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Figure 6.

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Kap121 is involved in the Rei1-dependent nuclear import of pre-60S factors. (A) KAP121 is a high copy number suppressor of rei1Δ. A rei1Δ strain was transformed with high copy number plasmids carrying distinct DNA inserts (REI1, REH1, KAP121, or no insert) and plated in 10−1 dilution series on synthetic medium without uracil at 23 or 30°C for 3 and 2 d, respectively. Generation times at 23°C were calculated from growth curves performed in liquid synthetic medium without uracil over a period of 30 h. The confidence intervals were calculated for a probability of 95%. (B) Overexpression of KAP121 is sufficient to overcome the import defect in rei1Δ strains. rei1Δ strains expressing ARX1-GFP, ALB1-GFP, or TIF6-GFP were transformed with vectors overexpressing REI1, KAP121, or nothing. The localization of Arx1-, Alb1-, or Tif6-GFP was detected after growth for 8 h in minimal medium without uracil at 23°C. (C) Kap121 copurifies with Arx1-TAP and Alb1-TAP. Tif6-, Rei1-, Arx1-, and Alb1-TAP–associated complexes were purified by TAP. The presence of Kap121 or Rlp24 in the eluates was assessed by Western blotting with specific antibodies against each of these proteins. The Ponceau red staining is shown on the bottom. (D) In the absence of Kap121, Arx1-GFP is blocked in the cytoplasm. KAP121 was placed under the control of the PGAL1 promoter in strains expressing ARX1-GFP or ALB1-GFP. The localization of Arx1-GFP or Alb1-GFP was detected in this mutant or in the corresponding wild-type strain after growth in glucose- containing medium for 16 h.

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