Cdc55p, the B-Type Regulatory Subunit of Protein Phosphatase 2A ...

MOLECULAR AND CELLULAR BIOLOGY, Feb. 1997, p. 620–626 0270-7306/97/$04.0010 Copyright q 1997, American Society for Microbiology

Vol. 17, No. 2

Cdc55p, the B-Type Regulatory Subunit of Protein Phosphatase 2A, Has Multiple Functions in Mitosis and Is Required for the Kinetochore/Spindle Checkpoint in Saccharomyces cerevisiae YANCHANG WANG

AND

DANIEL J. BURKE*

Department of Biology, University of Virginia, Charlottesville, Virginia 22903 Received 5 August 1996/Returned for modification 19 September 1996/Accepted 5 November 1996

Saccharomyces cerevisiae, like most eucaryotic cells, can prevent the onset of anaphase until chromosomes are properly aligned on the mitotic spindle. We determined that Cdc55p (regulatory B subunit of protein phosphatase 2A [PP2A]) is required for the kinetochore/spindle checkpoint regulatory pathway in yeast. ctf13 cdc55 double mutants could not maintain a ctf13-induced mitotic delay, as determined by antitubulin staining and levels of histone H1 kinase activity. In addition, cdc55::LEU2 mutants and tpd3::LEU2 mutants (regulatory A subunit of PP2A) were nocodazole sensitive and exhibited the phenotypes of previously identified kinetochore/ spindle checkpoint mutants. Inactivating CDC55 did not simply bypass the arrest that results from inhibiting ubiquitin-dependent proteolysis because cdc16-1 cdc55::LEU2 and cdc23-1 cdc55::LEU2 double mutants arrested normally at elevated temperatures. CDC55 is specific for the kinetochore/spindle checkpoint because cdc55 mutants showed normal sensitivity to g radiation and hydroxyurea. The conditional lethality and the abnormal cellular morphogenesis of cdc55::LEU2 were suppressed by cdc28F19, suggesting that the cdc55 phenotypes are dependent on the phosphorylation state of Cdc28p. In contrast, the nocodazole sensitivity of cdc55::LEU2 was not suppressed by cdc28F19. Therefore, the mitotic checkpoint activity of CDC55 (and TPD3) is independent of regulated phosphorylation of Cdc28p. Finally, cdc55::LEU2 suppresses the temperature sensitivity of cdc20-1, suggesting additional roles for CDC55 in mitosis. for bud morphogenesis, and for entry into mitosis (25). Given the potentially broad-spectrum substrates, the pleiotropic nature of the phenotype is reasonable. The two regulatory subunits of PP2A (A and B) provide substrate specificity to the catalytic subunits (41). There are two genes in S. cerevisiae that have significant identity with the regulatory B subunits of mammalian PP2A, called RTS1 and CDC55, and one gene that has significant identity with the regulatory A subunit, called TPD3 (6, 13, 37). Both CDC55 and TPD3 genes are essential at low temperatures but are dispensable at higher temperatures, and the RTS1 gene is essential only at the higher temperatures. When cdc55 and tpd3 mutants are grown at the low temperature, they arrest with aberrant bud morphologies, and therefore both genes have been implicated in cellular morphogenesis (13, 37). One interpretation is that the regulatory subunits modulate phosphatase activity toward the actin cytoskeleton and proteins required for bud morphogenesis. In this study, we provide evidence that CDC55 has multiple roles in mitosis. We have previously shown that cells having two mutations, one in a kinetochore component (ctf13) and the other in a component of the spindle checkpoint pathway, are severely compromised for growth (39). We used this synthetic phenotype to identify CDC55 as a component of the spindle checkpoint and implicate it in cell cycle regulation. Cdc55p function is limited to the spindle checkpoint and does not affect cell cycle progression in response to DNA damage or an inhibitor of DNA synthesis. We show that cdc55 affects the state of Cdc28p phosphorylation on the tyrosine residue that is implicated in cell cycle control. A mutation that abolishes the phosphorylation of Cdc28p does not affect the checkpoint regulation mediated through CDC55. However, the same mutation that abolishes the tyrosine phosphorylation of Cdc28p suppresses the cold sensitivity and aberrant bud morphologies of the cdc55 mutant. This finding suggests that the essential

Errors in DNA replication and in chromosome segregation can be corrected by checkpoint control mechanisms that arrest cell division at discrete points to allow for errors to be repaired (12, 29). Genes that function in specific checkpoints have been identified in Saccharomyces cerevisiae and Schizosaccharomyces pombe by mutations that uncouple events in the cell cycle (3, 5, 11, 17, 24). The assumption is that some of these mutations identify gene products that function in an intracellular signalling pathway that responds to a cellular error and transduces an inhibitory signal to the cell cycle machinery. The cell then arrests to repair the mistake. Molecular analysis of the checkpoint genes has shown that some of them encode protein kinases and implicates protein phosphorylation in the intracellular signal transduction pathway (9, 24, 31). Given a role for protein kinases, it is reasonable to assume that phosphoprotein phosphatases also function in the signalling pathway. Extensive genetic analyses of phosphoprotein phosphatases suggest that they play critical roles in mitosis and cellular morphogenesis (13, 20, 25, 27, 44). For example, the dualspecificity phosphatase (homolog of the S. pombe cdc251 gene) regulates the state of phosphorylation on the mitotic Cdk and is critical for mitotic timing (14). A variety of serine/ threonine protein phosphatases have also been implicated in mitosis in organisms from yeast to humans, but the precise functions are not known (25, 27, 44). One such phosphatase, protein phosphatase 2A (PP2A), is a heterotrimeric protein consisting of a catalytic subunit and two regulatory subunits (41). Catalytic subunits of PP2A are thought to function in mitosis in S. cerevisiae and are encoded by two genes, called PPH21 and PPH22, although some activity is provided by PPH3 (15, 25). Genetic analysis suggests that PPH21 and PPH22 are required for proper intracellular actin organization, * Corresponding author. Phone: (804) 982-5482. Fax: (804) 9824834. E-mail: [email protected]. 620

