The Identification of Wos2, a p23 Homologue That Interacts With Wee1 and Cdc2 in the Mitotic Control of Fission Yeasts

Corresponding author: Juan Jimenez, Departamento de Genética, Facultad de Ciencias, Universidad de Málaga, Campus Universitario de Teatinos, 29071 Málaga, Spain., jimmar{at}uma.es (E-mail)

Communicating editor: P. G. YOUNG

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Wee1 kinase inhibits entry into mitosis by phosphorylation of the Cdc2 kinase. Searching for multicopy suppressors that abolish this inhibition in the fission yeast, we have identified a novel gene, here named wos2, encoding a protein with significant homology to human p23, an Hsp90-associated cochaperone. The deletion mutant has a modest phenotype, being heat-shock sensitive. Using antibodies raised against bacterially produced protein, we determined that Wos2 is very abundant, ubiquitously distributed in the yeast cell, and its expression dropped drastically as cells entered into early stationary phase, indicating that its function is associated with cell proliferation. In proliferating cells, the amount of Wos2 protein was not subjected to cell cycle regulation. However, in vitro assays demonstrated that this Hsp90 cochaperone is potentially regulated by phosphorylation. In addition to suppressing Wee1 activity, overproduction of Wos2 displayed synthetic lethality with Cdc2 mutant proteins, indicating that this Hsp90 cochaperone functionally interacts with Cdc2. The level of Cdc2 protein and its associated H1 kinase activity under synthetic lethal conditions suggested a regulatory role for this Wos2-Cdc2 interaction. Hsp90 complexes are required for CDK regulation; the synergy found between the excess of Wos2 and a deficiency in Hsp90 activity suggests that Wos2 could specifically interfere with the Hsp90-dependent regulation of Cdc2. In vitro analysis indicated that the above genetic interactions could take place by physical association of Wos2 with the single CDK complex of the fission yeast. Expression of the budding yeast p23 protein (encoded by the SBA1 gene) in the fission yeast indicated that Wos2 and Sba1 are functionally exchangeable and therefore that properties described here for Wos2 could be of wide significance in understanding the biological function of cochaperone p23 in eukaryotic cells.

THE protein kinase encoded by cdc2 plays a key role in the mitotic control of all eukaryotic cells (NURSE 1990 ). In the fission yeast Schizosaccharomyces pombe, the activity of Cdc2 requires binding of the cdc13-encoded B-type cyclin, and the Cdc2-cyclin B complex is maintained in a repressed state during interphase by phosphorylation of the Cdc2 subunit on Tyr15 (GOULD and NURSE 1989 ). The major Tyr15 phosphorylation activity is provided by the Wee1 protein kinase (RUSSELL and NURSE 1987A ; MCGOWAN and RUSSELL 1993 ) whereas Cdc25 provides the predominant Tyr15 dephosphorylation activity (RUSSELL and NURSE 1986 ; GAUTIER et al. 1991 ), required to activate the Cdc2-cyclin B complex and subsequently to trigger mitosis.

The search for new elements involved in the regulation of Cdc2 by Wee1 in fission yeasts led to the identification of swo1, a gene encoding an Hsp90-like protein required for Wee1 activity (ALIGUE et al. 1994 ; MUNOZ and JIMENEZ 1999 ). Genetic analysis has shown that the Hsp90/Swo1 chaperone is also required for Cdc2 function (MUNOZ and JIMENEZ 1999 ), pointing toward Hsp90 as an important element in the regulation of these key cell cycle kinases.

Hsp90 is an abundant molecular chaperone essential to the establishment of many cellular regulation and signal transduction systems. In addition to playing a vital role in thermotolerance and stress responses, the cytoplasmic Hsp90s are essential for establishing the function of steroid hormone receptors, transcription factors, tyrosine and threonine/serine kinases, and tumor suppressors (PRATT 1997 ). In vitro, purified Hsp90 binds to denatured protein and displays antiaggregant properties. However, in vivo, Hsp90 activity involves a cohort of associated partners or cochaperones that participate in multiprotein complexes with Hsp90. Several discrete subcomplexes are associated with particular subclasses of target proteins, so that one of these subcomplexes contains Hsp90/Hsp70/Hip and p60/Sti1 (SMITH et al. 1993 ), while another contains one of several immunophilins/cyclophilins and the acidic protein p23 (JOHNSON and TOFT 1994 ).

