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. 1984 Aug;4(8):1542–1550. doi: 10.1128/mcb.4.8.1542

Membrane interactions of simian virus 40 large T-antigen: influence of protein sequences and fatty acid acylation.

U Klockmann, M Staufenbiel, W Deppert
PMCID: PMC368946  PMID: 6092917

Abstract

To sort out possible influences of protein sequences and fatty acid acylation on the plasma membrane association of simian virus 40 large T-antigen, we have analyzed the membrane interactions of carboxy-terminal fragments of large T-antigen, encoded by the adenovirus type 2 (Ad2+)-simian virus 40 hybrid viruses Ad2+ND1 and Ad2+ND2. The 28,000 (28K)-molecular-weight protein of Ad2+ND1 as well as the 42K and 56K proteins of Ad2+ND2 associate preferentially with membranous structures and were found in association with the membrane system of the endoplasmic reticulum and with plasma membranes. Neither the endoplasmic reticulum membrane- nor the plasma membrane-associated 28K protein of Ad2+ND1 is fatty acid acylated. We, therefore, conclude that fatty acid acylation is not necessary for membrane association of this protein and suggest that an amino acid sequence in this protein is responsible for its membrane interaction. In contrast, the 42K and 56K proteins of Ad2+ND2 in plasma membrane fractions contain fatty acid. However, the interaction of these proteins with the plasma membrane differs from that of the 28K protein of Ad2+ND1: whereas the 28K protein of Ad2+ND1 interacts stably with Nonidet P-40-soluble constituents of the plasma membrane, the 42K and 56K proteins of Ad2+ND2 are tightly bound to the Nonidet P-40-insoluble plasma membrane lamina. Thus, an amino acid sequence in the amino-terminal region of the 28K protein confers membrane affinity to these proteins, whereas a region between the amino-terminal end of the 42K protein of Ad2+ND2 and the amino-terminal end of the 28K protein of Ad2+ND1 contains a reactive site for fatty acid acylation. This posttranslational modification correlates with the stable association of the 42K and 56K proteins with the plasma membrane lamina. We suggest that the same sequences also mediate the proper plasma membrane association of large T-antigen in simian virus 40-transformed cells.

