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. 1996 Mar;8(3):363–373. doi: 10.1105/tpc.8.3.363

Transport of DNA into the nuclei of xenopus oocytes by a modified VirE2 protein of Agrobacterium.

B Guralnick 1, G Thomsen 1, V Citovsky 1
PMCID: PMC161106  PMID: 8721747

Abstract

We used Agrobacterium T-DNA nuclear transport to examine the specificity of nuclear targeting between plants and animals and the nuclear import of DNA by a specialized transport protein. Two karyophilic Agrobacterium virulence (Vir) proteins, VirD2 and VirE2, which presumably associate with the transported T-DNA and function in many plant species, were microinjected into Drosophila embryos and Xenopus oocytes. In both animal systems, VirD2 localized to the cell nuclei and VirE2 remained exclusively cytoplasmic, suggesting that VirE2 nuclear localization signals may be plant specific. Repositioning one amino acid residue within VirE2 nuclear localization signals enabled them to function in animal cells. The modified VirE2 protein bound DNA and actively transported it into the nuclei of Xenopus oocytes. These observations suggest a functional difference in nuclear import between animals and plants and show that DNA can be transported into the cell nucleus via a protein-specific pathway.

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Selected References

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  1. Binns A. N., Beaupré C. E., Dale E. M. Inhibition of VirB-mediated transfer of diverse substrates from Agrobacterium tumefaciens by the IncQ plasmid RSF1010. J Bacteriol. 1995 Sep;177(17):4890–4899. doi: 10.1128/jb.177.17.4890-4899.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chalfie M., Tu Y., Euskirchen G., Ward W. W., Prasher D. C. Green fluorescent protein as a marker for gene expression. Science. 1994 Feb 11;263(5148):802–805. doi: 10.1126/science.8303295. [DOI] [PubMed] [Google Scholar]
  3. Chelsky D., Ralph R., Jonak G. Sequence requirements for synthetic peptide-mediated translocation to the nucleus. Mol Cell Biol. 1989 Jun;9(6):2487–2492. doi: 10.1128/mcb.9.6.2487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Citovsky V., DE Vos G., Zambryski P. Single-Stranded DNA Binding Protein Encoded by the virE Locus of Agrobacterium tumefaciens. Science. 1988 Apr 22;240(4851):501–504. doi: 10.1126/science.240.4851.501. [DOI] [PubMed] [Google Scholar]
  5. Citovsky V., Knorr D., Zambryski P. Gene I, a potential cell-to-cell movement locus of cauliflower mosaic virus, encodes an RNA-binding protein. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2476–2480. doi: 10.1073/pnas.88.6.2476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Citovsky V., Warnick D., Zambryski P. Nuclear import of Agrobacterium VirD2 and VirE2 proteins in maize and tobacco. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3210–3214. doi: 10.1073/pnas.91.8.3210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Citovsky V., Zambryski P. Transport of nucleic acids through membrane channels: snaking through small holes. Annu Rev Microbiol. 1993;47:167–197. doi: 10.1146/annurev.mi.47.100193.001123. [DOI] [PubMed] [Google Scholar]
  8. Citovsky V., Zupan J., Warnick D., Zambryski P. Nuclear localization of Agrobacterium VirE2 protein in plant cells. Science. 1992 Jun 26;256(5065):1802–1805. doi: 10.1126/science.1615325. [DOI] [PubMed] [Google Scholar]
  9. Clever J., Yamada M., Kasamatsu H. Import of simian virus 40 virions through nuclear pore complexes. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7333–7337. doi: 10.1073/pnas.88.16.7333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Enenkel C., Blobel G., Rexach M. Identification of a yeast karyopherin heterodimer that targets import substrate to mammalian nuclear pore complexes. J Biol Chem. 1995 Jul 14;270(28):16499–16502. doi: 10.1074/jbc.270.28.16499. [DOI] [PubMed] [Google Scholar]
