FIG. 7.
Removal of a C-terminal domain of Cdc15 causes hyperactivation of MEN. (A) Strains were arrested for 3 h in G1 with the α-factor pheromone (5 μg/ml) and induced with 2% galactose for the last hour. Cells were synchronously released into YEP plus 2% raffinose and 2% galactose, and Dbf2 kinase activity and the amount of Dbf2 and Cdc28 (loading control) protein were determined at the indicated times. The following strains were used: A6131, A6130, A5568, and A5571. (B and C) Strains were grown to mid-log phase in YEP plus 2% raffinose and arrested in metaphase with 15 μg of nocodazole/ml for 1.5 h. An additional 7.5 μg of nocodazole/ml was added to maintain the metaphase arrest, and expression of GFP-CDC15 constructs was induced with 2% galactose. Samples were taken at indicated time points to determine the percentage of cells with Cdc14 released from the nucleolus. The image shown in panel C shows Cdc14 localization 90 min after galactose addition. Cdc14 staining is shown in red, and DNA is shown in blue. The following strains were used: A1411, A5965, A5966, and A5968.