Figure 2.

A permeabilized cell assay that measures nuclear export mediated by PKI and cytosolic factors. (A) The nuclei of digitonin-permeabilized HeLa cells were loaded with FITC-STV-NLS, washed with buffer, and further incubated with bPKI in the presence of buffer alone (Top), HeLa cell cytosol (3 mg/ml; Middle), or heat-treated HeLa cell cytosol (3 mg/ml; Bottom). The cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) to reveal the position of nuclei within each field. Middle shows that the addition of cytosol results in a significant loss of FITC-STV-NLS from HeLa cell nuclei, an effect that can be ascribed to heat-labile cytosolic proteins. (B) Permeabilized cells in the nuclear export assay are competent for NLS-mediated protein import. Allophycocyanin (APC) conjugated with SV40 NLS peptides (40), when added to export reactions, is imported into nuclei. This result shows that under the conditions of our assay, nuclei remain intact and NPCs are functional.