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Fission yeast WD-repeat protein pop1 regulates genome ploidy through ubiquitin-proteasome-mediated degradation of the CDK inhibitor Rum1 and the S-phase initiator Cdc18.

In fission yeast, maintenance of genome ploidy is controlled by at least two mechanisms. One operates through the Cdc2/Cdc13 kinase, which also involves the CDK inhibitor Rum1, and the other through the S-phase regulator Cdc18. By screening for sterile mutants that show increased ploidy, we have identified a new gene, pop1+, in mutants that become polyploid. The pop1 mutation shows a synthetic lethal interaction with the temperature-sensitive cdc2 or cdc13 mutation. In a pop1 mutant Rum1 and Cdc18 proteins become accumulated to high levels. The high ploidy phenotype in the pop1 mutant is dependent on the presence of the rum1+ gene, whereas the accumulation of Cdc18 is independent of Rum1. The predicted sequence of the Pop1 protein indicates that it belongs to a WD-repeat family with highest homology to budding yeast Cdc4, which participates in the ubiquitin-dependent pathway. Consistent with this notion, in a mutant of the 26S proteasome, higher molecular weight forms of Rum1 and Cdc18 are accumulated corresponding to polyubiquitination of these proteins. In the pop1 mutant, however, no ubiquitinated forms of these proteins are detected. Finally we show that Pop1 binds Cdc18 in vivo. We propose that Pop1 functions as a recognition factor for Rum1 and Cdc18, which are subsequently ubiquitinated and targeted to the 26S proteasome for degradation.

one case where the substrate (ornithine decarboxylase) is not ubiquitinated. Ubiquitin-dependent protein degradation has emerged as a primary mechanism that regulates crucial aspects of the cell cycle. Cyclin B is a classical substrate for the proteasome, which is abruptly degraded in anaphase. Other cyclins such as those of budding yeast and human cyclin E are also shown to be degraded by ubiquitin-dependent proteolysis Clurman et al. 1996;Won and Reed 1996). In addition, other important regulators for sister chromatid segregation in anaphase are degraded through the same proteasome pathway (Funabiki et al. 1996;Yamamoto et al. 1996).
Fission yeast usually proliferates as haploid cells with ploidy being maintained faithfully. Several conditions are known to cause diploidization or polyploidization, which can be classified into the following three types. The first type represents bypass of M phase, which takes place in conditions leading to inactivation of Cdc2/ Cdcl3 (B-type cyclin), an archetype cyclin-dependent protein kinase (CDK). This includes complete disruption of the cdcl3 ÷ gene, heat shock treatment of temperaturesensitive cdc2 or cdcl3 mutants, or ectopic overexpression of rural ÷, which encodes a CDK inhibitor (Broek et al. 1991;Hayles et al. 1994; Moreno and Nurse 1994; Correa-Bordes and . The second type comprises successive S phase in the absence of G1, G2, and M phases, which is driven by ectopic overexpression of cdcl8 ÷ encoding a critical regulator for the initiation of S phase (Kelly et al. 1993;Nishitani and Nurse 1995). The third type displays incomplete M phase, which is caused by the cutl mutation defective in chromosome disjunction (Creanor and Mitchison 1990;Uzawa et al. 1990).
The cellular amount of the Ruml protein is strictly regulated in a cell cycle-dependent manner such that it is extremely low in exponentially growing cells, whereas it is accumulated in those arresting at GI. Thus, the fluctuation of the Ruml protein amount ensures the activation of the Cdc2/Cdcl3 kinase at the G2/M boundary and inhibition of premature entry into M phase from the G1 phase (Moreno and Nurse 1994). It is, therefore, crucial to know how the level of Ruml is regulated in the cell cycle. Molecular mechanisms on regulation of the level of the Ruml protein, however, remain to be determined, except that it is known that it is not transcriptionally regulated: The amount of rural ÷ mRNA remains constant between exponentially growing cells and Gl-arrested cells. This result suggests that the level of Ruml protein may be regulated through protein turnover.
It is known that the level of cdcl8 ÷ mRNA as well as that of the protein is cell cycle-dependent, peaking at the GI/S boundary (Kelly et al. 1993;Nishitani and Nurse 1995;Muzi-Falconi et al. 1996). Cdcl8, which shows amino acid sequence similarity to budding yeast Cdc6 (Zhou et al. 1989), is crucial for the initiation of S phase and regulates coupling between Start and S phase and also between S phase and M phase. To initiate DNA replication at the correct timing, the Cdc 18 protein level must be high at the G~/S boundary, whereas to restrain rereplication at other phases of the cell cycle, the synthesized protein must somehow be inactivated after S phase. To achieve such a cell cycle-dependent fluctuation, the Cdcl8 protein would be expected to be shortlived and indeed it is shown to be (Muzi-Falconi et al. 1996). However, no molecular analysis has been addressed on the regulation of stability of the Cdcl8 protein.
In the present study, we have identified the popl ÷ gene, which is important for maintenance of ploidy; mutations of this gene result in appearance of diploid or polyploid cells. We have found that the Rum1 and Cdcl8 proteins are highly accumulated in the popl mutant, which accounts for increased ploidy of the popl mutant. The popl ÷ gene encodes a member of a WD-repeat family with the F-box motif that is involved in the ubiquitindependent proteolysis (Bai et al. 1996). We will show that the Ruml and Cdcl8 proteins are degraded through the proteasome pathway. Rum1 and Cdc18 are polyubiquitinated in the proteasome mutant, but not in the popl mutant. Finally Popl forms a complex with Cdcl8 in vivo. These findings show that the WD-repeat protein Pop 1 is a novel regulator of the cell cycle that is required for degradation of Rum l and Cdc 18 through the ubiquitin-proteasome pathway.

