Ca2+–synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion

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Abstract
In nerve terminals, exocytosis is mediated by SNARE proteins and regulated by Ca2+ and synaptotagmin-1 (syt). Ca2+ promotes the interaction of syt with anionic phospholipids and the target membrane SNAREs (t-SNAREs) SNAP-25 and syntaxin. Here, we have used a defined reconstituted fusion assay to determine directly whether syt–t-SNARE interactions couple Ca2+ to membrane fusion by comparing the effects of Ca2+–syt on neuronal (SNAP-25, syntaxin and synaptobrevin) and yeast (Sso1p, Sec9c and Snc2p) SNAREs. Ca2+–syt aggregated neuronal and yeast SNARE liposomes to similar extents via interactions with anionic phospholipids. However, Ca2+–syt was able to bind and stimulate fusion mediated by only neuronal SNAREs and had no effect on yeast SNAREs. Thus, Ca2+–syt regulates fusion through direct interactions with t-SNAREs and not solely through aggregation of vesicles. Ca2+–syt drove assembly of SNAP-25 onto membrane-embedded syntaxin, providing direct evidence that Ca2+–syt alters t-SNARE structure.