Distinct subunit functions and cell cycle regulated ... - CiteSeerX

1793

Journal of Cell Science 110, 1793-1804 (1997) Printed in Great Britain © The Company of Biologists Limited 1997 JCS3562

Distinct subunit functions and cell cycle regulated phosphorylation of 20S APC/cyclosome required for anaphase in fission yeast Hiroshi Yamada, Kazuki Kumada and Mitsuhiro Yanagida* Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606, Japan *Author for correspondence (e-mail: [email protected])

SUMMARY We show here that the fission yeast gene products Cut9 and Nuc2 are the subunits of the 20S complex, the putative APC (anaphase promoting complex)/cyclosome which contains ubiquitin ligase activity required for cyclin and Cut2 destruction. The assembly of Cut9 into the 20S complex requires functional Nuc2, and vice versa. The size of fission yeast APC/cyclosome is similar to that of higher eukaryotes, but differs greatly from that (36S) of budding yeast. The 20S complex is present in cells arrested at different stages of the cell cycle, and becomes slightly heavier in mitosis than interphase. Cut9 in the 20S complex is hyperphosphorylated specifically at the time of metaphase. The truncated forms of Cut9 block entry into mitosis, however.

INTRODUCTION The eukaryotic cell cycle progresses with the regulated activities of CDK/cyclin kinases (Murray and Hunt, 1993). While the Cdc2 and mitotic cyclin B kinase complex is activated in the G2/M transition by dephosphorylation at Tyr15 (Gould and Nurse, 1989; Nurse, 1990), cyclin B will be destroyed at mitotic exit by ubiquitin-dependent proteolysis, resulting in the loss of cyclin B-dependent Cdc2 kinase activities (Glotzer et al., 1991). Cyclin destruction in clam and frog eggs requires the ubiquitination pathway of cyclin B followed by targeting to the 26S proteasome (e.g. Murray, 1995). Consistently, fission and budding yeast mutations in the 26S proteasome subunits lead to the arrest in metaphase of cells containing the undegraded, activated Cdc2/cyclin B (Ghislain et al., 1993; Gordon et al., 1993, 1996). Ubiquitination of cyclin B consists of a series of steps: ubiquitin (Ub)-activating E1 enzyme, Ubconjugating E2 enzyme, and Ub-ligating E3 ligase (e.g. Hochstrasser, 1995). In the case of mitotic cyclins, the E3 Ubligation step is carried out by a large complex (King et al., 1995; Sudakin et al., 1995). This complex sediments approximately at 20S, and is called cyclosome or APC (anaphase promoting complex). We use the term APC/cyclosome throughout this paper since APC could have a different meaning in other literature and may also have a role other than that in anaphase, such as in DNA replication and cytokinesis (Kumada et al., 1995; Heichman and Roberts, 1996). The ubiquitin-ligase activity of 20S APC/cyclosome in vitro increases after activation of the extracts by M-phase kinase

The 20S assembly impaired in the cut9 mutant can be restored by elevating the level of a novel gene product Hcn1, similar to budding yeast Cdc26. Furthermore, deletion of protein kinase PKA (Pka1) suppresses the phenotype of the cut9 mutation and reduces phosphorylation of Cut9. In contrast, PP1 (Dis2) phosphatase mutation shows the reverse effect on the phenotype of cut9. The Cut9 subunit is likely to be a target for regulating APC/ cyclosome function through protein-protein interactions and phosphorylation. Key words: PKA, PP1, Mitosis, Sister chromatid separation, Metaphase, Anaphase

(King et al., 1995; Sudakin et al., 1995). The complex isolated from interphase cells has no ubiquitin ligase activity. If the complex is treated with Cdc2 kinase, it shows the ubiquitin ligase activity with a certain lag time. Purified 20S complex contains several polypeptides, two of which are similar to CDC16 and CDC27 in Saccharomyces cerevisiae (King et al., 1995; Tugendreich et al., 1995). These proteins contain the repeat motifs called TPR (tetratrichopeptide repeat), which are present in diverse proteins and may serve as regions for protein-protein interactions (reviewed by Goebl and Yanagida, 1991). S. cerevisiae Cdc16 and Cdc27 interact with each other (Lamb et al., 1994). Mutations in these proteins lead to a defect in G2/M progression (Pringle and Hartwell, 1981; Hartwell and Smith, 1985), and Clb cyclins were not destroyed in the mutants (Irniger et al., 1995): ubiquitination of Clb2 and Clb3 is defective in cdc16 and cdc27 mutants. These results demonstrate that the complex containing Cdc16 and Cdc27 is similar to the APC/cyclosome identified in clam and frog egg cells. The activities for destruction of Clbs in S. cerevisiae are high in G1 but low in G2 and M phase cells (Amon et al., 1994; Zachariae and Nasmyth, 1996). In fission yeast, the putative components of APC/cyclosome were initially identified by temperature-sensitive (ts) mutations, nuc2-663 and cut9-665, isolated by cytological screening (Hirano et al., 1986, 1988a). They displayed characteristic mitotic phenotypes (‘nuc’ stands for nuclear alteration, and ‘cut’, for cell untimely torn; Hirano et al., 1986, 1988a).