The p23 protein is one of the most abundant Hsp90-associated proteins whose function in living cells remains uncertain. This cochaperone was first identified in association with steroid receptors (SMITH et al. 1990 ) and was later shown to be a major component of Hsp90 complexes (JOHNSON and TOFT 1994 ). It is thought that p23 interacts with target polypeptides that are indirectly associated via Hsp90. However, in vitro folding assays have shown that p23 itself can function as a molecular chaperone that binds independently to proteins and prevents them from aggregating (BOSE et al. 1996 ; FREEMAN et al. 1996 ). In this report, we present a genetic and biochemical characterization of a p23 homologue from the fission yeast S. pombe. We show that the identified p23 cochaperone is required for heat-shock tolerance and plays a role in the activity of Wee1 and the regulation of Cdc2.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strains, media, and general methods:
The standard media and genetic procedures used in this work have been previously described (MORENO et al. 1991 ). The fission yeast S. pombe 972 h^- and 975 h⁺ haploid wild-type strains were used. The thermosensitive (ts) strains cdc2-33, cdc2-M26, cdc2-56, cdc2-L7, and cdc25-22 and the strains adh:wee1-50 (a strain overproducing the thermosensitive Wee1-50 protein from the adh promoter), CDC2Dm (in which the endogenous cdc2 is replaced by its Drosophila homologue), and swo1-w1 (partially defective in Hsp90 function) have been previously described (NURSE et al. 1976 ; NASMYTH and NURSE 1981 ; RUSSELL and NURSE 1987A , RUSSELL and NURSE 1987B ; BEJARANO et al. 1995 ; MUNOZ and JIMENEZ 1999 ). Double mutants were constructed by tetrad dissection. The strain nmt:wos2 (the wos2 gene is expressed from the nmt thiamine-repressible promoter; MAUNDRELL 1993 ) and wos2::ura4 (in which the wos2 gene is replaced by the ura4 gene) are described in this study. Yeast transformation was achieved using the electroporation procedure (PRENTICE 1992 ). Flow cytometry analysis was performed as described in SAZER and SHERWOOD 1990 . Cells were fixed with ethanol, stained with propidium iodine, and a Becton Dickinson FAC-Scan system was used. Fixed cells were also stained with 4', 6-diamidino-2-phenylindole (DAPI) and nuclei visualized in a Nikon microscope equipped with epifluorescence and a CCD camera. Calcofluor staining was used to visualize the cell wall and septum material. Double staining with DAPI and calcofluor was used to visualize nuclei and septum in the same cells. Under the conditions used, brightness of calcofluor is more intense than that of DAPI but the former photobleaches more rapidly. Thus, capturing fluorescent images early or late after excitation and using different exposure times allows us to visualize calcofluor and DAPI independently. Anti--tubulin (a generous gift from Dr. K. Gull) was used for microtubule localization (1/25 dilution) and anti-Wos2 polyclonal rabbit serum (1/400 dilution) for the localization of Wos2, following described procedures (ALFA et al. 1993 ). Bound antibodies were detected using fluorescein-conjugated secondary antibodies (Sigma, St. Louis; 1/40 dilution). The synchronous culture was prepared by means of the cdc25-22 ts mutation. A culture of a cdc25-22 strain was synchronized by restricting growth at 36° for 4 hr. The cells were then released into fresh medium at the permissive temperature of 25°, and aliquots were removed at 20-min intervals over 8 hr. The septation index was monitored in a Nikon microscope using dark-field microscopy. Protein samples were prepared for Western blot analysis of Wos2. For stationary culture experiments, cells were grown at 32° in minimal liquid medium until glucose (the limiting nutrient in the medium used) became exhausted. Samples of total RNA and protein were also used to visualize the wos2 mRNA and Wos2 protein, by standard Northern and Western blot analysis, respectively. Heat shock was achieved by transferring cultures exponentially growing at 25° in liquid media to 48° in a water bath. Aliquots were then taken at time intervals and placed in ice. Drops from these aliquots with equal number of cells were then placed in a YE plate to monitor growth after incubation for 2 days at 25°, or viable cells were counted by counting colonies formed from diluted samples plated in YE, incubated for 5 days at 25°.

Cloning of wos2:
An adh:wee1-50 leu1-32 strain was transformed with a S. pombe genomic library constructed in the pDB262 plasmid (WRIGHT et al. 1986 ). Prototrophic transformed cells were selected at 35° in minimal medium agar plates, and the colonies formed were replica plated into YE agar plates and incubated at 25°. Colonies growing under these conditions were microscopically screened, and one of them was selected because of the mitotic instability of the adh:wee1-50 suppressing activity, indicating that it is located in a plasmid. The plasmid was recovered from one of these colonies, and the suppressing activity localized to a 3.9-kb S. pombe genomic DNA fragment. This fragment was entirely sequenced in both strands and a single gene found (wos2) responsible for the suppression activity (GenBank accession no. L41166). This gene contained a single intron with conventional features found in S. pombe introns (PRAHALA et al. 1992 ). Using this 3.9-kb DNA fragment as a probe, wos2 was physically mapped to a single locus in chromosome I linked to mst1, between rad16 and nuc2.

Deletion of wos2:
To construct a wos2-deleted strain (wos2::ura4) an XbaI-AvaI fragment in wos2 was replaced with the ura4 gene in the 3.9-kb wos2 genomic clone. This construct was used to delete the wos2 gene in a diploid strain h⁺/h^- ade6-M210/ade6-M216 ura4-d18/ura4-d18. Tetrad dissection indicated that these diploids produced four viable meiotic spores. Stable ura⁺ diploids were selected, and Southern blot analysis indicated that the wos2 gene was deleted in these ura⁺ haploid spores. Southern blot analysis was carried out by digesting 1–2 µg DNA of the desired strain, separating the digested DNA on 0.9% agarose gels and blotting onto Hybond-N⁺ (Amersham, Piscataway, NJ) membranes according to the manufacturer's instructions. These membranes were hybridized in Church buffer at 65° (CHURCH and GILBERT 1984 ) and probed with wos2 DNA labeled by the random priming method. The filters were washed in 0.1x standard saline citrate (SSC)/0.1% SDS at 65° and autoradiographed or analyzed using a PhosphorImager system (Fuji).

Overexpression of wos2:
To isolate cDNAs coding for wos2, Escherichia coli colonies transformed with a cDNA library of S. pombe (MORENO and NURSE 1994 ) were probed with the 3.9-kb genomic fragment of wos2, and a variety of cDNAs were selected because of their different sizes. The different cDNAs were sequenced, and the largest one was subcloned into the pREP3X S. pombe expression vector (in this construct, the expression of wos2 is driven by the nmt1 thiamine repressible promoter) (MAUNDRELL 1993 ). To construct a stable strain overproducing wos2 (the nmt:wos2 strain), the promoter-cDNA-transcriptional stop construct was inserted into the pJK148 integrative plasmid. An h^- leu1-32 strain was transformed with this pJK-wos2 construct linearized within the leu1 gene, and a stable leu⁺ transformant was selected. Southern blot analysis indicated that a single copy of the construct was integrated at the leu1-32 locus. Similarly, the nmt:wos2 construction was subcloned into an ura4 integrative plasmid. An h^- ura4-D18 strain was transformed with this construction linearized within the wos2 gene, and stable ura⁺ transformants were selected. Southern blot analysis indicated that a single copy of the construction was integrated at the wos2 loci. These strains overexpressed wos2 when grown in minimal medium without thiamine. Samples were routinely taken 19 hr after induction. A total of 5 µg/ml thiamine was added to turn the nmt1 promoter off. The ura⁺ and leu⁺ markers were used to construct strains overexpressing wos2 in different genetic backgrounds or to select a strain overexpressing wos2 from two copies of the nmt:wos2 construction (2x nmt:wos2).

