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

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.

References

  1. Ungar, D. & Hughson, F.M. SNARE protein structure and function. Annu. Rev. Cell Dev. Biol. 19, 493–517 (2003).

    CAS  Article  Google Scholar 

  2. Sudhof, T.C. The synaptic vesicle cycle. Annu. Rev. Neurosci. 27, 509–547 (2004).

    Article  Google Scholar 

  3. Fasshauer, D., Otto, H., Eliason, W.K., Jahn, R. & Brunger, A.T. Structural changes are associated with soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex formation. J. Biol. Chem. 272, 28036–28041 (1997).

    CAS  Article  Google Scholar 

  4. Sutton, R.B., Fasshauer, D., Jahn, R. & Brunger, A.T. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution. Nature 395, 347–353 (1998).

    CAS  Article  Google Scholar 

  5. Fasshauer, D., Sutton, R.B., Brunger, A.T. & Jahn, R. Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs. Proc. Natl. Acad. Sci. USA 95, 15781–15786 (1998).

    CAS  Article  Google Scholar 

  6. Pelham, H.R., Banfield, D.K. & Lewis, M.J. SNAREs involved in traffic through the Golgi complex. Cold Spring Harb. Symp. Quant. Biol. 60, 105–111 (1995).

    CAS  Article  Google Scholar 

  7. Hanson, P.I., Heuser, J.E. & Jahn, R. Neurotransmitter release—four years of SNARE complexes. Curr. Opin. Neurobiol. 7, 310–315 (1997).

    CAS  Article  Google Scholar 

  8. Lin, R.C. & Scheller, R.H. Structural organization of the synaptic exocytosis core complex. Neuron 19, 1087–1094 (1997).

    CAS  Article  Google Scholar 

  9. Katz, B. The Release of Neurotransmitter Substances (Liverpool University Press, Liverpool, 1969).

  10. Sudhof, T.C. Synaptotagmins: why so many? J. Biol. Chem. 277, 7629–7632 (2002).

    CAS  Article  Google Scholar 

  11. Perin, M.S., Brose, N., Jahn, R. & Sudhof, T.C. Domain structure of synaptotagmin (p65). J. Biol. Chem. 266, 623–629 (1991).

    CAS  PubMed  Google Scholar 

  12. Tucker, W.C. & Chapman, E.R. Role of synaptotagmin in Ca2+-triggered exocytosis. Biochem. J. 366, 1–13 (2002).

    CAS  Article  Google Scholar 

  13. Ubach, J., Zhang, X., Shao, X., Sudhof, T.C. & Rizo, J. Ca2+ binding to synaptotagmin: how many Ca2+ ions bind to the tip of a C2-domain? EMBO J. 17, 3921–3930 (1998).

    CAS  Article  Google Scholar 

  14. Fernandez, I. et al. Three-dimensional structure of the synaptotagmin 1 C2B-domain: synaptotagmin 1 as a phospholipid binding machine. Neuron 32, 1057–1069 (2001).

    CAS  Article  Google Scholar 

  15. Chapman, E.R. & Davis, A.F. Direct interaction of a Ca2+-binding loop of synaptotagmin with lipid bilayers. J. Biol. Chem. 273, 13995–14001 (1998).

    CAS  Article  Google Scholar 

  16. Rufener, E., Frazier, A.A., Wieser, C.M., Hinderliter, A. & Cafiso, D.S. Membrane-bound orientation and position of the synaptotagmin C2B domain determined by site-directed spin labeling. Biochemistry 44, 18–28 (2005).

    CAS  Article  Google Scholar 

  17. Herrick, D.Z., Sterbling, S., Rasch, K.A., Hinderliter, A. & Cafiso, D.S. Position of synaptotagmin I at the membrane interface: cooperative interactions of tandem C2 domains. Biochemistry 45, 9668–9674 (2006).

    CAS  Article  Google Scholar 

  18. Li, L. et al. Phosphatidylinositol phosphates as co-activators of Ca2+ binding to C2 domains of synaptotagmin 1. J. Biol. Chem. 281, 15845–15852 (2006).

