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1 Department of Biophysics, Graduate School of Science
2 Department of Gene Mechanisms, Graduate School of Biostudies, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan
3 Bioinformatics Center, Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
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Abstract |
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850 are essential for viability. To obtain information on interactions among genes required for chromosome segregation, an approach called Strategy B was taken using mass transformation of the 1015 temperature-sensitive (ts) mutants that were made by random mutagenesis and transformed by plasmids carrying the genes for securin, separase, condensin, cohesin, kinetochore microtubule-binding proteins Dis1/Mtc1 or histones. Mutant strains whose phenotypes were either suppressed or inhibited by plasmids were selected. Each plasmid interacted positively or negatively with the average 14 strains. Identification of the mutant gene products by cloning revealed many hitherto unknown interactions. The interactive networks of segregation therefore may consist of genes with a variety of functions. For example, separase/Cut1 interacts with Cdc48/p97/VCP, which stabilizes securin and separase. Surprisingly, S. pombe cdc48 mutants displayed the mitotic phenotype highly similar to separase/cut1 mutants. This approach also provides a novel way of mutant isolation, resulting in two histone H2B strains and a cohesion mutant with a new phenotype. |
Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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We prepared 1015-ts (temperature-sensitive) strains of the fission yeast Schizosaccharomyces pombe by random mutagenesis, and their phenotypes were determined (Matsumura et al. 2003; Hayashi et al. 2004). These strains have been employed in this laboratory for mass gene cloning using a genomic DNA library (called Strategy A of the Mandala project). Mass gene cloning will result in identification of mutant genes and multicopy suppressors. Based on our preliminary results, approximately 700 distinct genes (covering 80% of the presumed essential genes) will be identified in the mutant collection. Another approach was mass transformation: plasmids that carried particular genes were introduced into 1015 strains and the colony formation of resulting transformants was tested (called Strategy B, Matsumura et al. 2003). Mass transformation leads to the selection of mutant strains that positively or negatively interact with the plasmid genes introduced. Results of mass transformation will be utilized to construct the interactive network, including mutant genes that are mostly essential and a variety of non-essential and essential genes obtained as high copy suppressors or inhibitors.
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Results |
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For constructing the network, one of nine plasmids (listed in Table 1) was introduced individually into the ts leu1 mutant collection, and such mass transformation was carried out many times. Leu+ transformants obtained were first plated at 26 °C, then re-plated at 26, 33 and 36 °C. Two types of transformants were selected: those able to form colonies at the semirestrictive 33 °C or the restrictive 36 °C (suppression by plasmid), and others unable to form colonies at the permissive 26 °C and the semipermissive 33 °C (inhibition by plasmid). The number of selected strains (Table 1) varied, ranging from 2 to 23 for suppression and 0 to 17 for inhibition, dependent on the plasmid genes introduced. The largest number of selected strains obtained was 23 for suppression and 17 for inhibition by separase plasmid pCUT1. The smallest number was only two for suppression and none for inhibition by plasmid pDIS1. The plasmid gene copy number per transformed cell was in the range of 10–20, but the increase of protein level measured was relatively small, 2–5-fold increase for Cut1, Cut2 and Dis1 (Matsumura et al. 2003).
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6.7% of the collection) emerged after mass transformation. In addition, the results of gene cloning and the genetic linkage analyses for the mutant strains indicated that some mutant strains were derived from the same genes. Thirty distinct genes therefore appeared in the gene network made by mass transformation. Detailed interaction results are described below. To estimate the frequencies of a secondary mutation, a number of original ts strains were back-crossed with wild-type and their cellular phenotypes at 36 °C in ts– cells were examined: recognizable changes were observed only infrequently (< 5%). In other estimations, we found that most ts strains inhibited by pCUT1 were back-crossed and found to be still inhibited by pCUT1. It was therefore unlikely in most strains reported here that the inhibition or the growth recovery by plasmid was as a result of the suppression of a second mutation. Positive interactions of pCUT1 with cdc48 and replication-related mutants
Plasmid carrying the separase gene pCUT1 suppressed the ts phenotypes of 23 strains (arrows in Fig. 1K). Gene cloning and genetic crossing established that 17 of them were cut1 mutants (linked to pCUT1 by the red arrows). An example micrograph of DAPI-stained cut1-119 cell reveals a typical defect in chromosome segregation (Fig. 1A). Separase mutations are most frequent (1.7% of the whole ts collection) as previously shown (Hirano et al. 1986). Securin plasmid pCUT2 partly rescued the ts phenotype of three cut1 mutants (119, 162 and 220; Fig. 1K): Overproduced Cut2 acts as a chaperone, stabilizing Cut1 mutant protein (Nagao et al. 2004).
