A fast, single-vesicle fusion assay mimics physiological SNARE requirements.

Abstract

Almost all known intracellular fusion reactions are driven by formation of trans-SNARE complexes 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 are required to explain the fast fusion which can occur in cells. Here we show that single vesicles containing the synaptic/exocytic v-SNAREs VAMP/synaptobrevin fuse rapidly with planar, supported bilayers containing the synaptic/exocytic t-SNAREs syntaxin-SNAP25. Fusion rates decreased dramatically when the number of externally oriented v-SNAREs per vesicle was reduced below 5-10, directly establishing this as the minimum number required for rapid fusion. Docking-to-fusion delay time distributions were consistent with a requirement that 5-11 t-SNAREs be recruited to achieve fusion, closely matching the v-SNARE requirement.