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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In Saccharomyces cerevisiae, the synthesis of inositol pyrophosphates is essential for vacuole biogenesis and the cell's response to certain environmental stresses. The kinase activity of Arg82p and Kcs1p is required for the production of soluble inositol phosphates. To define physiologically relevant targets of the catalytic products of Arg82p and Kcs1p, we used DNA microarray technology. In arg82Δ or kcs1Δ cells, we observed a derepressed expression of genes regulated by phosphate (PHO) on high phosphate medium and a strong decrease in the expression of genes regulated by the quality of nitrogen source (NCR). Arg82p and Kcs1p are required for activation of NCR-regulated genes in response to nitrogen availability, mainly through Nil1p, and for repression of PHO genes by phosphate. Only the catalytic activity of both kinases was required for PHO gene repression by phosphate and for NCR gene activation in response to nitrogen availability, indicating a role for inositol pyrophosphates in these controls. Arg82p also controls expression of arginine-responsive genes by interacting with Arg80p and Mcm1p, and expression of Mcm1-dependent genes by interacting with Mcm1p. We show here that Mcm1p and Arg80p chaperoning by Arg82p does not involve the inositol polyphosphate kinase activity of Arg82p, but requires its polyaspartate domain. Our results indicate that Arg82p is a bifunctional protein whose inositol kinase activity plays a role in multiple signalling cascades, and whose acidic domain protects two MADS-box proteins against degradation.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In yeast it is known that membrane-bound inositol lipids play important roles in adaptation to environmental stresses. More recently it has been shown that soluble inositol polyphosphates, especially inositol pyrophosphates play important roles in diverse cellular processes, such as cell wall maintenance, vacuolar morphogenesis, resistance to salt stress and also mediate homologous DNA recombination in yeast (Luo et al., 2001; Dubois et al., 2002). Arg82p and Kcs1p are two inositol polyphosphate kinases (Saiardi et al., 1999; 2000; York et al., 1999). Arg82p converts Ins(1,4,5)P3 to Ins(1,3,4,5)P4 and Ins(1,4,5,6)P4, which are converted to Ins(1,3,4,5,6)P5. Kcs1p converts Ins(1,3,4,5,6)P5 and InsP6 to different inositol pyrophosphates, PPInsP4, PPInsP5 and (PP)2InsP3 (Saiardi et al., 1999; 2000) (Fig. 1). Cells deleted of ARG82 or KCS1 genes display severe growth defect at high temperature, vacuolar fragmentation, increased leakiness of cellular phosphatase and hypersensitivity to salt stress (Dubois et al., 2002). A strain impaired in Arg82p kinase activity not only presents a strong reduction in Ins(1,3,4,5)P4, Ins(1,4,5,6)P4, Ins(1,3,4,5,6)P5 and InsP6 pools, but also a significant reduction of inositol pyrophosphates, whereas impairing Kcs1p kinase activity only decreases the synthesis of inositol pyrophosphates (Dubois et al., 2002). Consequently, the defects observed in both deleted strains would result from very low amounts of inositol pyrophosphates.

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Figure 1. Pathway for the synthesis of soluble inositol polyphosphates.

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Arg82p plays a second important role in the cell by stabilizing Mcm1p a protein essential for cell viability, and controlling G1/S and G2/M cell cycle transitions (Althoefer et al., 1995; Oehlen et al., 1996; McInerny et al., 1997), mating (Jarvis et al., 1989), osmotolerance (Kuo et al., 1997), recombination (Elble and Tye, 1992), minichromosome maintenance (Passmore et al., 1988) and arginine metabolism (Messenguy and Dubois, 1993). Arg82p stabilizes another MADS-box protein Arg80p, which is also required for the formation of a regulatory complex with the arginine sensor Arg81p, at the ‘arginine boxes’ present in the co-regulated arginine genes (Amar et al., 2000; El Bakkoury et al., 2000). Thus, for the arginine metabolism Arg82p acts as a protein chaperone, and Dubois et al. (2000; 2002) have shown that Arg82p kinase activity was not required for this function. However, it was not established whether the kinase activity of Arg82p was required for Mcm1p-dependent gene expression.

