Snare-Mediated Fusion Between Highly Curved and Un-Curved Membranes

During neurotransmitter release, curved synaptic vesicles fuse with the un-curved pre-synaptic plasma membrane, leading to the merger of two lipid bilayers and the release of neurotransmitters, a process that requires SNARE proteins in both membranes. Our previous modeling of this system employed two populations of highly curved vesicles (SUV), while others have used two populations of vesicles having ill-defined but likely not high curvature. In our studies, the v-SNARE synaptobrevin (SB), the t-SNARE syntaxin (SX), and SNAP-25 linked vesicles via a SNARE complex, but were unable to promote fusion without poly ethylene glycol (PEG) to force close membrane contact. We hypothesized that the geometry of the membranes may contribute to native synaptic vesicle fusion. Here we reconstitute SB into SUVs and SX into relatively uncurved (LUV) vesicles, whose composition, DOPC/DOPE/sphingomeylin/DOPS/cholesterol (32/25/15/8/20), models that of the native membranes. Lipid mixing (LM), contents mixing (CM) and leakage (L) time courses were fitted globally to 3- or 4-state sequential models, from which we obtained estimates of rate constants for conversion between states as well as probabilities of LM, CM and L for each state (Biophys. J., 2007, 92; 4012). In the absence of SNAREs, the mismatched curvature of the LUV-SUV system promotes more efficient and productive fusion events than fusion between SUVs (Biophys. J., 2010, 98, S1; 674a). LUVs containing SX (1:2250 P/L) and SUVs containing SB (1:950 P/L) still did not fuse in the absence of PEG. However with 6% PEG, the probability of CM was greatly shifted to the first step in the fusion process. The results suggest that with mismatched curvature, SNAREs may enhance rapid transient pore formation that precedes fully LM and final pore formation.Supported by NIGMS grants GM000678 to UNC and GM32707 to BRL.