Results
popl mutations lead to an increased ploidy and cell size Using flow cytometry (FACS), during the course of searching for sterile mutants because of a failure to arrest at G1 phase upon nutrient starvation we isolated a novel type of mutant that showed increased ploidy. Wild-type cells arrest at a 1C content of DNA under nitrogen starvation condition, which is prerequisite for conjugation ( Fig. 1A). In contrast, 14 mutants showed no G1 peak; instead, they exhibited increased ploidy. In particular, three mutants (364, 422, and 577) possessed a higher amount of DNA content such as 4C, 8C, and possibly more in a discrete manner ( Fig. 1 B-D, left), whereas the other 11 mutants showed 2C and 4C (data not shown). Also the forward scattering pattern that reflects cell size showed that, in proportion to the increase in DNA content, the cell size of these mutant cells was also increased ( Fig. 1 A-D, right). Genetic analysis indicated that all the three mutants were recessive and allelic, designated popl (polyploidy).
FACS analysis in the exponentially growing popl mutant showed a similar pattern to that seen in nitrogenstarved condition. Examples of cell morphology of the popl mutant stained with DAPI are shown in Figure 1E. Not only cell length but also cell width of these mutants is clearly larger than that of wild type. In addition, a single large nucleus was apparent, suggesting that onset of mitosis rather than cytokinesis is defective in the popl mutant. The cell size of the popl-364 mutant doubles on average compared with wild-type haploid cells, whereas that of diploid cells shows an -50% increase, consistent with the fact that some population of the popl mutant contains more than 4C D N A content (Fig. 1B). The generation time of the popl mutant is 30% longer than that of wild-type cells.
popl m u t a n t s are supersensitive to K-252a, a drug that causes over-replication To analyze genetically defective phenotypes of popl, the following experiments were performed. It has been reported that a drug K-252a causes overreplication when added to fission yeast cells as well as animal culture cells (Usui et al. 1991(Usui et al. , 1995. Thus, sensitivity to K-252a was examined. It was found that the popl mutant is supersensitive to K-252a (Table 1) but not to a related compound staurosporine (data not shown).
The popl mutations s h o w a synthetic lethal interaction with cdcl3 or cdc2 Next, synthetic phenotypes were examined between popl mutants and those in cdc2 or cdcl3. The popl mutations (-364, -577)were synthetically lethal when combined with temperature-sensitive cdcl3-117; dissection of 22 asci yielded no viable double mutants. We, however, sometimes obtained tiny colonies of double mutants after tetrad analysis, which were unable to propagate. Cells from these abortive colonies were extremely elongated with a single large nucleus, resembling cdcl3deficient cells (Hayles et al. 1994). Conversely, multicopy plasmids containing the cdcl3 ÷ gene were capable of suppressing partially the popl mutation (data not shown). These results suggest that either activity or level of the Cdc2/Cdcl3 kinase complexes is decreased in the popl mutant. A similar synthetic phenotype was also observed between popl and temperature-sensitive cdc2-33. A popl cdc2-33 mutant was not lethal at low temperature (26°C); however, the double mutant could not form colonies at 32°C at which either single mutant was capable of forming colonies. This result also supports the notion that the Cdc2/Cdcl3 kinase was reduced in the popl mutant. In contrast, the popl mutant did not show synthetic lethal phenotypes with the G1-arrest cdc10-i29 or G2-arrest cdc25-22 mutant.
The R u m l and CdcI8 proteins are accumulated in the popl m u t a n t As a first step in analyzing defective phenotypes of the p o p l mutant at a molecular level, the amount of various cell cycle regulators was examined by immunoblotting. Proteins examined include Cdc2, Cdcl3, Cdc18, and Ruml. Total cell extracts were prepared from wild-type, the p o p l mutant, A r u m l , and ruml+-overproducing cells, and immunoblotting was performed using respective antibodies. As shown in Figure 2A, it was found that the level of Rum 1 protein was increased significantly in the p o p l mutant (lane 2). As reported previously (Correa-Bordes and , the level of Rural protein in exponentially growing wild-type cells was extremely low and hardly detected (lane 1). In contrast, cells carrying ectopically overexpressed rural ÷ gene on plasmids produced a high level of Ruml protein (lane 4).
In addition to the Ruml protein, the level of Cdcl8 was also found to be accumulated in the p o p l mutant (Fig. 2B, lane 2). In contrast to an increased level of Ruml and Cdc18, the level of Cdcl3 and Cdc2 was not changed