Expression of the S. cerevisiae p23 homologue in the fission yeast:
A budding yeast homologue to wos2 was first identified as sequence YKL518, derived from the S. cerevisiae genome project (JACQUIER et al. 1992 ). More recently, this gene has been characterized as a p23 homologue named SBA1 (FANG et al. 1998 ). Oligonucleotides 5'-CGATTCATAATAGTCATGTCCG-3' and 5'-GCACTCCAGGTTGATTTGCTCC-3' were used to clone by PCR amplification the YKL518 open reading frame (ORF) of S. cerevisiae, and the resulting DNA fragment was subcloned into the pREP3x plasmid. This construct allows the nmt-driven expression of the amplified DNA in S. pombe.

Northern and Western blot analyses:
In Northern blot analysis, total RNA was prepared as described (MORENO et al. 1991 ) and probed with the 3.9-kb purified fragment containing the wos2 gene. Filters were analyzed using a PhosphorImager system (Fuji). Western blot procedures were performed by loading equivalent amounts of protein in SDS-polyacrylamide gels, and the blot was carried out using GeneScreen Plus membranes according to the manufacturer's (New England Nuclear, Boston) instructions. To obtain polyclonal antibodies against Wos2, the large cDNA encoding wos2 was subcloned in an E. coli expression vector (SAMBROOK et al. 1989 ); the E. coli-overproduced Wos2 protein was extracted from a polyacrylamide gel, and preparation of protein conjugates for rabbit immunization was as described (SAMBROOK et al. 1989 ). Western blots were made using serum (1/1000 dilution) against this gel-purified Wos2 protein overproduced in E. coli, anti--tubulin (Sigma; 1/10,000 dilution), or monoclonal anti-Cdc2 (1/1000 dilution) provided by Paul Nurse (ICRF, London). The blots were developed with an enhanced chemiluminescence method (ECL, Amersham).

Immunoprecipitation and H1 kinase assay:
Conventional experiments of immunoprecipitation in S. pombe extracts were carried out following described methods (MORENO et al. 1991 ). The immunoprecipitation of cell extracts obtained with 2 µl of anti-Wos2 or with 2 µl of anti-Cdc2 was used for histone H1 kinase assays as described in MORENO et al. 1991 . This procedure was also performed to assay Wos2 phosphorylation. An aliquot of each sample was used for Western blot analysis and Coomassie blue staining as controls.

[³⁵S]Methionine labeling and in vitro association assays:
For in vitro experiments, [³⁵S]methionine-labeled Wos2, Cdc2, and Cdc13 proteins were synthesized using the TnT Coupled Reticulocyte Lysate system kit (Promega, Madison, WI), according to the manufacturer's instructions. Aliquots of 2 µl of reticulocyte lysates containing the synthesized proteins were mixed, and immunoprecipitation with 1 µl of anti-Wos2, 1 µl of anti-Cdc2, or 1 µl of anti-Cdc13 antibodies was carried out as described in MORENO et al. 1991 . The labeled proteins were resolved by electrophoresis in polyacrylamide gels and visualized by autoradiography.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of wos2:
Wee1 encodes a tyrosine kinase that inhibits entry into mitosis by phosphorylation of the Cdc2 kinase. Overproduction of Wee1 causes a lethal G2 arrest. Since Wee1 is not essential for yeast growth or viability (RUSSELL and NURSE 1987A ), searching for multicopy suppressors of this lethal effect is a powerful selective procedure to isolate genes encoding negative regulators of Wee1. The adh:wee1-50 strain was used to this end. This strain overproduces a Wee1-50 thermosensitive protein (RUSSELL and NURSE 1987A ), which results in viable "wee" cells at 35°, where Wee1-50 is inactive, but yields a lethal cell cycle block upon incubation at 25°, where the protein becomes active. Using this strain, we isolated a 3.9-kb S. pombe DNA fragment from a genomic library, which, in a multicopy plasmid, rescued the lethality caused by the overproduction of Wee1-50. Subcloning and DNA sequencing indicated that a single gene (here named wos2, for wee1-50 overexpression suppressor) was responsible for the wee1-50 overexpression suppressor phenotype (Figure 1A). This gene physically mapped in chromosome I, between rad16 and nuc2. We also analyzed the suppression activity of wos2 using a different conditional system to overexpress wee1. Strains harboring a wee1 wild-type allele or a wee1-50 ts allele, subjected to the expression of the thiamine repressible promoter nmt (MAUNDRELL 1993 ), grow on thiamine-containing media but produce elongated arrested cells under derepressed conditions. When using these strains, we found that multicopy expression of wos2 suppressed the lethal arrest of the nmt:wee1-50 strain, too, indicating that the suppression activity of wos2 relied on the encoded Wee1-50 protein and not on the promoter used for its overexpression (Figure 1B).

View larger version (103K): In this window In a new window Download PPT slide   Figure 1. Identification of wos2. (A) Schematic representation of the genomic fragment responsible for the Wee1-50 suppression activity. The only gene in this fragment (wos2) contains a small intron (exons I and II are in boxes). The TATA box (), transcriptional ends (•), and restriction sites used in this work (XbaI-X-, DraI-D-, ClaI-C-, and AvaI-A-) are indicated. (B) Suppression activity of the cloned S. pombe wos2 gene. The DNA fragment containing wos2 was subcloned into the pON163 multicopy plasmid (pON163-wos2), and its suppression activity assayed in S. pombe cells overexpressing either the wee1-50 allele or the wild-type wee1 allele from the nmt promoter. Plasmid pON160 was used as a control. Suppression efficiently occurred in nmt:wee1-50 cells, yielding "wee" cells (on plasmid loss cells become elongated) as happens in the adh:wee1-50 strain, but cells overexpressing the wild-type wee1 gene were not suppressed by multicopy expression of wos2.

However, the multicopy expression of wos2 did not suppress the arrest caused by the wild-type protein, indicating that the Wee1-50 mutant version is more sensitive to the action of Wos2 (Figure 1B). Such interaction also occurred in a nim1-deleted background, the only upstream inhibitor of wee1 described in fission yeasts (RUSSELL and NURSE 1987B ), and consequently wos2 interacts with Wee1 in a nim1-independent manner.

