TY - JOUR
T1 - Ca 2+-independent syntaxin binding to the C 2B effector region of synaptotagmin
AU - Masumoto, Toshio
AU - Suzuki, Koichiro
AU - Ohmori, Iori
AU - Michiue, Hiroyuki
AU - Tomizawa, Kazuhito
AU - Fujimura, Atsushi
AU - Nishiki, Tei-ichi
AU - Matsui, Hideki
N1 - Funding Information:
We thank Dr. M. Takahashi (Kitasato University School of Medicine, Sagamihara, Japan) for the antibodies against synaptotagmin I, syntaxin 1, and synaptobrevin 2 and for the cDNAs of synaptotagmin I, syntaxin 1A, SNAP-25B, and synaptobrevin 2. We also thank Dr. S Kozaki (Osaka Prefecture University, Izumisano, Japan) for the antibodies against SNAP-25 and botulinum type B neurotoxin. This work was supported by JSPS KAKENHI ( 18800027 and 20590209 ) awarded to T.N.
PY - 2012/1
Y1 - 2012/1
N2 - Although synaptotagmin I, which is a calcium (Ca 2+)-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 Ca 2+ 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 coprecipitated with synaptotagmin I in the presence of ethylene glycol tetraacetic acid. The amount of coprecipitated proteins was significantly unaltered by the addition of Ca 2+ 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 Ca 2+-independent manner, and the binding was abolished in the presence of 1M NaCl. Synaptotagmin contains 2 Ca 2+-binding domains (C 2A, C 2B). Mutating the positively charged lysine residues in the putative effector-binding region of the C 2B domain, which are critical for transmitter release, markedly inhibited synaptotagmin-syntaxin binding, while similar mutations in the C 2A 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 C 2B domain effector region in a Ca 2+-independent fashion, providing biochemical evidence that synaptotagmin I binds SNARE complexes before Ca 2+ influx into presynaptic nerve terminals.
AB - Although synaptotagmin I, which is a calcium (Ca 2+)-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 Ca 2+ 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 coprecipitated with synaptotagmin I in the presence of ethylene glycol tetraacetic acid. The amount of coprecipitated proteins was significantly unaltered by the addition of Ca 2+ 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 Ca 2+-independent manner, and the binding was abolished in the presence of 1M NaCl. Synaptotagmin contains 2 Ca 2+-binding domains (C 2A, C 2B). Mutating the positively charged lysine residues in the putative effector-binding region of the C 2B domain, which are critical for transmitter release, markedly inhibited synaptotagmin-syntaxin binding, while similar mutations in the C 2A 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 C 2B domain effector region in a Ca 2+-independent fashion, providing biochemical evidence that synaptotagmin I binds SNARE complexes before Ca 2+ influx into presynaptic nerve terminals.
KW - Exocytosis
KW - Neurotransmitter release
KW - SNAP-25
KW - Synaptic vesicle
KW - Synaptobrevin
UR - http://www.scopus.com/inward/record.url?scp=82355187664&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=82355187664&partnerID=8YFLogxK
U2 - 10.1016/j.mcn.2011.09.007
DO - 10.1016/j.mcn.2011.09.007
M3 - Article
C2 - 22008253
AN - SCOPUS:82355187664
VL - 49
SP - 1
EP - 8
JO - Molecular and Cellular Neurosciences
JF - Molecular and Cellular Neurosciences
SN - 1044-7431
IS - 1
ER -