(A) Δcdc55 mutant cells exit mitosis in the presence of nocodazole. Wild-type (Y300) and Δcdc55 mutant (YYW28) cells in midlog phase were arrested at G1 phase with α-factor and then released into 30°C YPD medium containing 20 μg/ml nocodazole after sonication. Cells were harvested at 30-min intervals for budding index. The top panel shows the percentile of cells with extrabud and the bottom panel shows cells after 240-min incubation in the presence of nocodazole. The arrow indicates the rebudded Δcdc55 mutant cell in the presence of nocodazole. (B) Top, strain 409-2-6-3 (Δcdc55 URA3-tetO-112 LEU2-tetR-GFP) was arrested at G1 phase and then released into YPD medium containing 20 μg/ml nocodazole at 30°C. The picture was taken after 240-min incubation. The spots indicate GFP marked chromosome V. Bottom, the frequency of reduplicated chromosome in wild-type and Δcdc55 mutants. JBY583 (URA3-tetO-112 LEU2-tetR-GFP) and 409-2-6-3 (Δcdc55 URA3-teto-112 LEU2-tetR-GFP) were arrested at G1 phase and then released into YPD medium containing 20 μg/ml nocodazole. Cells were collected every 30 min and fixed for examination of the GFP signal. (C) The rebudding phenotype of Δcdc55 mutants is independent of Cdc28 Y19 phosphorylation. Top, the budding index of cells with indicated genotype in YPD medium at 30°C. Bottom, the percentage of rebudded cells in the presence of 20 μg/ml nocodazole. Strains (Y300, YYW28, 397-2-2, 412-13-3) were treated as described in Figure 2A.

Cdc14 is released from the nucleolus in Δcdc55 mutants in the presence of nocodazole. (A) A1411 (CDC14-HA) and 173-1-3 (Δcdc55 CDC14-HA) cells were arrested at G1 phase with α-factor and then released into YPD medium. Cells were collected every 20 min and fixed for Cdc14-HA staining. The budding index and the kinetics of Cdc14 release from the nucleolus are shown in the top and bottom panels, respectively. (B) The same strains were arrested at G1 phase and then released into YPD medium containing 20 μg/ml nocodazole at 30°C. The budding index and Cdc14 localization are shown. (C) The nuclear structure and Cdc14 localization of the 180 min samples in B are shown (top). The bottom of panel C shows the Net1 localization. G1-arrested NET1-myc and Δcdc55 NET1-myc cells were released into nocodazole medium. Cells were collected for Net1-myc staining. The nuclear structure and Net1 localization after 180-min incubation are shown. (D) Δcdc55 and Δpph21 Δpph22 mutants fail to prevent Cdc14 from releasing in the presence of nocodazole. Midlog phase of A1411, 173-1-3, and 472-7-2 were incubated in the presence of 20 μg/ml nocodazole at 30°C. Cells were collected after 3-h incubation for Cdc14 staining. (E) Hof1 phosphorylation is more pronounced in nocodazole-treated Δcdc55 mutants. G1-arrested 514-3-3 (HOF1-HA) and 514-2-3 (Δcdc55 HOF1-HA) cells were released into YPD medium containing 20 μg/ml nocodazole at 30°C. Cells were collected every 30 min for protein preparation. Hof1-HA protein was examined by Western blot analysis after SDS-PAGE.

Mitotic exit in Δcdc55 mutants depends on MEN pathway. (A) The localization of Cdc14 in Δslk19 and Δcdc55 Δslk19 mutants during unperturbed cell cycle progression. G1-arrested Δ464-3-2 and 450-3-1 were released into cell cycle at 30°C and 200 mM hydroxyurea was added into the medium at 80 min to block the next S phase. Cells were collected at 30 min interval for Cdc14 staining. (B) Left, the budding morphology of cells with indicated genotypes. Strains with indicated genotypes were arrested at G1 and released into YPD medium containing 20 μg/ml nocodazole at 30°C for the determination of budding index. Right, the localization of Cdc14 in the presence of nocodazole. Strains with indicated genotype were released into nocodazole medium after G1 arrest. Cells were collected every 30 min and subjected to immunofluorescence staining for Cdc14-HA. The percentage of cells with nucleolar localized Cdc14 was determined over time. (C) cdc15-2 CDC14-HA and cdc15-2Δ cdc55 CDC14-HA strains were arrested at G1 phase with α-factor and then released into YPD medium at 37°C. Budding index and the localization of Cdc14 were determined over time.

