Figure 9.

Perturbations in the Ran cycle can return Nup2p–GFP to the NPC in strains lacking Nup60p. (Ai) After initial image acquisition (START), the indicated strains were metabolically poisoned by azide/deoxyglucose treatment for 45 min (POISON). No change in the localization of Nup2p–GFP was observed. In contrast, Nup2p–GFP accumulated at the nuclear rim in strains lacking Nup60p after the 45-min poisoning treatment. The localization of Kap60p–GFP similarly accumulated at the nuclear rim under these conditions. Furthermore, a 10-min recovery period in glucose-containing media resulted in the return of Nup2p–GFP and Kap60p–GFP to their respective steady-state locales (RECOVERY). (Aii) Inactivation of the Ran-GTP exchange factor, Prp20p, enhances binding of Nup2p to alternative sites within the NPC. Nup2p–GFP was expressed in Δnup60,prp20-7 cells, grown at 23°C (START), shifted to 37°C for 90 min (TEMP. SHIFT), and then recovered at room temperature for 60 min (RECOVERY). Kap60p–GFP accumulated at the nuclear rim at the nonpermissive temperature, indicating a block in nuclear transport. Neither Nup49p–GFP nor Nup2p–GFP in otherwise wild-type cells appeared affected by this treatment; however, Nup2p–GFP signal returned to the nuclear rim and accumulated in the cytoplasm in a strain lacking Nup60p. This effect was reversible as growth at the permissive temperature restored the steady-state nuclear localization of Nup2p–GFP in the Δnup60 strain. (B) The alternative NPC docking site of Nup2p is likely at the cytoplasmic face of the NPC. Nup2p–pA in cells lacking Nup60p was localized by IEM of purified nuclei. We could find no signal was detected on the nuclear face of the NPC; however, there remained a pool of Nup2p–pA that remained associated with the NPC on the cytoplasmic face.