  11. Forbes D. J. Structure and function of the nuclear pore complex. Annu Rev Cell Biol. 1992;8:495–527. doi: 10.1146/annurev.cb.08.110192.002431. [DOI] [PubMed] [Google Scholar]
  12. Gall J. G., Murphy C., Callan H. G., Wu Z. A. Lampbrush chromosomes. Methods Cell Biol. 1991;36:149–166. [PubMed] [Google Scholar]
  13. Garcia-Bustos J., Heitman J., Hall M. N. Nuclear protein localization. Biochim Biophys Acta. 1991 Mar 7;1071(1):83–101. doi: 10.1016/0304-4157(91)90013-m. [DOI] [PubMed] [Google Scholar]
  14. Gerace L., Burke B. Functional organization of the nuclear envelope. Annu Rev Cell Biol. 1988;4:335–374. doi: 10.1146/annurev.cb.04.110188.002003. [DOI] [PubMed] [Google Scholar]
  15. Gietl C., Koukolíková-Nicola Z., Hohn B. Mobilization of T-DNA from Agrobacterium to plant cells involves a protein that binds single-stranded DNA. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9006–9010. doi: 10.1073/pnas.84.24.9006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gulizia J., Dempsey M. P., Sharova N., Bukrinsky M. I., Spitz L., Goldfarb D., Stevenson M. Reduced nuclear import of human immunodeficiency virus type 1 preintegration complexes in the presence of a prototypic nuclear targeting signal. J Virol. 1994 Mar;68(3):2021–2025. doi: 10.1128/jvi.68.3.2021-2025.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Herrera-Estrella A., Chen Z. M., Van Montagu M., Wang K. VirD proteins of Agrobacterium tumefaciens are required for the formation of a covalent DNA--protein complex at the 5' terminus of T-strand molecules. EMBO J. 1988 Dec 20;7(13):4055–4062. doi: 10.1002/j.1460-2075.1988.tb03299.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hicks G. R., Smith H. M., Shieh M., Raikhel N. V. Three classes of nuclear import signals bind to plant nuclei. Plant Physiol. 1995 Apr;107(4):1055–1058. doi: 10.1104/pp.107.4.1055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Howard E. A., Winsor B. A., De Vos G., Zambryski P. Activation of the T-DNA transfer process in Agrobacterium results in the generation of a T-strand-protein complex: Tight association of VirD2 with the 5' ends of T-strands. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4017–4021. doi: 10.1073/pnas.86.11.4017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Howard E. A., Zupan J. R., Citovsky V., Zambryski P. C. The VirD2 protein of A. tumefaciens contains a C-terminal bipartite nuclear localization signal: implications for nuclear uptake of DNA in plant cells. Cell. 1992 Jan 10;68(1):109–118. doi: 10.1016/0092-8674(92)90210-4. [DOI] [PubMed] [Google Scholar]
  21. Lanford R. E., Feldherr C. M., White R. G., Dunham R. G., Kanda P. Comparison of diverse transport signals in synthetic peptide-induced nuclear transport. Exp Cell Res. 1990 Jan;186(1):32–38. doi: 10.1016/0014-4827(90)90206-p. [DOI] [PubMed] [Google Scholar]
  22. Melchior F., Paschal B., Evans J., Gerace L. Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor. J Cell Biol. 1993 Dec;123(6 Pt 2):1649–1659. doi: 10.1083/jcb.123.6.1649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Michaud N., Goldfarb D. S. Multiple pathways in nuclear transport: the import of U2 snRNP occurs by a novel kinetic pathway. J Cell Biol. 1991 Jan;112(2):215–223. doi: 10.1083/jcb.112.2.215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Moore M. S., Blobel G. The GTP-binding protein Ran/TC4 is required for protein import into the nucleus. Nature. 1993 Oct 14;365(6447):661–663. doi: 10.1038/365661a0. [DOI] [PubMed] [Google Scholar]