1794 H. Yamada and others The nuc2 mutant was arrested at a metaphase-like stage at the restrictive temperature (36°C): highly condensed chromosomes and a short metaphase spindle were seen in arrested cells (Hirano et al., 1988a) containing high H1 kinase activity and undegraded Cdc13/cyclin B (our unpublished result). The septum was made in nuc2-663 at 36°C, however, suggesting that mutant cells were not entirely arrested in the metaphase state. nuc2 mutant cells cultured at 33°C frequently showed the cut phenotype in the absence of nuclear division (Kumada et al., 1995), suggesting that cytokinesis was no longer inhibited if Nuc2 was produced at a less restrictive temperature. The predicted amino acid sequence of Nuc2 (Hirano et al., 1988a) was similar to that of budding yeast Cdc27 (Sikorski et al., 1991). nuc2 mutant cells are sterile and arrested in the G2 phase rather than G1 under nitrogen starvation (Kumada et al., 1995). These sterile and G2-arrest phenotypes were suppressed by the deletion of the cig2+ gene (K. Kumada and M. Yanagida, unpublished result). Cig2 is one of the non-essential B-type cyclins in Schizosaccharomyces pombe (Connolly and Beach, 1994; Obara-Ishihara and Okayama, 1994), suggesting that nuc2-663 is defective in Cig2 destruction after nitrogen starvation at the permissive temperature. The nuc2-663 mutant is highly sensitive to caffeine at the permissive temperature (26°C), producing the mitotic phenotype in the presence of caffeine (Hirano et al., 1988a). As caffeine is an inhibitor for cAMP phosphodiesterase and causes the metaphase-like phenotype in nuc2-663, requirement of the inactivation of cAMP-dependent protein kinase A (PKA) for exiting mitosis was speculated (Hirano et al., 1988b). This has now been substantiated by the finding that Cut4 protein, a novel subunit essential for forming the 20S APC/cyclosome, is regulated by the cAMP/PKA inactivation pathway (Yamashita et al., 1996). cut4 mutation leads to a failure to form the 20S APC/cyclosome and consequently to ubiquitinate mitotic cyclin, and this failure can be restored by multicopy suppressors encoding cAMP phosphodiesterase or the regulatory subunit of PKA. An anaphase bypass phenotype was found in cut9-665 (Samejima and Yanagida, 1994): mutant cells entered mitosis with chromosome condensation and short spindle formation, then bypassed anaphase (that is, lacked sister chromatid separation), and continued to progress into post-anaphase events such as degradation of the spindle, reformation of the postanaphase cytoplasmic microtubule arrays, septation and cytokinesis. The cut9 mutant is thus defective in the initiation of anaphase and also in the restraint of post-anaphase events until the completion of anaphase. Cut9 protein was thus postulated to be a potential component of the feedback control system for anaphase and post-anaphase (Samejima and Yanagida, 1994). The cut9+ gene was essential for viability, and strongly interacted with three other genes, namely, nuc2+, scn1+ and scn2+. The cold sensitive (cs) scn1 and scn2 mutations, defective in late anaphase, can suppress the ts phenotype of cut9. The predicted amino acid sequence of Cut9 (Samejima and Yanagida, 1994) is similar to that of budding yeast Cdc16 (Icho and Wickner, 1987). In this study, we report that Nuc2 and Cut9 are essential to form the putative 20S APC/cyclosome, and describe their distinct behaviour in the cell cycle. A novel gene product Hcn1 which suppresses cut9 mutation is also reported.

MATERIALS AND METHODS Media, culture conditions and strains An S. pombe haploid wild-type 972 strain was used. ts and cs strains nuc2-663, cut9-665, cdc10-129, cdc17-K42, cdc22-C1 and cdc25-22, mts3-1 and nda3-KM311 were previously isolated (Hirano et al., 1986; Samejima and Yanagida, 1994; Nurse et al., 1976; Nasmyth and Nurse, 1981; Fernandez-Sarabia et al., 1993; Russell and Nurse, 1986; Gordon et al., 1993; Hiraoka et al., 1984). Deletion mutants of dis2+, pka1+ and cyr1+ were previously constructed (Ohkura et al., 1989; Maeda et al., 1990, 1994; Kinoshita et al., 1993). nda3-KM311 cells were integrated with the HA-tagged cut9+ gene and used for immunoprecipitation of Cut9 in the M phase-arrested cells. The integrated mutant cells showed the mitotic phenotype identical to that of nonintegrated nda3-KM311. Handling of S. pombe strains was as described by Gutz et al. (1974) and Moreno et al. (1991). The procedures described by Alfa et al. (1993) were followed for the block and release experiment of the cdc25 mutant. Temperature-sensitive strains were cultured at the restrictive temperature (36°C) for 4 hours except for cdc10 (3.5 hours), while cs mutants were incubated at 20°C for 8 hours. YPD is a rich medium containing 2% glucose, 1% yeast extract and 2% polypeptone. EMM2 is a minimal synthetic medium (Mitchison, 1970). Plasmids The full length cut9+ and truncated genes were overproduced using plasmid Rep1 containing the inducible promoter nmt1 (Maundrell, 1990). Plasmids M2, M3 and M5 were constructed using pRep2wee1+HAhis6 (a gift from Paul Russell) containing the ura4+ gene (Grimm et al., 1988) and pRep1 containing the LEU2 marker (Maundrell, 1990). The resulting plasmid pRep1/his6 HA containing the LEU2 marker was digested with NdeI and NotI, and ligated with the fragments made by the PCR method using appropriate primers. pCDC26 was a gift from H. Araki (Osaka University; Araki et al., 1992). pnmt1/CDC26 was constructed by ligating the whole coding region of CDC26 (made by PCR) with pRep1. Preparation of cell extracts, immunoprecipitation and immunoblotting Cells were disrupted by glass beads (40 seconds, 4 times), and were centrifuged at 5,000 rpm for 5 minutes or at 14,000 rpm for 20 minutes. Resulting supernatants were used as extracts. The modified HB buffer used contains 25 mM Tris-HCl, pH 7.5, 15 mM EGTA, 15 mM MgCl2, 60 mM β-glycerophosphate, 15 mM p-nitrophenylphosphate, 0.5 mM Na3VO4, 1 mM DTT, 0.1% NP-40, 0.1 mM NaF and protease inhibitors (1 mM PMSF, 1 µg/ml leupeptin, 2 µg/ml aprotinin, 10 µg/ml TPCK). Antibodies against Cut9 and Nuc2 were prepared previously (Hirano et al., 1988a; Samejima and Yanagida, 1994). Monoclonal antibody against tubulin (TAT1) and anti-PSTAIR antibodies were gifts from K. Gull, and M. Yamashita and Y. Nagahama, respectively (Woods et al., 1989). Immunoprecipitation was done according to the procedures of Stone et al. (1993). Modified HB buffer or TEG50X buffer, containing 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10% glycerol, 50 mM NaCl, 0.2% Triton X-100 and protease inhibitors (1 mM PMSF, 1 µg leupeptin and 2 µg/ml aprotinin), were used. Sucrose gradient centrifugation A linear sucrose gradient (15%-40%) made in HB buffer was run at 40,000 rpm for 12 hours at 4°C using a Beckman SW50.1 rotor. A sample of 1 mg protein was overlaid on top of the gradient (total 5.12 ml). Fractions (20, each 260-270 µl) were collected from each tube after centrifugation. The sedimentation markers used were bovine serum albumin (BSA, 4.5S) and thyroglobulin (16.5-19S; Peters et al., 1990). Alkaline phosphatase treatment Calf intestine alkaline phosphatase (Boehringer, cat no. 97075) was

