Figure 3.

Cdc25p destabilization and ubiquitination requires Clp1p and APC/C. (A) cdc10-V50 (KGY 1744) and cdc10-V50 clp1Δ (KGY 2752) cultures expressing pREP41myc-cdc25^C480S were grown in the absence of thiamine for 20 h at the permissive temperature (25°C), and then shifted to the nonpermissive temperature (36°C) for 3.5 h, at which point excess thiamine (4 μM) and cyclohexamide (100 μg/ml) were added to the cultures. Samples were taken at the indicated time points. Extracts were prepared and immunoblotted with 9E10 and anti-PSTAIR to detect Cdc25p and Cdk1p, respectively. (B) Percentage of G₁ cells from (A) as determined by Sytox Green (Molecular Probes) staining and flow cytometry. (C) In vivo ubiquitination assays. mts3-1 (KGY 574) (lane 1), mts3-1 cut2-myc (KGY 1923) (lane 3), mts3-1 cut2-myc lid1-6 (KGY 1948) (lane 4), mts3-1 cut2-myc clp1Δ (KGY 878) (lane 5), mts3-1 cdc25-myc (KGY 4366) (lane 7), mts3-1 lid1-6 cdc25-myc (KGY 100) (lane 8), mts3-1 skp1-A4 cdc25-myc (KGY 102) (lane 9), and mts3-1 clp1Δ cdc25-myc (KGY 110) (lane 10) strains transformed with pREP1-His₆-ubiquitin, and mts3-1 cut2-myc (KGY 1923) (lane 2) and mts3-1 cdc25-myc (KGY 4366) (lane 6) transformed with empty vector were grown at 25°C for 22 h in the absence of thiamine to induce His₆-ubiquitin expression and then shifted to 36°C for an additional 4 h. Samples were collected and extracts were prepared. Ubiquitin conjugates were purified on Ni-NTA beads, separated on SDS–PAGE, and immunoblotted with 9E10 antibodies to detect Cut2p and Cdc25p.