  25. Nigg E. A., Baeuerle P. A., Lührmann R. Nuclear import-export: in search of signals and mechanisms. Cell. 1991 Jul 12;66(1):15–22. doi: 10.1016/0092-8674(91)90135-l. [DOI] [PubMed] [Google Scholar]
  26. O'Neill R. E., Jaskunas R., Blobel G., Palese P., Moroianu J. Nuclear import of influenza virus RNA can be mediated by viral nucleoprotein and transport factors required for protein import. J Biol Chem. 1995 Sep 29;270(39):22701–22704. doi: 10.1074/jbc.270.39.22701. [DOI] [PubMed] [Google Scholar]
  27. Powers M. A., Forbes D. J. Cytosolic factors in nuclear transport: what's importin? Cell. 1994 Dec 16;79(6):931–934. doi: 10.1016/0092-8674(94)90024-8. [DOI] [PubMed] [Google Scholar]
  28. Rexach M., Blobel G. Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins. Cell. 1995 Dec 1;83(5):683–692. doi: 10.1016/0092-8674(95)90181-7. [DOI] [PubMed] [Google Scholar]
  29. Rihs H. P., Jans D. A., Fan H., Peters R. The rate of nuclear cytoplasmic protein transport is determined by the casein kinase II site flanking the nuclear localization sequence of the SV40 T-antigen. EMBO J. 1991 Mar;10(3):633–639. doi: 10.1002/j.1460-2075.1991.tb07991.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rihs H. P., Peters R. Nuclear transport kinetics depend on phosphorylation-site-containing sequences flanking the karyophilic signal of the Simian virus 40 T-antigen. EMBO J. 1989 May;8(5):1479–1484. doi: 10.1002/j.1460-2075.1989.tb03531.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Robbins J., Dilworth S. M., Laskey R. A., Dingwall C. Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell. 1991 Feb 8;64(3):615–623. doi: 10.1016/0092-8674(91)90245-t. [DOI] [PubMed] [Google Scholar]
  32. Shurvinton C. E., Hodges L., Ream W. A nuclear localization signal and the C-terminal omega sequence in the Agrobacterium tumefaciens VirD2 endonuclease are important for tumor formation. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11837–11841. doi: 10.1073/pnas.89.24.11837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  34. Tinland B., Hohn B., Puchta H. Agrobacterium tumefaciens transfers single-stranded transferred DNA (T-DNA) into the plant cell nucleus. Proc Natl Acad Sci U S A. 1994 Aug 16;91(17):8000–8004. doi: 10.1073/pnas.91.17.8000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tinland B., Hohn B. Recombination between prokaryotic and eukaryotic DNA: integration of Agrobacterium tumefaciens T-DNA into the plant genome. Genet Eng (N Y) 1995;17:209–229. [PubMed] [Google Scholar]
  36. Yamasaki L., Lanford R. E. Nuclear transport: a guide to import receptors. Trends Cell Biol. 1992 May;2(5):123–127. doi: 10.1016/0962-8924(92)90083-y. [DOI] [PubMed] [Google Scholar]
  37. Young C., Nester E. W. Association of the virD2 protein with the 5' end of T strands in Agrobacterium tumefaciens. J Bacteriol. 1988 Aug;170(8):3367–3374. doi: 10.1128/jb.170.8.3367-3374.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yusibov V. M., Steck T. R., Gupta V., Gelvin S. B. Association of single-stranded transferred DNA from Agrobacterium tumefaciens with tobacco cells. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):2994–2998. doi: 10.1073/pnas.91.8.2994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zambryski P., Tempe J., Schell J. Transfer and function of T-DNA genes from agrobacterium Ti and Ri plasmids in plants. Cell. 1989 Jan 27;56(2):193–201. doi: 10.1016/0092-8674(89)90892-1. [DOI] [PubMed] [Google Scholar]

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