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The role of Ppe1/PP6 phosphatase for equal chromosome segregation in fission yeast kinetochore

2003; Springer Nature; Volume: 22; Issue: 11 Linguagem: Inglês

10.1093/emboj/cdg266

1460-2075

Gohta Goshima,

Chromosomal and Genetic Variations

Article1 June 2003free access The role of Ppe1/PP6 phosphatase for equal chromosome segregation in fission yeast kinetochore Gohta Goshima Gohta Goshima Present address: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, 94107 USA Search for more papers by this author Osamu Iwasaki Osamu Iwasaki COE Research Project, Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto, 606-8502 Japan Search for more papers by this author Chikashi Obuse Chikashi Obuse Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara, 630-0101 Japan Search for more papers by this author Mitsuhiro Yanagida Corresponding Author Mitsuhiro Yanagida COE Research Project, Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto, 606-8502 Japan Search for more papers by this author Gohta Goshima Gohta Goshima Present address: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, 94107 USA Search for more papers by this author Osamu Iwasaki Osamu Iwasaki COE Research Project, Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto, 606-8502 Japan Search for more papers by this author Chikashi Obuse Chikashi Obuse Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara, 630-0101 Japan Search for more papers by this author Mitsuhiro Yanagida Corresponding Author Mitsuhiro Yanagida COE Research Project, Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto, 606-8502 Japan Search for more papers by this author Author Information Gohta Goshima2, Osamu Iwasaki1, Chikashi Obuse3 and Mitsuhiro Yanagida 1 1COE Research Project, Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto, 606-8502 Japan 2Present address: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, 94107 USA 3Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara, 630-0101 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:2752-2763https://doi.org/10.1093/emboj/cdg266 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Mis12 is a kinetochore protein essential for equal chromosome segregation and is evolutionarily conserved from yeast to human. In this study, we report the isolation and characterization of suppressors of the mis12 mutant in fission yeast. Our results indicate that Mis12 is negatively regulated by a highly conserved protein phosphatase Ppe1 (scSit4/dmPPV/hPP6) or its bound partner Ekc1 (scSAP), and it is positively regulated by a counteracting kinase Gsk3. Mass spectrometry analysis shows that at least two sites in Mis12 are phosphorylated. This mechanism of suppression occurs at the level of localization recovery of Mis12 to the kinetochore chromatin. Consistently, Mis12 and a subpopulation of Ppe1/Ekc1 were found to behave like non-histone-type chromatin-associating proteins in the chromatin fractionation assay. Mutant analysis of Ppe1 and Ekc1 revealed that they are important for faithful chromosome segregation, as the mutants exhibited unequal chromosome segregation similar to mis12 in the presence of a low concentration of tubulin poison. Ppe1/PP6 directly or indirectly modulates kinetochore chromatin protein Mis12 to ensure progression into normal anaphase. Introduction Equal segregation of chromosomes to two daughter cells is a process fundamental for genome inheritance. Centromeres are the chromosomal attachment sites for spindle microtubules during mitosis and meiosis. Proteins are located on the centromere DNAs to form a highly ordered complex structure, the kinetochore. The kinetochore has vital roles in ensuring the fidelity of chromosome segregation. We have employed the fission yeast Schizosaccharo myces pombe as an excellent model organism for the analyses of centromere/kinetochore structure and function. Centromeres of fission yeast (35–110 kb) are composed of two types of chromatin domains, specialized central chromatin and outer heterochromatin (e.g. Takahashi et al., 1992). The central domains contain 15 kb unique sequences and form the specialized chromatin, which