Properties of APC/cyclosome subunits 1795 used for treating immunoprecipitates made in extracts of nda3-311 integrated with the HA-tagged cut9+ gene by anti-HA antibodies. HAtagged Cut9-immunoprecipitates were washed three times with 50 mM Tris-HCl at pH 8.5 containing 0.1 mM EDTA and a protease inhibitor (1 mM PMSF). Phosphatase (60 units) was incubated with the beads for 60 minutes at 30°C. After washing 3 times, beads were run in an SDS-PAGE gel followed by immunoblotting. To inhibit CIAP, 1/2 volume of 2×HB buffer was added to the CIAP reaction buffer. Immunofluorescence microscopy The procedures described by Hagan and Hyams (1988) were followed.

RESULTS Cut9 and Nuc2 are enriched in the 20-23S fractions To examine sedimentation profiles of Cut9 and Nuc2, extracts of exponentially growing wild-type 972 cells at 26°C were made and run in a sucrose gradient centrifugation at 40,000 rpm for 12 hours at 4°C using a SW50.1 rotor; 20 fractions were collected from the top and analyzed by immunoblot using affinity-purified anti-cut9 and anti-nuc2 antibodies (Fig. 1A, αcut9 and α-nuc2, respectively). Both Cut9 and Nuc2 were enriched in the fractions around 20-23S (fractions 8 and 9). BSA and thyroglobulin were markers for 4.5S and 16.5-19S, respectively. A significant fraction (35%) of Cut9 was also present in slowly sedimenting fractions and peaked around 7S (fraction 3), while only a very small quantity (4%) of Nuc2 protein was found in the slowly sedimenting fractions, around 10S (fractions 5 and 6). Cut9 and Nuc2 are co-immunoprecipitated If Nuc2 and Cut9 are subunits of the same complex, they may be co-immunoprecipitated by antibodies. Immunoprecipitation

Fig. 1. Cut9 and Nuc2 are components of the 20S complex. (A) Extracts of wildtype 972 were run in a sucrose gradient at 40,000 rpm for 12 hours at 4°C. A total of twenty fractions were collected. Standard markers of BSA (4.5S) and thyroglobulin (16.5-19S) were used. Nuc2 protein detected by anti-nuc2 antibodies peaked at approximately at position 20S (fractions 8 and 9), while Cut9 detected by anti-cut9 antibodies distributed broadly, but also peaked around 20S. The leftmost lane is the immunoblot pattern of wild-type extracts. The second was the blank lane. (B) Immunoprecipitates by anti-cut9 (αcut9; lane 2) or anti-nuc2 (α-nuc2; lane 3) antibodies bound to beads were subject to SDS-PAGE and immunoblotted using anti-cut9 and anti-nuc2 antibodies. Lane 1 contained beads only.

Fig. 2. Cut9 and Nuc2 form the 20S complex in cell cycle mutants. S. pombe mutants cdc25-22 and mts3-1 were arrested in G2 and mitosis at 36°C for 4 hours, respectively, while cdc10-129 cells were blocked in G1 at 36°C for 3.5 hours. Cold-sensitive nda3-311 cells were arrested in mitosis at 20°C for 8 hours. Cell extracts prepared were run in sucrose gradient centrifugations as described in Fig. 1. Twenty fractions collected from each gradient were immunoblotted using anti-cut9 and anti-nuc2 antibodies. Note that the peak fractions of Cut9 and Nuc2 in extracts of mitotically arrested nda3 and mts3 mutants were shifted one fraction to the larger S values (fraction 9 and 10). Furthermore, Cut9 in the peak fractions showed the upper bands which are phosphorylated (see Fig. 3).

of wild-type cell extracts was done using anti-cut9 or anti-nuc2 antibodies, and resulting immunoprecipitates were analyzed by immunoblot (Fig. 1B). Immunocomplexes obtained by anticut9 (lane 2) contained Nuc2 as well as Cut9, while those obtained by anti-nuc2 (lane 3) contained Cut9 and Nuc2. Nuc2 and Cut9 are thus co-immunoprecipitated. The complex increases the S value in mitotically arrested cells To examine whether the amount of the 20S fractions varies depending upon cell cycle stage, four mutant extracts: cdc10129 arrested at the G1 phase, cdc25-22 blocked at G2, nda3KM311 (β-tubulin mutant) at mitotic prophase lacking a spindle and mts3-1 (proteasome subunit mutant; Gordon et al., 1993, 1996) at mitotic metaphase with a short spindle, were prepared and run in sucrose gradient centrifugation (Fig. 2). Mutants were cultured at the restrictive temperature (20°C for cs nda3-311 but 36°C for three other ts mutants) for 3.5-8 hours (4 hours for cdc25, mts3 and 3.5 hours for cdc10 mutants and 8 hours for nda3-311). Nuc2 and Cut9 proteins were present in the 20S complex fractions in all these mutant extracts with similar levels, suggesting that the complex is present in different cell cycle stages. Interestingly, the complex sedimented faster in mitotically arrested nda3 and mts3 mutants, one fraction being heavier than wild-type and interphase arrested mutant cells. This increase of the sedimentation rate (22-25S) in mitotically arrested cells was reproducible in a number of repeated centrifugations with the internal standard markers. The complex

