Transcriptional regulation of Saccharomyces cerevisiaeCYS3 encoding cystathionine γ-lyase
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- Hiraishi, H., Miyake, T. & Ono, B. Curr Genet (2008) 53: 225. doi:10.1007/s00294-008-0181-2
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In studying the regulation of GSH11, the structural gene of the high-affinity glutathione transporter (GSH-P1) in Saccharomyces cerevisiae, a cis-acting cysteine responsive element, CCGCCACAC (CCG motif), was detected. Like GSH-P1, the cystathionine γ-lyase encoded by CYS3 is induced by sulfur starvation and repressed by addition of cysteine to the growth medium. We detected a CCG motif (−311 to −303) and a CGC motif (CGCCACAC; −193 to −186), which is one base shorter than the CCG motif, in the 5′-upstream region of CYS3. One copy of the centromere determining element 1, CDE1 (TCACGTGA; −217 to −210), being responsible for regulation of the sulfate assimilation pathway genes, was also detected. We tested the roles of these three elements in the regulation of CYS3. Using a lacZ-reporter assay system, we found that the CCG/CGC motif is required for activation of CYS3, as well as for its repression by cysteine. In contrast, the CDE1 motif was responsible for only activation of CYS3. We also found that two transcription factors, Met4 and VDE, are responsible for activation of CYS3 through the CCG/CGC and CDE1 motifs. These observations suggest a dual regulation of CYS3 by factors that interact with the CDE1 motif and the CCG/CGC motifs.
KeywordsCYS3 Cystathionine γ-lyase Regulatory motifs Sulfur metabolism Saccharomyces cerevisiae
Genetic control of methionine biosynthesis has long been of interest (Thomas and Surdin-Kerjan 1997). Researchers in the de Robichon-Szulmajster Laboratory conducted an intensive study in Saccharomyces cerevisiae of methionine biosynthesis (Masselot and de Robichon-Szulmajster 1975), but its relationship to cysteine biosynthesis has remained obscure. S. cerevisiae can grow using methionine, homocysteine, cysteine, or glutathione as the sole sulfur source (Ono et al. 1984, 1988; Cherest and Surdin-Kerjan 1992). Subsequent work has determined that S. cerevisiae can reduce sulfate to form sulfide which is utilized to synthesize homocysteine (the sulfate assimilation pathway), and homocysteine-S is then converted to cysteine-S through cystathionine-S (the reverse trans-sulfuration pathway) (Ono et al. 1999). The reverse trans-sulfuration pathway consists of two enzymes, cystathionine β-synthase (βCTSase; EC 126.96.36.199) encoded by CYS4 and cystathionine γ-lyase (γCTLase; EC 188.8.131.52) encoded by CYS3.
In addition, we identified a high-affinity glutathionine transport system (GSH-P1) encoded by GSH11/HGT1/OPT1 in S. cerevisiae (Bourbouloux et al. 2000; Hauser et al. 2000; Miyake et al. 1998), and demonstrated that GSH11 is induced by sulfur starvation and repressed by addition of cysteine to the culture medium through a 5′-upstream sequence (CCGCCACAC), hereafter referred as the CCG motif (Miyake et al. 2002). In the course of that study, we identified VDE and seven additional proteins that bind to the CCG motif. The homing endonuclease VDE, which is produced by processing of the TFP1/VMA1 gene product (Hirata et al. 1990; Kane et al. 1990), is thought to function as a transcriptional activator of GSH11 because GSH11 is not expressed in a VDE-deleted strain, and the observed inability to express GSH11 is overcome by the introduction of the coding region of VDE or the entire VMA1 gene (Miyake et al. 2003). Given the observation that the activity of γCTLase increases markedly under sulfur starvation and decreases markedly when cells are grown in the presence of organic sulfur compounds such as glutathione, methionine, and cysteine (Ono et al. 1991), we decided to investigate the regulation of CYS3.
Materials and methods
Strains and plasmids
The S. cerevisiae strains used in this study were SF1-1C (MATa leu2 trp1 met17) and YPH500 (MATα ade2 lys2 his3 leu2 trp1 ura3), which are wild-type with respect to γCTLase activity and regulation. In the YOC2176 [MATα ade2 lys2 his3 leu2 trp1 ura3 VMA1-101 (vde-delta)] strain, the VDE region has been disrupted (Miyake et al. 2003) and we constructed the YPH500m4 (MATα ade2 lys2 his3 leu2 trp1 ura3 met4::CgHIS3) strain, by disrupting MET4 in the YPH500 strain. The Escherichia coli strain DH10B was used for amplification of plasmids. We used p69-2-1 (Ono et al. 1992) as a template for PCR amplification of CYS3, pBluescriptII SK+ (TOYOBO, Osaka, Japan) for sequencing, pMC1587 (containing the lacZ coding region (Casadaban et al. 1983)) for analysis of the CYS3 promoter region and pCgHIS3 (supplied by Dr. Harashima) for disruption of the MET4 gene.
