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Initial Research Project, Okinawa Institute of Science and Technology, Suzaki 12-22, Uruma, Okinawa, 904-2234 and Graduate School of Biostuides, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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How is the protease activity of separase regulated? If it is not strictly regulated, the premature separation of sister chromatids may occur. Alternatively or simultaneously, segregation of chromosomes may accompany with non-disjunction during mitosis. Separase is thus strictly repressed when the activity is not needed, and the process of separase activation must be effective and fast when necessary. Securin is not a simple inhibitor but seems to act as a chaperone of separase for generating the separase activity (Jallepalli et al. 2001; Hornig et al. 2002; Nagao et al. 2004).
Securin is absolutely required for chromosome segregation in fission yeast and fly (Funabiki et al. 1996a; Stratmann & Lehner 1996). It is also a recruitment factor for proper intracellular localization of separase in fission yeast (Kumada et al. 1998) and budding yeast (Jensen et al. 2001; Hornig et al. 2002). We showed that securin is bound to the non-conserved amino-terminal domain of separase (Kumada et al. 1998) and the same situation was reported for the budding yeast separase (Jensen et al. 2001; Hornig et al. 2002). In further studies using a fly (its separase consists of two proteins, THR and SSE) and budding yeast, securin is shown to be bound to the carboxy terminus of separase where the catalytic domain of protease exists. It is thus likely that the separase that is bound to securin takes the conformation in which the amino terminus is in close proximity to the carboxy catalytic domain (Jager et al. 2001; Hornig et al. 2002). It is unknown what kind of protein conformation the active separase takes. Evidence suggests that the active form may make a large conformational change upon dissociation from securin (Hornig et al. 2002). In addition to the structural regulation, phosphorylation of separase appears to be important. Higher eukaryotic separase is regulated by Cdc2 phosphorylation that represses the separase activity, and this repression is securin independent (Stemmann et al. 2001).
The regulation of separase activity by securin is mechanistically little understood. One reason is that the amino acid sequences of securin from diverse organisms show little sequence homology so that no hint of its function has been obtained from sequence comparison except for the common property that the amino terminus of securin contains the destruction box (RxxL) and the KEN box required for ubiquitin-mediated destruction (Cohen-Fix et al. 1996; Funabiki et al. 1996b; Zur & Brandeis 2001; Hagting et al. 2002; Leismann & Lehner 2003).
In fission yeast S. pombe, we previously showed that the carboxy terminus of securin/Cut2 physically interacts with the amino terminus of separase/Cut1 (Kumada et al. 1998). In fact, temperature sensitive (ts) mutations cut2-364 and cut2-447 resided in the carboxy terminus (364 allele is a nonsense mutation) so that the proper activation of separase seemed to fail in these ts mutant cells at the restrictive temperature (Funabiki et al. 1996a; Kumada et al. 1998). To gain more information on how securin regulates separase, we examined whether a particular region of securin, when mildly over-expressed, can inhibit sister chromatid separation. We found that the central 76 amino acids fragment of securin that did not contain the destruction motifs severely blocked sister chromatid separation. We will show that this is the third functional domain in securin essential for the generation of separase activity, and present mechanistic models to explain our findings in the context of separase protease inhibition and activation.
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Results |
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To examine the effect of overproduction, variously truncated securin/Cut2 fragments tagged with HA (hemaglutinin antigen) were constructed (Fig. 1A, left panel). The fragments were over-expressed under the inducible nmt1 promoter using REP81-based plasmids at 33 °C in the absence of thiamine (promoter, on). REP81 is the mildest promoter among others (Maundrell 1993). Over-expression of the full length securin (Cut2WT) was slightly inhibitory on wild-type colony formation, in comparison with vector plasmid (compare vector with WT, Fig. 1A, right panel). Over-expression of fragments that contained the central domain blocked colony formation. The shortest toxic fragment was 76 amino acid-long (81–156) and enriched in acidic amino acid clusters (red boxes): the region containing the 130th residue was essential for the toxicity. Over-expression of the indestructible 
80 fragment (e.g. 81–301) produced a negative dominant effect: 80 lacked the destruction boxes (DB1 and DB2, indicated by blue), and firmly bound to separase (Funabiki et al. 1996b; Kumada et al. 1998). This study was initiated to elucidate the cause of inhibition by fragment 81–156 and whether the mode of inhibition by the shortest fragment and 80 was identical. To confirm the increased cellular levels of 81–156 and 80, immunoblot was performed using anti-HA antibodies for cells after over-expression (10–12 h). The levels of overproduced proteins were comparable to that of wild-type protein (Cut2WT) (Fig. 1B).
