Advertisement

The Use of Permeabilized Cell Systems to Study Nuclear Transport

  • Amy M. Brownawell
  • James M. Holaska
  • Ian G. Macara
  • Bryce M. Paschal
Part of the Methods in Molecular Biology™ book series (MIMB, volume 189)

Abstract

Nucleocytoplasmic transport occurs through nuclear pore complexes (NPC), macromolecular structures which span the nuclear envelope (1). NPCs contain aqueous channels with a diameter of ~9 nm, allowing ions, metabolites, and small proteins to passively diffuse between the nucleus and the cytoplasm (2). Most proteins and RNAs, however, exceed the ~60-kDa diffusion limit and are transported through the NPC by mechanisms that are saturable (3), energy-dependent (4), and signal-mediated (6).

Keywords

Nuclear Import Nuclear Pore Complex Protein Kinase Inhibitor Nuclear Localization Sequence Potassium Acetate 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Davis, L. I. (1995) The nuclear pore complex. Annu. Rev. Biochem. 64, 865–896.PubMedCrossRefGoogle Scholar
  2. 2.
    Gorlich, D. and Kutay, U. (1999) Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15, 607–660.PubMedCrossRefGoogle Scholar
  3. 3.
    Goldfarb, D. S., Gariepy, J., Schoolnik, G., and Kornberg, R. D. (1986) Synthetic peptides as nuclear localization signals. Nature 322, 641–644.PubMedCrossRefGoogle Scholar
  4. 4.
    Newmeyer, D. D., Finlay, D. R, and Forbes, D. J. (1986) In vitro transport of a fluorescent nuclear protein and exclusion of non-nuclear proteins. J. Cell Biol. 103, 2091–2102.PubMedCrossRefGoogle Scholar
  5. 5.
    Adam, S. A., Marr, R. S., and Gerace, L. (1990) Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J. Cell Biol. 111, 807–816.PubMedCrossRefGoogle Scholar
  6. 6.
    Dingwall, C., Sharnick, S. V., and Laskey, R. A. (1982) A polypeptide domain that specifies migration of nucleoplasmin into the nucleus. Cell 30, 449–458.PubMedCrossRefGoogle Scholar
  7. 7.
    Lanford, R. E. and Butel, J. S. (1984) Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen. Cell 37, 801-813.Google Scholar
  8. 8.
    Lanford, R. E., Kanda, P., and Kennedy, R. C. (1986) Induction of nuclear transport with a synthetic peptide homologous to the SV40 T antigen transport signal. Cell 46, 575–582.PubMedCrossRefGoogle Scholar
  9. 9.
    Moore, M. S. and Blobel, G. (1992) The two steps of nuclear import, targeting to the nuclear envelope and translocation through the nuclear pore, require different cytosolic factors. Cell 69, 939–950.PubMedCrossRefGoogle Scholar
  10. 10.
    Melchior, F., Paschal, B., Evans, J., and Gerace, L. (1993) 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. 123, 1649–1659.PubMedCrossRefGoogle Scholar
  11. 11.
    Moore, M. S. and Blobel, G. (1993) The GTP-binding protein Ran/TC4 is required for protein import into the nucleus. Nature 365, 661–663.PubMedCrossRefGoogle Scholar
  12. 12.
    Moore, M. S. and Blobel, G. (1994) Purification of a Ran-interacting protein that is required for protein import into the nucleus. Proc. Natl. Acad. Set USA 91, 10,212–10,216.CrossRefGoogle Scholar
  13. 13.
    Paschal, B. M. and Gerace, L. (1995) Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62. J. Cell Biol. 129, 925–937.PubMedCrossRefGoogle Scholar
  14. 14.
    Adam, E. J. and Adam, S. A. (1994) Identification of cytosolic factors required for nuclear location sequence-mediated binding to the nuclear envelope. J. Cell Biol. 125 547–555.PubMedCrossRefGoogle Scholar
  15. 15.
    Gorlich, D., Prehn, S., Laskey, R. A., and Hartmann, E. (1994) Isolation ofa protein that is essential for the first step of nuclear protein import. Cell 79, 767–778.PubMedCrossRefGoogle Scholar
  16. 16.
    Chi, N. C., Adam, E. J., and Adam, S. A. (1995) Sequence and characterization of cytoplasmic nuclear protein import factor p97. J. Cell Biol. 130, 265–274.PubMedCrossRefGoogle Scholar
