Ca(2+)-synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion |
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Authors: | Bhalla Akhil Chicka Michael C Tucker Ward C Chapman Edwin R |
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Institution: | Howard Hughes Medical Institute, University of Wisconsin, 1300 University Avenue, SMI 129, Madison, Wisconsin, USA. |
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Abstract: | In nerve terminals, exocytosis is mediated by SNARE proteins and regulated by Ca(2+) and synaptotagmin-1 (syt). Ca(2+) 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 Ca(2+) to membrane fusion by comparing the effects of Ca(2+)-syt on neuronal (SNAP-25, syntaxin and synaptobrevin) and yeast (Sso1p, Sec9c and Snc2p) SNAREs. Ca(2+)-syt aggregated neuronal and yeast SNARE liposomes to similar extents via interactions with anionic phospholipids. However, Ca(2+)-syt was able to bind and stimulate fusion mediated by only neuronal SNAREs and had no effect on yeast SNAREs. Thus, Ca(2+)-syt regulates fusion through direct interactions with t-SNAREs and not solely through aggregation of vesicles. Ca(2+)-syt drove assembly of SNAP-25 onto membrane-embedded syntaxin, providing direct evidence that Ca(2+)-syt alters t-SNARE structure. |
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