A c c u m u l a t i o n of the R u m 1 protein in the popl m u t a n t is n o t attributable to an increased transcription
To address the question of how the Ruml protein is accumulated in the p o p l mutant, total RNAs were prepared from the same strains as used in immunoblotting and Northern analysis was performed with the rural ÷ gene as a probe. As shown in Figure 2F, in a clear contrast to the protein level, the amount of the r u m i ÷ mRNA was not increased in the p o p l mutant (cf. lanes 1 and 2), whereas a significant increase in the rural ÷ RNA was detected in cells where the rural ÷ gene was expressed ectopically (lane 4; note that the difference in the size of the rural ÷ mRNA because of ectopic expression). These results suggest that the increase in Rural protein in the p o p l mutant is not attributable to transcriptional induction of the rural ÷ gene; instead, it is a result of posttranscriptional or post-translational mechanisms (see below).
The popl m u t a n t is m o r e susceptible to a higher dosage of the ruml ÷ or cdcl8 ÷ gene If the accumulation of the Ruml and/or Cdcl8 proteins is responsible for the increased ploidy in the p o p l mutant, it is predicted that even moderate overexpression of the rural ÷ or c d c l 8 ÷ gene in the p o p l mutant would be lethal, whereas wild-type cells would be able to survive. To examine this possibility, the p o p l mutant was transformed with multicopy plasmids that contain the r u m 1 ÷ or cdcl 8 + gene under the thiamine-repressible n m t l promoter of either the strong (pREP3X) or crippled version (pREP41 and 81). We found that the p o p l mutant carrying either p R E P 3 X -r u m l ÷ or p R E P 4 1 -r u m l ÷ grew extremely slowly compared with those carrying an empty vector even in repressed condition ( Fig. 3A, left). In wild type, p R E P 3 X -r u m I ÷, not p R E P 4 1 -r u m l ÷, is toxic only in derepressed condition (Fig. 3A, middle). This result indicates that the p o p l mutant becomes highly susceptible to an increase of the rural ÷ gene dosage possibly because of the increase in Ruml protein level. An analogous susceptibility to an increased gene dosage was also observed in the c d c l 8 ÷ gene. The p o p l mutant carrying pREP41-cdc18 ÷ was capable of forming tiny colonies on plates in derepressed conditions, whereas wild-type cells carrying p R E P 4 1 -c d c l 8 ÷ could form colonies of size indistinguishable from those carrying a vector (Fig. 3A, left, and 3B, left and middle). Similarly, wild-type cells carrying p R E P 3 X -c d c l 8 ÷ managed to form tiny colonies, whereas no colonies were formed in the p o p l mutant carrying p R E P 3 X -c d c l 8 ÷. Thus p o p l becomes susceptible to an increased gene dosage of either r u m l ÷ or c d c l 8 ÷ consistent with the accumulation of Rural and Cdcl8 in the p o p l mutant. . This result demonstrates that increased ploidy in the p o p l mutant is attributable to the accumulation of a CDK inhibitor, R u m l . It should be noted that cell size of the popl r u m l double mutant is still slightly longer than that of either wild type or Arum 1 (Fig. 3C, cf. right and left panels). This phenomenon led us to pursue a relationship between Popl and Cdcl8.
The C d c l 8 protein a c c u m u l a t e s in the popl m u t a n t i n d e p e n d e n t l y of the R u r a l function It has been reported that cells carrying an increased dosage of the rural ÷ gene accumulate the Cdcl8 protein, although the molecular mechanisms remain to be determined (Jallepalli and Kelly 1996;Fig. 2B, lane 4). It is, therefore, interesting to see whether the Cdcl8 protein still accumulates in the p o p l rural double mutant. If the accumulation of Cdcl8 in the p o p l mutant is a secondary effect attributable to the accumulation of R u m l , the p o p l r u m l double mutant would contain a normal amount of Cdcl8. If, on the other hand, the amount of the Cdcl 8 protein is regulated by Pop 1 independently of R u m l , it would be expected that Cdcl8 still accumulates in the double mutant. Immunoblotting indicated that the latter is the case. The double mutant still contained a higher amount of Cdcl8, like a single p o p l mutant or cells containing ectopically expressed r u m 1 ÷ gene (Fig. 4A). This result shows that the accumulation of Cdcl8 in the popl mutant is not a secondary effect attributable to the accumulation of R u m l and that Popl regulates the amount of the Rum 1 and Cdc 18 proteins in an independent manner. In line with the independence of the accumulation of R u m l and Cdcl8 in the p o p l mutant, it was found that, like in a p o p l single mutant (Fig. 3B), p R E P 4 1 -c d c l 8 ÷ as well as p R E P 3 X -c d c l 8 ÷ is toxic in the popl rural double mutant (Fig. 4B). Cells of the p o p l r u m l double mutant carrying p R E P 4 1 -c d c l 8 ÷ became m u c h longer, whereas wild-type cells carrying the same plasmid did not (Fig.  4C). These results support further the notion that Cdcl8 accumulates in the p o p l mutant in the absence of R u m l and that the accumulation of Cdcl 8 does contribute phenotypes of the popl mutant, although minor compared with that of R u m l .