When searching for homologous sequences in DNA and protein databases, we found that Wos2 shares 30% identity to the human p23, and it is also homologous to the S. cerevisiae protein encoded by DNA sequence YKL518 (Figure 2A). This sequence was initially described from the genome sequencing project of this microorganism (JACQUIER et al. 1992 ), but very recently it has been identified as the budding yeast homologue to vertebrate p23 proteins (encoded by the SBA1 gene; FANG et al. 1998 ). Thus, Wos2 could identify a p23-like cochaperone of fission yeast. A detailed sequence analysis revealed that it contains a glycine-rich sequence common to Hsp70 and many other molecular chaperones (THODE et al. 1996 ).

View larger version (67K): In this window In a new window Download PPT slide   Figure 2. Sequence and physiological analysis of wos2 gene function. (A) Identity between the wos2-encoded protein and its counterparts from S. cerevisiae (Sba1/YKL518) and from the human p23 chaperone (p23 human; identical residues found in at least two sequences are indicated in shadow). (B) Western blot analysis using anti-Wos2 in wild-type cells (wos2) and cells deleted for the wos2 gene (wos2). (C) Involvement of wos2 in the heat-shock tolerance of S. pombe cells. Growth temperature of wild-type (wos2) or wos2-deleted (wos2) cells was shifted from 25° to 48°, and time-interval samples were taken to determine cell survival, either by plating a drop with equal number of cells (left) or by counting the number of cells per milliliter that form a colony (average and standard deviation of three independent experiments) (right). (D) Acid-base residue distribution of Wos2, Sba1/YKL518, and human p23 proteins.

Proteins involved in chaperone functions and the stress response are often required for heat-shock tolerance. To examine a possible role of wos2 in this response, we constructed a deletion mutant strain lacking Wos2 protein (Figure 2B). The wos2 deletion had no effect on cell growth or viability, but S. pombe cells deleted for wos2 were heat-shock sensitive (Figure 2C), indicating that Wos2 is actually a stress-related protein. Therefore, from the above combined results we conclude that Wos2 is likely a p23 protein in fission yeasts.

The putative protein sequence encoded by wos2 shows some other striking properties in common with YKL518/SBA1 and p23 homologues. The carboxyl-terminal half of these proteins has very few basic amino acids and, by contrast, contains clumps of acidic residues (Figure 2D). This poly-acidic sequence is also found in many other proteins, including cell cycle regulatory elements such as the S. cerevisiae Sic1 protein (SCHWOB et al. 1994 ) and a large number of cyclins, although the biological significance of this similarity is unknown.

Expression of wos2:
To further elucidate the biological function of wos2 and its possible regulation, we studied the expression of this gene in different phases of the S. pombe cell cycle and under different physiological conditions. No significant cell-cycle-dependent changes were observed in Wos2 during two rounds of synchronous cell divisions (Figure 3A), indicating that the level of this protein was not subject to oscillations during the cell cycle. The wos2 messenger RNA was extremely abundant in proliferating cells, being roughly >20-fold the level of cdc2 mRNA (assessed by PhosphorImager analysis of Northern blots). However, its expression dropped drastically as cells entered into early stationary phase (while the amount of cdc2 transcripts stayed constant; Figure 3B). The amount of Wos2 protein also decreased in starved cells (Figure 3B), indicating that its function is probably associated with cell proliferation. Immunofluorescence analysis revealed punctate staining of Wos2 in exponentially growing cells, which suggests that Wos2 is not a freely diffusible molecule but is restricted to dispersed macromolecular aggregates found in the nucleus and the cytoplasm (Figure 3C). As described for the expression of protein p23 in avian cells (JOHNSON et al. 1994 ), expression of Wos2 was not significantly induced by temperature, either by growth at 35° or by heat shock for 10–20 min at 48° (data not shown).

View larger version (42K): In this window In a new window Download PPT slide   Figure 3. Expression of wos2 in S. pombe cells. (A) Western blot analysis of Wos2 protein from protein samples of a synchronous culture followed for two generations. -Tubulin was analyzed as a control. The abundance of Wos2 (densitometric analysis) and the septation index in each sample of the synchronous culture are represented. Cells were synchronized by using a cdc25-22 mutant strain (see MATERIALS AND METHODS). Similar results were obtained in samples from cells synchronized by elutriation (not shown). (B) Northern blot analysis of wos2 mRNA in total RNA samples from proliferating (lane 1) and stationary starved (lane 2) cells (cdc2 mRNA was analyzed as a control). The abundance of Wos2 protein was also determined in the same cells by Western blot analysis. (C) Epifluorescence micrographs of wild-type (wt) and wos2-deleted cells (wos2) double stained with DAPI and anti-Wos2 antibody/fluorescein-conjugated secondary antibody (top micrographs). In our filter set the calcofluor signal is still seen in the fluorescein channel, and cells without DAPI staining were also used for the proper immunolocalization of Wos2 (bottom micrographs).

To further analyze the phenotype caused by the overexpression of wos2, we isolated cDNAs for this gene. Three different polyadenylated cDNAs were isolated, corresponding to the three transcripts observed in Northern blot analysis (Figure 3B). The largest wos2 cDNA was expressed under the control of the thiamine repressible nmt promoter, and the construct integrated at the leu1 locus in the S. pombe genome in a single copy. The genetic interactions previously described by using a multicopy plasmid system to overexpress the genomic wos2 gene took place as well upon wos2 overexpression from the nmt promoter in this strain. This nmt:wos2 strain was further used for genetic analysis.

Genetic interactions of wos2 and cdc2:
Interestingly, overproduction of Wos2 showed allelic interactions identical to those caused by a partial Hsp90 inactivation (MUNOZ and JIMENEZ 1999 ). In addition to suppressing Wee1-50 activity, the overexpression of wos2 produced a large proportion of elongated cells (~20%) in combination with the cdc2-33 ts allele at 25°, permissive temperature. These elongated cells were multinucleate and had multiple septa. The phenotype was exacerbated at 29°, while cells retained a wild-type phenotype under nmt:wos2 repression under this condition (Figure 4). At 29° in the absence of thiamine 100% of the cells were arrested and, as determined by flow cytometry analysis (Figure 4), ~70% of them were 4C or 8C (DNA content per cell) in agreement with the proportion of binucleate and tetranucleate cells observed under the microscope under these conditions.