VOL. 17, 1997

MITOTIC CHECKPOINT AND PHOSPHATASE 2A

TABLE 1. Genotypes of strains used in this study Strain

Relevant genotype

YPH1293R MATa ade2 his3 leu2 lys2 trp1 ura3 ctf13-30 1607-1-2 MATa ade2 lys2 ura3 cyh2 ctf13-30 bub1 794-9-4 MATa his3 leu2 lys2 trp1 ura3 ctf13-30 bub3:: LEU2 793-7-4 MATa ade2 his3 leu2 trp1 ura3 ctf13-30 mad1-1 792-3-1 MATa ade2 leu2 lys2 trp1 ura3 ctf13-30 mad2-2 1619-4-2 MATa leu2 trp1 ura3 ctf13-30 mad3 5943-4 MATa his7 lys5 ura3 can1 cyh2 GAL1 1634 MATa ade2 his3 leu2 lys2 trp1 ctf13-30 cdc55-100 TWY308 MATa trp1 ura3 mec1-1 CY5580 MATa leu2 ura3 cdc55::LEU2 CY1667 MATa his3 leu2 lys2 trp1 ura3 tpd3::LEU2 CY2580 MATa leu2 trp1 ura3 cdc28F19 (TRP1) 1638-2-2 MATa leu2 trp1 ura3 ctf13-30 cdc55::LEU2 1639-1-2 MATa ura3 cdc23-1 cdc55::LEU2 1640-2-1 MATa his3 trp1 ura3 cdc20-1 cdc55::LEU2 1652-2-1 MATa leu2 lys2 trp1 ura3 cdc55::LEU2 cdc28F19 1645-7-4 MATa his7 leu2 ura3 cdc55::LEU2 cdc16-1 406-2 MATa ade3 his7 leu2 ura3 cdc20-1 752-D13 MATa can1 cyh2 his7 lys5 ura3 cdc23-1 NIG1A5 MATa his7 leu2 ura3 cdc16-1

Source

P. Hieter This study This study This study This study This study Burke lab This study T. Weinert K. Arndt K. Arndt K. Arndt This study This study This study This study This study Burke lab Burke lab D. Koshland

function and the effect of cdc55 on cellular morphogenesis are mediated through CDC28. Finally, we show that a cdc55 mutation can suppress the temperature sensitivity of cdc20-1, suggesting that CDC55 has additional roles in mitosis. MATERIALS AND METHODS Yeast strains and media. The yeast strains used in this study are listed in Table 1. Strains were constructed by standard genetic methods (35). YM-1, YEPD, and SD media were as described elsewhere (10, 35). Benomyl was added to solid medium to 15 mg/ml from a 20-mg/ml stock in dimethyl sulfoxide (DMSO). Nocodazole (Sigma) was added to liquid medium YM-1 containing 1% DMSO to 10 mg/ml from a 3.3-mg/ml stock in DMSO. The DNA synthesis inhibitor hydroxyurea (Sigma) was added to YM-1 to 2.5 mg/ml. Mutant isolation. Strain YPH1293R was mutagenized with ethyl methanesulfonate to 10% survival and plated for single colonies on YEPD at 238C (permissive temperature for ctf13 mutant YPH1293R). Colonies were double replica plated onto YEPD plates and incubated at 328C for 2 days. Candidates that failed to grow 328C were selected for further analysis and transformed with plasmid pKF11 containing CTF13. The resulting transformants were tested for growth at 328C. Transformants that did not grow at 328C were considered temperature-sensitive mutants and were discarded. The candidates were crossed with ctf13 bub1, ctf13 bub3, ctf13 mad1, ctf13 mad2, and ctf13 mad3 double mutants. Those carrying alleles to known bub and mad mutations were identified by complementation of the 328C sensitivity and were discarded. Benomyl sensitivity of the remaining mutants was tested on YEPD plates containing 15 mg of benomyl per ml. Sensitivity to benzimidazoles was quantified by adding nocodazole, to a final concentration of 10 mg/ml, to cells growing in YM-1. Plating efficiency was determined by directly viewing of the cells spread on YEPD plates as described previously (39). Finally, candidates were treated with 10 mg of nocodazole per ml, and total histone H1 kinase activity was essentially as described previously (28). Phosphorylated histone H1 was visualized by autoradiography. Antitubulin immunofluorescence and DNA staining with 49,6-diamidino2-phenylindole (DAPI) were done as described previously (42). Cells were viewed and photographed as described earlier (34). Cells were stained with calcofluor as described previously (13). DNA analysis. A yeast genomic library in a YCp-based vector was used to clone the gene as described previously (32). Transformants were recovered by the lithium acetate method (7) and were selected on leucine-deficient synthetic complete medium plates at 238C and double replica plated to 328C. Transformants were identified, and plasmids were recovered from yeast and amplified in Escherichia coli as described previously (34). The complementing region was localized by Tn5 mutagenesis (34). Mutagenized plasmids were recovered from E. coli and tested in yeast for complementation. Transformants that failed to grow at 328C contained the plasmid in which the gene is disrupted by Tn5 insertion. The disrupted plasmid was cut with a restriction enzyme, a fragment, one end of the Tn5 transposon, was deleted, and the plasmid was recircularized. A synthetic oligonucleotide complementary to the end of Tn5 was designed to

621

prime DNA sequence reactions within the end of Tn5, and the DNA sequence of the complementing gene was determined. The DNA sequence was used to perform an on-line search of the nonredundant database at GenBank.

