Defects in Components of the Proteasome Enhance Transcriptional ...

MOLECULAR AND CELLULAR BIOLOGY, July 1999, p. 5155–5165 0270-7306/99/$04.0010 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 19, No. 7

Defects in Components of the Proteasome Enhance Transcriptional Silencing at Fission Yeast Centromeres and Impair Chromosome Segregation JEAN-PAUL JAVERZAT,1,2* GORDON MCGURK,1 GWEN CRANSTON,1 CHRISTIAN BARREAU,2 PASCAL BERNARD,2 COLIN GORDON,1 AND ROBIN ALLSHIRE1 Medical Research Council, Human Genetics Unit, Western General Hospital, Edinburgh EH4 2XU, Scotland, United Kingdom,1 and Institut de Biochimie et Ge´ne´tique Cellulaires, Centre National de la Recherche Scientifique, Unite´ Propre de Recherche 9026, 33077 Bordeaux Cedex, France2 Received 14 December 1998/Returned for modification 9 February 1999/Accepted 19 April 1999

Fission yeast centromeres are transcriptionally silent and form a heterochromatin-like structure essential for normal centromere function; this appears analogous to heterochromatin and position effect variegation in other eukaryotes. Conditional mutations in three genes designated cep (centromere enhancer of position effect) were found to enhance transcriptional silencing within centromeres. Cloning of the cep11 and cep21 genes by functional complementation revealed that they are identical to the previously described genes pad11 and mts21, respectively, which both encode subunits of the proteasome 19S cap. Like Mts2 and Mts4, epitopetagged Cep1/Pad1 localizes to or near the nuclear envelope throughout the cell cycle. The cep mutants display a range of phenotypes depending on the temperature. Silencing within the central domain of centromeres is increased at 36°C. This suggests that the proteasome is involved in regulating silencing and thus centromeric chromatin architecture, possibly by lowering the level of some chromatin-associated protein by ubiquitindependent degradation. This is the first report of defective proteasome function affecting heterochromatinmediated transcriptional silencing. At 36 and 32°C, the cep mutants lose chromosomes at an elevated rate, and at 18°C, the mutants are cryosensitive for growth. Cytological analysis at 18°C revealed a defect in sister chromatid separation while other mitotic events occurred normally, indicating that cep mutations might interfere specifically with the degradation of inhibitor(s) of sister chromatid separation. These observations suggest that 19S subunits confer a level of substrate specificity on the proteasome and raise the possibility of a link between components involved in centromere architecture and sister chromatid cohesion. activated, and anaphase proceeds (for recent reviews, see references 19 and 50). Proteins ubiquitinated by the APC are degraded by the proteasome, a large 28S protein complex made of a 20S proteolytic core particle (CP) and a 19S regulatory particle (RP). The crystal structure of the 20S complex from budding yeast has been determined previously (18). It consists of two A and two B rings, each made of seven different A and seven different B subunits. The active proteolytic sites are within the CP, and substrates are thought to enter the CP through the lumens located at each end of the cylinder. The CP alone can hydrolyze only small peptides and exists predominantly in a closed state, indicating that the ends of the channel are gated. Degradation of ubiquitinated proteins necessitates the binding of RP. In budding yeast, the RP contains at least 18 subunits and can be dissociated into two subparticles, the base and the lid (15). The base contains the six ATPase subunits and connects the RP to the CP. The base activates the CP for degradation of peptides and nonubiquitinated proteins, whereas the lid is required for ubiquitin-dependent degradation. It has been proposed that the lid selects ubiquitinated substrates and that the base gates the CP channel and unfolds the substrates. The centromere-kinetochore complex is a key element in the process of mitosis. It is defined as the nucleoprotein complex containing chromosomal factors and centromeric DNA. It fulfills mechanical functions such as sister chromatid cohesion, attachment to the spindle, and chromosome movement as well as the regulatory function of monitoring chromosome attachment to the spindle. In most eukaryotes, centromeric DNA consists of long arrays of repetitive DNA packaged into tran-

Mitosis is the process by which eukaryotic cells ensure the faithful transmission of their chromosomes. Many studies have shown that proteolysis governs mitotic progression by the orderly degradation of key proteins via the ubiquitin-proteasome pathway (see reference 50 for a recent review). Substrate specificity is conferred by a ubiquitin-protein ligase called the anaphase-promoting complex (APC) and associated regulatory proteins. After maturation-promoting factor drives cells into mitosis, the APC forms a complex with the substrate-specific activator Cdc20 to trigger the proteolysis of inhibitors of anaphase (Pds1 in Saccharomyces cerevisiae and Cut2 in Schizosaccharomyces pombe), thereby allowing the separation of sister chromatids and anaphase spindle elongation. Subsequently, APC binds another substrate-specific activator, Hct1/Cdh1, and the complex directs the destruction of AseI and the mitotic cyclin, thus allowing spindle disassembly and exit from mitosis. Mitotic progression is also controlled by a surveillance system, the spindle checkpoint, which prevents anaphase onset until chromosomes are properly attached to the spindle. Studies with yeasts and vertebrates indicate that the checkpoint consists of components able to sense unattached kinetochores and a signal transduction pathway which delays the onset of anaphase through inhibition of the APC. When the correct attachment is achieved, the negative signal disappears, APC is

* Corresponding author. Mailing address: Institut de Biochimie et Ge´ne´tique Cellulaires, CNRS UPR 9026, 1 rue Camille Saint Sae ¨ns, 33077 Bordeaux Cedex, France. Phone: (33) 556 99 90 26. Fax: (33) 556 99 90 67. E-mail: [email protected]. 5155

5156

MOL. CELL. BIOL.

JAVERZAT ET AL.

scriptionally silent, recombination cold, late-replicating heterochromatin. The exact function of heterochromatin is unknown, but kinetochores in higher eukaryotes generally form within centromeric heterochromatin (23). In the fission yeast, S. pombe, three regions of the genome are known to be assembled into heterochromatin. These are the silent mating-type loci, telomeres, and centromeres (2, 34, 46). Centromeres consist of inner (imr/B) and outer (otr/K1L) repetitive sequences arranged in a large inverted structure around a central core (44, 45) (see Fig. 1A). As is often the case for heterochromatic regions in higher eukaryotes, recombination (33) and transcription (2, 3) are repressed within fission yeast centromeres, and they are located at the nuclear periphery in interphase (12). The link between heterochromatin formation and centromere function has been clearly established for S. pombe. Mutations in clr1, clr2, clr3, clr4, rik1, and swi6 genes were shown to affect repression at the silent mating-type loci, at centromeres, and, to a limited extent, at telomeres (3, 11, 26, 47). In particular, mutations in clr4, rik1, and swi6 strongly affect silencing within the imr and otr regions, but not the central core, of the centromere and cause an elevated rate of chromosome loss. The swi61 gene encodes a protein with similarity to Drosophila melanogaster HP1 (27). HP1 localizes to pericentric heterochromatin, and mutations in HP1 suppress repression of marker genes lying in the vicinity of centromeric heterochromatin and apparently induce chromosome missegregation events (24). Both Swi6 and HP1 proteins contain a chromodomain and a chromo-shadow domain. It has been suggested that this class of proteins could act as adapter molecules to mediate heterochromatin formation (1, 38). Swi6 localizes to the three known silent domains of the fission yeast genome, centromeres, telomeres, and the mating-type region (9), and functional Clr4 and Rik1 are required for this localization (10). Clearly, heterochromatin assembly appears to be crucial for fission yeast centromere function, since the silencing defect caused by the lack of a functional clr4, rik1, or swi6 gene is correlated with defective movement of centromeres at anaphase and an elevated rate of chromosome loss (3, 9). It is very likely that many other structural components as well as regulatory factors are required to form normal centromeric heterochromatin and kinetochore assembly. Here a genetic screen for mutants which affect centromeric silencing within the central core region of S. pombe centromeres has been employed. The screen has identified a new class of mutants called cep (centromere enhancer of position effect). All cep mutants were found to be defective in mitotic chromosome segregation. They define three genes designated cep1, cep2, and cep3. The cep11 and cep21 genes were cloned and, surprisingly, were found to encode regulatory subunits of the proteasome complex. The cep31 gene remains to be identified and characterized. The results are discussed with respect to centromeric silencing and regulation of the metaphase-anaphase transition in fission yeast. MATERIALS AND METHODS Strains, media, transformation, and genetic techniques. All the strains used in this study are listed in Table 1. Media were essentially as described previously (2, 32). YEA refers to yeast extract medium (YE) supplemented with adenine; N/S refers to pombe minimum glutamate medium PMG containing adenine, uracil, histidine, and leucine; FOA is N/S but with the addition of FOA (5-fluoro-orotic acid) at 1 g/liter and URA2 is N/S medium without uracil. For transformation, a simplified version of the lithium acetate procedure was used (32). Standard genetic techniques were used (32). Comparative plating and serial dilution experiments were performed as described previously (2). Ch16 loss was measured by the half-sectoring assay method as previously described (3) with YE plates supplemented with a limiting amount (12 mg/liter) of adenine. Isolation of cep mutants. Strain FY312 was mutagenized with 2% ethyl methanesulfonate as described previously (32). Following ethyl methanesulfonate