The cell response to diverse stresses requires efficient MAP kinase signalling pathways (Banuett, 1998) which could be perturbed in strains lacking inositol pyrophosphates. To identify the regulatory network of Arg82p and Kcs1p, we conducted genome wide analysis. Among a set of genes whose expression was increased or decreased in strains deleted of ARG82 or KCS1 genes compared to wild-type strain, two families of genes emerged strikingly. In arg82Δ or kcs1Δ cells, genes controlled in response to the quality of the nitrogen source (NCR) were downregulated, whereas genes controlled by phosphate availability (PHO) were upregulated in high phosphate medium. In this study we also show that the control of NCR and PHO genes require the kinase domains of Arg82p and Kcs1p, but not the polyaspartate stretch in Arg82p involved in Mcm1p stabilization.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

In arg82Δ and kcs1Δ strains, NCR genes are downregulated and PHO genes are upregulated

To determine the specific regulation network of Arg82p and Kcs1p, we used DNA microarrays allowing to analyse changes in transcript abundance in arg82Δ strain compared to the same strain overexpressing ARG82 (ptetARG82) or to the wild-type strain, and in kcs1Δ strain compared to the same strain overexpressing KCS1 (ptetKCS1) or to the wild-type strain. Expression of 14 genes controlled in response to the quality of the nitrogen source was downregulated in strains deleted either of ARG82 or KCS1 genes. In contrast, expression of 15 other genes controlled in response to phosphate availability was upregulated in the presence of high phosphate concentration (Table 1). These microarrays did not identify other gene families whose expression was dependent on Arg82p or Kcs1p, not even genes involved in vacuole morphogenesis, salt resistance or cell wall integrity as might be expected from the phenotypes associated with arg82Δ and kcs1Δstrains.

Table 1. . New gene categories regulated by Arg82p and Kcs1p.
ORF identityGeneaRatio tet-ARG82/ arg82Δ ± SD aRatio tet-KCS1/ kcs1Δ ± SD
  • a

    . Expression of genes downregulated (ratios higher than 1), and upregulated (ratios lower than 1) in arg82Δ and kcs1Δ strains. The mean expression ratio was determined by microarray analysis comparing a strain overexpressing Arg82p (tet-ARG82) to arg82Δ strain, and a strain overexpressing Kcs1p (tet-KCS1) to kcs1Δ strain. Each value is the trimmed mean, excluding a single value from the top and bottom of the data set, for seven independent arrays.

YLR158cASP3-34.90 ± 1.125.10 ± 1.74
YLR160cASP3-44.89 ± 0.973.92 ± 1.65
YLR157cASP3-24.76 ± 0.994.67 ± 1.78
YLR155cASP3-13.40 ± 0.693.78 ± 1.06
YJL172wCPS12.70 ± 0.541.34 ± 0.15
YKR039wGAP12.66 ± 0.291.52 ± 0.32
YKR033c 2.64 ± 0.941.58 ± 0.23
YNL142wMEP22.58 ± 0.291.26 ± 0.17
YJR152wDAL52.51 ± 0.772.20 ± 0.54
YIR032cDAL31.82 ± 0.351.07 ± 0.21
YIR029wDAL21.78 ± 0.581.50 ± 0.20
YIR031cDAL71.74 ± 0.671.07 ± 0.12
YPR035wGLN11.70 ± 0.181.46 ± 0.16
YBR208cDUR1,21.52 ± 0.171.39 ± 0.24
YML123cPHO840.06 ± 0.020.12 ± 0.02
YAR071wPHO110.07 ± 0.010.40 ± 0.05
YHR215wPHO120.11 ± 0.040.47 ± 0.09
YBR296cPHO890.18 ± 0.090.58 ± 0.11
YPL019cVTC30.19 ± 0.040.27 ± 0.02
YER072wVTC10.25 ± 0.050.24 ± 0.03
YHR136cSPL20.26 ± 0.060.37 ± 0.03
YER072wNRF10.30 ± 0.040.42 ± 0.04
YJL117wPHO860.39 ± 0.010.61 ± 0.06
YDR281cPHM60.40 ± 0.080.97 ± 0.12
YDR481cPHO80.50 ± 0.050.68 ± 0.06
YGR233cPHO810.56 ± 0.030.67 ± 0.07
YFL004wVTC20.60 ± 0.130.67 ± 0.12
YPL110c 0.61 ± 0.150.91 ± 0.03
YJL012cVTC40.65 ± 0.080.54 ± 0.03

