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. 1989 Oct 1;109(4):1519–1528. doi: 10.1083/jcb.109.4.1519

Plasma membrane association of Acanthamoeba myosin I

PMCID: PMC2115813  PMID: 2793931

Abstract

Myosin I accounted for approximately 2% of the protein of highly purified plasma membranes, which represents about a tenfold enrichment over its concentration in the total cell homogenate. This localization is consistent with immunofluorescence analysis of cells that shows myosin I at or near the plasma membrane as well as diffusely distributed in the cytoplasm with no apparent association with cytoplasmic organelles or vesicles identifiable at the level of light microscopy. Myosin II was not detected in the purified plasma membrane fraction. Although actin was present in about a tenfold molar excess relative to myosin I, several lines of evidence suggest that the principal linkage of myosin I with the plasma membrane is not through F- actin: (a) KI extracted much more actin than myosin I from the plasma membrane fraction; (b) higher ionic strength was required to solubilize the membrane-bound myosin I than to dissociate a complex of purified myosin I and F-actin; and (c) added purified myosin I bound to KI- extracted plasma membranes in a saturable manner with maximum binding four- to fivefold greater than the actin content and with much greater affinity than for pure F-actin (apparent KD of 30-50 nM vs. 10-40 microM in 0.1 M KCl plus 2 mM MgATP). Thus, neither the MgATP-sensitive actin-binding site in the NH2-terminal end of the myosin I heavy chain nor the MgATP-insensitive actin-binding site in the COOH-terminal end of the heavy chain appeared to be the principal mechanism of binding of myosin I to plasma membranes through F-actin. Furthermore, the MgATP- sensitive actin-binding site of membrane-bound myosin I was still available to bind added F-actin. However, the MgATP-insensitive actin- binding site appeared to be unable to bind added F-actin, suggesting that the membrane-binding site is near enough to this site to block sterically its interaction with actin.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Adams R. J., Pollard T. D. Propulsion of organelles isolated from Acanthamoeba along actin filaments by myosin-I. Nature. 1986 Aug 21;322(6081):754–756. doi: 10.1038/322754a0. [DOI] [PubMed] [Google Scholar]
  2. Albanesi J. P., Fujisaki H., Hammer J. A., 3rd, Korn E. D., Jones R., Sheetz M. P. Monomeric Acanthamoeba myosins I support movement in vitro. J Biol Chem. 1985 Jul 25;260(15):8649–8652. [PubMed] [Google Scholar]
  3. Albanesi J. P., Fujisaki H., Korn E. D. A kinetic model for the molecular basis of the contractile activity of Acanthamoeba myosins IA and IB. J Biol Chem. 1985 Sep 15;260(20):11174–11179. [PubMed] [Google Scholar]
  4. Albanesi J. P., Fujisaki H., Korn E. D. Localization of the active site and phosphorylation site of Acanthamoeba myosins IA and IB. J Biol Chem. 1984 Nov 25;259(22):14184–14189. [PubMed] [Google Scholar]
  5. Albanesi J. P., Hammer J. A., 3rd, Korn E. D. The interaction of F-actin with phosphorylated and unphosphorylated myosins IA and IB from Acanthamoeba castellanii. J Biol Chem. 1983 Aug 25;258(16):10176–10181. [PubMed] [Google Scholar]
  6. 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]
  7. Brzeska H., Lynch T. J., Korn E. D. Localization of the actin-binding sites of Acanthamoeba myosin IB and effect of limited proteolysis on its actin-activated Mg2+-ATPase activity. J Biol Chem. 1988 Jan 5;263(1):427–435. [PubMed] [Google Scholar]
