Phosphorylated Stu2 ^S603 and Cdc28 activity are required for Stu2 nuclear import. (A) Exponentially growing stu2-AID cdc20-AID NUP2-mKate cells ectopically expressing GFP-GST fused constructs (GFP-GST, M2390; Stu2^592–607-GFP-GST, M2392) were treated with 500 µM auxin for 2 h to arrest cells in metaphase. Cells were fixed, and the Nup2-mKate and GFP signals were imaged. Left: Schematic of the constructs used. Right: Representative images of DIC, Nup2-mKate, and GFP signals for each strain with an outlined nucleus. Scale bar is 1 µm. (B) Exponentially growing stu2-AID cells ectopically expressing GFP-GST fused constructs (GFP-GST, M2441; NLS^SV40-GFP-GST, M2442; Stu2^592–607-GFP-GST, M2443; or Stu2^{592–607(KR/AA)}-GFP-GST, M2439) as well as NUP2-mKate were treated with α-factor for 2.5 h to arrest cells in G1 and then auxin for 30 min to degrade Stu2-AID. And exponentially growing stu2-AID cdc20-AID cells ectopically expressing GFP-GST fused constructs (GFP-GST, M2390; NLS^SV40-GFP-GST, M2391; Stu2^592–607-GFP-GST, M2392; or Stu2^{592–607(KR/AA)}-GFP-GST, M2437) as well as NUP2-mKate were treated with auxin for 2 h to degrade Stu2-AID and Cdc20-AID to arrest cells in metaphase. Cells were fixed, and Nup2-mKATE and -GFP-GST construct signals were imaged. The ratios of nuclear to cytoplasmic GFP intensities were quantified. Each data point represents this ratio for a single cell. Mean and SD are shown. n = 107–139 cells; P values were determined using two-tailed unpaired t tests. (C) Exponentially growing stu2-AID cdc20-AID cells ectopically expressing a GFP-GST fusion construct (Stu2^592–607, M2392; Stu2^{592–607(S603A)}, M2438; and Stu2^{592–607(S603E)}, M2726) were treated with auxin for 2 h to degrade Stu2-AID and Cdc20-AID to arrest cells in metaphase. Cells were fixed, and Nup2-mKATE and GFP signals were imaged. The ratios of nuclear to cytoplasmic GFP intensities were quantified. Each data point represents this ratio for a single cell. Bars represent median. Error bars are 95% confidence interval. n = 104–124 cells; P values were determined using a two-tailed unpaired. (D) Exponentially growing stu2-AID cells ectopically expressing full-length STU2-GFP alleles (STU2^WT-GFP, M2298 or stu2^S603A-GFP, M2351) were treated with α-factor for 2.5 h to arrest cells in G1 and then with auxin for 30 min to degrade Stu2-AID, or cells as above but with cdc20-AID (STU2^WT-GFP, M2208; or stu2^S603A-GFP, M2217) were treated with auxin for 2 h to degrade Stu2-AID and Cdc20-AID to arrest cells in metaphase. Cells were fixed, and Nup2-mKATE and Stu2-GFP signals were imaged. The ratios of nuclear to cytoplasmic GFP intensities were quantified. Each data point represents this ratio for a single cell. The median and the 95% confidence interval are shown. n = 100–165 cells; P values were determined using a two-tailed unpaired t test. (E) Exponentially growing stu2-AID Nup2-mKate cells with ectopic STU2-GFP and CDC28^WT (M5762) or cdc28-as1 (M5770) were treated with nocodazole for 2.5 h to arrest in mitosis. Auxin and 1NA-PP1 were added 30 min prior to harvesting; cells were fixed, and Nup2-mKate and GFP signals were imaged. The ratios of nuclear to cytoplasmic GFP intensities were quantified. Each data point represents this ratio for a single cell. Bars represent the mean of n = 100 individual measurements. Error bars are SEM. P values were determined using a two-tailed unpaired t test.