    CAS  Article  Google Scholar 

  19. Pang, Z.P., Shin, O.H., Meyer, A.C., Rosenmund, C. & Sudhof, T.C. A gain-of-function mutation in synaptotagmin-1 reveals a critical role of Ca2+-dependent soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex binding in synaptic exocytosis. J. Neurosci. 26, 12556–12565 (2006).

    CAS  Article  Google Scholar 

  20. Bai, J., Tucker, W.C. & Chapman, E.R. PIP2 increases the speed of response of synaptotagmin and steers its membrane-penetration activity toward the plasma membrane. Nat. Struct. Mol. Biol. 11, 36–44 (2004).

    CAS  Article  Google Scholar 

  21. Chapman, E.R., Hanson, P.I., An, S. & Jahn, R. Ca2+ regulates the interaction between synaptotagmin and syntaxin 1. J. Biol. Chem. 270, 23667–23671 (1995).

    CAS  Article  Google Scholar 

  22. Schiavo, G., Stenbeck, G., Rothman, J.E. & Sollner, T.H. Binding of the synaptic vesicle v-SNARE, synaptotagmin, to the plasma membrane t-SNARE, SNAP-25, can explain docked vesicles at neurotoxin-treated synapses. Proc. Natl. Acad. Sci. USA 94, 997–1001 (1997).

    CAS  Article  Google Scholar 

  23. Rickman, C. & Davletov, B. Mechanism of calcium-independent synaptotagmin binding to target SNAREs. J. Biol. Chem. 278, 5501–5504 (2003).

    CAS  Article  Google Scholar 

  24. Davis, A.F. et al. Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex onto membranes. Neuron 24, 363–376 (1999).

    CAS  Article  Google Scholar 

  25. Stevens, C.F. & Sullivan, J.M. The synaptotagmin C2A domain is part of the calcium sensor controlling fast synaptic transmission. Neuron 39, 299–308 (2003).

    CAS  Article  Google Scholar 

  26. Mackler, J.M., Drummond, J.A., Loewen, C.A., Robinson, I.M. & Reist, N.E. The C(2)B Ca(2+)-binding motif of synaptotagmin is required for synaptic transmission in vivo. Nature 418, 340–344 (2002).

    CAS  Article  Google Scholar 

  27. Fernandez-Chacon, R. et al. Synaptotagmin I functions as a calcium regulator of release probability. Nature 410, 41–49 (2001).

    CAS  Article  Google Scholar 

  28. Rhee, J.S. et al. Augmenting neurotransmitter release by enhancing the apparent Ca2+ affinity of synaptotagmin 1. Proc. Natl. Acad. Sci. USA 102, 18664–18669 (2005).

    CAS  Article  Google Scholar 

  29. Weber, T. et al. SNAREpins: minimal machinery for membrane fusion. Cell 92, 759–772 (1998).

    CAS  Article  Google Scholar 

  30. Schuette, C.G. et al. Determinants of liposome fusion mediated by synaptic SNARE proteins. Proc. Natl. Acad. Sci. USA 101, 2858–2863 (2004).

    CAS  Article  Google Scholar 

  31. Jahn, R. & Scheller, R.H. SNAREs–engines for membrane fusion. Nat. Rev. Mol. Cell Biol. 7, 631–643 (2006).

    CAS  Article  Google Scholar 

  32. Tucker, W.C., Weber, T. & Chapman, E.R. Reconstitution of Ca2+-regulated membrane fusion by synaptotagmin and SNAREs. Science 304, 435–438 (2004).

    CAS  Article  Google Scholar 

  33. Bhalla, A., Tucker, W.C. & Chapman, E.R. Synaptotagmin isoforms couple distinct ranges of Ca2+, Ba2+, and Sr2+ concentration to SNARE-mediated membrane fusion. Mol. Biol. Cell 16, 4755–4764 (2005).

    CAS  Article  Google Scholar 

  34. Bhalla, A., Chicka, M.C., Tucker, W.C. & Chapman, E.R. Ca(2+)-synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion. Nat. Struct. Mol. Biol. 13, 323–330 (2006).