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80 (Fig. 1C). The aster spindle microtubules (indicated by the arrows) were well developed. As cytokinesis occurred in the absence of nuclear division (bottom panel), the frequencies of the cut (cell untimely torn) phenotypes sharply increased (data not shown). The phenotypes were similar to 80 over-expression, which prevented the activation of separase (Funabiki et al. 1996b).
Quantitative data (Fig. 1D) showed that cells over-expressing 81–156 contained the partly elongated (3–6 µm long) anaphase spindle in the absence of chromosome segregation, whilst such a phenotype was not observed in cells carrying the vector plasmid. The frequencies of cells showing the block of chromosome segregation with the partly elongated spindle were also high for 80-expressing cells. Thus the lack of sister chromatid separation and the failure of full spindle extensions are the principal phenotypes of 81–156 over-expression. The similar phenotypes of 80 and 81–156 suggest that separase activation is prevented in cells that over-expressed 81–156.
Isolation of a separase mutant that overcomes the toxicity of securin 81–156
An attempt was made to screen resistant mutants against the toxicity of 81–156. To this end, pseudo-revertants that could grow in spite of the expressed 81–156 were isolated by spontaneous mutations: 690 revertants were obtained from 1.6 x 108 cells. They were then replica plated for screening for temperature sensitive (ts) strains. One ts strain (designated K73, and mapped to be in the cut1 locus, see below) was obtained by its resistant property against the toxicity at the permissive temperature (26 °C; Fig. 2A). We confirmed that the level of 81–156 in K73 after over-expression was similar to that in wild-type (data not shown). Under the over-expression of 81–156 at 26 °C (+ in Fig. 2B), 31% of cells in K73 containing the spindle showed normal chromosome segregation (filled column). The ability of K73 cells to overcome the toxicity of 81–156 during sister chromatid separation could explain the resistant property of K73.
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80: the resistance spectrum did not cover 80 (Fig. 2A). Tetrad dissection showed that the ts mutation of K73 was single and cosegregated with the property of resistance. A single recessive mutation thus confers both the ts and the resistant properties. The phenotypes of K73 at 36 °C for 2–4 h were observed (Fig. 2C) and found to be identical to the typical phenotype of separase/cut1 or seurin/cut2, displaying the archery bow of non-segregated chromosomes (Hirano et al. 1986; Uzawa et al. 1990).
For chromosome mapping of K73 mutation, isolation of genomic DNA fragments that complement the ts of K73, and random spore analysis were performed. K73 was tightly linked to cut1 locus. The mutation site of cut1-K73 was then determined by the integration rescue of ts phenotype (+ indicates the rescue as depicted in Fig. 2D). The cut1-K73 mutation site existed in the vicinity of six ts mutations in the cut1+ gene (Funabiki et al. 1996a). None of those previously reported ts cut1 mutants were resistant to the toxicity of 81–156, however. The resistant property of cut1-K73 against the 81–156 fragment was thus highly allele-specific.
The amino acid sequences of separase are conserved among certain fungal species (e.g. S. pombe, A. nidlans and N. crassa) in this central domain (Fig. 2E). Nucleotide sequencing of the genomic DNA derived from the mutant cut1-K73 indicated that the substitution occurred at 1004th from Phe to Ser (indicated by red). The position is conserved as the aromatic hydrophobic residues.
The 81–156 fragment locates throughout cell
To gain more information on how 81–156 brought about the toxicity, its intracellular location was determined. The myc-tagged 81–156 was expressed in wild-type cells and immunostained using anti-myc antibodies (Fig. 3A). Control securin/Cut2-myc (top panel) and indestructible 80-myc (middle panel) were seen in the nucleus throughout cell cycle. 80-myc cells displaying the cut phenotype lost the signal due to the cell breakage (indicated by the arrowhead). The (81–156)-myc was present in the whole cell throughout the cell cycle (bottom panel). Intracellular localization of 81–156 was thus not structurally specific, but seemed to be ubiquitous, probably because of its small size and hydrophilic property.
