Synaptotagmin 1 drives Trans SNARE Zippering with Ca2

In neurons, Synaptotagmin 1 (Syt1) is a major Ca2++-sensor that evokes vesicle fusion, a necessary event for the neurotransmitter release, in response to the Ca2++ signal. Although Syt1 is known to bind to the core fusion machinery SNAREpin the mechanism by which Syt1 regulates vesicle fusion is not known. Here we used site-specific fluorescence resonance energy transfer (FRET) to investigate the effects of Syt1 and Ca2+ on formation of trans SNAREpin between the complimentary SNAREs reconstituted to proteoliposomes. When the FRET pairs were attached at the N-termini of SNAREs C2AB, a soluble version of Syt1, had virtually no effect on the rate of the FRET change, indicating Syt1 does not interfere with the initial stage of SNARE complex formation that happens at the membrane-distal region. However, when the FRET pairs were placed at the membrane proximal region of the SNAREs C2AB accelerated the FRET change significantly in the presence of Ca2++, indicating C2AB/Ca2+ promotes the transition from the partially assembled SNARE complex to the fusion-competent SNAREpin. Thus, the results suggest that Syt1 and Ca2++ may drive SNARE zippering to facilitate membrane fusion.