INTRODUCTION

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Ubiquitin is a small (76-residue), abundant protein conserved in all eukaryotic cells. It exists in several cellular compartments, such as the cytosol, nucleus, and cell surface. It is well known that ubiquitin regulates the function and stability of target proteins through its posttranslational conjugation to target proteins. Before conjugation to target proteins, ubiquitin must be processed by a C-terminal hydrolase. The first step of the ubiquitin conjugation pathway is the ATP-dependent formation of a thioester bond between the conserved C-terminal glycine of processed ubiquitin and the active-site cysteine residue of an E1 ubiquitin-activating enzyme. The second step is the transfer of activated ubiquitin to the active-site cysteine of an E2 ubiquitin-conjugating enzyme. In the final step, the E2 enzyme may cooperate with an E3 ubiquitin protein ligase to form an isopeptide bond between the C-terminal glycine of ubiquitin and the -amino groups of lysine residues of target proteins. Ubiquitin covalently conjugated to target proteins can be removed by a ubiquitin isopeptidase (89).

Recently, a number of novel ubiquitin-like proteins were independently discovered in a number of species, suggesting that ubiquitin is part of a family of related proteins involved in the covalent modification of proteins. The first example of such a protein was the 15-kDa interferon-inducible, ubiquitin cross-reacting protein UCRP (25). UCRP contains two ubiquitin-related domains that are 43 and 62%, respectively, homologous to ubiquitin. It is conjugated to a number of unknown proteins and may serve as a trans-acting binding factor to direct the association of modified target proteins to intermediate filaments in the cytoplasm (25, 45). Other novel modification proteins such as Saccharomyces cerevisiae Rub1p and mammalian Nedd8 have also been reported (40, 44, 74). Rub1p and Nedd8 are also members of the ubiquitin-like protein family and display high homologies with ubiquitin, a probable functional homologue. Cdc53p/cullin, a subunit of the multifunctional SCF ubiquitin ligase, is a major substrate for Rub1p/Nedd8 conjugation (40, 74).

SUMO-1 was isolated as a protein covalently linked to RanGAP1, the Ran GTPase-activating protein of human cells (47). Modification by SUMO-1 targets the cytosolic RanGAP1 to the nuclear pore complex by promoting binding of the SUMO-1-modified RanGAP1 to RanBP2 (47, 48, 54, 75). In addition, it was suggested that the reversible modification of RanGAP1 by SUMO-1 had a regulatory role in the association of RanGAP1 with nuclear envelope (48). SUMO-1 was independently identified in two-hybrid screenings under names such as PIC1, which interacts with the PML component of nuclear multiprotein complex that is disrupted in acute promyelocytic leukemia (6), GMP1, which interacts with RanGAP1 (53), sentrin, which interacts with Fas/APO-1 or the tumor necrosis factor receptor 1 death domain (73), and UBL1, which interacts with the human Rad51/Rad52 proteins involved in DNA recombination and DNA double-strand break repair (81). There are at least two other proteins that are closely related to SUMO-1 (designated as SMT3C) (41), SMT3A (41) and SMT3B (10, 41, 49), in human cells, but there is only a single SUMO-1 homologue, Smt3p, in S. cerevisiae (33, 59). SMT3, an essential S. cerevisiae gene encoding a 11.5-kDa protein, was originally isolated as a multicopy suppressor of mutations in the MIF2 gene, which encodes a CENP-C-like centromere-binding protein (59). Smt3p is 48% identical to SUMO-1/PIC1/GMP1/sentrin/UBL1 and 17% identical to ubiquitin. Smt3p also becomes conjugated to several proteins posttranslationally (33). However, the precise function and target proteins of Smt3p remain to be determined.

The three-dimensional structure of SUMO-1 was determined (4). Although SUMO-1 has only 18% amino acid sequence identity with ubiquitin, its overall structure closely resembles that of ubiquitin. Lys48 of ubiquitin, required for the generation of ubiquitin polymers, is replaced by Gln at the corresponding position in SUMO-1 (Gln69). This explains why SUMO-1 has not been observed to form polymers (48). On the other hand, the positions of two C-terminal Gly residues required for isopeptide bond formation are conserved between ubiquitin and SUMO-1. The most prominent feature of SUMO-1 is its long and highly flexible N terminus, which protrudes from the core of the protein and which is absent in ubiquitin (4).

It has been proposed that several features of the ubiquitin pathway are conserved in the early steps of the Smt3p/SUMO-1 conjugation pathway (33). Like ubiquitin, Smt3p is proteolytically processed to expose its mature C terminus. Then, Smt3p is activated in an ATP-dependent manner by an activating enzyme made up of a heterodimer of Uba2p and Aos1p (33). Uba2p is a 71-kDa protein with high homology to the C-terminal regions of E1 ubiquitin-activating enzymes and displays conservation of the active-site cysteine residue participating in thioester formation. Aos1p is a 40-kDa protein with extensive similarity to the N-terminal regions of E1 ubiquitin-activating enzymes (33). A significant body of evidence indicates that the E2 enzyme for Smt3p/SUMO-1 conjugation is Ubc9p (78). A human Ubc9 homologue interacts with SUMO-1 in two-hybrid screening (80), and antibodies against the Xenopus laevis Ubc9 homologue can coimmunoprecipitate complexes containing SUMO-1-modified RanGAP1 (75). In addition, it was demonstrated that Ubc9p is the only E2-conjugating enzyme for Smt3p (32). UBC9 is an essential gene, and conditional ubc9^ts mutants arrest at the G₂/M of the cell cycle with concomitant accumulation of both B-type (78) and G₁ (5) cyclins. Recently, it was shown that modification of RanGAP1 by SUMO-1 requires the Ubc9 homologue in Xenopus eggs (76) and human cells (42). Although the Ubc9 E2-conjugating enzymes of Smt3p/SUMO-1 are related to those of ubiquitin, they do not share the same target proteins (32, 42). These results suggest that the two pathways are distinct. Recently, a protease specific for ubiquitin-like proteins, Ulp1p, which cleaves proteins modified by Smt3p and SUMO-1 but not by ubiquitin, was reported (43). Ulp1p-related proteins are conserved in many organisms. Ulp1p plays an essential role in the G₂/M phase of the cell cycle, indicating that Ulp1p-mediated Smt3p-protein deconjugation is required for cell cycle progression.

