Figure 3. Kap104 is specifically required for β-catenin nuclear accumulation in Saccharomyces cerevisiae.

(A) Schematic of the Anchor-Away assay mediated by the rapamycin-induced dimerization of nuclear transport receptor (NTR)-FKBP-rapamycin binding (FRBP and Pma1-FKBP12). Pma1 is a plasma membrane ATPase. (B) Deconvolved fluorescence images of cells with indicated FRB fusions expressing Xenopus β-catenin (665-782)-GFP treated with DMSO (vehicle) or rapamycin for 15 min. Heh2-mCherry was used as a nuclear envelope marker. White arrows indicate the nucleus. Scale bar is 5 µm. (C) Plot showing the ratio of mean nuclear to cytosolic fluorescence intensity of Xenopus β-catenin (665-782)-GFP in the 10 NTR-FRB strains treated with DMSO or rapamycin from 30 to 40 cells from three independent replicates. Red bar indicates the mean value with the SD. Experiments were performed three times. p-Values are from unpaired two-tailed t-test where ns is p>0.05 and ****p<0.0001. The data is uploaded as Figure 3—source data 1.

Figure 3—figure supplement 1. Sub-cellular localization of Xenopus β-catenin (665-782)-GFP in Anchor-Away strains in Saccharomyces cerevisiae.

Representative deconvolved fluorescence image of xβ-catenin (665-782)-GFP treated with DMSO (carrier) or rapamycin in the indicated nuclear transport receptor (NTR)-FKBP-rapamycin binding (FRB) strain. Heh2-mCherry was used as a nuclear membrane marker. Scale bar is 5 µm.

Figure 3—figure supplement 2. Anchor-Away cloning strategy in Saccharomyces cerevisiae.

(A) Schematic diagram of FKBP-rapamycin binding (FRB) tagging to individual endogenous nuclear transport receptors (NTRs) by homologous recombination. (B) Screening of NTR-FRB strains by colony PCR. Primers are listed in Supplementary file 3. (C) Positive NTR-FRB strains from the colony PCR in (B) were further tested for cell growth as some NTRs are essential for survival. No FRB and DMSO were used a negative controls.