Abstract
The high concentration of cholesterol in the plasma membrane relative to the endomembranes of eukaryotic cells allows the selective permeabilization of the plasma membrane with the glycoside digitonin leaving the intracellular membrane bound organelles intact. In this chapter, we describe the basic method to use digitonin permeabilized cells to reconstitute the transport of proteins containing nuclear localization signals into the nucleus. The assay requires only a target cell line that can be permeabilized with digitonin, a source of soluble transport factors, typically provided by the cytosol fraction of cultured cells, and a cargo protein of interest. No other specialized equipment is required other than a fluorescence microscope. The assay can be used to identify transport factors required to transport specific proteins, to study the regulation of protein transport, or to study nuclear protein transport under different conditions.
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References
Wente SR, Rout MP (2010) The nuclear pore complex and nuclear transport. Cold Spring Harb Perspect Biol 2:a000562
Kimura M, Imamoto N (2014) Biological significance of the importin-beta family-dependent nucleocytoplasmic transport pathways. Traffic 15:727–748
Marfori M, Mynott A, Ellis JJ, Mehdi AM, Saunders NF, Curmi PM, Forwood JK, Boden M, Kobe B (2011) Molecular basis for specificity of nuclear import and prediction of nuclear localization. Biochim Biophys Acta 1813:1562–1577
Adam SA, Marr RS, Gerace L (1990) Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J Cell Biol 111:807–816
Adam EJ, Adam SA (1994) Identification of cytosolic factors required for nuclear location sequence-mediated binding to the nuclear envelope. J Cell Biol 125:547–555
Chi NC, Adam EJ, Adam SA (1995) Sequence and characterization of cytoplasmic nuclear protein import factor p97. J Cell Biol 130:265–274
Gorlich D, Kostka S, Kraft R, Dingwall C, Laskey RA, Hartmann E, Prehn S (1995) Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope. Curr Biol 5:383–392
Imamoto N, Shimamoto T, Takao T, Tachibana T, Kose S, Matsubae M, Sekimoto T, Shimonishi Y, Yoneda Y (1995) In vivo evidence for involvement of a 58 kDa component of nuclear pore-targeting complex in nuclear protein import. EMBO J 14:3617–3626
Moore MS, Blobel G (1993) The GTP-binding protein Ran/TC4 is required for protein import into the nucleus. Nature 365:661–663
Moore MS, Blobel G (1994) Purification of a Ran-interacting protein that is required for protein import into the nucleus. Proc Natl Acad Sci U S A 91:10212–10216
Paschal BM, 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
Hu W, Jans DA (1999) Efficiency of importin alpha/beta-mediated nuclear localization sequence recognition and nuclear import. Differential role of NTF2. J Biol Chem 274:15820–15827
Kehlenbach RH, Dickmanns A, Kehlenbach A, Guan T, Gerace L (1999) A role for RanBP1 in the release of CRM1 from the nuclear pore complex in a terminal step of nuclear export. J Cell Biol 145:645–657
Kohler M, Speck C, Christiansen M, Bischoff FR, Prehn S, Haller H, Gorlich D, Hartmann E (1999) Evidence for distinct substrate specificities of importin alpha family members in nuclear protein import. Mol Cell Biol 19:7782–7791
Smith A, Brownawell A, Macara IG (1998) Nuclear import of Ran is mediated by the transport factor NTF2. Curr Biol 8:1403–1406
Furuta M, Kose S, Kehlenbach RH, Imamoto N (2014) Analysis of nucleocytoplasmic transport in digitonin-permeabilized cells under different cellular conditions. Methods Cell Biol 122:331–352
Tu LC, Musser SM (2011) Single molecule studies of nucleocytoplasmic transport. Biochim Biophys Acta 1813:1607–1618
Kimura M, Kose S, Okumura N, Imai K, Furuta M, Sakiyama N, Tomii K, Horton P, Takao T, Imamoto N (2013) Identification of cargo proteins specific for the nucleocytoplasmic transport carrier transportin by combination of an in vitro transport system and stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics. Mol Cell Proteomics 12:145–157
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Adam, S.A. (2016). Nuclear Protein Transport in Digitonin Permeabilized Cells. In: Shackleton, S., Collas, P., Schirmer, E. (eds) The Nuclear Envelope. Methods in Molecular Biology, vol 1411. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3530-7_29
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DOI: https://doi.org/10.1007/978-1-4939-3530-7_29
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-3530-7
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