RESULTS Ctf13p is one of three proteins that binds to the CDEIII sequence of yeast centromeres, and cells limited for Ctf13p grow slowly because the kinetochore/spindle checkpoint is activated (22, 39). Temperature-sensitive ctf13 mutants grow slowly at 328C, but double mutants with ctf13 and a spindle checkpoint mutation such as mad1, mad2, bub1, or bub3 are inviable at 328C (39). We identified CDC55 by using this synthetic lethal phenotype to search for additional checkpoint mutants by screening among mutagenized colonies of strain YPH280R (ctf13) for three phenotypes indicative of checkpoint mutants. First, we screened for colonies that could grow at 238C but not 328C (Fig. 1, row c); wild-type cells grew at both temperatures (Fig. 1, row e). To restrict our analysis to mutants that were temperature sensitive when limited for Ctf13p function, we transformed the candidate mutants with a plasmid containing CTF13 and tested for growth at 328C (Fig. 1, row b). Any CTF13-containing transformant that could not grow at 328C had an independent mutation causing temperature sensitivity and was not considered further. Second, we identified candidates, such as strain 1634 (Fig. 1, row c), that were benomyl sensitive by measuring viability after incubating them on agar medium containing a sublethal concentration of the drug. Mutations in genes that affect the kinetochore/spindle checkpoint result in cells that are more sensitive to benomyl than wild-type cells are (Fig. 1, row c) (17, 24). Third, we determined if the putative double mutants were delayed in the cell cycle by antitubulin immunofluorescent staining of cells. The ctf13 mutation in the parent strain YPH280R causes cells to delay at mitosis when incubated at the restrictive temperature. Double mutants that have ctf13 and either mad1, mad2, bub1, or bub3 do not delay because the kinetochore/spindle checkpoint is compromised (39). Therefore, if the putative mutants defined checkpoint components, they should also be incapable of delaying the cell cycle. Most ctf13 mutants (68%, n 5 200) had short spindles and undivided nuclei after a 3-h incubation at 388C because of the action of the kinetochore/spindle checkpoint (Fig. 2A). In contrast, some mutants, such as strain 1634 (Fig. 2B), had a reduced number of cells with short spindles and undivided nuclei (15%, n 5 200), suggesting that the double mutants could not maintain the ctf13-induced cell cycle delay. The microtubule staining in cells from strain 1634 was unusual in some respects. After 3 h at 388C, 68% of the cells were unbudded cells with a single spot of staining and looked

FIG. 1. Characterization of mitotic checkpoint mutants. Benomyl-sensitive checkpoint mutants are sensitive to a loss of ctf13 function. Cultures were grown to stationary phase, serially diluted 10-fold, spotted onto YEPD plates at 23 or 328C and onto YEPD plates containing benomyl, and incubated at 238C. Rows: a, strain 1634 (ctf13 cdc55-100 [CDC55]); b, strain 1634 (ctf13 cdc55-100 [CTF13]); c, strain 1634 (ctf13 cdc55-100); d, strain YPH280R (ctf13); e, strain 5943-4 (CTF13 CDC55). The arrow represents a contaminating bacterial colony.

622

WANG AND BURKE

FIG. 2. Checkpoint mutants cannot maintain a mitotic delay. (A and B) Spindle morphologies of cells after growth at the restrictive temperature. Cells of YPH1293R (ctf13) (A) and 1634 (ctf13 cdc55-100) (B) were incubated at 388C for 3 h, fixed with formaldehyde, and stained with an antitubulin antibody. (C) Histone H1 kinase assay. Nocodazole (10 mg/ml) was added to a culture of YPH1293R (ctf13) and 1634 (ctf13 cdc55-100) cells after growth to mid-log phase. The cell cultures were incubated at 238C, 25 ml of cells was removed after 3 and 5 h of incubation, and the cell extracts were used for H1 kinase assays. Lanes: 1, ctf13, 3 h; 2, ctf13, 5 h; 3, (ctf13 cdc55-100), 3 h; 4, (ctf13 cdc55-100), 5 h.

like cells in the G1 stage of the cell cycle. Another 16% of the cells had two spots of staining, suggesting that there were multiple spindle poles in a single cell lacking normal spindle structure. Only 1% of the cells contained long spindles and separated nuclei. We conclude that the cells from strain 1634 were unable to maintain a prolonged ctf13-induced G2 delay, and many of the cells continued onto the next cell cycle. Mutants that were temperature sensitive, benomyl sensitive, and unable to maintain the ctf13-induced cell cycle delay were considered further. We crossed all of the mutants to ctf13 mad1, ctf13 mad2, ctf13 mad3, ctf13 bub1, and ctf13 bub3 double mutants, selected diploids, and tested for complementation of the synthetic lethality by growth at 328C. We identified four alleles of BUB1, five alleles of BUB3, one allele of MAD1, two alleles of MAD2, and three alleles of MAD3. These mutants were not considered further. Finally, we prepared protein extracts from the candidate mutants and tested for histone H1 kinase activity. Cells that are arrested by nocodazole treatment or by mutations that trigger the kinetochore spindle checkpoint, such as ctf13, have high levels of histone H1 kinase activity (1, 36). In contrast, spindle checkpoint mutants are unable to arrest and maintain high levels of histone kinase when treated with nocodazole or when checkpoint mutation are combined with ctf13 (17, 24). We identified several mutants, like strain 1634, that were unable to maintain high levels of histone H1 kinase activity (Fig. 2C) when treated with nocodazole. We crossed the double mutants to obtain ctf13/ctf13 diploids and determined that growth at 328C was recessive in each case. The diploids were sporulated, and the resulting haploid spores were tested for 2:2 segregation of the 328C temperature sensitivity. After screening an initial 40,000 colonies, we retained four strains and showed by complementation that they carried three new checkpoint genes. We cloned the genes by transforming the strains with a YCp-based plasmid library and selecting for plasmids that complemented both the temperature sensitivity and the nocodazole sensitivity. Plasmids were recovered in E. coli and transformed into the original mutant strains to confirm that the complementing activity was plasmid linked.

