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. 1984 May 1;98(5):1796–1803. doi: 10.1083/jcb.98.5.1796

Ca2+-dependent binding of severin to actin: a one-to-one complex is formed

PMCID: PMC2113187  PMID: 6427234

Abstract

Severin is a protein from Dictyostelium that severs actin filaments in a Ca2+-dependent manner and remains bound to the filament fragments (Brown, S. S., K. Yamamoto, and J. A. Spudich , 1982, J. Cell Biol., 93:205-210; Yamamoto, K., J. D. Pardee , J. Reidler , L. Stryer , and J. A. Spudich , 1982, J. Cell Biol. 95:711-719). Further characterization of the interaction of severin with actin suggests that it remains bound to the preferred assembly end of the fragmented actin filaments. Addition of severin in molar excess to actin causes total disassembly of the filaments and the formation of a high-affinity complex containing one severin and one actin. This severin -actin complex does not sever actin filaments. The binding of severin to actin, measured directly by fluorescence energy transfer, requires micromolar Ca2+, as does the severing and depolymerizing activity reported previously. Once bound to actin in the presence of greater than 1 microM Ca2+, severin is not released from the actin when the Ca2+ is lowered to less than 0.1 microM by addition of EGTA. Tropomyosin, DNase I, phalloidin, and cytochalasin B have no effect on the ability of severin to bind to or sever actin filaments. Subfragment 1 of myosin, however, significantly inhibits severin activity. Severin binds not only to actin filaments, but also directly to G-actin, as well as to other conformational species of actin.

