Dominant Alleles of Saccharomyces cerevisiae CDC20 Reveal Its Role in Promoting Anaphase

Eric J. Schott and M. Andrew Hoyt
Genetics February 1, 1998 vol. 148 no. 2 599-610

Abstract

We identified an allele of Saccharomyces cerevisiae CDC20 that exhibits a spindle-assembly checkpoint defect. Previous studies indicated that loss of CDC20 function caused cell cycle arrest prior to the onset of anaphase. In contrast, CDC20-50 caused inappropriate cell cycle progression through M phase in the absence of mitotic spindle function. This effect of CDC20-50 was dominant over wild type and was eliminated by a second mutation causing loss of function, suggesting that it encodes an overactive form of Cdc20p. Overexpression of CDC20 was found to cause a similar checkpoint defect, causing bypass of the preanaphase arrest produced by either microtubule-depolymerizing compounds or MPS1 overexpression. CDC20 overexpression was also able to overcome the anaphase delay caused by high levels of the anaphase inhibitor Pds1p, but not a mutant form immune to anaphase-promoting complex- (APC-)mediated proteolysis. CDC20 overexpression was unable to promote anaphase in cells deficient in APC function. These findings suggest that Cdc20p is a limiting factor that promotes anaphase entry by antagonizing Pds1p. Cdc20p may promote the APC-dependent proteolytic degradation of Pds1p and other factors that act to inhibit cell cycle progression through mitosis.

ACCURATE chromosome segregation during eukaryotic cell division requires the precise assembly and function of the mitotic spindle. To ensure that replicated chromosomes are properly attached to a functioning spindle, the cell utilizes a surveillance-feedback mechanism, referred to as the spindle-assembly checkpoint (reviewed in Elledge 1996; Rudner and Murray 1996; Wells 1996). In the presence of spindle-assembly defects, this mechanism acts to inhibit progression into anaphase, the chromosome segregation stage of mitosis. It has been observed that the spindle-assembly checkpoint can respond to defects in the functions of microtubules, kinetochores, spindle pole structures, and spindle microtubule-based motor proteins.

Studies of the budding yeast Saccharomyces cerevisiae have revealed seven genes (BUB1, 2, and 3, MAD1, 2, and 3, and MPS1) whose functions are required to properly arrest cell cycle progression following spindle damage. Homologs of Mad2p and the Bub1p protein kinase have been localized to the kinetochores of vertebrate cells (Chenet al. 1996; Li and Benezra 1996; Taylor and McKeon 1997). In addition, the physical associations of S. cerevisiae Mad1p with Mad2p (cited in Rudner and Murray 1996) and Bub1p with Bub3p (Robertset al. 1994) have been demonstrated. These findings are consistent with the hypothesis that the signal indicating improperly attached chromosomes originates from kinetochores (Nicklas 1997). Although the site of action of the Mps1p protein kinase has not been determined, it is believed to act at an early step in the generation of the spindle-damage signal. Overexpression of MPS1 is able to block cell cycle progression prior to anaphase in a manner dependent upon the functions of the six MAD and BUB genes (Hardwicket al. 1996).

The mechanism by which the transduced spindle-damage signal blocks entry into anaphase is currently not known. Likely targets for this regulation are identified factors that control anaphase onset. Both entry into anaphase and subsequent exit from mitosis require the actions of the anaphase-promoting complex (APC, also known as the cyclosome), a multisubunit ubiquitin ligase (Kinget al. 1996). Ubiquitination mediated by the APC targets protein substrates to the degradative actions of the 26S proteasome. S. cerevisiae Pds1p, a target of APC-mediated degradation, acts as an anaphase inhibitor (Cohen-Fixet al. 1996; Yamamoto et al. 1996a,b). Cells deficient for Pds1p enter anaphase precociously or under conditions in which anaphase onset should be inhibited. Pds1p is normally degraded in anaphase in an APC-dependent manner, and nondegradable mutant forms inhibit anaphase onset. Therefore, it seems likely that a key regulatory event controlling anaphase initiation in S. cerevisiae is the degradation of Pds1p by the APC.

Entry into anaphase is coupled to proper assembly of the mitotic spindle. Kinesin-related Cin8p is the major mitotic motor protein responsible for spindle assembly and elongation in S. cerevisiae, but is not essential for viability because of the overlapping activities of other motors (Roofet al. 1992; Saunders and Hoyt 1992; Saunderset al. 1995). In a recent study, we identified a large number of mitosis-specific genes whose functions become essential in the absence of Cin8p (Geiseret al. 1997). A subset of these included the spindle-assembly checkpoint genes BUB1, 2, and 3, MAD1 and 2, and MPS1. In addition, mad3Δ caused a deleterious but nonlethal effect in cin8 cells. A reasonable explanation for this finding is that Cin8p-deficient cells require the spindle-assembly checkpoint to delay anaphase entry because their spindles are assembling inefficiently.

In addition to mutants deficient in previously characterized spindle-assembly checkpoint genes, a novel checkpoint mutant, PAC5-1, was identified in the screen for mutants that perish in the absence of CIN8 (Geiseret al. 1997). A property unique to this mutant allele is dominance for its lethal-with-cin8Δ phenotype, suggesting that it does not simply cause loss of function. In studies reported here, we demonstrate that PAC5-1 is a mutant allele of CDC20, a gene required for anaphase entry (Sethiet al. 1991; O'Tooleet al. 1997). (PAC5-1 is redesignated here as CDC20-50.) We demonstrate that CDC20-50 is dominant for the spindle-assembly checkpoint defect as well and that this effect could be mimicked by overexpression of CDC20. Overexpression of CDC20 could bypass the cell cycle arrest caused by microtubule-depolymerizing compounds or MPS1 overexpression. In addition, CDC20 overexpression was found to bypass the arrest caused by elevated levels of the anaphase inhibitor Pds1p, but not the arrest caused by a Pds1p mutant immune to APC-mediated destruction or the arrest caused by an APC mutant defect. These findings reveal a role for Cdc20p in promoting anaphase as an antagonist of Pds1p and are consistent with the recent observation that APC-mediated Pds1p degradation requires Cdc20p function (Visintinet al. 1997).