In this work, we describe the characterization of a Schizosaccharomyces pombe homologue of Smt3p/SUMO-1. We designated the gene encoding this S. pombe homologue pmt3⁺. Our results clearly showed that Pmt3p is required for a number of nuclear events including the control of telomere length and chromosome segregation.

	MATERIALS AND METHODS

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Fission yeast strains, media, and methods. Fission yeast strains were grown and used as described by Moreno et al. (62). All fission yeast strains used in this study are listed in Table 1. Standard genetic methods and staining with 4',6-diamidino-2-phenylindole (DAPI) were as described elsewhere (62). Cell number was determined with a Sysmex CDA-500 cell counter. Transformation of S. pombe was achieved by the lithium acetate (72) or the electroporation (35) method. Vegetative growth under nonselective conditions was in YPD (2% peptone, 1% yeast extract, 2% glucose) or YE (0.5% yeast extract, 3% glucose) medium, supplemented as required. The synthetic minimal medium used was EMM2 (60), supplemented as required. The thiamine-regulatable promoter nmt1 was repressed by adding 5 µg of thiamine per ml to EMM2 medium (55).

Cloning of a genomic fragment and the full-length cDNA of the pmt3⁺ gene. A genomic fragment of the pmt3⁺ gene was cloned by screening of an ordered S. pombe cosmid library (61) with the pmt3⁺ cDNA obtained by two-hybrid screening. The full-length cDNA of the pmt3⁺ gene was isolated by the screening of a ZapII S. pombe cDNA library (from about 5 × 10⁴ plaques) (34), using plaque hybridization with the pmt3⁺ cDNA obtained by two-hybrid screening as a probe. Sequencing was carried out on both strands by using an ABI PRISM dye terminator cycle sequencing ready reaction kit (Perkin-Elmer). Multiple alignments were carried out by using the CLUSTAL W program (86).

Construction of the pmt3 deletion strain. A 4.2-kb EcoRI genomic fragment containing the pmt3⁺ gene was subcloned from a pmt3⁺-containing cosmid after restriction mapping. The 1.8-kb ura4⁺ gene was inserted at the unique ApaLI site of a 4.2-kb pmt3⁺ genomic fragment which had been previously blunt ended and ligated to an XbaI linker. A 6.0-kb fragment carrying the pmt3::ura4 construct was transformed into diploid strain TP4-1D/TP4-5A (h⁺/h his2/+ leu1-32/leu1-32 ura4-D18/ura4-D18 ade6-M216/ade6-M210) (36). Stable transformants were isolated, and gene disruption was confirmed by Southern blot analysis. For analysis of the phenotype of haploids with a disruption of the pmt3⁺ gene, tetrad analyses were performed by standard methods (62).

Analysis of HU and UV responses. To test the response to hydroxyurea (HU), wild-type (HM123 [h leu1-32]), pmt3 (JN630 [h leu1-32 ura4-D18 pmt3::ura4]), cdc2-3w (JN632 [h leu1-32 cdc2-3w]), and cds1 (JN633 [h leu1-32 ura4-D18 cds1::ura4]) cells were grown to a mid-log phase (0.5 × 10⁷ to 1 × 10⁷ cells/ml) in YE medium at 30°C. HU was added to the culture to a final concentration of 12 mM at time zero. Portions were taken and plated on YE plates, and cell viability was determined (66).

Determination of minichromosome loss rates. Ch10 is a linear minichromosome that carries the sup3-5 tRNA gene, which suppresses the ade6-704 mutation (70). A colony color assay was performed to measure the stability of Ch10. S. pombe ade6-704 cells form red colonies. If the minichromosome Ch10 is stably maintained in the ade6-704 cells during colony formation, sup3-5 on Ch10 will suppress the ade6-704 mutation and the cells will form white Ade⁺ colonies. The rate of loss of Ch10 from wild-type (YM75 [h⁺ leu1-32 ura4-D18 ade6-704 Ch10]) and pmt3 (YM76 [h⁺ leu1-32 ura4-D18 pmt3::ura4 ade6-704 Ch10]) cells under normal conditions was calculated by two different methods (67). Each experiment was carried out at least four times.

Measurements of telomere length. S. pombe chromosome DNA was prepared by a glass bead-phenol extraction protocol (29). Chromosome DNA was digested with ApaI, resolved on a 1% agarose gel, and transferred onto a nylon membrane (GeneScreen Plus; NEN). The 0.3-kb ApaI-EcoRI fragment of pAMP2 (52), which contains the S. pombe telomeric repeat sequences, was used as a probe for telomeric repeat sequences in the Amersham ECL system.

