FIG. 7.

Impaired degradation of Clb5 in a ts rpn3 mutant. (A) Wild-type YE46 (RPN3) cells and rpn3 mutant YE100 (rpn3-4) cells were transformed with a centromeric plasmid carrying an HA-tagged CLB5 gene fused to the GAL1 promoter (pGAL-CLB5^HA). Cells were grown in selective medium with 2% raffinose to the early log phase, synchronized in G₁ with a mating pheromone, and shifted to 37°C. After transient expression of Clb5 for 60 min by the addition of galactose, cells were transferred to prewarmed glucose-containing medium without the pheromone. Samples taken before (lane −60) or after (lane 0) Clb5 induction and at the indicated times following the termination of Clb5 expression were analyzed by SDS-PAGE and immunoblotting with an anti-HA antibody (Clb5-HA₃). A Cdc28 immunoblot is shown as a loading control (α-PSTAIRE). Quantification of HA-tagged Clb5 immunoreactivity at the different times by densitometry is graphically represented. (B) G₁ arrest failure of the rpn3 mutant upon ectopic expression of Clb5. Cell samples from the experiment shown in panel A were subjected to flow cytometric analysis. 1N and 2N indicate cells with unreplicated and fully replicated nuclear DNA, respectively.