MATERIALS AND METHODS

Yeast strains and media: The S. cerevisiae strains used in these experiments are listed in Table 1. Strains MAY2830 and MAY2831 are cin8Δ cyh2 and carry a plasmid (pMA1208) with CIN8 and CYH2 (Geiseret al. 1997). The loss of pMA1208 allows cells to grow on media containing cycloheximide because of loss of the dominant CYH2 allele that causes sensitivity. The CDC20-50 allele, previously known as PAC5-1, was identified because it prevents the segregation of cycloheximide-resistant cells in the MAY2830/2831 background (Geiseret al. 1997). MAY4438 (a gift from M. Winey) carries a chromosomally integrated, galactose-inducible, and functional MPS1 tagged with myc (Hardwicket al. 1996). Strains expressing PDS1 or the destruction box mutant form pds1-mdb (Cohen-Fixet al. 1996) were created by transforming MAY2422 with pOC69 or pOC70 cut with EcoRI to direct integration into the LEU2 locus. Yeast transformations were carried out using the lithium acetate procedure (Gietzet al. 1992).

View this table:
TABLE 1

Yeast strains and plasmids

Rich (YPD), minimal (SD), and sporulation media were as described (Shermanet al. 1983). Where noted, methionine was added to 3 mm or omitted to induce expression from the MET25 promoter. For induction of the galactose promoter, cells were grown to log phase in minimal media containing 2% raffinose. Galactose was then added to 2%. To arrest cells in G1, α-factor (Bachem, Torrence, CA) was added to 6 μg/ml to log phase cells growing in rich media at pH 4.0. To arrest cells in S phase, hydroxyurea (Sigma, St. Louis) was added to 100 mm to log phase cells growing in minimal media at pH 5.8. The benzimidazole microtubule inhibitors benomyl (DuPont, Wilmington, DE) and noco-dazole (Sigma) were added to media at 70 μg/ml and 12 μg/ml. Cycloheximide was used at 5 μg/ml.

For the analysis of nuclear morphology by microscopy, cells were fixed in 70% ethanol and stained with 4,6-diamidino-2-phenylindole (DAPI) at 0.3 μg/ml.

Linkage analysis: The diploid strain created by mating MAY4366 and MAY4403 is heterozygous for CDC20-50 (PAC5-1) as well as the chromosome VII loci MAD1 and CYH2. Tetrad analysis of this diploid revealed the linkage of all three loci, with PAC5-1 approximately 8.5 cM from CYH2 (39 parental ditypes, eight tetratypes, and no nonparental ditypes). Less linkage was noted for PAC5-1 and MAD1 (32 parental ditypes, 15 tetratypes, and no nonparental ditypes). This indicated that PAC5-1 mapped 8.5 cM from CYH2, distal to MAD1, a position very close to CDC20.

DNA manipulations: Standard DNA manipulations techniques were utilized (Sambrooket al. 1989). Polymerase chain reaction (PCR) was performed with Vent polymerase according to the manufacturer's directions (New England Biolabs, Beverly, MA). CDC20 was amplified from strains MAY2830, ESY52, and MAY1715, using primers beginning at −181 and +30 relative to the CDC20 open reading frame in the Saccharomyces Genome Database. The 5′ primer was 5′-gctctagaCAGACTAAACCAGAGATC-3′ and the 3′ primer was 5′-cagccggcCATTATATGCCTTGACATG-3′ (lower case letters indicate nonyeast sequence). PCR-generated products were cloned into the SmaI site of pRS316 (Sikorski and Hieter 1989) to create pES26, pES25, and pES37, which carry CDC20, CDC20-50, and cdc20-1, respectively. All clones had the CDC20 open reading frame in the opposite orientation relative to the nucleotide numbering system of Sikorski and Hieter (1989). To put CDC20 alleles downstream from the MET25 promoter, HindIII fragments from the pRS316 CDC20 plasmids (3′ HindIII site from the pRS316 polylinker) were inserted into the HindIII site of p415-MET25 or p416-MET (Mumberget al. 1994). pES40 (carrying cdc20-1) was made by replacing the MscI-Bcl I fragment of pES34 with the corresponding fragment from pES37. pES39 (carrying cdc20-1, 50) was made by replacing the MscI-Bcl I fragment of pES33 with the corresponding fragment from pES37. All cloning junctions and mutant alleles were verified by sequencing.

Sequencing was conducted by the dideoxy chain termination method using the Sequenase kit (United States Biochemical, Cleveland) and analyzed on 6% polyacrylamide gels or by automated analysis by the Johns Hopkins Genetics Core Facility. Sequencing primers for CDC20 were designed based on the sequence found in the Saccharomyces Genome Data Base. CDC20 homologs were found using the BLAST computer program (Altschulet al. 1990). Sequence alignment for the CDC20 homologs was accomplished using the CLUSTAL W program (Thompsonet al. 1994).

Checkpoint assays: For assessment of budding during microtubule disruption, cells were released from α-factor arrest onto solid rich media containing 70 μg/ml benomyl. After 6 hr, cells were examined by light microscopy and the number of cell bodies per microcolony was determined for 200 microcolonies.

For assessment of DNA content during microtubule disruption, log phase cells in liquid media were treated with 12 μg/ml nocodazole. At 0 and 4 hr, samples were fixed in ethanol and stained with propidium iodide (Hutter and Eipel 1978). The DNA content of 10,000 cells was analyzed for each sample using an EPICS 753 flow cytometer.