Growth media and growth conditions
We used standard yeast growth media (Sherman et al. 1986) such as YPD (1% yeast extract, 2% peptone, and 2% glucose) and synthetic minimal (SD) medium. Sulfur-free (SF) medium was prepared by replacing the sulfate salts in the SD medium (Wickerham 1956) with the corresponding chloride salts (Ono et al. 1991). Where appropriate, supplements were added to the SD and SF media. For example, MET and CYS media were prepared by the addition of 100 μM methionine and 100 μM (derepression) or 10 mM (repression) cysteine. Escherichia coli was cultivated in LB broth (1% tryptone, 0.5% yeast extract, and 0.5% NaCl, pH 7.2) (Sambrook et al. 1989), with the addition of 50 μg/ml ampicillin when required. Solid media contained 2% agar. Saccharomyces cerevisiae and E. coli were grown at 30 and 37°C, respectively, and liquid cultures were incubated on a reciprocal shaker at 120 rpm.
Disruption of MET4
A DNA fragment containing the Candida glabrata HIS3 gene was amplified by PCR from pCgHIS3 using the primers: 5′-AGT CCC ACG AAG GCG ACT CAT ACA GCA CGG AAT TCA TAA AGT TGT AAA ACG ACG GCC AGT-3′ and 5′-ATG GAG CTT AGA AAA GAA GCC TCT GCT ACT ACA CCG TGC TCA CAG GAA ACA GCT ATG ACC-3′. The italicized sequences correspond to 10 bp downstream from the MET4 ATG initiation codon and 951 bp upstream from the TAG termination codon, respectively. PCR-amplification was performed for one cycle at 94°C for 1 min, 56°C for 30 s, and 1 min at 72°C, 30 cycles at 94°C for 30 s, 60°C for 30 s, and 1 min at 72°C, with a final extension step at 72°C for 7 min. The YPH500 strain was transformed with the 1.78 kb PCR product and His+ Met− transformants were obtained. We confirmed that the transformant YPH500m4 was met4− by complementation with the MET4-bearing plasmid pYCM4 (provided by Dr. Omura, Institute for Liquor Products, Suntory Ltd.).
Electrophoretic mobility shift assays
Saccharomyces cerevisiae SF1-1C was grown overnight in YPD and then transferred to either SF or fresh YPD media. After a 17 h incubation, the cells were harvested by centrifugation, washed once with homogenization buffer (20 mM Tris–HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol (DTT), 10% glycerol, 1 mM phenylmethylsulphonyl fluoride, 1 mM benzamidine–HCl) and resuspended in 0.3 ml of the same buffer with 0.4 ml glass beads. The cell suspension was subjected to ten cycles of shaking for 30 s and cooling in an ice bath for 60 s; cell debris was then removed by centrifugation. The supernatant was used as the cell extract.
Primers used for amplification of the 5′-upstream fragments of CYS3
AAT ACC CGG GTT CCA CCA GTG GGC CCA GTG
AAT ACC CGG GAG TGA CCG CCA CAC TGG ACC
AAT ACC CGG GTG GAC CCC ATA CCA CTT CTT
AAT ACC CGG GTA ACG CTA TGT AAT TCC ACC
AAT ACC CGG GAT TCA CGT GAT CTC AGC CAG
AAT ACC CGG GAG TAC AAT TGA GGC CTA TAC
AAT ACC CGG GCG CCA CAC TTT TTT TTC C
AAT ACC CGG GTT TTT TTT CCA TAA AAA T
AAT ACC CGG GCC GCC ACA CTT TTT TTT CC
CTG GCT GAG ATC ACG TGA AT
GGT GGA ATT ACA TAG CGT TA
TAA TTA TTA GTA ATG AAG GG
Competition experiments were performed as described above, with the addition of 100 pmol unlabeled oligonucleotides as the competitor. Four unlabeled double stranded fragments (URM-1, URM-2, URM-3, and URM-4) were custom-synthesized (Amersham Pharmacia Biotech).
Construction of plasmids for the lacZ reporter assay
Using p69-2-1 and the primers listed in Table 1, we PCR-amplified various fragments containing different segments of the 5′-upstream region of CYS3. The nucleotide sequence of the reverse primer (USC3-rev) encoded 12 amino acids from the N terminus of γCTLase and a BamHI site was designed near the 5′ end. This reverse primer was used in conjunction with forward primers to various segments of the 5′-upstream region of CYS3. Forward primers contained a SmaI site at the 5′ end. Although most PCR fragments were digested with SmaI and BamHI, and then ligated into the SmaI/BamHI site of pMC1587, fragments F20, F22 and F23 were digested with SmaI and ligated into the SmaI site in the F19 plasmid (Fig. 5). All gene constructs encoded proteins in which the first eight amino acids of β-galactosidase were replaced by the first 12 amino acids of γCTLase.