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Selected References

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  1. Anderson K. P., Klessig D. F. Posttranscriptional block to synthesis of a human adenovirus capsid protein in abortively infected monkey cells. J Mol Appl Genet. 1983;2(1):31–43. [PubMed] [Google Scholar]
  2. Avruch J., Wallach D. F. Preparation and properties of plasma membrane and endoplasmic reticulum fragments from isolated rat fat cells. Biochim Biophys Acta. 1971 Apr 13;233(2):334–347. doi: 10.1016/0005-2736(71)90331-2. [DOI] [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. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  5. Deppert W., Pates R. Cell surface location of simian virus 40-specific proteins on HeLa cells infected with adenovirus type 2-simian virus 40 hybrid viruses Ad2+ND1 and Ad2+ND2. J Virol. 1979 Aug;31(2):522–536. doi: 10.1128/jvi.31.2.522-536.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Deppert W. Simian virus 40 (SV40)-specific proteins associated with the nuclear matrix isolated from adenovirus type 2-SV40 hybrid virus-infected HeLa cells carry SV40 U-antigen determinants. J Virol. 1978 Apr;26(1):165–178. doi: 10.1128/jvi.26.1.165-178.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Deppert W., Walter G. Domains of simian virus 40 large T-antigen exposed on the cell surface. Virology. 1982 Oct 15;122(1):56–70. doi: 10.1016/0042-6822(82)90377-4. [DOI] [PubMed] [Google Scholar]
  8. Deppert W., Walter G., Linke H. Simian virus 40 tumor-specific proteins: subcellular distribution and metabolic stability in HeLa cells infected with nondefective adenovirus type 2-simian virus 40 hybrid viruses. J Virol. 1977 Mar;21(3):1170–1186. doi: 10.1128/jvi.21.3.1170-1186.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Deppert W., Walter G. Simian virus to (SV40) tumor-specific proteins in nucleus and plasma membrane of HeLa cells infected by adenovirus 2-SV40 hybrid virus Ad2+ND2. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2505–2509. doi: 10.1073/pnas.73.7.2505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fiers W., Contreras R., Haegemann G., Rogiers R., Van de Voorde A., Van Heuverswyn H., Van Herreweghe J., Volckaert G., Ysebaert M. Complete nucleotide sequence of SV40 DNA. Nature. 1978 May 11;273(5658):113–120. doi: 10.1038/273113a0. [DOI] [PubMed] [Google Scholar]
  11. Fulton A. B., Wan K. M., Penman S. The spatial distribution of polyribosomes in 3T3 cells and the associated assembly of proteins into the skeletal framework. Cell. 1980 Jul;20(3):849–857. doi: 10.1016/0092-8674(80)90331-1. [DOI] [PubMed] [Google Scholar]
  12. Garber E. A., Krueger J. G., Hanafusa H., Goldberg A. R. Only membrane-associated RSV src proteins have amino-terminally bound lipid. Nature. 1983 Mar 10;302(5904):161–163. doi: 10.1038/302161a0. [DOI] [PubMed] [Google Scholar]
  13. Goldman N., Brown M., Khoury G. Modification of SV40 T antigen by poly ADP-ribosylation. Cell. 1981 May;24(2):567–572. doi: 10.1016/0092-8674(81)90347-0. [DOI] [PubMed] [Google Scholar]
  14. Gooding L. R., O'Connell K. A. Recognition by cytotoxic T lymphocytes of cells expressing fragments of the SV40 tumor antigen. J Immunol. 1983 Nov;131(5):2580–2586. [PubMed] [Google Scholar]
  15. Jay G., Jay F. T., Chang C., Friedman R. M., Levine A. S. Tumor-specific transplantation antigen: use of the Ad2+ND1 hybrid virus to identify the protein responsible for simian virus 40 tumor rejection and its genetic origin. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3055–3059. doi: 10.1073/pnas.75.7.3055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jay G., Jay F. T., Chang C., Levine A. S., Friedman R. M. Induction of simian virus 40-specific tumour rejection by the Ad2+ND2 hybrid virus. J Gen Virol. 1979 Aug;44(2):287–296. doi: 10.1099/0022-1317-44-2-287. [DOI] [PubMed] [Google Scholar]
  17. Jay G., Jay F. T., Friedman R. M., Levine A. S. Biosynthesis, immunological specificity, and intracellular distribution of the simian virus 40-specific protein induced by the nondefective hybrid Ad2+ND1. J Virol. 1978 May;26(2):411–419. doi: 10.1128/jvi.26.2.411-419.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jay G., Jay F. T., Friedman R. M., Levine A. S. Simian virus 40-specific ribosome-binding proteins induced by a nondefective adenovirus 2-simian virus 40 hybrid. J Virol. 1977 Sep;23(3):692–699. doi: 10.1128/jvi.23.3.692-699.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Klockmann U., Deppert W. Acylated simian virus 40 large T-antigen: a new subclass associated with a detergent-resistant lamina of the plasma membrane. EMBO J. 1983;2(7):1151–1157. doi: 10.1002/j.1460-2075.1983.tb01560.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Klockmann U., Deppert W. Acylated simian virus 40-specific proteins in the plasma membrane of HeLa cells infected with adenovirus 2-simian virus 40 hybrid virus Ad2+ND2. Virology. 1983 Apr 30;126(2):717–720. doi: 10.1016/s0042-6822(83)80029-4. [DOI] [PubMed] [Google Scholar]
  21. Klockmann U., Deppert W. Acylation: a new post-translational modification specific for plasma membrane-associated simian virus 40 large T-antigen. FEBS Lett. 1983 Jan 24;151(2):257–259. doi: 10.1016/0014-5793(83)80081-7. [DOI] [PubMed] [Google Scholar]
