Reconstitution of Ca2+-regulated membrane fusion by synaptotagmin and SNAREs.
We investigated the effect of synaptotagmin I on membrane fusion mediated by neuronal SNARE proteins, SNAP-25, syntaxin, and synaptobrevin, which were reconstituted into vesicles. In the presence of Ca2+, the cytoplasmic domain of synaptotagmin I (syt) strongly stimulated membrane fusion when synaptobrevin densities were similar to those found in native synaptic vesicles. The Ca2+ dependence of syt-stimulated fusion was modulated by changes in lipid composition of the vesicles and by a truncation that mimics cleavage of SNAP-25 by botulinum neurotoxin A. Stimulation of fusion was abolished by disrupting the Ca2+-binding activity, or by severing the tandem C2 domains, of syt. Thus, syt and SNAREs are likely to represent the minimal protein complement for Ca2+-triggered exocytosis.
Tucker
Ward C
WC
Department of Physiology, University of Wisconsin, Madison, WI 53706, USA.
Weber
Thomas
T
Chapman
Edwin R
ER
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D005089:Exocytosis; D031541:Fluorescence Resonance Energy Transfer; D008051:Lipid Bilayers; D008055:Lipids / Q000032:analysis; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008561:Membrane Fusion; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D051381:Rats; D013572:Synaptic Vesicles / Q000737:chemistry / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D008051:Lipid Bilayers; D008055:Lipids; D008081:Liposomes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; C494209:Snap25 protein, mouse; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; C494311:Syt1 protein, mouse; C494312:Syt1 protein, rat; D050857:Synaptotagmins; D002118:Calcium