Reconstitution of nuclear import in permeabilized cells

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 464; p. 181
• Main Authors: Cassany, Aurelia, Gerace, Larry
• Format: Journal Article
• Language: English
• Published: United States 01.01.2009
• Subjects: Active Transport, Cell Nucleus - genetics; Active Transport, Cell Nucleus - physiology; HeLa Cells; Humans; Karyopherins - genetics; Karyopherins - metabolism; Protein Transport - genetics; Protein Transport - physiology; ran GTP-Binding Protein - genetics; ran GTP-Binding Protein - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism
• ISSN: 1940-6029
• DOI: 10.1007/978-1-60327-461-6_11

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 alpha, importin beta, 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.