displays a smeared nucleosome ladder after micrococcal nuclease (MNase) digestion. The outer regions produced the patterns of regular nucleosomal ladders (Polizzi and Clarke, 1991; Takahashi et al., 1992). This two-domain structure bears some resemblance to the higher eukaryotic centromeres (Blower and Karpen, 2001; Oegema et al., 2001). Stability tests of various artificial minichromosomes of fission yeast established that the central regions are essential for centromere function in both mitosis and meiosis, whereas outer repetitious regions make minor contributions in mitosis but are indispensable for meiotic chromosome segregation (e.g. Takahashi et al., 1992). Kinetochore microtubules as well as microtubule-associating proteins and spindle checkpoint proteins are likely to interact at the surface of central centromere regions during mitosis (Garcia et al., 2001; Nakaseko et al., 2001; Toyoda et al., 2002). Identification of proteins that bind to the central regions and form the structure for higher ordered kinetochore chromatin is, therefore, important for understanding mitotic kinetochore functions. Fission yeast mis6-302 and mis12-537 mutants exhibit unequal segregation of regular chromosomes when shifted to a restrictive temperature. In these mutants, sister chromatids were segregated to the daughter cells at anaphase without a long delay, but the segregation patterns of sister chromatids were random. Consequently, these mutations produced aneuploid cells with a high frequency. Localization studies using green fluorescent protein (GFP) and chromatin immunoprecipitation (ChIP) assays established that Mis6 and Mis12 are bound to the central centromere regions throughout the cell cycle. MNase digestion experiments indicated that these proteins are essential for forming the specialized chromatin of the central centromere regions (Saitoh et al., 1997; Goshima et al., 1999). However, no genetic or physical interactions have been found between Mis6 and Mis12. CENP-A, a histone H3 variant protein, is localized exclusively at centromere regions and plays an essential role in chromosome segregation from yeast to humans (Stoler et al., 1995; Howman et al., 2000; Takahashi et al., 2000; Blower and Karpen, 2001; Oegema et al., 2001). In fission yeast, spCENP-A is located to central centromere regions in a Mis6-dependent manner, and an spCENP-A mutant shows missegregation and central chromatin disruption phenotypes identical to mis6/mis12 (Takahashi et al., 2000). spCENP-A, therefore, appears to confer specialized nucleosomes to central DNAs. In other organisms so far investigated, Mis6 homologue depletion is unlikely to affect CENP-A localization at centromeres and, instead, CENP-A is indispensable for Mis6 recruitment to centromeres (Measday et al., 2002; Nishihashi et al., 2002; Goshima et al., 2003). CENP-A is also essential for kinetochore localization of other components in higher eukaryotes. These results lead to the model that CENP-A acts as a core nucleosome for the whole kinetochore chromatin architecture (reviewed by Smith, 2002). On the other hand, there has been little evidence that shows the interaction and obvious localization dependence between Mis12 and CENP-A, both of which locate in the central centromere regions. Localization of Mis12 was unaffected in the absence of CENP-A, and vice versa in fission yeast [temperature-sensitive (ts) mutant analyses] and HeLa cells [RNA interference (RNAi) analyses], indicating that the loading pathway for Mis12 is independent of CENP-A (Takahashi et al., 2000; Goshima et al., 2003). The reduction of human hMis12 or CENP-A by RNAi led to chromosome misalignment at metaphase, chromosome lagging in anaphase and micronuclei formation in interphase. These genetic and cytological results established that Mis12 is an important conserved kinetochore protein in an independent pathway forming kinetochore chromatin (Goshima et al., 2003). Although CENP-A's molecular characterization as well as its relationships to other kinetochore components have been well investigated, such knowledge was scarce for Mis12. The initial aim of this study was to identify proteins that interact directly