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Two strains (353 and 605) revealed the segregation defect highly similar to that observed in separase mutants at 36 °C (Fig. 1F–G). Separase plasmid pCUT1 strongly suppressed the ts phenotype at 36 °C (Fig. 1H). Interestingly, 353 and 605 were neither cut1 nor cut2 mutants. Cloning and genetic crossing established that the mutant gene was spac6f12.01 (designated Cdc48), highly similar (70% identity) to Cdc48/VCP/p97, an AAA ATPase, a ubiquitin-dependent chaperone required for various cellular activities (Patel & Latterich 1998). While the ts phenotype of 353 and 605 was fully suppressed at 36 °C by pCDC48 (Fig. 1H), securin plasmid pCUT2 produced the opposite effect, strongly inhibiting colony formation of 353 and 605 strains. Cut2 was over-expressed in cdc48-353 using a mild inducible promoter nmt1 in REP81 in the absence of thiamine (Fig. 1I,K; –Thi, promoter on). The same inhibitory effect was found for the other cdc48-605 mutant. A hypothesis is that polyubiquitinated Cut2 may have to interact with spCdc48 for its proper destruction in a dose-dependent manner so that the excess, polyubiquitinated Cut2, may become toxic in cdc48 mutant cells.
The phenotypic similarity between cut1 and cdc48 mutants was confirmed by immunofluorescence microscopy using anti-tubulin and anti-SPB antibodies (Fig. 1J). Chromosomes were physically stuck in the cdc48 mutant as in cut1. The mitotic spindle length in cdc48 (average 3.3 µm) at 36 °C was shorter than that of wild-type (average 5.1 µm) but similar to that of cut1 mutant (average 3.3 µm), whereas the spindle length at 26 °C of the mutants and the wild-type was the same. These results demonstrated that the segregation and spindle extension defects in cdc48 were indistinguishable from those seen in cut1 mutants (Kumada et al. 1998).
Chromosome segregation defects in S. pombe cdc48 mutants
SpCdc48 has two conserved AAA ATPase domains and is highly similar to budding yeast Cdc48 and human p97/VCP/hCdc48 (Fig. 2A). In order to obtain high-copy suppressor plasmids other than pCUT1, the S. pombe genomic library was employed and used for transformation of cdc48-353. Plasmids recovered from the Ts+ transformants were mostly pCDC48 or pCUT1, showing that the genetic suppression of cdc48 by pCUT1 was inherent. Note, however, that pCDC48 did not suppress cut1 mutants (data not shown): The suppression was unidirectional. Overproduced spCdc48 did not substitute mutant Cut1, whereas overproduced Cut1 seemed to largely overcome mutant spCdc48 protein dysfunction.
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The gene disruption experiment was carried out by one-step gene displacement: The cdc48+ gene was essential for cell viability (data not shown). The phenotype of cdc48 null was examined by germinating gene-disrupted spores. Germinated cells did not properly divide, or divided once or twice at 33 °C (Fig. 2C). Cells with aberrant mitotic phenotypes (condensed chromosomes, displaced nucleus, cut phenotypes) were frequent (40%). The mitotic phenotype of cdc48
null was severer than that of ts cdc48 mutants at 36 °C.
The stability of Cut1 is controlled by spCdc48 protein enriched in nuclear chromatin
Intracellular localization of spCdc48 was determined using the chromosomally integrated Cdc48-GFP gene expressed under the native promoter (Fig. 2D): The signal of Cdc48-GFP was highly intensive in nuclear chromatin and also present in cytoplasm. Although Cdc48/p97/VCP has been extensively studied in various organisms, its nuclear chromatin function is scarcely known. To examine whether the protein level of Cut1 and Cut2 was controlled by spCdc48, extracts of cdc48 mutant cultured at 26 °C (0 h) and shifted to 36 °C for 1–4 h were prepared. The level of Cut1 was reduced in two cdc48 mutants even at 26 °C and then further diminished at 36 °C after 4 h (Fig. 2E), suggesting that Cut1 needed Cdc48 for its maintenance. The level of Cut2 was also diminished at 26 °C, but did not significantly change after the shift to 36 °C. As control, the level of Cdc2 was examined and was kept constant.