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Selected References

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  1. Ames G. F. Resolution of bacterial proteins by polyacrylamide gel electrophoresis on slabs. Membrane, soluble, and periplasmic fractions. J Biol Chem. 1974 Jan 25;249(2):634–644. [PubMed] [Google Scholar]
  2. 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. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Bretscher A., Weber K. Villin is a major protein of the microvillus cytoskeleton which binds both G and F actin in a calcium-dependent manner. Cell. 1980 Jul;20(3):839–847. doi: 10.1016/0092-8674(80)90330-x. [DOI] [PubMed] [Google Scholar]
  4. Bretscher A., Weber K. Villin: the major microfilament-associated protein of the intestinal microvillus. Proc Natl Acad Sci U S A. 1979 May;76(5):2321–2325. doi: 10.1073/pnas.76.5.2321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brown S. S., Yamamoto K., Spudich J. A. A 40,000-dalton protein from Dictyostelium discoideum affects assembly properties of actin in a Ca2+-dependent manner. J Cell Biol. 1982 Apr;93(1):205–210. doi: 10.1083/jcb.93.1.205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Craig R., Szent-Györgyi A. G., Beese L., Flicker P., Vibert P., Cohen C. Electron microscopy of thin filaments decorated with a Ca2+-regulated myosin. J Mol Biol. 1980 Jun 15;140(1):35–55. doi: 10.1016/0022-2836(80)90355-1. [DOI] [PubMed] [Google Scholar]
  7. Craig S. W., Powell L. D. Regulation of actin polymerization by villin, a 95,000 dalton cytoskeletal component of intestinal brush borders. Cell. 1980 Dec;22(3):739–746. doi: 10.1016/0092-8674(80)90550-4. [DOI] [PubMed] [Google Scholar]
  8. Eisenberg E., Kielley W. W. Troponin-tropomyosin complex. Column chromatographic separation and activity of the three, active troponin components with and without tropomyosin present. J Biol Chem. 1974 Aug 10;249(15):4742–4748. [PubMed] [Google Scholar]
  9. Giffard R. G., Spudich J. A., Spudich A. Ca2+-sensitive isolation of a cortical actin matrix from Dictyostelium amoebae. J Muscle Res Cell Motil. 1983 Feb;4(1):115–131. doi: 10.1007/BF00711962. [DOI] [PubMed] [Google Scholar]
  10. Glenney J. R., Jr, Bretscher A., Weber K. Calcium control of the intestinal microvillus cytoskeleton: its implications for the regulation of microfilament organizations. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6458–6462. doi: 10.1073/pnas.77.11.6458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Glenney J. R., Jr, Kaulfus P., Weber K. F actin assembly modulated by villin: Ca++-dependent nucleation and capping of the barbed end. Cell. 1981 May;24(2):471–480. doi: 10.1016/0092-8674(81)90338-x. [DOI] [PubMed] [Google Scholar]
  12. Harafuji H., Ogawa Y. Re-examination of the apparent binding constant of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid with calcium around neutral pH. J Biochem. 1980 May;87(5):1305–1312. doi: 10.1093/oxfordjournals.jbchem.a132868. [DOI] [PubMed] [Google Scholar]
  13. Harris H. E., Weeds A. G. Plasma actin depolymerizing factor has both calcium-dependent and calcium-independent effects on actin. Biochemistry. 1983 May 24;22(11):2728–2741. doi: 10.1021/bi00280a022. [DOI] [PubMed] [Google Scholar]
  14. Hasegawa T., Takahashi S., Hayashi H., Hatano S. Fragmin: a calcium ion sensitive regulatory factor on the formation of actin filaments. Biochemistry. 1980 Jun 10;19(12):2677–2683. doi: 10.1021/bi00553a021. [DOI] [PubMed] [Google Scholar]
  15. Hinssen H. An actin-modulating protein from Physarum polycephalum. I. Isolation and purification. Eur J Cell Biol. 1981 Feb;23(2):225–233. [PubMed] [Google Scholar]
  16. Hinssen H. An actin-modulating protein from Physarum polycephalum. II. Ca++-dependence and other properties. Eur J Cell Biol. 1981 Feb;23(2):234–240. [PubMed] [Google Scholar]
  17. Houk T. W., Jr, Ue K. The measurement of actin concentration in solution: a comparison of methods. Anal Biochem. 1974 Nov;62(1):66–74. doi: 10.1016/0003-2697(74)90367-4. [DOI] [PubMed] [Google Scholar]
  18. Isenberg G., Aebi U., Pollard T. D. An actin-binding protein from Acanthamoeba regulates actin filament polymerization and interactions. Nature. 1980 Dec 4;288(5790):455–459. doi: 10.1038/288455a0. [DOI] [PubMed] [Google Scholar]
  19. KIELLEY W. W., HARRINGTON W. F. A model for the myosin molecule. Biochim Biophys Acta. 1960 Jul 15;41:401–421. doi: 10.1016/0006-3002(60)90037-8. [DOI] [PubMed] [Google Scholar]
  20. Korn E. D. Actin polymerization and its regulation by proteins from nonmuscle cells. Physiol Rev. 1982 Apr;62(2):672–737. doi: 10.1152/physrev.1982.62.2.672. [DOI] [PubMed] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. MacLean-Fletcher S. D., Pollard T. D. Viscometric analysis of the gelation of Acanthamoeba extracts and purification of two gelation factors. J Cell Biol. 1980 May;85(2):414–428. doi: 10.1083/jcb.85.2.414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Margossian S. S., Lowey S. Interaction of myosin subfragments with F-actin. Biochemistry. 1978 Dec 12;17(25):5431–5439. doi: 10.1021/bi00618a017. [DOI] [PubMed] [Google Scholar]