View larger version (91K): In this window In a new window Download PPT slide   Figure 4. Genetic interactions between wos2 and cdc2. Calcofluor and DAPI staining of cells harboring the nmt:wos2 construction in combination with different cdc2 alleles (cdc2-L7, cdc2-33 cdc2-M26, and CDC2Dm) incubated in media with thiamine (control) and without this vitamin (overexpressing wos2) at 29°. Calcofluor and DAPI staining are shown simultaneously in cells growing in the presence of thiamine. Flow cytometry analyses (DNA content per cell) of these strains incubated at 29° in media without thiamine are also shown. Populations containing 2C, 4C, and 8C represent mononucleate, binucleate, and tetranucleate cells, respectively.

A more drastic effect was observed when the overexpression of wos2 took place in a cdc2-M26 ts mutant, where >50% of the cells had condensed chromatin at 25° and many exhibited a novel septation defect. In these cells, the septum was initiated but exit of septum formation did not occur, and therefore the septum was continuously produced, sometimes along the cell (Figure 4). In some other cells, the septation pattern mimics that of S. pombe cdc16^- mutants (CHANG and NURSE 1993 ; FRANKHAUSER and SIMANIS 1994 ). The effect of wos2 over this cdc2-M26 allele was also enhanced with temperature, yielding 100% arrested cells at 29° and a high proportion of mononucleate cells with 2C DNA content (Figure 4).

A fission yeast strain in which the endogenous cdc2 gene was replaced by the Drosophila CDC2Dm homologue was also used (JIMENEZ et al. 1990 ). This Drosophila gene is functional in fission yeast, but exhibits a weak interaction with cdc2-regulatory proteins such as Wee1 and the B-type cyclin encoded by cdc13 (BEJARANO et al. 1995 ). The overexpression of wos2 in this CDC2Dm S. pombe strain was lethal at any temperature, producing 100% nonseptated Cdc^- cells with a G2 arrest (Figure 4).

The effects on these cdc2 alleles also occurred in a wee1-deficient background, indicating that this interaction is independent of wee1. The overexpression of wos2 in combination with other cdc2 alleles (such as cdc2-L7, cdc2-56, or "wee" alleles of cdc2), with ts alleles in cdc genes other than cdc2 (cdc10-129, cdc17-K42, cdc13-117, and cdc25-22 were tested), or in a wild-type background had no obvious effects on the cell cycle (see cdc2-L7 in Figure 4). Therefore, on the basis of this allele-specific interaction, we conclude that Wos2 functionally interacts with the Cdc2 mitotic machinery.

As assessed by Western blotting and histone H1 phosphorylation, the overproduction of Wos2 did not alter the level of Cdc2 protein or its kinase activity (Figure 5). In fact, the level of H1 phosphorylation slightly increased in cdc2-M26 cells overexpressing wos2 (Figure 5, lane 2), consistent with the mitotic arrest observed in this strain (Figure 4). These results suggest that Wos2 interferes with regulatory functions of Cdc2 rather than its stabilization or kinase activity.

View larger version (44K): In this window In a new window Download PPT slide   Figure 5. Effects of the overproduction of Wos2 in the abundance of Cdc2 and the level of Cdc2 kinase activity. Cellular proteins from wos2 (lane 1) and wos2-overexpressing cells (lane 2) in a cdc2-M26 background incubated at 29° (100% arrested cells) were separated on SDS-polyacrylamide gel, transferred to a membrane, and the abundance of Cdc2 and Wos2 in the membrane visualized by immunostaining (anti--tubulin was used as a loading control). Crude extracts of these cells were used to assay phosphorylation of added histone H1 in the presence of [-32P]ATP. Histone phosphorylation was determined by autoradiography.

Wos2 interferes with Hsp90 activity:
Wee1 and Cdc2 kinases are both highly dependent on Hsp90 activity for function (ALIGUE et al. 1994 ; MUNOZ and JIMENEZ 1999 ). Interestingly, all these genetic and biochemical effects caused by overexpression of Wos2 in cdc2 mutant cells resembled those caused by partial inactivation of the Hsp90 chaperone encoded by swo1 (MUNOZ and JIMENEZ 1999 ). This observation strongly suggests that an excess of Wos2 could diminish the activity of Hsp90 on its Hsp90-target kinases. To investigate this hypothesis we analyzed the effect caused by overproducing Wos2 in Hsp90-deficient cells. As shown in Figure 6, cells overproducing Wos2 and those partially defective for Hsp90 (swo1-w1) were indistinguishable from wild type at 25°. However, overexpression of wos2 was lethal in a swo1-w1 mutant background. This synergy between nmt:wos2 and swo1-w1 suggests that overproduction of Wos2 interferes with Hsp90 function in vivo. The synthetic cell cycle defect observed by overexpression of wos2 in swo1-w1 mutant cells (Figure 6) resembled that produced by overexpression of wos2 in cdc2-33 mutant cells (Figure 4). In both cases, identical septation deficiencies and abnormal distributions of DNA content per cell were obtained. This observation suggests that the excess of Wos2 could specifically interfere with the Hsp90-Cdc2 interaction.

View larger version (91K): In this window In a new window Download PPT slide   Figure 6. Genetic interactions between wos2 and swo1. Calcofluor and DAPI staining of cells overproducing Wos2 (nmt:wos2 in media without thiamine), cells partially deficient for Hsp90 activity (swo1-w1 at 25°), and cells overproducing Wos2 in a swo1-w1 background (nmt:wos2 swo1-w1 without thiamine at 25°). Flow cytometry analyses (DNA content per cell) of these cells are also shown. Populations containing 2C, 4C, and 8C represent mononucleate, binucleate, and tetranucleate cells, respectively.

