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. 1993 Aug;5(8):983–994. doi: 10.1105/tpc.5.8.983

Specific binding of nuclear localization sequences to plant nuclei.

G R Hicks 1, N V Raikhel 1
PMCID: PMC160333  PMID: 8400874

Abstract

We have begun to dissect the import apparatus of higher plants by examining the specific association of nuclear localization sequences (NLSs) with purified plant nuclei. Peptides to the simian virus 40 (SV40) large T antigen NLS and a bipartite NLS of maize were allowed to associate with tobacco and maize nuclei. Wild-type NLSs were found to compete for a single class of low-affinity binding sites having a dissociation constant (Kd) of approximately 200 microM. Peptides to mutant NLSs, which are inefficient in stimulating import, were poor competitors, as were reverse wild-type and non-NLS peptides. The NLS binding site was proteinaceous and resistant to extraction under conditions where pores were still associated. In addition, immunofluorescence and immunoelectron microscopy indicated that binding was at the nuclear envelope. Overall, plant nuclei may be an excellent system to identify components of the import apparatus.

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Selected References

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  1. Adam S. A., Gerace L. Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import. Cell. 1991 Sep 6;66(5):837–847. doi: 10.1016/0092-8674(91)90431-w. [DOI] [PubMed] [Google Scholar]
  2. Adam S. A., Lobl T. J., Mitchell M. A., Gerace L. Identification of specific binding proteins for a nuclear location sequence. Nature. 1989 Jan 19;337(6204):276–279. doi: 10.1038/337276a0. [DOI] [PubMed] [Google Scholar]
  3. Bednarek S. Y., Wilkins T. A., Dombrowski J. E., Raikhel N. V. A carboxyl-terminal propeptide is necessary for proper sorting of barley lectin to vacuoles of tobacco. Plant Cell. 1990 Dec;2(12):1145–1155. doi: 10.1105/tpc.2.12.1145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bossie M. A., DeHoratius C., Barcelo G., Silver P. A mutant nuclear protein with similarity to RNA binding proteins interferes with nuclear import in yeast. Mol Biol Cell. 1992 Aug;3(8):875–893. doi: 10.1091/mbc.3.8.875. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Breeuwer M., Goldfarb D. S. Facilitated nuclear transport of histone H1 and other small nucleophilic proteins. Cell. 1990 Mar 23;60(6):999–1008. doi: 10.1016/0092-8674(90)90348-i. [DOI] [PubMed] [Google Scholar]
  6. Chrispeels M. J., Raikhel N. V. Short peptide domains target proteins to plant vacuoles. Cell. 1992 Feb 21;68(4):613–616. doi: 10.1016/0092-8674(92)90134-x. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Davis L. I., Blobel G. Identification and characterization of a nuclear pore complex protein. Cell. 1986 Jun 6;45(5):699–709. doi: 10.1016/0092-8674(86)90784-1. [DOI] [PubMed] [Google Scholar]
  9. Denecke J., Goldman M. H., Demolder J., Seurinck J., Botterman J. The tobacco luminal binding protein is encoded by a multigene family. Plant Cell. 1991 Sep;3(9):1025–1035. doi: 10.1105/tpc.3.9.1025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dingwall C., Laskey R. A. Nuclear targeting sequences--a consensus? Trends Biochem Sci. 1991 Dec;16(12):478–481. doi: 10.1016/0968-0004(91)90184-w. [DOI] [PubMed] [Google Scholar]