Negative interactions of pCUT1 and pCUT2 with transcription factor and APC/C mutants
Separase plasmid-inhibited colony formation of 17 mutants. Five (48, 153, 234, 547 and 893) of them were previously reported: They were mutants of the APC/cyclosome (APC/C) subunits (Apc10, Cut9, Cut20 and Cut23) essential for polyubiquitination of cyclin and securin (Matsumura et al. 2003; see also Fig. 3G). Upon its activation, separase seemed to inhibit the APC/C function directly or indirectly. Ten strains were inhibited by pCUT2. Most of them were overlapped with those inhibited by pCUT1 (Fig. 3G). It is known that Cut2 acts as a chaperone for Cut1 (Nagao et al. 2004): high dosage Cut2 increases the level of Cut1. Overlapping may hence be explained by presuming that the inhibition by the dosage increase of Cut2/securin is effective through the higher Cut1/separase activity.
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Gene cloning and genetic analyses established that the gene for 891 was sak1+, previously identified as a high-dosage suppressor for PKA (protein kinase A) mutant (Wu & McLeod 1995). Sak1 is essential for viability and gene disruption results in aberrant cell shape and defect in cytokinesis. Similar phenotypes were found in sak1-891. The amino acid sequence of Sak1 resembles human transcription factor RFX (21% identity) and budding yeast Crt1/Rfx1 (23% identity): Crt1 was reported to interact with the general transcription factor TAF(II) (Huang et al. 1998; Li & Reese 2000). Consistent with the putative role in transcription, the Sak1-GFP chromosomally integrated with the native promoter revealed the GFP signals in nuclear chromatin throughout the cell cycle (Fig. 3F). As plasmid pSAK1 rescued the APC/C mutant cut9-234 (Matsumura et al. 2003), Sak1 may be related in ubiquitin-mediated anaphase-promoting proteolysis.
The other strain 879 was found to be a mutant in spbc146.01, encoding a protein similar to the budding yeast transcription factor Gal11 (Fassler & Winston 1989). As the cellular phenotype of S. pombe gal11 mutant at 36 °C was highly similar to that of sak1-891, Sak1 and spGal11 might share a similar function. The synthetic lethal interactions between pCUT1 and four transcription-related mutations should not be coincidental, as two of them, 838 and 891, were also synthetically lethal with the securin plasmid (Fig. 3G) The negative interaction seemed to require the active separase: colony formation of sak1-891 was not inhibited by pCUT1C1730A (Fig. 3H) that contained a single substitution C1730A causing the separase death (Matsumura et al. 2003).
We investigated the phenotypes of sak1-891 and cut9-234 carrying pCUT1, in order to gain information on how their colony formation was inhibited by pCUT1. Two notable features were seen in both strains (Fig. 3I). Multiple aberrant septa were formed in the middle of cells, while propidium iodide-stained nuclear chromatin was fragmented and scattered in whole cells. About 10% of cells showed these phenotypes, which were rarely (less than 1%) seen in control mutants with vector. The toxicity of pCUT1 appeared to be down-regulated by Sak1-mediated transcription or ubiquitin-mediated proteolysis so that the severe synthetic lethal phenotypes revealing fragmented nuclei emerged in these mutants.
Negative interactions of pCUT1 with cohesin, cytokinesis and TOR mutants
Two strains (407 and 897), interacting negatively with pCUT1 (but not with pCUT2), displayed mitotic phenotypes with condensed and lagging chromosomes (Fig. 4A,B), similar to those of cohesion mutants (Toyoda et al. 2002). Gene cloning and genetic mapping indeed established that 407 was a mutant of psc3+ (similar to Scc3) encoding an essential subunit of cohesin, while 897 was the mutant of psm1+ (similar to SMC1), an essential SMC subunit of cohesin (Tomonaga et al. 2000). As Rad21, another cohesin subunit, is the proteolysis target of Cut1, the excess Cut1 may result in a lethal effect on psc3-407 and psm1-897 in which the level of functional cohesin should be lower than in wild-type. Consistently, multicopy suppressor plasmid carrying rad21+ was isolated for psm1-897.