MOL. CELL. BIOL.

We mutagenized the plasmids with Tn5 to identify the complementing region of the DNA and sequenced from Tn5 into the complementing sequence. Two genes that we identified were novel and will be described elsewhere. In the remaining case, we found that Tn5 was inserted into a gene that was previously identified, the CDC55 gene, suggesting that the complementation in strain 1634 was due to Cdc55p. Cdc55p is homologous to the mammalian regulatory B subunit of the trimeric phosphoprotein phosphatase PP2A. CDC55 is essential only at low temperatures, and strains depleted of Cdc55p activity divide very slowly at 128C and become elongated with abnormal buds (13). We tested strain 1634 and determined that it was also cold sensitive and had an abnormal budding morphology (38). To confirm that the checkpoint mutant phenotype in strain 1634 resulted from a loss of CDC55, we tested the nocodazole sensitivity of strain CY5580 containing a cdc55::LEU2 deletion mutation. The data (Fig. 3) show that the cdc55 deletion mutation was nocodazole sensitive. The TPD3 gene encodes a homolog of the regulatory A subunit, which is also a subunit of PP2A. To determine if both regulatory subunits of PP2A functioned in the checkpoint, we tested the nocodazole sensitivity of strain CY1667, which has a tpd3::LEU2 deletion mutation. The data (Fig. 3) show that the tpd3 mutant, like the cdc55 mutants, was nocodazole sensitive. We constructed a ctf13 cdc55::LEU2 double-mutant strain (1638-2-2) and determined that the cells were unable to form colonies at 328C. In addition, the double mutants could not maintain the ctf13-induced cell cycle delay. Cells incubated at 388C had a low number of short spindles as determined from antitubulin staining, and protein extracts prepared from such cells had low levels of histone H1 kinase activity (38). Therefore, the cdc55 null mutant behaved similarly to strain 1634. To determine if strain 1634 had an allele of cdc55, we tested for complementation of the nocodazole sensitivity. Nocodazole sensitivities of a strain with cdc55::LEU2 and strain 1634 are both recessive. Furthermore, the mutations fail to complement the nocodazole sensitivity in diploids. We sporulated the noncomplementing diploid, dissected 10 tetrads, and determined that the benomyl sensitivity segregated 4:0. Therefore, strain 1634 has a new allele of CDC55 that we

FIG. 3. Plating efficiency of cells after growth in medium containing 10 mg of nocodazole per ml. Nocodazole was added to cell cultures to a final concentration of 10 mg/ml. Cells were removed at different times (hours), sonicated, and spread onto YEPD plates. The plating efficiency was counted after overnight incubation at 238C. Circles, wild-type strain 5943-4; triangles, ctf13 mad1 strain 793-7-4; squares, ctf13 cdc55-100 strain 1634; inverted triangles, cdc55::LEU2 strain CY 5580; diamonds, tpd3::LEU2 strain CY1667. All cells were incubated at 238C except tpd3::LEU2, cells, which were incubated at 308C. Data for strain 793-7-4 (mad1) are included as a positive control for nocodazole sensitivity.

VOL. 17, 1997

FIG. 4. Sensitivity of cdc55 cells to g radiation and hydroxyurea. (A) Sensitivity to g radiation. Cells were grown to 107/ml in YM-1, sonicated, then plated onto YEPD plates, and exposed to g radiation for various times. The plating efficiency was determined by counting the surviving colonies after overnight incubation at 238C. Triangles, wild-type strain 5943-4; circles, cdc55::LEU2 strain CY5580; squares, mec1-1 strain TWY308. (B) Sensitivity to hydroxyurea. Cells were grown to 107/ml, and hydroxyurea was added to a concentration of 2.5 mg/ml for different times (in hours). Aliquots were removed, sonicated, and spread onto YEPD to determine the plating efficiency. Squares, TWY308 mec1-1; circles, CY5580 (cdc55::LEU2).

have named cdc55-100. Strain 1634 (ctf13 cdc55-100) carrying the complementing CDC55 gene on a plasmid showed wildtype levels of resistance to benomyl (Fig. 1, row a) and grew as well as the ctf13 single mutant at 328C (Fig. 1, row d). These data implicate CDC55, and hence PP2A, in the spindle/kinetochore checkpoint. We determined if PP2A was required to arrest the cell cycle in response to DNA damage or to a drug that inhibits DNA synthesis by treating cells with g radiation and hydroxyurea. Cells of strain CY5580 (cdc55::LEU2) are resistant and remain viable when treated with increasing doses of g radiation (Fig. 4A). In contrast, mec1 mutants, defective for the DNA damage checkpoint (40), are quite sensitive (Fig. 4A). Similarly, cells of strain CY5580 (cdc55::LEU2) retained viability when incubated in hydroxyurea, but the mec1 mutant lost viability after short incubations in the drug (Fig. 4B). Lin and Arndt showed that a pph21 pph22 double mutant grows slowly because cells are limited for the mitotic function of the catalytic subunit of PP2A (25). pph21 pph22 double mutants do not dramatically affect bud morphology, but a pph21 pph22 mih1 triple mutant looks very similar to a cdc55 mutant (25). The MIH1 gene encodes the S. cerevisiae homolog of the S. pombe cdc251 gene, encoding the dual-specificity phosphatase that is a key regulator of CDC28/cdc21 (33). In addition, the cellular morphogenesis phenotype of the pph21 pph22 mih1 triple mutant is suppressed by cdc28F19, an allele of CDC28 that eliminates the site of inhibitory tyrosine phosphorylation. Therefore, cells that are limited for PP2A activity have abnormal morphologies when Cdc28p activity is inhibited by tyrosine phosphorylation. To determine if the phenotype of cdc55 was also dependent on CDC28, we constructed strain 1652-2-1 (cdc55::LEU2 cdc28F19) and compared the growth and morphology of cells after growth at 128C. The data (Fig. 5) show that cdc28F19 suppressed both the cold sensitivity and the abnormal bud morphology of cdc55. To determine if cdc28F19 suppressed the nocodazole sensitivity of cdc55, we measured the viability of cells from a cdc55 cdc28F19 strain after growth in the presence of nocodazole. The data (Fig. 6) show that the double mutant is as sensitive to nocodazole as the cdc55 single mutant, suggesting that cdc28F19 cannot revert the nocodazole sensitivity. We have recently identified two other genes, ZDS1 and ZDS2, in a different unrelated genetic screen. Interestingly, the zds1 zds2