Expression of Pmt3p and HA₃ tagging Pmt3p in S. pombe. The pmt3⁺ cDNA from the two-hybrid screen was digested with BamHI and XhoI and cloned into the BamHI-XhoI sites of pREP1 after inserting a XhoI linker into the SmaI site of the multicloning site downstream of the thiamine-regulatable nmt1 promoter (55). The resulting construct was termed pREP1x-pmt3. Tagging of Pmt3p with three copies of the hemagglutinin (HA) epitope was done as follows. The pmt3⁺ cDNA was amplified by PCR using oligonucleotide primers located at either end of the open reading frame, and the amplified DNA was digested with NotI and BamHI. Primers were 5'-TAGCGGCCGCATGTCTGAATCACCATCA-3' (a sense primer to create a NotI site at the start codon of pmt3⁺ cDNA; the NotI site is underlined) and 5'-CCGGATCCAAGGCATAGATGGGTGCA-3' (an antisense primer to create a BamHI site downstream of pmt3⁺ cDNA; the BamHI site is underlined). The NotI-BamHI fragment of pmt3⁺ cDNA was cloned into the NotI-BglII sites of the strongest nmt1 promoter (nmt) HA₃-tagging vector pSLF173 (23) after converting an ura4 marker to a LEU2 marker and into the weakest nmt1 promoter (nmt**) HA₃-tagging vector pSLF373 (23) after converting an ura4 marker to a LEU2 marker. The resulting constructs were termed pSLF173L-pmt3 and pSLF373L-pmt3, respectively.

Preparation of S. pombe cell lysates and Western blot analysis. Whole-cell extracts from S. pombe cells for Western analysis were prepared by the boiling sodium dodecyl sulfate (SDS)-glass bead method as described below (50). Ten milliliters of logarithmic-phase S. pombe cell cultures (about 5 × 10⁶/ml) was harvested. Pellets were washed twice with water, resuspended in 100 µl of water, and heated at 90°C for 5 min. Then 120 µl of 2× Laemmli buffer (4% SDS, 20% glycerol, 0.6 M -mercaptoethanol, 0.12 M Tris-HCl [pH 6.8]) containing 8 M urea was added to the samples, which were vigorously vortexed with an equal volume of acid-washed glass beads for 3 min and then heated again 90°C for 5 min. The whole-cell extracts were analyzed by SDS-polyacrylamide gel electrophoresis using 10% polyacrylamide gels and then transferred to Immobilon transfer membranes (Millipore) by using a wet-type transfer system. Anti-HA monoclonal antibody 12CA5 was purchased from BAbCo. Horseradish peroxidase-conjugated goat anti-mouse secondary antibody was purchased from Bio-Rad. Western blot detection was done with the ECL Plus system as described by the manufacturer (Amersham).

GFP tagging of Pmt3p. The plasmid for green fluorescent protein (GFP) tagging of Pmt3p was constructed as follows. For the GFP fusion at the N terminus of Pmt3p, GFP(S65A) (63) was amplified by PCR with a 5' primer and a 3' primer. Primers were 5'-CCGGATCCGGCGGCCGCATGAGTAAAGGAGAAGAA-3' [a sense primer to create a BamHI site before the start codon of GFP(S65A); the BamHI site is underlined] and 5'-GCGAATTCCTTTTGTATAGTTCATCCATGC-3' [an antisense primer to remove the stop codon of GFP(S65A) and to create an EcoRI site; the EcoRI site is underlined]. The PCR product of the GFP(S65A) fragment whose stop codon was removed was digested with BamHI and EcoRI (fragment 1). For preparation of the pmt3⁺ promoter region, the pmt3⁺ genomic DNA was amplified by PCR with a 5' primer and a 3' primer. Primers were 5'-GATATCTTGAATAACTTC-3' (a sense primer containing an EcoRV site, which is complementary to the 5'-terminal sequence shown in Fig. 1A; the EcoRV site is underlined) and 5'-GCGGATCCAGATACTATATAAAATC-3' (an antisense primer to create a BamHI site, which is complementary to the region [positions 25 to 9 in Fig. 1A]) upstream of the initiator ATG of pmt3⁺; the BamHI site is underlined). The PCR product of the pmt3⁺ promoter region was digested with EcoRV and BamHI (fragment 2). For preparation of the pmt3⁺ coding and 3' noncoding regions, the pmt3⁺ genomic DNA was amplified by PCR with a 5' primer and a 3' primer. Primers were 5'-GCGAATTCTGAATCACCATCAGC-3' (a sense primer to create an EcoRI site and to remove the initiating ATG codon of pmt3⁺, which is complementary to the region [positions +4 to +20 in Fig. 1A] just downstream of the initiator ATG of pmt3⁺; the EcoRI site is underlined) and 5'-AAGCTTCAAGAAAATTTAGC-3' (an antisense primer containing a HindIII site, which is complementary to the 3'-terminal sequence shown in Fig. 1A; the HindIII site is underlined). The PCR product of the pmt3⁺ coding and 3' noncoding regions was digested with EcoRI and HindIII (fragment 3). Fragments 1, 2, and 3 were cloned together into the EcoRV-HindIII site of pBluescript II KS+ (77) to construct a plasmid for GFP tagging of Pmt3p (pBS-GFPpmt3). The resulting plasmid, pBS-GFPpmt3, contained the native promoter of pmt3⁺ and the full pmt3⁺ coding region, whose N terminus was ligated in frame to the GFP gene. To integrate the GFP-tagged pmt3⁺ gene onto the genome, the EcoRV-HindIII fragment containing the pmt3⁺ promoter, the GFP-tagged pmt3⁺ coding region, and the 3' noncoding region of pBS-GFPpmt3 was isolated and cloned into the SmaI-HindIII site of an integration vector, pYC11 (13). The resulting plasmid, pYC11-GFPpmt3, was used for transformation of the wild-type strain (YM77 [h⁺ leu1-32 ura4-D18 ade6-704]), and stable Leu⁺ transformants (YM78 [h⁺ leu1-32 ura4-D18 ade6-704 pmt3⁺:pYC11- GFPpmt3]) were selected.

