Cdk1 activity is required for the secretion block in metaphase-arrested cdc20-1 cells. (A) The Bgl2 secretion block in cdc20-1 cells depends on Cdk1 activity. cdc20-1 and cdc20-1 cdk1-as cells were grown to early log phase at 25°C and shifted to 37°C for 1.5 h. Cells were then treated with DMSO (mock) or 15 µM 1NM-PP1 for 30 min. Internal and external pools of Bgl2 in cdc20-1 and cdc20-1 cdk1-as cells were examined by Western blotting. ADH served as a protein loading control. (B) The accumulation of internal Bgl2 was quantified in each group, with the accumulation in cdc20-1 cell treated with DMSO as 100%. Error bars represent standard deviation (n = 3). *, P < 0.01. (C) The cdc20-1 and cdc20-1 cdk1-as cells were grown to early log phase at 25°C and shifted to 37°C for 1.5 h. Cells were then treated with 15 µM 1NM-PP1 for 30 min and proceeded for thin-section EM. The cdc20-1 cells accumulated post-Golgi secretory vesicles at the restrictive temperature, whereas cdc20-1 cdk1-as did not. Bars, 1 µm. (D) Quantification of vesicle numbers per EM section in the cdc20-1 and cdc20-1 cdk1-as cells. Error bars represent standard error of the mean (n = 35). *, P < 0.01.