TABLE 1. S. pombe strains used in this study Strain

FY143 FY90 FY367 FY312 FY923 FY925 FY980 FY929 FY982 FY936 FY988 FY3451 FY3453 FY3455 FY524 FY3445 FY3447 FY3449 FY947 FY1007 FY1008 FY1009 FY1011 FY1012 FY1013 FY1015 FY1016 FY1521 FY1644 FY977 FY1123 FY1124 FY1125 FY1126 FY1127 FY1128 828 830 832

Genotype 2

h h1 leu1-32 ade6-210 h1 leu1-32 ade6-210 ura4-D18 h1 leu1-32 ade6-210 ura4-D18 TM1(NcoI)::ura41 h1 cep1-1 leu1-32 ade6-210 ura4-D18 TM1(NcoI)::ura41 h1 cep2-10 leu1-32 ade6-210 ura4-D18 TM1(NcoI)::ura41 h1 cep2-11 leu1-32 ade6-210 ura4-D18 TM1(NcoI)::ura41 h1 cep2-12 leu1-32 ade6-210 ura4-D18 TM1(NcoI)::ura41 h1 cep2-13 leu1-32 ade6-210 ura4-D18 TM1(NcoI)::ura41 h1 cep3-16 leu1-32 ade6-210 ura4-D18 TM1(NcoI)::ura41 h1 leu1-32 ade6-210 ura4-DS/E otr1L(dh/BglII)::ura4 oriI h1 cep1-1 leu1-32 ade6-210 ura4-DS/E otr1L(dh/BglII)::ura41 oriI h1 cep2-10 leu1-32 ade6-210 ura4-DS/E otr1L(dh/BglII)::ura41 oriI h1 cep3-16 leu1-32 ade6-210 ura4-DS/E otr1L(dh/BglII)::ura41 oriI h1 leu1-32 ade6-210 ura4-DS/E imr1R(aeHindIII)::ura41 h1 cep1-1 leu1-32 ade6-210 ura4-DS/E imr1R(aeHindIII)::ura41 h1 cep2-10 leu1-32 ade6-210 ura4-DS/E imr1R(aeHindIII)::ura41 h1 cep3-16 leu1-32 ade6-210 ura4-DS/E imr1R(aeHindIII)::ura41 h1 leu1-32 ade6-210 ura4-DS/E h2 cep1-1 leu1-32 ade6-210 ura4-DS/E h1 cep1-1 leu1-32 ade6-210 ura4-DS/E h1 cep2-10 leu1-32 ade6-210 ura4-DS/E h2 cep2-11 leu1-32 ade6-210 ura4-DS/E h1 cep2-11 leu1-32 ade6-210 ura4-DS/E h1 cep2-12 leu1-32 ade6-210 ura4-DS/E h1 cep2-13 leu1-32 ade6-210 ura4-DS/E h1 cep3-16 leu1-32 ade6-210 ura4-DS/E h1 cep1-HA-ura41 leu1-32 ade6-210 ura4-D18 h1 cep1cs-HA-ura41 leu1-32 ade6-210 ura4-DS/E h2 leu1-32 ade6-210 ura4-DS/E Ch16-216 h2 cep1-1 leu1-32 ade6-210 ura4-DS/E Ch16-216 h2 cep2-10 leu1-32 ade6-210 ura4-DS/E Ch16-216 h2 cep2-11 leu1-32 ade6-210 ura4-DS/E Ch16-216 h2 cep2-12 leu1-32 ade6-210 ura4-DS/E Ch16-216 h2 cep2-13 leu1-32 ade6-210 ura4-DS/E Ch16-216 h2 cep3-16 leu1-32 ade6-210 ura4-DS/E Ch16-216 h1 mts2-1 leu1-32 ade6-210 Ch16-216 h1 mts3-1 leu1-32 ade6-210 Ch16-216 h1 pad1-1 leu1-32 ade6-210 Ch16-216

treatment, cells were washed three times with 0.9% NaCl, resuspended in YEA at a density of 4 3 106 cells/ml, and allowed to grow at 32°C for 21 h, after which time the cell density was around 4 3 107 cells/ml. Cells were plated onto FOA plates at a density of 105 and 106 cells/plate and incubated for 4 days at 35°C. From a total of 107 cells plated, approximately 1,000 FOA-resistant colonies formed. To screen for cryosensitivity (cs), the 1,000 colonies were grown at 35°C for 3 days and then replica plated onto two N/S plates containing phloxin B and incubated at 18 and 32°C for 4 days. Six strains grew very poorly and stained dark pink at 18°C. The six mutant strains were backcrossed three times. For all mutants, tetrad analysis showed that FOA resistance and cs cosegregated and segregated 2:2 with wild type. To exclude marker-specific effects and the possibility that FOA resistance of the cep mutants was due to general drug resistance, repression of an ade61 marker gene inserted in the central core of centromere 1 (TM1NcoI::ade61) was also assayed. The cep1-1, cep2-10, and cep3-16 mutations resulted in red as opposed to pink wild-type colonies when plated on appropriate indicator plates (YE plus 1/10 adenine [data not shown]). On these plates, white colonies fully express ade61 whereas pink and red colonies indicate intermediate and more complete states of repression, respectively (2).