In order to validate the results obtained for NCR and PHO genes, we performed Northern blotting analysis using DAL5, DAL7, ASP3, PHO5, PHO11, PHO84, VTC3 and NRF1 probes. The results presented in Fig. 2 clearly confirmed that Arg82p and Kcs1p were required for NCR gene expression and for repression of phosphate-regulated genes. The mRNA levels of PHO and NCR genes were comparable in a wild-type strain and in a strain overexpressing ARG82 or KCS1, indicating that high overexpression did not exaggerate the influence of Kcs1p or Arg82p on the expression of NCR and PHO genes (Fig. 2). Because PHO5 is strongly regulated by phosphate (Toh-E et al., 1973), we included this gene in our mRNA analysis, although PHO5 was not identified in the microarray experiments indicating that some genes may escape detection in such experiments. It is noteworthy that in many cases there are discrepancies between the gene expression ratios from microarray analysis (Table 1) and the Northern blots, comparing wild-type and mutant strains. In our hands, the microarray technique seems often less sensitive than Northern blotting experiments.

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Figure 2. Northern blotting of NCR and PHO regulated genes in strains deleted or overexpressing ARG82 or KCS1 genes. Total RNAs were extracted from different strains grown on synthetic medium (see Experimental procedures) and 30 µg were analysed by Northern blotting with 32P-labelled DAL5, DAL7, ASP3, PHO5, PHO11, PHO84, VTC3, NRF1 and ACT1 (control) DNA probes. Strains were deleted of ARG82 (lanes 2 and 3) or KCS1 genes (lanes 4 and 5). arg82Δ strain was transformed with pCM262 (pURA3, lane 2) or pFV186 (pURA3, tet-ARG82, lane 3) plasmids. kcs1Δ strain was transformed with pCM262 (pURA3, lane 4) or pFV187 (pURA3, tet-KCS1, lane 5) plasmids (see Experimental procedures).

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To test the biological relevance of the effect of inositol pyrophosphates on the expression of PHO and NCR genes, we fused the PHO5 promoter to the coding sequence of HIS3 (pFV309) and the DAL7 promoter to the coding sequence of URA3 (pFV313). These plasmids were used to transform the control diploid strain BY4743 (ura3/ura3, his3/his3, leu2/leu2) and the kcs1Δ diploid strain (ura3/ura3, his3/his3, leu2/leu2, kcs1::kanMX4/kcs1::kanMX4). The expression of HIS3 or URA3 was tested by growth of the transformed strains in the absence of histidine or uracil respectively. At high phosphate concentration (see Experimental procedures), a growth condition which prevents expression of PHO genes, the absence of histidine led to a slower growth of the wild-type strain compared to the growth in the presence of histidine, and addition of 10 mM 3-amino triazole completely abolished the growth (Fig. 3A). In contrast, the kcs1Δ strain transformed with the plasmid expressing PHO5-HIS3 grew in the presence of 10 mM 3-amino triazole without histidine as well as in the presence of histidine, despite the fact that a kcs1Δ strain grows more poorly than the wild-type strain. These growth tests show that the amount of the HIS3 gene product in a kcs1Δ strain is sufficient to ensure growth without addition of histidine, and confirm the derepression of PHO5 in this mutant strain. The wild-type strain transformed with the plasmid expressing DAL7-URA3 (pFV313) grew well on M. ammonia or glutamine media, two repressive nitrogen sources, even in the absence of uracil. In contrast in a kcs1Δ background, the growth on M. ammonia was reduced and nearly abolished on glutamine as nitrogen source (Fig. 3B). This is consistent with the stronger repression of NCR gene expression by glutamine than by ammonia. The weak expression of DAL7-URA3 in the kcs1Δ strain grown on glutamine medium was confirmed by the ability of this strain to grow on a medium containing 5-fluoroorotic acid + uracil (data not shown). Thus, when inositol pyrophosphates are reduced, expression of an NCR-regulated gene such as DAL7 is sufficiently low to reduce the amount of URA3 product, leading to resistance on 5-FOA and to an auxotrophy on glutamine as nitrogen source.