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Two remaining strains 545 and 589 (Fig. 4D) with no obvious cell cycle phenotype were ste20 (alternatively called ste16) mutants (Hilti et al. 1999), while 803 displaying round-shaped cells (Fig. 4E) at 36 °C was a pop3/wat1 mutant (Kemp et al. 1997; Ochotorena et al. 2001). Wat1/Pop3 is a WD-40 protein, related to cell morphogenesis and genome stability. Molecular functions of Ste20 and Wat1/Pop3 are probably closely related to the TOR functions (Jacinto & Hall 2003), because the budding yeast putative homologues Tsc11 and Lst8 of Ste20 and Wat1, respectively, are the components of the TOR complex 2 that is involved in growth control and the signalling to the actin cytoskeleton (Loewith et al. 2002). Cells of wat1-803 isolated in this study and cultured at 26 and 36 °C were stained by phalloidin, an actin-specific fluorescent probe. Actin was localized normally at the growing ends in the rod shaped cells at 26 °C, whereas it was abnormally dispersed in round cells at 36 °C (Fig. 4F). Cellular functions of Cps1/Bgs1, Ste20 and Wat1 were therefore actin-related. Separase might be implicated in actin cytoskeleton, and these gene products might be related to the target so that the excess separase resulted inhibitory in the mutants.
Mutant strains that interact with condensin plasmids
To search for proteins that interact with condensin, pCUT3 and pCUT14 carrying the SMC subunit genes (Sutani et al. 1999) were employed for mass transformation. Seven mutant strains (10, 104, 199, 242, 296, 488 and 587) rescued by pCUT14 at 36 °C were determined to be cut14 alleles by gene cloning, following genetic analysis. They all showed the defect in mitotic chromosome condensation (two example strains in Fig. 4G). Three other strains (655, 105 and 641) weakly suppressed by pCUT14 were the mutants of DNA replication, orc5, top3 and cdc18, respectively (Fig. 4I). These were also suppressed by pCUT1 as described above.
Another plasmid pCUT3 scarcely interacted with any of the mutant strains. Only four, cut14-296, -488 and orc5-655, cdc18-641, were suppressed by pCUT3 (Fig. 4I). Similar results of suppression were obtained by pCUT14. In fission yeast, chromatin loading of MCM occurs in anaphase and requires ORC and Cdc18 (Kearsey et al. 2000). Condensin might be implicated in replication through such a recruitment process. Cnd2, a non-SMC condensin subunit, is required for DNA repair and a replication checkpoint as well as in mitotic chromosome condensation (Aono et al. 2002). Plasmid pCUT14 weakly inhibited colony formation of 347 (Fig. 4H), which showed the cell cycle arrest accompanying cell elongation. The 347 strain was the mutant for DNA polymerase, swi7/pol1.
Multiple interactions of plasmids pMTC1 and pDIS1
S. pombe has two related kinetochore microtubule binding proteins, Dis1 and Mtc1/Alp14 (Ohkura et al. 2001), required for sister chromatid separation at the restrictive temperature, but not absolutely required for cell viability. In the absence of Dis1 or Mtc1/Alp14, the spindle checkpoint was activated because of the lack of spindle-kinetochore interaction (Nakaseko et al. 2001). Their sequences are conserved in eukaryotes, similar to frog XMAP215, human tog and budding yeast Stu2 (McNally 2003). During mitosis, Dis1 and Mtc1 are bound to kinetochore chromatin and microtubules. Although data obtained by different organisms are not entirely consistent, the role of the Dis1-XMAP215 family may destabilize kinetochore microtubule for shortening in anaphase.
Mass transformation using plasmid pDIS1 showed that the interacting mutants were disapprovingly small in number: Only two strains (226 and 587) interacted with pDIS1. Strain 587 was determined to be a cut14 allele (Fig. 5J). Interaction between Dis1 and condensin has been known: a high-copy plasmid pCUT3 suppressed the phenotype of dis1 (Nakaseko et al. 1996). Dis1 and Cut3 became enriched in the central centromeres during mitosis (Nakaseko et al. 2001).
