Fig. 6.

Separase is necessary for proper Cdc55 phosphorylation. a Cdc55 phosphorylation is defective in separase mutants. Protein cell extracts were prepared in cycling cells from strains Y536, Y568 and Y567 incubated at 32 °C for the indicated times. Cdc55 phosphorylation was analyzed by western blot. Pgk1 is shown as loading control. b Impaired Cdc55 phosphorylation in anaphase in separase mutants. Strains Y564 and Y565 were arrested in metaphase, incubated at 32 °C for 2 h to inactivate the esp1-2 allele and release into anaphase. Cdc55 phosphorylation was analyzed by western blot. Ponceau staining is shown as loading control. A representative image of one of the experiments is depicted (top). Mean values and SEM of three independent experiments are presented (bottom). c Separase is not required for Zds1-induced Cdc55 phosphorylation. Strains Y888 and Y524 were arrested in metaphase by Cdc20 depletion, incubated at 32 °C for 2 h, and Zds1 ectopic expression was induced. d Premature Cdc14 release in separase mutants upon expression of the Cdc55 phospho-mimetic mutant, cdc55-ED. Strains Y2782, Y1561 and Y1481 were arrested in metaphase. Cdc14 release was analyzed by immunofluorescence. Mean values and SEM of three independent experiments are shown