RECONSTITUTION OF SNARE-MEDIATED FUSION WITH NATIVE AND ARTIFICIAL MEMBRANES

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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.