Fig. 4.

Low levels of Clb5 contribute to the dephosphorylation of Clb5-specific substrates during early anaphase. (A) The dephosphorylation of Sld2 in nocodazole-treated wild-type, cdc55Δ, and bfa1Δ mutants. The G₁-arrested cells were released into YPD medium containing 20 μg/ml nocodazole and incubated at 30°C. Cells were collected at 20 min intervals for the preparation of protein samples. The budding index and Sld2 protein modification are shown. (B) cdc15–2 and cdc15–2 spo12Δ cells exhibit low levels of Clb5, but high levels of Clb2. The G₁-arrested cells with indicated genotypes were released into YPD medium at 37°C. Cells were harvested every 20 min for protein preparation. Clb5-HA and Clb2-HA levels were determined after Western blotting and the protein levels of Pgk1 are shown as loading controls. (C) The outline of the experimental procedure for D and E. (D) CLB5 overexpression results in decreased Sld2 dephosphorylation. The cdc15–2 SLD2-myc cells with either a vector or a P_GAL-CLB5 plasmid were treated as described in C. Here, the modification of Sld2 after Western blotting is shown. (E) Overexpression of SIC1 leads to the dephosphorylation of Clb2 substrate Pol12 in cdc15–2 mutants. The cdc15–2 POL12-myc and cdc15–2 slk19Δ POL12-myc cells with either a vector or a P_GAL-SIC1 plasmid were treated as described in C. The modification of Pol12-myc protein is shown after Western blotting. (F) Stabilized Clb5 decreases Sld2 dephosphorylation in cdc15–2-arrested cells. The G₁-synchronized cells with indicated genotypes were released into YPD medium at 37°C. α-factor was added back to WT and clb5-dbΔ cell cultures after the majority of the cells became budded to block the second round of cell cycle. Cells were harvested to analyze the phosphorylation of Sld2.