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. 1985 Mar 1;100(3):727–735. doi: 10.1083/jcb.100.3.727

Isolation of an actin-binding protein from membranes of Dictyostelium discoideum

PMCID: PMC2113508  PMID: 3972891

Abstract

We prepared a probe of radiolabeled, glutaraldehyde cross-linked filamentous actin (F-actin) to study binding of actin to membranes of Dictyostelium discoideum. The probe bound to membranes or detergent extracts of membranes with a high affinity and in a saturable manner. The binding could be reduced by boiling of either the actin probe or the membranes, or by addition of excess native F-actin, but not by addition of an equivalent amount of bovine serum albumin, to the assay. The probe labeled several proteins when used to overlay sodium dodecyl sulfate gels of Dictyostelium membranes. One of these labeled proteins was a 24,000-mol-wt protein (p24), which was soluble only in the presence of a high concentration of sodium deoxycholate (5%, wt/vol) at room temperature or above. The p24 was purified by selective detergent extraction and column chromatography. When tested in a novel two-phase binding assay, p24 bound both native monomeric actin (G-actin) and F- actin in a specific manner. In this assay, G-actin bound p24 with a submicromolar affinity.

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Selected References

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  1. Albertsson P. A. Application of the phase partition method to a hydrophobic membrane protein, phospholipase A1 from Escherichia coli. Biochemistry. 1973 Jun 19;12(13):2525–2530. doi: 10.1021/bi00737a024. [DOI] [PubMed] [Google Scholar]
  2. Begg D. A., Rodewald R., Rebhun L. I. The visualization of actin filament polarity in thin sections. Evidence for the uniform polarity of membrane-associated filaments. J Cell Biol. 1978 Dec;79(3):846–852. doi: 10.1083/jcb.79.3.846. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ben-Ze'ev A., Duerr A., Solomon F., Penman S. The outer boundary of the cytoskeleton: a lamina derived from plasma membrane proteins. Cell. 1979 Aug;17(4):859–865. doi: 10.1016/0092-8674(79)90326-x. [DOI] [PubMed] [Google Scholar]
  4. Bourguignon L. Y., Singer S. J. Transmembrane interactions and the mechanism of capping of surface receptors by their specific ligands. Proc Natl Acad Sci U S A. 1977 Nov;74(11):5031–5035. doi: 10.1073/pnas.74.11.5031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Brown S. S., Malinoff H. L., Wicha M. S. Connectin: cell surface protein that binds both laminin and actin. Proc Natl Acad Sci U S A. 1983 Oct;80(19):5927–5930. doi: 10.1073/pnas.80.19.5927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Burridge K., Kelly T., Mangeat P. Nonerythrocyte spectrins: actin-membrane attachment proteins occurring in many cell types. J Cell Biol. 1982 Nov;95(2 Pt 1):478–486. doi: 10.1083/jcb.95.2.478. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carter-Su C., Pessin J. E., Mora R., Gitomer W., Czech M. P. Photoaffinity labeling of the human erythrocyte D-glucose transporter. J Biol Chem. 1982 May 25;257(10):5419–5425. [PubMed] [Google Scholar]
  9. Condeelis J. Isolation of concanavalin A caps during various stages of formation and their association with actin and myosin. J Cell Biol. 1979 Mar;80(3):751–758. doi: 10.1083/jcb.80.3.751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Edds K. T. Microfilament bundles. I. Formation with uniform polarity. Exp Cell Res. 1977 Sep;108(2):452–456. doi: 10.1016/s0014-4827(77)80056-6. [DOI] [PubMed] [Google Scholar]
  11. Gabbiani G., Chaponnier C., Zumbe A., Vassalli P. Actin and tubulin co-cap with surface immunoglobulins in mouse B lymphocytes. Nature. 1977 Oct 20;269(5630):697–698. doi: 10.1038/269697a0. [DOI] [PubMed] [Google Scholar]
  12. Geiger B. Membrane-cytoskeleton interaction. Biochim Biophys Acta. 1983 Aug 11;737(3-4):305–341. doi: 10.1016/0304-4157(83)90005-9. [DOI] [PubMed] [Google Scholar]
  13. Giuffre R. M., Tovell D. R., Kay C. M., Tyrrell D. L. Evidence for an interaction between the membrane protein of a paramyxovirus and actin. J Virol. 1982 Jun;42(3):963–968. doi: 10.1128/jvi.42.3.963-968.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Glenney J. R., Jr, Glenney P. The microvillus 110K cytoskeletal protein is an integral membrane protein. Cell. 1984 Jul;37(3):743–751. doi: 10.1016/0092-8674(84)90410-0. [DOI] [PubMed] [Google Scholar]
