Abstract
Exocytosis of neurosecretory vesicles is mediated by the SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor) proteins syntaxin-1, synaptobrevin and SNAP-25, with synaptotagmin functioning as the major Ca2+ sensor for triggering membrane fusion. Here we show that bovine chromaffin granules readily fuse with large unilamellar liposomes in a SNARE-dependent manner. Fusion is enhanced by Ca2+, but only when the target liposomes contain phosphatidylinositol-4,5-bisphosphate and when polyphosphate anions, such as nucleotides or pyrophosphate, are present. Ca2+-dependent enhancement is mediated by endogenous synaptotagmin-1. Polyphosphates operate by an electrostatic mechanism that reverses an inactivating cis association of synaptotagmin-1 with its own membrane without affecting trans binding. Hence, the balancing of trans- and cis-membrane interactions of synaptotagmin-1 could be a crucial element in the pathway of Ca2+-dependent exocytosis.
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Acknowledgements
We are indebted to G. Mieskes (Department of Neurobiology, Max Planck Institute for Biophysical Chemistry) for the arrangement of adrenal glands and logistical assistance. This work was supported by grants from the Alexander von Humboldt Foundation (to Y.P.) and the US National Institutes of Health (2 P01 GM072694-06A1 to R.J.).
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J.M.H. assisted in the generation of SNARE-containing large unilamellar liposomes and performed the light-scattering experiments. G.v.d.B. provided labeled proteins and assisted in the fluorescence anisotropy experiments. S.A. and M.H. provided purified synaptic vesicles. D.R. performed EM. Y.P. and R.J. designed the study and wrote the paper. Experiments were conducted mainly by Y.P. All authors discussed the results and commented on the manuscript.
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Park, Y., Hernandez, J., van den Bogaart, G. et al. Controlling synaptotagmin activity by electrostatic screening. Nat Struct Mol Biol 19, 991–997 (2012). https://doi.org/10.1038/nsmb.2375
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DOI: https://doi.org/10.1038/nsmb.2375
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