  24. Marston S., Weber A. The dissociation constant of the actin-heavy meromyosin subfragment-1 complex. Biochemistry. 1975 Aug 26;14(17):3868–3873. doi: 10.1021/bi00688a021. [DOI] [PubMed] [Google Scholar]
  25. Mooseker M. S., Graves T. A., Wharton K. A., Falco N., Howe C. L. Regulation of microvillus structure: calcium-dependent solation and cross-linking of actin filaments in the microvilli of intestinal epithelial cells. J Cell Biol. 1980 Dec;87(3 Pt 1):809–822. doi: 10.1083/jcb.87.3.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pardee J. D., Simpson P. A., Stryer L., Spudich J. A. Actin filaments undergo limited subunit exchange in physiological salt conditions. J Cell Biol. 1982 Aug;94(2):316–324. doi: 10.1083/jcb.94.2.316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pardee J. D., Spudich J. A. Mechanism of K+-induced actin assembly. J Cell Biol. 1982 Jun;93(3):648–654. doi: 10.1083/jcb.93.3.648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pardee J. D., Spudich J. A. Purification of muscle actin. Methods Cell Biol. 1982;24:271–289. doi: 10.1016/s0091-679x(08)60661-5. [DOI] [PubMed] [Google Scholar]
  29. Pollard T. D., Mooseker M. S. Direct measurement of actin polymerization rate constants by electron microscopy of actin filaments nucleated by isolated microvillus cores. J Cell Biol. 1981 Mar;88(3):654–659. doi: 10.1083/jcb.88.3.654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schacterle G. R., Pollack R. L. A simplified method for the quantitative assay of small amounts of protein in biologic material. Anal Biochem. 1973 Feb;51(2):654–655. doi: 10.1016/0003-2697(73)90523-x. [DOI] [PubMed] [Google Scholar]
  31. Schliwa M. Proteins associated with cytoplasmic actin. Cell. 1981 Sep;25(3):587–590. doi: 10.1016/0092-8674(81)90166-5. [DOI] [PubMed] [Google Scholar]
  32. Simpson P. A., Spudich J. A. ATP-driven steady-state exchange of monomeric and filamentous actin from Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4610–4613. doi: 10.1073/pnas.77.8.4610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Spudich J. A. Dictyostelium discoideum: methods and perspectives for study of cell motility. Methods Cell Biol. 1982;25(Pt B):359–364. doi: 10.1016/s0091-679x(08)61433-8. [DOI] [PubMed] [Google Scholar]
  34. Spudich J. A., Watt S. The regulation of rabbit skeletal muscle contraction. I. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem. 1971 Aug 10;246(15):4866–4871. [PubMed] [Google Scholar]
  35. Stryer L. Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem. 1978;47:819–846. doi: 10.1146/annurev.bi.47.070178.004131. [DOI] [PubMed] [Google Scholar]
  36. Sugino H., Hatano S. Effect of fragmin on actin polymerization: evidence for enhancement of nucleation and capping of the barbed end. Cell Motil. 1982;2(5):457–470. doi: 10.1002/cm.970020505. [DOI] [PubMed] [Google Scholar]
  37. Taylor D. L., Reidler J., Spudich J. A., Stryer L. Detection of actin assembly by fluorescence energy transfer. J Cell Biol. 1981 May;89(2):362–367. doi: 10.1083/jcb.89.2.362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Uyemura D. G., Brown S. S., Spudich J. A. Biochemical and structural characterization of actin from Dictyostelium discoideum. J Biol Chem. 1978 Dec 25;253(24):9088–9096. [PubMed] [Google Scholar]
  39. Wang L. L., Bryan J. Isolation of calcium-dependent platelet proteins that interact with actin. Cell. 1981 Sep;25(3):637–649. doi: 10.1016/0092-8674(81)90171-9. [DOI] [PubMed] [Google Scholar]
  40. Wang Y. L., Taylor D. L. Probing the dynamic equilibrium of actin polymerization by fluorescence energy transfer. Cell. 1981 Dec;27(3 Pt 2):429–436. doi: 10.1016/0092-8674(81)90384-6. [DOI] [PubMed] [Google Scholar]
  41. Weeds A. G., Taylor R. S. Separation of subfragment-1 isoenzymes from rabbit skeletal muscle myosin. Nature. 1975 Sep 4;257(5521):54–56. doi: 10.1038/257054a0. [DOI] [PubMed] [Google Scholar]
  42. Weeds A. Actin-binding proteins--regulators of cell architecture and motility. Nature. 1982 Apr 29;296(5860):811–816. doi: 10.1038/296811a0. [DOI] [PubMed] [Google Scholar]
  43. Woodrum D. T., Rich S. A., Pollard T. D. Evidence for biased bidirectional polymerization of actin filaments using heavy meromyosin prepared by an improved method. J Cell Biol. 1975 Oct;67(1):231–237. doi: 10.1083/jcb.67.1.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yamamoto K., Pardee J. D., Reidler J., Stryer L., Spudich J. A. Mechanism of interaction of Dictyostelium severin with actin filaments. J Cell Biol. 1982 Dec;95(3):711–719. doi: 10.1083/jcb.95.3.711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yin H. L., Hartwig J. H., Maruyama K., Stossel T. P. Ca2+ control of actin filament length. Effects of macrophage gelsolin on actin polymerization. J Biol Chem. 1981 Sep 25;256(18):9693–9697. [PubMed] [Google Scholar]
  46. Yin H. L., Zaner K. S., Stossel T. P. Ca2+ control of actin gelation. Interaction of gelsolin with actin filaments and regulation of actin gelation. J Biol Chem. 1980 Oct 10;255(19):9494–9500. [PubMed] [Google Scholar]

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