Fig. 4. Cdc28 binding activity and cellular localization of Clb2 mutants. (A) Cells were transformed with a control vector or the indicated constructs to direct expression of HA-tagged proteins under the control of the GAL1 promoter. Transformants were grown in SC-ura raffinose medium, arrested in M phase with nocodazole (15 µg/ml) for 2 hours before induction of protein expression by 2% galactose for 3 hours. Nocodazole arrest was used to avoid potential bias due to differences in cell cycle progression upon overexpression of the various Clb2 mutants. Cells were processed for immunoprecipitation with anti-HA antibodies and the immunoprecipitates were analyzed for their content of Clb2-HA and Cdc28 by western blotting and for H1-kinase activity (IP) as described in Fig. 3. Aliquots of the total extracts were also assayed for Clb2-HA and Cdc28 (TE). (B) Proportion of bud neck staining was estimated as described in Fig. 2. (C) The Clb2^KA,EA,FA mutation was expressed from the GAL1 promoter as GFP-fusion proteins and its cellular distribution followed by fluorescence microscopy as described in Fig. 1. (D) Cellular localization of Clb2-hpm, Clb2176-213, Clb2176-213-hpm mutants fused to GFP.