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To examine whether endogenous securin is destroyed in cells that over-expressed 81–156, we use the strain chromosomally integrated with the cut2-myc gene under the native promoter and transformed by plasmid pREP81-cut2(81–156)-HA. Resulting transformant cells could traverse mitosis in the presence of overproduced 81–156 fragment. As shown in Fig. 3B (left panel, stained by anti-Sad1 and DAPI; right panel, stained by anti-myc antibodies), endogenous Cut2 detected by anti-myc antibodies was degraded during mitosis in the presence of excess amounts of 81–156. Nevertheless sister chromatids were not separated in those mitotic cells, whilst the short spindle was present. Control cells overproducing 80 showed basically the same destruction pattern of endogenous Cut2-myc.
Separase/Cut1 is loaded on to the mitotic spindle
In interphase, separase/Cut1 was only faintly seen along cytoplasmic microtubules, but in mitosis its intense localization is seen on the mitotic spindle (Kumada et al. 1998). To examine whether separase/Cut1 was recruited to the mitotic spindle in the presence of the overproduced 81–156, the cut1-GFP gene expressed under the native promoter of chromosomally integrated gene was observed in cells overproducing 81–156 by pREP81-cut2(81–156)-HA. Immunofluorescence microscopy showed that Cut1-GFP signals were seen along the mitotic spindle (stained by anti-tubulin antibodies) as in wild-type cells (Fig. 3C). The loading of separase/Cut1 on to the spindle was thus normal in the excess 81–156. Basically the same observation was made for cells overproducing 80. Securin was shown to be the loading factor (Kumada et al. 1998; Jensen et al. 2001; Hornig et al. 2002) for separase so that endogenous securin was functional in spite of the presence of toxic 81–156.
The large complex of separase-securin maintains it size
It is known in fission yeast cells that the bulk of the securin-separase complex sediments as rather large complexes at 30S and 40S in sucrose gradient centrifugation (Funabiki et al. 1996a). Although the physiological meaning of the large complex formation is not understood, we attempted to determine whether the same large complex could be produced in cells overproducing the (81–156)-HA using sucrose gradient centrifugation. As shown in Fig. 3D, a sedimentation pattern (bottom panel) identical to that seen in the wild-type (top) was obtained: the size of the complex was not affected in the excess of toxic 81–156, which sediment very slowly (< 4S). Consistent with this result, in the immnoprecipitation experiment, approximately the same level of endogenous Cut2 was co-precipitated with Cut1-myc in cells overproducing the 81–156 compared to the control (data now shown). 81–156 fragment did not appear to interfere the association between Cut1 and endogenous Cut2.
The cleavage of cohesin Rad21 is abolished in the presence of 81–156
To determine whether the toxic 81–156 fragment inhibits the cleavage of cohesin Rad21, the synchronous culture was performed by the block and release of cdc25 mutant (Moreno et al. 1989) as depicted in Fig. 4A. S. pombe cdc25 mutant cells containing plasmid pREP81-cut280-HA or pREP81-cut2(81–156)-HA were first grown at 26 °C in the presence of thiamine (+), and then transferred to the culture not containing thiamine (–) at 26 °C for 12 h. At this time, the level of 81–156 was not high enough to inhibit sister chromatid separation. Then the culture was shifted to 36 °C so that cells became arrested at G2 phase. Mutant cells contained the excess amount of 81–156 after 4.25 h, and the synchronous advancement into the M phase occurred upon the temperature shift down to 26 °C. The protein band of the cleaved Rad21 product (indicated in the cleavage column of Fig. 4B and also in Fig. 4C at the time 50 min after the shift) was briefly seen in control cdc25 mutant culture (carrying the vector; top panel) within the period from 50 to 60 min after the shift down. Mitotic cyclin, Cdc13, was degraded at the same timing.
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80 or 81–156 (middle and bottom panels, respectively), however, the cleaved product of Rad21 was absent. Because Cdc13 cyclin degraded at the same timing to that for the wild-type vector, anaphase proteolysis was normal in these cells. Separase was thus inactive in cells overproducing 81–156 and 80. This is the first evidence that 80 as well as 81–156 prevented the activation of separase. An acidic stretch DIE is required for the toxicity of 81–156
To get a clue for generating the toxicity of 81–156, amino acid substitutions were introduced in the fragment, and resulting mutant fragments introduced into plasmid pREP81-HA were used for examining whether the toxicity was still retained. As shown in Fig. 5A, five constructs, EA1, EA2, AAA, EEE and APRRSA were made. EA1 and EA2 were substitutions in the highly acidic region, whilst three other substitutions represented mutations in the putative Cdc2, PKA and MAPK consensus phosphorylation sites present in the 81–156. The actual phosphorylation of S109PRR Cdc2 kinase site was verified using antibodies against phosphopeptide (Y. Kawasaki and M.Y., unpublished observation).
