Protein and lipidic intermediates of SNARE-mediated membrane fusion – IntermeSNARE

Many vital cellular processes, such as neuronal communication and insulin secretion, rely upon highly regulated fusion events between cargo containing vesicles and target membranes. The core of the fusion machinery consists of the assembly between vesicular (v-) SNARE and target (t-) SNARE proteins into a single complex, the SNAREpin, which brings the bilayers into close proximity and triggers their fusion. The assembly of SNAREs starts at their membrane-distal N-termini and proceeds toward their membrane-proximal C-termini (zipper model), a process that includes the passage through, at least, one stable intermediate binding state. How SNARE assembly is controlled spatially and temporally by the cell, how much energy is generated during SNAREpin folding, and how this energy is coupled to the fusion of two apposing bilayers are still very much open questions. Progressive assembly of cognate SNAREs may culminate in a release of energy sufficient to drive membrane merging, or alternatively, the assembling SNAREs may pass through a series of intermediates (potentially trapped and released through interactions with some regulatory proteins), each of which contributes enough energy to advance through the successive stages of membrane fusion. Characterization of these intermediates requires a capacity for (i) measuring the interactions between membrane-associated proteins at nanometer distance resolutions, and (ii) detecting local changes in the membrane structure. We propose to combine Surface Forces Apparatus (SFA) and Fluorescence Resonance Energy Transfer (FRET) technologies. The SFA directly measures the interaction energy between two facing functionalized membranes as a function of their separation distance and makes it possible to identify molecular rearrangements of interacting species during their association. FRET measurements can provide a description of protein assembly/disassembly pathways at the amino acid level, as well as a read-out of the lipid-mixing state of two apposing bilayers. These approaches will be applied to membrane-reconstituted SNAREs, in the absence or in the presence of various regulatory proteins and other identified inhibitors and activators of fusion, in an attempt to obtain a comprehensive description of the protein and lipidic intermediates of membrane fusion. We expect that the systems developed here will also be applicable to a wide variety of other integral membrane proteins which undergo significant conformational transitions during their normal cellular functions.