PP2A^Cdc55 counteracts Bfa1 phosphorylation.

<p>(A) Premature Bfa1 phosphorylation in metaphase in the absence of PP2A^Cdc55. Strains Y513 (<i>MATa MET-CDC20 CDC14-Pk₉ BFA1-HA₆</i>) and Y514 (as Y513, but <i>cdc55</i>Δ) were arrested in metaphase by Cdc20 depletion and released into synchronous anaphase by Cdc20 reintroduction. Bfa1 phosphorylation was analyzed by western blot. Pgk1 served as loading control. FACS profiles were used as a control of anaphase progression. At least 100 cells were scored at each time. Tubulin served as a loading control. (B) PP2A^Cdc55 catalytic activity is required to keep Bfa1 under-phosphorylated in metaphase. Strains Y1021 (<i>MATa Pk₃-CDC55 BFA1-HA₆</i>) and Y1020 (<i>MATa pph3</i>Δ <i>pph21</i>Δ <i>pph22-172 BFA1-HA₆</i>) were arrested in metaphase by nocodazole treatment and shifted to 37°C. (C) Zds1-dependent inactivation of PP2A^Cdc55 promotes Bfa1 phosphorylation. Strain Y597 (<i>MATa MET-CDC20 GAL1-Flag₃-ZDS1 CDC14-Pk₉ BFA1-HA₆</i>) was arrested in metaphase by Cdc20 depletion and Zds1 ectopic expression was induced by addition of galactose. Bfa1 phosphorylation and Zds1 expression levels were analyzed by western blot. (D) Overexpression of Cdc55 restrains Bfa1 phosphorylation. Strain Y1190 (<i>MATa</i> 4×<i>GAL1-CDC55 CDC14-Pk₉ BFA1-HA₆</i>) was arrested at G1 with α-factor and released into a synchronous cell cycle. Half of the culture was released into medium containing galactose to induce Cdc55 overexpression and the other half was released without galactose as a control. Bfa1 phosphorylation was analyzed by western blot and Pgk1 served as a loading control. FACS profiles were used as a control of cell cycle progression. (E) Cdc55 and Bfa1 interact. Co-immunoprecipitation of Cdc55 and Bfa1 was analyzed in protein extracts from strains Y1021 (<i>MATa Pk₃-CDC55 BFA1-HA₆</i>), Y1146 (<i>MATa pph3</i>Δ <i>pph21</i>Δ <i>pph22-172 Pk₃-CDC55 BFA1-HA₆</i>) and Y1145 (<i>MATa MET-CDC20 GAL1-Flag₃-ESP1 Pk₃-CDC55 BFA1-HA₆</i>). Strains Y1146 and Y1021 were treated at 37°C for 120 min. Strain Y1145 was arrested in metaphase by Cdc20 depletion, and separase ectopic expression was induced for 120 min. Protein extracts from strain Y1063 (<i>MATα BFA1-HA₆</i>) lacking a Pk epitope on Cdc55 served as a control.</p

A model for MEN regulation by PP2A^Cdc55.

<p>In early anaphase, FEAR-induced inactivation of the PP2A^Cdc55 promotes the first wave of Cdc14 release and contributes to the accumulation of the Cdc5-phosphorylated form of Bfa1. In this way, FEAR alleviates the inhibitory signal on Tem1 by promoting the Bfa1 phosphorylation and stimulates Cdc15 activity through the FEAR-induced Cdc14 released. However, the downstream MEN kinase Dbf2–Mob1 is kept inactive by Cdk1–Clb2 phosphorylation, restraining MEN activity. Mob1 phosphorylation is invariable despite the progressive decrease in Cdk1 activity because the counteracting phosphatase PP2A^Cdc55 is also downregulated in early anaphase. In late anaphase, the increase in Cdc14 activity and the decrease in Cdk1–Clb2 activity alleviate the Cdk1 inhibitory signal towards Dbf2–Mob1 and the MEN is fully active.</p

Premature Bfa1 inactivation does not provoke premature exit from mitosis.

