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1 Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan
2 Research Center, Asahi Glass Co. Ltd, Yokohama, Kanagawa 221-8755, Japan
3 National Research Institute of Brewing, Higashi-Hiroshima, Hiroshima 739-0046, Japan
Correspondence
Kaoru Takegawa
takegawa{at}ag.kagawa-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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met26 cells restored growth in the absence of adenine. HPLC analysis showed that total homocysteine content in met26 cells was higher than in other methionine auxotrophs and that introduction of the Sac. cerevisiae cystathionine pathway decreased total homocysteine levels. These data demonstrate that accumulation of homocysteine causes a defect in purine biosynthesis in the met26 mutant. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Marzluf, 1997; Thomas & Surdin-Kerjan, 1997).
In the fission yeast Schizosaccharomyces pombe, biosynthesis of cysteine and related metabolites has been examined extensively at the enzymic level. Brzywczy et al. (2002) reported that Sch. pombe possesses a specific variant pathway for de novo biosynthesis of cysteine. Whereas the budding yeast Saccharomyces cerevisiae synthesizes cysteine via the cystathionine pathway (homocysteine-cystathionine-cysteine; Fig. 1A, e and f), Sch. pombe synthesizes cysteine via the O-acetylserine pathway (serine-O-acetylserine-cysteine; Fig. 1B, g and h). We generated a cysteine auxotroph by blocking the O-acetylserine pathway as a means of investigating biosynthesis in Sch. pombe and have shown that cys1a encodes cysteine synthase, which catalyses the last step in the O-acetylserine pathway (Fig. 1B, h). As expected, a cys1a mutant exhibited cysteine auxotrophy (Fujita & Takegawa, 2004). In contrast to the cysteine biosynthetic pathway, methionine biosynthesis in Sch. pombe, including the steps of sulfur assimilation, have not been studied in great detail at either the enzymic or genetic level. While met6 has been identified as encoding homoserine O-acetyltransferase (Schweingruber et al., 1998) (Fig. 1, c), other genes involved in the methionine biosynthetic pathway are still unidentified.
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, d), was characterized. A met26 mutant exhibited not only methionine auxotrophy but also a growth requirement for adenine, i.e. a purine requirement. In contrast, other methionine auxotrophic mutants did not require adenine. Furthermore, a purine requirement was not observed in a methionine synthase mutant of Sac. cerevisiae. These data suggest that the purine requirement of the Sch. pombe met26 mutant correlates with an accumulation of homocysteine. We demonstrate that disruption of met26 causes homocysteine accumulation and that the accumulation is closely correlated with defective purine biosynthesis in Sch. pombe. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. The parent Sch. pombe strain ARC039 (Fujita & Takegawa, 2004) was provided by Asahi Glass Co. Ltd. Strain YF023 was generated through development of a new transformation system for Sch. pombe (Fujita et al., 2005). Briefly, strain YF023 was generated as follows. ARC039 was converted into an arginine and histidine auxotroph by disruption of arg12 and his2, respectively. Gene disruption was performed by repeated ura4 insertion into a target gene and subsequent positive selection using 5'-fluoroorotic acid. ade7 was generated by disrupting ade7 of ARC039 as described by Fujita et al. (2005). Sac. cerevisiae strains BY4742 and met6 (Clone ID 16396) were obtained from Open Biosystems. Synthetic minimal medium (MM) for Sch. pombe cells was used as described by Alfa et al. (1993). Basal MM medium was supplemented with 240 µg leucine ml–1, 75 µg ml–1 each of uracil, arginine, lysine, histidine and glutamic acid. Sac. cerevisiae strains were grown in synthetic defined (SD) medium (Wickerham, 1946). Basal SD medium was supplemented with 100 µg ml–1 each of leucine, uracil, lysine, histidine and tryptophan. A previously described electroporation method (Suga & Hatakeyama, 2001) was used to disrupt genes in Sch. pombe and a modified lithium acetate method (Morita & Takegawa, 2004) was used for plasmid transformation. Escherichia coli XL-1 Blue (Stratagene) was used for all cloning procedures.
