FIGURE 3:

Cdc6 phosphodegron for Cdc4. (A) GAL-CDC6-HA strains with various mutations (Δ370Δ371, Δ370, T370A, or T371A) were incubated in raffinose-containing medium and transferred to galactose-containing medium for 2 h to induce Cdc6 expression, and then cells were blocked with nocodazole. Cdc6 expression was suppressed by adding glucose. Protein samples were collected every 5 min and subjected to Western blot analysis to observe Cdc6-HA. Pgk1 was used as a loading control. Western blotting images were quantified. Results represent the average of three experiments, with error bars indicating SD. (B) Yeast two-hybrid analysis was performed to determine the binding between Cdc4p and the Cdc6p C-terminus. Full-length CDC4 in the pACT plasmid, containing the GAL4 activation domain (GAD), was cotransformed into L40 yeast strains along with the various CDC6 mutants in the pBTM116 plasmid fused to LexA. Transformants were assayed for β-galactosidase activity, as visualized in blue. Proteins were extracted from each strain (1–4), and Cdc6 protein levels were examined by Western blotting.