  17. 17.
    Gorlich, D., Kostka, S., Kraft, R., Dingwall, C., Laskey, R. A., Hartmann, E., et al. (1995) Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope. Curr. Biol. 5, 383–392.PubMedCrossRefGoogle Scholar
  18. 18.
    Radu, A., Blobel, G., and Moore, M. S. (1995) Identification of a protein complex that is required for nuclear protein import and mediates docking of import substrate to distinct nucleoporins. Proc. Natl. Acad. Sci. USA 92, 1769–1773.PubMedCrossRefGoogle Scholar
  19. 19.
    Wente, S. R. (2000) Gatekeepers of the nucleus. Science 288, 1374–1377.PubMedCrossRefGoogle Scholar
  20. 20.
    Bischoff, F. R. and Ponstingl, H. (1991) Catalysis of guanine nucleotide exchange on Ran by the mitotic regulator RCC1. Nature 354, 80–82.PubMedCrossRefGoogle Scholar
  21. 21.
    Bischoff, F. R., Krebber, H., Kempf, T., Hermes, I., and Ponstingl, H. (1995) Human RanGTPase-activating protein RanGAP1 is a homologue of yeast Rna1p involved in mRNA processing and transport. Proc. Natl. Acad. Sci. USA 92, 1749–1753.PubMedCrossRefGoogle Scholar
  22. 22.
    Hopper, A. K., Traglia, H. M., and Dunst, R. W. (1990) The yeast RNA1 gene product necessary for RNA processing is located in the cytosol and apparently excluded from the nucleus. J. Cell Biol. 111, 309–321.PubMedCrossRefGoogle Scholar
  23. 23.
    Ribbeck, K., Lipowsky, G., Kent, H. M., Stewart, M., and Gorlich, D. (1998) NTF2 mediates nuclear import of Ran. EMBO J. 17, 6587–6598.PubMedCrossRefGoogle Scholar
  24. 24.
    Smith, A., Brownawell, A., and Macara, I. G. (1998) Nuclear import of RanGDP is mediated by NTF2. Curr. Biol. 8, 1403–1406.PubMedCrossRefGoogle Scholar
  25. 25.
    Gorlich, D., Vogel, F., Mills, A. D., Hartmann, E., and Laskey, R. A. (1995) Distinct functions for the two importin subunits in nuclear protein import. Nature 377, 246–248.PubMedCrossRefGoogle Scholar
  26. 26.
    Moroianu, J., Hijikata, M., Blobel, G., and Radu, A. (1995) Mammalian karyo-pherin alpha 1 beta and alpha 2 beta heterodimers: alpha 1 or alpha 2 subunit binds nuclear localization signal and beta subunit interacts with peptide repeat-containing nucleoporins. Proc. Natl. Acad. Sci. USA 92, 6532–6536.PubMedCrossRefGoogle Scholar
  27. 27.
    Jakel, S. and Gorlich, D. (1998) Importin β, transportin, RanBP5 and RanBP7 mediate nuclear import of ribosomal proteins in mammalian cells. EMBO J. 17, 4491–4502.PubMedCrossRefGoogle Scholar
  28. 28.
    Jakel, S., Albig, W., Kutay, U., Bischoff, F. R., Schwamborn, K., Doenecke, D., et al. (1999) The importin beta/importin 7 heterodimer is a functional nuclear import receptor for histone H1. EMBO J. 18 2411–2423.PubMedCrossRefGoogle Scholar
  29. 29.
    Izaurralde, E., Kutay, U., Vonkobbe, C., Mattaj, I. W., and Gorlich, D. (1997) The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus. EMBO J. 16, 6535–6547.PubMedCrossRefGoogle Scholar
  30. 30.
    Rexach, M. and Blobel, G. (1995) Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins. Cell 83, 683–692.PubMedCrossRefGoogle Scholar
  31. 31.
    Gorlich, D., Pante, N., Kutay, U., Aebi, U., and Bischoff, F. R. (1996) Identification of different roles for RanGDP and RanGTP in nuclear protein import. EMBO J. 15, 5584–5594.PubMedGoogle Scholar
  32. 32.
    Adam, S. A. and Gerace, L. (1991) Cytosolic proteins that specifically bind nuclear location signals are receptors for nuclear import. Cell 66, 837–847.PubMedCrossRefGoogle Scholar
  33. 33.
    Adam, S. A., Sterne-Marr, R., and Gerace, L. (1992) Nuclear protein import using digitonin-permeabilized cells. Methods Enzymol. 219, 97–110.PubMedCrossRefGoogle Scholar
  34. 34.
    Gorlich, D., Henklein, P., Laskey, R. A., and Hartmann, E. (1996) A 41 amino acid motif in importin-alpha confers binding to importin-beta and hence transit into the nucleus. EMBO J. 15, 1810–1817.PubMedGoogle Scholar