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. Sch. pombe met26, met14 and met16 were disrupted using ura4 as a selective marker (Grimm et al., 1988). The 2·9, 0·85 and 0·98 kb DNA fragments, including the whole or a part of the ORF of met26, met14 and met16, respectively, were amplified from wild-type Sch. pombe genomic DNA and subcloned into pGEM-T Easy (Promega). met26 was disrupted by elimination of a 1·16 kb HindIII–HindIII fragment from the ORF followed by insertion of a ura4 cassette. met14/met16 was disrupted by insertion of a ura4 cassette into a HindIII site within a subcloned ORF fragment. Linearized DNA fragments carrying disrupted genes were used to transform parent strain ARC039 and stable ura4 transformants were selected. Genomic disruption of target genes was confirmed by PCR. Primers used are listed in Table 3. A met26 mutant, YF031, carrying arginine and histidine auxotrophies was also generated by disrupting met26 in strain YF023. Furthermore, YF041 was generated by disrupting ade7 in YF031.
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) as follows. Fragments (0·6 or 0·5 kb) of the upstream or downstream region of the met6 ORF were amplified from wild-type Sch. pombe genomic DNA. Upstream fragments were amplified using a sense primer carrying an XbaI site and an antisense primer carrying a BamHI site. Downstream fragments were amplified using a sense primer carrying a HindIII site and an antisense primer carrying an XhoI site (see Table 3). Amplified fragments were digested with the corresponding restriction enzymes and the XbaI–BamHI upstream fragment and HindIII–XhoI downstream fragment were inserted on either side of the loxP-ura4-loxP cassette. Linearized DNA fragments carrying the upstream and downstream region flanking the loxP-ura4-loxP cassette were used to transform parent strain ARC039. Disruption of the target gene was confirmed by PCR. Mutants were subsequently transformed with the Cre recombinase vector pRep41-Cre. Transformants were subcultured twice on YES plates and several clones that had lost LEU2 activity were picked. Most had lost ura4 activity through elimination of the marker due to loxP site-specific recombination.
Plasmid construction.
pAL-met26 was constructed by integrating full-length met26 into the NotI site of the multi-cloning site (MCS) of pAL-SK (+). pAL was a gift from Dr C. Shimoda (Osaka City University, Japan). A met26 fragment was amplified from wild-type Sch. pombe genomic DNA and subcloned into pGEM-T Easy. met26 fragments were released by digestion with NotI (pGEM-T Easy contains two NotI sites flanking the TA cloning site). met26 was amplified using a newly designed upstream primer met26S2 and the downstream primer met26AS was used to disrupt met26 (Table 3). pREP41-CYS4 and pREP4H-CYS3 were constructed by integrating full-length CYS4 or CYS3 into the NdeI/BamHI site of MCS of pREP41 (Basi et al., 1993) or pREP4H. pREP4H was constructed by replacing the LEU2 marker region of pREP41 with his2 as described by Fujita et al. (2005). Full-length CYS4 or CYS3 fragments were amplified from wild-type Sac. cerevisiae genomic DNA using an upstream primer carrying an NdeI site and a downstream primer carrying a BamHI site (Table 3).
Homocysteine analysis.
The total homocysteine content of each strain was determined using the homocysteine HPLC kit manufactured by Immundiagnostik. Briefly, cells were grown to mid-exponential phase (OD600=0·8–1·8) in YES or defined MM medium and were collected by centrifugation. Collected cells were washed with deionized water, freeze-dried and 20 mg of each strain was suspended in 600 µl 8 mM HCl in a microfuge tube and disrupted by vortexing after addition of 0·43–0·6 µm diameter glass beads (five 30 s pulses). After centrifugation, the supernatant was collected and an aliquot was subjected to fluorescence labelling. HPLC separation was performed on a TSK-GEL ODS-80Ts 4·6 (i.d.)x250 mm column (Tosoh). The mobile phase (1 ml min–1) was 2 % methanol containing 0·1 M acetic acid (pH 4·0) and fluorescence was detected using excitation and emission wavelengths of 385 and 515 nm, respectively. The homocysteine assay was performed on two independent samples and the results from only the first assay are shown because the values from the two experiments were nearly the same.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). As our original intent was to use met26 as a new auxotrophic marker for genetic manipulation of Sch. pombe, a host strain, YF041, carrying six markers (Table 1) was generated. Unexpectedly, overexpression of ade7 in this strain could not fully complement the adenine auxotrophy (data not shown). Subsequently, every strain that carried a met26 mutation was found to barely grow on adenine-free medium. These purine requirements were complemented by overexpression of full-length met26, suggesting that the phenotype was caused by the met26 mutation (Fig. 2).