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Plasmid pMTC1 rescued many ts strains (Fig. 5J), which included mis5-226 and cut14-587 described above. Two other strains (10 and 296) were cut14 alleles, while three other strains were found to be defective in mis4 (236 and 381) and psm1 (897), respectively, defective in cohesion. The elevated gene dosage of Mtc1 might ensure interaction between kinetochore and microtubules that was weakened in mis4 mutants. Gene cloning and genetic analysis showed that the strains 170 and 220 were implicated in proteolysis: 170 was an allele for Apc10, an essential APC/C subunit, while 220 was a separase cut1 mutant.
The strain 13 (Fig. 5B) was defective in Spp1/Pri1 an essential component of DNA primase required for an early step of DNA replication (Griffiths et al. 2001). The strain 510 (Fig. 5C) was defective in spbc31e1.05 that encodes a protein similar (35% identical) to budding yeast Gle1 implicated in RNA export and localized at the nuclear pores (Kendirgi et al. 2003). The strain 703 (Fig. 5D) was defective in Uba3, an activating enzyme for the ubiquitin-like modifier Nedd8 (Yashiroda & Tanaka 2003).
The strains 165 and 426 showed semiwee (short cell length) phenotypes (Fig. 5E,F), while 275 revealed pear-shaped cells and occasional unequal segregation (Fig. 5G). Strain 165 was found to be mutated in Ypt2, a GTP-binding protein (Miyake & Yamamoto 1990), while strain 426 was in Spac4f10.10c similar to budding yeast Mnn9 (Yip et al. 1994) and strain 275 was in Alp21/Sto1. Alp21 is a tubulin-specific chaperonin (Radcliffe et al. 1999) and required for spindle assembly that would explain the phenotype of 275. We found that strain 499 (Fig. 5H) was inhibited by pMTC1. This strain was determined to be defective in Cut20, one of the essential subunits for APC/C (Matsumura et al. 2003). The elevated dosage of Mtc1 therefore positively interacted with mutations in diverse cellular functions, including cytoskeletal regulation, DNA replication, chromosome condensation and proteolysis.
Interactions with cohesion-related pMIS4
Mis4 similar to budding yeast Scc2 is essential for establishing sister chromatid cohesion during the S phase and for recruitment of cohesin (Tomonaga et al. 2000). Mass transformation was carried out using plasmid pMIS4. The phenotype of 12 strains was suppressed. Three (236, 381 and 450, Fig. 5I) of them were mis4 mutants. Interestingly, 450 hardly showed the mitotic defect, and was not rescued by pMTC1. Four of seven cut14 alleles and two of 17 cut1 alleles were partly suppressed by pMIS4. The genes for three other strains, 228, 403 and 722, have not been cloned.
Mutants that interact with histone plasmids
S. pombe has three sets of paired histone H3–H4 genes, one paired H2A–H2B and one additional H2A gene. Mass transformation was performed using plasmids carrying the paired H3-H4 (hht3+ -hhf3+) and H2A-H2B (hta1+ -htb1+) genes. Three mutant strains (45, 226 and 718) were suppressed at 33 °C (but not at 36 °C) by plasmid pHHT3-HHF3. None of them was histone H3 or H4 mutation as expected from their redundancy. Gene cloning and the following genetic analysis showed that 45, 226 and 718 were, respectively, mutants in mis5/mcm6 and cdc18 essential for DNA replication. This suppression was allele specific as other cdc18 and mcm6/mis5 mutants were not suppressed.
Two mutant strains (72 and 223) were suppressed at 36 °C by plasmid pHTA1-HTB1. Cloning and genetic analysis established that these strains were mutants in histone H2B and suppressed by plasmid carrying the htb1+ gene, but not by plasmid carrying the hta1+ gene. These results were compatible with the concept that a single essential gene of H2B (htb1+) could give rise to the ts mutations. Although the coding region of H2B was short and highly conserved, they could produce two ts mutations.
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Discussion |
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Synthetic lethality of cohesin mutants with pCUT1 (but not by pCUT2) may fit an explanation that the excess separase activity is inhibitory in cohesin-reduced mutants. Consistently, separase-dead plasmid fails to inhibit the cohesin mutants. The elevated separase gene doses result in micronuclei formation and cytokinesis block in sak1 and cut9 mutants. These phenotypes were the reverse of those of separase-deficient ts mutants, in which cytokinesis occurred (the cut phenotype), while chromosomes were physically stuck. The active separase protease may be needed for restraining cytokinesis, consistent with the fact that the cut phenotype occurs frequently when separase-dead Cut1C1730A is overproduced in wild-type. Sak1 and Cut9 may down-regulate the excess separase for proper progression of mitosis. The implication of transcription factors is intriguing. It was previously reported that Cut1 was implicated in mRNA export (Azad et al. 2003).