The predicted Popl protein belongs to a WD-repeat protein f a m i l y
The p o p l ÷ gene was isolated by complementation using cdc2-33 popl-364 double mutant as a host (see Materials and Methods). A 2325-bp uninterrupted open reading frame (ORF) that encodes a protein of 775 amino acid residues was found (Fig. 5A). A comparison of the predicted amino acid sequence between Popl and known proteins shows that the Pop l protein is a member of WD-repeat proteins, which are composed of tandem repeats of the unit consisting of (GH-X23_41-WD) N4-8  (Neer et al. 1994; Fig. 5B, C). The Popl protein appears to contain seven repeats. A homology search against known databases (EMBL, PIR, and SWISS-PROT) indicates that the Popl protein shows the highest homology to Cdc4 from budding yeast Saccharomyces cerevisiae (Fong et al. 1986) and its putative homolog from Candida albicans (GenBank accession no. X96763). Overall identity among these proteins is not extremely high (30% between Popl and ScCdc4 and 35% between Popl and CaCdc4). It is, however, noteworthy that not only central WD-repeat motifs but also both amino-and carboxy-terminal flanking regions show a significant homology (Fig. 5B). These flanking regions of the Pop 1 protein do not show any homology to other members of WD-repeat proteins, In particular, Pop l contains the F-box motif, which is reported recently to exist in various proteins including Cdc4 and implicated in ubiquitin-mediated proteolysis (Fig. 5B, C; Bai et al. 1996). Functional exchangeability between Pop 1 and ScCdc4 has been performed; however, no positive results have been obtained (K. Kominami and T. Toda, unpubl.).

Gene disruption of the popl + gene
The popl ÷ gene was disrupted by one-step gene replacement method ( Fig. 5A; see Materials and Methods). The popl-deleted (z~popl) cells were viable and essentially the same phenotypes as the original mutants observed: increased ploidy and sterility. Thus, the popl + gene is not essential for viability but required for maintenance of ploidy.

Identification of the popl ÷ gene product
To identify the pop1 ÷ gene product, the pop1 ÷ gene was fused with a tag and introduced into the popl mutant. Immunoblotting using anti-hemagglutinin (HA) antibody showed that the Pop l protein was detected as a band of -90 kD. Approximately 40% of the Popl protein was found as an insoluble fraction (Fig. 6A, lanes 5,6). This might imply that some of the Popl protein forms a large structural complex inside the cell.
As a first step in determining the cell cycle-dependent regulation of the level of the Pop 1 protein, the amount of Pop l during the cell cycle was examined by arrest-release using the G2-arrest cdc25 mutant. No noticeable fluctuation of the Pop 1 protein in the cell cycle appeared to occur (Fig. 6B).