  11. Dombrowski J. E., Schroeder M. R., Bednarek S. Y., Raikhel N. V. Determination of the functional elements within the vacuolar targeting signal of barley lectin. Plant Cell. 1993 May;5(5):587–596. doi: 10.1105/tpc.5.5.587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dworetzky S. I., Feldherr C. M. Translocation of RNA-coated gold particles through the nuclear pores of oocytes. J Cell Biol. 1988 Mar;106(3):575–584. doi: 10.1083/jcb.106.3.575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Feldherr C. M., Kallenbach E., Schultz N. Movement of a karyophilic protein through the nuclear pores of oocytes. J Cell Biol. 1984 Dec;99(6):2216–2222. doi: 10.1083/jcb.99.6.2216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Garcia-Bustos J. F., Wagner P., Hall M. N. Nuclear import substrates compete for a limited number of binding sites. Evidence for different classes of yeast nuclear import receptors. J Biol Chem. 1991 Nov 25;266(33):22303–22306. [PubMed] [Google Scholar]
  16. Garcia-Bustos J. F., Wagner P., Hall M. N. Yeast cell-free nuclear protein import requires ATP hydrolysis. Exp Cell Res. 1991 Jan;192(1):213–219. doi: 10.1016/0014-4827(91)90178-w. [DOI] [PubMed] [Google Scholar]
  17. Gu Z., Moerschell R. P., Sherman F., Goldfarb D. S. NIP1, a gene required for nuclear transport in yeast. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10355–10359. doi: 10.1073/pnas.89.21.10355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hall M. N., Hereford L., Herskowitz I. Targeting of E. coli beta-galactosidase to the nucleus in yeast. Cell. 1984 Apr;36(4):1057–1065. doi: 10.1016/0092-8674(84)90055-2. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Kalderon D., Richardson W. D., Markham A. F., Smith A. E. Sequence requirements for nuclear location of simian virus 40 large-T antigen. Nature. 1984 Sep 6;311(5981):33–38. doi: 10.1038/311033a0. [DOI] [PubMed] [Google Scholar]
  21. Kalinich J. F., Douglas M. G. In vitro translocation through the yeast nuclear envelope. Signal-dependent transport requires ATP and calcium. J Biol Chem. 1989 Oct 25;264(30):17979–17989. [PubMed] [Google Scholar]
  22. Keegstra K. Transport and routing of proteins into chloroplasts. Cell. 1989 Jan 27;56(2):247–253. doi: 10.1016/0092-8674(89)90898-2. [DOI] [PubMed] [Google Scholar]
  23. Lanford R. E., Butel J. S. Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen. Cell. 1984 Jul;37(3):801–813. doi: 10.1016/0092-8674(84)90415-x. [DOI] [PubMed] [Google Scholar]
  24. Lassner M. W., Jones A., Daubert S., Comai L. Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal. Plant Mol Biol. 1991 Aug;17(2):229–234. doi: 10.1007/BF00039497. [DOI] [PubMed] [Google Scholar]
  25. Lee W. C., Mélèse T. Identification and characterization of a nuclear localization sequence-binding protein in yeast. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8808–8812. doi: 10.1073/pnas.86.22.8808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lee W. C., Zabetakis D., Mélèse T. NSR1 is required for pre-rRNA processing and for the proper maintenance of steady-state levels of ribosomal subunits. Mol Cell Biol. 1992 Sep;12(9):3865–3871. doi: 10.1128/mcb.12.9.3865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Markland W., Smith A. E., Roberts B. L. Signal-dependent translocation of simian virus 40 large-T antigen into rat liver nuclei in a cell-free system. Mol Cell Biol. 1987 Dec;7(12):4255–4265. doi: 10.1128/mcb.7.12.4255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Meier U. T., Blobel G. A nuclear localization signal binding protein in the nucleolus. J Cell Biol. 1990 Dec;111(6 Pt 1):2235–2245. doi: 10.1083/jcb.111.6.2235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Meier U. T., Blobel G. Nopp140 shuttles on tracks between nucleolus and cytoplasm. Cell. 1992 Jul 10;70(1):127–138. doi: 10.1016/0092-8674(92)90539-o. [DOI] [PubMed] [Google Scholar]
  30. Moore M. S., Blobel G. The two steps of nuclear import, targeting to the nuclear envelope and translocation through the nuclear pore, require different cytosolic factors. Cell. 1992 Jun 12;69(6):939–950. doi: 10.1016/0092-8674(92)90613-h. [DOI] [PubMed] [Google Scholar]
