A Fast, Single-Vesicle Fusion Assay Mimics Physiological SNARE Requirements

SNARE proteins play a central role in nearly all intracellular fusion reactions; fusion is thermodynamically driven by formation of trans-SNARE complexes (SNAREpins) through pairing of vesicle-associated v-SNAREs with complementary t-SNAREs on target membranes. However, the number of SNARE complexes required for fusion is unknown and there is controversy about whether additional proteins maybe required to account for the speed with which fusion can occur in cells. Small unilamellar vesicles containing the synaptic/exocytic v-SNAREs VAMP/synaptobrevin fuse rapidly with planar, supported bilayers containing the synaptic/exocytic t-SNARE syntaxin-SNAP25, with single fusion events occurring in ∼10 ms to seconds. However, in previous reports the SNAP25 subunit of the t-SNARE was not required, or an artificial peptide was needed, raising questions about the physiological relevance of these results. We now include a lipid-linked polymer chain in both bilayers whose size and density mimic that of bulk membrane proteins that need to be cleared before fusion in physiological settings. The result is that SNAP25 dependence is restored but rapid fusion (mean delay after docking of 130 ms) remains. A dramatic drop in the overall fusion rate occurs as the number of v-SNAREs per vesicle is reduced below a threshold of 5-10 externally-oriented v-SNAREs per vesicle, directly establishing this as the minimum number required for rapid fusion. The distribution of delay times for fusion following docking by the first SNAREpin appears to be limited by the time required for additional t-SNAREs to diffuse to the docking site, and implies that 5-10 t-SNAREs must be recruited to achieve fusion, closely matching the v-SNARE requirement.