The Rural and Cdcl8 proteins are accumulated in the proteasome-deficient m u t a n t
In budding yeast Cdc4 is involved in ubiquitin-dependent proteolysis of a CDK inhibitor, Sicl (Schwob et al. 1994). Bearing this in mind, the possibility that Ruml and Cdcl8 are degraded through the proteasome-mediated pathway was examined. To this end, the temperature-sensitive rots3-1 mutant that is defective in subunit 14 of the 26S proteasome regulatory complex (Gordon et al. 1996) was used. Total cell extracts were prepared from wild type and rots3-1, which were incubated at restrictive temperature (35.5°C) and were immunoblotted using anti-Rum1 and anti-Cdc18 antibodies. As shown in Figure 7A, it was found that both Ruml and Cdcl8 proteins became accumulated in the rots3-1 mutant at 35.5°C (lanes 5-8). It is noteworthy that even at 0 hr, the Ruml protein could be detected in the rots3-1 mutant (lane 5). The extent of the accumulation of the Ruml protein was higher than that of the Cdcl8 protein, probably because of cell cycle-dependent transcriptional regulation of the cdcl8 ÷ gene (Nishitani and Nurse 1995;Muzi-Falconi et al. 1996). This result strongly suggests that the Rum 1 and Cdc 18 proteins are degraded through the 26S proteasome-dependent pathway and the Popl protein is involved in degradation of these two proteins.
The Rural and Cdcl 8 proteins are polyubiquitinated but not in the popl m u t a n t If Rum1 and Cdcl8 are degraded through the 26S proteasome, it would be expected that these proteins become polyubiquitinated in the proteasome mutants. If so, it   (ScCdc4) and C. albicans Cdc4 homolog (CaCdc4). Values represent percentage of identity of amino acids. Note that the third WD repeat in Popl and CaCdc4 is shown with ovals as the number of amino acids in these repeats is larger (see Fig. 5C). (C) A comparison of amino acid sequences of Popl, ScCdc4, and CaCdc4. Identical amino acids are shown in closed squares; conservative amino acids are shown in gray squares. The position of the WD repeats and F-box is also shown.
would be interesting to see what happens in the popl mutant. To examine this possibility, a tagged version of R u m l (Rural-HA) was overproduced in the rots3-1 or popl mutant and ubiquitination of R u m l was examined (see Materials and Methods). Immunoblotting with anti-HA antibody against extracts prepared from mts3-1 incubated at 26°C (Fig. 7B, lane 3) or 35.5°C (lane 2) revealed that, in addition to a predicted size of Rum 1-HA (38 kD), higher molecular mass forms were detected in a discrete pattern. To show ubiquitination of Rum 1-HA, the same filter was immunoblotted with anti-ubiquitin antibody. As expected, a similar, if not identical, band pattern of the higher molecular mass forms (emphasized with horizontal bars) was detected with anti-ubiquitin antibody (lanes 5,6). It should be noted that anti-ubiquitin antibody did not recognize a band of 38 kD, which is nonubiquitinated, showing specificity of anti-ubiquitin antibody. The ubiquitinated forms of R u m l -H A were more abundant in the mts3-1 mutants incubated at the restrictive temperature (35.5°C, lane 5) than at the permissive temperature (26°C, lane 6). This result demonstrates clearly that Rum l is polyubiquitinated and degraded by the ubiquitin-26S proteasome pathway.
In contrast to a pattern in the mts3-1 mutant shown above, no higher molecular mass forms of Rum 1-HA were detected, and, more important, no ubiquitinated bands were present in the popl mutant (Fig. 7B, lanes  1,4). Control immunoblotting with anti-Cdc2 antibody showed a similar level of the Cdc2 proteins with no higher molecular mass forms (Fig. 7B, bottom).
In addition to R u m l , similar results were obtained in Cdcl 8; myc-Cdcl 8 was polyubiquitinated in the rots3-1 mutant, but not in the popl mutant (Fig. 7C). These results establish the notion that Pop l is required for ubiquitination and degradation of Rum1 and Cdcl8.

The Popl protein forms a complex with Cdcl8
To define the biochemical function of the Pop l protein more precisely, the possibility of physical interaction between Pop 1 and Cdc 18 was examined. Cell extracts were prepared from cells that expressed HA-tagged Popl (Popl-HA) and myc-tagged Cdc18 (myc-Cdcl8) (Fig. 8, lane 1), and immunoprecipitation was performed with anti-HA antibody in the presence or absence of competi-

D i s c u s s i o n
In this study we have identified a novel cell cycle regulator, Pop 1, as a critical d e t e r m i n a n t for the m a i n t e n a n c e of genome ploidy. In a popl m u t a n t the CDK inhibitor R u m l and the S-phase regulator C d c l 8 a c c u m u l a t e to high level. In contrast, the popl rural double m u t a n t no longer shows increased ploidy, suggesting that the popl m u t a n t phenotype is a result of the a c c u m u l a t i o n of the R u m l protein. We have s h o w n that the R u m l and C d c l 8 proteins are a c c u m u l a t e d in the m u t a n t defective in one of the subunits of the 26S proteasome. Moreover, these proteins contain higher molecular mass polyubiquitinated forms w i t h discrete band patterns. In contrast, in the popl m u t a n t , no ubiquitinated forms of these pro-  1-4) 1,2) or with anti- ubiquitin (lanes 3,4). Again, 70-kD nonubiquitinated form (myc-Cdcl8) was not detected with anti-ubiquitin antibody. be a component or regulator of the ubiquitin-proteasome-dependent pathway that specifically recognizes a CDK inhibitor and an S-phase regulator.