In vitro systems have been used to demonstrate that vertebrate p23 physically interacts with target proteins (BOSE et al. 1996 ; FREEMAN et al. 1996 ). To assay for physical interactions, [³⁵S]methionine-labeled Wos2 and Cdc2 were synthesized separately using reticulocyte lysates, and association of these two proteins was assayed by coimmunoprecipitation in mixed lysates. As shown in Figure 7A, anti-Wos2 antibodies coimmunoprecipitated labeled Cdc2 from mixed extracts and, reciprocally, monoclonal anti-Cdc2 copurified Wos2. The anti-Cdc2 antibody was highly specific for the S. pombe Cdc2 (Figure 7A); therefore, the existence of Wos2 in anti-Cdc2-purified proteins from Wos2-Cdc2 mixed extracts suggests that Wos2 physically interacts with Cdc2 in vitro. The relatively low amount of Wos2 coimmunoprecipitated with anti-Cdc2 indicates that these two proteins interact with low affinity, as has been previously described for other chaperones (SMITH 1993 ; BOSE et al. 1996 ; FREEMAN et al. 1996 ). Polyclonal anti-Wos2 antibodies nonspecifically bind a mild amount of Cdc2 (perhaps through reticulocyte p23 protein), but in equivalent amounts of synthesized Cdc2 protein, the presence of Wos2 significantly increased the level of Cdc2 coimmunoprecipitated with anti-Wos2, in support of a Cdc2-Wos2 association.

View larger version (76K): In this window In a new window Download PPT slide   Figure 7. Physical association of Wos2 and Cdc2 determined by coimmunoprecipitation assays from labeled proteins synthesized in reticulocyte extracts. (A) Immunoprecipitation of Wos2 and/or Cdc2 using anti-Wos2 or anti-Cdc2 from extracts containing Wos2, Cdc2, or both proteins, as indicated. The anti-Wos2 identified a single Wos2 protein. The anti-Cdc2 recognized Cdc2 and two additional truncated polypeptides, generated by translation of cdc2 mRNAs from internal methionine codons. (B) Immunoprecipitation of Wos2, Cdc2, and/or Cdc13 proteins with anti-Wos2 or anti-Cdc13 from mixed extracts containing Wos2 and Cdc13 or Wos2, Cdc2, and Cdc13, as indicated.

The cdc13-encoded cyclin binds to Cdc2 with high affinity to form a Cdc2/cyclin B complex. As shown in Figure 7B, Wos2 was not found in anti-Cdc13 immunoprecipitates from mixed extracts containing synthesized Wos2 and Cdc13, but it was copurified when Cdc2 was also added; reciprocally, Cdc13 also coimmunoprecipitated using anti-Wos2 in this extract (Figure 7B). These results confirm that Wos2 may associate with the Cdc2 kinase and with Cdc2/cyclin complexes. Thus, an excess of Wos2 could reduce Hsp90 function in vivo by competitive binding to common target kinases.

We failed to demonstrate such physical interaction by measuring Cdc2 kinase activity (H1 phosphorylation) in immunoprecipitates of Wos2 obtained from yeast crude extracts, but surprisingly, the immunoprecipitated Wos2 protein was efficiently phosphorylated in this assay (Figure 8). This phosphorylation took place in the absence of Cdc2 and Wee1 kinase activity (assayed at 37° in cdc2-33 and wee1-50 genetic backgrounds) and in cells lacking the Spc1 MAP kinase (deleted for the spc1 gene; SHIOZAKI and RUSSELL 1995 ). The nature of this kinase activity is now being investigated, but despite its source, it demonstrates that Wos2 may be phosphorylated in vitro, according to the large number of putative phosphorylation sites that can be found on its protein sequence; phosphorylation sites were also described earlier for the avian p23 (JOHNSON et al. 1994 ).

View larger version (45K): In this window In a new window Download PPT slide   Figure 8. In vitro phosphorylation of Wos2 protein. Histone H1 and [-32P]ATP were added to anti-Wos2 immunoprecipitates from wild-type cells (wos2) or from cells deleted for wos2 (wos2) to assay for protein phosphorylation. Proteins were separated on SDS-polyacrylamide gel and the amount of Wos2 was determined by Western blot analysis (a), protein phosphorylation was determined by autoradiography (b), and Coomansie blue staining was used to visualize the added H1 histone (c).

The physiological expression of wos2 is extremely high in proliferating cells (Figure 5). Thus, the additional expression of wos2 driven by the nmt promoter could not be sufficient to produce a significant effect in wild-type cells growing exponentially. To overproduce a significantly higher level of Wos2, a strain containing two copies of the nmt:wos2 construction (2x nmt:wos2) was used. This 2x nmt:wos2 strain behaved like a wild-type strain in media with thiamine, but in the absence of this vitamin, the overexpression of wos2 was lethal, yielding ~75% multinucleate cells, most of them binucleate (Figure 9A). According to flow cytometry analysis of this strain, most of the cells were 2C (Figure 9B), suggesting that each individual nucleus of these cells was actually 1C in DNA content. The nuclei of binucleate cells were decondensed and the interphase microtubule array re-established (Figure 9C), indicating that cells had exited from mitosis, but the nuclei remained adjacent, indicating that exit from mitosis occurred prior to maximal microtubule elongation. Therefore, higher level of expression of wos2 in a wild-type background allowed the exit from mitosis, but delayed progression through the early G1 phase.

View larger version (53K): In this window In a new window Download PPT slide   Figure 9. DAPI staining (A) and flow cytometry analysis (B) of S. pombe cells harboring two copies of the nmt:wos2 construction under repressed (+thiamine) and derepressed (-thiamine) conditions. Cells are as wild type in the presence of thiamine, but in media lacking this vitamin wos2 produces abnormal cells containing 1, 2, or 4 nuclei with a 1C DNA content each. Cells with many nuclei may be also found (top micrograph). Microtubule array was also analyzed in these wos2-arrested cells (C) using monoclonal anti--tubulin serum to localize microtubule (left) and DAPI to visualize nuclei (right); (DAPI was also used in the -tubulin staining).

