Synaptotagmin C2B Domain Regulates Ca2+-triggered Fusion in Vitro (original) (raw)

Abstract

Synaptotagmin (syt) 1 is localized to synaptic vesicles, binds Ca 2؉ , and regulates neuronal exocytosis. Syt 1 harbors two Ca 2؉-binding motifs referred to as C2A and C2B. In this study we examine the function of the isolated C2 domains of Syt 1 using a reconstituted, SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor)-mediated, fusion assay. We report that inclusion of phosphatidylethanolamine into reconstituted SNARE vesicles enabled isolated C2B, but not C2A, to regulate Ca 2؉-triggered fusion. The isolated C2B domain had a 6-fold lower EC 50 for Ca 2؉-activated fusion than the intact cytosolic domain of Syt 1 (C2AB). Phosphatidylethanolamine increased both the rate and efficiency of C2AB-and C2B-regulated fusion without affecting their abilities to bind membraneembedded syntaxin-SNAP-25 (t-SNARE) complexes. At equimolar concentrations, the isolated C2A domain was an effective inhibitor of C2B-, but not C2AB-regulated fusion; hence, C2A has markedly different effects in the fusion assay depending on whether it is tethered to C2B. Finally, scanning alanine mutagenesis of C2AB revealed four distinct groups of mutations within the C2B domain that play roles in the regulation of SNARE-mediated fusion. Surprisingly, substitution of Arg-398 with alanine, which lies on the opposite end of C2B from the Ca 2؉ /membrane-binding loops, decreases C2AB t-SNARE binding and Ca 2؉-triggered fusion in vitro without affecting Ca 2؉triggered interactions with phosphatidylserine or vesicle aggregation. In addition, some mutations uncouple the clamping and stimulatory functions of syt 1, suggesting that these two activities are mediated by distinct structural determinants in C2B.

Loading...

Loading Preview

Sorry, preview is currently unavailable. You can download the paper by clicking the button above.

References (49)