Identification of Popl as a critical regulator of ploidy control
Despite extensive screening for diplodizing mutants, the popl mutants have not been identified before (Broek et al. 1991;Hayles et al. 1994). We reason that this is because of sterility and meiosis-deficiency of the popl mutants. Previous screening was based on the assumption that diplodized mutants would undergo meiosis and form spores, which was used to enrich such increased ploidy mutants. As shown in this study, we isolated the pop l mutants from our collections of sterile mutants using FACS. Sterility of the popl mutants could be attributable to inability of the popl mutant to arrest at G 1 upon nitrogen starvation, which is prerequisite for conjugation (Kumada et al. 1995). Failure of meiosis might be related to the accumulation of Ruml in the popl mutant, which would block the meiotic activation of Cdc2/ Cdcl3 complexes. Alternatively, proteins other than Ruml and Cdcl8 may become accumulated in the popl mutant and might need to be degraded for meiotic progression. The nonlethal phenotype of the popl disruptant is not surprising, as it has been reported that fission yeast cells can tolerate being pentaploid (Molnar and Sipiczki 1993). We have shown that Rum 1 protein is accumulated in the popl mutant, but the amount is far less than that found in lethal rum l+~overproducing cells where the rum l ÷ gene is expressed ectopically. The amount of Ruml protein accumulated in the popl mutant would be insufficient to inhibit mitoses completely. In addition, the amount of Cdcl8 protein found in the popl mutant appears to be insufficient to drive successive S phase, as the popl ruml double mutant no longer shows a high ploidy phenotype. We suspect that only some proportion of the popl cells bypass M phase and rereplicate in each cell cycle. Alternatively, it is possible that in the popl mu-tant the overreplication occurs only in some cell cycles at a frequency that does not affect the ability to obtain colonies. Because the popl mutants we have isolated are not conditional, at present it is difficult to estimate precisely the frequency of rereplication per cell cycle in the mutant.

Targets of Popl-dependent proteolysis: Rum 1 and Cdcl8
We suspected initially that a sole target of Popl-dependent proteolysis might be Ruml as the defective phenotypes of the popl rum 1 double mutant are modest; FACS analysis indicated a normal ploidy (2C). However, subsequent analyses using the popl ruml double mutant have convinced us that the accumulation of the two proteins in the popl mutant is a parallel process. Popl regulates protein stability of Rum 1 and Cdcl 8 in an independent manner.
Regarding targets for Pop l, we cannot conclude definitely that Rural and Cdcl8 are the only proteins regulated by Popl. As shown here, the high ploidy phenotypes are suppressed in the popl rum 1 double mutants. We have found several other phenotypes of the popl mutant in addition to increased ploidy, including a low viability in stationary phase, supersensitivity to UV exposure, and a high rate of chromosome loss. The former two phenotypes are also suppressed by the rum 1 disruption (K. Kominami and T. Toda, unpubl.), suggesting that the major targets of Popl-dependent proteolysis are Rum 1 and Cdc 18.

Proteasome, ubiquitin, and Popl
Our data show that Ruml and Cdcl8 are degraded through the proteasome-dependent pathway. It is interesting to point out that, in line with this notion, mts3-1 mutant cells have been reported to rereplicate their DNA at restrictive temperature (Gordon et al. 1996). Although it was interpreted previously that overreplication is not a result of a bypass of M phase, phenotypic analysis of polyploidization in the mts3-1 mutant needs to be reexamined in view of the accumulation of Rum l and Cdcl8. On the other hand, in the rots2-1 mutant, which is defective in subunit 4 of the 26S proteasome (Gordon et al. 1993), neither Ruml nor Cdcl8 is accumulated (K. Kominami and T. Toda, unpubl.). It is, therefore, possible that Ruml and Cdcl 8 are degraded by proteasome in a Popl-dependent and 26S subunit-specific manner.
We have shown that Pop l forms a complex with Cdcl 8. This may suggest that a primary function of Popl is to recognize and bind specific substrates that are then ubiquitinated and targeted to proteasome for degradation. This is consistent with the results, which show that no ubiquitination of Rum 1 and Cdcl 8 occurs in the popl mutant. Popl may act as a tether between substrates and the E2/E3 complexes, or Pop1 may be a component of E3. Recent analysis shows that budding yeast Clns and Sicl become phosphorylated before ubiquitination and degradation Schneider et al. 1996;Willems et al. 1996). Cdc18 and Ruml might need to be phosphorylated in order for Pop l to recognize and bind.