Sensitivity to DNA damage was assessed by spotting a dilution series of cells suspended in water onto rich agar and then by exposing the plates to increasing doses of UV light (0–300 J/m2) using a Stratalinker (Stratagene, La Jolla, CA). Exposed plates were kept in the dark and incubated for 2 days at 26° and then evaluated for growth. The same dilution series of cells was also spotted onto rich media agar containing 0.01–0.16% methyl methanesulfonate (MMS; from Sigma) and incubated for 3 days prior to evaluation of growth. For cells expressing CDC20 or CDC20-50 from the MET25 promoter, log phase cultures were transferred into minimal media lacking methionine for 1.5 hr prior to the DNA-damaging treatment described above.

RESULTS

CDC20-50 confers a dominant checkpoint defect: Cells deleted for CIN8 grow at wild-type rates at 26°, but undergo prolonged M phase compared to wild-type cells, presumably because of reduced spindle function (Hoytet al. 1992; Saunderset al. 1995). The CDC20-50 mutation (previously referred to as PAC5-1) was identified in a screen for mutants that make CIN8 essential at 26° (Geiseret al. 1997). A cin8Δ cyh2 strain, carrying CIN8 on a plasmid with the counter-selectable marker CYH2, grew well on cycloheximide plates, indicating that it was able to survive loss of the CYH2-CIN8 plasmid (Figure 1A). An isogenic strain that also carries the CDC20-50 mutation was unable to segregate cycloheximide-resistant cells, indicating that they were inviable without the CIN8 plasmid and that the combination of cin8Δ with CDC20-50 is lethal. In CIN8 cells (i.e., in cells with a CIN8 plasmid), CDC20-50 did not cause a defect in growth rate. A similar behavior was exhibited by the bub2-71 spindle-assembly checkpoint mutant identified in the same screen as CDC20-50 (Geiseret al. 1997). A cin8Δ/cin8Δ cyh2/cyh2 diploid, carrying the CIN8-CYH2 plasmid, was able to grow on cycloheximide. Isogenic strains that were either homozygous or heterozygous for CDC20-50 were unable to segregate cycloheximide-resistant cells, indicating that they were inviable without the CIN8 plasmid. The inviability of CDC20/CDC20-50 heterozygotes on cycloheximide media indicated that the lethality conferred by CDC20-50 in the absence of CIN8 is a dominant phenotype. The dominance of CDC20-50 was unique among the pac (perish in the absence of CIN8) mutants previously identified (Geiseret al. 1997).

Figure 1.
Figure 1.

Dominant phenotypes of CDC20-50. (A) Lethality of the CDC20-50 cin8Δ combination. Haploid (top) and diploid (bottom) strains with the genotype indicated on the left were spotted onto rich media with or without 5 μg/ml cycloheximide (cyh) and incubated 2 days at 26°. These strains are also cin8Δ cyh2 (diploids are homozygous) and carry pMA1208 (CIN8 CYH2). The inability to grow on cycloheximide media indicates that the cells cannot survive loss of the CIN8 plasmid. The spot on the right for each sample is a tenfold dilution of the spot on the left. (B) CDC20-50 cells produce extra buds on benomyl-containing media. Haploid (top) and diploid (bottom) cells of the indicated genotype were released from α-factor synchronization onto rich solid media containing 70 μg/ml benomyl. After 6 hr, the numbers of cell bodies per microcolony was determined by microscopy; 4+ indicates four or more cell bodies per microcolony.

Exposing cells to microtubule inhibitors such as benomyl disrupts their mitotic spindles and causes them to arrest in the cell cycle with a preanaphase morphology (large-budded, mononucleate, and with a G2 DNA content). A hallmark phenotype of spindle-assembly checkpoint mutants is continued bud emergence in the presence of microtubule inhibitors. Although we had preliminary evidence that CDC20-50 confers a spindle-assembly checkpoint defect, it was of interest to determine whether this effect was also dominant. Spindle-assembly checkpoint function was assessed in haploid and diploid CDC20-50 mutants by plating cells onto solid media containing a high concentration of benomyl (Figure 1B). Prior to plating, the cells were synchronized in G1 by the α-factor mating pheromone; transfer to the benomyl-containing media released the cells from the α-factor block. After 6 hr, the plates were examined microscopically and the bud morphology of cells was determined. A majority of cells of wild-type haploid and diploid strains arrested with the characteristic large-budded morphology (≥80%). Haploid CDC20-50 cells bypassed the large-budded arrest; over 50% of cells formed one or more extra buds. The extent to which the CDC20-50 strain bypassed the arrest was similar to the bub2-71 spindle-assembly checkpoint mutant strain. In addition, both CDC20-50/CDC20-50 and CDC20/CDC20-50 diploid strains formed extra buds in the presence of high concentrations of benomyl, indicating that CDC20-50 is dominant for the multibudding phenotype as well. The extent to which CDC20-50 diploids bypassed the large-budded arrest was similar to that seen with CDC20-50 haploids. Note that the checkpoint defect is not dependent on the cin8Δ mutation.

Another characteristic of spindle-assembly checkpoint mutants is aberrant initiation of DNA synthesis in the presence of spindle damage, resulting in a greater than G2 DNA content. To examine this, we added the microtubule inhibitor nocodazole to liquid exponential cultures of CDC20-50, bub2-71, and wild-type cells. At 0 and 4 hr after nocodazole addition, samples were removed and processed for flow cytometric analysis of DNA content (Figure 2). The results indicated that, like bub2-71, many CDC20-50 mutant cells performed additional DNA replication steps in the presence of nocodazole-induced microtubule damage, while wild-type cells arrested with a G2 DNA content.

Cloning of the CDC20-50 allele: Initial evidence that PAC5-1 is an allele of CDC20 was obtained from crosses to test its linkage to known spindle-assembly checkpoint genes. Analysis of 47 tetrads from a diploid in which MAD1, CYH2, and PAC5-1 (all on chromosome VII) were heterozygous indicated that PAC5-1 was 8.5 cM centromere distal to CYH2, a position corresponding to that of the CDC20 gene (see materials and methods).

Figure 2.
Figure 2.

