Disrupting both PPH21 and PPH22 in cdc55-null cells suppresses the morphological defect. Wild-type, cdc55Δ, pph21Δ cdc55Δ, pph22Δ cdc55Δ, and pph21Δ pph22Δ cdc55Δ strains were grown on YPD plates at 30°C for 1 day and then were shifted to 16°C for 2 days. Cells were harvested, washed, fixed, and then microscopically examined using Nomarski optics. (a) Wild-type cells; (b) cdc55Δ cells; (c) pph21Δ cdc55Δ cells; (d) pph22Δ cdc55Δ cells; (e) pph21Δ pph22Δ cdc55Δ cells.

SWE1 expression is essential for the morphological defect of cdc55-null cells. cdc55Δ and swe1Δ cdc55Δ strains were grown on YPD plates at 30°C for 1 day and then shifted to 16°C for 2 more days. Cells were harvested, washed, fixed, and then microscopically examined using Nomarski optics. (a) cdc55Δ cells at 16°C; (b) swe1Δ cdc55Δ cells at 16°C.

Swe1 mRNA levels in S- and M-phase-arrested wild-type and cdc55-null cells. Total RNA was extracted from cells that were treated in the same manner as those shown in Fig. 4A. The RNAs were separated on 1% agarose gels and transferred to filters for Northern blot analysis (upper panel). The probe for Swe1 mRNA was generated by random primer labeling a 0.6-kb HindIII/XbaI fragment from the SWE1 ORF. As an indication of relative lane loading, the lower panel shows the ethidium bromide-stained gel prior to transfer. Lanes 1, 3, and 5: wild-type cells as cycling, HU-arrested, and nocodazole-arrested samples, respectively; lanes 2, 4, and 6: cdc55Δ cells as cycling, HU-arrested, and nocodazole-arrested samples, respectively.

Disrupting PPH21 and PPH22 in a cdc55-null strain lowers Swe1p levels in metaphase-arrested cells. Wild-type, cdc55Δ and pph21Δ pph22Δ cdc55Δ cells (all expressing MYC-tagged SWE1 genes) were grown at 30°C in YPD medium to early log phase. Wild-type and cdc55-null cells were arrested with nocodazole (20 μg/ml) for 3 h. pph21Δ pph22Δ cdc55Δ cells (Δ21Δ22Δ55) were treated with nocodazole for 5 h because of the slow growth rate of these cells. The arrest phenotype of all cells was microscopically confirmed prior to their harvest. Western analyses of the proteins isolated from these cells were carried out using anti-MYC antibody to measure Swe1p levels (upper panel) and anti-PSTAIR antibody to detect Cdc28p and Pho85p in order to determine relative protein loading in each lane (lower panel). Lane 1, wild-type cells; lane 2, cdc55Δ cells; lane 3, pph21Δ pph22Δ cdc55Δ cells.

Mih1p levels in wild-type and cdc55-null cells are similar. To determine Mih1p levels, wild-type (i.e., CDC55) and cdc55-null strains expressing an MIH1 gene tagged with 12 copies of the MYC epitope were constructed (see Materials and Methods). Such cells growing in YPD medium at 30°C were collected as unsynchronized cycling cells (lanes 1 and 2), cells arrested in S phase with 0.2 M HU for 3 h (lanes 3 and 4), or cells arrested in mitosis with 20 μg of nocodazole (noc) per ml for 3 h (lanes 5 and 6). Total cell proteins were extracted as described above, separated by SDS-PAGE, transferred to filters, and immunodecorated using the 9E10 anti-MYC antibody to detect Mih1p (a). The lower half of the gel not used for the protein transfer was stained with Coomassie blue to show the relative protein loading. To determine whether the heterogeneity of Mih1p gel mobility seen in (a) was due to differential phosphorylation of Mih1p, proteins were extracted from HU-arrested wild-type and cdc55Δ cells, immunoadsorbed to protein A beads, and treated with alkaline phosphatase (see Materials and Methods). Proteins released from the protein A beads were subjected to a Western blot analysis (b) as above. (−p), untreated controls; (+p), alkaline phosphatase treated.

Normal Swe1p degradation is suppressed in mitosis-arrested cdc55-null cells. To determine the relative rates of degradation of Swe1 mRNA in wild-type and cdc55-null cells, strains expressing chromosomally integrated SWE1-MYC₁₂ genes driven by a GAL1 promoter (see Materials and Methods) were developed. Such cells were grown in YP raffinose to early log phase and then arrested in nocodazole (20 μg/ml) for 3 h. The arrest phenotype was confirmed by microscopic analysis. Galactose was added to each culture to 2% for 30 min to transiently induce SWE1 transcription. Cells were then collected by centrifugation, washed, and resuspended in YPD medium containing nocodazole in order to suppress any further Swe1 mRNA production and to maintain mitotic arrest. Cells were harvested at 0, 25, 50, and 90 min after repression of transcription. Total RNA was extracted, and a Northern analysis (as in Fig. 5) was carried out (a, upper panel). The ethidium bromide-stained agarose gel prior to transfer served as a loading control (a, lower panel). Based on the above results, we measured Swe1p degradation in the following way. Swe1 mRNA was transiently induced as above. Beginning at 50 min after the SWE1 expression had been suppressed by glucose (defined as t = 0), cells were collected at intervals and the levels of Swe1p were determined (b) as in Fig. 3. Western blots were scanned and quantitated, the levels of Swe1p were plotted (c), and the degradation rates of Swe1p in wild-type and cdc55Δ cells were calculated.