The SBA1 gene has been shown recently to code for the budding yeast p23 protein (FANG et al. 1998 ). Oligonucleotides were designed to PCR amplify to coding sequence of this gene, and its nmt-driven expression in the fission yeast demonstrated that SBA1 was functionally identical to the S. pombe wos2, efficiently suppressing the cell cycle block caused by the overexpression of Wee1-50, showing the same allele-specific interactions with cdc2 mutants, producing binucleate cells at higher levels of expression, and complementing the heat-shock sensitivity in a S. pombe strain with the endogenous wos2 gene deleted. We therefore conclude that wos2 and SBA1 are functionally homologous. This fact, joint to the in vivo interaction of wos2 with a Drosophila Cdc2 protein, suggests that the described function for wos2 may be universal in eukaryotic cells.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Hsp90 is an abundant and highly conserved molecular chaperone. It exists in several discrete subcomplexes that together comprise the Hsp90 chaperone machinery. One of these subcomplexes contains several immunophilins and the acidic protein p23 (JOHNSON and TOFT 1994 ). The wos2 DNA sequence predicts a gene product homologous to this p23 cochaperone. In addition to sequence identity, both p23 and Wos2 migrate more slowly in denaturing gels than would be predicted from their size (21 kD for Wos2), suggesting some shared structural features (JOHNSON et al. 1994 ). Along with a significant sequence and structural similarity, Wos2 displayed biological properties previously described for p23 in higher eukaryotes. For instance, it is potentially regulated by phosphorylation (Figure 8); it is heat shock related (Figure 2); and it is very abundant and constitutively expressed in proliferating cells (Figure 3). Furthermore, the S. cerevisiae p23/Sba1 protein works in S. pombe as a functional homologue to Wos2. Taken together, these data indicate that Wos2 is a fission yeast homologue of vertebrate p23 proteins.

The p23 cochaperone was first identified in association with the progesterone receptor (SMITH et al. 1990 ), and was later shown to be an Hsp90-binding protein (JOHNSON et al. 1994 ). More recently, p23 has also been found to associate via Hsp90 with other proteins including transcription factors, heme-regulated eIF-2alpha kinase, and viral reverse transcriptase (NAIR et al. 1996 ; HU et al. 1997 ; XU et al. 1997 ). The synthetic cell cycle defects caused by overproduction of Wos2 in specific Wee1 and Cdc2 mutants (Figure 1 and Figure 4) provide direct evidence that p23 also interacts with these two cell cycle kinases.

Such interaction led to loss of wee1-50 activity but the effect on Cdc2 was different. In this later case, overexpression of wos2 allowed for normal levels of Cdc2 kinase (Figure 5), but impeded Cdc2 functions required for the cell to undergo mitosis and cytokinesis in specific Cdc2 mutant backgrounds (Figure 4). Phenotypes of nmt:wos2 cdc2-M26 synthetic lethal mutants resembled phenotypes displayed by dominant lethal mutants in cdc2 that identify Cdc2 regulatory elements (LABIB et al. 1995 ). We suggest that Wos2 interferes with regulatory functions in the Cdc2 complex. In agreement with this observation, other yeast Hsp90 cochaperones such as Ydj1, Cdc37, Cpr7, and Sti1 emerge as important elements in the cell cycle control and regulation of CDK activity (XU and LINDQUIST 1993 ; GERBER et al. 1995 ; KIMURA et al. 1995 , KIMURA et al. 1997 ; DUINA et al. 1996 ; WEISMAN et al. 1996 ; YAGLOM et al. 1996 ; CHANG et al. 1997 ).

The p23 protein has been characterized as a component of many Hsp90-containing heterocomplexes, and consequently, interactions of p23 with target proteins are thought to occur indirectly via its association to Hsp90 (BOHEN 1998 ). However, it has been shown that purified p23 can bind independently to target polypeptides (BOSE et al. 1996 ; FREEMAN et al. 1996 ). Our in vitro analysis is also consistent with a physical interaction between Wos2 and Cdc2 (Figure 7). Thus, p23 could exist in Hsp90 complexes, but this cochaperone itself could also interact with specific polypeptides. Interestingly, the excess of Wos2 showed phenotypes and allele-specific interactions identical to mutants with reduced activity in the swo1-encoded Hsp90 chaperone (ALIGUE et al. 1994 ; MUNOZ and JIMENEZ 1999 ), suggesting that Wos2 could compete binding of Hsp90 to common target substrates. Thus, the allele-specific interaction of Wos2 with Cdc2 may reflect a less tight interaction between mutant Cdc2-M26 and Cdc2-33 proteins with Hsp90 complexes (more easily perturbed by Wos2). The synthetic lethality caused by the excess of Wos2 in a Hsp90-deficient background is in agreement with this suggestion (Figure 6). Furthermore, incubation of these two cdc2 alleles at 36° for 4 hr disassembles the mitotic Cdc2 machinery, while this treatment had no effect on other cdc2 ts alleles (BROEK et al. 1991 ), indicating that Cdc2 complexes are particularly dependent on chaperone proteins in these two cdc2 alleles. Both mutations lie in the central region of Cdc2 containing the regulatory thr167 residue (MACNEILL et al. 1991 ). Phosphorylation of this residue is essential to enhance cyclin binding and drive the conformational changes that result in maximal kinase activation (RUSSO et al. 1996 ). Interestingly, certain mutations in this thr167 residue resemble the phenotype of yeast cells overproducing Wos2 in Cdc2-33 or Cdc2-M26 mutant backgrounds (GOULD et al. 1998 ).

In wild-type cells higher levels of wos2 overexpression were required to display a cell cycle deficiency, but in this genetic background it caused an early G1 block and impeded cell separation. This Wos2-mediated interference suggests that G1 regulatory elements also require the activity of a p23-Hsp90 complex in S. pombe cells. In budding yeasts, Cdc37 is required for the association of Cdc28/Cdc2 with multiple cyclins (GERBER et al. 1995 ), and Ydj1 has a direct role in the phosphorylation of the G1 cyclin Cln3 by Cdc28 (YAGLOM et al. 1996 ). Thus, Wos2 could also interfere with the assembly of Cdc2 with S. pombe G1 cyclins. Consistent with this idea, the G1 arrest caused by overexpression of wos2 displayed a phenotype similar to that described for S. pombe cells disrupted for the cig1 and cig2 genes (CONNOLLY and BEACH 1994 ). Conflicting data for cig1 and cig2/cyc17 have obscured the function of these proteins, but cig2/cyc17 emerges as a G1 cyclin (MARTIN-CASTELLANOS et al. 1996 ; MONDESERT et al. 1996 ).