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Fig. 3. Cut9 is phosphorylated during mitosis. (A) Extracts of wildtype (972) and mutants were immunoblotted using anti-cut9, antinuc2 and anti-PSTAIR (cdc2) antibodies. Cut9 in mitotically-arrested nda3, mts3, dis1 and dis2 produced the upper bands. The upper bands were weak in mts3, and could be seen only after a longer exposure. (B) Immunoprecipitates made by anti-HA antibodies were incubated in the presence (+) or the absence (−) of alkaline phosphatase (CIAP), and subject to SDS-PAGE followed by immunoblotting using anti-cut9 antibodies. The upper bands disappeared after phosphatase treatment. If phosphatase inhibitor (PPaseInh) was added in advance, the upper bands remained. Nuc2 was an internal control of the immunoprecipitates. (C) cdc25-22 cells were blocked in the G2-phase at 36°C and released to 26°C. Synchronized cells were taken and the cell plate index (CPI), the spindle index and the cell number were obtained by calicoflour stain, anti-tubulin staining and plating at 26°C, respectively. The spindle index peaked twice at 60 minutes and 200-220 minutes after the temperature shift. (D) Immunoblot of synchronized cells using anticut9, anti-nuc2, and PSTAIR antibodies. The upper bands of Cut9 peaked twice at 60 and 200-220 minutes, coinciding with timing of the peaks for the spindle index.

appears to undergo compositional and/or conformational changes at the mitotic stage. Upper bands of Cut9 in the 20S complex are phosphorylated forms Cut9 was found to produce the slowly migrating, upper electrophoretic bands in mitotically-arrested nda3 and mts3 extracts (Fig. 2). These upper bands were produced only in the 20S complex (fractions 9 and 10), suggesting that this alteration of Cut9 occurred after Cut9 was assembled into the large

complex. Densitometric analysis indicated that approximately 40% of Cut9 in the 20S fractions was in the upper, slowly migrating bands. Nuc2 protein did not show such upper bands. To determine which mutations would lead to the formation of the upper bands for Cut9, various mutant extracts were prepared and immunoblotted using anti-cut9 antibodies (Fig. 3A). Only mitotically blocked mutants produced the upper bands (nda3, dis1, dis2 and mts3; Hiraoka et al., 1984; Nabeshima et al., 1995; Ohkura et al., 1989; Gordon et al., 1996; the upper bands in mts3 were relatively weak and could be seen only after long exposure). The dis2 mutant lacks PP1 phosphatase activity, while dis1 is defective in spindle formation. In these mitotically arrested cells, H1 kinase activities were high and sister chromatids did not separate. To examine whether the upper bands were made by protein phosphorylation, HA-tagged Cut9 in extracts of nda3-311 mutant integrated with the HA-tagged cut9+ gene was immunoprecipitated by anti-HA antibodies, followed by incubation in the presence (+) or the absence (−) of alkaline phosphatase (CIAP, Fig. 3B). The upper bands were completely eliminated by phosphatase treatment (the second lane). If phosphatase inhibitor (PPaseInh) was added prior to this treatment, the upper bands remained (lane 3), indicating that their disappearance depended upon the phosphatase treatment. nda3 mutant cells immunoprecipitated by polyclonal anti-cut9 antibodies produced identical results (data not shown). The level of Nuc2 in the immunoprecipitates did not change before and after phosphatase treatment, suggesting that proteolysis during the treatment was negligible. Cut9 was thus phosphorylated. Multiple upper bands suggested the presence of multiple phosphorylation sites in Cut9. Cut9 is hyperphosphorylated at the M-phase in synchronous culture To determine whether this Cut9 hyperphosphorylation actually occurs during the progression of mitosis, the block and release experiment was done using cdc25 mutant cells blocked in the G2 phase at 36°C and then released by the shift to 26°C (Fig. 3C). The first and second mitoses determined by the spindle index (% cells containing the spindle) took place 60 and 200220 minutes after the release, respectively. The Cut9 upper bands seen by immunoblot using extracts of synchronized cells precisely coincided with timing of the maximal spindle index (Fig. 3D). The levels of Cut9 and Nuc2 were roughly the same during the cell cycle. Cut9 in the 20S APC/cyclosome complex is hence stage-specific phosphorylated during the cell cycle. Cut9 and Nuc2 sediment anomalously in cut9 and nuc2 mutants To examine the effect of cut9 and nuc2 mutations on the sedimentation profiles of Cut9 and Nuc2, sucrose gradient centrifugations of mutant extracts were performed. Extracts of nuc2-663 and cut9-665 were made from cells growing at the permissive temperature (26°C, 0 hours) or cells transferred to the restrictive temperature (36°C) for 4 hours (Fig. 4A). In nuc2-663, Cut9 and Nuc2663 sedimented around 20S in extracts prepared at 26°C (0 hours), but hardly sedimented at 20S in extracts prepared at 36°C; both proteins at 36°C broadly distributed in sucrose gradient centrifugation. In cut9-665 extracts, the major fraction of Nuc2 was found to be slowly and broadly sedimenting even at 26°C. The relative level of

Properties of APC/cyclosome subunits 1797 plasmid or pHCN1 were made and run in a sucrose gradient (Fig. 5C, /vector and /pHCN1). The 20S complex in cut9-665 carrying pHCN1 monitored by anti-cut9 and anti-nuc2 antibodies was found to be fully restored at 26°C (36°C, 0 hours). At 36°C (6 hours), a considerable fraction of Cut9 and Nuc2 formed the 20S complex. This was in striking contrast to cut9665, which carried the vector plasmid at 36°C (upper panels), in which Cut9 hardly sedimented in the 20S fractions. These results strongly suggested that the elevated gene dosage of hcn1+ could facilitate the assembly of mutant Cut9665 protein into the 20S APC/cyclosome. Hcn1 might have a chaperon-like activity. We examined whether the S. cerevisiae CDC26 can also suppress the cut9-665 mutation. The coding region of CDC26 was therefore ligated with the nmt1 promoter in pRep1 and the resulting plasmid pnmt1/CDC26 was introduced into cut9-665 (Fig. 5D). In the absence of thiamine (the condition to overproduce Cdc26 protein), transformants could grow well at 36°C, indicating that overproduced Cdc26 could suppress cut9665. Multicopy plasmid carrying the CDC26 gene with the native promoter failed to suppress cut9-665.