The interactive modes of spCdc48 with securin and separase were unique among other genes identified in this study. Securin plasmid negatively, while separase plasmids positively, interact with cdc48 mutants. Cdc48/p97/VCP is a member of the AAA family of ATPase required for cell division and implicated in a variety of cellular functions, including ubiquitin–proteasome dependent ER-associated degradation system (Patel & Latterich 1998; Lord et al. 2002; Thoms 2002; Cao et al. 2003; Fu et al. 2003). It is thought to be a molecular chaperone, interacts with polyubiquitin proteins and may facilitate unfolding and/or folding of the interacting proteins, dependent upon conditions, and may be important for linking ubiquitinated protein to 26S proteasome. It would be reasonable to assume that Cdc48 interacts with polyubiquitinated securin, that in turn stabilizes separase, and that the defective phenotypes of cdc48 mutants are mainly because of the unstable separase at the restrictive temperature. This would explain why the suppression was unidirectional: pCDC48 failed to rescue cut1 mutants. Although Cdc48 in higher eukaryotes is thought to play very diverse multiple cellular roles, its vital role in rapidly dividing cells may be in chromosome segregation and cytokinesis. In fission yeast, the prominent cellular defects in cdc48 in both ts and gene disrupted cells are in chromosome segregation and cytokinesis. In this microbe, the duration of G2 shares > 50% of the generation time so that the inactivation of Cdc48 may immediately (within 60 min) affect the progression of mitosis.
We consider a hypothesis that Cdc48 protects separase through interacting dynamically with polyubiquitinated securin. A mechanism might exist for separase being able to escape from the degradation by 26S proteasome when its partner securin is degraded, and Cdc48 might be involved in such mechanism. Cdc48, known to act as a quality control chaperone, may act as a stabilizer of polyubiquitinated securin. Over-expressed pCUT2 could therefore inhibit the cdc48 mutant. Rescue of the cdc48 mutant by pCUT1 (but not vice versa) suggests that separase is downstream of Cdc48 and also that separase is the main downstream target of Cdc48 in rapidly dividing cells. Further work is necessary for understanding the actual role of Cdc48. The present study sheds light on a novel functional link between Cdc48, securin and separase.
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Experimental procedures |
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The procedures described by Takahashi et al. (1994) were followed. Briefly, N-methyl-N'-nitrosoguanidine was used for the mutagenesis of a haploid strain h– leu1. Replica plating was carried out for the complete YPD plate at 26 and 36 °C. Colonies that failed to grow at 36 °C were collected. They were all streaked and single colonies were selected and examined for their ts phenotype. By examining over 200 000 colonies, 1015 ts strains were isolated.
Screening of mutant strains by plasmids
Plasmids pCUT1, pCUT2, pCUT1 C1730A, pCUT3, pCUT14 (Matsumura et al. 2003), pDIS1 (Nabeshima et al. 1995) and pMTC1 (Nakaseko et al. 2001) were previously described. pHHT3-HHF3 and pHTA1-HTB1 were made in this study. Transformation was performed by the lithium method. A fixed amount of plasmid DNA (500 ng/tube) was used for each transformation. The collection of S. pombe ts strains (T. Hayashi et al., submitted) was employed for transformation. They were individually cultured in the YPD medium and the mid-log phase of each culture with an appropriate cell concentration (4 x 106 1 x 107/mL) was used for transformation. The resulting cells were plated at 26 °C on the EMM2 plate for 4–7 days, depending on the speed of colony formation. Colonies formed were plated at 26, 33 and 36 °C on synthetic EMM2 plates and at 36 °C on complete YPD plates, and the degree of colony formation was compared between plates.
Mutant phenotype observation
DAPI staining of ts mutants
Mutant cells were grown at 26 °C, then at 36 °C for 2–5 h in liquid YPD culture. Cells were fixed by 2.5% glutaraldehyde for 20 min on ice, washed three times by saline phosphate buffer and observed under a fluorescence microscope after staining with DAPI (25 µg/mL).