623

FIG. 5. The cold sensitivity and abnormal morphology of cdc55 cells were suppressed by cdc28F19 mutation. (A) Cells were grown to saturation at 238C, and serial 10-fold dilutions were spotted onto a YEPD plate and incubated at 128C for 10 days. Rows: a, cdc55::LEU2 cdc28F19; b, cdc55::LEU2; c, cdc28F19. (B) Cells were grown to 107/ml and shifted to 128C for 24 h. The cells were observed after staining with calcofluor.

(zds for zillions of different screens) double mutant is cold sensitive and has a multibudded phenotype very similar to that of a cdc55 mutant (2, 45). In addition, the cold sensitivity and abnormal bud morphologies of the zds1 zds2 double mutant are also suppressed by cdc28F19 (4). This finding suggests that a loss of Zds1p and Zds2p affects the state of phosphorylation of Cdc28p. However, zds1 zds2 double mutants are not nocodazole sensitive. Therefore, not all mutants that influence tyrosine phosphorylation of Cdc28p are components of the kinetochore/spindle checkpoint. Cells arrested by the kinetochore/spindle checkpoint have high levels of Clb2p-dependent Cdc28p kinase activity and sister chromatids that are unseparated. Somehow the checkpoint inactivates the ubiquitin-dependent proteolytic activity that has been termed the anaphase-promoting complex (APC) and that normally degrades cyclins and destroys putative cohesive proteins that hold sister chromatids together (18, 43). It was possible that the kinetochore/spindle checkpoint was functional in cdc55 mutants but that somehow the mutants were able to bypass the requirement for an active APC. For example, if the cohesive proteins that hold sister chromatids together were regulated by phosphorylation/dephosphorylation, then inactive cohesion proteins may accumulate in cdc55 mutants treated with nocodazole and sister chromatids would

FIG. 6. Nocodazole sensitivity of cdc55 single mutants and cdc55 cdc28F19 double mutants. Cells were grown in medium containing 10 mg of nocodazole per ml for different times at 238C. Cells were sonicated and spread onto YEPD plates to determine the plating efficiency. Circles, wild-type strain 5943-4; squares, cdc55::LEU2 strain CY5580; triangles, cdc55::LEU2 cdc28F19 strain 1652-2-1.

624

WANG AND BURKE

FIG. 7. A cdc55 deletion mutation suppresses the temperature sensitivity of cdc20-1. Serial 10-fold dilutions of CDC55 CDC20 (row a), cdc20-1 (row b), and cdc20-1 cdc55::LEU2 (row c) strains were spotted onto YEPD plates and incubated at different temperature for 2 days (348C) or 3 days 238C.

separate. If that is the case, then separating chromatids should be independent of activating the APC. To test this, we constructed strains with two mutations; cdc55::LEU2 and either cdc16-1 or cdc23-1, temperature-sensitive mutations in genes that encode components of the APC (18). In both cases, the double mutants were temperature sensitive and arrested with the terminal phenotypes of cdc16 and cdc23 strains, and in neither case was the viability of the double mutants different from the viability of the individual single mutants. Therefore, cdc55 mutants cannot continue in the cell cycle when the APC is inhibited. We tested other cdc mutants to try and gain insights into other possible functions for Cdc55p. We found that the temperature sensitivity of cdc20-1 could be suppressed by cdc55:: LEU2 (Fig. 7). CDC20 encodes a protein that contains WD-40 repeats and affects mitotic spindle assembly (30, 34). The terminal morphology of cdc20 mutants is similar to that of cdc16 and cdc23 mutants; they arrest after DNA replication but prior to nuclear division. Therefore, CDC55 has multiple functions in the cell cycle. Cdc55p affects bud morphogenesis, the kinetochore/spindle checkpoint and mitotic spindle assembly. DISCUSSION We have applied a new screen for kinetochore/spindle checkpoint mutants and have identified an allele of the CDC55 gene. The new allele of cdc55 confers a phenotype similar to those conferred by a cdc55 deletion mutation and a tpd3 deletion mutation, suggesting that the regulatory subunits of PP2A have more diverse functions than previously suspected. The cold sensitivity and the morphological phenotypes of cdc55 strains are genetically separable from the checkpoint phenotype because the former could be suppressed by cdc28F19 but the latter could not. Furthermore, the cell cycle response to DNA damage and a DNA synthesis inhibitor is unaffected by cdc55, suggesting a unique role for Cdc55p in the kinetochore/spindle checkpoint. Additional roles for Cdc55p in mitosis are suggested by suppression of the temperature sensitivity of cdc20 by cdc55::LEU2. Two previous screens identified strains with a nonoverlapping set of checkpoint mutations (bub and mad) on the basis of the inability to arrest in response to nocodazole (17, 24). Identifying two nonoverlapping set of mutations suggested that there were more genes to be identified. We used a different approach for identifying checkpoint mutants based on our previous observation that certain mad and bub mutants were synthetically lethal in combination with ctf13 (39). Our screen was successful in that we identified alleles of the bub and mad mutants in addition to four mutations that define three additional checkpoint genes. Molecular analysis showed that one of the genes was CDC55, which had previously been implicated in cellular morphogenesis (13). Three data suggest that CDC55 is part of the kinetochore/

