Reconstitution of Nuclear Import in Permeabilized Cells
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.
KeywordsNuclear protein import Digitonin-permeabilized cells Recombinant protein expression Recombinant protein purification Shuttling nuclear transport factors Karyopherins Importins Ran
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.
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