Ca 2+-independent syntaxin binding to the C 2B effector region of synaptotagmin

Toshio Masumoto, Koichiro Suzuki, Iori Ohmori, Hiroyuki Michiue, Kazuhito Tomizawa, Atsushi Fujimura, Tei-ichi Nishiki, Hideki Matsui

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1-8
Number of pages8
JournalMolecular and Cellular Neuroscience
Volume49
Issue number1
DOIs
Publication statusPublished - Jan 2012

Fingerprint

Syntaxin 1
Synaptotagmins
SNARE Proteins
Synaptotagmin I
Qa-SNARE Proteins
Synaptic Vesicles
Proteins
Synaptotagmin II
Synaptosomal-Associated Protein 25
Vesicle-Associated Membrane Protein 2
R-SNARE Proteins
HEK293 Cells
Egtazic Acid
Exocytosis
Presynaptic Terminals
Brain
Immunoblotting
Lysine
Glutamic Acid
Calcium

Keywords

  • Exocytosis
  • Neurotransmitter release
  • SNAP-25
  • Synaptic vesicle
  • Synaptobrevin

ASJC Scopus subject areas

  • Molecular Biology
  • Cellular and Molecular Neuroscience
  • Cell Biology

Cite this

Ca 2+-independent syntaxin binding to the C 2B effector region of synaptotagmin. / Masumoto, Toshio; Suzuki, Koichiro; Ohmori, Iori; Michiue, Hiroyuki; Tomizawa, Kazuhito; Fujimura, Atsushi; Nishiki, Tei-ichi; Matsui, Hideki.

In: Molecular and Cellular Neuroscience, Vol. 49, No. 1, 01.2012, p. 1-8.

Research output: Contribution to journalArticle

Masumoto, Toshio ; Suzuki, Koichiro ; Ohmori, Iori ; Michiue, Hiroyuki ; Tomizawa, Kazuhito ; Fujimura, Atsushi ; Nishiki, Tei-ichi ; Matsui, Hideki. / Ca 2+-independent syntaxin binding to the C 2B effector region of synaptotagmin. In: Molecular and Cellular Neuroscience. 2012 ; Vol. 49, No. 1. pp. 1-8.
@article{fb7be59190e64bc8a5e65e60c0aab43a,
title = "Ca 2+-independent syntaxin binding to the C 2B effector region of synaptotagmin",
abstract = "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.",
keywords = "Exocytosis, Neurotransmitter release, SNAP-25, Synaptic vesicle, Synaptobrevin",
author = "Toshio Masumoto and Koichiro Suzuki and Iori Ohmori and Hiroyuki Michiue and Kazuhito Tomizawa and Atsushi Fujimura and Tei-ichi Nishiki and Hideki Matsui",
year = "2012",
month = "1",
doi = "10.1016/j.mcn.2011.09.007",
language = "English",
volume = "49",
pages = "1--8",
journal = "Molecular and Cellular Neurosciences",
issn = "1044-7431",
publisher = "Academic Press Inc.",
number = "1",

}

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

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 -