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The Janus-faced nature of the C2B domain is fundamental for synaptotagmin-1 function

Nature Structural & Molecular Biology volume 15, pages 1160–1168 (2008)Cite this article

Abstract

Synaptotagmin-1 functions as a Ca2+ sensor in neurotransmitter release and was proposed to act on both the synaptic vesicle and plasma membranes through interactions involving the Ca2+ binding top loops of its C2 domains and the Ca2+-independent bottom face of the C2B domain. However, the functional importance of the C2B domain bottom face is unclear. We now show that mutating two conserved arginine residues at the C2B domain bottom face practically abolishes synchronous release in hippocampal neurons. Reconstitution experiments reveal that Ca2+–synaptotagmin-1 can dramatically stimulate the rate of SNARE-dependent lipid mixing, and that the two-arginine mutation strongly impairs this activity. These results demonstrate that synaptotagmin-1 function depends crucially on the bottom face of the C2B domain and strongly support the notion that synaptotagmin-1 triggers membrane fusion and neurotransmitter release by bringing the vesicle and plasma membranes together, much like the SNAREs do but in a Ca2+-dependent manner.

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Figure 1: Two highly conserved arginine residues at the bottom face of the synaptotagmin-1 C2B domain.
Figure 2: The bottom face of the synaptotagmin-1 C2B domain is crucial for fast Ca2+-triggered neurotransmitter release.
Figure 3: The bottom face of the synaptotagmin-1 C2B domain regulates probability and Ca2+ sensitivity of release.
Figure 4: The R398Q R399Q mutation impairs synaptotagmin-1–membrane interactions.
Figure 5: Ca2+ and synaptotagmin-1 induce a drastic increase in the rate of SNARE-mediated lipid mixing that is abolished by the R398Q R399Q mutation.
Figure 6: Relative fusion-stimulation activities of synaptotagmin-1 fragments.
Figure 7: Proposed mode of how the synaptotagmin-1 C2B domain and the SNARE complex cooperate in Ca2+-triggered membrane fusion.

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References

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

    Article  Google Scholar 

  2. Rizo, J., Chen, X. & Arac, D. Unraveling the mechanisms of synaptotagmin and SNARE function in neurotransmitter release. Trends Cell Biol. 16, 339–350 (2006).

    Article  CAS  Google Scholar 

  3. Chapman, E.R. How does synaptotagmin trigger neurotransmitter release? Annu. Rev. Biochem. 77, 615–41 (2008).

    Article  CAS  Google Scholar 

  4. Sutton, R.B., Davletov, B.A., Berghuis, A.M., Sudhof, T.C. & Sprang, S.R. Structure of the first C2 domain of synaptotagmin I: a novel Ca2+/phospholipid-binding fold. Cell 80, 929–938 (1995).

    Article  CAS  Google Scholar 

  5. Shao, X., Davletov, B.A., Sutton, R.B., Sudhof, T.C. & Rizo, J. Bipartite Ca2+-binding motif in C2 domains of synaptotagmin and protein kinase C. Science 273, 248–251 (1996).

    Article  CAS  Google Scholar 

  6. 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).

    Article  CAS  Google Scholar 

  7. Shao, X., Fernandez, I., Sudhof, T.C. & Rizo, J. Solution structures of the Ca2+-free and Ca2+-bound C2A domain of synaptotagmin I: does Ca2+ induce a conformational change? Biochemistry 37, 16106–16115 (1998).

    Article  CAS  Google Scholar 

  8. 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).

    Article  CAS  Google Scholar 

  9. 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).

    Article  CAS  Google Scholar 

  10. Zhang, X., Rizo, J. & Sudhof, T.C. Mechanism of phospholipid binding by the C2A-domain of synaptotagmin I. Biochemistry 37, 12395–12403 (1998).

    Article  CAS  Google Scholar 

  11. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  13. 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).

    Article  CAS  Google Scholar 

  14. Brunger, A.T. Structure and function of SNARE and SNARE-interacting proteins. Q. Rev. Biophys. 38, 1–47 (2005).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. Bhalla, A., Chicka, M.C., Tucker, W.C. & Chapman, E.R. Ca2+–synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion. Nat. Struct. Mol. Biol. 13, 323–330 (2006).