or indirectly with Mis12 through genetic screening. To this end, extragenic and high copy suppressors were isolated for the fission yeast mis12 mutant. Through the analyses of extragenic suppressors, Mis12 was found to be under the control of protein phosphorylation–dephosphorylation by a counteracting kinase and phosphatase. Results Suppression of mis12-537 by mutations in conserved proteins Ppe1 and Ekc1 We attempted to isolate extragenic or high dosage suppressors for mis12-537 in the hope that identification of suppressor gene products would produce an understanding of the regulation of Mis12 kinetochore function. Among 500 spontaneously isolated pseudo-revertants of the mis12-537 mutant, 24 strains were found to be cold sensitive (cs) and could grow at 36°C but failed at 22°C (Goshima et al., 1999). These extragenic suppressors are designated ekc (extragenic suppressor of kinetochore) mutants. Genetic analysis by crossing (Materials and methods) established that these 24 mutant alleles were derived from only two genetic loci (designated ekc1 and ekc2). As shown in Figure 1A, the double mutants ekc1 mis12 and ekc2 mis12 formed colonies at 36°C, while single segregants ekc1 and ekc2 failed to form colonies at 22°C. Curiously, all the ekc mutant strains invariably revealed pear-shaped cells at any temperature (Figure 1B). Regulation of cell morphology may be defective in ekc mutant cells. Figure 1.Extragenic suppression of mis12-537 by ppe1 and ekc1 mutants. (A) Left: double mutants mis12-537 ekc1-163, mis12-537 ekc2-213 and mis12-537 Δppe1 could form colonies at 36°C, whereas single mis12-537 did not. Right: single ekc1 and ekc2 mutants were cold sensitive and unable to form colonies at 22°C. (B) ekc1-163 is pear-shaped even at the permissive temperature. Bar, 10 μm. (C) Amino acid sequence alignment of S.pombe Ekc1, S.cerevisiae SAP190 and Homo sapiens KIAA0685. Identical residues are boxed, and similar residues are hatched. Saccharomyces cerevisiae has four SAPs (SAP4, 190, 185 and 155), while human has three similar sequences (KIAA0685, KIAA1115 and KIAA1558). (D) Sequence alignment of S.pombe Ppe1 phosphatase, S.cerevisiae Sit4, H.sapiens PP6, D.melanogaster PPV and S.pombe Ppa2 and Ppe2/SPBC26H8.05. (E) Exponentially growing wild-type, ekc1, ppe1 and mis12 cells were spotted after dilution onto YPD plate that contained 0.2 or 0.4 μg/ml staurosporine, and incubated at 33°C. (F) The strain containing the integrated Ekc1-Myc was immunoprecipitated by anti-Myc antibodies (lanes 1–3) and anti-Ppe1 antibodies (lanes 7–9), and immunoblotting was used to detect the three proteins Ekc1, Ppe1 and Mis12 in the resulting precipitates. Beads without antibodies (lanes 10 and 11) or the strain without myc epitopes (lanes 4–6) were used as control. I, input; S, supernatant; P, immunoprecipitate. Equal amounts of I and S were loaded, whereas immunoprecipitates were 6- (Px6) or 5-fold (Px5) concentrated. Download figure Download PowerPoint The ekc1+ gene was cloned from a genomic DNA library using transformation of the cs phenotype of the ekc1 mutant. Ekc1 was identical to SPCC663.01, an uncharacterized gene. The gene product contains 851 amino acids and is 30–37% identical to the budding yeast SAPs (SAP190, SAP185, SAP155 and SAP4), which are phosphatase modulators and bind to Sit4 phosphatase as a positive regulator (Luke et al., 1996). The SAP proteins are conserved from yeast to humans (Figure 1C), and although Saccharomyces cerevisiae and human have four and three SAPs, respectively, the genome of Schizosaccharomyces pombe has only one. We speculated that Ekc2 might be identical to Ppe1 phosphatase (Matsumoto and Beach, 1993; Shimanuki et al., 1993), the fission yeast orthologue of scSit4/hPP6/dmPPV (Mann et al., 1993; Bastians and Ponstingl, 1996; Cohen, 1997). The cs phenotype of the ekc2-213 mutant was indeed rescued by a multicopy plasmid carrying the authentic ppe1+ gene. Subsequent crossing between ekc2-213 and the Δppe1 deletion mutant confirmed that ekc2 was allelic to ppe1. Consistently, the double mutant Δppe1 mis12 could grow at 36°C (Figure 1A, left panel). The deletion of ppe1+ is known to cause the cs phenotype