Reduced levels of Clb2 protein in the clb5Δ mutant. Wild-type cells (US2340) and clb5Δ cells carrying CDC14-GFP integrated at the CDC14 locus (US2337) were arrested in G₁ with α factor and then released into YEP+Glu medium at a permissive temperature. Samples were analyzed for the budding index, nuclear division, and Clb2 and Cdc28 protein levels.

(A) Cdc14-GFP is not prematurely dispersed in clb5Δ cells. Wild-type (left panels) and clb5Δ (right panels) samples at various times are shown. The graphs depict the extents of nuclear division and Cdc14-GFP dispersion. DAPI, 4′,6′-diamidino-2-phenylindole. (B) Insufficient phosphorylation of Hct1 in a clb5Δ strain. Wild-type (US2352) and clb5Δ (US2354) cells carrying GAL-HA₃-HCT1 were arrested in G₁ by α-factor treatment in YEP+Raff+Gal at 24°C. The cultures were filtered, washed, and resuspended in YEP+Glu at 24°C. Samples were analyzed for DNA content, nuclear division index, Cdc28 protein levels, and Hct1 phosphorylation. (C) Inefficient phosphorylation of Hct1 in clb3Δ clb4Δ clb5Δ cells. clb3Δ clb4Δ clb5Δ cells carrying MET3-HA₃-HCT1 on a 2μm plasmid (US2438) were synchronized in G₁ by α-factor treatment for 3 h (the first 1.5 h in YEP+Raff+Met and the second 1.5 h in −Met+Raff medium). The culture was divided into two halves; one-half of the cells were resuspended in YEP+Raff+Met, and the other half were resuspended in YEP+Gal+Met. Samples were analyzed for DNA content, Hct1 phosphorylation, and Clb2 and Cdc28 protein levels.

(A) Cln2 expression does not result in Clb2 accumulation. clb3Δ clb4Δ clb5Δ GAL-CLB5 (US2417) cells were arrested in G₁ with α factor in YEP+Raff. The culture was filtered, and cells were resuspended in YEP+Raff for 2 h. Galactose (2%) was then added to induce the synthesis of Clb5 for 2 h. In parallel, clb3Δ clb4Δ clb5Δ GAL-CLB5 MET3-HA₃-CLN2 (US2439) cells, synchronized in G₁ by α-factor treatment in YEP+Glu+Met, were filtered and resuspended in YEP+Glu+Met for 2 h. Cells were then transferred to −Met+Glu medium to induce the synthesis of HA₃-Cln2 for 2 h. Samples were analyzed for Clb2 and Cdc28 protein levels and CLB5, CLB2, and CLN2 RNA levels. (B) Overexpression of Cln2 is unable to restore viability in clb3Δ clb4Δ clb5Δ cells. clb3Δ clb4Δ clb5Δ GAL-CLB5 cells were plated on either glucose (upper right) or galactose (upper left) plates, whereas clb3Δ clb4Δ clb5Δ GAL-CLB5 MET3-HA₃-CLN2 cells were plated on −Met+Glu medium. Plates were photographed after 3 days at 24°C.

A schematic depicting the interaction between APC and Clb proteins and the resulting oscillations in Clb2 abundance (see the text for details).

(A) clb3Δ clb4Δ clb5Δ cells arrest in G₂/M with low levels of CLB2 transcript and Clb2 protein. Wild-type cells (US356) and clb3Δ clb4Δ clb5Δ GAL-CLB5::TRP1 cells (US2417) were synchronized in G₁ by α-factor treatment and then released in YEP+Glu medium at 24°C. Samples were collected at 20-min intervals and analyzed for the state of nuclear division, CLB2 and CLN1 RNA transcripts, and the levels of Clb2 and Cdc28 proteins. (B) clb3Δ clb4Δ clb5Δ cells arrest in G₂/M without a mitotic spindle. Left panels, wild-type cells with long mitotic spindles in the 100-min sample from the experiment described for panel A; right panels, clb3Δ clb4Δ clb5Δ cells in 100- and 240-min samples, clearly lacking mitotic spindles. DAPI, 4′,6′-diamidino-2-phenylindole. (C) clb3Δ clb4Δ clb5Δ cells survive in the presence of MET3-CLB2-HA₃. The clb3Δ clb4Δ clb5Δ GAL-CLB5::TRP1 control cells (US2417) were plated on either YEP+Gal (top left sector) or YEP+Glu (top right sector) plates. clb3Δ clb4Δ clb5Δ GAL-CLB5::TRP1 cells carrying MET3-CLB2-HA₃ on a CEN plasmid (US2435) were plated on a −Met+Glu plate (bottom right sector). The plates were incubated at 24°C and photographed after 48 h. (D) Clb2 is unstable in S phase in clb3Δ clb4Δ clb5Δ cells. Wild-type cells (US2404) and clb3Δ clb4Δ clb5Δ cells carrying MET3-CLB2-HA₃ on a CEN plasmid (US2435) were arrested in early S phase by growth in YEP+Glu+Met medium containing HU (30 mg/ml). After 3 h, the cultures were filtered and cells were resuspended in −Met+Glu+HU for 40 min. Methionine was added to the cultures to repress transcription, and samples, collected for a further 90 min, were analyzed for Clb2-HA₃ and Cdc28 protein levels.

(A) Deletion of HCT1 relieves G₂/M arrest in clb3Δ clb4Δ clb5Δ cells. clb3Δ clb4Δ clb5Δ GAL-CLB5::TRP1 (US2417) and clb3Δ clb4Δ clb5Δ hct1Δ GAL-CLB5::TRP1 (US2440) cells were treated with nocodazole (15 μg/ml) for 3.5 h in YEP+Gal at 24°C. The cells were washed and resuspended in YEP+Glu. Clb2 and Cdc28 protein levels were monitored in samples collected at 20-min intervals. (B) Deletion of HCT1 allows clb3Δ clb4Δ clb5Δ cells to grow on YEP-Glu medium. (C) Deletion of HCT1 relieves G₂/M arrest in clb3Δ clb4Δ clb5Δ cells. The left panels show cells from the experiment described for panel A. clb3Δ clb4Δ clb5Δ cells form a mitotic spindle within 40 min after the release from nocodazole arrest but eventually are blocked in G₂/M of the subsequent cycle without mitotic spindles (260-min sample). The right panels show that clb3Δ clb4Δ clb5Δ hct1Δ cells have reassembled spindles (80-min sample) after the release and continue to progress through the next cycle as indicated by the presence of mitotic spindles of various sizes (180-min sample). Noc, nocodazole; DAPI, 4′,6′-diamidino-2-phenylindole.

The oscillatory pattern in Clb2 protein levels is maintained even when CLB2 transcription is constitutive. Wild-type (US356) (♦) and clb2Δ ADH-CLB2 (US164) (○) cells, synchronized in G₁ by α-factor treatment in YEP+Glu at 24°C, were released into YEP+Glu medium. The state of mitotic spindles and the levels of Clb2 and Cdc28 proteins and CLB2 transcript are shown.