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Mutant strains whose cut2 gene contained the same EA1 or EA2 substitution mutation were made and designated cut2-EA1 or cut2-EA2, respectively. Whereas the cut2-EA1 mutant grew like the wild-type (cut2-WT) at both 26 and 36 °C, the cut2-EA2 mutant displayed the ts phenotype so that no colony formed at 36 °C (Fig. 5D). The strain cut2-EA2 was found to be defective in the mitotic anaphase (Nagao et al. 2004). In addition, interphase DNA damage repair was greatly impaired due to the impaired cleavage of Rad21. The essential sequence DIE in full length Cut2 was thus required for the toxic effect in the fragment, whilst the nonessential ELE in full length did not produce toxicity in the fragment. The DIE motif is hence functionally important in the full length securin/Cut2.
A separase cleavage site is made near the DIE sequence
Inhibition of separase by the DIE-containing securin fragment might implicate the interaction of fragment with the catalytic site of separase. We wondered whether a cleavage site, similar to that in cohesin, could be made in the vicinity of the DIE motif. To examine this possibility, substitution mutations GRD or GRS that were consensus sequences for the separase cleavage site were introduced into Cut2 (Fig. 6A, bottom): MPP was substituted to GRD or GRS (top). In extracts of wild-type cells ectopically expressing Cut2GRD-HA or Cut2GRS-HA, the cleaved products were observed specifically for GRD and GRS by anti-Cut2 antibodies (Fig. 6B, indicated by arrow). Such a product band was not seen for the non-substituted Cut2WT-HA in wild-type cells expressing the non-substituted wild-type Cut2WT-HA. The appearance of the cleaved product was dependent on the separase activity as the band was hardly produced in cut1-T693 mutant cells expressing Cut2GRD or Cut2GRS (right panel). These results suggested that the DIE region of Cut2 resembled the cleavage site of separase and could actually become the site of cleavage if GRD or GRS sequence was properly introduced in its vicinity.
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To determine whether the 81–156 fragment physically interacted with separase/Cut1, full length Cut2, the 81–156 fragment and the EA2 mutated 81–156 fragment, all of which were tagged with HA, were ectopically expressed in wild-type cells, and analyzed by immunprecipitation using anti-HA antibodies (Fig. 6C). Under the experimental condition, the relatively high level of control Cut1-myc was coimmunoprecipitated with full length Cut2-HA. In contrast, a small amount of Cut1-myc was coimmunoprecipitated with the HA-tagged fragment Cut2(81–156)-HA. Little Cut1-myc, however, was coimmunoprecipitated with the AIA mutated fragment Cut2(81–156)EA2-HA, so that the physical interaction between the fragment and Cut1 appeared to be DIE motif dependent. We interpreted these results to show that the interaction between the Cut2 fragment and Cut1 was only dynamic. These results suggest the central domain of Cut2 around the DIE motif might be capable of interacting with the catalytic site of separase.
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Discussion |
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80 Cut2 firmly binds to separase (our unpublished observation) so that separase is never activated. How then can 81–156 be so strongly inhibitory to separase? In sharp contrast to full length Cut2, immunoprecipitation shows that the level of the complex formation between 81–156 and Cut1 is rather low. The 81–156 fragment is found in whole cells, probably not firmly binding to any cellular materials, whilst localization of 80 Cut2 is abundant in the nucleus, identical to that of full length securin until anaphase. We postulate that the inhibitory interaction between 81–156 and separase is transient and/or dynamic. An explanation for the failure to obtain the stable complex with separase is that the excessive 81–156 inhibits separase in a dynamic fashion after the destruction of endogenous securin, and the resulting separase is either denatured or alters to an inactive form. It is known that active separase, a > 200 kDa large protease, is rather unstable. The protease activity of separase in vitro can be only briefly sustained (Uhlmann et al. 2000).
