Figure 12.

Nuclear import capacity is decreased in temperature-arrested nup57-E17 cells. (A) To assay nuclear import, wt (SWY519) and nup57-E17 (SWY1587) cells transformed with a plasmid expressing NLS-β-galactosidase under GAL10 control (pNLS-E1) were analyzed. Cells grown in 2% raffinose media at 37°C for 2.5 h were induced for NLS-β-galactosidase expression by addition of 2% galactose, and growth at 37°C was continued for 3.5 h. Cells were fixed and processed for indirect immunofluorescence microscopy. Localization of the reporter was determined using mAbs against β-galactosidase as described in MATERIALS AND METHODS. (B) Export of poly(A)⁺ RNA was not strongly inhibited. wt (SWY809), gle1–4 (SWY1186), and nup57-E17 (SWY1586) cells were grown at 23°C, shifted to 37°C for 4 h, and processed for in situ hybridization with a digoxigenin-oligo(dT)₃₀ probe. Rhodamine-conjugated anti-digoxigenin antibodies were used to localize probe binding. Exposure and printing times are identical for wt and mutant cells in a given experiment. Coincident DAPI staining is shown. Bar, 5 μm (A); 15 μm (B).