Similarity to other systems: Budding yeast Cdc4, Sicl, and Cdc6, and human p27 K~pl
It is intriguing that in budding yeast Cdc4 is also involved in degradation of a CDK inhibitor, Sicl, through ubiquitin-mediated proteolysis. In the temperature-sensitive cdc4 or cdc34 mutant that is defective in ubiquitin-conjugating enzyme Ubc3 (Goebl et al. 1988), the Sicl protein as well as Cdc6 accumulates (Schwob et al. 1994;Piatti et al. 1996). In parallel with Ruml, Sic1 becomes stabilized in G1, which prevents premature activation of Cdc28/B-type cyclins (Clbl-6) complexes. Furthermore, like the popl mutant, overproduction of the B-type cyclin Clb4 is capable of suppressing partially the temperature-sensitive cdc4-1 mutant (Bai et al. 1994). Although overall homology between Pop1 and Cdc4 is not extremely high (30%), it is worth pointing out that Cdc4 is a protein that shows the highest homology in the entire budding yeast genome. In addition, the F-box motif that appears to be required for a physical interaction between Cdc4 and Skpl is also conserved in Popl. Thus it is very likely that Pop l is a fission yeast homolog of Cdc4. Skpl has been shown to be also required for ubiquitin-mediated proteolysis of Cln2, Clb5, and Sic1 (Bai et al. 1996). Cdc34 (Ubc3), Cdc53, and Skpl are shown to act closely with Cdc4 and Sic1 (Schwob et al. 1994;Mathias et al. 1996;King et al. 1996). It is interesting to see whether in fission yeast these homologs function with Popl to regulate Ruml and Cdcl8.
In spite of the parallels between Popl-Ruml and Cdc4-Sicl, defective phenotypes of the popl and cdc4 mutants look very different. For example, the CDC4 gene is essential and mutant cells arrest in G1 with multiple buds, whereas popl ÷ is not essential and the mutant leads to overreplication. In addition, in budding yeast, mutants in subunits of the anaphase promoting complex (APC) give rise to polyploidy, implying that this complex is involved in degrading proteins that results in polyploidy when stabilized (Heichman and Roberts 1996). This phenotypic difference, however, could be explained in the following manner. Sic l inhibits Cdc28 kinase activities associated with all six B-type cyclins (Clbl-6) responsible for an S-phase progression (Donovan et al. 1994;Nugroho and Mendenhall 1994;Schwob et al. 1994). As Cln cyclins are stabilized in the cdc34 mutant (Deshaies et al. 1995: Blondel andMann 1996), the cdc4 mutant cells continue budding without DNA replication. In contrast, Ruml inhibits Cdc2/Cdcl3 and partially Cdc2/Cig2, but not Cdc2/Cigl (Correa-Bordes and Fisher and Nurse 1996;Martin-Castellanos et al. 1996). Thus even though a high level of Ruml is present, S phase still occurs owing to activities of Cdc2/ Cigl,2, which can compensate for loss of the S-phase function of B cyclins (Fisher and Nurse 1996;Martin-Castellanos et al. 1996). A common function of Pop1 and Cdc4 can, therefore, be summarized as degradation of CDK inhibitors Rum1 and Sicl and possibly S-phase regulators Cdcl 8 and Cdc6. An interesting question then is whether fission yeast has CDK inhibitors other than Ruml that inhibit Cdc2/Cigl,2 or Pucl, another Gl-type cyclin (Forsburg and Nurse 1991).
A further analogy can been seen in mammalian systems where a CDK inhibitor, p27 Kip1, has been shown to be degraded through ubiquitin-dependent proteolysis (Pagano et al. 1995). Interestingly, it has been shown that Ubc3 (and Ubc2) is responsible for degradation of p27 Kip1. In animal cells, CDK inhibitors that inhibit specifically Cdc2/cyclin A or B have not been identified. There is, however, circumstantial evidence that supports the existence of such CDK inhibitors (e.g., Waldman et al. 1996). In opposition to the current view of the cellular function of CDK inhibitors (Sherr and Roberts 1995), the phenotypes of the popl mutant leading to Ruml and Cdcl8 accumulating are apparently analogous to those of tumor cells, which also result in increased ploidy, genome instability, and failure to differentiate (Hartwell and Kastan 1994). Identification of novel CDK inhibitors as well as a Popl homolog in mammalian cells would be a future issue to explore evolutionarily conserved functions of a WD-repeat protein.

Strains, media, genetic techniques, and nomenclatures
The Schizosaccharomyces pombe strains used in this study are listed in Table 2. HM123 was used as wild type unless otherwise stated. Standard procedures for S. pombe genetics were followed according to Moreno et al. (1991). Low nitrogen minimal medium (0.25% NH4C1 instead of 0.5%) was used for nitrogen starvation experiments. Plates contained 1.6% agar. All the cultures were grown at 30°C unless otherwise stated. Gene disruptions are abbreviated as the gene preceded by/~, such as z~popl.
Isolation of the popl mutants h 9° strain (TP114-2A, Table 1) was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine. Sterile mutants (1008) were first isolated by iodine vapor treatment from -200,000 mutagenized colonies. Each sterile mutant was grown in low nitrogen medium for 3 days at 26°C and processed for FACS as described previously (Kumada et al. 1995).

Allelism tests
A cross between each mutant and wild type or a pairwise cross among mutants was performed using protoplast fusion method (Moreno et al. 1991). Crosses between the popl mutants (364, 422, and 577) and wild type (HM143) showed that a mutation in the three mutants is recessive and segregates as a single chromosomal locus. In addition, all the three mutants (KK200, 422, and 577) did not complement each other, demonstrating that they were derived from mutations in the same locus.

Cloning of the pop 1 + gene
The popl-364 cdc2-33 strain (KK201) was used to isolate the popl ÷ gene. Briefly the double mutant was transformed with an S. pombe genomic library constructed in a vector pDB248. LEU ÷ transformants that could form colonies at 32°C but not at 36°C were selected. In total 14,000 colonies were screened and three transformants were obtained. Three plasmids that contained different but overlapping insert DNAs (pKK101, 103, and 105) were obtained.

