Fig. 3. Localization of truncated forms of Clb2. (A) Construction of truncated forms of Clb2. Positions of truncations and internal deletion are shown relative to the cyclin fold of Clb2 (started at residues 259). (B) Cdc28 binding and activation properties of truncated Clb2 mutants and chimera. Wild-type cells were transformed with a control vector or indicated constructs allowing expression of HA-tagged protein from the GAL1 promoter and grown in SC-ura raffinose medium. Expression of the HA-tagged proteins was induced by addition of 2% galactose for 3 hours. Cell lysate were processed for immunoprecipitation with anti-HA antibodies. Clb2-HA (top panel) and Cdc28 (middle panel) in the immunoprecipitates were assayed by western blotting with anti-HA and anti-PSTAIRE antibodies respectively. The bottom panel shows an autoradiogram of the Clb2-associated histone H1 kinase activity present in the immunoprecipitates. (C) Bud neck localization of the truncated forms of Clb2. To make the comparison more accurate, the proportion of bud neck staining was estimated only in large budded cells with separated nuclei, a stage where the staining is best visualized with the wild-type protein. At least 150 cells were counted for each strain. (D) Cellular localization of truncated mutants fused to GFP. Expression of the GFP-fusion proteins from the GAL1 promoter was as described in Fig. 1.

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