Fluorescence microscopy. Immunofluorescent staining and in situ hybridization of cells were done according to a protocol provided by Y. Chikashige (11), which is a modification of the protocols of Chikashige et al. (12) and Dernburg et al. (17). For GFP-Pmt3-expressing cells, cells were fixed with 3% formaldehyde at room temperature for 60 min. TAT1 mouse monoclonal anti--tubulin antibody (90) and anti-Sad1 rabbit polyclonal antibody (27) were used to stain microtubule and spindle pole bodies (SPB), respectively. Secondary antibodies used were goat anti-mouse immunoglobulin G (IgG) Cy3-conjugated antibody, goat anti-rabbit Cy3-conjugated antibody (Jackson Laboratory), goat anti-rabbit Oregon green-conjugated antibody (Molecular Probes), and donkey anti-mouse Cy5-conjugated antibody (Amersham). DNA was stained with DAPI (1 µg/ml). Microscopic images were obtained by using a Delta Vision system (Applied Precision) with an Olympus oil immersion objective lens (Dplan Apo 100/NA1.3). Several z-axis sections of 0.1-µm intervals were combined by the quick projection program to avoid overlooking any signals within a cell.

Nucleotide sequence accession number. The nucleotide sequence of pmt3⁺ has been deposited in the GenBank database under accession no. AB017187.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Identification of S. pombe Smt3p/SUMO-1 homologue. Two-hybrid screening of an S. pombe cDNA library was done with an accessory factor of DNA polymerase , PCNA, as bait. Two novel cDNAs were isolated, one of which was found to encode a homologue of the S. cerevisiae Smt3p after DNA sequencing. The SMT3 gene was originally isolated as a multicopy suppressor of the S. cerevisiae mif2^ts mutation (59) and was suggested to be a novel type ubiquitin-related factor. Therefore, we named this gene pmt3⁺ (S. pombe homologue of SMT3). Although we confirmed the in vivo interaction between PCNA and Pmt3p by immunoprecipitation (data not shown), we have found no genetic relationship between PCNA and Pmt3p.

View larger version (60K): [in this window] [in a new window]   FIG. 1.   (A) Nucleotide sequence of S. pombe pmt3+ gene and predicted amino acid sequence of Pmt3p. The predicted amino acid sequence is shown in single-letter code below the nucleotide sequence. Intron sequences are shown in lowercase letters. Intron consensus sequences (92) are underlined. An in-frame stop codon upstream of the putative initiator ATG is also underlined. (B) Sequence alignment of a novel ubiquitin-like SUMO-1 family. Multiple alignment between S. pombe Pmt3p, S. cerevisiae Smt3p, human SMT3A (41), SMT3B (49), SMT3C (SUMO-1) (47), and Caenorhabditis elegans SMT3 (14) was done with the CLUSTAL W algorithm (86). Asterisks under amino acids indicate exact matches between the six aligned sequences. Dashes within the coding sequence indicate spaces inserted to achieve an optimal match. A bracket indicates the conserved diglycine, the expected site of precursor cleavage. (C) Sequence alignment of ubiquitin and ubiquitin-like proteins. Multiple alignment between S. pombe Pmt3p, S. cerevisiae Rub1p (44), mouse Nedd8 (39), and S. cerevisiae ubiquitin was done with the CLUSTAL W algorithm (86). Asterisks under amino acids indicate exact matches between the four sequences. Dashes within the coding sequence indicate spaces inserted to achieve an optimal match. A bracket indicates the conserved diglycine. An arrow indicates the site of the Lys48 of ubiquitin required to generate ubiquitin polymer.

Gene disruption of the pmt3⁺ gene and phenotypes of pmt3 cells. Gene disruption of pmt3⁺ was carried out as described in Materials and Methods. Precise replacement of one of the two chromosomal copies of pmt3⁺ in an S. pombe diploid strain by the pmt3::ura4 fragment was confirmed by Southern blot analysis of genomic DNA (Fig. 2A and C). All dissected tetrad spores of the Ura⁺ diploid gave two Ura colonies and two tiny Ura⁺ colonies (Fig. 2B). Thus, the pmt3 cells were viable but grew more slowly than wild-type cells (the doubling time of pmt3 cells was about 5 h at 30°C in YE medium) (Fig. 2E). DAPI staining and microscopic analysis indicated that about 23% of pmt3 cells display aberrant cellular and nuclear morphologies (Fig. 2D); 6% were generally elongated (Fig. 2D-1), 4% showed a displaced nucleus (Fig. 2D-2 and 2D-3), and 2% showed enucleated daughter cells (Fig. 2D-4), indicating that cells had undergone cytokinesis without prior completion of mitosis. In addition, the chromatin of 4% of the cells was highly condensed (Fig. 2D-5 and 2D-6), indicating strong mitotic delay. Furthermore, 7% of the cells displayed the typical cut phenotype where cells remain connected by threads of chromatin (Fig. 2D-7 and 2D-8). These phenotypes are reminiscent of the constant-cut phenotypes of rad31⁺ and hus5⁺ mutants or disruptants (3, 21, 79) (see Discussion). Missegregation of the chromosomes was also confirmed by flow cytometric (fluorescence-activated cell sorting [FACS]) analysis. Wild-type cells showed the normal 2C DNA content peak. The profile of pmt3 cells showed a decrease of the number of cells with 2C DNA content and an increase in the number of cells with a DNA content greater or less than 2C DNA content (at times 2 and 0 h in Fig. 3C). This profile is consistent with the faulty chromosome segregation observed by DAPI staining.

