Synaptotagmin I and Ca2+ promote half fusion more than full fusion in SNARE-mediated bilayer fusion

Synaptic membrane fusion, which is necessary for neurotransmitter release, may be mediated by SNAREs and regulated by synaptotagmin (Syt) and Ca2+. Fusion of liposomes mediated by reconstituted SNAREs produces full fusion and hemifusion, a membrane structure in which outer leaflets are mixed but the inner leaflets remain intact. Here, using the liposome fusion assay, it is shown that Syt promoted both hemifusion and full fusion in a Ca2+-dependent manner. Syt · Ca2+ increased hemifusion more than full fusion, modulating the ratio of hemifusion to full fusion. Unlike the case of neuronal SNAREs, stimulation of fusion by Syt · Ca2+ was not seen for other SNAREs involved in trafficking in yeast, indicating that the Syt · Ca2+ stimulation was SNARE-specific. We constructed hybrid SNAREs in which transmembrane domains were swapped between neuronal and yeast SNAREs. With these hybrid SNAREs, we demonstrated that the interaction between the SNARE motifs of neuronal proteins and Syt · Ca2+ was required for the stimulation of fusion.