The putative site of interaction in separase for the DIE motif essential for the toxicity and the interaction with separase in the 81–156 fragment may be not exposed outside in the complex that is bound to endogenous securin, but may become accessible to the fragment immediately after securin destruction (Fig. 7B). Isolation of the separase mutant K73 resistant to the toxicity of 81–156 suggests that DIE in the fragment may physically interact with the site in the vicinity of cut1-K73 mutation site (1004Phe). In fact, 1004Phe residue is within a small (
200 amino acids) region where seven ts separase mutants were generated and predicted to be a helix-rich structure (leucine zipper; Uzawa et al. 1990 and our unpublished observation). The coiled coil with adjacent helix breaking residues would bend and reach the distant catalytic site of separase that is located in the carboxy end. If so, 1004Phe locating in the middle of the coiled coil possibly directly interacts with the catalytic site. Note that the sequence conservation around 1004Phe (Fig. 2E) is relatively high, and the presumed coiled coil may be a specific interaction site.
The sequence predictions indicate that the long amino-terminal region of fly separase is alpha-alpha superhelical folds similar to ARM/HEAT repeats (Jager et al. 2004; Viadiu et al. 2005). The hinge is postulated between the amino terminal half and the carboxy terminal conserved regions (Jager et al. 2001; Nagao & Yanagida 2002). Discovery of the regulation of separase activity by CDK in vertebrate (Stemmann et al. 2001) fits well to the hinge hypothesis as the phosphorylation site S1126 in human separase is close to the hinge. Phosphorylation may alter configuration of the hinge so that the hinge activates the separase activity. The hinge is not identified in fungal separases, but the coiled coil containing 1004Phe is present in the middle of separase near the potential hinge region.
How then does the central domain of securin act? The 81–156 sequence is enriched in proline, acidic, and serine/threonine residues. Another characteristic is the alternation of the basic and acidic residues that are clustered. Similar sequence features are found in the securin of budding yeast Pds1 and human PTTG. The 81–156 may take random coil-like amorphous conformation, and can become a fully stretched structure when it is specifically bound to separase. It has phosphorylation sites for Cdc2, Polo, MAPK and PKA. Full length securin containing the 127AIA mutation substituted from 127DIE abolishes interaction with separase (Nagao et al. 2004). Consistently, 127AIA mutation in the 81–156 fragment causes the loss of toxicity. These results strongly suggest that the central domain of securin is the site for regulating separase. Separase is a very large protein so that the greater part of securin except for the amino terminal destruction motifs may participate in binding to separase. Recently, electron microscopic structure of the human separase-securin complex is resolved: securin binds in an extended fashion along separase, rather than in distinct sites (Viadiu et al. 2005) as illustrated in Fig. 7B,C.
It is still possible, however, that the 81–156 fragment affects separase indirectly through other factors such as activator/stabilizer of separase (e.g. AAA ATPase Cdc48; Yuasa et al. 2004) or cleavage enhancer (e.g. polo kinase; Alexandru et al. 2001) that might physically interact with the 81–156 fragment (Fig. 7C). The fragment might be interfered with the unfolding activity of Cdc48 toward separase. Polo kinase phosphorylates the cleavage site and is known to enhance the cohesin cleavage.
How is the interaction between securin and separase connected to separase regulation? In budding yeast and human, securin seems to inhibit separase by preventing the access of substrates to the active site of separase (Hornig et al. 2002; Waizenegger et al. 2002). It is conceivable that securin is a non-cleavable pseudosubstrate that inhibits separase by directly binding to its active site. Alternatively, securin shields separase's active site through inducing a conformational change in separase that blocks substrate access indirectly. We speculate that the toxic central fragment of securin acts on separase as an inhibitor when the destruction of securin occurs (Fig. 7B). Mutation analyses indicate that specific sequence 127DIE129 is essential for producing the toxic effect. Although the sequence identical to the consensus for separase cleavage is not present in the central domain of securin, a part of this domain resembles the cleavage site of separase and may directly interact with the catalytic site of separase. Consistent with this hypothesis, mutations in Cut2 that contain a complete separase recognition site (127DIEYMP to 127DIEYGR) led to cleavage in a separase-dependent manner. Separase activity is known to be inhibited by the peptide inhibitors based on the cleavage site sequences (Uhlmann et al. 2000; Waizenegger et al. 2002). The sequence including 127DIE in Cut2/securin appears to mimic the cleavage sites in Rad21/Scc1 and blocks the substrate entry to the active site of separase. Considering that the separase activity is impaired in cut2-EA2 mutant, the same residues required for separase inhibition may also act as a template for the formation of catalytic pocket.
It is now possible to speculate how securin can become a chaperon and an inhibitor of separase. Securin may stabilize separase by direct interaction and inhibits the premature activation of separase activity until its destruction. The chaperon and inhibitor activity seems to be inseparable in securin. In short, we show that the central domain of securin has a functionally essential specific sequence that may directly interact with the catalytic region of separase. This central securin domain is unrelated to destruction by poyubiquitination, but essential for the activation of separase.