VOL. 19, 1999

CENTROMERIC SILENCING AND CENTROMERE FUNCTION

Cytological techniques. Immunostaining and fluorescence in situ hybridization (FISH) were performed as described previously (10). Antihemagglutinin (antiHA) monoclonal antibody 12CA5 (Babco) was used at a 1/100 dilution. Polyclonal anti-HA (Babco) was cleaned up by preabsorption three times against total S. pombe proteins from a strain lacking the HA epitope immobilized on a membrane filter by Western blotting. The anti-a-tubulin monoclonal antibody TAT1 (51) was a gift from Keith Gull. A Zeiss Axioplan fluorescence microscope coupled to a Photometrics camera and IP Lab Spectrum software with Digital Scientific extensions were used for the capture and analysis of images. Western blotting. Total proteins were extracted in radioimmunoprecipitation assay buffer as described previously (32). Samples containing 50 mg of total protein were electrophoresed on duplicate sodium dodecyl sulfate–10% polyacrylamide gels. One gel was Coomassie blue stained to check for equal loading, and the other was electroblotted onto a polyvinylidene difluoride membrane (Bio-Rad). The filter was incubated with a 1:1,000 dilution of the anti-HA monoclonal antibody 16B12 (Babco). Bound antibody was detected with alkaline phosphatase-conjugated sheep anti-mouse immunoglobulin G with 5-bromo-4chloro-3-indolyl phosphate–nitroblue tetrazolium as a substrate. Plasmids. The S. pombe cDNA library was provided by C. J. Norbury and B. Edgar (Imperial Cancer Research Fund, Cell Cycle Group, Oxford, United Kingdom). In this library, cDNAs are directionally cloned into pREP3X, an S. pombe vector in which expression is under the control of the thiamine-repressible nmt1 promoter (29). A fission yeast genomic library in pUR19 with the S. pombe ura41 gene as the selection marker was provided by A. M. Carr (Medical Research Council Cell Mutation Unit, Brighton, United Kingdom). Isolation of the cep11 and cep21 genes. The cep11 and cep21 genes were isolated by complementation of the cryosensitive phenotype of the mutant strains. Strain FY1008 (cep1-1) was transformed with the S. pombe cDNA library. Transformants were selected on minimal medium without thiamine at 32°C, and colonies were replica plated onto the same medium and incubated at 18°C for 7 days. Two colonies formed at 18°C. Plasmid DNA was recovered in Escherichia coli DH5a by the extraction procedure previously described (5). Sequence data from the ends of the inserts showed that both plasmids contained the same cDNA. Database searches with the BLAST program (4) showed that the cep11-complementing cDNA was derived from the previously cloned pad11 gene (41). The complementing plasmid (pREPcep11) linearized within cDNA sequences at the single NruI site was integrated into FY947 (cep11). Integration by homologous recombination was checked by Southern blotting of genomic DNA extracted from the transformants (data not shown). Genetic analysis of crosses between integrant strains and FY1007 (cep1-1) showed that the LEU2 marker from the plasmid was tightly linked (,1 centimorgan [cM]) to the cep1 locus. The cep21 gene was cloned by complementation of the cs phenotype of FY1013 with a genomic library. All complementing plasmids were found to contain the same genomic region. Plasmid pUR19cep21 contained the smallest DNA insert. The plasmid linearized within the 5-kb insert at the single NcoI site was transformed into FY947 (cep21). Integration by homologous recombination was checked by Southern blotting of genomic DNA extracted from the transformants. Genetic analysis of crosses between integrant strains and FY1011 (cep211) showed that the ura41 marker from the plasmid was tightly linked to the cep2 locus. Several attempts to isolate the cep31 gene as described above by complementation of the cryosensitive phenotype failed; the cep31 gene remains to be characterized. A PCR-based strategy was used to identify mutations within the cep2 coding sequence. Two colonies from the four cep2 mutants and a wild-type control strain were picked and subjected to PCR with the following primer pair: C699 (top strand, in the 20-bp intron starting after A of the initiating codon, ATG) and D858 (positions 1297 to 1279). PCR products were sequenced on both strands with primers C699 (as described above), D757 (positions 243 to 262), D790 (positions 613 to 633), K61 (positions 938 to 957), D858 (positions 1297 to 1279), C822 (positions 1137 to 1127), C657 (positions 711 to 690), and J345 (positions 334 to 311). Samples were run on an Applied Biosystems sequencer, and sequences were compared with the wild type. Generation of HA-tagged cep1 alleles. The cep1 locus of strain FY1521 carries a cep11 allele extended with three copies of the HA sequence. The construction of FY1521 is described elsewhere (37). The same procedure was used to tag the cep1-1 allele. Plasmid pUC19ura4-cep1-3HA (37) was linearized at the single SmaI site lying within cep1 sequences and integrated into FY1008 (cep1-1) to give FY1644 (cep1cs-HA).

RESULTS Isolation of enhancer-of-centromere-position-effect mutants. The overall organization of DNA sequences within fission yeast centromere 1 (cen1) is shown in Fig. 1A. As previously demonstrated, insertion of the marker gene ura41 within centromeric sequences results in repression of ura41 gene expression (2, 3). This can be visualized by assaying the ability of the marked strains to grow on selective (URA2) and counterselective (FOA) plates (see Fig. 3). Whatever the site of ura41

5157

FIG. 1. Strategy utilized to screen for mutants which enhance transcriptional repression within the central domain of S. pombe centromeres. (A) Schematic representation of S. pombe cen1. (B) Screening procedure. Details are given in the text. wt, wild type; EMS, ethyl methanesulfonate.

insertion within cen1, the reporter gene is always transcriptionally repressed. However, repression is never complete, since all integrant strains eventually form colonies on medium lacking uracil. The level of repression is dependent on the site of insertion, therefore defining domains within the centromere (3). For example, ura41 is more tightly repressed when inserted within the otr and imr repeats than when inserted within the central core cnt1. In addition, expression of ura41 from within the central core is temperature sensitive: increased temperature allows more growth on URA2 selective medium, but growth on FOA is inhibited. These growth characteristics are indicative of increased expression from the central core at higher temperatures. This property was exploited to perform a screen for mutations which enhance transcriptional repression (silencing) within the central core of fission yeast centromeres. The different steps of the screen are depicted in Fig. 1B. The strain FY312 (2) carries ura41 within cnt1 [TM1(NcoI):: ura41]. FY312 is able to form colonies on FOA plates at 25°C, whereas this ability is lost at 35°C. To screen for enhanced repression, mutants conferring FOA resistance (FOAr) at 35°C were isolated. From approximately 107 cells plated, about 1,000 colonies formed on FOA plates at 35°C. Colonies were picked and further screened for cs for growth at 18°C. Six mutants were obtained. Mutations affecting spindle integrity or centromere function might be expected to be sensitive to microtubule-destabilizing drugs such as methyl benzimidazol-2-yl carbamylate (MBC); therefore, subsequent to isolation all cep mutants were tested for the ability to grow in the presence of MBC. Surprisingly, the six cep mutant strains were found to be resistant to MBC (Fig. 2). Dissection of tetrads resulting from

5158

JAVERZAT ET AL.