Fig. 4. The 20S complex fails to form in nuc2 and cut9 mutants. (A) Extracts of nuc2-663 and cut9-665 mutants grown at 26°C (upper panels) and then shifted to 36°C for 4 hours (lower panels) were run in sucrose gradient centrifugation as described in Fig. 1. Immunoblot patterns of each fraction using anti-cut9 and anti-nuc2 antibodies are shown. An additional lower band seen for anti-nuc2 antibodies in cut9-665 is probably a proteolytic form of Nuc2. (B) Extracts of nuc2-663 mutant carrying multicopy plasmid pNUC2 grown at 36°C for 4 hours were run in sucrose gradient centrifugation, and fractions were immunoblotted as described above.

Cut9665 mutant protein in the 20S fractions decreased at 36°C (4 hours). When nuc2-663 mutant carrying multicopy plasmid pNUC2 (pNC106; Hirano et al., 1986) was grown at 36°C for 4 hours and the extract was run in sucrose gradient centrifugation, Cut9 sedimented in the 20S fraction (Fig. 4B). Nuc2 sedimented in the 20S complex and also existed abundantly around 10S due to overproduction by plasmid. Therefore, the assembly of Cut9 into the 20S complex required functional Nuc2, and vice versa. High copy suppressor pHCN1 restores the 20S complex in cut9-665 We isolated a multicopy plasmid, pHCN1, carrying the 2.5 kb long S. pombe genomic sequence (Fig. 5A), which suppressed the ts phenotype of cut9-665 at 36°C (Fig. 5B). Nucleotide sequencing indicated that its predicted amino acid sequence encodes a small protein which contains 80 amino acids and is similar to the S. cerevisiae Cdc26 (Zachariae et al., 1996). The gene is designated hcn1+ (high copy suppressor for cut nine) To examine whether pHCN1 affects 20S complex formation in the cut9 mutant, extracts of cut9-665 carrying the vector

Genetic interactions of cut9-665 with PKA and PP1 mutations As an initial effort toward understanding cell cycle stage specific phosphorylation of Cut9, we examined genetic interactions between cut9-665 and a large number of mutations in protein kinases, phosphatases or related proteins by crossing. We found only two types of significant genetic interactions. One was with mutations related to type 1 protein phosphatase (PP1) and the other was with PKA. Genetic crossing indicated that the double mutant cut9-665 dis2-11 was lethal. Dis2 is one of the two PP1 catalytic subunits (Dis2 and Sds21) in fission yeast, and cs dis2-11 mutation is semi-dominant (Ohkura et al., 1989; Yamano et al., 1994). PP1 is essential for the progression from metaphase to anaphase in fission yeast: the double deletion ∆dis2 ∆sds21 cells are blocked at metaphase (Ishii et al., 1996). Synthetic lethality of cut9-665 dis2-11 is thus consistent with a hypothesis that Cut9 and PP1 catalytic subunits share an essential function. In dis2-11, the 20S complex is normally produced (data not shown), and Cut9 is hyperphosphorylated (Fig. 3A). Deletion of dis2+ (∆dis2) is viable at 26, 30, 33 and 36°C as the other Sds21 catalytic subunit is functional (Fig. 6A). We found that the double mutant cut9-665 ∆dis2 was viable, but unable to form a colony even at 30°C and produced only tiny colonies at 26°C. The double mutant cells arrested at 36°C showed condensed chromosomes and a short spindle (data not shown). The pka1+ gene codes for the catalytic subunit of PKA (Maeda et al., 1994; Yu et al., 1994). The deletion of pka1+ (∆pka1) was viable (Maeda et al., 1994; Fig. 6B). The double mutant ∆pka1 cut9-665, however, produced colonies at 33°C, while the single cut9 mutant did not produce colonies at this temperature (Fig. 6B). The cyr1+ gene encodes adenylate cyclase (Maeda et al., 1990; Young et al., 1989), and the deletion of cyr1+ (∆cyr1) was viable (Maeda et al., 1990). The double mutant ∆cyr1 cut9-665 produced colonies at 33°C, whereas the single cut9 mutant could not form a colony at this temperature (Fig. 6C). The two double mutants ∆pka1 cut9 and

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Fig. 5. 20S complex is restored in cut9-665 carrying plasmid pHCN1. (A) Isolation of the hcn1+ gene as a multicopy suppressor for cut9-665. Plasmid pKK7-06 suppressed the ts phenotype of cut9-665. Subcloning and subsequent nucleotide sequencing identified an open reading frame (indicated by the open arrow) containing 80 amino acids (DDBJ/GenBank accession number AB001373). The ability of suppression was retained in plasmid pOP-010 containing the hcn1+ gene placed in the downstream of the nmt promoter (REP1). The N-terminal region of Hcn1 is similar to that of budding yeast Cdc26, a component of APC/cyclosome (Zachariae et al., 1996). (B) Multicopy plasmid pHCN1 suppressed cut9-665 at 33 and 36°C. (C) Extracts of cut9-665 carrying the vector plasmid grown at 26°C (36°C, 0 hours) and shifted to 36°C for 6 hours were run in sucrose gradient centrifugation. Both Cut9 and Nuc2 proteins failed to form the 20S peaks (fractions 8 and 9) after 6 hours at 36°C. Extracts of cut9-665 carrying high-copy suppressor plasmid pHCN1 grown at 26°C (36°C, 0 hours) and shifted to 36°C for 6 hours were run in sucrose gradient centrifugation. Both Cut9 and Nuc2 in extracts made at 26°C formed the sharp peak around 20S (fractions 8 and 9). Even at 36°C, the 20S fractions formed by Cut9 and Nuc2 were quite distinct. (D) Overexpression of the Saccharomyces cerevisiae CDC26 gene under the nmt1 promoter (pnmt1/CDC26) in the absence of thiamine (−Thi) suppressed cut9-665 at 36°C. The nmt1 promoter was induced in the absence of thiamine.