Nucleotide sequence determination
The nucleotide sequence of a 2.8-kb SmaI-BglII fragment (Fig.  5A) was determined with the dideoxy method (Sanger et al. 1977). These sequence data are available from EMBL/GenBank/ DDBJ under accession no. Y08391.

Gene disruption
Two kinds of plasmids were constructed, an insertion and a complete deletion. For insertion, a 1.8-kb ura4 ÷ fragment was inserted in an internal SacI site of pKK103 (Fig. 5A). For a deletion method, a 2.2-kb BglII fragment that encompasses 94% of ORF was deleted and replaced with a 1.8-kb ura4 + fragment. A diploid strain (CHP428/429) was used to disrupt the popl ÷ gene.
Stable Ura + transformants were picked and the disruption of the popl ÷ gene was verified by PCR and then tetrad analysis was performed. We obtained four viable spores where uracil auxotrophy segregates 2:2 and Ura ÷ segregants showed polyploid phenotype. The popl-364 mutant did not complement with Apopl, demonstrating that the gene we cloned is popl ÷.
Construction and expression of the tagged popl ÷ and ruml ÷ genes The tagged version of the popl ÷ gene was constructed as follows. The entire popl ÷ gene was amplified by PCR using two oligonucleotides; the 5' oligonucleotide is 5'-GGGCATAT-GAATGAGAAGAAAAAAGA-3' where the NdeI site is underlined and the 3' oligonucleotide is 5'-GGGGCGGCCGC-TAGAAGATTTAGTAGATCCAA-3' where the NotI site is underlined. A PCR-amplified 2342-bp fragment was subcloned into pREP41 (HA-6His) (provided by Dr. J. Millar, National Institute for Medical Research, London, UK) to yield pREP41-popl+-HA/6His, which expresses the gene under the control of a partially crippled version of the nmtl thiamine-repressible promoter (Basi et al. 1993) and contains six His and two repeats of a peptide derived from the influenza HA. pREP42-popl +-HA/ 6His was made by subcloning a 2.4-kb NdeI-BamHI fragment from pREP41-popl+-HA/6His into pREP42. Tagging of 6His-HA in the carboxyl terminus did not interfere with the normal function of the Popl protein as pREP41-popl+-HA/6His suppressed the popI mutation. A 2279-bp BglII-BamHI fragment containing HA/6His was replaced with a 2.2-kb BglII fragment of pKK103, where the popl+-HA/6His was regulated under a natural promoter. The tagged version of the rumi ÷ gene (pREP41-rum 1 +-HA~ 6His) was constructed in a manner similar to popl ÷-HA/6His except that the following primers were used: 5'-GGGCATATGGAACCTTCAACACCAC-3' and 5'-GGGG-GGGGGCGGCCGCTTCGTAATAAATTGTGCCTGT-3'.

Immunochemical assays
Antibodies used in this study were following; anti-Cdc2, anti-Cdcl3 (provided by Dr. H. Yamano, ICRF), anti-Rum1 (affinitypurified, a gift from Dr. J. Correa-Bordes, ICRF), anti-Cdcl8 (af-finity-purified, a gift from Dr. H. Nishitani, ICRF), anti-tubulin antibody (T-5168, Sigma), anti-HA (16B12, BAbCO), anti-myc (9El0, ICRF), and anti-ubiquitin monoclonal antibody (MAB 1510, Chemicon Inc.). Fission yeast cell extracts were prepared as described (Moreno et al. 1991). To detect the Ruml protein in total cell extracts, cells were boiled for 5 min before breakage with glass beads (Correa-Bordes and . To detect ubiquitination of Ruml and Cdcl8, cell extracts were prepared as follows essentially according to Watanabe et al. (1997). Cells were rapidly killed by addition of the ice-cold extraction buffer (50 mM Tris-HC1 at pH 7.4, 1 mM EDTA, 10 mM ~-mercaptoethanol, 0.5 mM PMSF) containing 10% of trichloroacetic acid (TCA) and disrupted with glass beads. After washing with acetone five times, the proteins were solubilized by boiling (3 min) in the extraction buffer containing 1% SDS and 0.1 ~M ubiquitin aldehyde (kindly provided by Dr. Keiji Tanaka, The Tokyo Metropolitan Institute of Medical Science, japan) to prevent deubiquitination. The supernatant was mixed with an equal amount of the HB buffer (Moreno et al. 1991). Standard procedures for immunoblotting were followed (Harlow and Lane 1988) except that CAPS (3-cyclohexylamino-1-propanesulfonic acid) buffer was used to detect proteins <50 kD (Mat-sudaira 1987). For immunoprecipitation, cell extracts (2.5 mg) were mixed with anti-myc antibody or anti-HA antibody, followed by protein G-Sepharose beads (Pharmacia). HA-peptide (40 ~g) was added to extracts in a competitor experiment. Antiubiquitin antibody was used in 1:2500 dilution. Horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Bio-Rad) and a chemiluminescence system (ECL, Amersham) were used to detect bound antibody.