<p>(A) Cdc15 phosphorylation in the absence of Cdc55. Strains Y2961 (<i>MATa MET-CDC20 CDC14-Pk₉ CDC15-HA₆</i>) and Y528 (as Y2961, but <i>cdc55</i>Δ) were released into a synchronous anaphase by Cdc20 depletion and reintroduction. Cdc15 phosphorylation was analyzed by western blot. The budding index was used to monitor cell cycle progression. Pgk1 served as a loading control. (B) Cdc14 activity in <i>cdc55</i>Δ mutant cells. Strains Y2810 (<i>MATa MET-CDC20 CDC14-</i>Pk₉) and Y2118 (as Y2810, but <i>cdc55</i>Δ) were arrested in metaphase by Cdc20 depletion. Anaphase samples were taken 20 min after release when more than 80% of the cells showed anaphase spindles. Phosphatase activity from immunopurified Cdc14 was measured as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003966#s4" target="_blank">Materials and Methods</a>. Means and standard deviations are shown. (C) The MEN is not prematurely active in the absence of Cdc55. Strains Y547 (<i>MATa MET-CDC20 CDC14-Pk₉ HA₃-CDH1</i>) and Y548 (as Y547, but <i>cdc55</i>Δ) were released into synchronous anaphase by Cdc20 depletion and reintroduction. Cdh1 phosphorylation and Cdc5, Clb2 and Sic1 proteins levels were analyzed by western blot.</p

Bfa1 localizes asymmetrically at the dSPB in the absence of PP2A^Cdc55 activity.

<p>(A) Cdc55 deletion causes premature Bfa1 asymmetric localization at the dSPB in metaphase-arrested cells. Strains Y559 (<i>MATa MET-CDC20 CDC14-Pk₉ BFA1-eGFP</i>) and Y560 (as Y559, but <i>cdc55</i>Δ) were arrested in metaphase by Cdc20 depletion. Percentages of equal, high/low and single distribution of Bfa1-eGFP were determined. At least 100 cells were scored for each strain. (B) Bfa1 localization becomes asymmetric upon Zds1-dependent inactivation of PP2A^Cdc55. Strain Y875 (<i>MATα MET-CDC20 GAL1-Flag₃-ZDS1 CDC14-Pk₉ BFA1-mCherry SPC42-YFP</i>) was arrested in metaphase by Cdc20 depletion and galactose was added to induce Zds1 overexpression. Asymmetric Bfa1-mCherry signal was quantified as the SPB relative intensity ratio as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003966#s4" target="_blank">Materials and Methods</a>. (C) Premature Bfa1 asymmetric localization in the absence of Cdc55 in a metaphase-to-anaphase transition. Strains Y951 (<i>MATa MET-CDC20 CDC28F19 CDC14-myc₉ BFA1-mCherry SPC42-YFP</i>) and Y1017 (as Y951, but <i>cdc55</i>Δ) were arrested in metaphase by Cdc20 depletion and released into synchronous anaphase by Cdc20 reintroduction. Time-lapse microscopy was used to visualize Bfa1-mCherry localization dynamics. Bfa1 SPB ratios were measured during mitosis progression (n = 15 for WT; n = 10 for <i>cdc55</i>Δ). The distance between the two SPBs was used to calculate the spindle length. (D) Premature Bfa1 asymmetric localization in the absence of Cdc55 in a synchronous cell cycle after G1 release. Strains Y876 (<i>MATa CDC28F19 CDC14-myc₉ BFA1-mCherry SPC42-YFP</i>) and Y1006 (as Y876, but <i>cdc55</i>Δ) were arrested at G1 with α-factor and released into a synchronous cell cycle. Time-lapse microscopy was performed as in (C) (n = 26 for WT; n = 25 for <i>cdc55</i>Δ). Scale bar, 2 µm.</p

Cdk1– Clb2 inhibitory signal restrains MEN activation until anaphase.

<p>(A) Premature Cdk1-Clb2 localization at the SPB during metaphase in the absence of Cdc55. Strains Y985 (<i>MATa MET3-CDC20 CDC28F19 CDC14- myc₉ Cdk1-eGFP BFA1-mCherry</i>) and Y967 (as Y985, but <i>cdc55</i>Δ) were arrested in metaphase by Cdc20 depletion. At least 50 cells were scored for each strain. (B) Mob1 dephosphorylation upon Cdk1 inhibition. Strain Y1032 (<i>MATa MET3-CDC20 CDC28-as1 GAL1-Flag₃-ZDS1 MOB1-HA₆</i>) were arrested in metaphase by Cdc20 depletion and Zds1 was induced. After 180 min of Zds1 induction, 1 µM of 1NM-PP1 drug was added to inhibit Cdk1 to half of the culture and DMSO was added to the other half as a control. Scale bar, 2 µm.</p

Downregulation of PP2A^Cdc55 promotes Cdc15 activation.

