RECONSTITUTION OF SNARE-MEDIATED FUSION WITH NATIVE AND ARTIFICIAL MEMBRANES

SNARE蛋白在膜融合中起着关键作用。介导神经元胞吐的圈套蛋白包括囊泡蛋白Synaptobrevin/VAMP和质膜驻留蛋白Synaxin 1和SNAP-25。圈套经历了一个与主要构象变化相关的组装-拆解周期。膜之间陷阱的组装被认为催化了双层的融合。然而，用SNARS重组的脂质体融合动力学缓慢，到目前为止，还没有任何已知的控制胞吐或其他细胞内融合反应的调控步骤是由纯化的成分重组的。在这里，我们打算详细地研究蛋白质脂质体与神经元SNARE蛋白的融合，或者与含有相应SNARs的脂质体融合，或者与含有相应SNARs的生物膜融合为内源蛋白。已经开发了灵敏的分析方法，可以同时测量囊泡对接和融合。初步结果表明，含有SNARE的脂质体的融合率强烈地依赖于膜中SNARs的构象状态。首先，我们将确定含有Synaptobrevin的脂质体与含有Synaxin 1和SNAP-25的脂质体融合动力学的条件，包括构象和低聚状态以及调节蛋白如Synaptopagmin 1、Complins和MUNC-18的影响。其次，我们将深入研究Synaxin L/SNAP-25脂质体与突触小泡的融合，重点确定在初步实验中观察到的增强融合动力学的因素。第三，我们将研究含有突触素的脂质体与血浆倒置草坪的结合和融合。 来源于神经内分泌细胞的膜。

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.