and pear-shaped or round cells at both permissive and restrictive temperatures (Shimanuki et al., 1993). Ppe1 is a protein phosphatase that is highly conserved from yeast to human (Figure 1D). Indeed, the human orthologue PP6 could suppress the phenotype of budding yeast sit4 and fission yeast ppe1 mutants (Bastians and Ponstingl, 1996). The amino acid sequence of Ppe1 is similar (51% identical) to that of type 2A phosphatase Ppa2 (Kinoshita et al., 1990), but Δppa2, the deletion mutant of Ppa2, did not suppress the ts phenotype of mis12-537 (data not shown). Schizosaccharomyces pombe has another highly conserved protein phosphatase designated Ppe2 (SPBC26H8.05), the orthologue of human and fly PP4 (Helps et al., 1998), and budding yeast Pph3 (Ronne et al., 1991). It is highly similar (53% identity) to Ppe1, but the deletion mutant Δppe2 that was viable did not suppress the ts phenotype of mis12-537 (data not shown). The ts phenotype of mis6-302 was not suppressed by Δppe1. The suppression of mis12-537 by the Δppe1 mutant was thus quite specific. While Δppe1 was hypersensitive to staurosporine, a potent kinase inhibitor (Toda et al., 1991; Shimanuki et al., 1993), ekc1 mutants were similarly sensitive to the drug (Figure 1E). To determine if there was a physical interaction between Ekc1 and Ppe1, we performed immunoprecipitation using polyclonal anti-Ppe1 antibodies and monoclonal anti-myc antibodies for a strain chromosomally integrated with the Ekc1-Myc gene (Figure 1F). Quantitative analysis indicated that 20% of the total Ekc1-Myc was precipitated by anti-myc antibody (comparison between lanes 1 and 3). In the precipitates, 20% of Ppe1 was co-precipitated (lanes 1 and 3). Similar levels of Ppe1 and Ekc1-Myc co-precipitation were obtained for the precipitates made by anti-Ppe1 antibodies (lanes 7 and 9). However, Mis12 protein was not detected in the precipitates with either anti-Ppe1 or anti-Ekc1-Myc antibodies under the experimental conditions used (lanes 3 and 9). Ekc1 thus stably binds to Ppe1, but not to Mis12. As the functional link between Ppe1–Ekc1 and kinetochore is a novel aspect, we investigated the mitotic roles of Ppe1–Ekc1. Ekc1 is enriched in the nucleus To determine its localization, Ekc1 tagged with yellow fluorescent protein (YFP) at the C-terminus was chromosomally integrated in wild type and observed (Figure 2A). The Ekc1–YFP signal was enriched in the nucleus in both interphase and mitosis. The signal was also found in the cytoplasm, except for vacuole-like structures. The nuclear signals of Ekc1 closely resembled that of nuclear chromatin in interphase (indicated by the arrow), whereas the mitotic nuclear signals (indicated by the arrowhead) were more diffuse in the whole nucleus, suggesting that a pool of Ekc1 might be dissociated from nuclear chromatin during mitosis. Figure 2.Nuclear-enriched localization of Ekc1–YFP and Ppe1–GFP. Chromosomally integrated Ekc1–YFP (A, green) and Ppe1–GFP (phosphatase-dead) (B, green) showed nuclear chromatin-enriched localization. 4′,6-diamidino-2-phenylindole (DAPI) was used for DNA staining (red) without fixation. Bars, 10 μm. Download figure Download PowerPoint To determine the localization of Ppe1, GFP was fused to the C-terminus of Ppe1, and the resulting Ppe1–GFP gene was chromosomally integrated with the native promoter. The Ppe1–GFP signal was indistinguishable from Ekc1–YFP localization (Figure 2B); the signal was enriched at the nuclear chromatin and seen as diffused in cytoplasm except for vacuole-like regions. However, the integrant strain showed cs growth and a cell shape defect. As we could not determine Ppe1 localization by immunofluorescence using polyclonal anti-Ppe1 antibodies, it remains to be determined whether the localization of functional Ppe1 was identical to that of Ppe1–GFP shown here. The ectopically expressed N-terminus of Ppe1 (amino acids 1–94) tagged with GFP showed a chromatin-enriched localization, similar to full-length Ppe1–GFP (data not shown). It is hence likely that native Ppe1 is targeted to chromatin by its N-terminal domain. Microtubule inhibitor causes unequal segregation in ekc mutants In ekc1 and ekc2 (and also