Flow cytometric DNA content analysis. Nocodazole was added (12 μg/ml) to exponentially growing cultures of the indicated genotypes. At 0 and 4 hr, samples were prepared for propidium iodide DNA staining and analysis by flow cytometry. The arrows point to cells with a greater than G2 DNA content.

To establish that PAC5-1 is an allele of CDC20, we PCR amplified and cloned the CDC20 gene from both wild-type and PAC5-1 strains (see materials and methods). A 2043 basepair fragment was amplified that included the CDC20 open reading frame plus 161 base pairs 5′ and 49 base pairs 3′. Because the CDC20-50 mutation is dominant, we reasoned that a centromere plasmid carrying CDC20-50 should also confer lethality to cin8Δ cells. When a CDC20 clone derived from the PAC5-1 strain (labeled pCDC20-50 in Figure 3) was transformed into a strain that carried CIN8 on a CYH2 plasmid, it caused a cycloheximide-sensitive phenotype similar to that caused by the original PAC5-1 mutation (Figure 3A). Neither vector alone nor a PCR clone of CDC20 derived from a wild-type strain caused a cycloheximide-sensitive phenotype. Therefore, PAC5-1 (CDC20-50) is a mutant allele of CDC20 (also see next section).

As a test of CDC20 function, the PCR-derived clones of both CDC20 and CDC20-50 were assayed for their ability to complement the recessive temperature sensitivity of the cdc20-1 loss-of-function mutant (Figure 3B). As expected, CDC20 complemented the growth of the cdc20-1 mutant at nonpermissive temperatures. The CDC20-50 clone was equal to the CDC20 clone in its ability to complement cdc20-1, even at temperatures as high as 37°. This was not unexpected, since strains carrying a genomic CDC20-50 mutation were able to grow at 37° as well (data not shown).

CDC20-50 carries a mutation in a residue conserved among CDC20 homologs: To determine the nature of the CDC20-50 mutation, the inserts of plasmids carrying CDC20 and CDC20-50 were sequenced, and the sequences were compared to that of CDC20 (YGL116W) in the Saccharomyces Genome Data Base. The 2043 base pair insert of the plasmid carrying CDC20 was identical to the Saccharomyces Genome Data Base sequence for the CDC20 open reading frame and surrounding sequence. The insert of the plasmid carrying CDC20-50 differed from the CDC20 plasmid by a single nucleotide substitution within the CDC20 open reading frame. In CDC20-50, a G → A transition at base pair 1506 changes a glycine codon (GGA) to an arginine codon (AGA), corresponding to amino acid 446 of Cdc20p.

Figure 3.
Figure 3.

Phenotypic analysis of cloned CDC20 alleles from wild-type and PAC5-1 strains. (A) Dominant lethality caused by the CDC20 allele derived from the PAC5-1 strain combined with cin8Δ. Strain MAY2830 [cin8Δ cyh2 pMA1208 (CIN8 CYH2)] was transformed with a vector control (pRS316) or from a PCR fragment clone derived from a CDC20 strain (pCDC20) or a PAC5-1 strain (pPAC5). Transformants were spotted onto rich media with or without 5 mg/ml cycloheximide (cyh) and incubated 2 days at 26°. (B) Clones derived from CDC20 and PAC5-1 strains complement the temperature-sensitive cdc20-1 allele. The three plasmids described in (A) were transformed into the cdc20-1 strain MAY1715. Transformants were spotted onto rich media and incubated for 2 days at 26° or 35°.

In its carboxyl-terminal end, Cdc20p contains seven copies of a motif known as the WD repeat (Sethiet al. 1991). Originally identified as a repeated motif in the β-subunit of trimeric guanine nucleotide-binding proteins, the approximately 40 amino acid WD motif is also found in many other proteins from a wide variety of organisms (Neeret al. 1994). In Cdc20p, Gly446 falls within the fifth WD repeat. When Cdc20p is aligned with homologs from other organisms (including human), it is seen that Gly446 corresponds to a residue conserved in all identified relatives (Figure 4). Most of the other WD repeats of Cdc20p and its homologs have a glycine or a small uncharged amino acid at the corresponding position.

Figure 4.
Figure 4.

CDC20 mutant allele sequence changes. (Top) Linear representation of the positions of the WD repeats in S. cerevisiae Cdc20p and homologs from S. cerevisiae (Hct1p/Cdh1p), human (p55CDC), Drosophila (fizzy) and Schizosaccharomyces pombe (slp1). (Bottom) Amino acid sequence line-ups of the fifth and seventh WD repeats of Cdc20p and homologs showing the sequence changes caused by the CDC20-50 and cd20-1 mutations.

The dominant CDC20-50 effect is eliminated by a loss-of-function mutation: The finding that CDC20-50 was dominant and had full CDC20 function suggested that CDC20 activity was necessary for the checkpoint defect of CDC20-50. We investigated whether a gene that carries both CDC20-50 (dominant gain-of-function) and cdc20-1 (recessive temperature-sensitive loss-of-function) mutations would display the CDC20-50 checkpoint defect.

To construct the desired double mutant, it was first necessary to determine the sequence change of the cdc20-1 mutation (see materials and methods). The sequence of cdc20-1 differed from the wild type by one nucleotide: a single G → A transition at position 1810, which changes codon 544 from Gly to Arg, within the seventh WD repeat motif (Figure 4). Note that this Gly to Arg change is in a position within the seventh WD repeat adjacent to the position of the CDC20-50 Gly to Arg change within the fifth WD repeat.

We combined the cdc20-1 mutation with the CDC20-50 allele by replacement of a restriction fragment carrying the 1810 G → A mutation of cdc20-1 for the same region of a CDC20-50 clone. The double change mutant allele is referred to as cdc20-1, 50. This allele was placed downstream of the MET25 promoter to create PMET → cdc20-1, 50. For comparison, PMET → CDC20-50 and PMET → cdc20-1 were similarly constructed. The MET25 promoter is induced approximately ninefold by growth in the absence of methionine and retains some basal activity in the presence of methionine (Mumberget al. 1994). In addition, all constructs possess 76 base pairs 5′ from the translation initiation codon, an amount sufficient to allow CDC20 complementation of the cdc20-1 mutant (Sethiet al. 1991). Therefore, although these constructs are inducible, they are not strongly repressed in noninducing conditions (in the presence of methionine).

