(A) eS26, but not Tsr2:eS26 or Tsr2, interacts with Kap123, Kap104 and Pse1. Recombinant, GST-Kap123, GST-Kap104, GST-Pse1 and GST alone were immobilized on Glutathione Sepharose and incubated with purified 3.4 µM Tsr2, 4 µM Tsr2:eS26, or E. coli lysate containing ∼4 µM eS26FLAG in PBSKMT combined with competing E. coli lysates for 1 hr at 4°C. After washing with PBSKMT, bound proteins were eluted in SDS sample buffer and separated by SDS-PAGE. Proteins were visualized by Coomassie Blue staining or Western analyses using indicated antibodies. L = input. GST-tagged importins are indicated with asterisks. (B) Nuclear uptake of GFP-eS26 is impaired in kap123Δ and kap104Δ mutants. Strains expressing GFP-eS26 were grown in synthetic media at 25°C (ts-mutants: pse1-1 and kap104Δ) or 30°C to mid-log phase. Ts-mutant strains were then shifted to 37°C for 4 hr and localization of GFP-eS26 was analyzed by fluorescence microscopy. Percentage of cells displaying cytoplasmic mislocalization of the GFP-eS26 fusion is indicated. Scale bar = 5 µm. (C) Tsr2-3xGFP is targeted to the nucleus by Kap123. Importin mutant strains expressing Tsr2-3xGFP were grown in synthetic media at 25°C (ts-mutants: pse1-1 and kap104Δ) or 30°C to mid-log phase. Pse1-1 and kap104Δ cells were then shifted to 37°C for 4 hr. PGAL1-RPS26Arps26bΔ cells containing Tsr2-3xGFP were grown for 15 hr in glucose containing media. Localization of Tsr2-3xGFP was analyzed by fluorescence microscopy. Scale bar = 5 µm. (D) RanGTP (His6-Gsp1Q71L-GTP) does not efficiently release eS26 from Kap123 and Pse1. GST-importin:eS26FLAG complexes immobilized on Glutathione Sepharose were incubated with either buffer alone or with 1.5 µM RanGTP or 3 nM 3′-end of 18S rRNA for 1 hr at 4°C. Washing, elution, and visualization were performed as in (A). GST-tagged importins are indicated with asterisks. (E) Tsr2 efficiently dissociates the Kap123:eS26FLAG complex. The GST-Kap123: eS26FLAG complex immobilized on Glutathione Sepharose was incubated with either buffer alone or with 1.5 µM or 375 nM RanGTP or 1.5 µM or 375 nM Tsr2. Samples were withdrawn at the indicated time points. Washing, elution, and visualization were performed as in (A). GST-tagged Kap123 is indicated with an asterisk. (F) eS26 stably associates with Tsr2 after its release from Kap123. Left panel indicates the experimental setup as flowchart. Immobilized GST-Kap123:eS26FLAG complex was incubated with 1.5 µM His6-Tsr2 or buffer alone. As shown in the flowchart, the supernatant was incubated with Ni-NTA Agarose for 1 hr at 4°C (IP-Sup). Washing, elution, and visualization were performed as in (A). GST-tagged Kap123 is indicated with an asterisk.