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Synaptotagmin activates membrane fusion through a Ca2+-dependent trans interaction with phospholipids

Nature Structural & Molecular Biology volume 14, pages 904–911 (2007)Cite this article

Abstract

Synaptotagmin-1 is the calcium sensor for neuronal exocytosis, but the mechanism by which it triggers membrane fusion is not fully understood. Here we show that synaptotagmin accelerates SNARE-dependent fusion of liposomes by interacting with neuronal Q-SNARES in a Ca2+-independent manner. Ca2+-dependent binding of synaptotagmin to its own membrane impedes the activation. Preventing this cis interaction allows Ca2+ to trigger synaptotagmin binding in trans, accelerating fusion. However, when an activated SNARE acceptor complex is used, synaptotagmin has no effect on fusion kinetics, suggesting that synaptotagmin operates upstream of SNARE assembly in this system. Our results resolve major discrepancies concerning the effects of full-length synaptotagmin and its C2AB fragment on liposome fusion and shed new light on the interactions of synaptotagmin with SNAREs and membranes. However, our findings also show that the action of synaptotagmin on the fusion-arrested state of docked vesicles in vivo is not fully reproduced in vitro.

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Figure 1: The C2AB fragment of synaptotagmin accelerates liposome fusion mediated by neuronal SNAREs in the presence of Ca2+.
Figure 2: The assembly rate of SNARE complexes on liposomes reconstituted with neuronal Q-SNAREs is not influenced by the C2AB fragment of synaptotagmin.
Figure 3: Ca2+-dependent enhancement of fusion by the C2AB fragment of synaptotagmin is specific for neuronal Q-SNAREs syntaxin-1A and SNAP-25A, but not for the neuronal R-SNARE synaptobrevin-2.
Figure 4: Ca2+-dependent enhancement of fusion by the C2AB fragment of synaptotagmin depends on acidic phospholipids in the R-SNARE membrane.
Figure 5: Ca2+-dependent enhancement of fusion by the C2AB fragment of synaptotagmin requires an intact Ca2+-binding site in the C2A but not the C2B domain.
Figure 6: Effects of full-length synaptotagmin inserted in the R- or Q-SNARE membrane on the fusion rate mediated by neuronal SNAREs (standard fusion reaction).
Figure 7: Effects of Ca2+ on the fusion of synaptobrevin liposomes containing full-length synaptotagmin with neuronal Q-SNARE liposomes in the presence or absence of acidic phospholipids.
Figure 8: Fusion of synaptobrevin-containing liposomes with liposomes containing a stabilized Q-SNARE acceptor complex is not accelerated by synaptotagmin.
Figure 9: Schematic summary of the interactions between synaptotagmin, SNAREs and membranes containing acidic phospholipids in SNARE-mediated liposome fusion.

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Acknowledgements

We thank U. Ries for expert technical assistance, M. Holt for help with calcium measurements, S. Pabst for cloning, and P. Burkhardt and I. Bethani for valuable comments on the manuscript. Ca2+-binding mutants of the full-length protein were kind gifts from T. Südhof (Howard Hughes Medical Institute, University of Texas Southwestern). This work was supported by US National Institutes of Health grant P01 GM072694 (to R.J.) and the Boehringer Ingelheim Fonds (to A.S.).

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  1. Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen, D-37077, Germany

    Alexander Stein, Anand Radhakrishnan, Dietmar Riedel, Dirk Fasshauer & Reinhard Jahn

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Stein, A., Radhakrishnan, A., Riedel, D. et al. Synaptotagmin activates membrane fusion through a Ca2+-dependent trans interaction with phospholipids. Nat Struct Mol Biol 14, 904–911 (2007). https://doi.org/10.1038/nsmb1305

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