FIG. 2. Cold sensitivity and MBC resistance of cep mutants. Cells grown at 32°C were harvested, and about 103 cells were spotted onto complete medium (YEA) with or without MBC (20 mg/liter). Incubation times were 7 days at 18°C and 3 days at 32°C. WT, wild type.

crosses between mutants and FY312 showed cosegregation of MBCr and FOAr with the cs phenotype and 2:2 segregation versus wild type, thus demonstrating that, for all six mutants, a single mutation was responsible for all phenotypes. All mutations were found to be recessive to wild-type alleles in heterozygous diploids with respect to the cs phenotype. By the same criteria, complementation groups were made by analysis of diploid strains carrying pairwise combinations of cep mutations. Three complementation groups were found, defining three loci: cep1 (one mutant allele, cep1-1), cep2 (four mutant alleles, cep2-10 to cep2-13), and cep3 (one mutant allele, cep316). The serial dilution growth assay shown in Fig. 3 shows the effect of cep mutations on the repression exerted in the central domain of cen1. In a wild-type background, repression of ura41 within cnt1 allows growth on FOA at 32°C (Fig. 3, top panel, FY312). However, this ability is lost when cells are incubated at 36°C, showing that repression of ura41 expression within the centromere is alleviated by increased temperatures (Fig. 3, middle panel, FY312). In contrast, all cep mutants can form colonies on FOA at 36°C. Consistent with enhancement of the repression within the centromere, the mutant colonies formed on URA2 plates at 36°C were smaller than wild-type colonies formed on the same plate, whereas no difference could be seen on the N/S plate (Fig. 3, middle panel). In the presence of a fully expressed ura41 gene, all cep mutants were as FOA sensitive as were wild-type cells (data not shown). In addition, the cep mutations were also found to enhance the repression of the ade61 gene from within cnt1, giving rise to red rather than pink colonies (data not shown; see Materials and Methods). Therefore, FOA resistance at 36°C does not result from a general drug resistance phenotype but is indeed a consequence of reduced ura41 gene expression from within cen1 central core. Growth of wild-type strains with ura41 inserted at the distal end of otr1 (FY988) or between the Ala and Gln tRNA genes of imr1 (FY524) on FOA is also inhibited at 36°C in comparison to 32°C (Fig. 3, bottom panel) (3). This presumably reflects looser repression at these sites. The cep1-1, cep2-10, and cep3-16 mutations were also found to enhance repression of these ura41 insertions as demonstrated by increased growth on FOA at 36°C (Fig. 3, bottom panel). This strengthens further the link between centromeric silencing and these cep mutants. The effects of cep mutations are not centromere 1 specific since all six mutations also showed the same

MOL. CELL. BIOL.

effect on the expression of ura41 from the central domain of cen3 (data not shown). From the above results, we concluded that cep mutants are enhancers of transcriptional repression within fission yeast centromeres. Mutations in the cep genes alter the fidelity of chromosome transmission. We have previously shown that clr4, rik1, and swi6 mutants affect centromeric silencing and the fidelity of transmission of chromosomes, pointing out the link between centromeric silencing and centromere function (3, 9, 10). As cep mutations modify centromeric silencing, we asked whether chromosome stability was also affected. The rate of chromosome loss was estimated by scoring the mitotic loss rate of the 530-kb Ch16 linear minichromosome (28) at 32 and 36°C (Table 2) by the half-sectoring assay method (3). In wild-type cells, the Ch16 loss rate is less than 0.1% of cell divisions (Table 2) (3, 35). As shown in Table 2, the minichromosome was lost at an elevated rate in all cep mutants. The most dramatic increase in loss rate is seen in cep1 and cep3 mutants, where the loss rate per division is up to 80- to 100-fold higher than that of wild type; a similar elevated rate was observed for rik1, clr4, and swi6 mutants (3). For cep2 mutants, the loss rate is moderate but still significantly higher than that in wild type (5- to 15fold increase). As previously observed for suppressors of centromeric silencing, enhancers of centromeric repression within the fission yeast centromere also cause an elevated rate of chromosome loss, suggesting a role for cep genes in centromere function. Mutations in all three cep genes cause defective mitosis at restrictive temperature. The cep mutants lose chromosomes at elevated rates at the permissive temperature and are cs for growth. It is possible that the cs phenotype results from a failure in centromere function during mitosis. To investigate this possibility, we performed cytological analysis of mutant cells upon shift to the restrictive temperature. Cells were grown to early log phase at the permissive temperature (32°C) and shifted to 18°C. At a 1.5-h interval, samples were removed for determination of survival over time and cytological analysis. 49,6-Diamidino-2-phenylindole (DAPI) and antitubulin antibody staining was used to visualize nuclear chromatin and microtubules, respectively. Approximately 200 to 300 cells were observed for each time point. Based on the absence or presence of the mitotic spindle, cells were classified to be in interphase, metaphase and early anaphase (spindle , 5 mm) or late anaphase (spindle . 5 mm). When shifted from 32 to 18°C, wild-type and mutant cells nearly stopped dividing, as shown by the very small proportion of metaphase cells (Fig. 4A), but after 4.5 h, the population was composed of about 12 to 16% metaphase cells (Fig. 4A and D, 4.5 h). Therefore, the shift from 32 to 18°C appeared to synchronize cells to some extent. For wild type, the fraction of metaphase cells decreased after 4.5 h but remained high until 6 h for all three mutants, indicating that mutant cells spent more time in metaphase than did wild type. For all mutant strains, this wave of mitosis was characterized by the presence of abnormal late anaphases (Fig. 4D, 6 h) in which chromosomes fail to segregate properly to the spindle poles. Lagging chromosomes were not observed in wild-type cells or mutant cells at the permissive temperature (data not shown). After 6 h at 18°C, the frequency of defective anaphases in cep mutants was very high, averaging 52, 46, and 58% of late-anaphase cells for cep1, cep2, and cep3 mutants, respectively. At later time points in the experiment, telophase cells with lagging chromosomes were seen, and even septated cells with micronuclei which presumably arose from the failure of a chromosome to reach the pole before the exit from mitosis (Fig. 4D, 9 h). Twelve hours after the shift, mitotic cells be-

VOL. 19, 1999


5159

FIG. 3. (Top and middle) Mutations in cep1, cep2, and cep3 genes enhance repression of the ura4 gene when placed within the central domain of cen1. (Bottom) cep mutations also enhance silencing within the outer (otr) and inner (imr) repeats of centromere 1. Cells grown at 32°C in N/S medium were serially diluted (one-fifth); spotted onto URA2, FOA, and N/S plates; and incubated at 32 or 36°C. About 2 3 104 cells were plated in the highest-density spots. Strains and location of the ura4 gene are as indicated. wt, wild type.

came very rare for cep1-1 and cep2-12 mutants, and the population was mainly composed of septated and interphase cells (Fig. 4D, 12 h). At a later time point, 24 h, it was confirmed that cep1-1 and cep2-12 were arrested as interphase-like cells (data not shown). In contrast, cep3-16 cells attempted to go through a second round of mitosis but were arrested with a

short, deformed spindle. These cells first appeared 12 h after the shift (Fig. 4D, 12 h) and represented 38.5% of the cell population after 24 h. For all mutant strains, the cell number increased moderately after the shift (Fig. 4B), and after 12 h, cells virtually stopped dividing. The cell number increased about 1.8-fold for cep1-1

TABLE 2. Effects of cep mutations on the fidelity of transmission of the Ch16 linear minichromosome at 32 and 36°C 32°C

36°C

Strain

Background

Chromosome loss per division (%)a

Fold increase in loss rate



FY977 FY1123 FY1124 FY1125 FY1126 FY1127 FY1128

Wild type cep1-1 cep2-10 cep2-11 cep2-12 cep2-13 cep3-16

0.05 (2/4,078) 5.94 (268/4,510) 0.33 (8/2,436) 0.56 (13/2,329) 0.32 (8/2,503) 0.77 (19/2,463) 4.18 (234/5,599)

1 119 7 11 6 15 84

0.08 (2/2,516) 1.18 (21/1,774) NDb NDb 0.82 (9/1,093) 0.47 (3/642) 6.3 (12/191)

1 15 NDb NDb 10 6 80

a The rate of minichromosome loss was determined by the half-sectoring assay as described previously (3). The total number of colonies examined for each strain is given in parentheses. b ND, not determined.

5160

JAVERZAT ET AL.

MOL. CELL. BIOL.