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Figure 3. Growth tests showing the effect of a kcs1 deletion on the expression of PHO and NCR-dependent reporter genes. A. Tenfold serial dilutions of cells from the diploid strain (BY4743, ura3, leu2, his3) transformed with plasmid pFV309, expressing gene HIS3 under the control of PHO5 promoter, and the diploid kcs1Δ strain (4743kcs1Δ) transformed with the same plasmid, were plated and incubated at 30°C for 4 days on the different media indicated. AT means 3-amino triazole. B. Tenfold serial dilutions of cells from the diploid strain (BY4743, ura3, leu2, his3) transformed with plasmid pFV313, expressing gene URA3 under the control of DAL7 promoter, and the diploid kcs1Δ strain (4743kcs1Δ) transformed with the same plasmid, were plated and incubated at 30°C for 4 days on the different media indicated.

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Influence of Arg82p and Kcs1p on PHO and NCR gene expression under different physiological conditions

The PHO regulatory system consists of at least five PHO-specific regulatory proteins, the Pho2p and Pho4p transcriptional activators, the Pho80p-Pho85p cyclin-cyclin dependent protein kinase (CDK) complex, and the Pho81 CDK inhibitor. When the Pi concentration in the medium is high, Pho85p kinase is active and hyperphosphorylates Pho4p, which is maintained in the cytoplasm (Kaffman et al., 1994; Schneider et al., 1994; Oshima, 1997). At low Pi concentration Pho81p inhibits the Pho80p-Pho85p kinase preventing the phosphorylation of Pho4p which is then preferentially localized in the nucleus, where together with Pho2p it activates target gene transcription (Kaffman et al., 1994; Komeili and O’Shea, 1999). To determine whether Arg82p and Kcs1p are required for repression of PHO genes by phosphate, we have measured the mRNA levels after growth of wild-type, arg82Δ and kcs1Δ strains on media containing high or low phosphate concentrations (Fig. 4). In a kcs1Δ strain, expression of PHO11 gene is high, independently of the phosphate concentration in the growth medium, and is comparable to its expression in a wild-type strain at low phosphate concentration. This indicates that Kcs1p is necessary for repression of PHO genes by phosphate. In an arg82Δ strain, the level of PHO11 mRNA is high only at high phosphate concentration. The weak amount of PHO11 mRNA at low phosphate concentration in the arg82Δ strain is consistent with the data presented recently by Steger et al. (2003) showing a role for Arg82p, but not for Kcs1p, in the regulation of chromatin remodelling at PHO5 promoter.