MOL. CELL. BIOL.

spindle checkpoint. First, ctf13 cdc55 double mutants cannot maintain a mitotic delay when limited for Ctf13p function. Second, the cdc55 mutants are nocodazole sensitive. Third, the cdc55 mutants cannot maintain high levels of histone H1 kinase activity when kinetochore function is impaired. Some role for protein phosphatases in the kinetochore/spindle checkpoint is expected, given the identification of protein kinases and phosphorylated proteins in the checkpoint pathway. Bub1p is a protein kinase, and Mad1p is a phosphoprotein that becomes phosphorylated in response to nocodazole (9, 31). Our data suggest that the regulatory subunits of PP2A are also required for the checkpoint pathway. Regulatory subunits can have either a negative or a positive effect on the catalytic subunit of PP2A, depending on the substrate (41). We do not know, a priori, whether a loss of Cdc55p causes PP2A hyperactivity or inactivity resulting in the checkpoint phenotype. One possibility is that one of the components of the signal transduction pathway must be dephosphorylated to function. Cdc55p may direct PP2A to dephosphorylate the hypothetical protein when the checkpoint is active. In this way, Cdc55p would act as a positive effector. The alternative explanation is that Cdc55p has an inhibitory function. If chromosomes are not attached to the spindle, the checkpoint is activated by the action of protein kinases that inhibit anaphase. When all of the chromosomes are aligned on the spindle, the signal must be extinguished so that cells may proceed to anaphase. PP2A may be required to reverse the effect of the protein kinases. To prevent the premature reversal of the signal, Cdc55p would inhibit PP2A as long as the checkpoint is activated. When the chromosomes are properly attached to the spindle, Cdc55p may become inactive, and PP2A would become active and alleviate the signal to arrest cell division. The role of Cdc55p in this model is to promote both the arrest when chromosomes are unattached and the recovery from arrest when it is time to proceed into anaphase. Genetic analysis of PP2A has suggested mitotic functions in both S. pombe and Drosophila melanogaster (8, 26, 44). The aar1 gene of D. melanogaster encodes the B subunit of PP2A and affects both spindle structure and anaphase resolution of sister chromatids (8, 26). Interestingly, chromosomes often lag during anaphase in the aar1 mutant, suggesting that anaphase was initiated before chromosomes were attached to the spindle. The regulatory subunit of PP2A may also be required for the spindle/kinetochore checkpoint in D. melanogaster. There are also effects on the mitotic spindle structure in aar1 mutants (8). The abnormal resolution of chromosomes may be due to the combination of aberrant spindle function and inactivity of the kinetochore/spindle checkpoint in aar1 mutants, suggesting that the regulatory subunit has multiple roles in mitosis. Previous data suggest that Cdc55p plays a role in cellular morphogenesis by controlling the pattern of cell surface growth and the formation of septa, perhaps through an interaction with Bem2p (19). We found that the essential function of CDC55 and the abnormal bud morphology of a cdc55 mutant could be suppressed by preventing tyrosine phosphorylation of Cdc28p by using the allele cdc28F19. The role of phosphorylation of tyrosine residue 19 of Cdc28p in S. cerevisiae is different from the role in other organisms where the phosphorylation on the cognate tyrosine residue of the Cdk1 is required for mitotic timing. However, in S. cerevisiae, the tyrosine phosphorylation is the target of a morphogenetic checkpoint that coordinates bud growth with nuclear division (23). Cells that do not bud will delay at nuclear division, presumably to allow sufficient time for the bud to form. Nuclear division is delayed because cells accumulate the phosphorylated form of Cdc28p. cdc28F19 may suppress the cold sensitivity and abnormal mor-

VOL. 17, 1997

phology of cdc55 because the mutant phenotype is due to the morphogenetic checkpoint. At reduced temperatures, cdc55 mutants bud abnormally and probably activate the checkpoint. Bud growth continues, although it is abnormal and the checkpoint signals for tyrosine 19 phosphorylation that inactivates Cdc28p. Budded cells depleted of Cdc28p activity will continue polarized growth at bud tips and form elongated cells, and they look similar to cdc55 cells that are incubated at the restrictive temperature (13). Alternatively, Cdc55p may be required to maintain the dephosphorylated form of Cdc28p in the normal mitotic cycle. At reduced temperatures, cells divide slowly and perhaps are cold sensitive for a process required to complete the G2-to-M transition. In the absence of Cdc55p, sufficient quantities of the tyrosine-phosphorylated Cdc28p may accumulate and the cells, delayed in G2/M, shift to the polarized growth. Interestingly, there are other mutants that have phenotypes similar to those of cdc55 and tpd3 mutants and are suppressed by cdc28F19. For example, several investigators have identified the ZDS1 and ZDS2 genes, suggesting a large number of unrelated genetic interactions (2, 45). We have found that the zds1 zds2 double mutant has a morphology strikingly similar to that of cdc55 and tpd3 mutants and is also suppressed by cdc28F19. Comparison of Zdsp sequences with the available databases has provided no clues as to function, but they are clearly not related to PP2A or any of the regulatory subunits. Either they are also required for bud formation or they maintain the dephosphorylated form of Cdc28p. In addition, pph21 pph22 mih1 triple mutants look morphologically similar to cdc55 mutants and are suppressed by cdc28F19 (25). The effect of Cdc55p on Cdc28p appears to be independent of the checkpoint because cdc55 cdc28F19 mutants are nocodazole sensitive. However, cdc55 is pleiotropic, and the nocodazole sensitivity may be more complex than just the effect on the checkpoint. The activated kinetochore/spindle checkpoint presumably prevents anaphase by inhibiting ubiquitindependent proteolysis by the APC so that cyclin B levels remain high and sister chromatids remain cohesive (16, 18). We propose that Cdc55p is a component of the checkpoint and does not affect a downstream target of the APC because cdc55 cannot bypass mutations in CDC16 and CDC23, genes that encode components of the APC. cdc55 suppresses the temperature sensitivity of cdc20-1. This finding suggests a role for protein phosphorylation in the function of CDC20. Genetic interactions between CDC20 and the two protein kinases Cdc28p and Cdc5p have been described (21, 45). It is clear that cdc55 cannot completely bypass the essential requirement for Cdc20p because cdc55::LEU2 suppresses the temperature sensitivity of cdc20-1 at 348C but cannot suppress the temperature sensitivity at the fully restrictive temperature (368C) (38). Therefore, the lack of Cdc55p improves the function of a partially active Cdc20p. Perhaps Cdc20p is active as a phosphoprotein that is inactivated by PP2A in a CDC55-dependent manner. In a cdc20 mutant growing at 348C, there may be little of the active phosphoprotein, and in the cdc20 cdc55 double mutant, more of the phosphorylated form accumulates, which results in suppression. Regardless of the molecular explanation for the suppression, the genetic interactions extend the roles of CDC55 and suggest that it functions at multiple times and places in the cell cycle. In addition to the documented roles in cellular morphogenesis, Cdc55p has important roles in mitosis and in kinetochore/ spindle checkpoint regulation.


