Nature Structural & Molecular Biology 13, 323 - 330 (2006)
Published online: 26 March 2006; | doi:10.1038/nsmb1076
Ca2+–synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusionAkhil Bhalla1, 2, 3, 4, Michael C Chicka1, 2, 4, Ward C Tucker2
& Edwin R Chapman1, 21
Howard Hughes Medical Institute, University of Wisconsin, 1300 University Avenue, SMI 129, Madison, Wisconsin, USA. 2
Department of Physiology, University of Wisconsin, 1300 University Avenue, SMI 129, Madison, Wisconsin, USA. 3
Molecular and Cellular Pharmacology Program, University of Wisconsin, 1300 University Avenue, SMI 129, Madison, Wisconsin, USA. 4
These authors contributed equally to this work.
Correspondence should be addressed to Edwin R Chapman chapman@physiology.wisc.edu 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.
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