Assay for β-galactosidase activity
Cells were incubated in CYS (SF medium supplemented with 10 mM cysteine) or SF media for 17 h, then harvested and resuspended in 1 ml of Z buffer (Sherman et al. 1986), followed by treatment with 0.05 ml chloroform and 0.05 ml 0.1% SDS. β-galactosidase activity was measured at 30°C as the mean rate of hydrolysis of o-nitrophenyl-β-d-galactopyranoside (ONPG) to o-nitrophenol (ONP) and galactose. β-galactosidase activity was calculated using the formula A420 × 1,000 min−1 ml−1 OD600−1.
Electrophoretic mobility shift assays using segments from the 5′-upstream region of CYS3
lacZ reporter assays
Analysis of 5′-upstream region of CYS3
β-Galactosidase specific activities (U min−1 OD600−1)
0 mM Cys (D)
10 mM Cys (R)
0 mM Cys (D)
10 mM Cys (R)
0 mM Cys (D)
10 mM Cys (R)
617.4 ± 116
309.1 ± 54
446.3 ± 26
292.4 ± 71
42.7 ± 6.2
29.5 ± 8.3
2.9 ± 0.2
2.8 ± 0.9
26.2 ± 7.7
26.0 ± 1.4
31.2 ± 6.6
17.4 ± 5.3
10.4 ± 2.7
17.3 ± 1.2
522.6 ± 40
97.1 ± 12
166.8 ± 22
121.0 ± 29
1.5 ± 3.6
18.0 ± 3.0
429.4 ± 42
146.4 ± 46
161.1 ± 16
120.2 ± 27
36.5 ± 6.5
25.2 ± 5.6
241.1 ± 41
181.1 ± 34
167.5 ± 29
197.2 ± 37
97.1 ± 1.5
71.7 ± 4.0
586.6 ± 61
216.4 ± 47
386.3 ± 76
278.9 ± 55
28.5 ± 8.9
21.2 ± 3.4
642.8 ± 61
216.1 ± 16
390.5 ± 50
298.9 ± 81
35.1 ± 2.7
24.8 ± 9.5
245.1 ± 18
277.5 ± 27
183.4 ± 18
197.0 ± 19
111.9 ± 31
57.7 ± 17
568.4 ± 32
279.1 ± 63
374.5 ± 64
271.8 ± 49
104.0 ± 15
60.3 ± 2.2
In contrast, the F18 construct (containing the CGC motif in addition to the two TATA boxes) exhibited 80% the activity of the F07 construct in the absence of cysteine, and 30% the activity of F07 in the presence of 10 mM cysteine, indicating that the CGC motif is responsible for strong activation in the absence of cysteine, and strong repression in its presence (5-fold induction). Since the F21 construct (containing the CCG motif located in the position of the CGC motif) behaved similarly to the F18 construct, it is possible that, in closer proximity to the coding region, the CCG motif can function in a similar manner to the CGC motif (2.9-fold induction). The F20 construct (containing the CCG motif and its adjacent downstream regions) exhibited approximately half the activity of the F21 construct in the absence of cysteine. Notably, the F20 construct contains not only the CCG motif but also the adjacent regions. Therefore, we concluded that either the sequence(s) adjacent to the CCG motif are inhibitory or the distance between the motif and its target gene is critical for the activator function of the CCG motif.
As observed in comparison of the F10, F08, and F23 constructs, the presence of the CDE1 motif plus either the CGC motif or the CCG motif caused a high level of constitutive expression of the target gene. In contrast, the CCG and CGC motifs alone are responsible for a high level of inducible expression.
