1. Exit from Mitosis in Budding Yeast
Foong May Yeong, Hong Hwa Lim, Padmashree C.G. , Uttam Surana 
Molecular Cell 
Volume 5, Issue 3, Pages (March 2000)
DOI: /S (00)80444-X

2. Figure 1 Requirement of Cdc20 Function for Exit from Mitosis
(A) Exit from mitosis in cdc15–2 (US106) cells released from telophase arrest (control). cdc15–2 cells were arrested in telophase at 37°C for a total of 5 hr (see arrest scheme in [B]). The cells were then filtered and resuspended in YPD medium at 24°C. Samples withdrawn at the indicated time points were analyzed for the budding index, Clb2-associated kinase activity (PhosphorImager units), Clb2 and Cdc28 protein levels, and CDC20 and SIC1 RNAs. The FACS profile shows DNA content at 2.5 and 5 hr.
(B) Cells lacking Cdc20 function fail to exit mitosis. cdc15–2 cdc20Δ:LEU2 GAL-CDC20::TRP1 (US1976) cells were subjected to the following experimental regime: Cycling culture→2.5 hr in gal, 37°C→2.5 hr in glu, 37°C (Cdc20 depletion)→2 hr in Raff, 24°C (Restoration of Cdc15 function). The culture was then divided into two halves; glucose was added to one half to keep the CDC20 transcription repressed, while galactose was added to the other half to induce Cdc20 expression. Samples were withdrawn at various time points and analyzed as in (A).
Molecular Cell 2000 5, DOI: ( /S (00)80444-X)

3. Figure 2
Overexpression of Sic1 Induces Degradation of Clb2 in the cdc15–2 cdc20Δ Mutant
cdc15–2 cdc20Δ:LEU2 cells carrying four copies of GAL-SIC1-cmyc3 integrated at the URA3 locus and MET-HA3-CDC20 on TRP1 selectable CEN plasmid (US2091) were incubated for 2 hr at 36°C in glucose medium lacking methionine. The culture was filtered, washed, and resuspended in raffinose medium containing methionine (0.24 mg/ml) and maintained at 36°C for another 2.5 hr. The culture was then divided into two halves, washed, filtered, and resuspended in methionine medium containing either glucose or galactose at 24°C. Samples were withdrawn at the indicated times and analyzed for state of budding, DNA content, Clb2-associated kinase activity (PhosphorImage units), and the level of Clb2 and Cdc28 proteins. The photomicrographs show the morphology of cells at the end of 4.5 hr after the shift to 24°C.
Molecular Cell 2000 5, DOI: ( /S (00)80444-X)

4. Figure 3
Clb2 Is Partially Degraded prior to Mitotic Exit in an Hct1-Independent Manner
(A) Partial proteolysis of Clb2 and inactivation of Clb2-associated kinase activity during progression to telophase. cdc15–2 CDC28-cmyc3-TRP1 CLB2-HA3-HIS3 (US1186) cells were synchronized in G1 by α factor (0.8 μg/ml) treatment for 3.5 hr at 24°C. The cells were then filtered, washed, and then released into YPD medium at 36°C. Samples were withdrawn every 15 min and analyzed for the state of mitotic spindles, DNA content, Clb2-associated kinase activity (PhosphorImage units), and Cdc28-associated Clb2 and Cdc28 protein (internal control).
(B) Cell cycle progression in wild-type cells released from G1 block. Cells carrying tagged version of CDC28 and CLB2 (CDC28-cmyc3-TRP1 and CLB2-HA3-HIS3) (US1165) at their respective loci were synchronized in G1 using α factor (0.8 μg/ml) for 3.5 hr at 24°C. The cells were then filtered, washed, and then released into YPD medium at 24°C. Samples withdrawn at various time points were analyzed as in (A).
(C) The partial loss of Cdc28-Clb2 kinase activity during progression to telophase does not require Hct1 function. cdc15–2 hct1Δ:URA3 (US2072) cells were synchronized in S phase with hydroxyurea (HU, 30 mg/ml) for 3.5 hr at 24°C. The cells were filtered, washed, and released into YPD medium at 36°C. Samples were withdrawn every 15 min and analyzed as in (A).
Molecular Cell 2000 5, DOI: ( /S (00)80444-X)