<p>(A) Cdc15 is dephosphorylated upon Zds1-dependent inactivation of PP2A^Cdc55. Strain Y603 (<i>MATa MET-CDC20 GAL1-Flag₃-ZDS1 CDC14-Pk₉ CDC15-HA₆</i>) was arrested in metaphase by Cdc20 depletion and galactose was added to induce Zds1 expression. Cdc15 phosphorylation and Zds1 expression levels were analyzed by western blot. (B) Zds1-dependent inactivation of PP2A^Cdc55 induces Cdc15 asymmetric localization. Strain Y1014 (<i>MATα MET-CDC20 GAL1-Flag₃-ZDS1 CDC14-Pk₉ CDC15-eGFP BFA1-mCherry</i>) was arrested in metaphase by Cdc20 depletion and Zds1 expression was induced. At least 50 cells were scored for each strain. (C) Cdc55 deletion causes premature Cdc15 asymmetric localization in metaphase. Strains Y984 (<i>MATa MET-CDC20 CDC14-myc₉ CDC15-eGFP BFA1-mCherry</i>) and Y966 (as Y984, but <i>cdc55</i>Δ) were arrested in metaphase by Cdc20 depletion and the percentage of cells with asymmetric Cdc15-eGFP was quantified. At least 50 cells were scored for each strain. (D) Cdc15 premature asymmetric localization in the absence of Cdc55 in a metaphase-to-anaphase transition. The same strains as in (C) were released into a synchronous anaphase by Cdc20 depletion and reintroduction and followed by time-lapse microscopy (n = 14 for WT; n = 10 for <i>cdc55</i>Δ). Scale bar, 2 µm.</p

PP2A^Cdc55 regulates Mob1 phosphorylation.

<p>(A) MS² spectrum of peptide MpSPVLTpTPK, which includes the dually phosphorylated Ser and Thr residues. Peptide sequence and assigned fragment ions <i>y</i> (in blue) and <i>b</i> (in yellow) are indicated (pRS site probabilities = 98.8%; determined by Proteome Discoverer Software v 1.3). (B) Cdc55 counteracts Mob1 phosphorylation. Strains Y1109 (<i>MATa MET-CDC20 CDC14-Pk₉ eGFP</i>-<i>MOB1</i>) and Y1110 (as Y1109, but <i>cdc55</i>Δ) were released into a synchronous anaphase by Cdc20 depletion and reintroduction. Mob1 phosphorylation was analyzed by western blot. (C) Cdc55 and Mob1 interact. Co-immunoprecipitation of Cdc55 and Mob1 was analyzed in protein extracts from Y1106 (<i>MATa Pk₃-CDC55 MOB1-HA₆</i>). Protein extracts from strain Y1104 (<i>MATa MOB1-HA₆</i>) lacking a Pk epitope on Cdc55 served as a control.</p

PP2A^Cdc55 inactivation is insufficient to induce Dbf2–Mob1 activation.

<p>(A) Mob1 phosphorylation is not affected by PP2A^Cdc55 downregulation after Zds1 induction. Strain Y807 (<i>MATα MET-CDC20 GAL1-Flag₃-ZDS1 CDC14-Pk₉ MOB1-HA₆</i>) was arrested in metaphase by Cdc20 depletion and Zds1 expression was induced. Mob1 phosphorylation and Zds1 expression levels were analyzed by western blot. Anaphase (anaph) sample from a synchronous culture served as a Mob1 dephosphorylation control. (B) Mob1 is not loaded onto the SPB upon Zds1-dependent inactivation of PP2A^Cdc55. Strain Y913 (<i>MATα MET-CDC20 GAL1-Flag₃-ZDS1 CDC14-Pk₉ BFA1-mCherry MOB1-eGFP</i>) was arrested in metaphase by Cdc20 depletion and galactose was added to induce Zds1 overexpression. At least 50 cells were scored for each strain. (C) Mob1 loading onto the SPB is restricted to anaphase in the absence of Cdc55. Strains Y1023 (<i>MATa MET-CDC20 CDC28F19 CDC14-myc₉ MOB1-eGFP BFA1-mCherry</i>) and Y1076 (as Y1023, but <i>cdc55</i>Δ) were released into a synchronous cell cycle after α-factor arrest in G1 and followed by time-lapse microscopy (n = 10 for WT; n = 10 for <i>cdc55</i>Δ). Scale bar, 2 µm.</p