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Vol. 17, Issue 1, 80-89, January 2006

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Phosphatase 2A Negatively Regulates Mitotic Exit in Saccharomyces cerevisiae

Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL 32306

Submitted December 22, 2004; Revised June 16, 2005; Accepted July 21, 2005
Monitoring Editor: Tim Stearns

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
In budding yeast Saccharomyces cerevisiae, Cdc5 kinase is a component of mitotic exit network (MEN), which inactivates cyclin-dependent kinase (CDK) after chromosome segregation. cdc5-1 mutants arrest at telophase at the nonpermissive temperature due to the failure of CDK inactivation. To identify more negative regulators of MEN, we carried out a genetic screen for genes that are toxic to cdc5-1 mutants when overexpressed. Genes that encode the B-regulatory subunit (Cdc55) and the three catalytic subunits (Pph21, Pph22, and Pph3) of phosphatase 2A (PP2A) were isolated. In addition to cdc5-1, overexpression of CDC55, PPH21, or PPH22 is also toxic to other temperature-sensitive mutants that display defects in mitotic exit. Consistently, deletion of CDC55 partially suppresses the temperature sensitivity of these mutants. Moreover, in the presence of spindle damage, PP2A mutants display nuclear localized Cdc14, the key player in MEN pathway, indicative of MEN activation. All the evidence suggests the negative role of PP2A in mitotic exit. Finally, our genetic and biochemical data suggest that PP2A regulates the phosphorylation of Tem1, which acts at the very top of MEN pathway.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
The key driving force for cell division in all eukaryotic cells is the conserved cyclin-dependent kinase (CDK). CDK activity fluctuates during the cell cycle, peaking at the S and M phases and dropping at the G1 phase. Although the high CDK activity is required for DNA duplication and chromosome segregation, the low CDK activity at late M and G1 phase is essential for the loading of DNA replication complexes onto replication origins (Noton and Diffley, 2000; Lengronne and Schwob, 2002). Thus, cells have developed a signal transduction pathway, named the mitotic exit network (MEN), to inactivate CDK after mitosis (Morgan, 1999). The components of the MEN pathway include protein kinases Cdc5, Cdc15, Dbf2, a GTPase Tem1, a phosphatase Cdc14, and a Dbf2 binding protein Mob1 (Jaspersen et al., 1998; Luca and Winey, 1998; Lee et al., 2001a). Phosphatase Cdc14, the key player in the MEN pathway, dephosphorylates Cdh1, an activator for APC (anaphase promoting complex), and subsequently activates the degradation of a B-type cyclin, Clb2. Cdc14 also enhances the stability of Sic1 protein, which acts as a CDK inhibitor (Visintin et al., 1998). During most of the cell cycle, Cdc14 is localized in the nucleolus, but its translocation out of the nucleolus in anaphase and telophase allows it to encounter its substrates, such as Cdh1 and Sic1. All the components in the MEN pathway are required for the release of Cdc14 from the nucleolus, suggesting that Cdc14 acts downstream of MEN pathway (Shou et al., 1999; Visintin et al., 1999).

The regulation of MEN activity is achieved through the multilayer control of Tem1, a small GTPase that localizes at the spindle pole body (SPB) and acts on the very top of the MEN pathway. Tem1's activator, the GTPase exchange factor (GEF) Lte1, exhibits daughter-cell specific localization. Thus, SPB-localized Tem1 is activated after it encounters Lte1 upon the entrance of SPB into daughter cells (Bardin et al., 2000). Before that, Tem1 is kept inactive by a two-component GTPase-activating factor (GAP) composed of Bfa1 and Bub2 (Alexandru et al., 1999; Geymonat et al., 2002, 2003). Protein kinase Cdc5 has also been implicated in MEN signaling by regulating Bfa1/Bub2. It phosphorylates one of the GAP components, Bfa1, and frees Tem1 from the inhibition by Bfa1 (Hu et al., 2001; Lee et al., 2001b). Tem1-GTP is believed to activate a downstream protein kinase Cdc15, which then activates the protein kinase Dbf2 in a manner dependent on the Dbf2 associated factor Mob1 (Komarnitsky et al., 1998; Mah et al., 2001). We have identified a new mechanism that inactivates MEN through the induction of Amn1 protein upon MEN activation. Amn1 binds to Tem1 and abolishes its association with the downstream target Cdc15 (Wang et al., 2003). Thus the cooperation of Tem1's cellular localization, Bfa1/Bub2 GAP activity, and the cell cycle regulated appearance of Amn1, limits the functional window of MEN to late M and early G1 phase.

Protein phosphatase 2A regulates a significant array of cellular events. This holoenzyme consists of a catalytic subunit, C, and two regulatory subunits, A and B (Millward et al., 1999). In budding yeast, PPH21, PPH22, and PPH3 encode the catalytic subunits of PP2A, and CDC55 encodes one of the regulatory B subunits (Healy et al., 1991; Ronne et al., 1991; Evans and Stark, 1997). In the presence of nocodazole, a drug that disrupts spindle structures, sister chromatids separate in cdc55 mutants, suggesting a function for PP2A in anaphase entry (Minshull et al., 1996; Wang and Burke, 1997). The phenotype of cdc55 mutants seems to be the result of increased Cdc28 phosphorylation. In S. cerevisiae, the CDK kinase activity is inhibited in G1 and early S phase by Swe1 kinase-dependent phosphorylation at tyrosine 19 of Cdc28 (Booher et al., 1993). cdc55 mutants are unable to keep a high CDK activity in nocodazole-treated cells because of the inhibitory phosphorylation of Cdc28. The premature sister chromatid separation and the cold sensitivity phenotypes in cdc55 mutants are suppressed by the CDC28F19 mutation, in which Cdc28 is resistant to the phosphorylation by Swe1 (Minshull et al., 1996; Wang and Burke, 1997). Consistently, Swe1 protein level is increased in cdc55 mutants because of the compromised Swe1 protein degradation (Yang et al., 2000). Thus, the accumulation of Swe1 in cdc55 mutants results in Cdc28 phosphorylation, which may contribute to the known phenotypes of cdc55 mutants.

