Nuclear Protein Import in a Permeabilized Cell Assay (original) (raw)

Abstract

An in vitro nuclear import assay, using digitonin-permeabilized cells, grown on cover slips, and supplemented with exogenous cytosol and fluorescent-transport ligand, was developed in our laboratory several years ago (1). For a general introduction into this technique and references for alternative assays developed by other laboratories, see refs. 2 and 3. Although the original assay faithfully reproduces transport, quantitation by methods such as densitometry on photographic negatives or ACAS (anchored cell analysis and sorting) interactive laser cytometry is a rather time-consuming process. Another disadvantage of the original assay involving attached cells is that the number of cells per assay cannot easily be controlled. Both problems are circumvented by using a modification of the import assay recently developed by Paschal and Gerace (4), and described in detail below. Here, suspension cells are used as the source for permeable cells, and relative transport rates are determined rapidly by flow cytometry (an ELISA-based quantitative assay that allows determination of molar transport rates is described in ref. 5).

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References

  1. Adam, S. A., Sterne-Marr, R. E., and Gerace, L. (1990) Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J. Cell Biol. 111, 807–816.
    Article PubMed CAS Google Scholar
  2. Adam, S. A., Sterne-Marr R. E., and Gerace, L. (1992) Nuclear protein import using digitonin-permeabilized cells, in Methods in Enzymology, vol. 219, Reconstitution of Intracellular Transport (Rothman J. E., ed.), Academic, San Diego, CA, pp. 97–110.
    Chapter Google Scholar
  3. Marshallsay, C. (1995) In vitro nucleo-cytoplasmic transport. Mol. Biol. Reports 20, 163–71.
    Article CAS Google Scholar
  4. 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.
    Article PubMed CAS Google Scholar
  5. Melchior, F., Sweet, D. J., and Gerace, L. (1995) Analysis of Ran/TC4 function in nuclear protein import, in Methods Enzymology, vol. 257, Academic, San Diego, CA, pp. 279–291.
    Google Scholar
  6. 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, 499–509.
    Article PubMed CAS Google Scholar
  7. Colbeau, A., Nachbaur, J., and Vignais, P. M. (1971) Biochim. Biophys. Acta 249, 462.
    Article PubMed CAS Google Scholar
  8. Görlich, D., Prehn, S., Laskey, R. A., and Hartmann, E. (1994) Isolation of a protein that is essential for the first step of nuclear protein import. Cell 79, 767–778.
    Article PubMed Google Scholar
  9. 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.
    Article PubMed CAS Google Scholar
  10. Forbes, D. J. (1992) Structure and function of the nuclear pore complex. Annu. Rev. Cell Biol. 8, 495–527.
    Article PubMed CAS Google Scholar
  11. Melchior, F., Guan, T., Yokoyama, N., Nishimoto, T., and Gerace, L. (1995) GTP hydrolysis by Ran occurs at the nuclear pore complex in an early step of protein import. J. Cell Biol. 131, 571–581.
    Article PubMed CAS Google Scholar

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Author information

Authors and Affiliations

  1. Department of Cell Biology, Scripps Research Institute, La Jolla, UK
    Frauke Melchior

Authors

  1. Frauke Melchior
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Editors and Affiliations

  1. Hannah Research Institute, Scotland, UK
    Roger A. Clegg

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© 1998 Humana Press Inc.

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Melchior, F. (1998). Nuclear Protein Import in a Permeabilized Cell Assay. In: Clegg, R.A. (eds) Protein Targeting Protocols. Methods in Molecular Biology™, vol 88. Humana Press. https://doi.org/10.1385/0-89603-487-9:265

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