Nocodazole-treated cdc55Δ cells remain arrested in mitosis. To determine whether nocodazole-treated cdc55-null cells maintain a mitotic arrest, we examined the metabolism of Pds1p and Cln2p in these cells and compared it to that seen in wild-type controls. To examine Pds1p initially, isogenic wild-type (CDC55) and cdc55Δ strains, each transformed with a plasmid carrying an HA-tagged PDS1 gene behind a GAL1 promoter (see Materials and Methods), were grown in raffinose medium to early log phase. Nocodazole was then added to the culture medium to 20 μg/ml to arrest cell growth, and 3 h later, galactose was added to induce the accumulation of HA-tagged Pds1p. After 2 h, cells were collected by centrifugation, washed once, and then, to repress further PDS1 transcription, resuspended in dextrose medium containing nocodazole to maintain mitotic arrest. Cell samples were collected immediately and at 0.5-h intervals for 2 h after PDS1 turnoff. Total cell proteins (see Materials and Methods) were separated by SDS-PAGE, transferred to nitrocellulose filters, and immunoblotted using 12CA5 anti-HA antibody to detect Pds1p (A). The lower half of the gel not used for the protein transfer was stained with Coomassie blue to show the relative protein loading in each lane. Endogenous Pds1p levels were determined in CDC55 and cdc55-null strains at various times during an extended exposure to nocodazole or after a release from a 3-h nocodazole arrest (B). Wild-type and cdc55-null cells, each expressing an HA-tagged PDS1 gene (see Materials and Methods), were grown to early log phase in YPD medium. Nocodazole was added to both cultures, and aliquots of cells were collected at 0, 1, 2, and 3 h. After 3 h in nocodazole, the remaining wild-type and cdc55Δ cultures were split in half. One-half of the cells remained in YPD containing nocodazole. The other cells were collected, washed, and then resuspended in fresh YPD containing no nocodazole to allow cells to enter the cell cycle. Aliquots of cells were collected 20, 40, 60, 80, and 100 min later. Western analyses were carried out to measure Pds1p levels (upper panels), and those portions of the gels not used for protein transfer were stained to show the relative loading in each lane. Lanes 1 through 4, cells in nocodazole at 0, 1, 2, and 3 h; lanes 5 through 9, cells at 20, 40, 60, 80, or 100 min after nocodazole release; lanes 10 through 14, cells in nocodazole at 20, 40, 60, 80, or 100 min after the initial 3-h drug incubation. Cln2p levels were determined in CDC55 and cdc55-null strains at various times during an extended exposure to nocodazole or after release from a 3-h nocodazole arrest (C). Wild-type and cdc55-null cells, each expressing an HA-tagged CLN2 gene (see Materials and Methods), were grown to early log phase in YPD medium. Nocodazole was added to both cultures, and aliquots of cells were collected at 0, 1, 2, and 3 h. After 3 h in nocodazole, the remaining wild-type and cdc55Δ cultures were split in half. One-half of the cells remained in YPD containing nocodazole. The other cells were collected, washed, and then resuspended in fresh YPD containing no nocodazole to allow cells to reenter the cell cycle. Aliquots of cells were collected 1, 2, and 3 h later. Western blot analyses were carried out to measure Cln2p levels (upper panels), and that portion of the gel not used for protein transfer was stained to show the relative loading in each lane. Lanes 1 through 7, cells in nocodazole at 0, 1, 2, 3, 4, 5, and 6 h; lanes 8 through 11, cells at 0, 1, 2, and 3 h after nocodazole release at 3 h. Note: lanes 4 and 8 contain the same protein samples.

