title: Two synaptobrevin molecules are sufficient for vesicle fusion in central nervous system synapses.
abstract: Exocytosis of synaptic vesicles (SVs) during fast synaptic transmission is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly formed by the coil-coiling of three members of this protein family: vesicle SNARE protein, synaptobrevin 2 (syb2), and the presynaptic membrane SNAREs syntaxin-1A and SNAP-25. However, it is controversially debated how many SNARE complexes are minimally needed for SV priming and fusion. To quantify this effective number, we measured the fluorescence responses from single fusing vesicles expressing pHluorin (pHl), a pH-sensitive variant of GFP, fused to the luminal domain of the vesicular SNARE syb2 (spH) in cultured hippocampal neurons lacking endogenous syb2. Fluorescence responses were quantal, with the unitary signals precisely corresponding to single pHluorin molecules. Using this approach we found that two copies of spH per SV fully rescued evoked fusion whereas SVs expressing only one spH were unable to rapidly fuse upon stimulation. Thus, two syb2 molecules and likely two SNARE complexes are necessary and sufficient for SV fusion during fast synaptic transmission.
authors.affiliation: Department of Membrane Biophysics, Max-Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
authors.forename: Raunak
authors.initials: R
authors.lastname: Sinha
authors.forename: Saheeb
authors.initials: S
authors.lastname: Ahmed
authors.forename: Reinhard
authors.initials: R
authors.lastname: Jahn
authors.forename: Jurgen
authors.initials: J
authors.lastname: Klingauf
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002490:Central Nervous System / Q000187:drug effects / Q000378:metabolism*; D005089:Exocytosis / Q000187:drug effects; D005453:Fluorescence; D018628:Gene Dosage / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology; D008561:Membrane Fusion / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D009435:Synaptic Transmission / Q000187:drug effects; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism*; D050988:Vesicle-Associated Membrane Protein 2 / Q000172:deficiency / Q000378:metabolism*; D051040:Vesicle-Associated Membrane Protein 3 / Q000172:deficiency / Q000378:metabolism
chemical_list: C485187:PHluorin; D011993:Recombinant Fusion Proteins; D050988:Vesicle-Associated Membrane Protein 2; D051040:Vesicle-Associated Membrane Protein 3; C494808:vesicle-associated membrane protein 2, mouse; C494953:vesicle-associated membrane protein 3, mouse; D049452:Green Fluorescent Proteins; D002118:Calcium