Δppe1) mutants, chromosomes appeared to be equally segregated. As these mutants were found to be hypersensitive to the tubulin poison, thiabendazole (TBZ), at the permissive temperature, 33°C (Figure 3A), we examined whether chromosome segregation was still equal if TBZ was added to the culture medium. When treated with 20 μg/ml TBZ, ekc1-163 and Δppe1mutant cells produced a high frequency (52 and 48% of mitoses, respectively) of unequal segregation of chromosomes at 33°C (Figure 3B). Wild-type controls at 33°C exhibited only 11% unequal segregation under this condition. The occurrence of mis-segregation was verified by light microscopy using CEN1–GFP (Nabeshima et al., 1998) as shown in Figure 3C. Two sister centromere signals (arrowheads) were often found in one daughter nucleus in both mutants. The role of Ppe1–Ekc1 phosphatase in equal sister chromatid separation thus became evident when microtubules were inhibited in semi-permissive concentrations of TBZ. The TBZ sensitivities of the double mutants ekc1 mis12 and Δppe1 mis12 were similar to those of the single ekc mutant (Figure 3A and B). Figure 3.Chromosome missegregation in ekc1 and Δppe1 mutants in the presence of TBZ. (A) Moderate hypersensitivity of ekc1-163, Δppe1, mis12, ekc1-163 mis12 and Δppe1 mis12 mutants to TBZ, a microtubule-destabilizing drug. Exponentially growing cells were diluted and spotted onto YPD plates that contained 20 μg/ml TBZ, and incubated at 33°C. nda2-KM52, an α-tubulin mutant, was used as a control hypersensitive mutant. (B) Unequal chromosome segregation frequently occurred in ekc1-163, Δppe1, ekc1 mis12 and Δppe1 mis12 mutant cells in the presence of TBZ. Wild-type and mutant cells were cultured at 33°C, and TBZ (20 μg/ml) was added (time 0). The frequency of dividing cells did not change after TBZ addition, whereas the frequency of mutant cells displaying unequal nuclear division increased to 48–55% (of binucleate cells) after 4 h. (C) Mis-segregation of sister chromatids in ekc1-163 and Δppe1 was confirmed through the use of cen1[lys1]–GFP, a cen1-proximal DNA marker (arrowheads) (33°C, 4 h after TBZ addition). Bar, 10 μm. Download figure Download PowerPoint Kinetochore localization of Mis12 is restored in the double mutant ekc1 mis12 To investigate how the phenotype of mis12-537 was rescued by ekc mutations, we determined the localization of mutant Mis12 protein in an ekc1 mutant background (Figure 4A). For this purpose, we constructed a strain (designated Mis12ts–GFP) in which the wild-type mis12+ gene was replaced with the GFP-tagged mutant mis12-537ts (G52E) gene. This strain exhibited the phenotypes of ts growth and unequal chromosome segregation at 36°C, just like the original mis12-537 strain. Mis12ts–GFP cells were cultured at the permissive temperature (26°C) and then shifted to the restrictive temperature, 36°C (time 0). Cells were observed by microscopy without fixation after 2, 4 and 8 h (Figure 4A and B). The dot-like localization of Mis12-537ts–GFP mutant protein that was clearly seen in 60% of the cells before the temperature shift up disappeared after 4 h at 36°C (<5%). Control cells expressing Mis12wt–GFP protein showed a high frequency of dot-like localization (∼60%) at both 26 and 36°C. Note that GFP observation was carried out at a single focal plane, so that the signals of GFP could not be seen in a fraction of cells in which the centromeres were out of focus. To test the effect of the ekc1 mutation, Mis12ts–GFP was observed in the ekc1-163 background. The ekc1 Mis12ts–GFP double mutant was cultured at 36°C, and the GFP signal was observed. The dot-like localization of mutant Mis12 protein was restored, strongly suggesting that localization of Mis12 is under the control of Ekc1. Figure 4.Restoration of Mis12 mutant protein localization in the ekc1-163 background. (A) The dot-like localization of Mis12 mutant protein was restored in the double mutant mis12 ekc1. Images represent Mis12–GFP and Mis12-537–GFP expressed in the wild-type and ekc1 background cells (36°C, 8 h). (B) The frequencies of the centromeric dot-like appearance of Mis12ts–GFP were restored in ekc1 mutant cells. Mis12ts–GFP signals in the wild-type