Metaphase-arrested cdc55-null cells contain more Swe1p than metaphase-arrested wild-type cells. Wild-type and cdc55-null strains expressing an SWE1 gene tagged with 12 copies of the MYC epitope were constructed (see Materials and Methods). Such cells growing in YPD medium at 30°C were collected as unsynchronized cycling cells (lanes 1 and 2), cells arrested in S phase with 0.2 M HU for 3 h (lanes 3 and 4), or cells arrested in mitosis with 20 μg of nocodazole (noc) per ml for 3 h (lanes 5 and 6). Total cell proteins were extracted and analyzed (A) as in Fig. 1, except that we used the monoclonal antibody 9E10 to detect the MYC-tagged protein. The lower half of the gel not used for the protein transfer was stained with Coomassie blue to show the relative protein loading. To see whether the phenomenon of premature sister chromatid separation exhibited by nocodazole-treated cdc55Δ cells was related to increased Swe1p levels, we determined (B) the relative levels of Swe1p in wild-type (CDC55CDC28), CDC55CDC28^VF, cdc55ΔCDC28, and cdc55ΔCDC28^VF strains, each expressing a MYC-tagged SWE1 gene (see Materials and Methods for details). Such cells, growing in YPD medium at 30°C, were collected from asynchronous cultures (cycling) or from cultures that had been treated with nocodazole (noc) for either 3 or 6 h; Western analyses were carried out as above. To be able to metaphase arrest wild-type (CDC55) and cdc55-null cells not using drug treatment, we created CDC55 and cdc55Δ strains expressing SWE1:MYC₁₂ and also carrying the gene cdc23-1. The presence of the latter gene allows inactivation of the APC by raising the temperature of the culture to 35°C. Cultures of the two strains were grown at 25°C in YPD and asynchronous early-log-phase cells were collected (lanes 1 and 2), cultures were treated with HU for 3 h before cell collection (lanes 5 and 6), or cultures were shifted to 35°C for 3 h before cell collection (lanes 3 and 4). Total proteins were collected, and Western blot analyses were carried out to determine Swe1p levels (C). As another way to determine if the excess Swe1p in the metaphase-arrested cdc55-null cells was caused by the lethal effects of the nocodazole or elevated temperature treatments (D), the same wild-type and cdc55-null cells expressing SWE1:MYC₁₂ used in panel A were arrested for 3 h with HU and then washed into fresh YPD medium containing no drug (defined as t = 0). Cells were collected at intervals following the S-phase release for Western blot analysis and for microscopically monitoring cell cycle progression as in Fig. 3. In order to magnify the electrophoretic heterogeneity of the MYC₁₂-tagged Swe1p, SDS-PAGE was carried out using 6% gels rather than the standard 10%. Upper panels, percentages of cells in telophase; middle panels, Swe1p levels; lower panels, loading controls.

Measuring Mih1 phosphatase activity in wild-type and cdc55-null cells. To test the ability of the anti-phospho-Tyr15-Cdc2 antibody to detect S. cerevisiae phospho-Tyr19-Cdc28, total proteins were isolated from growing, unsynchronized cells expressing either an integrated HA-tagged Cdc28 or an integrated Cdc28-HA in which tyrosine 19 was converted to a phenylalanine (Y19F-Cdc28HA). Western blot analyses (A) were carried out as in Fig. 2, first using anti-phospho-Tyr15-Cdc2 antibody (upper panel) and, after washing the filter, using anti-HA antibody 12CA5 (lower panel). To compare the in vivo phosphatase activities of Mih1p in wild-type, cdc55Δ, and mih1Δ strains (all carrying integrated CDC28:HA genes), cells growing in YPD at 30°C were arrested with HU for 3 h. Cells were then collected by centrifugation, washed, and treated in one of two ways. One-fifth of the cells were resuspended in fresh YPD without HU. At various times thereafter, aliquots of cells were fixed and stained with DAPI. By microscopic examination, the percentages of large-budded cells with two separated nuclei (telophase cells) in each population were determined (B, upper panels). These samples were used to monitor the recovery of cells from S-phase arrest and their subsequent entry into and passage through telophase. The other four-fifths of the cells were resuspended in YPD plus nocodazole so that the cells released from S phase arrest would arrest again at the next metaphase. Aliquots of cells were withdrawn at the time of S-phase release (t = 0) and at various times thereafter (60, 90, 120, 150, and 180 min). At the end of the experiment, cells were stained with DAPI to make certain they were still arrested at metaphase as large-budded cells with a single nucleus. Total cell protein extracts were prepared, and a Western blot analysis was performed using the anti-phospho-Tyr15-Cdc2 antibody (B, middle panels). Subsequently, the filter was washed and reprobed with anti-PSTAIR monoclonal antibody to reveal the amount of Cdc28-HA protein in each lane (B, lower panels). To compare the in vitro Mih1 phosphatase activity of wild-type and cdc55Δ cells arrested in mitosis, Mih1p was immunoadsorbed to protein A beads from lysates of cells (either cdc55Δ or CDC55) expressing a MYC-tagged form of Mih1p (see Materials and Methods). As negative controls, similar immunoadsorptions were carried out on CDC55 and cdc55Δ strains expressing a normal (i.e., nontagged) MIH1 gene. The protein A beads were added to a soluble protein extract prepared from an mih1ΔCDC28:HA strain that had been treated with nocodazole for 3 h (see Materials and Methods for details). The phospho-Y19-Cdc28p in such extracts served as the substrate for the protein A-associated Mih1 phosphatase activity. Following protein A bead addition, the extracts were incubated with agitation at 23°C, and samples were withdrawn at 15-min intervals and, as above, analyzed for phospho-Y19-Cdc28 levels and Cdc28p levels (C). The resulting Western blots were quantitated, and the data for phospho-Y19-Cdc28 were plotted (D). To test the effects of the enzyme assay incubation on the quantity and quality of Mih1p recovered, aliquots of the protein A beads containing Mih1-myc from CDC55 (wt) and cdc55Δ (Δc) strains were collected before (one-tenth of the total) and after (one-sixth of the original total) the enzyme assay incubation. The released Mih1p was then subjected to a Western blot analysis (E).