    Article  CAS  Google Scholar 

  17. Dai, H., Shen, N., Arac, D. & Rizo, J.A. Quaternary SNARE–synaptotagmin–Ca2+–phospholipid complex in neurotransmitter release. J. Mol. Biol. 367, 848–863 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. Mackler, J.M., Drummond, J.A., Loewen, C.A., Robinson, I.M. & Reist, N.E. The C2B Ca2+-binding motif of synaptotagmin is required for synaptic transmission in vivo. Nature 418, 340–344 (2002).

    Article  CAS  Google Scholar 

  20. Nishiki, T. & Augustine, G.J. Dual roles of the C2B domain of synaptotagmin I in synchronizing Ca2+-dependent neurotransmitter release. J. Neurosci. 24, 8542–8550 (2004).

    Article  CAS  Google Scholar 

  21. Robinson, I.M., Ranjan, R. & Schwarz, T.L. Synaptotagmins I and IV promote transmitter release independently of Ca2+ binding in the C2A domain. Nature 418, 336–340 (2002).

    Article  CAS  Google Scholar 

  22. Fernandez-Chacon, R. et al. Structure/function analysis of Ca2+ binding to the C2A domain of synaptotagmin 1. J. Neurosci. 22, 8438–8446 (2002).

    Article  CAS  Google Scholar 

  23. 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).

    Article  CAS  Google Scholar 

  24. Rickman, C. et al. Conserved prefusion protein assembly in regulated exocytosis. Mol. Biol. Cell 17, 283–294 (2006).

    Article  CAS  Google Scholar 

  25. 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).

    Article  CAS  Google Scholar 

  26. Mackler, J.M. & Reist, N.E. Mutations in the second C2 domain of synaptotagmin disrupt synaptic transmission at Drosophila neuromuscular junctions. J. Comp. Neurol. 436, 4–16 (2001).

    Article  CAS  Google Scholar 

  27. Arac, D. et al. Close membrane-membrane proximity induced by Ca2+-dependent multivalent binding of synaptotagmin-1 to phospholipids. Nat. Struct. Mol. Biol. 13, 209–217 (2006).

    Article  CAS  Google Scholar 

  28. Geppert, M. et al. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79, 717–727 (1994).

    Article  CAS  Google Scholar 

  29. Rosenmund, C. & Stevens, C.F. Definition of the readily releasable pool of vesicles at hippocampal synapses. Neuron 16, 1197–1207 (1996).

    Article  CAS  Google Scholar 

  30. Tang, J. et al. A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis. Cell 126, 1175–1187 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  34. Parlati, F. et al. Rapid and efficient fusion of phospholipid vesicles by the α-helical core of a SNARE complex in the absence of an N-terminal regulatory domain. Proc. Natl. Acad. Sci. USA 96, 12565–12570 (1999).

    Article  CAS  Google Scholar 

  35. Stein, A., Radhakrishnan, A., Riedel, D., Fasshauer, D. & Jahn, R. Synaptotagmin activates membrane fusion through a Ca2+-dependent trans interaction with phospholipids. Nat. Struct. Mol. Biol. 14, 904–911 (2007).

    Article  CAS  Google Scholar 

  36. Ubach, J. et al. The C2B domain of synaptotagmin I is a Ca2+-binding module. Biochemistry 40, 5854–5860 (2001).

    Article  CAS  Google Scholar 

  37. 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).

    Article  CAS  Google Scholar 

  38. Chicka, M.C., Hui, E., Liu, H. & Chapman, E.R. Synaptotagmin arrests the SNARE complex before triggering fast, efficient membrane fusion in response to Ca2+. Nat. Struct. Mol. Biol. 15, 827–835 (2008).

    Article  CAS  Google Scholar 

  39. Bowen, M.E., Weninger, K., Ernst, J., Chu, S. & Brunger, A.T. Single-molecule studies of synaptotagmin and complexin binding to the SNARE complex. Biophys. J. 89, 690–702 (2005).

    Article  CAS  Google Scholar 

  40. Rizo, J. & Rosenmund, C. Synaptic vesicle fusion. Nat. Struct. Mol. Biol. 15, 665–674 (2008).

    Article  CAS  Google Scholar 

  41. Shao, X. et al. Synaptotagmin-syntaxin interaction: the C2 domain as a Ca2+-dependent electrostatic switch. Neuron 18, 133–142 (1997).

