membrane-resident proteins syntaxin 1 and SNAP-25. SNAREs undergo an assembly-disassembly cycle that is associated with major conformational changes. Assembly of SNAREs between membranes is thought to catalyze the fusion of bilayers. However, fusion of liposomes reconstituted with SNAREs proceeds with slow kinetics, and so far none of the regulatory steps known to control exocytosis or other intracellular fusion reactions has been reconstituted from purified components. Here we propose to characterize in detail the fusion of proteoliposomes with neuronal SNARE proteins either with liposomes containing the corresponding SNAREs or with biological membranes containing the corresponding SNAREs as endogenous proteins. Sensitive assays have been developed allowing for measuring both vesicle docking and fusion. Preliminary results have shown that the fusion rate of SNARE-containing liposomes strongly depends on the conformational state of the SNAREs in the membrane. First, we will define the conditions that determine the fusion kinetics of liposomes containing synaptobrevin with those containing syntaxin 1 and SNAP-25, including the influence of conformational and oligomeric states and of regulatory proteins such as synaptotagmin 1, complexins, and Munc-18. Second, we will study in depth the fusion of syntaxin l/SNAP-25 liposomes with synaptic vesicles, with an emphasis on identifying the factors responsible for the enhanced fusion kinetics observed in preliminary experiments. Third, we will investigate the binding and fusion of synaptobrevin-containing liposomes with inverted lawns of plasma membranes derived from neuroendocrine cells.