The PMET → CDC20 constructs were tested for their ability to confer CDC20-complementing activity to a cdc20-1 strain and for their ability to dominantly kill cin8Δ cells (Figure 5). When transformed into a temperature-sensitive cdc20-1 mutant, only the PMET → CDC20-50 construct was able to efficiently relieve the growth defect at 33° (Figure 5A). Partial complementation at 33° was provided by PMET → cdc20-1, but only when methionine was omitted from the media to induce high expression of cdc20-1. The PMET → cdc20-1, 50 construct provided no complementing activity, indicating that this double mutant provided even less CDC20 activity than cdc20-1. Since we do not possess the ability to measure Cdc20p protein levels, we could not directly test whether the cdc20-1, 50 form was stably expressed. However, we did note that this allele uniquely caused a deleterious phenotype. Expression of PMET → cdc20-1, 50 induced by methionine-less media inhibited the growth of cdc20-1 cells at 26° (data not shown). The other CDC20 forms did not cause this effect. This indicates that the product of the cdc20-1, 50 allele is probably stably expressed. When transformed into cin8Δ cells carrying a CIN8 CYH2 plasmid, PMET → CDC20-50 prevented segregation of cycloheximide-resistant cells, but PMET → cdc20-1, 50 did not (Figure 5B). (The temperature-sensitive effects of PMET → cdc20-1 on cycloheximide resistance will be discussed below.)

Figure 5.
Figure 5.

The cdc20-1 mutation eliminates the effects of the CDC20-50 mutation. (A) Abilities of CDC20 alleles to complement the temperature sensitivity of cdc20-1. The PMET plasmid vector or constructs in which the indicated CDC20 alleles were placed downstream of the promoter were transformed into the cdc20-1 strain MAY1715. Transformants were spotted onto minimal media with or without 3 mM methionine and incubated at the indicated temperatures. (B) Abilities of CDC20 alleles to dominantly cause lethality in the absence of CIN8. The same plasmids tested in (A) were transformed into MAY2830 (cin8Δ cyh2 pMA1208 [CIN8 CYH2]). Transformants were tested for their ability to segregate cycloheximide-resistant cells by spotting onto minimal media lacking uracil (to select for the PMET plasmid) and methionine (to induce expression from PMET). A reduced nonpermissive temperature of 33° was used for this experiment to demonstrate the ability of overexpressed cdc20-1 to partially complement the chromosomal cdc20-1 allele (row 3 in [A]).

In summary, the combination of both CDC20-50 and cdc20-1 changes in the same gene product abrogates the dominant cin8Δ synthetic lethality caused by the CDC20-50 form. Therefore, the gain of function exhibited by the CDC20-50 product requires some aspect of normal Cdc20p function.

Overexpression of CDC20 causes a spindle-assembly checkpoint defect: The temperature-sensitive mutant cdc20-1 is unable to enter anaphase at the restrictive temperature. The effect of the CDC20-50 mutation is inappropriate progression through mitosis when spindles are damaged. The findings that CDC20-50 is dominant, provides CDC20-complementing function, and causes a phenotype that appears to be the opposite to that of loss of function led us to examine whether overexpression of CDC20 may cause consequences similar to that of CDC20-50. CDC20, expressed from the PMET promoter induced by omitting methionine from the media, was found to cause lethality in combination with cin8Δ (Figure 6). This effect required full induction since the addition of methionine to the media now permitted the appearance of cycloheximide-resistant cells. PMET → CDC20-50 caused cycloheximide sensitivity independent of methionine, consistent with our conclusion that it represents an overactive form and does not require overexpression for its effect. Significantly, PMET → cdc20-1 also caused lethality with cin8Δ, but only in the absence of methionine and only at 26° (Figure 5B). At 33°, this effect was eliminated, consistent with the temperature-sensitivity of the cdc20-1 product. None of the PMET → CDC20 forms caused slow growth of wild-type cells on media lacking methionine, although slow growth caused by CDC20 expression from a higher-level galactose-inducible promoter has been reported (Lim and Surana 1996).

Figure 6.
Figure 6.

Overexpression of CDC20 causes a phenotype similar to that of CDC20-50. (A) Lethality of PMET-expressed CDC20 in the absence of CIN8. MAY2830 (cin8Δ cyh2 pMA1208 [CIN8 CYH2]) was transformed with PMET vector only or by PMET driving expression of CDC20 or CDC20-50. Transformants were spotted onto minimal media lacking uracil (to select for the PMET plasmid) and with or without cycloheximide and methionine as indicated. (B) Extra bud formation on benomyl-containing media caused by PMET-driven expression of CDC20. Wild-type cells transformed with the indicated plasmid genotype were released from a-factor synchronization onto solid minimal media lacking uracil (to select for the PMET plasmid) and methionine (to induce expression from PMET) and containing 70 μg/ml benomyl.

Using the benomyl arrest assay, PMET → CDC20 and PMETCDC20-50 were tested for the ability to cause cells to bypass the spindle-assembly checkpoint (Figure 6B). Wild-type haploid cells carrying PMET → CDC20, PMET → CDC20-50 or vector only were arrested in G1 with α-factor and then released onto benomyl-containing solid media (70 μg/ml) lacking methionine. After 6 hr, the proportion of cells that had arrested with a single large bud or had produced more than one bud was determined. A high percentage of cells carrying vector only arrested with a large-budded morphology on benomyl; only 11% produced cell groupings with three or more cell bodies. In contrast, a high number of cells overexpressing CDC20 or CDC20-50 produced extra buds indicating bypass of the benomyl-induced cell cycle arrest; 40% of PMET → CDC20 cells produced extra buds as did 55% of PMET → CDC20-50 cells. In summary, both the perish-in-absence-of-CIN8 phenotype and the spindle checkpoint defect of CDC20-50 could be mimicked simply by increased expression of CDC20.