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Experimental procedures |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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S. pombe strains used were derived from haploid h– 972 and h+ 975. cut1 alleles, cdc25-22, cut2-WT, and cut2-EA2 were previously described (Moreno et al. 1989; Funabiki et al. 1996a; Nagao et al. 2004). The complete YPD and the minimal EMM2 media were used (Mitchison 1970). Thiamine (final concentration, 2 µM) was added to repress the nmt1 promoter. Epitope or GFP tagging of the endogenous cut1 or cut2 genes were chromosomally integrated as previously described (Funabiki et al. 1996a, 1996b). The deletion and/or mutated cut2 genes tagged with HA3 or myc8 epitope at the C-terminus were cloned into pREP81 vector (Maundrell 1993) by PCR and verified by nucleotide sequencing. To integrate cut2(81–156)-HA under the control of the nmt1 promoter in REP41 at the leu1 locus, the integration vector pBSleu constructed by the procedure similar to that for pINT5 (Fankhauser & Simanis 1994) was used. Construction of cut2-EA1 mutant was described as previously (Nagao et al. 2004).
Isolation of a temperature-sensitive mutant resistant to the expression of Cut2(81–156)
Wild-type (h– leu1) cells carrying plasmid pREP81-cut2(81–156)-GFP were plated on the plate containing thiamine-deficient EMM2 at 26 °C. Six hundred and ninety pseudorevertants were obtained from 1.6 x 108 cells, in which one strain (K73) did not form colonies on the complete YPD plate (the repressive condition) at 36 °C. Isolation of genomic DNA fragments that complemented the temperature sensitivity of K73 and following genetic linkage analysis showed that the K73 mutation was tightly linked to cut1. Re-transformation followed by tetrad dissection indicated that a single mutation in K73 was responsible for both temperature sensitivity and resistant to the toxic expression of Cut2(81–156) in cut1-K73 (PD:TT:NPD = 9:0:0). Determination of the mutation site of cut1-K73 followed the procedures reported previously (Funabiki et al. 1996a).
Other techniques
Cell extracts were prepared using glass beads in the KB buffer (Nagao et al. 2004). For detecting the cleaved Rad21 or Cut2 protein bands, cell extracts were made in 10% trichloroacetic acid. Immunoprecipitation and sucrose gradient centrifugation were performed as previously described (Funabiki et al. 1996a; Tomonaga et al. 2000) except for the use of KB or KBH buffer (50 mM HEPES-KOH at pH 7.5, 5 mM EDTA, 60 mMß-glycerophosphate, 0.1% NP-40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 2 mg/mL pepstatin A, and complete EDTA-free (Roche)) in cell extraction. DAPI staining was carried as described (Adachi & Yanagida 1989). The procedures for immunofluorescence microscopy were previously described (Hagan & Hyams 1988), and cells were fixed by glutaraldehyde and paraformaldehyde. For cells expressing Cut1-GFP, fixation by methanol at –80 °C for 30 min was employed. Antibodies used were anti-Cdc13 (Stone et al. 1993), anti-Cut1 (Uzawa et al. 1990), anti-Cut2 (Funabiki et al. 1996b), anti-Rad21 (Birkenbihl & Subramani 1992), anti-Sad1 (Hagan & Yanagida 1995), anti-PSTAIR that was used for detection of Cdc2 (a gift from Dr Y. Nagahama), TAT1 for staining alpha-tubulin (a gift from Dr K. Gull), anti-HA 16B12 (Babco), and anti-myc 9E10 (Calbochem), and anti-GFP (Roche).
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: yanagida{at}kozo.lif.kyoto-u.ac.jp, nagao{at}irp.oist.jp
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Received: 31 May 2005
Accepted: 11 December 2005
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) and aberrant segregation (
) in cells with spindle are shown. Wild-type (left) and cut1-K73 mutant (right) cells carrying vector (–) or pREP81-cut2(81–156)-HA (+) were observed at 26 °C 15 h after absence of thiamine. (C) The phenotype of cut1-K73 at 36 °C for 2–4 h. Nuclear chromatin DNA was stained by DAPI. The bar, 10 µm. (D) The integration rescue (
-HA IP pellet). Asterisk and dot indicate a contaminant and IgG light chain band, respectively.