Here we report that PP2A is also involved in mitotic exit regulation. Overproduction of PP2A components is toxic to temperature-sensitive mutants that have defects in mitotic exit. cdc55 mutants exit mitosis in the presence of spindle damage, as judged by the appearance of extra buds and nuclear-localized Cdc14. PP2A and Bfa1/Bub2 may negatively regulate mitotic exit in parallel pathways because cdc55 bfa1 and cdc55 bub2 double mutants are synthetic sick and exhibit more frequent nuclear localized Cdc14. PP2A may regulate mitotic exit by promoting Tem1 protein dephosphorylation. Thus, we identified a new layer of regulation for mitotic exit, involving PP2A.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Yeast Strains, Growth, and Media
The genotype and sources of relevant yeast strains are listed in Table 1. All the strains listed are isogenic with W303 derived Y300 strain. All strains were constructed using standard genetic crosses. YYW28 was made by using a PCR-based method (Longtine et al., 1998). To arrest yeast cells at G1 phase, 5 µg/ml -factor was added into midlog cell cultures (OD₆₀₀ = 0.4) and the cultures were incubated for 2.5 h. To release them into cell cycle, the cell cultures were centrifuged and washed once with H₂O. Nocodazole was purchased from ICN (Costa Mesa, CA) and was used at 20 µg/ml in a final concentration of 1% dimethyl sulfoxide.

Cytological Techniques
Immunofluorescence straining was done after formaldehyde (3.7%) fixation for 15 min. Cells were treated with zymolase for 15 min and then stained with anti-HA antibody (1:100; CRP, Berkeley, CA) overnight at 4°C on 14-well slides after methanol/acetone treatment. Afterward, cells were stained with FITC-conjugated secondary antibody and DAPI and then visualized under immunofluorescence microscope (Zeiss, Thornwood, NY).

Protein Techniques
Two milliliters of cell culture was used to prepare protein samples for time-course experiments. Cells were collected in tubes with screw caps after being centrifuged and 50 µl of 20% TCA and glass beads were added. Cells were broken by using beads beater for 2 min. Protein was precipitated by centrifuge at 3000 rpm for 2 min after glass beads were removed. Equal volumes (50 µl) of 1 M Tris-base and protein-loading buffer were added. Dissolved protein samples were boiled for 5 min.

Two hundred milliliters of cell culture (OD₆₀₀ = 0.4) was used for Tem1 protein immunoprecipitation. Cells were collected by centrifugation and washed once with water and then resuspended in 0.5 ml RIPA buffer supplied with protease and phosphatase inhibitors. Cells were broken with beads beater and the cell debris was removed after centrifuge at 14,000 rpm for 20 min at 4°C. Anti-myc antibody, 8 µl, (from CRP) was added into the cell extract and the tube was shaken for 1.5 h at 4°C. Then 50 µl of anti-mouse IgG agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) was added and shaken for 1.5 h. The beads were washed with 1x phosphatase buffer for three times and resuspended in 60 µl 1x phosphatase buffer. The beads were then used for phosphatase treatment. In our experiment, NaF, -glyceral phosphate, and Na₃VO₄ were used as phosphatase inhibitor.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Genetic Screen for Genes Lethal to cdc5-1 When Overexpressed
Cdc5 kinase promotes mitotic exit by phosphorylating Bfa1 and leading to its disassociation from Tem1 (Hu et al., 2001). To further understand the regulation of mitotic exit, we carried out a genetic screen for genes that cause lethality in cdc5-1 Ts mutants when overexpressed (Wang et al., 2003). A P_GAL-cDNA library was introduced into cdc5-1 mutants and the transformants were selected on URA dropout plates (Liu et al., 1992). From a total of 12,000 transformants, 10 plasmids exhibited a high-dosage lethal phenotype in cdc5-1 mutants. The recovered plasmids contain AMN1, CLB1, CDC55, PPH21, PPH22, and PPH3 genes. The negative role of AMN1 in mitotic exit regulation was reported previously (Wang et al., 2003). Like Clb2, Clb1 could be another APC^Cdh1 substrate and its overproduction will deteriorate the growth of cdc5-1 mutants in which the function of APC^Cdh1 is compromised (Charles et al., 1998; Shirayama et al., 1998). In addition to AMN1 and CLB1, we also isolated several genes that encode the subunits of PP2A, including the B-regulatory subunit Cdc55 and the catalytic subunits Pph21, Pph22, and Pph3.

Cdc5 kinase phosphorylates Bfa1 and promotes mitotic exit (Hu et al., 2001). Also it phosphorylates cohesin Scc1 and facilitates its cleavage by the separase Esp1 (Alexandru et al., 2001). The lethality in cdc5-1 mutants caused by the overexpression of PP2A subunits may result from the negative effects of PP2A on mitotic exit or on other Cdc5 related cell cycle processes. Thus we examined if overexpression of PP2A components is toxic to other temperature-sensitive mutants of the MEN pathway. P_GAL-CDC55, P_GAL-PPH21, P_GAL-PPH22, P_GAL-PPH3, and a control vector were introduced into tem1-3 and mob1-77 mutants that have defects in mitotic exit (Shirayama et al., 1994; Luca and Winey, 1998). The growth of the transformants was examined after incubation on both glucose and galactose plates at the permissive temperature. Overexpression of CDC55, PPH21, and PPH22 were lethal not only to cdc5-1, but also to tem1-3 and mob1-77 (Figure 1A and unpublished data). As overexpression of PP2A components is toxic to the Ts mutants that have defects in mitotic exit, it is likely that PP2A plays a negative role in mitotic exit. In our assays, overexpression of PPH3 was toxic to cdc5-1 mutants, but not to tem1-3 and mob1-77 (unpublished data). We also noticed that high dosages of CDC55, PPH21, PPH22, but not PPH3 resulted in slow growth of wild-type cells (Figure 1A).