Involvement of the homing nuclease VDE in the regulation of CYS3
To ascertain the involvement of VDE in regulation of CYS3, lacZ reporter constructs were introduced into the YOC2176 strain (a vde-delta derivative of YPH500; Table 2, 2nd column). In the absence of cysteine, F07 (containing all three motifs of interest) exhibited somewhat lower activity in the Δvde mutant (YOC2176) than in the wild-type strain (YPH500); that is, the fold induction by F07 was 2× for the wild-type strain and 1.5× for the Δvde mutant. In the presence of cysteine, there was no detectable difference between the two strains. A more significant difference was observed for the F18 (CGC motif) and F21 (CCG motif) constructs. In the absence of cysteine, both constructs exhibited approximately one-third the activity in the Δvde mutant as in the wild-type strain. In contrast, there was no detectable difference between the strains in the presence of cysteine. F22 (CDE1 motif) caused slightly lower gene expression in the Δvde mutant, relative to the wild-type strain, in the absence of cysteine. F10 showed approximately two-thirds the level of gene expression in the Δvde mutant relative to the wild-type strain in the absence of cysteine, while both strains exhibited similar levels of gene expression in the presence of 10 mM cysteine. Therefore, VDE may play a role in activation of CYS3. In the absence of cysteine, similar β-galactosidase activities were observed with the F07, F10, F08, and F23 constructs. The latter three constructs contain the CDE1 motif plus either the CGC (F10 and F08) or CCG motif (F23); the level of gene expression due to these constructs was reduced by the addition of cysteine. In contrast, the F18 (CGC motif alone), F20 and F21 (CCG motif alone), and F22 (CDE1 motif alone) constructs induced intermediate levels of gene expression, and the expression level was not affected by the addition of cysteine. Hence, the expression of CYS3 via the CGC motif or the CCG motif is weaker in the Δvde mutant than in the wild-type strain.
These results suggest the following conclusions: (1) the CGC and CCG motifs are equally effective for CYS3 activation, and this activation is not subjected to repression by cysteine; (2) the CDE1 motif alone exhibits a level of activation similar to the CGC and CCG motifs; and (3) the CGC and CCG motifs increase activation in the presence of the CDE1 motif, and this activation is subject to repression by cysteine. The wild-type strain (YPH500) and the vde-delta strain (YOC2176) exhibited a marked difference in activation mediated by the CGC (F18) or CCG (F21) motif and by combining the CDE1 motif with either the CGC (F10 and F08) or CCG (F23) motif. Based on the present observations, together with our previous finding that VDE binds the CCG motif (Miyake et al. 2003), we contend that VDE plays a role in activation of CYS3 via the CGC and CCG motifs.
Role of MET4 in the regulation of CYS3
In order to investigate the role of MET4 in regulation of CYS3, we constructed the met4 mutant YPH500m4 and performed lacZ reporter assays using this strain (Table 2, the 3rd column). In all assays, the level of β-galactosidase activity was low, suggesting that MET4 is required for regulation mediated by each of the three motifs. However, the F22 construct (CDE1 motif alone) exhibited a higher level of induction in the met4 mutant (YPH500m4) than in the wild-type strain (YPH500); that is, the fold induction for construct F22 was 0.9× for the wild-type strain and 1.9× for the met4 mutant. This result suggests that an auxiliary transcription factor other than Met4 is involved in expression of CYS3 under sulfur starvation conditions.
In this study, we examined the roles of three putative regulatory motifs present in the 5′-upstream region of the S. cerevisiae CYS3 gene, CGC (−193 to −186), CDE1 (−217 to −210), and CCG (−311 to −303). We found that the CCG and CGC motifs play roles in expression of the target gene and that differences observed between the motifs are largely attributable to their proximity to the target gene. As the CGC motif (CGCCACAC) is only one nucleotide shorter than the CCG motif (CCGCCACAC), we suggest that the functional body of the motifs is identical and should be referred to as the CCG/CGC motif. Activation by the CCG/CGC motif is repressed by 10 mM cysteine, and this response is lowered substantially in the absence of VDE, suggesting that VDE binds directly or indirectly to the CCG/CGC motif to activate the target gene. We have also found that a high concentration of cysteine (10 mM) is necessary for repression of the CYS3 gene. Our result is consistent with the results reported by Hansen and Johannesen (2000), in that cysteine repression of the sulfate assimilation pathway genes MET14 and MET25 occurred at a concentration of 10 mM cysteine. Thus, quite high levels of cysteine appear to be needed for repression of the CYS3, MET14, and MET25 genes.
In this investigation, we focused on the CCG/CGC and CDE1 regulatory motifs. However, CYS3 also possesses a Met31/Met32 binding site (MBS motif; −198 to −193) (Blaiseau et al. 1997; Chiang et al. 2006). Our data (Table 2) indicate that the CCG/CGC motif regulates expression of CYS3, but the MBS motif does not. In the 5′-upstream region of CYS3, there are also regulatory motifs that appear to be involved in the stress response, such as the yAP-1 response element (YRE motif; −235 to −229) (Lee et al. 1999) and the Xbp1 binding site (XBS motif; −166 to −161) (Mai and Breeden 2000). Since glutathione plays a central role in the cellular response to oxidative stress and is synthesized using cysteine as a substrate, there appears to be a link between expression of CYS3 and the roles of these stress response motifs. This line of investigation remains for future studies.
This work was supported by funds from the Bio-Venture project and the 21st century COE program of Ritsumeikan University. We are grateful to S. Harashima (Osaka University) and F. Omura (Institute for Liquor Products, Suntory Ltd) for their kind gifts of plasmids containing Candida glabrata HIS3 and MET4, respectively.
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