background became diffused after 4 h. (C) Schizosaccharomyces pombe cell extracts of wild-type and mutant cells cultured at 36°C were prepared at 2 h intervals. Immunoblotting of extracts was performed using anti-Mis12 and control anti-PSTAIR antibodies. Download figure Download PowerPoint Immunoblotting of mis12-537 extracts prepared from the culture at the restrictive temperature, 36°C, showed that Mis12-537 mutant protein was less stable than that of the wild-type control, and that the instability was not restored to the wild-type level in the double mutant ekc1 mis12-537 (Figure 4C). This result strongly suggested that mutant Mis12 protein gained the ability to be incorporated into the centromere in the background of ekc1 mutation at 36°C. Mis12, Ppe1 and Ekc1 are bound to chromatin The above results suggested that Ppe1 and Ekc1 might directly or indirectly regulate Mis12. To examine whether wild-type and mutant Mis12 proteins behave as chromatin proteins and to study how ekc mutations affect the behaviour of Mis12, a chromatin fractionation assay was introduced for spheroplasted cell lysates of S.pombe (schematized in Figure 5A). Cell lysates (designated whole-cell extract; WCE) were centrifuged and separated into soluble (Sup) and pellet (PT) fractions. Whole chromatin DNA and chromatin-interacting proteins remained in the PT fraction at this stage. This pellet was treated by either MNase (Figure 5A, left) or NaCl extraction (right). After MNase treatment for 10–30 min, the average length of chromatin DNA was fragmented to <10 kb (Figure 5B, right panel lanes 3 and 5), resulting in the release of chromatin-associated proteins to the Sup (S). Hence, in this assay, those proteins that were first in the PT and then released into the S fraction after MNase treatment were defined as chromatin-associated proteins. Figure 5.Chromatin fractionation assay of Mis12, CENP-A, Ekc1 and Ppe1 proteins. (A) Schematic representation of the chromatin fractionation assay employed in the present study. See text for explanations. (B) Left: immunoblotting of tubulin (TUB), Ekc1-Myc, Ppe1, Mis12 and spCENP-A in various fractions: WCE, whole-cell extracts; PT, the pellet fraction after spheroplasting; S, supernatant; and P, pellet after MNase treatment for 0, 10 and 30 min. Four-fold concentrated proteins were loaded in the PT, S and P lanes. Mis12, CENP-A, Ekc1-Myc and Ppe1 were found to be solubilized after MNase digestion. Coomassie Blue staining is shown at the bottom. Right: DNA samples extracted before or after MNase digestion (0, 10 and 30 min) were electrophoresed in an agarose gel, followed by ethidium bromide staining. Lane 1, S at 0 min; lane 2, P at 0 min; lane 3, S at 10 min; lane 4, P at 10 min; lane 5, S at 30 min; lane 6, P at 30 min; lane 7, size marker. (C) The PT fraction was washed with the buffer containing 0–1000 mM NaCl followed by centrifugation. Four-fold concentrated proteins were loaded in the PT, S and P lanes. Approximately 30% of Mis12 was solubilized by treatment with 300 mM NaCl. Most Ppe1 and Ekc1-Myc was solubilized in 300 mM NaCl. Coomassie Blue staining is shown at the bottom. (D–F) The same NaCl treatment experiment as (C) was performed for mis12-537 (D), ekc1 mis12-537 (E) and Δppe1 mis12-537 (F) mutants. (G) Recovery of centromere chromatin structure by ekc1 mutation. Nuclear chromatin was prepared from spheroplasts of wild type and mis12, ekc1 and ekc1 mis12 mutants cultured at 36°C for 8 h. Chromatin was digested with MNase for 0, 1, 2, 4 and 8 min (left to right lanes). Digested DNAs were electrophoresed in an agarose gel. Southern hybridization using the central centromere DNA probe imr1 was performed. The size markers are shown on the left. Download figure Download PowerPoint Supernatants and pellets were analysed by SDS–PAGE, followed by immunoblot and also Coomassie Blue staining (Figure 5B, left panel). Simultaneously, DNAs before and after the MNase treatment were electrophoresed in an agarose gel (right panel). Coomassie Blue staining indicated that most proteins were in the Sup at the first stage, and the PT proteins almost completely went into the S fraction after MNase treatment. Digested