Figure 2. Rkm1 accompanies the transport... | Rockefeller University Press

Figure 2.

$Rkm1 accompanies the transport of Rpl23 to the nucleus. (A) The localization of Rkm1-GFP was visualized in wild-type and GAL::RPL23. Overnight cells were subcultured in a medium containing 2% galactose for 2 h. Cultures were kept in the galactose, or 2% glucose was added for another 4 h before examination with fluorescence microscopy. (B) GST-Kap121 or GST-Kap123 was incubated with uL14, Rkm1, or both at 4°C for 1 h. After three times of wash, the proteins were eluted with 1X SDS sample buffer and analyzed by Coomassie blue staining and western blotting. (C) To visualize the localization of Rkm1-GFP, wild-type and kap121ts at log phase were shifted to 37°C for 2 h, and wild-type and kap123Δ were cultured at 30°C. (D) Nmd3-GFP and Rkm1-GFP localizations were monitored in wild-type and crm1T539C incubated with LMB (0.1 μg/ml) for 30 min. Hoechst was used to stain the nucleus. The intensity ratios between nucleus and cytoplasm were calculated in 15 cells and analyzed with Student’s t test (***P < 0.001). (E) GST-Kap121/Kap123 in complex with Rkm1 or Rkm1/uL14 was immobilized on the glutathione beads. Bcp1 was added and incubated for another hour at 4°C. The remaining amounts of Rkm1 and uL14 on the beads were analyzed by Western blotting. (F) Cell extracts from Rkm1-TAP were fractioned through 7–47% sucrose gradients. Each fraction was precipitated with TCA and analyzed by anti-TAP, anti-Bcp1, anti-eL8, and anti-uL14 antibodies. The corresponding sedimentation peaks of the ribosomal subunits were indicated above. Source data are available for this figure: SourceData F2.$

Rkm1 accompanies the transport of Rpl23 to the nucleus. (A) The localization of Rkm1-GFP was visualized in wild-type and GAL::RPL23. Overnight cells were subcultured in a medium containing 2% galactose for 2 h. Cultures were kept in the galactose, or 2% glucose was added for another 4 h before examination with fluorescence microscopy. (B) GST-Kap121 or GST-Kap123 was incubated with uL14, Rkm1, or both at 4°C for 1 h. After three times of wash, the proteins were eluted with 1X SDS sample buffer and analyzed by Coomassie blue staining and western blotting. (C) To visualize the localization of Rkm1-GFP, wild-type and kap121ts at log phase were shifted to 37°C for 2 h, and wild-type and kap123Δ were cultured at 30°C. (D) Nmd3-GFP and Rkm1-GFP localizations were monitored in wild-type and crm1T539C incubated with LMB (0.1 μg/ml) for 30 min. Hoechst was used to stain the nucleus. The intensity ratios between nucleus and cytoplasm were calculated in 15 cells and analyzed with Student’s t test (***P < 0.001). (E) GST-Kap121/Kap123 in complex with Rkm1 or Rkm1/uL14 was immobilized on the glutathione beads. Bcp1 was added and incubated for another hour at 4°C. The remaining amounts of Rkm1 and uL14 on the beads were analyzed by Western blotting. (F) Cell extracts from Rkm1-TAP were fractioned through 7–47% sucrose gradients. Each fraction was precipitated with TCA and analyzed by anti-TAP, anti-Bcp1, anti-eL8, and anti-uL14 antibodies. The corresponding sedimentation peaks of the ribosomal subunits were indicated above. Source data are available for this figure: SourceData F2.