Fig. 3. Phosphorylation of Spc110p dependent on CDK consensus sites. (A) Western blot analysis of yeast extracts from wild-type cells expressing HA₃-Spc110p sampled during a time-course synchrony experiment. Low-mobility bands corresponding to phosphorylated Spc110p appeared as cells initiated bud emergence (45 minutes after the release from G1). Phosphorylation peaked at metaphase (see Friedman et al., 1996). Numbers indicate time in minutes after release from an -factor-induced block; a, extract from asynchronous cells. (B) Effect of overexpression of the phosphatase Cdc14p on Spc110p phosphorylation. An overnight cell culture in YEPRaffinose (R) was synchronised by addition of 15 µg/ml nocodazole (R + Noc). During the arrest, the culture was divided and dextrose (D) or galactose (G) was added for repression or induction of GAL1:CDC14, respectively. Extracts were obtained from cell samples collected after 1, 2 and 3 hours. 90% of cells were held prior to anaphase in all samples analysed (not shown). Asterisk indicates a crossreacting band. (C) Effect of overexpression of Clb5p on Spc110p phosphorylation. Cells of the indicated strains were synchronised in YEPRaffinose by addition of 15 µg/ml nocodazole (R + Noc). Cells were released from the arrest by transferring to either YEPDextrose (D) or YEPGalactose (G) medium to repress or induce GAL1:CLB5, respectively. Extracts were prepared after incubation for 2 or 4 hours. (D) Western blot analysis of extracts from asynchronous cells expressing wild-type Spc110p (WT), Spc110^36Ap (S36A), Spc110^91Ap (S91A) and Spc110^36A91Ap (S36A S91A). c, untagged control. (E) Western blot analysis of extracts from synchronous cell populations, expressing the indicated versions of Spc110p, obtained by release from an -factor-induced arrest. Phosphorylation was delayed in the case of the Spc110^91Ap until completion of spindle assembly (75-90 minutes, not shown). Phosphorylation of Spc110^36Ap was diminished at later time points reaching a lower maximal level by 75 minutes. The double substituted mutant combined both effects. Cell-cycle analysis accompanying this experiment is shown in supplementary material Fig. S2. HA₃-epitope tagged Spc110p and HIS₆-tagged Cdc14p were detected as described in Materials and Methods.