  8. Carboni J. M., Conzelman K. A., Adams R. A., Kaiser D. A., Pollard T. D., Mooseker M. S. Structural and immunological characterization of the myosin-like 110-kD subunit of the intestinal microvillar 110K-calmodulin complex: evidence for discrete myosin head and calmodulin-binding domains. J Cell Biol. 1988 Nov;107(5):1749–1757. doi: 10.1083/jcb.107.5.1749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Clarke B. J., Hohman T. C., Bowers B. Purification of plasma membrane from Acanthamoeba castellanii. J Protozool. 1988 Aug;35(3):408–413. doi: 10.1111/j.1550-7408.1988.tb04118.x. [DOI] [PubMed] [Google Scholar]
  10. Collins J. H., Borysenko C. W. The 110,000-dalton actin- and calmodulin-binding protein from intestinal brush border is a myosin-like ATPase. J Biol Chem. 1984 Nov 25;259(22):14128–14135. [PubMed] [Google Scholar]
  11. Coluccio L. M., Bretscher A. Calcium-regulated cooperative binding of the microvillar 110K-calmodulin complex to F-actin: formation of decorated filaments. J Cell Biol. 1987 Jul;105(1):325–333. doi: 10.1083/jcb.105.1.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Conzelman K. A., Mooseker M. S. The 110-kD protein-calmodulin complex of the intestinal microvillus is an actin-activated MgATPase. J Cell Biol. 1987 Jul;105(1):313–324. doi: 10.1083/jcb.105.1.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Côté G. P., Albanesi J. P., Ueno T., Hammer J. A., 3rd, Korn E. D. Purification from Dictyostelium discoideum of a low-molecular-weight myosin that resembles myosin I from Acanthamoeba castellanii. J Biol Chem. 1985 Apr 25;260(8):4543–4546. [PubMed] [Google Scholar]
  14. Fujisaki H., Albanesi J. P., Korn E. D. Experimental evidence for the contractile activities of Acanthamoeba myosins IA and IB. J Biol Chem. 1985 Sep 15;260(20):11183–11189. [PubMed] [Google Scholar]
  15. Fukui Y., Yumura S., Yumura T. K., Mori H. Agar overlay method: high-resolution immunofluorescence for the study of the contractile apparatus. Methods Enzymol. 1986;134:573–580. doi: 10.1016/0076-6879(86)34122-3. [DOI] [PubMed] [Google Scholar]
  16. Gadasi H., Korn E. D. Evidence for differential intracellular localization of the Acanthamoeba myosin isoenzymes. Nature. 1980 Jul 31;286(5772):452–456. doi: 10.1038/286452a0. [DOI] [PubMed] [Google Scholar]
  17. Hagen S. J., Kiehart D. P., Kaiser D. A., Pollard T. D. Characterization of monoclonal antibodies to Acanthamoeba myosin-I that cross-react with both myosin-II and low molecular mass nuclear proteins. J Cell Biol. 1986 Dec;103(6 Pt 1):2121–2128. doi: 10.1083/jcb.103.6.2121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hammer J. A., 3rd, Albanesi J. P., Korn E. D. Purification and characterization of a myosin I heavy chain kinase from Acanthamoeba castellanii. J Biol Chem. 1983 Aug 25;258(16):10168–10175. [PubMed] [Google Scholar]
  19. Hawkes R., Niday E., Gordon J. A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem. 1982 Jan 1;119(1):142–147. doi: 10.1016/0003-2697(82)90677-7. [DOI] [PubMed] [Google Scholar]
  20. Jung G., Korn E. D., Hammer J. A., 3rd The heavy chain of Acanthamoeba myosin IB is a fusion of myosin-like and non-myosin-like sequences. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6720–6724. doi: 10.1073/pnas.84.19.6720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Korn E. D., Atkinson M. A., Brzeska H., Hammer J. A., 3rd, Jung G., Lynch T. J. Structure-function studies on Acanthamoeba myosins IA, IB, and II. J Cell Biochem. 1988 Jan;36(1):37–50. doi: 10.1002/jcb.240360105. [DOI] [PubMed] [Google Scholar]