A recently reported study found that CDC20 overexpressed from a high-level galactose-inducible promoter caused increased sensitivity to UV irradiation (Lim and Surana 1996). This finding suggested that the normal G2 delay imposed by the DNA damage response checkpoint could be eliminated by high levels of Cdc20p. However, we were unable to detect any increased sensitivity to DNA damage caused by UV irradiation or MMS for cells expressing PMET → CDC20 or PMET → CDC20-50 (see materials and methods). It is possible that the difference between these findings is the result of the increased strength of the PGAL promoter relative to the PMET promoter (Mumberget al. 1994). Cells expressing CDC20-50 from its normal chromosomal locus and promoter were also no more sensitive to these DNA-damaging treatments than CDC20 cells (data not shown).

Overexpression of CDC20 bypasses the preanaphase arrest caused by overexpressed MPS1: Damaged spindles generate a signal that is translated into a preanaphase cell cycle arrest by the spindle-assembly checkpoint gene products. The observation that overproduced Mps1p could induce a similar preanaphase arrest, dependent upon the BUB and MAD gene products, suggested that it functions upstream in the signal transduction pathway (Hardwicket al. 1996). We investigated whether overexpressed CDC20 could bypass the arrest caused by MPS1 overexpression.

A strain carrying an MPS1 gene under the control of a high-level, galactose-inducible PGAL promoter was transformed with PMET → CDC20 or a vector plasmid for control. Cells were grown in raffinose-containing media (plus methionine, but lacking leucine to select for the plasmid) and synchronized in S phase with the DNA synthesis inhibitor hydroxyurea. For the last 30 min of hydroxyurea treatment, galactose was added to the media to induce MPS1 expression and methionine was omitted to induce CDC20 expression. The cells were then released from the hydroxyurea block into galactose-containing media lacking methionine. At intervals, samples were removed, fixed, and stained with DAPI and observed microscopically to determine whether nuclear division had occurred (Figure 7A). In addition, at the point of release from hydroxyurea, a sample was spread onto solid galactose media lacking methionine, allowing us to score bud emergence (Figure 7B). The cells carrying the vector plasmid were inhibited from nuclear division and new bud emergence for the course of the experiment. In contrast, the cells carrying the PMET → CDC20 plasmid efficiently entered anaphase, divided their nuclei, and created new buds. A very similar effect was exhibited by cells carrying a PMET → CDC20-50 plasmid (data not shown). Therefore, the preanaphase arrest caused by overexpression of MPS1 can be bypassed by overexpression of CDC20.

Overexpression of CDC20 overcomes the preanaphase arrest caused by overexpressed PDS1, but not loss of APC function: Loss of CDC20 prevents entry into anaphase (Sethiet al. 1991; O'Tooleet al. 1997), and we have demonstrated that overexpression of CDC20 causes inappropriate entry into anaphase. Entry into anaphase normally requires the APC-mediated degradation of Pds1p (Cohen-Fixet al. 1996). It is possible, therefore, that Cdc20p exerts its effects through either the APC or Pds1p. To examine this possibility, we tested the ability of overexpressed CDC20 to bypass the preanaphase arrest caused by either loss of APC function or overexpression of PDS1.

A cdc16-1 strain, temperature sensitive for the function of the APC (Zachariae and Nasmyth 1996; Zachariaeet al. 1996), and a CDC16 strain were transformed with PMET → CDC20 or a vector plasmid for control. Cells were synchronized with hydroxyurea in methionine-containing media (minus uracil, to hold the plasmid) at 26°. Fifteen minutes prior to release from the hydroxyurea block, the cells were transferred to media lacking methionine (to induce CDC20 expression) and the incubation temperature was raised to 37°. The cells were released from the hydroxyurea into the same media at 37°. Samples were removed at time intervals, stained with DAPI, and observed to determine whether they had performed nuclear division (Figure 8). The CDC16 cells efficiently performed nuclear division within 1.5 hr, while the cdc16-1 cells were inhibited for the course of the experiment. The presence of the PMET → CDC20 plasmid did not allow the cdc16-1 cells to enter anaphase at 37°. Therefore, CDC20, overexpressed under our experimental conditions, was unable to bypass loss of APC function. The expression of CDC20-50 also did not cause cdc16-1 or cdc23-1 (encodes another component of the APC) cells to overcome their preanaphase arrest (data not shown).

Figure 7.
Figure 7.

Overexpressed CDC20 overcomes the preanaphase arrest caused by MPS1 overexpression. MAY4438, carrying a chromosomally integrated PGAL → MPS1, was transformed with PMET vector only (□) or a PMET → CDC20 plasmid (○). Transformants were grown in minimal raffinose media and synchronized with hydroxyurea. For the last 30 min of hydroxyurea treatment, galactose was added to the media to induce MPS1 expression and methionine was omitted to induce CDC20 expression. The cells were then released from the hydroxyurea block into liquid and solid galactose-containing media lacking methionine at 26°. (A) The liquid media cells were fixed at timepoints, stained with DAPI, and observed for nuclear division. Percent divided nuclei indicates the percentage of total cells with two DAPI-staining chromosomal masses. (B) The solid media cells were examined by microscopy for bud emergence. The percent cells with extra buds is the percent microcolonies with three cell bodies.

