Abstract
Synaptic-vesicle exocytosis is mediated by the vesicular Ca2+ sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using PC12 cells from Rattus norvegicus and artificial supported bilayers, we show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca2+ through PIP2. This interaction allows both Ca2+-binding sites of synaptotagmin-1 to bind to phosphatidylserine in the vesicle membrane upon Ca2+ triggering. We determined the crystal structure of the C2B domain of synaptotagmin-1 bound to phosphoserine, allowing development of a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca2+ influx bringing the vesicle membrane close enough for membrane fusion.
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Acknowledgements
We thank V. Belov (Max Planck Institute for Biophysical Chemistry, Göttingen, Germany) for the KK114-maleimide dye and D. Cafiso (University of Virginia, Charlottesville, West Virginia, USA) for 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 (SFB803). We thank A. Schönle (Max Planck Institute for Biophysical Chemistry, Göttingen, Germany) for the software ImSpector. X-ray diffraction data were collected at beamline X10SA at the Swiss Light Source, and we thank the beamline staff for their help during data collection.
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G.v.d.B., A.H., D.M. and R.J. designed and performed the experiments and wrote the paper. E.I., D.F. and K.K. performed the crystallographic structure determination. H.J.R. and H.G. performed the molecular dynamics simulations. S.M. and U.D. synthesized the peptide. S.W.H., V.M. and C.E. contributed to the microscopy and discussed data. All authors contributed to the manuscript.
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Honigmann, A., van den Bogaart, G., Iraheta, E. et al. Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment. Nat Struct Mol Biol 20, 679–686 (2013). https://doi.org/10.1038/nsmb.2570
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DOI: https://doi.org/10.1038/nsmb.2570
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