  1. Katz, B., and Miledi, R. (1965) Proc. R. Soc. Lond. B. Biol. Sci. 161, 496 -503
  2. Rothman, J. E. (1994) Nature 372, 55-63
  3. Chapman, E. R. (2008) Ann. Rev. Biochem. 77, 615-641
  4. Perin, M. S., Brose, N., Jahn, R., and Sudhof, T. C. (1991) J. Biol. Chem. 266, 623-629
  5. Chapman, E. R., and Davis, A. F. (1998) J. Biol. Chem. 273, 13995-14001
  6. Bai, J., Wang, P., and Chapman, E. R. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 1665-1670
  7. Bai, J., Tucker, W. C., and Chapman, E. R. (2004) Nat. Struct. Mol. Biol. 11, 36 -44
  8. Bhalla, A., Tucker, W. C., and Chapman, E. R. (2005) Mol. Biol. Cell 16, 4755-4764
  9. Schiavo, G., Stenbeck, G., Rothman, J. E., and Sollner, T. H. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 997-1001
  10. Chapman, E. R., Hanson, P. I., An, S., and Jahn, R. (1995) J. Biol. Chem. 270, 23667-23671
  11. Bai, J., Wang, C. T., Richards, D. A., Jackson, M. B., and Chapman, E. R. (2004) Neuron 41, 929 -942
  12. Lynch, K. L., Gerona, R. R., Larsen, E. C., Marcia, R. F., Mitchell, J. C., and Martin, T. F. (2007) Mol. Biol. Cell 18, 4957-4968
  13. Shin, O. H., Rhee, J. S., Tang, J., Sugita, S., Rosenmund, C., and Sudhof, T. C. (2003) Neuron 37, 99 -108
  14. Shin, O. H., Maximov, A., Lim, B. K., Rizo, J., and Sudhof, T. C. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 2554 -2559
  15. Shin, O. H., Rizo, J., and Sudhof, T. C. (2002) Nat. Neurosci. 5, 649 -656
  16. Popov, S. V., and Poo, M. M. (1993) Cell 73, 1247-1249
  17. Chicka, M. C., Hui, E., Liu, H., and Chapman, E. R. (2008) Nat. Struct. Mol. Biol. 15, 827-835
  18. Weber, T., Zemelman, B. V., McNew, J. A., Westermann, B., Gmachl, M., Parlati, F., Sollner, T. H., and Rothman, J. E. (1998) Cell 92, 759 -772
  19. Tucker, W. C., Weber, T., and Chapman, E. R. (2004) Science 304, 435-438
  20. Bhalla, A., Chicka, M. C., Tucker, W. C., and Chapman, E. R. (2006) Nat. Struct. Mol. Biol. 13, 323-330
  21. Mackler, J. M., Drummond, J. A., Loewen, C. A., Robinson, I. M., and Reist, N. E. (2002) Nature 418, 340 -344
  22. Robinson, I. M., Ranjan, R., and Schwarz, T. L. (2002) Nature 418, 336 -340
  23. Stevens, C. F., and Sullivan, J. M. (2003) Neuron 39, 299 -308
  24. Nishiki, T., and Augustine, G. J. (2004) J. Neurosci. 24, 8542-8550
  25. Littleton, J. T., Stern, M., Perin, M., and Bellen, H. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10888 -10892
  26. Loewen, C. A., Lee, S. M., Shin, Y. K., and Reist, N. E. (2006) Mol. Biol. Cell 17, 5211-5226
  27. Li, L., Shin, O. H., Rhee, J. S., Arac, D., Rah, J. C., Rizo, J., Sudhof, T., and Rosenmund, C. (2006) J. Biol. Chem. 281, 15845-15852
  28. Stein, A., Radhakrishnan, A., Riedel, D., Fasshauer, D., and Jahn, R. (2007) Nat. Struct. Mol. Biol. 14, 904 -911
  29. Martens, S., Kozlov, M. M., and McMahon, H. T. (2007) Science 316, 1205-1208
  30. Chapman, E. R., An, S., Edwardson, J. M., and Jahn, R. (1996) J. Biol. Chem. 271, 5844 -5849
  31. Bowen, M. E., Weninger, K., Ernst, J., Chu, S., and Brunger, A. T. (2005) Biophys. J. 89, 690 -702
  32. Shao, X., Li, C., Fernandez, I., Zhang, X., Sudhof, T. C., and Rizo, J. (1997) Neuron 18, 133-142
  33. Fernandez, I., Ubach, J., Dulubova, I., Zhang, X., Sudhof, T. C., and Rizo, J. (1998) Cell 94, 841-849
  34. Wang, P., Wang, C. T., Bai, J., Jackson, M. B., and Chapman, E. R. (2003) J. Biol. Chem. 278, 47030 -47037
  35. Sutton, R. B., Davletov, B. A., Berghuis, A. M., Sudhof, T. C., and Sprang, S. R. (1995) Cell 80, 929 -938
  36. Sutton, R. B., Fasshauer, D., Jahn, R., and Brunger, A. T. (1998) Nature 395, 347-353
  37. Fuson, K. L., Montes, M., Robert, J. J., and Sutton, R. B. (2007) Biochemistry 46, 13041-13048
  38. Desai, R. C., Vyas, B., Earles, C. A., Littleton, J. T., Kowalchyck, J. A., Martin, T. F., and Chapman, E. R. (2000) J. Cell Biol. 150, 1125-1136
  39. Breckenridge, W. C., Gombos, G., and Morgan, I. G. (1972) Biochim. Bio- phys. Acta 266, 695-707
  40. Breckenridge, W. C., Morgan, I. G., Zanetta, J. P., and Vincendon, G. (1973) Biochim. Biophys. Acta 320, 681-686
  41. Tucker, W. C., Edwardson, J. M., Bai, J., Kim, H. J., Martin, T. F., and Chapman, E. R. (2003) J. Cell Biol. 162, 199 -209
  42. Szoka, F., Jr., and Papahadjopoulos, D. (1980) Annu. Rev. Biophys. Bioeng. 9, 467-508
  43. Rickman, C., and Davletov, B. (2003) J. Biol. Chem. 278, 5501-5504
  44. Fontaine, R. N., Harris, R. A., and Schroeder, F. (1980) J. Neurochem. 34, 269 -277
  45. Chernomordik, L., and Kozlov, M. (2003) Ann. Rev. Biochem. 72, 175-207
  46. Dai, H., Shen, N., Arac, D., and Rizo, J. (2007) J. Mol. Biol. 367, 848 -863
  47. Nalefski, E. A., and Falke, J. J. (1996) Protein Sci. 5, 2375-2390
  48. M. B., and Chapman, E. R. (2006) Ann. Rev. Biophys. Biomol. Struct. 35, 135-160
  49. C2B Domain of Synaptotagmin 1 Regulates SNARE-mediated Fusion NOVEMBER 14, 2008 • VOLUME 283 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 31775