Figure 8.

NLS-mediated nuclear protein uptake and poly(A)⁺ RNA export in thermosensitive nup49-313 cells. (A) The thermosensitive nup49-313 mutant was transformed with pGAL-NLS-GFP-lacZ (classical NLS of SV-40 large T antigen) and transformants were selected on SDC (−ura) plates. Transformants were grown in selective synthetic raffinose (−ura) medium at 20°C. One-half of the culture was left at 20°C (right panel), the other half was shifted for 11 h to 37°C (left panel). Cells from both cultures were collected by centrifugation, transfered into in SGal (−ura) medium and incubated for 5 h at 20°C to induce pGAL-NLS-GFP-lacZ expression. Cells were finally inspected in the fluorescence microscope. Note that at both temperatures the nuclear import reporter accumulates in the nucleus. (B) Nuclear export of poly(A)⁺ RNA in nup49-313/L25-GFP and L25-GFP cells. Cells were grown at the indicated temperatures (14 h at 33°C; 14 h at 33°C, and 4 h at 20°C; 18 h at 20°C) before they were fixed and processed for in situ hybridization using a fluorescently labeled oligo dT-probe.