View larger version (59K): [in this window] [in a new window]   Figure 1. (A) Overexpression of PP2A components is lethal to mutants defective in mitotic exit. Saturated cell cultures were 10-time serial diluted and then spotted onto plates containing either glucose (Glu) or galactose (Gal). The plates were incubated at 25°C for 3 d. (B) and (C) cdc55 mutant suppresses the temperature-sensitive phenotype of some mutants in the MEN pathway. The saturated cultures of strains with indicated genotype were 10-fold serial diluted and spotted onto YPD plates and incubated at indicated temperatures for 3 d.

cdc55 Mutants Exhibit Premature Mitotic Exit Phenotype
Bfa1 and Bub2 are required for preventing mitotic exit, and bfa1 or bub2 mutants rebud in the presence of spindle disruption (Hoyt et al., 1991; Li, 1999). If Cdc55 plays a negative role in mitotic exit, we expect that cdc55 mutants will exhibit a similar phenotype. Thus, we examined the cell cycle progression of cdc55 mutants in the presence of nocodazole, a microtubule-depolymerizing drug that disrupts the spindle structure. G1-arrested wild-type and cdc55 mutant cells were released into 30°C YPD medium containing 20 µg/ml nocodazole. In wild-type cells, disruption of the spindle structure activates the spindle checkpoint and arrests cells at metaphase. After incubation for 3 h, however, cdc55 mutant cells began to rebud. After 4-h incubation in the presence of nocodazole, 30% of cdc55 mutant cells exhibited extrabuds while wild-type cells were still arrested as large budded cells, indicating that cdc55 mutant cells might exit mitosis (Figure 2A).

View larger version (28K): [in this window] [in a new window]   Figure 2. (A) cdc55 mutant cells exit mitosis in the presence of nocodazole. Wild-type (Y300) and cdc55 mutant (YYW28) cells in midlog phase were arrested at G1 phase with -factor and then released into 30°C YPD medium containing 20 µg/ml nocodazole after sonication. Cells were harvested at 30-min intervals for budding index. The top panel shows the percentile of cells with extrabud and the bottom panel shows cells after 240-min incubation in the presence of nocodazole. The arrow indicates the rebudded cdc55 mutant cell in the presence of nocodazole. (B) Top, strain 409-2-6-3 (cdc55 URA3-tetO-112 LEU2-tetR-GFP) was arrested at G1 phase and then released into YPD medium containing 20 µg/ml nocodazole at 30°C. The picture was taken after 240-min incubation. The spots indicate GFP marked chromosome V. Bottom, the frequency of reduplicated chromosome in wild-type and cdc55 mutants. JBY583 (URA3-tetO-112 LEU2-tetR-GFP) and 409-2-6-3 (cdc55 URA3-teto-112 LEU2-tetR-GFP) were arrested at G1 phase and then released into YPD medium containing 20 µg/ml nocodazole. Cells were collected every 30 min and fixed for examination of the GFP signal. (C) The rebudding phenotype of cdc55 mutants is independent of Cdc28 Y19 phosphorylation. Top, the budding index of cells with indicated genotype in YPD medium at 30°C. Bottom, the percentage of rebudded cells in the presence of 20 µg/ml nocodazole. Strains (Y300, YYW28, 397-2-2, 412-13-3) were treated as described in Figure 2A.

We also generated cdc55 mutants with GFP marked chromosome V to examine sister chromatid separation and chromosome reduplication (Michaelis et al., 1997). As reported previously, a significant portion of cdc55 mutant cells showed separated chromatids in the presence of nocodazole. After 4-h incubation, we noticed that 10% cdc55 mutant cells contained more than two GFP dots, indicating that chromosomes were reduplicated (Figure 2B). The results suggest that cdc55 mutant cells are able to exit mitosis and finish the second round of DNA replication in the presence of nocodazole.

The Premature Mitotic Exit Phenotype in cdc55 Mutants Is Independent of Swe1
cdc55 mutant cells exhibit increased Cdc28 phosphorylation at tyrosine 19 (Minshull et al., 1996). Moreover, CDC28F19 mutant that lacks inhibitory phosphorylation site on Cdc28 suppress the cold sensitivity and premature sister separation in cdc55 mutants (Minshull et al., 1996; Wang and Burke, 1997; Yang et al., 2000). Therefore, we tested if the hyperphosphorylation of Cdc28 protein in cdc55 mutants also contributes to its premature mitotic exit phenotype. For this purpose, we generated cdc55 CDC28F19 double mutants. As reported earlier, the double mutants did not display abnormal bud morphology. To determine if the double mutants exit mitosis, G1-synchronized wild-type, CDC28F19, cdc55, and cdc55 CDC28F19 cells were released into YPD medium containing 20 µg/ml nocodazole and the budding indexes were determined. Interestingly, similar to cdc55 single mutants, cdc55 CDC28F19 double mutants also began rebud after incubation in the presence of nocodazole for 3 h (Figure 2C, bottom). The examination of unperturbed cell cycle progression in these mutants did not show any dramatic discrepancy except that the cdc55 mutant exhibited slower cell cycle progression (Figure 2C, top). We also found that swe1 cdc55 double mutants were able to rebud in the presence of nocodazole (unpublished data). This results argue against the hypothesis that increased phosphorylation of Cdc28 by Swe1 contributes to the premature mitotic exit in cdc55 mutants.