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, c) (Schweingruber et al., 1998) was disrupted as described in Methods. Homologues of Sac. cerevisiae MET14, encoding adenylylsulfate kinase (EC2.7.1.25; Fig. 1, a), and MET16, encoding 3'-phosphoadenylylsulfate reductase (EC1.8.4.8; Fig. 1, b) were found by searching the Sch. pombe genomic database, yielding SPAC1782.11, designated met14, and SPAC13G7.06, designated met16. The deduced amino acid sequences of met14 and met16 exhibit 65 and 55 % identity with those of Sac. cerevisiae Met14p and Met16p, respectively. Disruptants were generated and were confirmed to be methionine auxotrophs, indicating that met14 and met16 function in methionine biosynthesis. The met6, met14 and met16 mutants were then tested for adenine auxotrophy with met26 serving as a control (Fig. 3). Only the met26 mutant was found to be an adenine auxotroph. A Sac. cerevisiae met6 strain lacking a functional methionine synthase was also tested and was found not to exhibit adenine auxotrophy, in contrast to the equivalent Sch. pombe met26 strain (Fig. 3).
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-synthase (EC4.2.1.22; Fig. 1A, e) and CYS3, encoding cystathionine 
-lyase (EC4.4.1.1; Fig. 1A, f), were overexpressed in the Sch. pombe met26 mutant under the control of the nmt1 (no message in thiamine) promoter (Basi et al., 1993). Strain YF031 (Table 1) was used as a host for this experiment because an additional selective marker was needed. As a preliminary experiment, Sac. cerevisiae Cys4p and Cys3p were tested for activity in Sch. pombe. CYS4 and CYS3 were co-expressed in the Sch. pombe cysteine auxotroph cys1a, which lacks the O-acetylserine pathway (Fujita & Takegawa, 2004). Co-expression of CYS4 and CYS3 in the cys1a mutant restored cysteine prototrophy, indicating functional expression in this strain (data not shown). Strain YF031 co-expressing CYS4 and CYS3 no longer exhibited a purine requirement, but was still auxotrophic for methionine (Fig. 4, lower row). As expected, the purine requirement persisted in the presence of 5 µg thiamine ml–1, which suppressed nmt1-promoter-driven CYS4 and CYS3 expression (Fig. 4, upper row).
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]. These results demonstrate that the met26 mutant indeed accumulates a high level of homocysteine.
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; Klein & Favreau, 1988; Zonneveld & van der Zanden, 1995). In Sch. pombe, defects in ade6 and ade7 lead to accumulation of the intermediate phosphoribosylaminoimidazole (AIR) and phosphoribosylaminoimidazole carboxylate (CAIR), respectively (see Fig. 5a). These intermediates form a characteristic red pigment after transport into the vacuole through the action of a glutathione conjugate pump (Chaudhuri et al., 1996). Parental strain ARC039, the met26 mutant and the met26 ade7 double mutant (YF041) did not form a red colour when grown in YE medium, whereas the ade7 mutant did (Fig. 5b). This result indicates that the met26 mutation inhibits red pigment formation of the ade7 mutant under adenine-limiting conditions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, d) not only causes methionine auxotrophy, but also blocks purine biosynthesis in Sch. pombe. In this study, met26 was identified as the Sch. pombe methionine synthase gene. A met26 mutant exhibited a distinct growth defect on adenine-free medium, suggesting a defect in de novo purine biosynthesis (Fig. 2). A purine requirement was not observed in other Sch. pombe methionine auxotrophs tested, i.e. met14, met16 and met6 mutants. Furthermore, a purine requirement was not observed in a Sac. cerevisiae met6 mutant, lacking a functional methionine synthase (Fig. 3). These results suggested the possibility that the defect in purine biosynthesis in the met26 mutant was caused by intracellular accumulation of homocysteine. In Sch. pombe, intracellular accumulation of homocysteine would result from loss of methionine synthase as methionine synthase is the sole enzyme which converts homocysteine into another metabolite, whereas an alternative homocysteine-consuming pathway exists in Sac. cerevisiae (Brzywczy et al., 2002). This speculation was strongly supported by the observation that introduction of the Sac. cerevisiae cystathionine pathway (Fig. 1A, e–f) into the Sch. pombe met26 mutant complemented the growth defect on adenine-free medium (Fig. 4). met26 cells co-expressing Sac. cerevisiae CYS4 and CYS3 were able to grow on the adenine-free medium, although their growth rate was considerably lower than that of the parent strain ARC039. The lower growth rate of the CYS3- and CYS4-co-expressing met26 strain may be due to the resultant overproduction of cysteine. High levels of cysteine have been reported to be inhibitory to a variety of micro-organisms (Maw, 1961; Allen & Hussey, 1971; Kari et al., 1971).