View larger version (55K): [in this window] [in a new window]   FIG. 2.   Gene disruption of the pmt3+ gene. (A) Diagrammatic representation of disruption of the pmt3+ gene by one-step gene replacement. The ura4+ gene was used to disrupt the coding region of pmt3+ as indicated, and the resulting plasmid with the disrupted pmt3+ gene was used to transform the wild-type diploid strain. Hatched boxes indicate the three exons of the pmt3+ gene. (B) Tetrad analysis of a heterozygous diploid. A diploid which consisted of one copy of wild-type pmt3+ and its disrupted allele was manipulated and incubated on a YE plate supplemented with adenine at 30°C for 4 days. (C) Demonstration of pmt3 deletion by genomic Southern blot analysis. DNA was prepared from wild-type (+/+) and the heterozygous (+/) diploid strains, digested with EcoRI, separated by agarose gel electrophoresis, blotted onto a nylon membrane, and probed with the 4.2-kb EcoRI fragment containing the pmt3+ gene. The 4.2-kb band represents the pmt3+ locus; the 6.0-kb bands represent the pmt3::ura4 allele, in which the ura4+ gene was inserted into the ApaLI site of pmt3+. M, size markers. (D) DAPI staining of pmt3 cells at the permissive temperature (30°C). Several phenotypes in addition to size heterogeneity can be seen: 1, elongated cell; 2 and 3, displaced nuclei; 4, nucleate daughter cell; 5 and 6, highly condensed chromosomes; 7 and 8, typical cut abortive mitosis. (E) Various phenotypes of pmt3 cells. pmt3 cells are hypersensitive to high temperature, HU, and MMS. Wild-type (HM123) and pmt3 (JN630) cells were spotted onto YE and YE plates containing 5 mM HU or 0.0025% MMS and incubated for 4 days at 30 or 36°C.

View larger version (35K): [in this window] [in a new window]   FIG. 3.   UV and HU sensitivity of pmt3 cells. (A) UV sensitivity of pmt3 cells. Viabilities of wild-type (HM123) and pmt3 (JN630) cells after UV irradiation were measured. Cells in mid-log phase were plated and exposed to UV at a dose of 0 to 150 J/m2. Sample plates were incubated, and cell viabilities were determined. UV sensitivity of pmt3 cells could be completely restored by introducing the plasmid expressing pmt3+. (B) HU sensitivity of pmt3 cells. Viabilities of wild-type (HM123), pmt3 (JN630), cdc2-3w (JN632), and cds1 (JN633) cells after incubation for 0 to 8 h at 30°C in YE medium in the presence of 12 mM HU were determined. Cells were grown to mid-log phase in YE medium at 30°C. HU was added to the culture at a final concentration of 12 mM at time zero. Samples were withdrawn at the indicated times and plated on YE plate, and cell viability was determined. (C) DNA contents of wild-type (HM123) and pmt3 (JN630) cells after HU treatment described for panel B were analyzed by flow cytometry. At various time points, cells were fixed and stained by propidium iodide, and their DNA contents were determined by FACS analysis.

Requirement of Pmt3p for chromosome segregation. Since the DAPI staining and FACS profile described above suggested faulty segregation of chromosomes in pmt3 cells, we tested the sensitivity of pmt3 cells to the microtubule-destabilizing agent thiabendazole (TBZ). TBZ is known to bind tubulin molecules and to inhibit their polymerization. Increased sensitivity to microtubule-destabilizing benzimidazole compounds is a characteristic of defective microtubule components or microtubule-interacting proteins. A plate concentration of 10 µg of TBZ per ml severely inhibited the growth of the pmt3 cells but had little effect on the growth of wild-type cells (Fig. 4A). This result also suggested that pmt3⁺ may be involved in assembly of the microtubule system.

View larger version (52K): [in this window] [in a new window]   FIG. 4.   (A) TBZ sensitivity of pmt3 cells. Wild-type (HM123) and pmt3 (JN630) cells were streaked on YE and YE plates containing TBZ (10 µg/ml) and incubated for 4 days at 30°C. (B) Frequency of minichromosome loss in wild-type and pmt3 cells. The genomic pmt3+ gene was disrupted in a strain carrying the minichromosome. Single colonies of isogenic wild-type (YM75) and pmt3 (YM76) cells were grown at 30°C. See Materials and Methods for an explanation of minichromosome loss rate.

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Striking increase in telomere length in pmt3 cells. We found another interesting phenotype in pmt3 cells, that loss of Pmt3p function caused a striking increase in telomere length. S. pombe chromosomes I and II contain ApaI restriction sites which are located on the centromere-proximal side of the telomeric repeats (84). When the chromosome DNA of wild-type cells was digested with ApaI and analyzed for telomere length by Southern blot analysis with a telomeric repeat DNA fragment as the probe, a band smear of about 0.3 kb, which corresponds to the length of the telomeric repeat in wild-type cells, was detected (Fig. 5, lane 1). As shown in lane 2, disruption of the pmt3⁺ gene caused elongation of telomeres up to a length intermediate (0.8 to 1.2 kb) between that of the wild-type and taz1 cells (15), although the signal intensities were not quantitatively estimated in this experiment.

View larger version (49K): [in this window] [in a new window]   FIG. 5.   Southern blot analysis of telomere length in wild-type and pmt3 cells. Genomic DNA was extracted, digested with ApaI, and separated on 1% agarose gels. DNAs were blotted onto a nylon membrane and probed with the 0.3-kb EcoRI-ApaI fragment of pAMP2. Positions of DNA size markers and their sizes are given on the left. Brackets indicate the positions of wild-type telomeres and the heterogeneous telomeres of pmt3 cells. I, the point where expression of the pmt3+ gene was induced; R, the point where expression of the pmt3+ gene was rerepressed. Lane M, BstEII-digested bacteriophage lambda DNA; lane 1, wild-type (HM123); lane 2, pmt3 (JN630); lanes 3 to 10, pmt3 cells transformed with pSFL373L-pmt3 carrying pmt3+ cDNA under control of the nmt** promoter. Time courses of the restoration of wild-type telomere length after induction of pmt3+ gene expression and telomere elongation after repression of pmt3+ gene expression are shown in lanes 3 to 10. DNA was isolated before induction (0 generation; lane 3) and 18 (lane 4), 36 generations (lane 5), 54 (lane 6), 72 (lane 7), and 90 generations (lane 8) after induction. pmt3+ gene expression was repressed again at point R. Lanes 9 and 10, 18 and 36 generations after repression. The lower panel indicates expression levels of Pmt3p detected by Western blot analysis using monoclonal antibody 12CA5.

