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ID 52344
FullText URL
Author
Masumoto, Toshio
Suzuki, Koichiro
Ohmori, Iori
Michiue, Hiroyuki Kaken ID
Tomizawa, Kazuhito
Fujimura, Atsushi
Nishiki, Tei-ichi Kaken ID
Abstract
Although synaptotagmin I, which is a calcium (Ca2+)-binding synaptic vesicle protein, may trigger soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated synaptic vesicle exocytosis, the mechanisms underlying the interaction between these proteins remain controversial, especially with respect to the identity of the protein(s) in the SNARE complex that bind(s) to synaptotagmin and whether Ca2+ is required for their highly effective binding. To address these questions, native proteins were solubilized, immunoprecipitated from rat brain extracts, and analyzed by immunoblotting. SNARE complexes comprising syntaxin 1, 25-kDa synaptosomal-associated protein (SNAP-25), and synaptobrevin 2 were coprecipitzted with synaptotagmin I in the presence of ethylene glycol tetraacetic acid. The amount of cop recipitated proteins was significantly unaltered by the addition of Ca2+ to the brain extract. To identify the component of the SNARE complex that bound to synaptotagmin, SNARE was coexpressed with synaptotagmin in HEK293 cells and immunoprecipitated. Syntaxin, but not SNAP-25 and synaptobrevin, bound to synaptotagmin in a Ca2+-independent manner, and the binding was abolished in the presence of 1 M NaCl. Synaptotagmin contains 2 Ca2+-binding domains (C(2)A, C2B). Mutating the positively charged lysine residues in the putative effector-binding region of the C2B domain, which are critical for transmitter release, markedly inhibited synaptotagmin-syntaxin binding, while similar mutations in the C(2)A domain had no effect on binding. Synaptotagmin-syntaxin binding was reduced by mutating multiple negatively charged glutamate residues in the amino-terminal half of the syntaxin SNARE motif. These results indicate that synaptotagmin I binds to syntaxin 1 electrostatically through its C2B domain effector region in a Ca2+-independent fashion, providing biochemical evidence that synaptotagmin I binds SNARE complexes before Ca2+ influx into presynaptic nerve terminals.
Keywords
Neurotransmitter release
Synaptic vesicle
Exocytosis
SNAP-25
Synaptobrevin
Published Date
2012-01
Publication Title
Molecular and Cellular Neuroscience
Volume
volume49
Issue
issue1
Start Page
1
End Page
8
ISSN
1044-7431
Content Type
Journal Article
Official Url
http://www.sciencedirect.com/science/article/pii/S1044743111002181
Related Url
http://ousar.lib.okayama-u.ac.jp/metadata/52246
language
英語
Copyright Holders
(C) 2011 Elsevier Inc. All rights reserved.
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author
Refereed
True
DOI
Web of Science KeyUT