Figure 5. Stabilized Cdc20 allows bypass of the spindle assembly checkpoint.
(A) APC/C reactions were performed using purified unlabeled Cdc20 (WT), Cdc20-0K, Cdc20-5K, or a mock translation (−) incubated with APC/C (5 nM), 35S-securin and E1/E2(Ubc4)/methyl-ubiquitin.
(B) Reactions were performed using purified 35S-Cdc20 or 35S-Cdc20-5K, plus APC/C (1 nM [+] or 5 nM [++]). Recombinant His6-Cdh1 was purified from baculovirus-infected insect cells.
(C) Asynchronous log-phase cultures of strains carrying CDC20 or CDC20-5K at the endogenous locus were arrested in G1 with 1 μg/ml α-factor (αF) for 3 h. α-factor was washed out and cells were harvested at the indicated times. α-factor was re-added when a majority of the cells had budded. Samples were analyzed by western blotting with anti-Cdc20, anti-Myc (securin), and anti-Cdk1 (as a loading control). Similar results were observed by flow cytometry analysis of DNA content (data not shown).
(D) Asynchronous log-phase cultures of CDC20, CDC20-5K, or mad2Δ cells were arrested with α-factor and released into media containing 60 μg/ml benomyl. Cells were harvested at the indicated times and α-factor was re-added when a majority of the cells had budded. Samples were analyzed by western blotting.
(E) CDC20 or CDC20-5K strains, carrying a non-destructible securin mutant, PDS1- db, under the control of the GAL promoter, were arrested in benomyl in galactose-containing media to induce a metaphase arrest. 100 μg/ml Cycloheximide (CHX) was added and samples were analyzed by western blotting.
(F) Purified unlabeled Cdc20 (left) or Cdc20-5K (right) was pre-incubated with the indicated Mad2 concentration before addition of APC/C (5 nM), E1/E2(Ubc4)/methyl-ubiquitin, and purified 35S-securin. The (−) control represents background activity and was subtracted from activity in the presence of exogenous activator. The zero concentration was plotted on a log scale as 0.01 μM. Similar results were obtained in three independent experiments.
(G) Purified unlabeled Cdc20 (left) or Cdc20-5K (right) was pre-incubated with the indicated Mad3-Bub3 concentration and APC/C (5 nM). Reactions were started by the addition of 35S-securin and E1/E2(Ubc4)/methyl-ubiquitin. Results are representative of two independent experiments.