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The Nucleus pp 181-205 | Cite as

Reconstitution of Nuclear Import in Permeabilized Cells

  • Aurelia Cassany
  • Larry Gerace
Part of the Methods in Molecular Biology book series (MIMB, volume 464)

Abstract

The trafficking of protein and RNA cargoes between the cytoplasm and the nucleus of eukaryotic cells, which is a major pathway involved in cell regulation, is mediated by nuclear transport sequences in the cargoes and by shuttling transport factors. The latter include receptors (karyopherins) that recognize the cargoes and carry them across the nuclear pore complex (NPC), and the small GTPase Ran, which modulates karyopherin–cargo binding. Nuclear import can be studied in vitro using digitonin-permeabilized cells, which are depleted of shuttling transport factors. Nuclear import can be reconstituted in the permeabilized cells with exogenous cytosol or with purified recombinant transport factors, and can be quantified by light microscopy of fluorescently labeled cargoes or by immunofluorescence staining. Here we describe procedures for in vitro nuclear import in permeabilized mammalian cells, and for the preparation of recombinant transport factors (importin α, importin β, importin 7, transportin, Ran, NTF2) and other reagents commonly used in the assay. This assay provides means to characterize the molecular mechanisms of nuclear import and to study the import requirements of specific cargoes.

Keywords

Nuclear protein import Digitonin-permeabilized cells Recombinant protein expression Recombinant protein purification Shuttling nuclear transport factors Karyopherins Importins Ran 

Notes

Acknowledgments

The writing of this chapter was supported by the National Institutes of Health (NIH) grant AI55729 to LG. AC was supported by a fellowship from the French Foundation for Medical Research (FRM), SPE20041102385. We are grateful to Geza Ambrus-Aikelin for comments on the manuscript. We thank the postdocs of the Gerace lab for their protocols.

References

  1. 1.
    Fahrenkrog, B., Koser, J., and Aebi U. (2004) The nuclear pore complex: a jack of all trades? Trends Biochem. Sci. 29, 175-182PubMedCrossRefGoogle Scholar
  2. 2.
    Mosammaparast, N. and Pemberton, L.F. (2004) Karyopherins: from nuclear-transport mediators to nuclear-function regulators. Trends Cell Biol. 14, 547-556PubMedCrossRefGoogle Scholar
  3. 3.
    Macara, I.G. (2001) Transport into and out of the nucleus. Microbiol. Mol. Biol. Rev. 65, 570-594PubMedCrossRefGoogle Scholar
  4. 4.
    Pemberton, P.L. and Paschal B. (2005) Mechanisms of receptor-mediated nuclear import and nuclear export. Traffic 6, 187-198PubMedCrossRefGoogle 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, 807-816PubMedCrossRefGoogle Scholar
  6. 6.
    Chan, R.C. and Forbes, D.J. (2005) In vitro study of nuclear assembly and nuclear import using Xenopus egg extracts. In: Xenopus protocols (Liu, J.X., Humana Press, Totowa, NJ, pp. 289-300Google Scholar
  7. 7.
    Colbeau, A., Nachbaur, J., and Vignais, P.M. (1971) Enzymatic characterization and lipid composition of rat liver subcellular membrane. Biochim. Biophys. Acta 249, 462-492PubMedCrossRefGoogle Scholar
  8. 8.
    Melchior F. (1998) Nuclear protein import in a permeabilized cell assay, in Protein targeting protocols (Clegg, R.A., Humana Press, Totowa, NJ, pp. 265-273CrossRefGoogle Scholar
  9. 9.
    Kehlenbach, R.H. and Gerace L. (2002) Analysis of nuclear protein import and export in vitro using fluorescent cargoes, in GTPase protocols (Manser, E. and Leung, T., ed), Humana Press, Totowa, NJ, pp. 231-245Google Scholar
  10. 10.
    Paraskeva, E., Izaurralde, E., Bischoff, F.R., Huber, J., Kutay, U., Hartmenn, E., Luhrmann, R., and Gorlich, D. (1999) CRM1-mediated recycling of snurportin 1 to the cytoplasm. J. Cell Biol. 145, 255-264PubMedCrossRefGoogle Scholar
  11. 11.
    Kalderon, D., Roberts, B. L., Richardson, W. D., and Smith, A.E. (1984) A short amino acid sequence able to specify nuclear location. Cell 39, 499-509PubMedCrossRefGoogle Scholar
  12. 12.
    Dingwall, C. and Laskey, R.A. (1991) Nuclear targeting sequences-a consensus? Trends Biochem. Sci. 16, 478-481Google Scholar
  13. 13.
    Lyman, S.K., Guan, T., Bednenko, J., Wodrich, H., and Gerace, L. (2002) Influence of cargo size on Ran and energy requirements for nuclear protein import. J. Cell Biol. 159, 55-67PubMedCrossRefGoogle Scholar
  14. 14.
    Weis, K., Mattaj, I.W., and Lamond, A.I. (1995) Identification of hSRP1 alpha as a functional receptor for nuclear localization sequences. Science 268, 1049-1053PubMedCrossRefGoogle Scholar
  15. 15.
    Jakel, S., Albig, W., Kutay, U., Bischoff, F.R., Schwamborn, K., Doenecke, D., and Gorlich, D. (1999) The importin beta/importin 7 heterodimer is a functional nuclear import receptor for histone H1. EMBO J. 18, 2411-2423PubMedCrossRefGoogle Scholar
  16. 16.
    Pollard, V.W., Michael, W.M., Nakielny, S., Siomi, M.C., Wang, F., and Dreyfuss G. (1996) A novel receptor-mediated nuclear import pathway. Cell 86, 985-994PubMedCrossRefGoogle Scholar
  17. 17.
    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, 1649-1659PubMedCrossRefGoogle Scholar
  18. 18.
    Paschal, B. 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, 925-937PubMedCrossRefGoogle Scholar
  19. 19.
    Paine, P.L., Moore, L.C., and Horowitz S.B. (1975) Nuclear envelope permeability. Nature 254, 109-114PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Aurelia Cassany
    • 1
  • Larry Gerace
    • 1
  1. 1.Department of Cell and Molecular BiologyThe Scripps Research InstituteLa JollaUSA

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