Fig. 3.

Cdc55 phosphorylation alters Cdc14 release from the nucleolus. a Cdc55 phosphorylation is impaired in the non-phosphorylable cdc55-2A mutant. Protein extracts from strains Y1211, Y1215, Y1216 and Y1217 arrested in metaphase by Cdc20 depletion were prepared. Cdc55 phosphorylation was analyzed by western blot. Pgk1 protein levels are shown as loading control. A representative experiment of one of the assays is presented (top). Quantifications of the slower and faster Cdc55 migration isoforms are depicted (bottom). Mean values and SEM are shown. b Cdc14 release is impaired during early anaphase in non-phosphorylable Cdc55 mutants. Strains Y1211, Y1215, Y1216, and Y1217 were arrested in metaphase by Cdc20 depletion and released into synchronous anaphase by Cdc20 re-introduction. Mitosis progression was monitored by FACS analysis of DNA content (up). Cdc14 release from the nucleolus and anaphase spindles were monitored by immunofluorescence (middle). Representative images of cells in early anaphase with a spindle length of 3–5 μm are depicted in c. Scale bar, 2 μm. d Cdc14 nucleolar release from b was analyzed as a function of spindle length. The strain Y3034 was used as a FEAR mutant control. At least 50 cells were scored for each spindle length. e Mutation of the T174A within Cdc55 impaired Cdk1–Clb2 phosphorylation in vitro. Strain Y564 was arrested in metaphase by Cdc20 depletion, Clb2 was purified by immunoprecipitation, and the Cdc28–Clb2 kinase assay was performed using bacteria-purified GST-Cdc55(1–193) and GST-Cdc55(1–193)-T174A as substrates. A representative experiment is depicted (left). Mean values and SEM of four independent immunoprecipitations are shown (right). f Cdc14 is prematurely released during metaphase in Cdc55 phospho-mimetic mutants. Strains Y879, Y1211, Y1212, Y1213, and Y1214 expressing Cdc55 phospho-mimetic mutants of the Cdk1 consensus sites within Cdc55 were arrested in metaphase by Cdc20 depletion. Cdc14 release was checked by immunofluorescence. At least 100 cells were scored for each strain. Mean values and SD of three independent experiments are shown. Photographs are of cells in early anaphase with a spindle length of 3–5 μm (g). Scale bar, 2 μm