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Ca 2+ -triggered SNARE-mediated membrane fusion is essential for neuronal communication. The speed of this process is of particular importance because it sets a time limit to cognitive and physical activities. In this work, we expand the proteoliposome-to-supported bilayer (SBL) fusion assay by successfully incorporating synaptotagmin 1 (Syt1), a major Ca 2+  sensor. We report that Syt1 and Ca 2+  together can elicit more than a 50-fold increase in the number of membrane fusion events when compared with membrane fusion mediated by SNAREs only. What is remarkable is that ~55% of all vesicle fusion events occurs within 20 ms upon vesicle docking. Furthermore, pre-binding of Syt1 to SNAREs prior to Ca 2+  inhibits spontaneous fusion, but intriguingly, this leads to a complete loss of the Ca 2+  responsiveness. Thus, our results suggest that there is a productive and a non-productive pathway for Syt1, depending on whether there is a premature interaction between Syt1 and SNAREs. Our results show that Ca 2+  binding to Syt1 prior to Syt1's binding to SNAREs may be a prerequisite for the productive pathway. The successful reconstitution of Syt1 activities in the physiological time scale provides new opportunities to test the current mechanistic models for Ca 2+ -triggered exocytosis.

Productive and Non-productive Pathways for Synaptotagmin 1 to Support Ca2+-Triggered Fast Exocytosis