625

ACKNOWLEDGMENTS This work was supported by NIH grant GM 40334. We thank Kim Arndt, Ted Weinert, Doug Koshland, and Phil Heiter for providing strains. We also thank Penny Tavormina, Ted Weinert, and Danny Lew for many useful discussions and Penny Tavormina for comments on the manuscript. REFERENCES 1. Amon, A., U. Surana, I. Muroff, and K. Nasmyth. 1992. Regulation of p34CDC28 tyrosine phosphorylation is not required for entry into mitosis in S. cerevisiae. Nature 355:368–371. 2. Bi, E., and J. R. Pringle. 1996. ZDS1 and ZDS2, genes whose products may regulate Cdc42p in Saccharomyces cerevisiae. Mol. Cell. Biol. 16:5264–5275. 3. Carr, A. M. 1994. Radiation checkpoints in model systems. Int. J. Radiat. Biol. 66:S133–S139. 4. Clark, D. J. 1996. A genetic analysis of high copy suppressors of cdc20-1 in the yeast Saccharomyces cerevisiae. Thesis. University of Virginia, Charlottesville. 5. Enoch, T., and P. Nurse. 1990. Mutation of fission yeast cell cycle control genes abolishes dependence of mitosis on DNA replication. Cell 60:665–673. 6. Evangelista, C. C., A. M. Rodriguez Torres, M. P. Limbach, and R. S. Zitomer. 1996. Rox3 and Rts1 function in the global stress response pathway in baker’s yeast. Genetics 142:1083–1093. 7. Gietz, D., A. St. Jean, R. A. Woods, and R. H. Schiestl. 1992. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 20:1425. 8. Gomes, R., R. E. Karess, H. Okhura, D. M. Glover, and C. E. Sunkel. 1993. Abnormal anaphase resolution (aar): a locus required for progression through mitosis in Drosophila. J. Cell Sci. 104:1–11. 9. Hardwick, K. G., and A. W. Murray. 1995. Mad1p, a phosphoprotein component of the spindle assembly checkpoint in budding yeast. J. Cell Biol. 131:709–720. 10. Hartwell, L. 1967. Macromolecular synthesis in temperature sensitive mutants of yeast. J. Bacteriol. 93:1662–1670. 11. Hartwell, L., T. Weinert, L. Kadyk, and B. Garvik. 1994. Cell cycle checkpoints, genomic integrity, and cancer. Cold Spring Harbor Symp. Quant. Biol. 59:259–263. 12. Hartwell, L. H., and M. B. Kastan. 1994. Cell cycle control and cancer. Science 266:1821–1828. 13. Healy, A. M., S. Zolnierowicz, A. E. Stapleton, M. Goebl, A. A. DePaoliRoach, and J. R. Pringle. 1991. CDC55, a Saccharomyces cerevisiae gene involved in cellular morphogenesis: identification, characterization, and homology to the B subunit of mammalian type 2A protein phosphatase. Mol. Cell. Biol. 11:5767–5780. 14. Hoffmann, I., and E. Karsenti. 1994. The role of cdc25 in checkpoints and feedback controls in the eukaryotic cell cycle. J. Cell Sci. 18:75–79. 15. Hoffmann, R., S. Jung, M. Ehrmann, and H. W. Hofer. 1994. The Saccharomyces cerevisiae gene PPH3 encodes a protein phosphatase with properties different from PPX, PP1 and PP2A. Yeast 10:567–578. 16. Holloway, S. L., M. Glotzer, R. W. King, and A. W. Murray. 1993. Anaphase is initiated by proteolysis rather than by the inactivation of maturationpromoting factor. Cell 73:1393–1402. 17. Hoyt, M. A., L. Totis, and B. T. Roberts. 1991. S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell 66:507–517. 18. Irniger, S., S. Piatti, C. Michaelis, and K. Nasmyth. 1995. Genes involved in sister chromatid separation are needed for b-type cyclin proteolysis in budding yeast. Cell 81:269–278. 19. Kim, Y. J., L. Francisco, G. C. Chen, E. Marcotte, and C. S. Chan. 1994. Control of cellular morphogenesis by the Ip12/Bem2 GTPase-activating protein: possible role of protein phosphorylation. J. Cell Biol. 127:1381–1394. 20. Kinoshita, N., H. Yamano, H. Niwa, T. Yoshida, and M. Yanagida. 1993. Negative regulation of mitosis by the fission yeast protein phosphatase ppa2. Genes Dev. 7:1059–1071. 21. Kitada, K., A. L. Johnson, L. H. Johnston, and A. Sugino. 1993. A multicopy suppressor gene of the Saccharomyces cerevisiae G1 cell cycle mutant gene dbf4 encodes a protein kinase and is identified as CDC5. Mol. Cell. Biol. 13:4445–4457. 22. Lechner, J., and J. Carbon. 1991. A 240 kd multisubunit protein complex, CBF3, is a major component of the budding yeast centromere. Cell 64:717– 725. 23. Lew, D. J., and S. I. Reed. 1995. A cell cycle checkpoint monitors cell morphogenesis in budding yeast. J. Cell Biol. 129:739–749. 24. Li, R. and A. W. Murray. 1991. Feedback control of mitosis in budding yeast. Cell 66:519–531. 25. Lin, F. C., and K. T. Arndt. 1995. The role of Saccharomyces cerevisiae type 2A phosphatase in the actin cytoskeleton and in entry into mitosis. EMBO J. 14:2745–2759. 26. Mater-Jaekel, R. E., H. Ohkura, R. Gomes, C. E. Sunkel, S. Bumgartner, B. A. Hemmings, and D. M. Glover. 1993. The 55-Kda regulatory subunit of the Drosophila protein phosphatase 2A is required for anaphase. Cell 72: 621–633.