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Figure 4. Northern blotting of PHO11 gene in strains deleted of ARG82 or KCS1 genes after growth on media containing high (H) or low phosphate (L) concentrations. Total RNAs were extracted from different strains grown on synthetic medium + 25 µg per ml uracil (see Experimental procedures) and 30 µg were analysed by Northern blotting with 32P-labelled PHO11 and ACT1 (control) DNA probes. Lanes 1 and 2, wild-type strain BY4709, lanes 3 and 4, strain 4709arg82Δ, lanes 5 and 6 strain 4709kcs1Δ. These strains were grown on high phosphate (1.5 mg ml−1) (lanes 1, 3, 5) or low phosphate (30 µg ml−1) (lanes 2, 4, 6).

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Expression of nitrogen catabolic genes requires the two GATA activators, Nil1p/Gat1p in response to a deficiency of glutamate, and Gln3p in response to a deficiency of glutamine. In the presence of a preferred nitrogen source, Gln3p is sequestered in the cytoplasm by Ure2p (reviewed in Magasanik and Kaiser, 2002). To test whether Arg82p and Kcs1p also regulate the expression of nitrogen-regulated genes dependently on nitrogen availability, we have measured the mRNA levels of a series of NCR genes, after growth on different nitrogen sources and in different mutant strains. Figure 5 represents the results for two such regulated genes, ASP3 and CPS1. We chose three growth conditions, ammonia medium (am) on which only Nil1p is active, glutamine medium (gln) on which Nil1p and Gln3p are inactive, and after a two hour shift from glutamine to proline medium (pro) on which both regulators are active. As expected, expression of ASP3 and CPS1 is higher in cells grown on ammonia medium than on glutamine, and are fully derepressed on proline (lanes 1, 2, 3). In an arg82Δ strain, there is a significant reduction of ASP3 and CPS1 expression on ammonia medium, a slight reduction of expression on glutamine, and almost no effect on proline (lanes 10, 11, 12). It is noteworthy that in strains deprived of Gln3p or Nil1p, there is still a strong derepression of ASP3 and CPS1 on proline medium (lanes 6 and 9). In contrast, the simultaneous deletion of gln3Δ and nil1Δ, of gln3Δ and arg82Δ or of nil1Δ and arg82Δ, abolished almost entirely ASP3 and CPS1 expression on all three tested media (lanes 13–21). However, for CPS1 there is still a slight derepression in the strain nil1Δ, arg82Δ after growth on proline medium (lane 21). These results and those obtained for other nitrogen regulated genes, DAL7, MEP2, GAP1 and GLN1 (data not shown) favour the idea that Arg82p (and probably Kcs1p) is required to fully activate NCR-regulated genes by Nil1p, and to a lesser extent by Gln3p.

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Figure 5. Northern blotting of CPS1 and ASP3 genes in strains deleted of ARG82, GLN3, NIL1 genes and in strain containing double deletions, after growth on media containing ammonia (am), glutamine (gln) or proline (pro) as nitrogen source. Total RNAs were extracted from different strains grown on synthetic medium + 25 µg per ml uracil (see Experimental procedures) and 30 µg were analysed by Northern blotting with 32P-labelled CPS1, ASP3 and ACT1 (control) DNA probes. Wild-type strain BY4709 (lanes 1, 2, 3), strain 4709gln3Δ (lanes 4–6), strain 4709nil1Δ (lanes 7–9), strain 4709arg82Δ (lanes 10–12), strain 03167b (gln3Δ, nil1Δ) (lanes 13–15), strain 4709gln3Δ, arg82Δ (lanes 16–18) and 4709nil1Δ, arg82Δ (lanes 19–21). The strains were grown on ammonia medium (lanes 1, 4, 7, 10, 13, 16 and 19), on glutamine medium (lanes 2, 5, 8, 11, 14, 17 and 20), or after a 2 h shift from glutamine to proline medium (lanes 3, 6, 9, 12, 15, 18 and 21).

