Studying the Effects of Inositol Pyrophosphates in an In Vitro Vesicle–Vesicle Fusion Assay

In a recent in vitro vesicle fusion study in which we monitored the fusion of reconstituted SNARE and synaptotagmin-containing proteoliposomes, we discovered that inositol pyrophosphate (5-IP7) is a potent inhibitor of neuronal exocytosis. We found that the inhibitory effect of 5-IP7, which is 10 times more potent than those of IP6 and 1-IP7, requires direct interaction with synaptotagmin. Here, we provide a detailed protocol for proteoliposome preparation and bulk observation of proteoliposome fusion based on FRET signals.