start-ver=1.4
cd-journal=joma
no-vol=49
cd-vols=
no-issue=1
article-no=
start-page=1
end-page=8
dt-received=
dt-revised=
dt-accepted=
dt-pub-year=2012
dt-pub=201201
dt-online=
en-article=
kn-article=
en-subject=
kn-subject=
en-title=
kn-title=Ca2+-independent syntaxin binding to the C2B effector region of synaptotagmin
en-subtitle=
kn-subtitle=
en-abstract=
kn-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.
en-copyright=
kn-copyright=

en-aut-name=MasumotoToshio
en-aut-sei=Masumoto
en-aut-mei=Toshio
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=1
ORCID=

en-aut-name=SuzukiKoichiro
en-aut-sei=Suzuki
en-aut-mei=Koichiro
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=2
ORCID=

en-aut-name=OhmoriIori
en-aut-sei=Ohmori
en-aut-mei=Iori
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=3
ORCID=

en-aut-name=MichiueHiroyuki
en-aut-sei=Michiue
en-aut-mei=Hiroyuki
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=4
ORCID=

en-aut-name=TomizawaKazuhito
en-aut-sei=Tomizawa
en-aut-mei=Kazuhito
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=5
ORCID=

en-aut-name=FujimuraAtsushi
en-aut-sei=Fujimura
en-aut-mei=Atsushi
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=6
ORCID=

en-aut-name=NishikiTei-ichi
en-aut-sei=Nishiki
en-aut-mei=Tei-ichi
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=7
ORCID=

en-aut-name=MatsuiHideki
en-aut-sei=Matsui
en-aut-mei=Hideki
kn-aut-name=
kn-aut-sei=
kn-aut-mei=
aut-affil-num=8
ORCID=

affil-num=1
en-affil=
kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Physiol

affil-num=2
en-affil=
kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Physiol

affil-num=3
en-affil=
kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Physiol

affil-num=4
en-affil=
kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Physiol

affil-num=5
en-affil=
kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Physiol

affil-num=6
en-affil=
kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Physiol

affil-num=7
en-affil=
kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Physiol

affil-num=8
en-affil=
kn-affil=Okayama Univ, Grad Sch Med Dent & Pharmaceut Sci, Dept Physiol
en-keyword=Neurotransmitter release
kn-keyword=Neurotransmitter release
en-keyword=Synaptic vesicle
kn-keyword=Synaptic vesicle
en-keyword=Exocytosis
kn-keyword=Exocytosis
en-keyword=SNAP-25
kn-keyword=SNAP-25
en-keyword=Synaptobrevin
kn-keyword=Synaptobrevin
END