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Inositol polyphosphate kinase activity of Arg82p or Kcs1p is required for NCR and phosphate-regulated gene expression

Arg82p and Kcs1p contain several well conserved amino acid sequences among IP kinases, such as the IP binding site and the SSLL domain (Fig. 6). Mutations in these regions abolished the IP kinase activity of each protein, without affecting the stability of the mutated proteins (Dubois et al., 2002). The polyaspartate stretch of Arg82p is required for the control of arginine metabolism, but not for the kinase function (Dubois et al., 2000). Kcs1p presents two putative leucine zipper motifs, which could be involved in protein–protein interactions. To discriminate which domains of these proteins were required for expression of nitrogen and phosphate genes, messenger RNA levels were determined in strains mutated in the different domains (IP binding site SSLL and polyaspartate stretch for Arg82p, and SLL and leucine zipper motifs for Kcs1). Mutations affecting kinase activity of Arg82p (D131A or S257A, S258A, L259A, L260A, Fig. 6, lanes 4, 5), or Kcs1p (S887A, L888A, L889A, lane 8) reduced expression of DAL5, DAL7 and ASP3 genes, whereas they derepressed expression of PHO11, VTC3, NRF1 genes. In contrast, mutations which did not impair Arg82p or Kcs1p kinase activity (arg82Δ282-303, lane 3; kcs1L794A,L801A-L857A,L864A, lane 9) did not affect expression of these genes.

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Figure 6. Northern blotting of NCR and PHO regulated genes in strains mutated in ARG82 or KCS1 genes. A. Schematic representation of Arg82p and Kcs1p. The open bars represent the Arg82p and Kcs1p proteins, with the localization of the different domains and mutations analysed. The numbers correspond to the position of the amino acids in the proteins. B. Total RNAs were extracted from different strains grown on synthetic medium (see Experimental procedures) and 30 µg were analysed by Northern blotting with 32P-labelled DAL5, DAL7, ASP3, PHO11, VTC3, NRF1 and ACT1 (control) DNA probes. Strains were deleted of ARG82 (lanes 1–5) or KCS1 genes (lanes 6–9). arg82Δ strain was transformed with pFV145 (pURA3, ARG82, lane 1), pFL38 (pURA3, lane 2), pFV160 (pURA3, arg82Δasp, lane 3), pFV148 (pURA3, arg82D131 A, lane4), pFV215 (pURA3, arg82SLL, lane 5) plasmids. kcs1Δ strain was transformed with pFV241 (pURA3, KCS1, lane 6), pFL38 (pURA3, lane 7), pFV217 (pURA3, kcs1SLL, lane 8), pFV198 (pURA3, kcs1-L1L2, lane 9) plasmids (see Experimental procedures).

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Based on amino acid sequences comparison we have identified the S. pombe orthologues of Arg82p and Kcs1p. SpArg82p could be responsible for the inositol multikinase activity reported by Ongusaha et al. (1998). We cloned these two genes and expressed them under the dependence of the tet promoter in S. cerevisiae arg82Δ and kcs1Δ strains respectively. It is noteworthy that spArg82p does not contain the aspartate-rich region and did not restore the control of arginine metabolism impaired in an S. cerevisiae arg82Δ strain (unpubl. data). In contrast, overexpression of spArg82p restored nitrogen gene expression and phosphate gene repression in an arg82Δ strain (Fig. 7, lane 6). Similarly, overexpression of spKcs1p complemented the defect in gene expression resulting from loss of Kcs1p (lane 3).

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Figure 7. Suppression of defects in PHO and NCR gene expression associated with deletion of ARG82 or KCS1 genes. Total RNAs were extracted from different strains grown on synthetic medium (see Experimental procedures) and 30 µg were analysed by Northern blotting with 32P-labelled DAL7, ASP3, PHO11, VTC3 and ACT1 (control) DNA probes. Strains were deleted of KCS1 (lanes 1–3) or ARG82 genes (lanes 4–8). kcs1Δ strain was transformed with pCM262 (pURA3, lane 1), pFV187 (pURA3, tetKCS1sc, lane 2), pFV236 (pURA3, tetKCS1sp, lane 3) plasmids (see Experimental procedures). arg82Δ strain was transformed with pCM262 (pURA3, lane 4), pFV186 (pURA3, tetARG82sc, lane 5), pFV234 (pURA3, tetARG82sp, lane 6), pFV187 (pURA3, tetKCS1sc, lane 7), pFV188 (pURA3, tetMCM1, lane 8).