Calcofluor and propidium iodide staining
Cells were fixed by 70% ethanol for 15 min at room temperature, washed three times by saline phosphate buffer, treated with 1 mg/mL Rnase A and observed under a fluorescence microscopy after staining with calcofluor (2.5 µg/mL) and propidium iodide (1 µg/mL).
Immunofluorescence microscopy
The previously described procedures (Hagan & Hyams 1988; Hagan & Yanagida 1995) were followed. Tubulin was stained with anti-TAT1 antibody (Woods et al. 1989) and anti-mouse IgG Alexa 546 antibody (Molecular Probes). SPB was stained with anti-Sad1 antibody (Hagan & Yanagida 1995) and anti-rabbit IgG Alexa 488 antibody (Molecular Probes).
GFP fluorescence microscopy
To obtain GFP-tagged sak1+ or cdc48+ strains, we created an NotI site at the termination codon of either sak1+ or cdc48+ and used them for fusion with the GFP(S65T) gene at the carboxyl termini. We integrated the resultant DNA fragments on to the chromosome of S. pombe haploid cells by replacing the endogenous sak1+ or cdc48+ gene using an S. pombe ura4+ gene or an Saccharomyces cerevisiae LEU2 gene as a selective marker. The microscope method was previously described (Sutani et al. 1999).
Gene cloning of ts strains
An S. pombe genomic DNA library containing the Sa. cerevisiae LEU2 gene as the selection marker was used. Plasmids were recovered from Ts+ Leu+ transformants. Nucleotide sequencing was carried out for each plasmid, the BLAST technique was used for the obtained sequences, and the region covered by the inserted genomic sequence was determined. Subcloning was carried out when necessary.
Preparation of cell extracts for immunoblotting
S. pombe cells were disrupted by glass beads (15 s, four times). Resulting extracts were centrifuged at 2000 g for 3 min. The extraction buffer used contains 25 mM Tris-HCl (pH 7.5), 15 mM EGTA, 60 mM ß-glycerophosphate, 1 mM DTT, 0.1% NP-40, 10% glycerol and 1 mM phenylmethylsulfonyl fluoride (PMSF). Anti-VCP(Cdc48) and PSTAIRE antibodies were gifts from A. Kakizuka (Hirabayashi et al. 2001) and Y. Nagahama (Yamashita et al. 1992). Antibodies against Cut1 and Cut2 were as previously described (Uzawa et al. 1990; Kumada et al. 1998).
Gene disruption of cdc48+
One-step gene replacement was used. An N-terminal portion and 5'-upstream fragment of S. pombe cdc48+ (700 bp) were amplified by the PCR method with the oligonucleotides 5'-CCGCTCGAGGATATTTAAAACACATTAAATTCC-3' and 5'-CCCAAGCTTTCGACCATGCACCATGCGC-3'. Also a C-terminal portion and 3'-downstream fragment of cdc48+ (800 bp) were amplified by the PCR method with the oligonucleotides 5'-ATAAGAATGCGGCCGCTGAATCCGTCAAAACTCTTTC-3' and 5'-CCCAAGCTTAAACTGCCACCTTAATGCATG-3'. Each fragment was ligated with each end of the S. pombe ura4+ gene. Then, S. pombe diploid (h+/h– leu1/leu1 ura4/ura4 ade6-216/ade6-210 his2/+) cells were transformed with the resultant DNA fragment. The genomic DNA of stable Ura+ transformants was analysed by Southern hybridization to confirm the disruption of the cdc48+ gene. To examine the phenotype of gene-disrupted cells, heterozygous diploid cells were sporulated at 26 °C. Ura+ (gene disrupted) spores were germinated in EMM2 liquid medium lacking uracil at 33 °C. Germinated cells were collected after 20 h, fixed with 2.5% glutaraldehyde, and stained with DAPI (25 µg/mL).
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Acknowledgements |
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Footnotes |
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Present addresses:aHuman Genome Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan;
bDivision of Cell Biology, Institute of Life Science, Kurume University, 2432-3 Aikawa-machi, Kurume, Fukuoka, 839-0861, Japan
* Correspondence: E-mail: yanagida{at}kozo.lif.kyoto-u.ac.jp |
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Received: 11 June 2004
Accepted: 10 August 2004
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