VOL. 19, 1999


TABLE 3. Frequency of abnormal anaphase cells with unseparated chromosome III sister chromatids Background

No. of abnormal anaphase cellsa with a single FISH signalb

No. of abnormal anaphase cellsa with two FISH signalsb

Frequency of chromosome III nondisjunction (%)

cep1-1 cep2-12 cep3-16

21 10 22

4 10 7

84 50 76

a Anaphase cells (spindle length . 5 mm) with DAPI-stained material in the spindle midzone. b Chromosome III was visualized with an rDNA FISH probe.

and cep2-12 and 2.2-fold for cep3-16, suggesting that mutant cells could complete only a single round of mitosis and cytokinesis at the restrictive temperature. Cell viability decreased with time for all mutants (Fig. 4C). As shown by the cytological analysis (Fig. 4D), cell death is likely to be the consequence of chromosome missegregation events. For the cep3-16 mutant, at an additional time point, 24 h, it was revealed that survival was close to zero, showing that the metaphase-like arrest seen for cep3-16 cells is an irreversible event. Further cytological analysis of the phenotypes of cep1, cep2, and cep3 mutants was performed by examining the positions of centromeres by FISH. Cells were refixed and hybridized with a probe detecting all three centromeres. As seen in Fig. 4E, centromeres lag on late-anaphase spindle in all three mutants. The behavior of centromeres was found to be very variable from one cell to another; in some cells, more than three spots of fluorescence could be detected on the spindle, suggesting that sister chromatids separated correctly but were deficient in their poleward movement. However, in many cells, centromeric DNA remained clustered either in the middle of the spindle or at only one pole, suggesting a defect in sister chromatid disjunction and/or centromere function (Fig. 4E). The abnormal DAPI staining of cells shown in Fig. 4D (6 h) is also consistent with a failure in sister chromatid separation. To further investigate this point, we asked whether sister chromatids of a given chromosome were separated in anaphase cells with lagging chromosomes. Tubulin-stained cells from the 6-h point were hybridized with a ribosomal DNA (rDNA) probe which specifically decorates chromosome III. As shown in Fig. 4F and Table 3, a single FISH signal was often detected in anaphase cells with lagging chromosomes, indicating that chromosome III sister chromatids have failed to separate even though these cells have progressed into mid- to late anaphase. The above-described cytological analyses show that cep mutants at the restrictive temperature experience a defective mitosis in which chromosomes fail to separate properly at anaphase while other mitotic events such as spindle elongation and mitotic exit occur normally. FISH analyses revealed that

5161

sister chromatids of aberrantly segregating chromosomes were often not separated. This suggests that cep mutations cause an inefficient, late, or delayed disjunction of sister chromatids, which prevents chromosomes from moving toward the poles at anaphase. The cep1 and cep2 genes encode regulatory subunits of the proteasome. The cep11 and cep21 alleles were cloned by complementation of the cs phenotype with a cDNA library and a genomic library, respectively. Strains in which the complementing plasmid was integrated by homologous recombination within insert sequences were made. Genetic analyses showed that homologous recombination had occurred at cep1 and cep2 loci, thus demonstrating that cloned sequences contained the cep11 and cep21 genes. Partial sequence determination and database searches revealed that both complementing DNAs contained known S. pombe genes. The cep1 gene was found to be identical to pad11, an essential gene recently shown to encode a regulatory subunit of the proteasome (37, 41). Similarly, the 5-kb insert containing the cep21 allele was found to contain mts21, an essential gene which encodes another regulatory component of the proteasome (16). Point mutations within the mts2 coding sequence were found in all four cep2 mutant alleles, resulting in the following amino acid substitutions: D291N (cep2-10), G194D (cep2-11), G194D (cep2-12), and R353H (cep2-13). These data confirm that cep2 and mts2 define a single gene. Cellular localization of Cep1. The cep1 locus of strain FY1521 carries cep11-HA, a fully functional cep1 allele tagged with three copies of the HA epitope at the C terminus (37). The cs allele of cep1-1 was also tagged with the three-HA tag (cep1cs-HA, strain FY1644). As shown in Fig. 5, anti-HA antibodies readily detect a protein of the expected size (39 kDa) in extracts from FY1521 and FY1644, whereas no signal was detected in an untagged cep11 strain. The intensity of the signal appears lower in cep1cs-HA. A time course experiment after shifting to the restrictive temperature showed that the level of Cep1cs-HA remained constant with time, showing that the cs does not result from the loss of the protein (data not shown). In fixed FY1521 cells, staining with anti-HA antibodies resulted in the images shown as a montage in Fig. 6. In interphase cells, the Cep1-HA protein (in red) is found all around the nucleus (left panel). Triple labeling with anti-a-tubulin (green), anti-Cep1-HA (red), and DAPI (DNA, blue) allowed the visualization of Cep1-HA localization throughout mitosis (Fig. 6A). In early mitosis (metaphase), Cep1-HA remained around the nuclear periphery, while during anaphase, a bridge of Cep1-HA was seen to extend between the two separating chromosome masses. This pattern is consistent with Cep1-HA being located at or near the nuclear envelope throughout the entire cell cycle. Localization of defective Cep1cs-HA in FY1644 at 18°C was indistinguishable from that of wild-type

FIG. 4. Phenotypes of cep1, cep2, and cep3 mutants at the restrictive temperature. (A to C) Wild-type, cep1-1, cep2-12, and cep3-16 cells were grown to early log phase at 32°C and transferred at 18°C, and samples were taken for analysis every 1.5 h as described in the text. When shifted to the cold, cells from wild-type and cep mutants stopped dividing (A), but after 4.5 h, growth resumed, and a wave of mitosis was observed. Unlike the wild-type control, cep mutant cells divided only once at 18°C (B) and lost viability (C). (D) Cytological analysis by DAPI (red, pseudocolor) and antitubulin staining (green). Row 1 shows that early mitotic cells (spindle length , 5 mm) were first observed 4.5 h after transfer at 18°C. Rows 2 and 3 show that wild-type cells proceeded normally through mitosis (leftmost panels) but that all three cep mutants experienced defective chromosome segregation. After 6 h (row 2), about 50% of anaphase cells displayed lagging and/or aberrantly segregated chromosomes, and later (9 h [row 3]) abnormal telophase and septated cells were observed. After 12 h (row 4), cep1 and cep2 mutants eventually arrested as interphase-like cells while cep3-16 cells attempted a second mitosis but arrested as metaphase-like cells (rightmost panel). (E) Centromere FISH analysis of anaphase cells. The leftmost panel shows a wild-type cell with the centromeres (red signal) separated at both ends of the anaphase spindle (green). In most cases, the FISH signals in cep mutant cells were clustered at only one pole or in the midzone of the anaphase spindle, indicating a defect in sister chromatid separation. (F) rDNA FISH analysis of anaphase cells showing the absence of separation of chromosome III sister chromatids in cep mutants. Bars, 5 mm.

5162

JAVERZAT ET AL.

FIG. 5. Western blot detection of HA-tagged Cep1 and Cep1cs proteins. Equal amounts of total proteins from the indicated strains were loaded in the lanes. The blot was probed with monoclonal anti-HA antibody.