cdc55 Mutant Suppresses AMN1 Overexpression Phenotype
Overexpression of AMN1 gene slows down cell cycle progression because of its inhibition of mitotic exit (Wang et al., 2003). We predicted that AMN1 overexpression phenotype would be alleviated in mutants with hyperactive MEN. To test this, a vector and a P_GAL-AMN1 plasmid were transformed into wild-type, bfa1, bub2, mad1, and cdc55 mutants. As expected, cells with P_GAL-AMN1 plasmid grew slowly on the plates with galactose because of the slower mitotic exit. The AMN1 overexpression phenotype was suppressed by bfa1 or bub2 deletion, consistent with the negative role of Bfa1/Bub2 complex in mitotic exit regulation (Figure 3A). Similarly, we found that the cdc55 mutant also suppressed the AMN1 overexpression phenotype (Figure 3A). However, deletion of MAD1, a spindle checkpoint gene that acts in a different branch from Bfa1/Bub2, could not suppress the AMN1 overexpression phenotype. In response to spindle damage, Mad1, together with other spindle checkpoint components, prevents the activation of APC^Cdc20 (Hwang et al., 1998), whereas Bfa1/Bub2 complex inhibits mitotic exit by keeping Tem1 from activation (Alexandru et al., 1999). Because bfa1, bub2, and cdc55 mutants are all able to suppress the AMN1 overexpression phenotype, it is likely that mitotic exit pathways are up-regulated in cdc55 mutants.

View larger version (44K): [in this window] [in a new window]   Figure 3. (A) cdc55 mutant suppresses AMN1 overexpression phenotype. Strains (Y300, YFH240, YYW28, and YYW81) were transformed with either a CEN-URA3 vector or a PGAL-AMN1 plasmid. Saturated cultures of the transformants were 10-fold serial diluted and spotted onto plates containing glucose or galactose and incubated at 30°C for 2 d. (B) Mutations in catalytic subunits of PP2A result in benomyl sensitivity. Saturated cultures of strains with indicated genotype (Y300, YYW28, Y2475, and Y2762) were 10-fold serial diluted and spotted onto YPD and benomyl (15 µg/ml) plates and incubated at 25°C for 3 d. (C) pph21 pph22 mutants suppress AMN1 overexpression phenotype. Y300 and Y2762 were transformed with either a CEN-URA3 vector or a PGAL-AMN1 plasmid. The transformants were treated as described in Figure 3A. (D) tpd3 mutant suppresses AMN1 overexpression phenotype. 483-15-1 (tpd3::KAN) was transformed with either a vector or a PGAL-AMN1 plasmid and the growth of the transformants was examined as described.

Loss of Function of PP2A Leads to Mitotic Exit in cdc55 Mutants
Cdc55 may function as a negative regulator of PP2A, because the accumulation of Swe1 protein in cdc55 mutants is suppressed when PPH21 and PPH22 are deleted (Yang et al., 2000). Therefore, it is not clear whether the loss or the gain of function of PP2A in cdc55 mutants leads to the premature mitotic exit. Because overexpression of either Cdc55 or catalytic subunits Pph21, Pph22 is toxic to mutants in MEN pathway, it is likely that both Cdc55 and the catalytic subunits of PP2A negatively regulates mitotic exit. If that is the case, mutations in PP2A catalytic subunits will result in a cdc55-like phenotype. Previous results indicate that cdc55 is sensitive to microtubule disassembly drugs. We therefore examined the growth of pph21 single and pph21 pph22 double mutants on plates containing 15 µg/ml benomyl, a microtubule depolymerizing drug like nocodazole. pph21 single mutants exhibited benomyl sensitivity similar to that of wild-type cells, but pph21 pph22 double mutants failed to form colonies on benomyl plates (Figure 3B). Because both cdc55 and pph21 pph22 mutants are sensitive to microtubule-disassembling drugs, the inactive PP2A in cdc55 mutants might contribute to its sensitivity to benomyl.

We have shown that cdc55 mutants suppress the AMN1 overexpression phenotype, presumably because of the hyperactive mitotic exit pathways. pph21 pph22 double mutants should alleviate AMN1 overexpression phenotype as well if mitotic exit pathways are hyperactive in the double mutants. Therefore, a vector and a P_GAL-AMN1 were introduced into pph21 pph22 double mutants and the growth of the transformants was examined on plates containing either glucose or galactose. pph21 pph22 mutants containing P_GAL-AMN1 grew much better than wild-type cells (Figure 3C). Similarly, we examined if loss of the A regulatory subunit (Tpd3) of PP2A also exhibited hyperactive MEN activity, and we found that deletion of TPD3 also suppressed AMN1 overexpression phenotype (Figure 3D). Thus, we reason that the regulatory and catalytic subunits of PP2A negatively regulate mitotic exit and the premature mitotic exit phenotype in cdc55 mutants results from the loss of function of PP2A.

