Direct interaction of SNARE complex binding protein synaphin/complexin with calcium sensor synaptotagmin 1.
Although the binding of synaphin (also called complexin) to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is critical for synaptic vesicle exocytosis, the exact role of synaphin remains unclear. Here, we show that synaphin directly binds to synaptotagmin 1, a major Ca(2+) sensor for fast neurotransmitter release, in a 1:1 stoichiometry. Mapping of the synaphin site involved in synaptotagmin 1 binding revealed that the C-terminal region is essential for the interaction between these two proteins. Binding was sensitive to ionic strength, suggesting the involvement of charged residues in the C-terminus region. Mutation of the seven consecutive glutamic acid residues (residues 108-114) at the C-terminal region of synaphin to alanines or glutamines resulted in a dramatic reduction in synaptotagmin 1 binding activity. Furthermore, a peptide from the C-terminus of synaphin (residues 91-124) blocked the binding of synaptotagmin 1 to synaphin, an effect that was abolished by mutating the consecutive glutamic acid residues to alanine. Immunoprecipitation experiments with brain membrane extracts showed the presence of a complex consisting of synaphin, synaptotagmin 1, and SNAREs. We propose that synaphin recruits synaptotagmin 1 to the SNARE-based fusion complex and synergistically functions with synaptotagmin 1 in mediating fast synaptic vesicle exocytosis.
Tokumaru
Hiroshi
H
Faculty of Pharmaceutical Sciences at Kagawa, Tokushima Bunri University, Kagawa 769-2193, Japan. tokumaruh@kph.bunri-u.ac.jp
Shimizu-Okabe
Chigusa
C
Abe
Teruo
T
D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D018076:DNA, Complementary; D005089:Exocytosis / Q000235:genetics / Q000502:physiology*; D047468:Immunoprecipitation; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D051381:Rats; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D013572:Synaptic Vesicles / Q000235:genetics / Q000378:metabolism*; D050863:Synaptotagmin I / Q000235:genetics / Q000378:metabolism*
D033942:Adaptor Proteins, Vesicular Transport; D018076:DNA, Complementary; D009419:Nerve Tissue Proteins; D050600:SNARE Proteins; D050863:Synaptotagmin I; C095993:complexin II