    CAS  Article  Google Scholar 

  35. Mahal, L.K., Sequeira, S.M., Gureasko, J.M. & Sollner, T.H. Calcium-independent stimulation of membrane fusion and SNAREpin formation by synaptotagmin I. J. Cell Biol. 158, 273–282 (2002).

    CAS  Article  Google Scholar 

  36. Struck, D.K., Hoekstra, D. & Pagano, R.E. Use of resonance energy transfer to monitor membrane fusion. Biochemistry 20, 4093–4099 (1981).

    CAS  Article  Google Scholar 

  37. Hayashi, T. et al. Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J. 13, 5051–5061 (1994).

    CAS  Article  Google Scholar 

  38. Antonin, W., Holroyd, C., Tikkanen, R., Honing, S. & Jahn, R. The R-SNARE endobrevin/VAMP-8 mediates homotypic fusion of early endosomes and late endosomes. Mol. Biol. Cell 11, 3289–3298 (2000).

    CAS  Article  Google Scholar 

  39. Pobbati, A.V., Stein, A. & Fasshauer, D. N- to C-terminal SNARE complex assembly promotes rapid membrane fusion. Science 313, 673–676 (2006).

    CAS  Article  Google Scholar 

  40. Rickman, C. et al. Synaptotagmin interaction with the syntaxin/SNAP-25 dimer is mediated by an evolutionarily conserved motif and is sensitive to inositol hexakisphosphate. J. Biol. Chem. 279, 12574–12579 (2004).

    CAS  Article  Google Scholar 

  41. Loewen, C.A., Lee, S.M., Shin, Y.K. & Reist, N.E. C2B polylysine motif of synaptotagmin facilitates a Ca2+-independent stage of synaptic vesicle priming in vivo. Mol. Biol. Cell 17, 5211–5226 (2006).

    CAS  Article  Google Scholar 

  42. Yoon, T.Y., Okumus, B., Zhang, F., Shin, Y.K. & Ha, T. Multiple intermediates in SNARE-induced membrane fusion. Proc. Natl. Acad. Sci. USA 103, 19731–19736 (2006).

    CAS  Article  Google Scholar 

  43. Arac, D. et al. Close membrane-membrane proximity induced by Ca(2+)-dependent multivalent binding of synaptotagmin-1 to phospholipids. Nat. Struct. Mol. Biol. 13, 209–217 (2006).

    CAS  Article  Google Scholar 

  44. Martens, S., Kozlov, M.M. & McMahon, H.T. How synaptotagmin promotes membrane fusion. Science 316, 1205–1208 (2007).

    CAS  Article  Google Scholar 

  45. Margittai, M., Otto, H. & Jahn, R. A stable interaction between syntaxin 1a and synaptobrevin 2 mediated by their transmembrane domains. FEBS Lett. 446, 40–44 (1999).

    CAS  Article  Google Scholar 

  46. Fasshauer, D., Antonin, W., Margittai, M., Pabst, S. & Jahn, R. Mixed and non-cognate SNARE complexes. Characterization of assembly and biophysical properties. J. Biol. Chem. 274, 15440–15446 (1999).

    CAS  Article  Google Scholar 

  47. Brandhorst, D. et al. Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity. Proc. Natl. Acad. Sci. USA 103, 2701–2706 (2006).

    CAS  Article  Google Scholar 

  48. Sieber, J.J., Willig, K.I., Heintzmann, R., Hell, S.W. & Lang, T. The SNARE motif is essential for the formation of syntaxin clusters in the plasma membrane. Biophys. J. 90, 2843–2851 (2006).

    CAS  Article  Google Scholar 

  49. Takamori, S. et al. Molecular anatomy of a trafficking organelle. Cell 127, 831–846 (2006).

    CAS  Article  Google Scholar 

  50. Schagger, H. & von Jagow, G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368–379 (1987).

    CAS  Article  Google Scholar 

  51. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).

    CAS  Article  Google Scholar 

  52. Avery, J. et al. A cell-free system for regulated exocytosis in PC12 cells. J. Cell Biol. 148, 317–324 (2000).

    CAS  Article  Google Scholar 

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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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Correspondence to 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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