    Article  CAS  Google Scholar 

  42. Giraudo, C.G., Eng, W.S., Melia, T.J. & Rothman, J.E. A clamping mechanism involved in SNARE-dependent exocytosis. Science 313, 676–680 (2006).

    Article  CAS  Google Scholar 

  43. Schaub, J.R., Lu, X., Doneske, B., Shin, Y.K. & McNew, J.A. Hemifusion arrest by complexin is relieved by Ca2+–synaptotagmin I. Nat. Struct. Mol. Biol. 13, 748–750 (2006).

    Article  CAS  Google Scholar 

  44. Huntwork, S. & Littleton, J.T. A complexin fusion clamp regulates spontaneous neurotransmitter release and synaptic growth. Nat. Neurosci. 10, 1235–1237 (2007).

    Article  CAS  Google Scholar 

  45. Zimmerberg, J., Akimov, S.A. & Frolov, V. Synaptotagmin: fusogenic role for calcium sensor? Nat. Struct. Mol. Biol. 13, 301–303 (2006).

    Article  CAS  Google Scholar 

  46. Parsegian, V.A., Rand, R.P. & Stamatoff, J. Perturbation of membrane structure by uranyl acetate labeling. Biophys. J. 33, 475–477 (1981).

    Article  CAS  Google Scholar 

  47. Winiski, A.P., McLaughlin, A.C., McDaniel, R.V., Eisenberg, M. & McLaughlin, S. An experimental test of the discreteness-of-charge effect in positive and negative lipid bilayers. Biochemistry 25, 8206–8214 (1986).

    Article  CAS  Google Scholar 

  48. Ubach, J., Garcia, J., Nittler, M.P., Sudhof, T.C. & Rizo, J. Structure of the Janus-faced C2B domain of rabphilin. Nat. Cell Biol. 1, 106–112 (1999).

    Article  CAS  Google Scholar 

  49. Deak, F. et al. Rabphilin regulates SNARE-dependent re-priming of synaptic vesicles for fusion. EMBO J. 25, 2856–2866 (2006).

    Article  CAS  Google Scholar 

  50. Xue, M. et al. Distinct domains of complexin I differentially regulate neurotransmitter release. Nat. Struct. Mol. Biol. 14, 949–958 (2007).

    Article  CAS  Google Scholar 

  51. Lois, C., Hong, E.J., Pease, S., Brown, E.J. & Baltimore, D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295, 868–872 (2002).

    Article  CAS  Google Scholar 

  52. Chen, X. et al. SNARE-mediated lipid mixing depends on the physical state of the vesicles. Biophys. J. 90, 2062–2074 (2006).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank H. Deng and H. Chen for technical assistance, R. Atkinson for assistance in confocal microscopy, C. Lois (Massachusetts Institute of Technology) and R. Nehring (Baylor College of Medicine) for providing the lentiviral vectors, T. Südhof (Stanford University) for providing synaptotagmin-1 heterozygous mice and R. Castillejos (Harvard University) for providing silicon masters for molding microchannels. This work was supported by the Baylor College of Medicine Mental Retardation and Developmental Disabilities Research Center, and by National Institutes of Health grants NS50655 (C.R.), NS40944 (J.R.) and GM65364 (G. M. Whitesides, Harvard University).

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  1. Mingshan Xue and Cong Ma: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Neuroscience, Baylor College of Medicine, One Baylor Plaza, Houston, 77030, Texas, USA

    Mingshan Xue & Christian Rosenmund

  2. Department of Biochemistry University of Texas Southwestern Medical Center, Dallas, 6000 Harry Hines Boulevard, Dallas, 75390, Texas, USA

    Cong Ma, Timothy K Craig & Josep Rizo

  3. Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, 6000 Harry Hines Boulevard, Dallas, 75390, Texas, USA

    Cong Ma, Timothy K Craig & Josep Rizo

  4. Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, 77030, Texas, USA

    Christian Rosenmund

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Correspondence to Christian Rosenmund or Josep Rizo.

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Xue, M., Ma, C., Craig, T. et al. The Janus-faced nature of the C2B domain is fundamental for synaptotagmin-1 function. Nat Struct Mol Biol 15, 1160–1168 (2008). https://doi.org/10.1038/nsmb.1508

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