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Fig. 6. Genetic interactions of cut9-665 with PP1 and PKA mutations. (A) The double mutant cut9-665 ∆dis2 constructed by crossing formed small colonies at 26 and hardly at 30°C, while single cut9 and ∆dis2 produced colonies at these temperatures. (B) The double mutant cut9-665 ∆pka1 formed by crossing produced colonies at 33°C, whereas single cut9-665 formed few colonies at the same temperature. (C) The double mutant cut9-665 ∆cyr1 produced colonies at 33°C, whereas the single cut9 mutant scarcely produced colonies at this temperature. (D) Single cut9-665 and double cut9665 ∆pka1 mutants were cultured at 33°C for 4 hours. Extracts of these mutants were run in sucrose gradient centrifugation and immunoblotted using anti-cut9 and anti-nuc2 antibodies. The 20S complex fractions were clearly formed in the double mutant, but only poorly in the single mutant extracts. The total amount of Cut9 and Nuc2 in the two extracts was the same.

∆cyr1 cut9 thus behaved similarly, indicating that the decrease of PKA activity could partly suppress the cut9 mutation. We examined whether the above partial suppression of cut9665 by ∆pka1 affected the level of 20S APC/cyclosome formation. The single cut9-665 and the double mutant cut9665 ∆pka1 were cultured at 33°C for 4 hours, and the extracts were run by sucrose gradient centrifugation. The level of 20S APC/cyclosome complex formed in the double mutant cells

Fig. 7. Block and release experiment of cdc25 ∆pka1 and cdc25 ∆cyr1. The double mutant cells were cultured at 36°C for 4 hours 15 minutes and then transferred to 26°C. Cells were taken at 20 minutes intervals, and extracts were made for immunoblot and measurement of H1 kinase. The activity of H1 kinase and the frequency of cells containing the short spindle (metaphase cells) in the extracts of cdc25 pka1 are shown in A. Immunoblot patterns of the extracts (cdc25 pka1 and cdc25 cyr1) using anti-cut9, -cdc13 and -cdc2 PSTAIR antibodies are shown in B. Control mitotic extracts of nda3 mutant (the rightmost lanes) shows the Cut9 upper bands, the positions of which were higher than that seen in the double mutants.

was considerably higher than that of the single cut9-665 (Fig. 6D). The above results suggested that formation of the 20S complex was inhibited by PKA but might require PP1. These results indicated that Cut9 and Dis2 might share similar functions in anaphase whereas Cut9 and PKA perform opposing functions. Reduced Cut9 phosphorylation in mitotic cells deleting pka1+ or cyr1+ To examine hyperphosphorylation of Cut9 in ∆pka1 or ∆cyr1 cells, cell cycle block and release experiments were done using

1800 H. Yamada and others the double mutants cdc25 ∆pka1 or cdc25 ∆cyr1 constructed in the present study (Fig. 7A,B). The double mutant cells were blocked at 36°C for 4 hours 15 minutes (cdc25 mutation causes block at G2) and then released to 26°C. Cells were taken at every 20 minutes and cultured for 4 hours. The level of H1 kinase activity and the frequency of cells with a short mitotic spindle, both of which peaked in metaphase, were measured (Fig. 7A), and immunoblotting was done using antibodies against Cut9, Cdc13 or PSTAIR (Fig. 7B). The upper Cut9 band was present in mitotic cells of the double mutant, but intensity of the band was greatly reduced in comparison with the upper band intensity of Cut9 in single cdc25 cells (Fig. 3D); densitometric analysis indicated that the ratio of band intensities between the upper and lower bands was more than tenfold smaller than that in single cdc25 mutant cells. Cut9 hyper-

phosphorylation thus occurred in a cell cycle dependent manner but was diminished in ∆pka1 or ∆cyr1 cells. Overexpression of truncated Cut9 leads to cell cycle arrest The full length cut9+ gene was placed downstream of the inducible nmt promoter (Rep1; Maundrell, 1990) and overexpressed in the absence of thiamine. Overproduction of full length Cut9 by pC9R1 was confirmed by immunoblotting (data not shown). The overproduction produced no phenotype in wild-type, and normal colonies were made (Fig. 8A,B). This result is in sharp contrast to the phenotype of overproduced Nuc2, which blocks cytokinesis but not nuclear division, resulting in the production of giant cells containing a large number of nuclei (Kumada et al., 1995). Excess Nuc2 is thus

A A

Int.I

Int.II

1

671 1

2

3

4

5

6

7

8

9

plasmid name

vector

C9R1

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++

pCUT9-75

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++

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pREP1

HAhis6

M2

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HAhis6

M3

pREP1

HAhis6

M5

pREP1

M2 0.6

pREP1

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10

56 271 BgII

202 287 491

Fig. 8. Elevated levels of N-truncated Cut9 lead to the G2-arrest. (A) Construction of truncated cut9 genes. All of these genes were placed downstream of the strong inducible promoter nmt1 in Rep1. Overproduction effects of truncated genes were examined for whether they were able to form colonies in the absence of thiamine (−Thi): + and ++ indicate colony formation (++ is normal in size). − indicates no colony formation. NA, not tested (these plasmids do not have the nmt promoters). Truncated genes were also tested for whether they were able to suppress ts cut9-665 or cut9 null (∆cut9): ++, complementation; −, no complementation; ND, not determined. Nterminal 56 and C-terminal 69 amino acids were dispensable (Samejima and Yanagida, 1994), as pCUT9-75 and pCUT9-76 suppressed cut9 null. (B) Plasmids M2 and M3 carrying the Nterminally truncated Cut9 strongly inhibited colony formation of wild-type cells (HM123) in the absence of thiamine, while the full length Cut9 (C9R1), the Nterminal fragment (NB) and the Cterminal fragment (M5) showed no inhibition effect. (C) Wild-type cells (HM123) carrying plasmid M3 were cultured at 33°C for 20 hours in the absence of thiamine. Cells stained by DAPI and anti-tubulin antibodies showed the interphase microtubule network and interphase nuclear chromatin structure. Bar, 10 µm.

403

202

BamHI

287

BamHI

BamHI

HAhis6

C9R1 his6HA

pREP1

pCUT9-76

pDB248

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ts complementation

-

++ ++

colony formation (-Thi.)