Measurement of total homocysteine indicated that intracellular levels in the met26 mutant were higher than in the parent or in the other methionine auxotrophs (Table 4). These data demonstrate that high levels of homocysteine impair purine biosynthesis via direct or indirect mechanisms. In mammals, homocysteine has been reported to induce vascular disease via disturbance of various biological processes, and excessive levels in plasma are closely correlated with cardiovascular disease (Aguilar et al., 2004). Homocysteine induces endothelial dysfunction through an inhibition of lysyl oxidase (LOX) and repression of LOX expression (Raposo et al., 2004). Topal et al. (2004) reported that homocysteine induced oxidative stress, which causes vascular damage by inactivating endothelial NO synthase through a reduction of the cofactor tetrahydrobiopterin in human endothelial cells. Homocysteine also diminishes prostacyclin production through inhibition of cyclooxygenase activity (Quere et al., 1995). In Sac. cerevisiae, homocysteine has been reported to interfere with ergosterol biosynthesis (Hatanaka et al., 1974; Parks & Casey, 1995). The methionine biosynthetic pathway is linked indirectly to purine biosynthesis via metabolism of folic acid (Fig. 5a), suggesting a metabolic interaction between the methionine and purine biosynthetic pathways. Holmes & Appling (2002) reported that intracellular accumulation of the purine intermediate 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) interferes with methionine biosynthesis in Sac. cerevisiae. Therefore, a negative feedback loop between the methionine and purine biosynthetic pathways involving the inhibitory effect of homocysteine may exist in Sch. pombe.
While the present study demonstrates an inhibition of purine biosynthesis by elevated homocysteine, the specific target of this inhibition remains unknown. Preliminary analysis using a fission yeast DNA microarray indicated no repression of purine biosynthetic gene expression in met26 mutant cells, consistent with inhibition occurring at the post-translational level (data not shown). The observation that the met26 mutation inhibits accumulation of red pigment in ade7 cells under adenine-limiting conditions indicates that homocysteine accumulation interferes with a step prior to the series of reactions catalysed by the enzymes encoded by ade6 and ade7 (Fig. 5b). Further work to determine the target for homocysteine inhibition is in progress.
We also tested whether homocysteine accumulation can block purine biosynthesis in Sac. cerevisiae. A met6 cys4 double mutant unable to metabolize homocysteine (see Fig. 1A) was generated and tested for auxotrophies. Whereas the Sac. cerevisiae met6cys4 double mutant was not an adenine auxotroph, it did exhibit methionine and cysteine auxotrophy (data not shown). Intriguingly, intracellular accumulation of homocysteine was not observed in the met6cys4 double mutant when grown in adenine-free SD medium, but a remarkable accumulation of homocysteine was observed when grown in YPD medium (data not shown). It was therefore unclear whether homocysteine accumulation interferes with purine biosynthesis in Sac. cerevisiae. Further investigation is needed to elucidate the mechanism of suppression of homocysteine accumulation in the met6cys4 double mutant grown in adenine-free medium.
To our knowledge, this study describes the first case of inhibition of purine biosynthesis by excessive levels of homocysteine in eukaryotic cells. Homocysteine accumulation in a methionine synthase mutant was found to occur specifically in Sch. pombe that possesses a variant pathway of sulfur metabolism. Elucidation of the mechanisms responsible for the homocysteine-inducible defect in purine biosynthesis in Sch. pombe may shed light on the mode of action of homocysteine and related metabolites in higher eukaryotes.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 1 August 2005;
revised 11 October 2005;
accepted 27 October 2005.
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