Distinct Mechanisms Control the Stability of the Related S-Phase Cyclins Clb5 and Clb6

N-terminal deletions hyperstabilize Clb6. (A) ClustalW alignment of Clb5 and Clb6. Black boxes indicate identities. Gray boxes indicate putative phosphorylation sites (S/T-P) in Clb6. Clb5 D-box is shown. Arrows demarcate the start of Clb6 N-terminal deletions. (B) The relative stability of HA-tagged Clb6 N-terminal deletions. CLB6 transcription was induced from the GAL1 promoter by treating asynchronous cultures (in YEP-raffinose) with 0.1% galactose for 25 min. Cells were then harvested, and at t = 0 min, cells were resuspended in YEP-dextrose containing 1 mg/ml cycloheximide. Wild type (wt), peak levels of endogenous Clb6 (cells collected from synchronous population expressing CLB6-HA from its own promoter, 15 min after release from α-factor); no tag, cell lysates from cells expressing Clb6 without an HA epitope tag; PSTAIRE, loading control. The asterisk indicates an HA background band.

Clb6p hyperstabilized in cdc4-3 and cdc34-3 mutant cells at restrictive temperatures and in the presence of a proteosome inhibitor. (A and B) The relative stability of HA-tagged Clb6 in (A) wild-type (wt) cells versus cdc4-3 sic1Δ mutants at 33°C and (B) wild-type cells versus cdc34-3 sic1Δ mutants at 35°C. (C) The relative stability of HA-tagged Clb6 in wild-type cells versus sic1Δ cells. The promoter shutoff experiments whose results are shown in panels A and B were performed as described in the legend to Fig. 2 except that cultures were shifted to restrictive temperatures for 30 min and then treated with galactose at restrictive temperatures for 30 min as follows: wt/CLB6-HA cells, 0.2% galactose; cdc4-3 sic1Δ/CLB6-HA cells, 0.8% galactose; and cdc34-3 sic1Δ/CLB6 cells, 1% galactose. In panel C, cells were harvested from YEP-sucrose and grown in YEP-galactose for 30 min. Times indicated are minutes after resuspension in YEP-dextrose containing cycloheximide. wt, peak levels of endogenous Clb6. (D) Stability of HA-tagged Clb6 in cells treated or untreated with MG132. Mid-log-phase cultures in YEP-sucrose were treated with 50 μM MG132 in dimethyl sulfoxide (+) or dimethyl sulfoxide alone (−) and then grown at 30°C for 1 h. Galactose (0.5%) was then added to induce Clb6 expression from the GAL1 promoter for 30 min. Times indicated are minutes after resuspension in YEP-dextrose containing cycloheximide. (E) Slow-mobility forms of Clb6 appear in the presence of the proteasome inhibitor MG132 and the deubiquitinase inhibitor NEM. Cells expressing an HA-tagged Clb6 from the GAL1 promoter were untreated (−) or treated (+) with 100 μM MG132 in YEP-sucrose for 45 min. Clb6-HA expression was then induced by the addition of galactose for 45 min. Total protein was then isolated in the presence (+) or absence (−) of NEM. (D and E) Strains had the gene encoding the ABC transporter Pdr5 deleted to decrease the rate at which MG132 was pumped out of cells. PSTAIRE, loading control. The asterisk indicates an HA background band.

Clb6 is a phospho-protein and is hyperstabilized when putative phosphorylation sites are mutated to nonphosphorylatable residues. (A) Multiple species of Clb6 are observed in whole-cell lysates. Lysates were untreated (−) or treated (+) with lambda phosphatase (phosphatase) in the presence (+) or absence (−) of sodium fluoride and of sodium orthovanadate (inhibitors). The asterisk indicates slow-mobility bands. (B and C) The relative stability of wild-type Clb6 and Clb6 proteins bearing phosphorylation site mutations. The experiment was carried out as described in the legend to Fig. 2 except that cultures were treated with 0.05% galactose for 20 min. Clb6 (wt) and the S6A, S6AT39A, and Δ3P mutants are shown (B). Clb6 (wt) and the Δ3P and S147A mutants are shown (C). Times indicated are minutes after resuspending cells in YEP-dextrose containing cycloheximide. wt, peak levels of endogenous Clb6; no tag, cell lysates from cells expressing Clb6 without an HA epitope tag; PSTAIRE, loading control. The asterisk in panels B and C indicates an HA background band.

Both Cdc28 and Pho85 regulate the stability of Clb6. (A) Stability of Clb6-HA in cells expressing the analogue-sensitive allele cdc28-as1. Log-phase asynchronous cultures in YEP-sucrose were harvested, and Clb6-HA expression was induced in YEP-galactose medium for 45 min. Cultures were then either untreated (−) or treated (+) with 5 μM 1-NM-PP1 for 30 min. Times indicated are minutes after terminating expression by resuspending cells in YEP-dextrose containing cycloheximide. (B) Stability of Clb6-HA in cells with the PHO85 gene deleted. Cultures were treated as described in the legend to Fig. 2. (C) Stability of Clb6-HA in cdc28-as1 pho85Δ cells. cdc28-as1 pho85Δ cultures were either untreated (−) or treated (+) with 5 μM 1-NM-PP1 as described for panel A. wt, wild type; PSTAIRE, loading control.

Clb6Δ3P expressed from the endogenous CLB6 promoter is hyperstable and suppresses the S-phase defect of clb5Δ cells. Wild-type Clb6 at its genomic locus was replaced by Clb6-HA or Clb6Δ3P-HA in clb5Δ cells. Clb6-HA (A) and Clb6Δ3P-HA (B) protein levels over time in a synchronous population of cells released from α-factor arrest. Times indicated are minutes after release from alpha factor. (C) Flow cytometric analysis of DNA content and budding index for the experiments whose results are shown in panels A and B. (D) Flow cytometric analysis of asynchronous, log-phase cultures. The percentage of cells in S phase is shown for each histogram. PSTAIRE, loading control.