PP2A Regulates Cdc14 Localization
Phosphatase Cdc14 localizes in the nucleolus during most of the cell cycle (Shou et al., 1999; Visintin et al., 1999). After MEN activation, Cdc14 is released into the nucleus so that Cdc14 is able to dephosphorylate its substrates and promote the inactivation of CDK (Visintin et al., 1998). Thus, the localization of Cdc14 has been used as a molecular marker for mitotic exit. We tested the possibility that PP2A inhibits mitotic exit through the regulation of Cdc14 localization. The localization of Cdc14 in wild-type and cdc55 mutant cells was examined in the presence of nocodazole. CDC14-HA and cdc55 CDC14-HA strains in midlog phase were synchronized at G1 and then released into 30°C YPD medium containing 20 µg/ml nocodazole. The cells were harvested and subjected to immunofluorescence staining. As expected, the majority of wild-type cells exhibited nucleolar localized Cdc14, indicating that mitotic exit pathways were inactive. In contrast, cdc55 mutant cells showed nuclear localized Cdc14 beginning at 90 min after G1 release. After 120 min, almost all of the cdc55 mutant cells showed nuclear localized Cdc14 in the presence of nocodazole (Figure 4, B and C). However, in the absence of nocodazole, cdc55 mutant exhibited normal cell cycle-regulated Cdc14 localization, except that cdc55 mutants showed slower cell cycle progression (Figure 4A). The nuclear localized Cdc14 in cdc55 mutants in the presence of nocodazole could be a result of deformed nucleolar structure. To clear this issue, we examined the localization of Net1, a protein localized in the nucleolus through the cell cycle. G1 synchronized NET1-myc and cdc55 NET1-myc cells were released into YPD medium containing nocodazole. In contrast to Cdc14, both wild-type and cdc55 mutant cells exhibited nucleolar localized Net1 protein in the presence of nocodazole (Figure 4C, bottom), suggesting that the nuclear localized Cdc14 in cdc55 mutants is not a result of deformed nucleolus. Thus, cdc55 mutants fail to keep mitotic exit pathways inactive in the presence of nocodazole and the premature mitotic exit in cdc55 mutants is resulted from Cdc14 release from the nucleolus.

View larger version (45K): [in this window] [in a new window]   Figure 4. Cdc14 is released from the nucleolus in cdc55 mutants in the presence of nocodazole. (A) A1411 (CDC14-HA) and 173-1-3 (cdc55 CDC14-HA) cells were arrested at G1 phase with -factor and then released into YPD medium. Cells were collected every 20 min and fixed for Cdc14-HA staining. The budding index and the kinetics of Cdc14 release from the nucleolus are shown in the top and bottom panels, respectively. (B) The same strains were arrested at G1 phase and then released into YPD medium containing 20 µg/ml nocodazole at 30°C. The budding index and Cdc14 localization are shown. (C) The nuclear structure and Cdc14 localization of the 180 min samples in B are shown (top). The bottom of panel C shows the Net1 localization. G1-arrested NET1-myc and cdc55 NET1-myc cells were released into nocodazole medium. Cells were collected for Net1-myc staining. The nuclear structure and Net1 localization after 180-min incubation are shown. (D) cdc55 and pph21 pph22 mutants fail to prevent Cdc14 from releasing in the presence of nocodazole. Midlog phase of A1411, 173-1-3, and 472-7-2 were incubated in the presence of 20 µg/ml nocodazole at 30°C. Cells were collected after 3-h incubation for Cdc14 staining. (E) Hof1 phosphorylation is more pronounced in nocodazole-treated cdc55 mutants. G1-arrested 514-3-3 (HOF1-HA) and 514-2-3 (cdc55 HOF1-HA) cells were released into YPD medium containing 20 µg/ml nocodazole at 30°C. Cells were collected every 30 min for protein preparation. Hof1-HA protein was examined by Western blot analysis after SDS-PAGE.

We also examined the localization of Cdc14 in pph21 pph22 double mutants in the presence of nocodazole. Asynchronized cells were incubated in YPD medium containing 20 µg/ml nocodazole for 3 h and Cdc14 localization was examined. Like cdc55 mutants, 50% of pph21 pph22 double mutant cells exhibited nuclear localized Cdc14, whereas almost all the wild-type cells showed nucleolar localized Cdc14 (Figure 4D). These data support our conclusion that the B regulatory subunit Cdc55 and the catalytic subunits Pph21 and Pph22 act together to prevent the activation of mitotic exit pathways in the presence of nocodazole.

To determine the mitotic exit in molecular level, we analyzed the phosphorylation of Hof1 in wild-type and cdc55 mutants in the presence of nocodazole. Hof1 is a phosphoprotein required for cytokinesis. The phosphorylation of Hof1 depends on the functional MEN pathway as its phosphorylation is blocked in dbf2-2, cdc14-1, and cdc15-2 mutants (Vallen et al., 2000). Thus, the phosphorylation status of Hof1 protein could be used as a marker of MEN activation. HOF1-HA and cdc55 HOF-HA strains were arrested at G1 and then released into YPD medium containing nocodazole. cdc55 mutants show more phosphorylated Hof1 protein than wild-type cells in the presence of nocodazole, supporting the notion that MEN pathway is hyperactive in cdc55 mutants (Figure 4E).

PP2A Controls Mitotic Exit Independent of Bfa1/Bub2
Our data indicate that PP2A plays a negative role in mitotic exit and Bfa1/Bub2 does so as well. We have demonstrated that protein kinase Cdc5 phosphorylates Bfa1 and promotes mitotic exit (Hu et al., 2001). One reasonable model is that PP2A dephosphorylates Bfa1 and keeps Bfa1 active. If that is the case, mutations in PP2A will result in the hyperphosphorylation and inactivation of Bfa1. To test this model, we first constructed a cdc55 BFA1-HA strain and the phosphorylation of Bfa1 protein was examined in synchronized wild-type and cdc55 mutant cells. As with wild-type cells, cdc55 mutants exhibited cell cycle-regulated Bfa1 phosphorylation, and increased hyperphosphorylated Bfa1 was not observed in cdc55 mutants (Figure 5A). We also examined the phosphorylation of Bfa1 in cells overexpressing CDC55. The results indicate that CDC55 overexpression does not change the Bfa1 phosphorylation profiles (Figure 5A). Therefore, PP2A does not appear to inhibit mitotic exit through dephosphorylation of Bfa1.