(A) Overexpression of PP2A components is lethal to mutants defective in mitotic exit. Saturated cell cultures were 10-time serial diluted and then spotted onto plates containing either glucose (Glu) or galactose (Gal). The plates were incubated at 25°C for 3 d. (B) and (C) Δcdc55 mutant suppresses the temperature-sensitive phenotype of some mutants in the MEN pathway. The saturated cultures of strains with indicated genotype were 10-fold serial diluted and spotted onto YPD plates and incubated at indicated temperatures for 3 d.

(A) Δcdc55 mutant suppresses AMN1 overexpression phenotype. Strains (Y300, YFH240, YYW28, and YYW81) were transformed with either a CEN-URA3 vector or a P_GAL-AMN1 plasmid. Saturated cultures of the transformants were 10-fold serial diluted and spotted onto plates containing glucose or galactose and incubated at 30°C for 2 d. (B) Mutations in catalytic subunits of PP2A result in benomyl sensitivity. Saturated cultures of strains with indicated genotype (Y300, YYW28, Y2475, and Y2762) were 10-fold serial diluted and spotted onto YPD and benomyl (15 μg/ml) plates and incubated at 25°C for 3 d. (C) Δpph21 Δpph22 mutants suppress AMN1 overexpression phenotype. Y300 and Y2762 were transformed with either a CEN-URA3 vector or a P_GAL-AMN1 plasmid. The transformants were treated as described in Figure 3A. (D) Δtpd3 mutant suppresses AMN1 overexpression phenotype. 483-15-1 (tpd3::KAN) was transformed with either a vector or a P_GAL-AMN1 plasmid and the growth of the transformants was examined as described.

CDC55 exhibits synthetic phenotype with BFA1 and BUB2. (A) G1-arrested BFA1-HA (YFH286) and Δcdc55 BFA1-HA (283-2-4) cells were released into 30°C YPD medium. Cells were taken every 20 min to prepare protein extracts. The phosphorylation status of Bfa1 were determined by Western blot analysis with anti-HA antibodies. In the bottom panel, cdc14-1 BFA1-HA cells with a vector or a P_GAL-CDC55 plasmid were grown to midlog phase in the raffinose medium at 25°C followed by the following treatments: 1) to add galactose and incubate for 1 h at 25°C; 2) to add galactose and incubate for 1 h at 25°C and then shift to 34°C for 3 h; 3) to shift to 34°C for 3 h; and 4) to shift to 34°C for 3 h and then add galactose and incubate at 34°C for 1 h. The phosphorylation of Bfa1 protein is shown after Western blotting with anti-HA antibody. (B) Δcdc55 Δbfa1 and Δcdc55 Δbub2 double mutants display slow-growth phenotype. Cells with indicated genotype were incubated at 30°C for 2 d. (C) The localization of Cdc14 in asynchronized Δcdc55 Δbfa1 (443-5-1) double mutants. (D) Cell cycle-regulated Cdc14 localization in Δcdc55 (173-1-3), Δbub2 (221-1-1) and Δcdc55 Δbub2 (482-7-4) mutants. Cells were arrested at G1 phase with α-factor and then released into YPD medium at 30°C. Hydroxyurea, 200 mM, was added into the medium at 80 min when majority of the cells were large budded in order to block the release of Cdc14 from the nucleolus during the next cell cycle.

PP2A regulates mitotic exit through Tem1 modification. (A) Δcdc55 deletion partially suppresses the mitotic exit defects of tem1-3 mutants. G1-arrested tem1-3, tem1-3 Δcdc55, cdc15-2, and cdc15-2 Δcdc55 mutants were released into 37°C YPD medium and the budding morphology was examined over time. The percentage of rebudded cells is shown. (B) Tem1 is a phosphoprotein. Tem1-myc fusion protein was immunoprecipitated from the cell lysate of cdc14-1 TEM1-myc incubated at 36°C for 2 h. The precipitates were incubated with λ phosphatase at 30°C for 30 min in the presence or absence of phosphatase inhibitors. Proteins were separated with 10% SDS-PAGE and then subjected to Western blotting with anti-myc antibody. (C) Δcdc55 mutation leads to hyperphosphorylation of Tem1. 504-2-1 (TEM1-13myc) and 504-1-3 (Δcdc55 TEM1-13myc) were arrested at G1 phase and then released into 30°C YPD medium containing 20 μg/ml nocodazole. Protein samples were prepared every 20 min with TCA method. Proteins were separated by SDS-PAGE followed by Western blot analysis. Top, the percentage of large budded cells; bottom, the modification of Tem1 protein. (D) Protein samples from 0-, 80-, and 140-min time points in C were loaded side by side and the band shift of Tem1 protein is shown after Western blot analysis.