Abstract
Synaptotagmin-1 triggers Ca2+-sensitive, rapid neurotransmitter release by promoting interactions between SNARE proteins on synaptic vesicles and the plasma membrane. How synaptotagmin-1 promotes this interaction is unclear, and the massive increase in membrane fusion efficiency of Ca2+-bound synaptotagmin-1 has not been reproduced in vitro. However, previous experiments have been performed at relatively high salt concentrations, screening potentially important electrostatic interactions. Using functional reconstitution in liposomes, we show here that at low ionic strength SNARE-mediated membrane fusion becomes strictly dependent on both Ca2+ and synaptotagmin-1. Under these conditions, synaptotagmin-1 functions as a distance regulator that tethers the liposomes too far from the plasma membrane for SNARE nucleation in the absence of Ca2+, but while bringing the liposomes close enough for membrane fusion in the presence of Ca2+. These results may explain how the relatively weak electrostatic interactions between synaptotagmin-1 and membranes substantially accelerate fusion.
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Acknowledgements
We thank A. Stein and U. Ries for protein purification and comments. G.v.d.B. is financed by the Human Frontier Science Program. This work was supported by the US National Institutes of Health (P01 GM072694 to R.J.) and the Deutsche Forschungsgemeinschaft (SFB755 to S.T. and S.H.; SFB803 to K.M., J.H.R., M.H., U.D., H.G. and R.J.).
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S.T. and S.H. performed the flow cytometry experiments. J.H.R. and H.G. performed the MD simulations. M.H. purified the synaptic vesicles. Thermophoresis data were from K.M. and U.D. D.R. performed the EM. G.v.d.B. performed all other experiments. G.v.d.B. and R.J. designed the study and wrote the paper. All authors discussed the results and commented on the manuscript.
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van den Bogaart, G., Thutupalli, S., Risselada, J. et al. Synaptotagmin-1 may be a distance regulator acting upstream of SNARE nucleation. Nat Struct Mol Biol 18, 805–812 (2011). https://doi.org/10.1038/nsmb.2061
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DOI: https://doi.org/10.1038/nsmb.2061
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