Cep1-HA (data not shown). Thus, the mutant phenotypes of cep1-1 do not result from gross mislocalization of Cep1. Chromosome segregation in other mutants with defective proteasome function. The mts2-1, mts3-1, and mts5-1 (pad1-1) temperature-sensitive (dead at 36°C) mutations in components of the proteasome have been described previously (16, 17, 37). To test if elevated rates of chromosome loss are a general property of defective proteasome function, the minichromosome Ch16 was crossed into mts2-1, mts3-1, and pad1-1 backgrounds, and the rate of minichromosome loss was assessed at the permissive temperature (25°C) and semipermissive temperature (30°C). As can be seen from Table 4, Ch16 stability is not affected at the permissive temperature and the loss rate remains low at 30°C even though colony growth was affected. All fission yeast proteasome mutants isolated confer MBC resistance at permissive temperatures (32°C for cep mutants and 25°C for mts mutants), indicating that the gene products are not fully functional even under permissive conditions. However, only cep alleles show elevated chromosome loss rates in these conditions. Thus, chromosome loss is not a general phenotype associated with defective proteasome function but appears to be specific to the pad1 (cep1-1) and mts2 (cep2) alleles isolated in this study.

MOL. CELL. BIOL.

property, MBC resistance, is also shared with fission yeast strains bearing mutations in genes encoding other components of the proteasome such as mts3 and mts4 (16, 17, 37, 49), and overexpression of the human pad1 homolog, POH1, results in similar phenotypes (43). The similarity in phenotypes of all the cep mutants and defects in other proteasome subunits strongly suggest that cep31 might encode another component of the proteasome. This hypothesis awaits the isolation of the cep31 gene. However, attempts to clone cep31 by the normal approach of complementing cep3-16 cs have so far failed, suggesting that multicopy cep31 could be deleterious to the cell. Mutations in 19S cap proteasome subunits impair chromosome segregation. The proteasome is known to degrade critical regulatory proteins involved in distinct cellular processes. In fission yeast, temperature-sensitive mutations in mts21, mts31, mts41, and pad11 genes cause cells to be transiently arrested at the metaphase stage of mitosis. At the restrictive temperature, cells accumulate with a short spindle and undivided nucleus. The spindle never elongates to the length of an anaphase spindle but rather is degraded. Septation occurs without nuclear division, forming an anucleate cell (16, 17, 37, 49). It has been shown that cells with defective Mts2 are unable to degrade ubiquitinated proteins, consistent with the idea that the metaphase arrest results from a failure to hydrolyze APC substrates (16). The cs alleles of mts21 and pad11 isolated in this study show somewhat different mitotic phenotypes. Unlike the heat-sensitive alleles, cep1-1 and cep2-12 cells did not accumulate with a short spindle but instead showed a rather normal spindle elongation process and went through anaphase, telophase, and cytokinesis and eventually arrested in interphase. However, the segregation of chromosomes was defective. In roughly half of anaphase cells, chromosomes were found to lag on the spindle, a situation which is rarely seen with wild-type cells. FISH analyses revealed that aberrantly segregating chromosomes had often failed to separate their sister chromatids even in mid- to late anaphase, indicating that cep mutants are defective in sister chromatid separation. Similarly, cells expressing nondegradable Cut2 block sister chromatid separa-

DISCUSSION Two cep genes encode structural components of the proteasome. In this study, we report the results of a screen for mutations which enhance silencing from within the central domain of fission yeast centromeres. The primary screen with silencing as a sole selection gave a large number of putative mutations. An additional screen for cold sensitivity reduced the number to six cep (centromere enhancer of position effect) mutations. They define three genes, two of which were cloned and identified by sequence analyses. The cep11 gene was found to be identical to pad11, encoding an essential regulatory subunit of the proteasome (37, 41). Similarly, cep21 is identical to mts21, encoding another essential component of the 19S regulatory cap of the 26S proteasome (16). In addition to the phenotypes described, the cep1, cep2, and cep3 mutants were also found to be resistant to the microtubule poison MBC. This

FIG. 6. Cellular localization of Cep1. Cells from strain FY1521 (cep11-HA) were grown to early log phase and then fixed and stained with anti-HA (red), antitubulin (green), and DAPI (blue). (A) Montage showing the localization of Cep1 through the cell cycle. For each cell, the corresponding panels are placed one below the other. From left to right, an example is shown of cells at various stages of the cell cycle from interphase to cytokinesis. (B) Anti-HA signal alone. Bar, 5 mm.

VOL. 19, 1999


5163

TABLE 4. Loss rates of the Ch16 minichromosome in proteasome mutants at permissive (25°C) and semipermissive (30°C) temperatures 25°C Strain

FY977 828 831 833

30°C

Background





Wild type mts2-1 mts3-1 pad1-1

0.07 (4/5,384) 0.07 (5/6,950) 0.08 (7/9,133) 0.09 (3/3,466)

1 1 1 1

0.05 (3/6,579) 0.17 (10/6,017) 0.27 (17/6,279) NDb

1 3 5 NDb

a The rate of minichromosome loss was determined by the half-sectoring assay as described previously (3). The total number of colonies examined for each strain is given in parentheses. b ND, not determined because the survival of pad1-1 cells was very low at this temperature.

tion, but importantly, spindle elongation is also inhibited (13, 14, 25). As spindle elongation is clearly not affected in cep1 and cep2 mutants, it seems likely that Cut2 proteolysis occurs but that another, unknown target (perhaps centromeric) is not degraded, leading to the chromatid separation defect observed in late anaphase (see below). Detection of changes in Cut2 protein levels is clearly difficult with mutants which are not completely defective in proteasome function and might just slightly alter Cut2 dynamics. This scenario implies that the defect in proteolysis is restricted to certain APC substrates while others would be correctly processed, indicating that 19S subunits confer a level of substrate specificity in the degradation pathway, as previously hypothesized by Rechsteiner et al. (39), Dubiel et al. (8), and McDonald and Byers (30). In budding yeast, different 19S cap subunits apparently localize to the nucleus (30). In mammalian cells, proteasomes are both cytoplasmic and nuclear with some cell cycle-specific changes. In particular, proteasomes were found to localize around chromosomes during mitosis (36). However, a recent study with green fluorescent protein-tagged proteasomes in living human fibrosarcoma cells indicates that proteasomes are primarily located in the cytoplasm and the nucleus, are excluded from the nucleoli, and have little association with the nuclear envelope or perinuclear region (40). This is in sharp contrast with data obtained for fission yeast and might reflect the fact that the nuclear envelope does not break down during fungal mitosis. The Cep1/Pad1-HA protein cofractionates with the 19S proteasome cap (37) and here was found to localize predominantly at or near the nuclear periphery throughout the cell cycle. The Mts2p (Cep2) 19S cap subunit also has a perinuclear localization and colocalizes with Cep1-HA (48). These are the first reports of proteasome localization in fission yeast. It is not known if every 26S proteasome in the cell actually contains Pad1 (Cep1) and Mts2 (Cep2) proteins, leaving the possibility that only a subpopulation of 26S proteasomes is detected with these reagents. The existence of distinct subpopulations of proteasomes was suggested by experiments showing that levels of 19S ATPases vary differentially during development (7). It is therefore possible that Pad1 (Cep1)- and Mts2 (Cep2)-containing proteasomes define a category of 26S complexes with specific mitotic functions. Immunoelectron microscopy has since indicated that Pad1/Cep1-HA is predominantly located at the inner face of the nuclear envelope (48). Such a localization allows proteasomes to be in close proximity to chromosomes and therefore might aid the efficient in situ degradation of APC substrates such as proteins involved in sister chromatid cohesion. Centromeric silencing and the ubiquitin-proteasome pathway. The mutants isolated in this study display different phenotypes depending on the temperature. At the restrictive tem-