Pmt3p-protein conjugation in S. pombe. The results mentioned above suggest that Pmt3p is required in multiple cellular events. We speculated that Pmt3p exerts its function through its covalent attachment to target proteins like its homologues SUMO-1 and Smt3p. To confirm that S. pombe Pmt3p is conjugated to cellular proteins in vivo, Pmt3p was amino-terminally tagged with three copies of the HA epitope. The gene encoding this fusion protein was placed under the control of the inducible nmt1 promoter (pSLF173L-pmt3). Western blot analysis using an anti-HA antibody revealed the expression in pmt3 cells of HA₃-Pmt3p in the presence of thiamine. Migration of the HA₃-tagged Pmt3p was far slower than that predicted from the sequence. This is consistent with the previous reports that both mammalian SUMO-1 and S. cerevisiae Smt3p show anomalously slow migration on SDS-gels (6, 33, 48). In addition, several other anti-HA-reactive proteins with sizes ranging from about 40 to more than 175 kDa were observed as distinct bands (Fig. 6). Considering the pleiotropic phenotypes of pmt3 cells, these bands most likely represent HA₃-Pmt3p conjugated to proteins of different sizes whose identities remain to be determined, although some of the bands may be degradation products of the higher-molecular-mass species. We also detected a similar but less clear conjugation pattern in the strain with the integrated GFP-Pmt3 fusion protein described below (data not shown).

View larger version (46K): [in this window] [in a new window]   FIG. 6.   Pmt3p-protein conjugation in vivo. pmt3 cells were transformed with a plasmid carrying the gene for HA3-tagged Pmt3p (3 × HA-Pmt3p) under control of the inducible nmt1 promoter (pSLF173L-pmt3) and a control vector (pSLF173L). The strains were grown in EMM2 medium supplemented with thiamine to repress the overexpression of HA3-Pmt3p. Protein extracts were prepared as described in Materials and Methods and subjected to Western blot analysis with anti-HA antibody 12CA5. High-molecular-mass Pmt3p-protein conjugates are indicated by a bracket.

Localization of Pmt3p. Various phenotypes of pmt3 cells described above predict that several nuclear proteins are regulated by conjugation of Pmt3p. To determine the intracellular localization of Pmt3p, GFP (63, 69) was fused to the N terminus of Pmt3p. The fusion gene under the control of the pmt3⁺ promoter was integrated into wild-type cells (see Materials and Methods). We confirmed that the pmt3 phenotypes were rescued by expression of the GFP-Pmt3 fusion protein, indicating that this fusion protein was functional (data not shown). The green fluorescence of the fusion protein was observed predominantly in the nucleus. Interestingly, intense signals appeared as one or more spots in the nuclei. To identify the nature of the spots, the cells expressing the fusion protein were fixed and stained with anti-Sad1 antibody, which recognizes SPB. The pattern of GFP fluorescence in the fixed cells was not significantly different from that of the live cells (data not shown). In interphase cells, the SPB signals always colocalized with the fluorescent spots. In Fig. 7A, one of the two spots of the green fluorescence signal can be seen to be colocalized with the anti-Sad1 signal. To examine the localization of GFP-Pmt3p during mitosis, cells expressing the fusion protein were fixed and stained with the anti--tubulin antibody. The GFP-Pmt3p spots were not detected at the spindle poles during prometaphase and metaphase. Instead, a relatively bright region or intense spots were observed between the spindle poles (Fig. 7B). After anaphase, GFP-Pmt3p spots reappeared at the spindle poles. The behavior of the bright signals at the SPB is reminiscent of centromeres which cluster and associate with SPB throughout the cell cycle except around metaphase in S. pombe (24). We compared centromere positioning in wild-type and pmt3 cells by using a combination of immunofluorescence and in situ hybridization with a probe specific for centromeric repeated sequences. The positioning of centromeres in pmt3 cells was indistinguishable from that in wild-type cells (Fig. 7C), suggesting that Pmt3p is not essential for centromere clustering at the SPB. Another feature of GFP-Pmt3p localization is that the GFP signal, which was dispersed throughout the nucleus in interphase, seemed to be excluded from chromosome DNA by condensed chromosomes during mitosis (Fig. 7B). These observations indicate that Pmt3p may be required for kinetochore and/or SPB functions during chromosome segregation.