View larger version (43K): [in this window] [in a new window]   Figure 5. CDC55 exhibits synthetic phenotype with BFA1 and BUB2. (A) G1-arrested BFA1-HA (YFH286) and cdc55 BFA1-HA (283-2-4) cells were released into 30°C YPD medium. Cells were taken every 20 min to prepare protein extracts. The phosphorylation status of Bfa1 were determined by Western blot analysis with anti-HA antibodies. In the bottom panel, cdc14-1 BFA1-HA cells with a vector or a PGAL-CDC55 plasmid were grown to midlog phase in the raffinose medium at 25°C followed by the following treatments: 1) to add galactose and incubate for 1 h at 25°C; 2) to add galactose and incubate for 1 h at 25°C and then shift to 34°C for 3 h; 3) to shift to 34°C for 3 h; and 4) to shift to 34°C for 3 h and then add galactose and incubate at 34°C for 1 h. The phosphorylation of Bfa1 protein is shown after Western blotting with anti-HA antibody. (B) cdc55 bfa1 and cdc55 bub2 double mutants display slow-growth phenotype. Cells with indicated genotype were incubated at 30°C for 2 d. (C) The localization of Cdc14 in asynchronized cdc55 bfa1 (443-5-1) double mutants. (D) Cell cycle-regulated Cdc14 localization in cdc55 (173-1-3), bub2 (221-1-1) and cdc55 bub2 (482-7-4) mutants. Cells were arrested at G1 phase with -factor and then released into YPD medium at 30°C. Hydroxyurea, 200 mM, was added into the medium at 80 min when majority of the cells were large budded in order to block the release of Cdc14 from the nucleolus during the next cell cycle.

cdc55 mutant exhibits SWE1-dependent abnormal morphology. The synthetic phenotype between cdc55 and bfa1, bub2 may come from the combination of abnormal morphology and the spindle checkpoint defects. To test this, we constructed cdc55 bub2 swe1 triple mutants. Even though swe1 suppressed the abnormal bud morphology, the synthetic slow growth phenotype of cdc55 bub2 could not be suppressed by the absence of SWE1. This observation indicates that SWE1-dependent abnormal morphology in cdc55 mutant does not contribute to the poor growth phenotype of cdc55 bfa1 and cdc55 bub2 double mutants, consistent with the notion that Swe1 accumulation in cdc55 mutants is not related to mitotic exit regulation. We also examined the cell cycle-regulated localization of Cdc14 in synchronized cdc55 bub2 swe1 triple mutants. Because the mutant cells are very sick, only some of the cells responded to -factor treatment. Thus, only the cells with shmoo morphology were counted for this experiment. We failed to observe proper cell cycle-regulated Cdc14 localization in the triple mutants. Even in the G1-arrested cells, 40% of the triple mutant cells exhibited nucleolar localized Cdc14, whereas almost all the cdc55 and bub2 single mutant cells exhibited nucleolar localized Cdc14 (Figure 5D). On the basis of these results, we conclude that the synthetic phenotype between CDC55 and BFA1/BUB2 is likely due to constitutively activated mitotic exit pathways. The results also suggest that PP2A and Bfa1/Bub2 control mitotic exit in an independent manner and the presence of either one of them is sufficient for a successful mitosis.

FEAR Pathway May Not Be Required for the Mitotic Exit in cdc55 Mutants
What is the target of PP2A that is related to mitotic exit? Both FEAR and MEN pathways control mitotic exit by regulating Cdc14 localization. The FEAR (Cdc fourteen early anaphase release) network promotes Cdc14 release from the nucleolus during early anaphase (Stegmeier et al., 2002). We next addressed the possibility that PP2A regulates mitotic exit by inhibiting the FEAR pathway. If the premature mitotic exit phenotype in cdc55 mutants is a result of hyperactive FEAR, deletion of SLK19, which encodes one of the FEAR components, should suppress the cdc55 mutant phenotype. We generated cdc55 slk19 double mutants to examine their rebudding phenotype in the presence of nocodazole. It appeared that the rebudding phenotype of cdc55 in the presence of nocodazole was partially suppressed by the slk19 mutation (Figure 6B). After 5-h incubation in the presence of nocodazole, 20% of cdc55 single mutant cells exhibited rebudding morphology. However, only 10% of cdc55 slk19 double mutant cells rebudded.

View larger version (41K): [in this window] [in a new window]   Figure 6. Mitotic exit in cdc55 mutants depends on MEN pathway. (A) The localization of Cdc14 in slk19 and cdc55 slk19 mutants during unperturbed cell cycle progression. G1-arrested 464-3-2 and 450-3-1 were released into cell cycle at 30°C and 200 mM hydroxyurea was added into the medium at 80 min to block the next S phase. Cells were collected at 30 min interval for Cdc14 staining. (B) Left, the budding morphology of cells with indicated genotypes. Strains with indicated genotypes were arrested at G1 and released into YPD medium containing 20 µg/ml nocodazole at 30°C for the determination of budding index. Right, the localization of Cdc14 in the presence of nocodazole. Strains with indicated genotype were released into nocodazole medium after G1 arrest. Cells were collected every 30 min and subjected to immunofluorescence staining for Cdc14-HA. The percentage of cells with nucleolar localized Cdc14 was determined over time. (C) cdc15-2 CDC14-HA and cdc15-2 cdc55 CDC14-HA strains were arrested at G1 phase with -factor and then released into YPD medium at 37°C. Budding index and the localization of Cdc14 were determined over time.