perature, sister chromatid separation is defective. At the permissive temperature of 36°C, centromeric silencing is enhanced and the Ch16 minichromosome is lost at an elevated rate. As cep1 and cep2 mutations affect components of the proteasome, it seems reasonable to assume that the mutant phenotypes result from defective proteolysis. A key question is whether the cep1 and cep2 chromosome segregation and silencing phenotypes result from the altered degradation of a single protein or whether there are other protein targets which are not properly processed and cause the enhancement of silencing and elevated chromosome loss. In favor of there being a single target, several studies indicate that ubiquitination of certain chromatin proteins is required for efficient silencing and chromosome segregation. Loss of a deubiquitinating activity (Ubp3) which associates with the S. cerevisiae silencing protein Sir4 in vitro results in an increased level of repression of the silent HML, HMR, and telomeric genes (31). Similarly, reduced dosage of a gene encoding a putative Drosophila deubiquitinating enzyme also acts to enhance repression of a variegating gene embedded in centric heterochromatin (20). Conversely, mutation of the S. cerevisiae RAD6 gene, which encodes a ubiquitin-conjugating activity and can ubiquitinate histones H2A, H2B, and H3 in vitro, results in defective transcriptional silencing at HML and HMR and at telomeres (21). Recently, Singh et al. (42) have demonstrated that the putative S. pombe Rad6 ubiquitin-conjugating enzyme homolog, Rhp6, is required to maintain repression of the silent mat2 and mat3 loci in mating-type switchingcompetent cells. Again in Drosophila, mutation of the UbcD1 gene, encoding a putative ubiquitin-conjugating enzyme, leads to a high incidence of visible telomere-telomere associations during anaphase (6). Several of these alleles of UbcD1 also alleviate silencing phenotypes associated with centromeric and telomeric heterochromatin (14a). Therefore, ubiquitination of certain chromatin proteins appears to be required for silencing and chromosome segregation. It is not known, however, if the effect on silencing is mediated through the degradation of the ubiquitinated proteins or whether ubiquitin serves solely as a regulatory posttranslational modification of chromatin proteins without targeting them for proteolysis. The finding that proteasome mutants enhance centromeric repression implies that silencing might indeed be regulated by ubiquitinationdependent proteolysis, and it is clearly of interest to identify the putative centromere protein target(s), for example, through the isolation of suppressors of the centromeric silencing phenotypes of cep mutants. In this context, it is worth noting that the S. cerevisiae kinetochore component p58Ctf13 undergoes transient ubiquitination and proteasome-mediated degradation during kinetochore assembly (22). In a screen designed to isolate factors affecting centromere architecture with transcriptional silencing within fission yeast

5164

JAVERZAT ET AL.

centromeres as a primary assay, we have identified components of the proteasome as being effectors of centromere silencing. This strongly suggests that some component of silent centromeric chromatin is subject to regulation by proteolytic degradation. Further investigation will reveal how defective proteasome function leads to enhanced repression of marker genes residing within fission yeast centromeres. ACKNOWLEDGMENTS We thank A. Carr and C. Norbury for providing S. pombe libraries, K. Gull for the gift of TAT1 monoclonal antibody, and M. Yanagida for the gift of FISH probes and the Ch16 minichromosome. Thanks go to P. Perry, N. Davidson for the photographic work, and A. Pidoux for useful comments on the manuscript. J.-P.J. was supported by a Travelling Fellowship from The Wellcome Trust and an EC Human Capital and Mobility Award to R.A. Core support for this work was provided by the Medical Research Council of Great Britain to R.A. Work in Bordeaux, France, was supported by the Centre National de la Recherche Scientifique. P.B. was supported by a grant from the Ministe`re de la Recherche et de l’Enseignement Supe´rieur. REFERENCES 1. Aasland, R., and A. F. Stewart. 1995. The chromo shadow domain, a second chromo domain in heterochromatin-binding protein 1, HP1. Nucleic Acids Res. 23:3163–3173. 2. Allshire, R. C., J.-P. Javerzat, N. J. Redhead, and G. Cranston. 1994. Position effect variegation at fission yeast centromeres. Cell 76:157–169. 3. Allshire, R. C., E. R. Nimmo, K. Ekwall, J.-P. Javerzat, and G. Cranston. 1995. Mutations derepressing silent centromeric domains in fission yeast disrupt chromosome segregation. Genes Dev. 9:218–233. 4. Altshul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403–410. 5. Beach, D., M. Piper, and P. Nurse. 1982. Construction of a Schizosaccharomyces pombe gene bank in a yeast bacterial shuttle vector and its use to isolate genes by complementation. Mol. Gen. Genet. 187:326–329. 6. Cenci, G., R. B. Rawson, G. Belloni, D. H. Castrillon, M. Tudor, R. Petrucci, M. L. Goldberg, S. A. Wasserman, and M. Gatti. 1997. UbcD1, a Drosophila ubiquitin-conjugating enzyme required for proper telomere behavior. Genes Dev. 11:863–875. 7. Dawson, S. P., J. E. Arnold, N. J. Mayer, S. E. Reynolds, M. A. Billett, C. Gordon, L. Colleaux, P. M. Kloetzel, K. Tanaka, and R. J. Mayer. 1995. Developmental changes of the 26 S proteasome in abdominal intersegmental muscles of Manduca sexta during programmed cell death. J. Biol. Chem. 270:1850–1858. 8. Dubiel, W., K. Ferrell, and M. Rechsteiner. 1995. Subunits of the regulatory complex of the 26S protease. Mol. Biol. Rep. 21:27–34. 9. Ekwall, K., J.-P. Javerzat, A. Lorentz, H. Schmidt, G. Cranston, and R. Allshire. 1995. The chromodomain protein Swi6: a key component at fission yeast centromeres. Science 269:1429–1431. 10. Ekwall, K., E. R. Nimmo, J.-P. Javerzat, B. Borgstrom, R. Egel, G. Cranston, and R. Allshire. 1996. Mutations in the fission yeast silencing factors clr41 and rik11 disrupt the localisation of the chromo domain protein Swi6p and impair centromere function. J. Cell Sci. 109:2637–2648. 11. Ekwall, K., and T. Ruusala. 1994. Mutations in rik1, clr2, clr3 and clr4 genes asymmetrically derepress the silent mating-type loci in fission yeast. Genetics 136:53–64. 12. Funabiki, H., I. Hagan, S. Uzawa, and M. Yanagida. 1993. Cell cycle-dependent specific positioning and clustering of centromeres and telomeres in fission yeast. J. Cell Biol. 121:961–976. 13. Funabiki, H., H. Yamano, K. Kumada, K. Nagao, T. Hunt, and M. Yanagida. 1996. Cut2 proteolysis required for sister-chromatid separation in fission yeast. Nature 381:438–441. 14. Funabiki, H., H. Yamano, K. Nagao, H. Tanaka, H. Yasuda, T. Hunt, and M. Yanagida. 1997. Fission yeast Cut2 required for anaphase has two destruction boxes. EMBO J. 16:5977–5987. 14a.Gatti, M. Personal communication. 15. Glickman, M. H., D. M. Rubin, O. Coux, I. Wefes, G. Pfeifer, Z. Cjeka, W. Baumeister, V. A. Fried, and D. Finley. 1998. A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome and eIF3. Cell 94:615–623. 16. Gordon, C., G. McGurk, P. Dillon, C. Rosen, and N. D. Hastie. 1993. Defective mitosis due to a mutation in the gene for a fission yeast 26S protease subunit. Nature 366:355–357. 17. Gordon, C., G. McGurk, M. Wallace, and N. D. Hastie. 1996. A conditional lethal mutant in the fission yeast 26 S protease subunit mts31 is defective in