626

WANG AND BURKE

27. Mayer-Jaekel, R. E., H. Ohkura, P. Ferrigno, N. Andjelkovic, K. Shiomi, T. Uemura, D. M. Glover, and B. A. Hemmings. 1994. Drosophila mutants in the 55 kDa regulatory subunit of protein phosphatase 2A show strongly reduced ability to dephosphorylate substrates of p34cdc2. J. Cell Sci. 107:2609–2616. 28. Moreno, S., J. Hayles, and P. Nurse. 1989. Regulation of p34cdc2 protein kinase during mitosis. Cell 58:361–372. 29. Murray, A. W. 1995. The genetics of cell cycle checkpoints. Curr. Opin. Genet. Dev. 5:5–11. 30. O’Toole, E., D. N. Mastronarde, T. H. Giddings, M. Winey, D. J. Burke, and J. R. McIntosh. Three dimensional analysis and ultrastructural design of mototic spindles form the cdc20 mutant of Saccharomyces cerevisiae. Mol. Biol. Cell, in press. 31. Roberts, B. T., K. A. Farr, and M. A. Hoyt. 1994. The Saccharomyces cerevisiae checkpoint gene BUB1 encodes a novel protein kinase. Mol. Cell. Biol. 14:8282–8291. 32. Rose, M. D., P. Novick, J. H. Thomas, D. Botstein, and G. R. Fink. 1987. A Saccharomyces cerevisiae genomic plasmid bank based on a centromerecontaining shuttle vector. Gene 60:237–243. 33. Russell, P., S. Moreno, and S. I. Reed. 1989. Conservation of mitotic controls in fission and budding yeasts. Cell 57:295–303. 34. Sethi, N., M. C. Monteagudo, D. Koshland, E. Hogan, and D. J. Burke. 1991. The CDC20 gene product of Saccharomyces cerevisiae, a beta-transducin homolog, is required for a subset of microtubule-dependent cellular processes. Mol. Cell. Biol. 11:5592–5602. 35. Sherman, F., G. R. Fink, and J. B. Hicks. 1983. Laboratory course manual for methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 36. Sorger, P. K., and A. W. Murray. 1992. S-phase feedback control in budding

MOL. CELL. BIOL.

37.

38. 39.

40.

41. 42.

43.

44.

45.

yeast independent of tyrosine phosphorylation of p34cdc28. Nature 355:365– 368. van Zyl, W., W. Huang, A. A. Sneddon, M. Stark, S. Camier, M. Werner, C. Marck, A. Sentenac, and J. R. Broach. 1992. Inactivation of the protein phosphatase 2A regulatory subunit A results in morphological and transcriptional defects in Saccharomyces cerevisiae. Mol. Cell. Biol. 12:4946–4959. Wang, Y. 1996. Unpublished observations. Wang, Y., and D. J. Burke. 1995. Checkpoint genes required to delay cell division in response to nocodazole respond to impaired kinetochore function in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol. 15:6838–6844. Weinert, T. A., G. L. Kiser, and L. H. Hartwell. 1994. Mitotic checkpoint genes in budding yeast and the dependence of mitosis on DNA replication and repair. Genes Dev. 8:652–665. Wera, S., and B. A. Hemmings. 1995. Serine/threonine protein phosphatases. Biochem. J. 311:17–29. Williamson, D. H., and D. J. Fennell. 1975. The use of fluorescent DNAbinding agent for detecting and separating yeast mitochondrial DNA. Methods Cell Biol. 12:335–351. Yamamoto, A., V. Guacci, and D. Koshland. 1996. Pds1p, an inhibitor of anaphase in budding yeast, plays a critical role in the APC and checkpoint pathway(s). J. Cell Biol. 133:99–110. Yanagida, M., N. Kinoshita, E. M. Stone, and H. Yamano. 1992. Protein phosphatases and cell division cycle control. Ciba Found. Symp. 170:130– 140. Yu, Y., Y. W. Jiang, R. J. Wellinger, K. Carlson, J. M. Roberts, and D. J. Stillman. 1996. Mutations in the homologous ZDS1 and ZDS2 genes affect cell cycle progression. Mol. Cell. Biol. 16:5254–5263.