Loss of the SBA1-encoded p23 protein in budding yeasts did not result in any growth phenotype, indicating that this Hsp90 cochaperone is dispensable under normal growth conditions (FANG et al. 1998 ). This result contrasts with the relative importance of Hsp90 for cell growth (BORKOVICH et al. 1989 ). In our study, we have shown that S. pombe cells lacking Wos2 were heat-shock sensitive (Figure 2), revealing for the first time an in vivo role for p23/Wos2 in the stress response. In vitro chaperone assays have shown that purified p23 maintains early unfolded proteins in a folding-competent state, but in contrast to Hsp90, this cochaperone is unable to refold them (BOSE et al. 1996 ; FREEMAN et al. 1996 ). Surprisingly, p23/Sba1/Wos2 is a ubiquitous protein constitutively expressed at very high level (Figure 3 and Figure 5; JOHNSON et al. 1994 ; FANG et al. 1998 ), while expression of other chaperones such as Hsp90 requires time after stress induction (ALIGUE et al. 1994 ). Thus, from a physiological point of view, we suggest that Wos2 acts in vivo as a "first aid chaperone" that maintains cell proteins in a folding-competent state when environmental changes take place until Hsp90 and other chaperones accumulate and initiate the refolding tasks. Overall, Wos2 could play a dual role in living cells: at normal growth temperatures Wos2 could be involved in the regulation of key cell cycle kinases such as Wee1 and Cdc2. Conditions that cause general protein damage can divert Wos2 from its normal targets to other partially denatured proteins.

Although constitutively expressed, the biological function of Wos2 could be regulated by phosphorylation. Immunopurification of Wos2 led to the copurification of an associated protein kinase with efficient Wos2-phosphorylation activity (Figure 8). Interestingly, Hsp90 is able to autophosphorylate on serine and threonine residues (CSERMELY et al. 1995 ). Furthermore, purified Brassica Hsp90 protein could also phosphorylate other protein substrates (PARK et al. 1998 ). Therefore, since p23 is known to be Hsp90 associated, we cannot exclude the possibility that p23 could be an in vivo substrate of this putative Hsp90 phosphorylation activity.

In conclusion, our results suggest that Wos2 is a p23 homologue. The genetic and biochemical analysis described here establishes a novel role for this p23 homologue in the control of the Wee1 and Cdc2 cell cycle kinases and also as a general chaperone in living cells. The functional conservation of the assayed homologue from budding yeasts supports the idea that roles for wos2 described in this study may be of universal significance.

	FOOTNOTES

¹ Present address: 310 Turker Hall, University of Missouri, Columbia, MO 65211-7400.

	ACKNOWLEDGMENTS

We thank Lucia Cruzado and Inmaculada Florido for excellent technical assistance, Andrés Garzón, Karin Labib, Mark L. Edgley, and Stuart MacNeill for useful discussions, Sergio Moreno, Jaime Correa, and Paul Nurse for providing antibodies, plasmids, and libraries, Juan Pérez for his help in preparing anti-Wos2 antiserum, and Elmar Maier for providing membranes for physical mapping of wos2. This work was supported by grants from the Spanish Ministerio de Educación y Cultura (PB96-0681) and Junta de Andalucía.

Manuscript received March 1, 1999; Accepted for publication August 11, 1999.

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				F. Forafonov, O. A. Toogun, I. Grad, E. Suslova, B. C. Freeman, and D. Picard p23/Sba1p Protects against Hsp90 Inhibitors Independently of Its Intrinsic Chaperone Activity Mol. Cell. Biol., May 15, 2008; 28(10): 3446 - 3456. [Abstract] [Full Text] [PDF]

				S. Huard, R. T. Elder, D. Liang, G. Li, and R. Y. Zhao Human Immunodeficiency Virus Type 1 Vpr Induces Cell Cycle G2 Arrest through Srk1/MK2-Mediated Phosphorylation of Cdc25 J. Virol., March 15, 2008; 82(6): 2904 - 2917. [Abstract] [Full Text] [PDF]

				V. T. George, G. Brooks, and T. C. Humphrey Regulation of Cell Cycle and Stress Responses to Hydrostatic Pressure in Fission Yeast Mol. Biol. Cell, October 1, 2007; 18(10): 4168 - 4179. [Abstract] [Full Text] [PDF]

				A. K. Lovgren, M. Kovarova, and B. H. Koller cPGES/p23 Is Required for Glucocorticoid Receptor Function and Embryonic Growth but Not Prostaglandin E2 Synthesis Mol. Cell. Biol., June 15, 2007; 27(12): 4416 - 4430. [Abstract] [Full Text] [PDF]

				I. Grad, T. A. McKee, S. M. Ludwig, G. W. Hoyle, P. Ruiz, W. Wurst, T. Floss, C. A. Miller III, and D. Picard The Hsp90 Cochaperone p23 Is Essential for Perinatal Survival Mol. Cell. Biol., December 1, 2006; 26(23): 8976 - 8983. [Abstract] [Full Text] [PDF]

				D. M. Price, Z. Jin, S. Rabinovitch, and S. D. Campbell Ectopic Expression of the Drosophila Cdk1 Inhibitory Kinases, Wee1 and Myt1, Interferes With the Second Mitotic Wave and Disrupts Pattern Formation During Eye Development Genetics, June 1, 2002; 161(2): 721 - 731. [Abstract] [Full Text] [PDF]

				N. Tahbaz, J. B. Carmichael, and T. C. Hobman GERp95 Belongs to a Family of Signal-transducing Proteins and Requires Hsp90 Activity for Stability and Golgi Localization J. Biol. Chem., November 9, 2001; 276(46): 43294 - 43299. [Abstract] [Full Text] [PDF]

				A. J. Weaver, W. P. Sullivan, S. J. Felts, B. A. L. Owen, and D. O. Toft Crystal Structure and Activity of Human p23, a Heat Shock Protein 90 Co-chaperone J. Biol. Chem., July 21, 2000; 275(30): 23045 - 23052. [Abstract] [Full Text] [PDF]