  31. Newmeyer D. D., Forbes D. J. An N-ethylmaleimide-sensitive cytosolic factor necessary for nuclear protein import: requirement in signal-mediated binding to the nuclear pore. J Cell Biol. 1990 Mar;110(3):547–557. doi: 10.1083/jcb.110.3.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Newmeyer D. D., Forbes D. J. Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation. Cell. 1988 Mar 11;52(5):641–653. doi: 10.1016/0092-8674(88)90402-3. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Restrepo M. A., Freed D. D., Carrington J. C. Nuclear transport of plant potyviral proteins. Plant Cell. 1990 Oct;2(10):987–998. doi: 10.1105/tpc.2.10.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Roberts B. Nuclear location signal-mediated protein transport. Biochim Biophys Acta. 1989 Aug 14;1008(3):263–280. doi: 10.1016/0167-4781(89)90016-x. [DOI] [PubMed] [Google Scholar]
  36. Shi Y., Thomas J. O. The transport of proteins into the nucleus requires the 70-kilodalton heat shock protein or its cytosolic cognate. Mol Cell Biol. 1992 May;12(5):2186–2192. doi: 10.1128/mcb.12.5.2186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shieh M. W., Wessler S. R., Raikhel N. V. Nuclear targeting of the maize R protein requires two nuclear localization sequences. Plant Physiol. 1993 Feb;101(2):353–361. doi: 10.1104/pp.101.2.353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Silver P. A. How proteins enter the nucleus. Cell. 1991 Feb 8;64(3):489–497. doi: 10.1016/0092-8674(91)90233-o. [DOI] [PubMed] [Google Scholar]
  39. Snow C. M., Senior A., Gerace L. Monoclonal antibodies identify a group of nuclear pore complex glycoproteins. J Cell Biol. 1987 May;104(5):1143–1156. doi: 10.1083/jcb.104.5.1143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sterne-Marr R., Blevitt J. M., Gerace L. O-linked glycoproteins of the nuclear pore complex interact with a cytosolic factor required for nuclear protein import. J Cell Biol. 1992 Jan;116(2):271–280. doi: 10.1083/jcb.116.2.271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stochaj U., Silver P. Nucleocytoplasmic traffic of proteins. Eur J Cell Biol. 1992 Oct;59(1):1–11. [PubMed] [Google Scholar]
  42. Tinland B., Koukolíková-Nicola Z., Hall M. N., Hohn B. The T-DNA-linked VirD2 protein contains two distinct functional nuclear localization signals. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7442–7446. doi: 10.1073/pnas.89.16.7442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Varagona M. J., Schmidt R. J., Raikhel N. V. Monocot regulatory protein Opaque-2 is localized in the nucleus of maize endosperm and transformed tobacco plants. Plant Cell. 1991 Feb;3(2):105–113. doi: 10.1105/tpc.3.2.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Varagona M. J., Schmidt R. J., Raikhel N. V. Nuclear localization signal(s) required for nuclear targeting of the maize regulatory protein Opaque-2. Plant Cell. 1992 Oct;4(10):1213–1227. doi: 10.1105/tpc.4.10.1213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Willmitzer L., Wagner K. G. The isolation of nuclei from tissue-cultured plant cells. Exp Cell Res. 1981 Sep;135(1):69–77. doi: 10.1016/0014-4827(81)90300-1. [DOI] [PubMed] [Google Scholar]
  46. Yamasaki L., Kanda P., Lanford R. E. Identification of four nuclear transport signal-binding proteins that interact with diverse transport signals. Mol Cell Biol. 1989 Jul;9(7):3028–3036. doi: 10.1128/mcb.9.7.3028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. van der Krol A. R., Chua N. H. The basic domain of plant B-ZIP proteins facilitates import of a reporter protein into plant nuclei. Plant Cell. 1991 Jul;3(7):667–675. doi: 10.1105/tpc.3.7.667. [DOI] [PMC free article] [PubMed] [Google Scholar]

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