5. Figure 4
Partial Proteolysis of Clb2 during Progression to Telophase Is Dependent Both on APC and Cdc20 Activities
(A) Clb2-associated kinase activity in cdc23–1 pds1–1 (US2009) cells remains high. cdc23–1 pds1–1 cells were synchronized in G1 by α factor (1 μg/ml) treatment for 3.5 hr at 24°C after which they were filtered, washed, and released into YPD meduim at 36°C. Samples were analyzed every 10 min to determine the state of nuclear division, DNA content, and the levels of Cdc28-Clb2 kinase activity, Clb2 and Cdc28 proteins.
(B) Clb2-associated kinase activity in cdc20–1 pds1–1 (US1236) cells remains high. cdc20–1 pds1–1 cells were treated and analyzed as in (A).
Molecular Cell 2000 5, DOI: ( /S (00)80444-X)

6. Figure 5
Cdc28-Clb2 Kinase Inactivation and Degradation of Clb2 Are Biphasic
cdc15–2 CDC28-cmyc3-TRP1 CLB2-HA3-HIS3 (US1186) cells were first synchronized in G1 by α factor (0.8 μg/ml) for 3.5 hr at 24°C and then released into pheromone-free YPD medium at 36°C. Samples were withdrawn every 15 min for 3.5 hr, after which the cells were again filtered and released into YPD medium at 24°C. Samples withdrawn at every 10 min were analyzed for state of mitotic spindle, budding index (in 24°C samples), DNA content (data not shown), SIC1 RNA, and the Cdc28-Clb2 kinase activity. The levels of Cdc28-associated Clb2 and Cdc28 protein were determined at selected time points as indicated (lowest panel).
Molecular Cell 2000 5, DOI: ( /S (00)80444-X)

7. Figure 6 Cdc20 Mediates Clb2 Degradation at Higher Levels of Clb2
(A) cdc15–2 cells carrying four copies of GAL-CLB2 integrated at TRP1 locus (US2018) were arrested in late telophase at 36°C (raffinose). Three samples, separated by 10 min intervals, were taken prior to induction of CLB2 for 2 hr (by addition of 2% galactose). Cycloheximide (1 mg/ml) and 2% glucose were added to inhibit Clb2 induction. Samples were taken at the indicated times and analyzed for DNA content, Clb2-associated kinase activity, and the levels of Clb2 and Cdc28 proteins. As a control, cdc15–2 cells (US106) were subjected to the same treatment. The levels of kinase activity that Clb2 and Cdc28 proteins represented in the graphs are relative to their levels in the 0 min sample.
(B) Overexpression of Cdc20 induces limited Clb2 degradation in nocadazole-arrested cells. cdc15–2 cells carrying GAL-HA3-CDC20 on a CEN vector (US1338) were arrested in nocodazole (15 μg/ml) at 24°C. Galactose was added to induce Cdc20 expression. The DNA content, Clb2-associated kinase activity, and the levels of HA3-Cdc20, Clb2, and Cdc28 proteins were monitored in samples withdrawn at every 15 min. The FACS profile shows continued arrest with 2 N DNA content during the experiment.
Molecular Cell 2000 5, DOI: ( /S (00)80444-X)

8. Figure 7
Cdc28-Clb5 Kinase Activity in Telophase in the Absence of Cdc20 Function
Cdc15 cdc20Δ cells kept alive by one copy of GAL-CDC20 integrated at the TRP1 locus (US1976) were arrested in telophase and then released in a manner identical to that described in Figure 1. Cells were analyzed for Cdc28-Clb2 and Cdc28-Clb5 activities using Clb2- and Clb5-specific antibodies.
Molecular Cell 2000 5, DOI: ( /S (00)80444-X)