  15. Gruenstein E., Rich A., Weihing R. R. Actin associated with membranes from 3T3 mouse fibroblast and HeLa cells. J Cell Biol. 1975 Jan;64(1):223–234. doi: 10.1083/jcb.64.1.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Helenius A., Simons K. Solubilization of membranes by detergents. Biochim Biophys Acta. 1975 Mar 25;415(1):29–79. doi: 10.1016/0304-4157(75)90016-7. [DOI] [PubMed] [Google Scholar]
  17. Jacobson B. S. Actin binding to the cytoplasmic surface of the plasma membrane isolated from Dictyostelium discoideum. Biochem Biophys Res Commun. 1980 Dec 31;97(4):1493–1498. doi: 10.1016/s0006-291x(80)80034-9. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Lehrer S. S. Damage to actin filaments by glutaraldehyde: protection by tropomyosin. J Cell Biol. 1981 Aug;90(2):459–466. doi: 10.1083/jcb.90.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lehto V. P., Vartio T., Badley R. A., Virtanen I. Characterization of a detergent-resistant surface lamina in cultured human fibroblasts. Exp Cell Res. 1983 Feb;143(2):287–294. doi: 10.1016/0014-4827(83)90053-8. [DOI] [PubMed] [Google Scholar]
  21. Luna E. J., Fowler V. M., Swanson J., Branton D., Taylor D. L. A membrane cytoskeleton from Dictyostelium discoideum. I. Identification and partial characterization of an actin-binding activity. J Cell Biol. 1981 Feb;88(2):396–409. doi: 10.1083/jcb.88.2.396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Luna E. J., Goodloe-Holland C. M., Ingalls H. M. A membrane cytoskeleton from Dictyostelium discoideum. II. Integral proteins mediate the binding of plasma membranes to F-actin affinity beads. J Cell Biol. 1984 Jul;99(1 Pt 1):58–70. doi: 10.1083/jcb.99.1.58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mooseker M. S., Tilney L. G. Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells. J Cell Biol. 1975 Dec;67(3):725–743. doi: 10.1083/jcb.67.3.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Nicolson G. L. Trans-membrane control of the receptors on normal and tumor cells. II. Surface changes associated with transformation and malignancy. Biochim Biophys Acta. 1976 Apr 30;458(1):1–72. doi: 10.1016/0304-419x(76)90014-7. [DOI] [PubMed] [Google Scholar]
  25. Ostlund R. E., Leung J. T., Kipnis D. M. Muscle actin filaments bind pituitary secretory granules in vitro. J Cell Biol. 1977 Apr;73(1):78–87. doi: 10.1083/jcb.73.1.78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  28. Petrini M., Emerson D. L., Galbraith R. M. Linkage between surface immunoglobulin and cytoskeleton of B lymphocytes may involve Gc protein. Nature. 1983 Nov 3;306(5938):73–74. doi: 10.1038/306073a0. [DOI] [PubMed] [Google Scholar]
  29. Schleicher M., Gerisch G., Isenberg G. New actin-binding proteins from Dictyostelium discoideum. EMBO J. 1984 Sep;3(9):2095–2100. doi: 10.1002/j.1460-2075.1984.tb02096.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Snabes M. C., Boyd A. E., 3rd, Bryan J. Detection of actin-binding proteins in human platelets by 125I-actin overlay of polyacrylamide gels. J Cell Biol. 1981 Sep;90(3):809–812. doi: 10.1083/jcb.90.3.809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Spudich J. A. Biochemical and structural studies of actomyosin-like proteins from non-muscle cells. II. Purification, properties, and membrane association of actin from amoebae of Dictyostelium discoideum. J Biol Chem. 1974 Sep 25;249(18):6013–6020. [PubMed] [Google Scholar]
  33. Sundqvist K. G., Ehrnst A. Cytoskeletal control of surface membrane mobility. Nature. 1976 Nov 18;264(5583):226–231. doi: 10.1038/264226a0. [DOI] [PubMed] [Google Scholar]
  34. Toh B. H., Hard C. C. Actin co-caps with concanavalin A receptors. Nature. 1977 Oct 20;269(5630):695–697. doi: 10.1038/269695a0. [DOI] [PubMed] [Google Scholar]
  35. Utsumi K., Okimasu E., Morimoto Y. M., Nishihara Y., Miyahara M. Selective interaction of cytoskeletal proteins with liposomes. FEBS Lett. 1982 May 17;141(2):176–180. doi: 10.1016/0014-5793(82)80041-0. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. 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]

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