(A) slx9-1 genetically interacts with factors involved in 40S pre-ribosome export. slx9-1 is synthetically lethal with mex67∆loop, mtr2∆loop116-137, or yrb2∆ and strongly synthetically enhanced with rrp12-GFP. Strains containing the indicated WT and mutant alleles were spotted in 10-fold serial dilutions on 5-FOA-SD or SD and grown at 20–30°C for 3–6 days. (B) Slx9 shuttles between the nucleus and the cytoplasm. Cells expressing Enp1-GFP, Gar1-GFP, or Slx9-GFP were mated with kar1-1 cells expressing Nup82-mCherry. The resulting heterokaryons were analyzed by fluorescence microscopy. Scale bar = 5 µm. (C) Slx9 directly binds to RanGTP. GST-Slx9 or GST-Ssb1C was immobilized on GSH-Sepharose before incubating with either buffer alone or buffer containing 2 µM His6-RanQLGTP, 50 nM Crm1-His6 or 2 µM His6-RanQLGTP, and 50 nM Crm1-His6. After washing, bound proteins were eluted in LDS sample buffer, separated by SDS-PAGE and visualized by Coomassie staining or Western blotting using the indicated antibodies. L = input. (D) Slx9 specifically interacts with the GTP-bound form of Ran. GST-Slx9, GST-Yrb1, or GST-Ntf2 was immobilized on GSH-Sepharose and incubated with buffer alone or 2 µM His6-Ran loaded with GDP or GTP. Analysis of the eluted proteins was carried out as described in (C). L = input. (E) Slx9-1 binding to RanGTP is impaired. Top: GST-Slx9 or GST-Slx9-1 immobilized on GSH-Sepharose was incubated with buffer alone or 2 µM His6-RanQLGTP. Analysis of the eluted proteins was carried out as described in (C). L = input. Bottom: bar graph depicts the bound His6-RanQLGTP Western blot signal normalized to GST-Slx9 and GST-Slx9-1 levels, respectively. Four independent experiments were performed and Western blots were quantified by software ImageJ (Version 1.44o). Error bars (S.D.) are indicated.