Transient overexpression of PDS1 has been observed to cause a preanaphase delay (Cohen-Fixet al. 1996). This study also reported that transient overexpression of the pds1-mdb mutant form, altered within the “destruction box” sequence required for ubiquitination and subsequent degradation, caused a more pronounced delay in anaphase onset. To examine whether overexpression of CDC20 could alleviate this delay, strains expressing the PDS1 forms from PGAL were transformed with PMET → CDC20 or a vector plasmid. Following hydroxyurea synchronization in raffinose plus methionine media, cells were transferred for 2 hr to media containing hydroxyurea and galactose (to induce expression of PDS1) and lacking methionine (to induce expression of CDC20). Cells were released from the hydroxyurea block by transfer to liquid or solid glucose media (to repress further PDS1 expression) lacking methionine. Liquid culture samples were removed and examined for nuclear division (Figure 9A). In addition, the cells spread onto solid media were observed for bud emergence (Figure 9B). A delay in nuclear division and bud emergence caused by overexpressed PDS1 could be detected under these conditions (compare PGAL → PDS1 [PMET vector] with WT [PMET vector] in Figure 9, A and B). The extent of the PDS1-induced delay was reduced in cells that overexpressed CDC20; PGAL → PDS1 (PMET → CDC20) cells entered anaphase and budded approximately 30 min earlier than PGAL → PDS1 (PMET vector) cells. Therefore, the preanaphase delay caused by Pds1p could be relieved by overexpression of Cdc20p. In contrast, expression of pds1-mdb caused a strong block to anaphase onset that was not relieved by the overexpression of CDC20. Interestingly, overexpression of CDC20 produced a slight but reproducible increase in the rate of nuclear division and bud emergence in the wild-type cells as well.

Figure 8.
Figure 8.

CDC20 overexpression did not overcome the preanaphase arrest caused by cdc16-1. CDC16 strain MAY2422 and cdc16-1 strain MAY2096 were transformed with PMET vector and PMET → CDC20 plasmids and synchronized with hydroxyurea. Fifteen minutes prior to release from the hydroxyurea block, the cells were transferred to liquid media lacking methionine (to induce CDC20 expression) and the incubation temperature was raised to 37°. The cells were released from the hydroxyurea into the same media at 37°. Samples were removed at time intervals, fixed, stained with DAPI, and observed. The percent of total cells that are large-budded (bud 50% diameter of mother or greater) and have one DAPI-staining chromosomal mass is displayed. Symbols: ▪, cdc16-1 (PMET vector); ●, cdc16-1 (PMET → CDC20 plasmid); □, CDC16 (PMET vector); ○, CDC16 (PMET → CDC20 plasmid).

Figure 9.
Figure 9.

Overexpressed CDC20 overcomes the preanaphase arrest caused by PDS1 overexpression. MAY5197 and MAY5198 carry galactose-inducible chromosomal PDS1 and pds1-mdb (mutant destruction box) alleles, respectively. MAY2422 is a wild-type strain that expresses PDS1 from its normal promoter. These strains were transformed with PMET vector or PMET → CDC20 plasmids. Transformants were synchronized with hydroxyurea in raffinose media and were transferred for 2 hr to media containing hydroxyurea and galactose (to induce expression of PDS1) and lacking methionine (to induce expression of CDC20). Cells were released from the hydroxyurea block by transfer to liquid or solid glucose media (to repress further PDS1 expression) lacking methionine at 26°. Liquid and solid media cells were observed for nuclear division (A) and extra bud formation (B), respectively, as described in the legend for Figure 7. In addition, the plating efficiencies of these cells were examined prior to and following the 2 hr galactose addition. For all six genotypes, the plating efficiency of the treated cells remained high, indicating that neither this protocol nor bypass of the Pds1p delay induced lethality. The data presented are for a representative experiment. The relative order of anaphase entry and bud emergence depicted here was reproduced in three additional repeats of this protocol. Symbols: ○, wild-type strain (PMET → CDC20 plasmid); □, wild-type strain (PMET vector); ●, PGAL → PDS1 strain (PMET → CDC20 plasmid); ▪, PGAL → PDS1 strain (PMET vector); ▵, PGAL → pds1-mdb strain (PMET → CDC20 plasmid); ▴, PGAL → pds1-mdb strain (PMET vector).

DISCUSSION

The actions of the spindle-assembly checkpoint prevent cell cycle progression from metaphase into anaphase in response to mitotic spindle defects. We have identified and characterized the CDC20-50 mutant allele that caused S. cerevisiae cells to bypass this cell cycle arrest in a dominant fashion. In contrast, loss of CDC20 function (e.g., caused by the recessive temperature-sensitive cdc20-1 allele) caused cells to arrest prior to anaphase with duplicated chromosomes and an assembled bipolar spindle (Sethiet al. 1991; O'Tooleet al. 1997). A similar defect in cell cycle progression has been noted for loss of function of Drosophila fizzy, a CDC20 homolog (Dawsonet al. 1995; Sigristet al. 1995). The dominance and inappropriate cell cycle progression exhibited by CDC20-50 suggested that it may represent a gain-of-function allele. Indeed, we were able to mimic the phenotypes of CDC20-50 by overexpressing CDC20. It seems likely, therefore, that the Cdc20-50p form is overactive, producing aberrantly high levels of what is a normal Cdc20p function. The Cdc20-50p change, affecting a conserved amino acid within the fifth of seven WD repeats, may create an intrinsically overactive protein or may destroy an interaction with a function that negatively regulates Cdc20p activity.

The ability of overexpressed CDC20 to promote entry into anaphase suggests that it acts as a limiting activator of anaphase. Our findings indicate that a likely target of Cdc20p regulation is the anaphase inhibitor Pds1p. Entry into anaphase requires the APC-mediated proteolytic degradation of Pds1p (Cohen-Fixet al. 1996). We found that the delay to anaphase entry caused by transient high levels of Pds1p could be relieved by overexpression of CDC20. This suggests that Cdc20p acts as an antagonist of Pds1p. The observed antagonism required that Pds1p be susceptible to APC-mediated destruction. The block to anaphase progression caused by a destruction box mutant form of Pds1p, immune to the actions of the APC, could not be relieved by overexpression of CDC20. It seems most likely, therefore, that Cdc20p can promote the destruction of Pds1p in an APC-dependent fashion. Consistent with this hypothesis is our finding that the metaphase arrest caused by loss of APC function could not be bypassed by CDC20 overexpression. Pds1p is stabilized in APC-mutant cells (Cohen-Fixet al. 1996). If a role of Cdc20p is to direct the APC to Pds1p, high levels of Cdc20p would not be expected to compensate for an APC defect. In agreement with our findings is the recently reported observation that Pds1p is stabilized in cdc20-1 cells and destabilized in cells overexpressing CDC20 (Visintinet al. 1997).