∆cut9 complementation

++ ++

++

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+ ++ ++ ++ NA

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Properties of APC/cyclosome subunits 1801 an inhibitor of septum formation. Full length Cut9 overproduced in nuc2-663 was severely inhibitory, however, suggesting interaction between Cut9 and Nuc2. Colony formation was strongly inhibited when the Nterminal region of Cut9 was deleted and resulting truncated proteins M2 and M3 were overproduced: M2 and M3 deleted the N-terminal 201 and 286 amino acids, respectively (Fig. 8A and B). Cells arrested by plasmid M3 were examined by DAPI and anti-tubulin antibody staining (Fig. 8C). Unexpectedly, cells were blocked in interphase (cells were elongated, containing a single nucleus with cytoplasmic microtubules) rather than in mitosis. These were G2-arrested cells, as they contained 2C DNA and low H1 kinase activity (data not shown). This Ntruncated overexpression phenotype was probably due to a negative dominant effect, and resembled neither the phenotypes of ts cut9, nuc2 nor overproducing Nuc2. The N-terminal region of Cut9 might be a regulatory domain specifically required for cell cycle regulation of Cut9 function. DISCUSSION Nuc2 and Cut9 are essential for APC/cyclosome formation This study shows that Cut9 and Nuc2 are subunits for the 20S complex, equivalent to the 20S APC/cyclosome required for anaphase promoting proteolysis (King et al., 1995; Sudakin et al., 1995). Consistently, the degradation of Cdc13, the fission yeast M phase cyclin, and Cut2, a nuclear and spindle protein essential for sister chromatid separation, requires Cut9 (Funabiki et al., 1996). Another subunit of the 20S complex is Cut4 (Yamashita et al., 1996), which is similar to Aspergillus BimE and mouse tsg24 (Starborg et al., 1994). Two recent publications also show that the BimE homologs of budding yeast and frog are the subunits of APC/cyclosome (Zachariae et al., 1996; Peters et al., 1996). The proposed relationships of the

gene products described in this paper are depicted in Fig. 9. In spite of highly conserved amino acid sequences of the subunits so far identified, the S value (36S) of budding yeast APC/cyclosome is strikingly larger than that (20S) of fission yeast and higher eukaryotes. It is of considerable interest to identify the reason for this very large size difference. Sucrose gradient centrifugation experiments in this and another study (Yamashita et al., 1996) indicate that the mature 20S complex is present in neither nuc2, cut4 nor cut9 mutants. cut4-533 is extremely sensitive to heavy metals such as Cd, Ni or Co, and shows medium-dependent growth (Yamashita et al., 1996). The heavy metal stress caused the degradation of the 20S complex in cut4-533. However, the defects of cut4 were fully restored by the increased gene dosage of cAMP dependent phosphodiesterase or the regulatory subunit of PKA, which would lead to the inactivation of the cAMP/PKA pathway. In this study, we showed that the phenotypes of the cut9 mutant were also suppressed by the deletion of PKA. The PKA inactivation pathway thus appeared to stabilize the 20S complex through direct or indirect interactions with Cut9 and Cut4 (see below), resulting in the restoration of their role in anaphase progression. Post-translational modification of APC/cyclosome during the cell cycle Detailed analyses by sucrose gradient centrifugations suggested that the APC/cyclosome existed throughout the cell cycle at an approximately identical level. However, the sedimentation rate increased approximately 10% in mitotically arrested nda3-311, mts3-1 (Fig. 2) and dis2-11 (our unpublished result). The complex was likely to contain an additional subunit(s) or to change its conformation during mitosis. Phosphorylation of the 20S subunits in mitotically arrested cells might induce a structural change in the complex. We showed that Cut9 protein produced multiple upper bands during mitosis, and this band shift was due to phosphorylation. Interphase (Anaphase)

Metaphase

PP1 PKA

22-24S

20-22S

Nuc2

Nuc2 Cut4 Cut9

Cut9 P P P

APC/cyclosome

Fig. 9. Fission yeast Nuc2 and Cut9 are the essential subunits of the 20S APC/cyclosome. The complex is required for anaphase proteolysis. PP1, type 1 protein phosphatase and PKA, protein kinase A directly or indirectly control phosphorylation of Cut9 which occurs only in the complex at metaphase. The deletion of PKA or the dosage increase of a novel gene hcn1+ suppress the failure of 20S complex assembly in the cut9 mutant cell. pHCN1, the multicopy suppressor plasmid for the cut9 mutant and Hcn1 is similar to budding yeast Cdc26; Cut4, another subunit of the 20S complex; ∆dis2, dis2-11, PP1 mutations. P, phosphorylation.