DNA was detected in the solubilized fraction (lanes 3 and 5). Immunoblot using anti-Mis12 antibody revealed that wild-type Mis12 was first found in PT but was completely released to S after MNase digestion. spCENP-A behaved similarly to Mis12. As a control, we found tubulin to be exclusively in the Sup after cell lysis. These results established that most Mis12 and spCENP-A were chromatin bound. Ppe1 and Ekc1-Myc were present initially in both Sup and PT, and their subpopulation in the PT moved to S after MNase treatment, indicating that the subfractions of Ppe1 and Ekc1-Myc were chromatin bound. To estimate the concentration of NaCl required for extracting Mis12 from chromatin, PT was washed with buffer containing various concentrations of NaCl (Figure 5C). After 10 min on ice, samples were centrifuged and separated into soluble (S) and pellet (P). Proteins, such as histones, tightly bound to DNA and chromatin were not dissociated from DNA by the low salt treatment. The PT fraction was washed with 100–1000 mM NaCl-containing buffer. Coomassie Blue staining indicated that most proteins in the PT were solubilized by 300 mM NaCl. Thirty to forty percent of Mis12 was solubilized after extraction with 300 mM NaCl, and almost all of the protein was solubilized by 1000 mM NaCl. spCENP-A was more resistant than Mis12 to the salt treatment, and it remained in P after 300 mM NaCl treatment. Ppe1 and Ekc1 were solubilized after 200–300 mM NaCl, similar to Mis12. These properties indicated that Mis12, Ppe1 and Ekc1 behaved in a manner similar to non-histone chromatin proteins, and associated fairly strongly with chromatin but less tightly than histone proteins. ekc mutations restore the Mis12-containing inner centromere chromatin We performed the chromatin assay using mis12-537 (Figure 5D), ekc1 mis12-537 (Figure 5E) and Δppe1 mis12-537 (Figure 5F) strains (36°C, 6 h), and found that Mis12-537 protein was more resistant to NaCl extraction in the presence of functional Ppe1 and Ekc1 (Figure 5D). Unlike wild-type Mis12, mutant Mis12-537 protein detected in the PT was not solubilized by 300 mM NaCl treatment. Further, more than half of the Mis12-537 protein remained in the pellet fraction even after treatment with 1000 mM NaCl, conditions that solubilized CENP-A. In sharp contrast, most of the Mis12-537 protein became solubilized by 200–300 mM NaCl treatment in the background of ekc1-163 or Δppe1 (Figure 5E and F). Down-regulated Ppe1–Ekc1 thus dramatically recovered the salt solubility property of Mis12-537 to that of wild-type Mis12. We then examined whether ekc1 mutation restores the specialized centromere chromatin in the central regions. Southern hybridization was performed after MNase digestion of chromatin DNAs using the inner centromere sequence (imr) as a probe. The smeared centromere chromatin pattern present in wild-type and single ekc1 mutant cells after MNase treatment was abolished in mis12-537 at 36°C, but restored in the ekc1 mis12 double mutant cells cultured at 36°C (Figure 5G). The outer repetitive heterochromatin was not affected by either ekc1 or mis12, and displayed regular nucleosomal ladders at 36°C (data not shown). The functional defect of Ekc1 could thus result in restoration of the central centromere chromatin structure in mis12-537 mutant. The dosage increase of Gsk3 kinase suppresses the phenotype of mis12-537 Extragenic suppressor analysis identified Ppe1 as a phosphatase that interacts with Mis12. We next screened for high gene dosage suppressors for the ts phenotype of mis12-537 using two S.pombe genomic DNA libraries based on the multicopy plasmid vector. A number of high gene dosage suppressors were found, including Gsk3, Ssp1, Spi1, Sds23 and Mts2 (Matsumoto and Beach, 1991; Gordon et al., 1993; Matsusaka et al., 1995; Ishii et al., 1996; Plyte et al., 1996). Gsk3 and Ssp1 are described below, and other suppressors will be described elsewhere. A strong suppressor gene obtained from both libraries resembled the mammalian GSK-3β kinase. Gsk3 kinase in fission yeast is non-essential for cell viability, but does suppress cytokinesis-defective cdc14 when overexpressed [it was also called Skp1 (shaggy kinase