Because the budding index could not give us a clearcut answer, we further analyzed mitotic exit in cdc55 slk19 mutants by examining the localization of Cdc14. G1-arrested wild-type, cdc55, and cdc55 slk19 mutants cells with HA-tagged CDC14 were released into YPD medium either with or without 20 µg/ml nocodazole. In the absence of nocodazole, slk19 and slk19 cdc55 mutants exhibited cell cycle-regulated Cdc14 localization (Figure 6A). In the presence of nocodazole, Cdc14 localized in the nucleus in wild-type cells, whereas cdc55 mutants exhibited nuclear-localized Cdc14. cdc55 slk19 double mutants also showed nuclear localized Cdc14, but with delayed kinetics (Figure 6B, right). It appeared that defects in FEAR pathway delayed the mitotic exit process in cdc55 mutants. One explanation is that PP2A inhibits mitotic exit partially through its inhibition of FEAR pathway. Alternately, the premature release of Cdc14 in cdc55 mutants has nothing to do with FEAR pathway, but the defects of this pathway slow down the mitotic exit process in cdc55 mutants. Thus, PP2A must regulate mitotic exit pathway other than FEAR.

View larger version (30K): [in this window] [in a new window]   Figure 7. PP2A regulates mitotic exit through Tem1 modification. (A) cdc55 deletion partially suppresses the mitotic exit defects of tem1-3 mutants. G1-arrested tem1-3, tem1-3 cdc55, cdc15-2, and cdc15-2 cdc55 mutants were released into 37°C YPD medium and the budding morphology was examined over time. The percentage of rebudded cells is shown. (B) Tem1 is a phosphoprotein. Tem1-myc fusion protein was immunoprecipitated from the cell lysate of cdc14-1 TEM1-myc incubated at 36°C for 2 h. The precipitates were incubated with phosphatase at 30°C for 30 min in the presence or absence of phosphatase inhibitors. Proteins were separated with 10% SDS-PAGE and then subjected to Western blotting with anti-myc antibody. (C) cdc55 mutation leads to hyperphosphorylation of Tem1. 504-2-1 (TEM1-13myc) and 504-1-3 (cdc55 TEM1-13myc) were arrested at G1 phase and then released into 30°C YPD medium containing 20 µg/ml nocodazole. Protein samples were prepared every 20 min with TCA method. Proteins were separated by SDS-PAGE followed by Western blot analysis. Top, the percentage of large budded cells; bottom, the modification of Tem1 protein. (D) Protein samples from 0-, 80-, and 140-min time points in C were loaded side by side and the band shift of Tem1 protein is shown after Western blot analysis.

PP2A May Regulate Tem1 Protein Phosphorylation
To answer if the mitotic exit in cdc55 mutants depends on MEN functions, the cell cycle progression of tem1-3 and tem1-3 cdc55 was also examined at 37°C. Unlike cdc15-2, the mitotic exit defects in tem1-3 mutants were partially suppressed by deletion of CDC55. After 4-h incubation, >30% of tem1-3 cdc55 double mutants exited mitosis, as indicated by the appearance of extrabuds; however, all the cdc15-2 cdc55 double mutants were arrested as large budded cells (Figure 7A). This result is consistent with our observation that cdc55 mutation partially suppresses the temperature sensitivity of tem1-3 mutants when incubated at 30°C (Figure 1B). Because cdc55 suppresses the mitotic exit defects in tem1-3 mutants, PP2A might function as a negative regulator of Tem1.

It has been shown that Tem1 exhibits cell cycle-regulated modification, but the nature of this modification remains unclear. Therefore, we first examined if phosphorylation contributes to the band shift of Tem1 protein. Protein samples were prepared with cdc14-1 TEM1-myc strain incubated at 36°C for 2 h, as Tem1 exhibited more slow-migrating forms in cdc14-1-arrested cells. After immunoprecipitation with anti-myc antibody, Tem1 protein was subjected to protein phosphatase treatment in the presence or absence of phosphatase inhibitors. We found that the majority of the slow-migrating forms of Tem1 disappeared after phosphatase treatment (Figure 7B), indicating that Tem1 is a phosphoprotein.

Then we asked if the Tem1 protein phosphorylation is regulated by PP2A. We examined the phosphorylation status of Tem1 in wild-type and cdc55 mutant cells, and it was very clear that cdc55 mutants exhibited more modified Tem1 protein in asynchronized cells (Figure 7D). The phosphorylation of Tem1 was also examined in synchronized TEM1–13myc and cdc55 TEM1–13myc cells. G1-arrested cells were released into YPD medium containing 20 µg/ml nocodazole at 30°C. In G1-arrested cells, we observed more phosphorylated Tem1 in cdc55 mutants. As cells entered S-phase, there were fewer phosphorylated Tem1 proteins. We noticed the appearance of a slow-migrating Tem1 band in cdc55 mutants at 100 min; however, the phosphorylated Tem1 did not appear until 140 min after G1 release in wild-type cells (Figure 7C). When we ran the protein samples from wild-type and cdc55 mutant cells side by side, it was clear that cdc55 mutants exhibited more phosphorylated Tem1 protein under various conditions (Figure 7D), indicating that loss of function of PP2A enhances Tem1 phosphorylation. Given the fact that Cdc55 is a component of phosphatase, a reasonable model is that PP2A dephosphorylates Tem1 and inhibits its functions in mitotic exit.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Previous data indicate that budding yeast Cdc55 regulates bud morphology and sister chromatid separation. Here we report that PP2A also plays a negative role in mitotic exit and several pieces of evidence support this conclusion. First, overexpression of some of the PP2A components is toxic to mutants in which the MEN pathway is compromised. Second, cdc55 mutants exhibit extrabuds and nuclear localized Cdc14 in the presence of nocodazole. Moreover, the cdc55 deletion mutant could suppress the toxicity resulting from the overproduction of Amn1, which acts as a negative regulator of MEN. Finally, Hof1, a protein required for cytokinesis and its phosphorylation, depends on activated MEN pathway and exhibits more phosphorylated forms in cdc55 mutants in the presence of nocodazole. These results are consistent with our earlier observation that cdc55 mutants suppress the temperature sensitivity of cdc20-1 mutants (Wang and Burke, 1997). HCT1/CDH1 was identified as a high copy suppressor of cdc20–1 mutants (Schwab et al., 1997). We reason that the hyperactive MEN pathway in cdc55 mutants activates APC^Cdh1, which overcomes the requirement of Cdc20.

