Ca(2+)-independent syntaxin binding to the C(2)B effector region of synaptotagmin.
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(2)A, C(2)B). Mutating the positively charged lysine residues in the putative effector-binding region of the C(2)B 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 C(2)B 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.
Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama-shi, Okayama 700-8558, Japan.
Toshio
T
Masumoto
Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama-shi, Okayama 700-8558, Japan.
Koichiro
K
Suzuki
Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama-shi, Okayama 700-8558, Japan.
Iori
I
Ohmori
Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama-shi, Okayama 700-8558, Japan.
Hiroyuki
H
Michiue
Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama-shi, Okayama 700-8558, Japan.
Kazuhito
K
Tomizawa
Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama-shi, Okayama 700-8558, Japan.
Atsushi
A
Fujimura
Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama-shi, Okayama 700-8558, Japan. Electronic address: nishiki@md.okayama-u.ac.jp.
Tei-Ichi
TI
Nishiki
Department of Physiology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama-shi, Okayama 700-8558, Japan.
Hideki
H
Matsui
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D051381:Rats; D050600:SNARE Proteins / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism; D050863:Synaptotagmin I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D050827:Syntaxin 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D050600:SNARE Proteins; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; D050827:Syntaxin 1; D002118:Calcium