  22. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  23. 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]
  24. Lanford R. E., Butel J. S. Antigenic relationship of SV40 early proteins to purified large T polypeptide. Virology. 1979 Sep;97(2):295–306. doi: 10.1016/0042-6822(79)90341-6. [DOI] [PubMed] [Google Scholar]
  25. Lewis A. M., Jr, Levin M. J., Wiese W. H., Crumpacker C. S., Henry P. H. A nondefective (competent) adenovirus-SV40 hybrid isolated from the AD.2-SV40 hybrid population. Proc Natl Acad Sci U S A. 1969 Aug;63(4):1128–1135. doi: 10.1073/pnas.63.4.1128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lewis A. M., Jr, Rowe W. P. Studies of nondefective Adenovirus 2-Simian virus 40 hybrid viruses. 8. Association of Simian virus 40 transplantation antigen with a specific region of the early viral genome. J Virol. 1973 Oct;12(4):836–840. doi: 10.1128/jvi.12.4.836-840.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lewis A. M., Jr, Rowe W. P. Studies on nondefective adenovirus-simian virus 40 hybrid viruses. I. A newly characterized simian virus 40 antigen induced by the Ad2+ND 1 virus. J Virol. 1971 Feb;7(2):189–197. doi: 10.1128/jvi.7.2.189-197.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Magee A. I., Schlesinger M. J. Fatty acid acylation of eucaryotic cell membrane proteins. Biochim Biophys Acta. 1982 Nov 30;694(3):279–289. doi: 10.1016/0304-4157(82)90008-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mann K., Hunter T., Walter G., Linke H. Evidence for simian virus 40 (SV40) coding of SV40 T-antigen and the SV40-specific proteins in HeLa cells infected with nondefective adenovirus type 2-SV40 hybrid viruses. J Virol. 1977 Oct;24(1):151–169. doi: 10.1128/jvi.24.1.151-169.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mescher M. F., Jose M. J., Balk S. P. Actin-containing matrix associated with the plasma membrane of murine tumour and lymphoid cells. Nature. 1981 Jan 15;289(5794):139–144. doi: 10.1038/289139a0. [DOI] [PubMed] [Google Scholar]
  31. Meyer D. I., Krause E., Dobberstein B. Secretory protein translocation across membranes-the role of the "docking protein'. Nature. 1982 Jun 24;297(5868):647–650. doi: 10.1038/297647a0. [DOI] [PubMed] [Google Scholar]
  32. Michel M. R., Schwyzer M. Messenger ribonucleoproteins of cells infected by simian virus 40 contain large T-antigen. Eur J Biochem. 1982 Dec;129(1):25–32. doi: 10.1111/j.1432-1033.1982.tb07016.x. [DOI] [PubMed] [Google Scholar]
  33. Reddy V. B., Thimmappaya B., Dhar R., Subramanian K. N., Zain B. S., Pan J., Ghosh P. K., Celma M. L., Weissman S. M. The genome of simian virus 40. Science. 1978 May 5;200(4341):494–502. doi: 10.1126/science.205947. [DOI] [PubMed] [Google Scholar]
  34. Scheidtmann K. H., Echle B., Walter G. Simian virus 40 large T antigen is phosphorylated at multiple sites clustered in two separate regions. J Virol. 1982 Oct;44(1):116–133. doi: 10.1128/jvi.44.1.116-133.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Soule H. R., Butel J. S. Subcellular Localization of simian virus 40 large tumor antigen. J Virol. 1979 May;30(2):523–532. doi: 10.1128/jvi.30.2.523-532.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Staufenbiel M., Deppert W. Different structural systems of the nucleus are targets for SV40 large T antigen. Cell. 1983 May;33(1):173–181. doi: 10.1016/0092-8674(83)90346-x. [DOI] [PubMed] [Google Scholar]
  37. Staufenbiel M., Deppert W. Intermediate filament systems are collapsed onto the nuclear surface after isolation of nuclei from tissue culture cells. Exp Cell Res. 1982 Mar;138(1):207–214. doi: 10.1016/0014-4827(82)90107-0. [DOI] [PubMed] [Google Scholar]
  38. Sternberger L. A., Hardy P. H., Jr, Cuculis J. J., Meyer H. G. The unlabeled antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem. 1970 May;18(5):315–333. doi: 10.1177/18.5.315. [DOI] [PubMed] [Google Scholar]
  39. Tevethia S. S., Tevethia M. J., Lewis A. J., Reddy V. B., Weissman S. M. Biology of simian virus 40 (SV40) transplantation antigen (TrAg). IX. Analysis of TrAg in mouse cells synthesizing truncated SV40 large T antigen. Virology. 1983 Jul 30;128(2):319–330. doi: 10.1016/0042-6822(83)90259-3. [DOI] [PubMed] [Google Scholar]
  40. Van Roy F., Fransen L., Fiers W. Phosphorylation patterns of tumour antigens in cells lytically infected or transformed by simian virus 40. J Virol. 1981 Oct;40(1):28–44. doi: 10.1128/jvi.40.1.28-44.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Walter G., Martin H. Simian virus 40-specific proteins in HeLa cells infected with nondefective adenovirus 2-simian virus 40 hybrid viruses. J Virol. 1975 Nov;16(5):1236–1247. doi: 10.1128/jvi.16.5.1236-1247.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Walter G., Scheidtmann K. H., Carbone A., Laudano A. P., Doolittle R. F. Antibodies specific for the carboxy- and amino-terminal regions of simian virus 40 large tumor antigen. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5197–5200. doi: 10.1073/pnas.77.9.5197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Zorn G. A., Anderson C. W. Adenovirus type 2 expresses fiber in monkey-human hybrids and reconstructed cells. J Virol. 1981 Feb;37(2):759–769. doi: 10.1128/jvi.37.2.759-769.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]

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