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Taken together, these data indicate that the IP kinase activity of Arg82p and Kcs1p is essential for the expression of nitrogen genes and for repression of phosphate-regulated genes by phosphate, suggesting a role for inositol pyrophosphates in these regulatory pathways.

We have shown previously that overexpression of Kcs1p in the arg82Δ background, rescued vacuolar morphology and resistance to salt stress, whereas the growth rate and cell wall integrity were improved but not fully restored (Dubois et al., 2002). Our Northern blot analysis with RNAs extracted from the arg82Δ strain overexpressing Kcs1p, showed that expression of phosphate regulated genes (PHO11, VTC3) was repressed but that nitrogen regulated genes (ASP3, DAL7) remained downregulated (Fig. 7, lane 7). arg82Δ strain overexpressing Kcs1p contains substantial levels of higher inositol polyphosphates such as (PP)2InsP4 and PP-InsP5 (Dubois et al., 2002). Synthesis of such products restored fully the repression of PHO genes, but was not sufficient to restore expression of NCR genes.

Mcm1p-dependent gene expression requires the polyaspartate domain of Arg82p but not its IP kinase activity

Previous work had shown that the only function of Arg82p in the control of the metabolism of arginine was to recruit and stabilize in the nucleus the two MADS-box proteins Mcm1p and Arg80p (Dubois et al., 2000; El Bakkoury et al., 2000). Overexpression of Mcm1p in an arg82Δ strain partially restored Mcm1-dependent functions, such as mating and control of cell cycle genes (Dubois and Messenguy, 1994; El Bakkoury et al., 2000). To test whether the IP kinase activities of Arg82p and Kcs1p were required for expression of Mcm1p-dependent genes, RNAs were extracted from strains arg82Δ, arg82Δ transformed with pARG82, parg82D131 A, parg82Δasp, ptetspARG82, ptetKCS1, or ptetMCM1, and from strains kcs1Δ and kcs1Δ transformed with pKCS1. These RNAs were probed with MFα1, an α-specific gene, whose expression is Mcm1p-dependent. In contrast to Arg82p, Kcs1p was not required for the expression of this gene (Fig. 8, lanes 1 and 2). A mutation in Arg82p (D131A) abolishing its IP kinase activity had no effect on the expression of MFα1 (lane 6), whereas the deletion of the polyaspartate residues (lane 5) impaired its expression. Overexpresssion of Mcm1p, but not of Kcs1p nor spArg82p, restored MFα1 RNA levels (lanes 8, 9, 10). It is noteworthy that overexpression of Mcm1p in an arg82Δ strain had no effect on NCR or phosphate-regulated genes (Fig. 7, lane 8).

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Figure 8. Northern blotting of MFα1 gene in different kcs1 or arg82 mutant strains, and in arg82Δ strain overexpressing different proteins. Total RNAs were extracted from different strains grown on synthetic medium (see Experimental procedures) and 30 µg were analysed by Northern blotting with 32P-labelled MFα1 and ACT1 DNA probes. Strains were deleted of KCS1 (lanes 1 and 2) or ARG82 genes (lanes 3–10). kcs1Δ strain was transformed with pFV241 (pURA3, KCS1, lane 1), pCM262 (pURA3, lane 2), plasmids (see Experimental procedures). arg82Δ strain was transformed with pFV145 (pURA3, ARG82, lane 3), pCM262 (pURA3, lane 4), pFV160 (pURA3, arg82Δasp, lane 5), pFV148 (pURA3, arg82D131 A, lane 6), pFV186 (pURA3, tetARG82sc, lane 7), pFV234 (pURA3, tetARG82sp, lane 8), pFV187 (pURA3, tetKCS1sc, lane 9), pFV188 (pURA3, tetMCM1, lane 10).