It has been shown that Swe1 protein accumulates in cdc55 mutants and either swe1 deletion or CDC28F19 mutation can suppress the abnormal bud morphology in cdc55 mutants (Wang and Burke, 1997; Yang et al., 2000). However, neither swe1 deletion nor CDC28F19 mutation could suppress the mitotic exit in cdc55 mutants in the presence of nocodazole. Other observations also argue against the role of Swe1 in mitotic exit. We noticed that cdc55 swe1 double mutant cells are capable of reduplicating their chromosomes in the presence of nocodazole (Wang, personal observation). Moreover, the sickness of cdc55 bub2 double mutants could not be rescued by swe1 deletion. Also, pph21 pph22 double mutants are capable of exiting mitosis without accumulating Swe1. Apparently, the premature mitotic exit phenotype in cdc55 mutants has nothing to do with Swe1-dependent Cdc28 phosphorylation. It is likely that PP2A has many substrates and the defects in the phosphorylation of different substrates contribute to the complex phenotype of cdc55 mutants.

Swe1 protein accumulates in cdc55 mutants and deletion of PPH21 and PPH22 suppress Swe1 accumulation, suggesting that Cdc55 deregulates PP2A activity. But that is not the case for Cdc55 in the regulation of mitotic exit. First, pph21 pph22 double mutants exhibit benomyl sensitivity, as do cdc55 mutants. Moreover, either cdc55 or pph21 pphh22 mutants suppress the AMN1 overexpression phenotype and both mutants exhibit nuclear localized Cdc14 in the presence of nocodazole, suggesting that mitotic exit pathways are hyperactive in both cdc55 and pph21 pph22 mutants. Consistently, deletion of TPD3, the A regulatory subunit of PP2A, also leads to less sensitivity to AMN1 overexpression. All these results indicate that both regulatory and catalytic subunits of PP2A are required for the negative regulation of mitotic exit pathways. It is the loss of function of PP2A in cdc55 mutants that causes premature mitotic exit.

Bfa1 and Bub2 also negatively regulate mitotic exit by forming a complex with Tem1, a key player in the MEN pathway (Pereira et al., 2000). Because Bfa1 is a phosphoprotein and its phosphorylation promotes mitotic exit (Hu et al., 2001), a reasonable model is that PP2A dephosphorylates Bfa1 to inhibit mitotic exit. However, our results argue against this model. The synthetic phenotype of cdc55 bfa1 and cdc55 bub2 double mutants indicates that PP2A and Bfa1/Bub2 might act in concert and have additive effects on mitotic exit. Inactivation of both pathways results in deregulated Cdc14 localization and premature mitotic exit, which leads to the aberrant mitosis and sickness of the double mutants.

Both FEAR and MEN pathways promote mitotic exit by stimulating Cdc14 release from the nucleolus. Our result indicates that defective FEAR pathway fails to block the mitotic exit completely in cdc55 mutants, because cdc55 slk19 double mutants are still able to rebud and release Cdc14 from the nucleolus in the presence of nocodazole. It is unlikely that PP2A negatively regulates mitotic exit by inhibiting FEAR pathway. MEN could be the target of PP2A, based on the following observations. We have shown that AMN1 inhibits mitotic exit by binding to Tem1. Deletion of either CDC55 or PPH21 PPH22 suppresses AMN1 overexpression phenotype. Deletion of CDC55 also partially suppresses the temperature sensitivity of cdc5-1 and tem1-3, indicating that MEN is hyperactive in cdc55. However, cdc15-2 mutation completely suppresses the mitotic exit in cdc55 mutants when incubated at the restrictive temperature, suggesting that MEN is indispensable for the mitotic exit in cdc55 mutants. Importantly, cdc55 mutants show increased Tem1 protein phosphorylation. Therefore, the hyperactive MEN in cdc55 mutants may result from the change of Tem1 protein modification. It is possible that a protein kinase phosphorylates Tem1 and activates MEN pathway, whereas PP2A dephosphorylates Tem1 and keeps it inactive. Defective PP2A will result in the increase of hyperphosphorylated Tem1, which promotes mitotic exit. In summary, we have identified a new layer of regulation of MEN, involving PP2A. In collaboration with Bfa1/Bub2 and Amn1, PP2A ensures cell cycle-regulated localization of Cdc14, which is essential for a successful mitosis.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
We thank Drs. Stephen Elledge, Angelica Amon, Jeff Bahant, Kyung Lee, Raymond Deshaies, Erfei Bi, James Broach, and John Pringle for plasmids and strains. We thank Dr. Daniel Burke for sharing the unpublished data. We thank Yanping Sun for preparing the media and buffers for the experiments. We also thank Dr. Stephen Elledge for his comments and Dr. Lloyd Epstein for thorough reading of this manuscript. This work was support by American Heart Association Scientist Development Grant 0435276B to Y.W. Y.W. was also supported by The Leukemia and Lymphoma Society Special Fellowship.

	Footnotes

 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E04–12–1109) on August 3, 2005.

Address correspondence to: Yanchang Wang (yanchang.wang{at}med.fsu.edu).

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