  22. Korn E. D., Weisman R. A. Phagocytosis of latex beads by Acanthamoeba. II. Electron microscopic study of the initial events. J Cell Biol. 1967 Jul;34(1):219–227. doi: 10.1083/jcb.34.1.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Korn E. D., Wright P. L. Macromolecular composition of an amoeba plasma membrane. J Biol Chem. 1973 Jan 25;248(2):439–447. [PubMed] [Google Scholar]
  24. 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]
  25. Lehto V. P., Virtanen I. Immunolocalization of a novel, cytoskeleton-associated polypeptide of Mr 230,000 daltons (p230). J Cell Biol. 1983 Mar;96(3):703–716. doi: 10.1083/jcb.96.3.703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lynch T. J., Albanesi J. P., Korn E. D., Robinson E. A., Bowers B., Fujisaki H. ATPase activities and actin-binding properties of subfragments of Acanthamoeba myosin IA. J Biol Chem. 1986 Dec 25;261(36):17156–17162. [PubMed] [Google Scholar]
  27. Lynch T. J., Brzeska H., Korn E. D. Limited tryptic digestion of Acanthamoeba myosin IA abolishes regulation of actin-activated ATPase activity by heavy chain phosphorylation. J Biol Chem. 1987 Oct 5;262(28):13842–13849. [PubMed] [Google Scholar]
  28. Maruta H., Gadasi H., Collins J. H., Korn E. D. Multiple forms of Acanthamoeba myosin I. J Biol Chem. 1979 May 10;254(9):3624–3630. [PubMed] [Google Scholar]
  29. Maruta H., Korn E. D. Acanthamoeba cofactor protein is a heavy chain kinase required for actin activation of the Mg2+-ATPase activity of Acanthamoeba myosin I. J Biol Chem. 1977 Dec 10;252(23):8329–8332. [PubMed] [Google Scholar]
  30. Maruta H., Korn E. D. Acanthamoeba myosin II. J Biol Chem. 1977 Sep 25;252(18):6501–6509. [PubMed] [Google Scholar]
  31. Mooseker M. S. Organization, chemistry, and assembly of the cytoskeletal apparatus of the intestinal brush border. Annu Rev Cell Biol. 1985;1:209–241. doi: 10.1146/annurev.cb.01.110185.001233. [DOI] [PubMed] [Google Scholar]
  32. Pollard T. D., Korn E. D. Acanthamoeba myosin. I. Isolation from Acanthamoeba castellanii of an enzyme similar to muscle myosin. J Biol Chem. 1973 Jul 10;248(13):4682–4690. [PubMed] [Google Scholar]
  33. Pollard T. D., Korn E. D. Acanthamoeba myosin. II. Interaction with actin and with a new cofactor protein required for actin activation of Mg 2+ adenosine triphosphatase activity. J Biol Chem. 1973 Jul 10;248(13):4691–4697. [PubMed] [Google Scholar]
  34. Pollard T. D., Korn E. D. Electron microscopic identification of actin associated with isolated amoeba plasma membranes. J Biol Chem. 1973 Jan 25;248(2):448–450. [PubMed] [Google Scholar]
  35. 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]
  36. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ueno T., Korn E. D. Isolation and partial characterization of a 110-kD dimer actin-binding protein. J Cell Biol. 1986 Aug;103(2):621–630. doi: 10.1083/jcb.103.2.621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ulsamer A. G., Wright P. L., Wetzel M. G., Korn E. D. Plasma and phagosome membranes of Acanthamoeba castellanii. J Cell Biol. 1971 Oct;51(1):193–215. doi: 10.1083/jcb.51.1.193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wetzel M. G., Korn E. D. Phagocytosis of latex beads by Acahamoeba castellanii (Neff). 3. Isolation of the phagocytic vesicles and their membranes. J Cell Biol. 1969 Oct;43(1):90–104. doi: 10.1083/jcb.43.1.90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Yumura S., Mori H., Fukui Y. Localization of actin and myosin for the study of ameboid movement in Dictyostelium using improved immunofluorescence. J Cell Biol. 1984 Sep;99(3):894–899. doi: 10.1083/jcb.99.3.894. [DOI] [PMC free article] [PubMed] [Google Scholar]

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