MOL. CELL. BIOL. metaphase to anaphase transition. J. Biol. Chem. 271:5704–5711. 18. Groll, M., L. Ditzel, J. Lowe, D. Stock, M. Bochtler, H. D. Bartunik, and R. Huber. 1997. Structure of 20S proteasome from yeast at 2.4 A resolution. Nature 386:463–471. 19. Hardwick, K. G. 1998. The spindle checkpoint. Trends Genet. 14:1–4. 20. Henchoz, S., F. De Rubertis, D. Pauli, and P. Spierer. 1996. The dose of a putative ubiquitin-specific protease affects position-effect variegation in Drosophila melanogaster. Mol. Cell. Biol. 16:5717–5725. 21. Huang, H., A. Kahana, D. E. Gottschling, L. Prakash, and S. W. Liebman. 1997. The ubiquitin-conjugating enzyme Rad6 (Ubc2) is required for silencing in Saccharomyces cerevisiae. Mol. Cell. Biol. 17:6693–6699. 22. Kaplan, K. B., A. A. Hyman, and P. K. Sorger. 1997. Regulating the yeast kinetochore by ubiquitin-dependent degradation and Skp1p-mediated phosphorylation. Cell 91:491–500. 23. Karpen, G. H., and R. C. Allshire. 1997. The case for epigenetic effects on centromere identity and function. Trends Genet. 13:489–496. 24. Kellum, R., and B. M. Alberts. 1995. Heterochromatin protein 1 is required for correct chromosome segregation in Drosophila embryos. J. Cell Sci. 108:1419–1431. 25. Kumada, K., T. Nakamura, K. Nagao, H. Funabiki, T. Nakagawa, and M. Yanagida. 1998. Cut1 is loaded onto the spindle by binding to Cut2 and promotes anaphase spindle movement upon Cut2 proteolysis. Curr. Biol. 8:633–641. 26. Lorentz, A., L. Heim, and H. Schmidt. 1992. The switching gene swi6 affects recombination and gene expression in the mating-type region of Schizosaccharomyces pombe. Mol. Gen. Genet. 233:436–442. 27. Lorentz, A., K. Ostermann, O. Fleck, and H. Schmidt. 1994. Switching gene swi6, involved in repression of silent mating-type loci in fission yeast, encodes a homologue of chromatin-associated proteins from Drosophila and mammals. Gene 143:139–143. 28. Matsumoto, T., S. Murakami, O. Niwa, and M. Yanagida. 1990. Construction and characterization of centric and acentric linear minichromosomes in fission yeast. Curr. Genet. 18:323–330. 29. Maundrell, K. 1990. nmt1 of fission yeast. J. Biol. Chem. 265:10857–10864. 30. McDonald, H. B., and B. Byers. 1997. A proteasome cap subunit required for spindle pole body duplication in yeast. J. Cell Biol. 137:539–553. 31. Moazed, D., and D. Johnson. 1996. A deubiquitinating enzyme interacts with SIR4 and regulates silencing in S. cerevisiae. Cell 86:667–677. 32. Moreno, S., A. Klar, and P. Nurse. 1991. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194:795–823. 33. Nakaseko, Y., Y. Adachi, S. Funahashi, O. Niwa, and M. Yanagida. 1986. Chromosome walking shows a highly homologous repetitive sequence present in all the centromere regions of fission yeast. EMBO J. 5:1011–1021. 34. Nimmo, E. R., G. Cranston, and R. C. Allshire. 1994. Telomere-associated chromosome breakage in fission yeast results in variegated expression of adjacent genes. EMBO J. 13:3801–3811. 35. Niwa, O., T. Matsumoto, Y. Chikashige, and M. Yanagida. 1989. Characterization of Schizosaccharomyces pombe minichromosome deletion derivatives and a functional allocation of their centromere. EMBO J. 8:3045–3052. 36. Palmer, A., G. G. Mason, J. M. Paramio, E. Knecht, and A. J. Rivett. 1994. Changes in proteasome localization during the cell cycle. Eur. J. Cell Biol. 64:163–175. 37. Penney, M., C. Wilkinson, M. Wallace, J. P. Javerzat, K. Ferrell, M. Seeger, W. Dubiel, S. McKay, R. Allshire, and C. Gordon. 1998. The pad11 gene encodes a subunit of the 26 S proteasome in fission yeast. J. Biol. Chem. 273:23938–23945. 38. Platero, J. S., T. Hartnett, and J. C. Eissenberg. 1995. Functional analysis of the chromo domain of HP1. EMBO J. 14:3977–3986. 39. Rechsteiner, M., L. Hoffman, and W. Dubiel. 1993. The multicatalytic and 26 S proteases. J. Biol. Chem. 268:6065–6068. 40. Reits, E. A. J., A. M. Benham, B. Plougastel, J. Neefjes, and J. Trowsdale. 1997. Dynamics of proteasome distribution in living cells. EMBO J. 16:6087– 6094. 41. Shimanuki, M., Y. Saka, M. Yanagida, and T. Toda. 1995. A novel essential fission yeast gene pad11 positively regulates pap1(1)-dependent transcription and is implicated in the maintenance of chromosome structure. J. Cell Sci. 108:569–579. 42. Singh, J., V. Goel, and A. J. Klar. 1998. A novel function of the DNA repair gene rhp6 in mating-type silencing by chromatin remodeling in fission yeast. Mol. Cell. Biol. 18:5511–5522. 43. Spataro, V., T. Toda, R. Craig, M. Seeger, W. Dubiel, A. L. Harris, and C. Norbury. 1997. Resistance to diverse drugs and ultraviolet light conferred by overexpression of a novel human 26 S proteasome subunit. J. Biol. Chem. 272:30470–30475. 44. Steiner, N. C., K. M. Hahnenberger, and L. Clarke. 1993. Centromeres of the fission yeast Schizosaccharomyces pombe are highly variable genetic loci. Mol. Cell. Biol. 13:4578–4587. 45. Takahashi, K., S. Murakami, Y. Chikashige, H. Funabiki, O. Niwa, and M. Yanagida. 1992. A low copy number central sequence with strict symmetry and unusual chromatin structure in fission yeast centromere. Mol. Biol. Cell 3:819–835. 46. Thon, G., A. Cohen, and A. J. Klar. 1994. Three additional linkage groups

VOL. 19, 1999


that repress transcription and meiotic recombination in the mating-type region of Schizosaccharomyces pombe. Genetics 138:29–38. 47. Thon, G., and A. J. Klar. 1992. The clr1 locus regulates the expression of the cryptic mating-type loci of fission yeast. Genetics 131:287–296. 48. Wilkinson, C. R., M. Wallace, M. Morphew, P. Perry, R. Allshire, J. P. Javerzat, J. R. McIntosh, and C. Gordon. 1998. Localization of the 26S proteasome during mitosis and meiosis in fission yeast. EMBO J. 17:6465– 6476. 49. Wilkinson, C. R., M. Wallace, M. Seeger, W. Dubiel, and C. Gordon. 1997.

5165

Mts4, a non-ATPase subunit of the 26 S protease in fission yeast, is essential for mitosis and interacts directly with the ATPase subunit Mts2. J. Biol. Chem. 272:25768–25777. 50. Wolf, D. A., and P. K. Jackson. 1998. Cell cycle: oiling the gears of anaphase. Curr. Biol. 8:636–639. 51. Woods, A., T. Sherwin, R. Sasse, T. H. MacRae, A. J. Baines, and K. Gull. 1989. Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies. J. Cell Sci. 93: 491–500.