View larger version (39K): [in this window] [in a new window]   FIG. 7.   Cellular localization of the Pmt3p and centromere positioning. (A and B) Cells expressing the GFP-Pmt3 fusion protein (YM78 [h+ leu1-32 ura4-D18 ade6-704 pmt3+:pYC11-GFPpmt3]) were grown exponentially in EMM2 supplemented with adenine and uracil at 26°C. The cells were fixed with formaldehyde and stained with anti-Sad1 rabbit polyclonal antibody followed by Cy3-conjugated anti-rabbit IgG secondary antibody (A) or anti--tubulin mouse monoclonal antibody followed by Cy3-conjugated anti-mouse IgG secondary antibody (B). Fluorescence images for a given cell: 1, DAPI; 2, GFP; 3, Cy3; 4, merged images of blue for DAPI, green for GFP, and red for Cy3; 5, GFP image before deconvolution. (C) Wild-type cells (HM123) or pmt3 (JN630) cells were grown exponentially in EMM2 supplemented with leucine at 26°C. The cells were fixed by the formaldehyde-glutaraldehyde method and stained with anti-Sad1 rabbit polyclonal antibody followed by the Oregon green-conjugated anti-rabbit IgG secondary antibody and with anti--tubulin mouse monoclonal antibody followed by the Cy5-conjugated anti-mouse IgG secondary antibody. The immunostained cells were fixed with formaldehyde again, and in situ hybridization probed with Cy3-labeled pRS140 which contains a centromeric repeated sequence of S. pombe (13), was carried out. Fluorescence images for a given cell: 1, DAPI; 2, Oregon green; 3, Cy3; 4, Cy5; 5, merged images of blue for DAPI, green for Oregon green, red for Cy3, and white for Cy5. The images were not deconvolved except for images 5 of panels A and B. All images are shown at the same magnification. Scale bar, 5 µm.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Members of the Smt3p/SUMO-1 family are homologous to ubiquitin and play major regulatory roles in such processes as the control of cellular protein localization and/or protein-protein interaction through their posttranslational conjugation to several proteins. However, the precise nature of the processes controlled by Smt3p/SUMO-1 remains obscure. In this work, we isolated an S. pombe homologue of the Smt3p/SUMO-1 family. We showed that Pmt3p is conjugated to target proteins like Smt3p/SUMO-1 and that Pmt3p modification is involved in multiple processes such as chromosome segregation and telomere length maintenance.

Our genetical analyses showed that the pmt3⁺ gene is not essential for viability, although pmt3-disrupted cells grew more slowly and showed several phenotypes. Unlike humans, which have at least three SUMO-1 homologues (41), there is only a single homologue (Smt3p) in S. cerevisiae. S. cerevisiae SMT3 is an essential gene, and its temperature-sensitive mutant displays G₂/M defects (58). We speculate that this difference in cell viability between S. cerevisiae SMT3 and S. pombe pmt3⁺ may be dependent on the nature of the modified proteins; i.e., one or more of the Smt3p-modified proteins may be essential for cell viability in S. cerevisiae but not in S. pombe. Alternatively, there might be multiple SUMO-1 homologues having functions overlapping that of Pmt3p in S. pombe.

In S. cerevisiae, a heterodimer of Uba2p and Aos1p is an ATP-dependent Smt3p-activating enzyme (33) and Ubc9p is the only E2-conjugating enzyme for Smt3p (32). UBA2, AOS1, and UBC9 are all essential genes, consistent with the absolute requirement for the SMT3 function described above (5, 32, 33). On the other hand, in S. pombe, the rad31⁺ and hus5⁺ genes were isolated as a UBA-related AOS1 homologue (79) and as a UBC9 homologue (3), respectively. Recently, we characterized another UBA-related gene in S. pombe, the S. cerevisiae UBA2 homologue, and named it fub2⁺ (fission yeast UBA2) gene (84a). These fub2⁺, rad31⁺, and hus5⁺ genes are all dispensable for cell growth, although each of the disruptants grew slowly, showed aberrant mitosis, and displayed increased sensitivities to factors such as high temperature, HU, TBZ, UV, and MMS, characteristics that are entirely consistent with the phenotypes of pmt3 cells (3, 79, 84a). These facts strongly suggest that in S. pombe, Pmt3p may be activated by a heterodimer of Fub2p and Rad31p, the E1-activating enzyme for Pmt3p, and that the activated Pmt3p is transferred to the active-site cysteine of Hus5p, the E2-conjugating enzyme for Pmt3p.

The fact that pmt3 cells were strikingly sensitive to HU, UV, and MMS suggested that pmt3⁺ may be essential for the checkpoint coupling mitosis to the completion of DNA replication and the DNA damage response. Unlike the checkpoint rad mutants, however, pmt3 cells showed only a moderate loss of viability after treatment with HU (Fig. 3B). Microscopic analysis of the cell morphology indicated that treated pmt3 cells were more elongated than untreated pmt3 cells and that there was no increase in the frequency of cut cells during the first few hours after treatment with HU (data not shown). Furthermore, FACS analysis showed that pmt3 cells can arrest the cell cycle after exposure to HU (Fig. 3C). These results suggest that pmt3⁺ is involved in the DNA damage tolerance process rather than the checkpoint itself. The cut phenotype of pmt3 cells may be attributed to a defect in chromosome segregation because the cut phenotype is also observed in mutants with defects in chromosome segregation (28) and pmt3 cells are defective in chromosome segregation as discussed below. Consistent with this notion, similar results were reported for rad31⁺ and hus5⁺, and both genes have been shown to be required for the DNA damage tolerance process (3, 79). Therefore, it is tempting to speculate that Pmt3p may directly or indirectly target and modify a protein involved in the recovery process after checkpoint arrest.