PDS1 function is also required to prevent DNA-damaged cells from entering anaphase prematurely (Yamamotoet al. 1996b). We also note the recent report demonstrating that overexpression of CDC20 allows cells with damaged DNA, caused by a temperature-sensitive cdc13 mutation, to enter anaphase (Lim and Surana 1996). It is possible that CDC20-induced degradation of Pds1p in the cdc13 cells promoted aberrant anaphase onset.

Cells deficient for PDS1 enter anaphase inappropriately but do not exit mitosis into the G1 phase. In nocodazole-treated pds1 cells, sister chromatids can disjoin, but new bud emergence or DNA replication rounds remain inhibited (Yamamotoet al. 1996b). However, we observed new bud emergence and DNA replication in nocodazole-treated cells expressing CDC20-50 or overexpressing CDC20, demonstrating exit from M phase and progression through G1. This indicates that there must exist additional targets of activated Cdc20p beyond Pds1p. Exit from M phase requires the APC-mediated destruction of the mitotic cyclin proteins (Suranaet al. 1993), and normal spindle disassembly requires the APC-mediated destruction of the spindle protein Ase1p (Juanget al. 1997). Recent studies demonstrated that destruction of Ase1p and the Clb2p mitotic cyclin occur under the control of HCT1/CDH1, an S. cerevisiae CDC20 homolog (Schwabet al. 1997; Visintinet al. 1997). It is possible that additional targets must be degraded as well to complete exit from mitosis. While the degradation of these targets may not normally be under CDC20 control, nocodazole-treated cells overexpressing CDC20 were able to exit M phase and therefore were able to overcome all regulatory obstacles to progression. Perhaps under these conditions, Cdc20p can direct the APC to targets beyond its normal scope. Indeed, it was reported that although the mito-tic cyclin Clb2p is not stabilized in cdc20-1 cells, Clb2p is destabilized in cells overexpressing CDC20 (R. Visintin, S. Prinz and A. Amon, unpublished results). Functional overlap between CDC20 and HCT1/CDH1 was suggested by the observation that overexpression of HCT1/CDH1 could suppress the temperature sensitivity of cdc20-1 (Schwabet al. 1997). Perhaps overexpressed CDC20 can lead to the destruction of protein targets normally under the control of HCT1/CDH1.

We have demonstrated that overexpression of CDC20 can bypass the cell cycle arrest caused by the spindle-assembly checkpoint. The mutant phenotypes of the seven spindle-assembly checkpoint genes characterized to date (BUB1, 2, 3, MAD1, 2, 3, and MPS1) suggest that they act as negative regulators of anaphase onset and cell cycle progression. In contrast, the phenotypes of CDC20 loss- and gain-of-function mutants indicate that it must be an activator of anaphase. Our findings suggest the interesting possibility that Cdc20p is actually the target of regulation for the spindle-assembly checkpoint. In this formulation, the checkpoint gene products would act upstream of Cdc20p to transduce the spindle-damage signal and to inhibit the Cdc20p anaphase-promoting function. CDC20-50 or overexpression of CDC20 may create forms that are insensitive to the cycle-arresting signals produced by the checkpoint mechanism. However, our data do not rule out the possibility that Cdc20p function is unrelated to spindle-assembly checkpoint function. High levels of Cdc20p may simply be able to drive anaphase entry in a manner such that the arrest signal originating from the checkpoint mechanism is bypassed. The MPS1-encoded protein kinase is believed to act at an early step in the spindle-damage signaling pathway, and its overexpression appears to mimic normal pathway activation (Hardwicket al. 1996). We found that CDC20 overexpression could overcome the metaphase arrest caused by overexpression of MPS1. This could indicate that Cdc20p antagonizes Mps1p function, perhaps in a manner similar to its antagonism of Pds1p. It seems more likely, however, that the MPS1-induced arrest resembles that caused by spindle disruption and that both produce a similar signal that is ignored or bypassed by the downstream-acting overexpressed Cdc20p.

In summary, we have found that the CDC20 product behaves like a limiting factor regulating the entry into anaphase. CDC20 homologs exist throughout the eukaryotes, including Drosophila fizzy, which also appears to be required for anaphase entry (Dawsonet al. 1995; Sigristet al. 1995). The metaphase-anaphase transition is a critical regulatory point in the eukaryotic cell cycle. At this stage, numerous regulatory influences reporting the readiness of the cell to segregate chromosomes must be integrated and acted upon. It seems likely that Cdc20p homologs perform a central role in this important regulatory mechanism.

Acknowledgments

The authors thank Penny Tavormina, Dan Burke, Mark Winey, and Orna Cohen-Fix for the gifts of strains and DNAs; Angelika Amon and Wolfgang Seufert for the communication of unpublished findings; and Orna Cohen-Fix, Cindy Dougherty, Katie Farr, John Geiser, and Penny Tavormina for helpful discussions and comments on the manuscript. This work was support by National Institutes of Health grant GM-49363 awarded to M.A.H.

Footnotes

  • Communicating editor: M. D. Rose

  • Received August 13, 1997.
  • Accepted October 10, 1997.

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Volume 148 Issue 2, February 1998

Genetics: 148 (2)

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Dominant Alleles of Saccharomyces cerevisiae CDC20 Reveal Its Role in Promoting Anaphase

Eric J. Schott and M. Andrew Hoyt
Genetics February 1, 1998 vol. 148 no. 2 599-610
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