  35. 35.
    Kutay, U., Izaurralde, E., Bischoff, F. R., Mattaj, I. W., and Gorlich, D. (1997) Dominant-negative mutants of importin-beta block multiple pathways of import and export through the nuclear pore complex. EMBO J. 16, 1153–1163.PubMedCrossRefGoogle Scholar
  36. 36.
    Nemergut, M. E. and Macara, I. G. (2000) Nuclear import of the Ran exchange factor, RCC1, is mediated by at least two distinct mechanisms. J. Cell Biol. 149, 835–850.PubMedCrossRefGoogle Scholar
  37. 37.
    Michael, W. M., Eder, P. S., and Dreyfuss, G. (1997) The K nuclear shuttling domain: a novel signal for nuclear import and nuclear export In the hnRNP K protein. EMBO J. 16, 3587–3598.PubMedCrossRefGoogle Scholar
  38. 38.
    Yokoya, F., Imamoto, N., Tachibana, T., and Yoneda, Y. (1999) Beta-catenin can be transported into the nucleus in a Ran-unassisted manner. Mol. Biol. Cell 10, 1119–1131.PubMedGoogle Scholar
  39. 39.
    Holaska, J. M. and Paschal, B. M. (1998) A cytosolic activity distinct from Crm1 mediates nuclear export of protein kinase inhibitor in permeabilized cells. Proc. Natl. Acad. Sci. USA 95, 14,739–14,744.PubMedCrossRefGoogle Scholar
  40. 40.
    Kehlenbach, R. H., Dickmanns, A., and Gerace, L. (1998) Nucleocytoplasmic shuttling factors including Ran and Crm1 mediate nuclear export of NFAT in vitro. J. Cell Biol. 141, 863–874.PubMedCrossRefGoogle Scholar
  41. 41.
    Love, D. C., Sweitzer, T. D., and Hanover, J. A. (1998) Reconstitution of HIV-1 rev nuclear export: independent requirements for nuclear import and export. Proc. Natl. Acad. Sci. USA 95, 10, 608–10,613.Google Scholar
  42. 42.
    Englmeier, L., Olivo, J. C., and Mattaj, I. W. (1999) Receptor-mediated substrate translocation through the nuclear pore complex without nucleotide triphosphate hydrolysis. Curr. Biol. 9, 30–41.PubMedCrossRefGoogle Scholar
  43. 43.
    Plafker, K. and Macara, I. G. (2000) Facilitated nucleocytoplasmic shuttling of the Ran binding protein RanBP1. Mol. Cell. Biol. 20, 3510–3521.PubMedCrossRefGoogle Scholar
  44. 44.
    Paschal, B. M. (1998) Assay of nuclear protein import in permeabilized cells using flow cytometry, in Cell Biology: A Laboratory Handbook (Academic Press, San Diego, CA), pp. 305–313.Google Scholar
  45. 45.
    Newmeyer, D. D. and Wilson, K. L. (1991) Egg extracts for nuclear import and nuclear assembly reactions. Methods Cell Biol. 36, 607–634.PubMedCrossRefGoogle Scholar
  46. 46.
    Dingwall, C. and Palacios, I. (1998) In vitro systems for the reconstitution of snRNP and protein nuclear import. Methods Cell Biol. 53, 517–543.PubMedCrossRefGoogle Scholar
  47. 47.
    Lounsbury, K. M., Richards, S. A., Carey, K. L., and Macara, I. G. (1996) Mutations within the Ran/TC4 gtpase-effects on regulatory factor interactions and subcellular localization. J. Biol. Chem. 271, 32,834–32,841.PubMedCrossRefGoogle Scholar
  48. 48.
    Welch, K., Franke, J., Kohler, M., and Macara, I. G. (1999) RanBP3 contains an unusual nuclear localization signal that is imported preferentially by importin-alpha3. Mol. Cell. Biol. 19, 8400–8411.PubMedGoogle Scholar
  49. 49.
    Black, B. E., Levesque, L., Holaska, J. M., Wood, T. C., and Paschal, B. M. (1999) Identification of an NTF2-related factor that binds Ran-GTP and regulates nuclear protein export. Mol. Cell. Biol. 19, 8616–8624.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Amy M. Brownawell
    • 1
  • James M. Holaska
    • 2
  • Ian G. Macara
    • 1
  • Bryce M. Paschal
    • 3
  1. 1.Department of Pharmacology, Center for Cell SignalingUniversity of VirginiaCharlottesville
  2. 2.Department of MicrobiologyUniversity of VirginiaCharlottesville
  3. 3.Department of Biochemistry and Molecular Genetics, Center for Cell SignalingUniversity of VirginiaCharlottesville

Personalised recommendations