Ca(2+)-synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion.
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.
Howard Hughes Medical Institute, University of Wisconsin, 1300 University Avenue, SMI 129, Madison, Wisconsin, USA.
Akhil
A
Bhalla
Michael C
MC
Chicka
Ward C
WC
Tucker
Edwin R
ER
Chapman
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005089:Exocytosis; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D008081:Liposomes; D008561:Membrane Fusion / Q000502:physiology*; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009411:Nerve Endings / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000235:genetics / Q000378:metabolism; D050863:Synaptotagmin I / Q000235:genetics / Q000378:metabolism*
D005656:Fungal Proteins; D008081:Liposomes; D011994:Recombinant Proteins; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; C494312:Syt1 protein, rat; D002118:Calcium