pHCN1 pka1

AAA AAA AAA Cut9

ts nuc2 ts cut9

AAA AAA AA AAA Nuc2

In cut9 and nuc2 mutants

Cut4

Ub-M-cyclin Ub-Cut2

1802 H. Yamada and others Cut9 unbound to the 20S complex was not the substrate of phosphorylation. The 20S complex was possibly regulated in a cell cycle specific manner by phosphorylation and dephosphorylation. Subunits of the Xenopus APC/cyclosome also showed Cdc2 kinase dependent phosphorylation (King et al., 1995; Peters et al., 1996). The ubiquitin ligase activity of APC/cyclosome requires phosphorylation dependent upon Cdc2 kinase (Sudakin et al., 1995), while phosphatase treatment inactivates the ligase activity of the activated APC/cyclosome (Peters et al., 1996). Cut9 has no obvious site for Cdc2 kinase, however. The cut9 mutation showed reverse genetic interactions with ∆pka1 and dis2 mutants. Consistently, in ∆pka1 cells, Cut9 mitotic phosphorylation was greatly reduced, whereas in dis2-11, Cut9 was hyperphosphorylated. Cut9 might be directly or indirectly phosphorylated or dephosphorylated by PKA or PP1, respectively. Definitive identification of the kinase or phosphatase directly involved, however, requires further study, particularly for determination of the phosphorylation sites in Cut9. One hypothesis is that phosphorylated Cut9 activates the ubiquitin ligase activity in metaphase, while dephosphorylated Cut9 leads to the reduction of the activity. This hypothesis implies that the activated ubiquitin ligase might not be sufficient for the onset of anaphase. Polyubiquitinated proteins are not degraded in metaphase (Klotzbücher et al., 1996). To initiate proteolysis, a reaction other than ubiquitin ligation may be required; the decrease of deubiquitination activity is one possibility (Hochstrasser, 1995). Inactivation of ubiquitin ligase in the 20S complex is other possibility. A protein kinase responsible for phosphorylating Cut9 might exist within the APC/cyclosome and be regulated during metaphase/anaphase progression. Alternatively, Cut9 bound to the 20S complex may take a specific conformation accessible to a protein kinase in metaphase, but not to a phosphatase. The TPR repeat motif present in Cut9 is thought to provide flexibility in protein conformation and play a role in protein-protein interactions. Interactions between PKA and the APC/cyclosome found in this study are intriguing. Suppression of the cut9 mutant by ∆pka1 is probably due to stabilization of the the 20S complex which contains mutant Cut9. However, mitotic Cut9 phosphorylation is greatly reduced in ∆pka1, possibly resulting in the reduction of ubiquitin ligase activity in the 20S complex. This would destabilize metaphase but facilitate anaphase progression. One possible hypothesis to explain the role of PKA is that PKA activity is regulated during mitosis and controls the activation and inactivation of the 20S complex through phosphorylation of Cut9. PKA may not directly phosphorylate Cut9: Cut9 in the 20S complex isolated from G2-arrested cdc25 mutant did not show the slowly migrating upper bands when incubated with PKA (our unpublished result). It is unknown whether Nuc2 is phosphorylated; no upper band was found for Nuc2, but radiolabeling is needed to determine phosphorylation of Nuc2. Cooperative but distinct Nuc2 and Cut9 functions Association of Nuc2 and Cut9 to the 20S complex seemed to be mutually dependent. Cut9 and Nuc2 may directly associate as their budding yeast counterparts do (Lamb et al., 1994; Zachariae et al., 1996). Strong genetic interactions are observed between cut9-665 and nuc2-663 (Samejima and Yanagida, 1994; Ishii et al., 1996). Functions of Nuc2 and Cut9

in the formation of 20S complex are probably cooperative, as cold-sensitive suppressors scn1 and scn2 act on both cut9 and nuc2 (Samejima and Yanagida, 1994), and the elevated gene dosage of Sds23 suppresses both nuc2-663 and cut9-665 (Ishii et al., 1996). Overproduction effects of Nuc2 and Cut9 are strikingly different, however. Overproduced Nuc2 inhibits septation, whereas N-truncated Cut9 blocks the transition from G2 to M. A possible explanation for this difference is that Nuc2 and Cut9 proteins unbound to the 20S complex perform distinct functions. Nuc2 and Cut9 might interact with different proteolysis substrates for ubiquitin ligation, and dominant negative effects due to association of excess Nuc2 or Cut9 with different targets might result in different phenotypes. Nuc2 and Ntruncated Cut9 or Cut9 and N-truncated Cut9 were simultaneously overproduced in wild-type cells, but the toxic effect of single overproduction was not suppressed: no colony was formed in both cases (our unpublished result). Sucrose gradient centrifugation of wild-type extracts containing overproduced N-truncated Cut9 showed that a significant fraction of Nuc2 was apparently unbound to the 20S complex (unpublished result), consistent with a hypothesis that N-truncated Cut9 inhibits 20S complex formation through aberrantly interacting with Nuc2. High copy suppressors for cut9-665 While plasmid pHCN1 fully suppresses cut9-665, it does not suppress nuc2-663. The predicted amino acid sequence of Hcn1 resembles budding yeast Cdc26, which was shown to be a component of the 36S APC/cyclosome, possibly implicated in the chaperoning activity (Zachariae et al., 1996). Functional similarity between Hcn1 and Cdc26 was further confirmed by suppression of cut9-665 by overproduced Cdc26. The question of how an elevated gene dosage of hcn1+ can restore the assembly of the 20S complex in cut9-665 remains to be investigated, but direct and specific interaction between Cut9 and Hcn1 is plausible. Another high copy suppressor for cut9-665 was pSds23 (Ishii et al., 1996). This plasmid has a broad specificity for rescuing various mutations in PP1 and the APC/ cyclosome, however. Regulation of the 20S complex by PP1 and PKA Genetic evidence suggests that PKA and PP1 interact with Cut9. Fission yeast PP1 is required for the transition from metaphase to anaphase (Ishii et al., 1996). PP1 and Cut9 possibly share an essential function as the double mutant is lethal. It is unknown at which stage in anaphase promoting proteolysis, PP1 is actually implicated. Our preliminary in vitro experiments suggested that PP1 was not directly involved in dephosphorylation of Cut9 phosphorylated in nda3 mutant. Although the wild-type extracts contained the activity dephosphorylating Cut9, the phosphatase activity was still present in the extracts of PP1-deficient mutant. The kinase and phosphatase directly acting on Cut9 remain to be determined, although they are likely to be downstream of PKA and PP1 (Fig. 9). Fission yeast PKA is non-essential for viability, and only one catalytic subunit gene pka1+ has been found so far (Maeda et al., 1994; Yu et al., 1994). The involvement of PKA in APC/cyclosome formation is also shown by studying the cut4 mutation (Yamashita et al., 1996). PKA is inhibitory to APC/cyclosome formation in the cut4 mutant,

Properties of APC/cyclosome subunits 1803 consistent with the present result. Involvement of PKA in ubiquitin-dependent anaphase proteolysis was clearly demonstrated in the Xenopus cycling extract (Grieco et al., 1996). Involvement of PP1 for dephosphorylation of Cut9 is evidenced only by genetic interaction and is less clear than PKA for phosphorylation. In short, Cut9, an essential subunit of the 20S complex, interacts directly or indirectly with several proteins including Nuc2, Hcn1, Scn1, Scn2, PKA and PP1, as shown in the present and previous studies. Phosphorylation of Cut9 bound to the 20S complex is regulated during mitosis. Protein-protein interactions and phosphorylation/dephosphorylation may thus be the major ways for regulating APC/cyclosome function in the cell cycle. We thank Drs Masayuki Yamamoto and Hiroyuki Araki for plasmids and strains, and Drs K. Gull, M. Yamashita and Y. Nagahama for antibodies. This work was supported by a Specially Promoted Research grant from the Ministry of Education, Science and Culture of Japan, and a CREST grant of the Japan Science and Technology Corporation.

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(Received 10 March 1997 - Accepted 28 May 1997)