Telomeres are specialized nucleoprotein structures at the ends of eukaryotic chromosomes which have been shown to be essential for maintaining chromatin integrity. However, considerably less research has been dedicated to understanding telomere structure and the mechanism of telomere regulation in S. pombe. S. pombe telomeres consist of 200 to 300 bp of DNA with the repeat unit consensus sequence 5'-(TTACAG)_1-8-3' on the 3'-end strand (18, 84). Unexpectedly, we found that the large and rapid elongation of telomere length in the pmt3⁺ disruptant cells suggests that Pmt3p functions in the regulation of telomere length (Fig. 5). This is the first demonstration of involvement of one of the Smt3p/SUMO-1 family proteins in telomere length maintenance. Our preliminary results showing that the S. cerevisiae ubc9^ts mutant displays no telomere elongation at the restrictive temperature suggest that the Smt3p ubiquitin-like pathway does not play an important role in the control of telomere length at least in S. cerevisiae (84a). Three possibilities may be considered. (i) Pmt3p might modify and inhibit a negative regulator of telomere length. A candidate for a negative regulator of telomere length is the S. pombe telomere-binding protein, Taz1p. Disruption of the taz1⁺ gene causes a massive increase in telomere length and a defect of telomere loci silencing (15). However, we could not detect any Pmt3p-dependent modification of Taz1p in vivo by Western blot analysis or any silencing defect at telomeric loci in pmt3 cells (data not shown). Thus, these results predict the existence of a negative regulator other than Taz1p which would be inhibited by Pmt3p modification. (ii) Telomerase-independent, recombination-dependent mechanism of telomere repeat synthesis has been observed in S. cerevisiae undergoing senescence (46, 56). The observation that telomere length was immediately reelongated to the level of pmt3 cells after the repression of pmt3⁺ expression (Fig. 5) suggests the involvement of this mode of recombination. Therefore, it is also possible that certain factors involved in recombination are modified, resulting in the inhibition of the telomere elongation in this recombinational mode. The S. pombe homologues of human Rad51 and Rad52, which were shown to interact with both SUMO-1 and human Ubc9 by the two-hybrid assay, respectively (37, 80, 81), are attractive candidates for modification and inhibition of recombinational activity by Pmt3p. (iii) It is known that specific DNA replication mutations affect telomere length in S. cerevisiae (1, 7, 9, 82). We have found that telomeres are strikingly elongated in S. pombe pol^ts and pol^ts mutants (84a). Thus, it is tempting to speculate that Pmt3p may control telomere length by interacting with PCNA, an accessory factor of DNA polymerase , because Pmt3p was originally isolated as a PCNA-interacting factor in our two-hybrid screening assay.

The defect of chromosome segregation in pmt3 cells seems to be plausible because the S. cerevisiae SMT3 gene was originally isolated as a high-copy-number suppressor of mutations in MIF2, which encodes a CENP-C like centromere-binding protein (59). Furthermore, a previous report on the human homologue to S. cerevisiae Ubc9p suggested that it interacts with three subunits of S. cerevisiae centromere DNA-binding core complex, CBF3 (31). Thus, it appears very likely that the function of Smt3p/Ubc9p in the G₂/M phase of S. cerevisiae may be related to its ability to regulate centromere proteins involved in chromosome segregation during mitosis. Our observation that the GFP-Pmt3 fusion protein is predominantly colocalized with the SPB with which clusters of centromeres associate and that this colocalization disappears during prometaphase and metaphase is reminiscent of the requirement for Pmt3p in kinetochore functions (Fig. 7A and B). It is also possible that Pmt3p modifies SPB proteins that release the conjugated Pmt3p moiety at mitosis or proteins that disappear from the SPB as a result of dissociation or degradation. There are many proteins associated with the SPB in S. pombe (2, 8, 20, 26, 27, 30, 38, 51, 64, 68, 71, 83, 85, 88, 91). Their activities change dynamically at mitosis and are involved in chromosome separation and/or cytokinesis. Our GFP-Pmt3p fusion system may provide a useful means to identify target proteins involved in chromosome segregation.

The nature of the additional GFP-Pmt3p spots that did not overlap with the anti-Sad1 signal is unknown. The number and position of the dots varied depending on the cell under observation. The GFP-Pmt3p spots may be related to mammalian PML bodies, which are the major targets of SUMO-1 conjugation (19, 65). The diffuse nature of GFP-Pmt3p fluorescence in interphase nuclei (Fig. 7A) may indicate Pmt3p involvement in DNA replication, repair, and/or recombination, a notion that is also supported by the phenotypes of pmt3 cells. The diffuse green fluorescence moved to the DAPI-negative part of the nucleus at mitosis (Fig. 7B). This behavior suggests that Pmt3p may be involved in the regulation of condensation and decondensation of chromosomes or the nuclear matrix association of interphase chromatin.

A distinct nuclear rim localization, consistent with the localization of SUMO-1-modified RanGAP1 (47, 53), can be observed in Cos-7 cells expressing low levels of HA-tagged SUMO-1 (48), and the human homologue of Ubc9 colocalizes with RanGAP1 at the nuclear envelope (42). These observations indicate that RanGAP1 is the major substrate of SUMO-1 modification in mammalian cells. Furthermore, it was shown that mutation of the Drosophila semushi gene, which encodes a Ubc9 homologue, blocks nuclear import of Bicoid during embryogenesis (22). These results suggest the possibility that the pleiotropic phenotype observed in the pmt3 cells is caused by the abnormal nuclear transport. However, Rna1p, the S. cerevisiae RanGAP1 homologue, is not detected in modified form by Western blotting using antibodies against Rna1p (16), indicating that Rna1p is unlikely to be a substrate of the Smt3p modification pathway. Using the GFP-Pmt3 fusion protein, we could not detect any specific GFP staining of the nuclear envelope in S. pombe (Fig. 7A), suggesting that the putative S. pombe RanGAP1 homologue may not be a substrate of the Pmt3p modification pathway. It should be noted that neither S. cerevisiae (87) nor S. pombe (57) RanGAP1 homologue has the conserved C-terminal domain that includes the site of SUMO-1 ligation (54).

We have identified the pmt3⁺ gene by two-hybrid screening using S. pombe PCNA as bait. We have confirmed that Pmt3p can interact with PCNA in vivo by immunoprecipitation using anti-PCNA antibodies (data not shown). Therefore, we believe that Pmt3p and PCNA may directly or indirectly interact each other. However, we cannot rule out the possibility that Pmt3p can transiently modify PCNA or that PCNA associates or cooperates with a protein modified by Pmt3p, as was shown to be the case for the association between RanBP2 and SUMO-1-modified RanGAP1 (47, 48, 75). PCNA is required for DNA replication, for DNA repair, and maybe for the recovery pathway after DNA replication arrest induced by DNA synthesis inhibitors or DNA damage. Pmt3p may directly or indirectly modify and regulate PCNA in the recovery pathway.