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All these data show that expression of Mcm1p-dependent genes does not necessitate the production of inositol pyrophosphates in contrast to NCR and phosphate-regulated genes. Thus the role of Arg82p in Mcm1-mediated gene expression is to interact with and stabilize Mcm1p, as overexpression of this protein can compensate for the lack of Arg82p.

The polyaspartate domain of Arg82p was shown to be crucial for its role in the arginine regulation (Qiu et al., 1990; Dubois et al., 2000) indicating that this region could be important for the interaction between Arg82p and the MADS-box proteins Mcm1p and Arg80p. To address this point, two-hybrid assays were used to examine the interaction between Arg80p or Mcm1p and arg82p mutated proteins (arg82D131 A, arg82Δ282-303). Deletion of the polyaspartate domain and not mutation impairing the IP kinase activity caused a significant decrease in the interaction with Mcm1p or Arg80p (Table 2). In addition we examined the stability of Mcm1p and Arg80p in the wild-type and mutated arg82 strains by Western blot. Mcm1p was produced under the control of tet promoter and was detected using antibodies raised against purified GST-Mcm1p, whereas Arg80p tagged at its C-terminal end with V5 epitope, was produced under the control of GAL10 promoter, and detected with anti-V5 antibodies. Protein stability was measured after addition of cycloheximide to the culture (20 µg per ml). The stability of Mcm1p and Arg80p was impaired in the arg82Δasp strain to the same extent as in a arg82Δ strain. In contrast, impairing the IP kinase activity of Arg82p (mutation D131A) had no effect on the stability of the two MADS-box proteins (Fig. 9).

Table 2. . Ability of mutated GAD-arg82 proteins to interact with GBD-Arg80p and GBD-Mcm1p.
Hybridβ-Galactosidase specific activity (nmol of o-nitrophenyl-β-d- galactopyranoside hydrolysed min−1 mg−1 of protein)
GBD-Arg80p (pME46)GBD-Mcm1p (pNA51)
  1. Transcription activation of the lacZ gene was estimated by determination of β-galactosidase activity in S. cerevisiae strain HY co-transformed with plasmids expressing GBD-Arg80p (pME46) or GBD-Mcm1p (pNA51) and the different wild-type or mutated GAD-arg82p fusions. Values are the mean of three independent measurements with variations less than 15%.

GAD (pACTII) 2 2
GAD-Arg82p (pME18)2326
GAD-arg82Δasp (pNA14) 2 2
GAD-arg82D131A (pFV174)1720
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Figure 9. Determination of Arg80p or Mcm1p stability in wild-type and mutated arg82 strains, using Western blotting. Aliquots of exponentially growing cells were taken at different times (indicated in the figure) after the addition of 20 µg ml−1 cycloheximide at time 0. Proteins were extracted, electrophoresed on 10% SDS-polyacrylamide gel and electrotransferred to Hybond membrane. Each lane contains about 25 µg protein.Strain 4719arg82Δ (ura3, trp1, arg82::kanMX4) was transformed with pFL39 (TRP1), pFV194 (TRP1, ARG82), pFV196 (TRP1, arg82D131 A) and pFV197 (TRP1, arg82Δasp) plasmids. A. Arg80p protein tagged with V5 epitope (see Experimental procedures) was visualized with anti-V5 antibodies. To detect Arg80p, the above strains were also transformed with plasmid pFV260 (pGAL10-ARG80-V5, 2µ, URA3), and cells were grown on 1% galactose. B. Mcm1p was visualized with anti-GST-Mcm1 antibodies. To detect Mcm1p, the above strains were also transformed with plasmid pFV188 (tetMCM1, 2µ, URA3).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Recent studies have shown that inositol pyrophosphates are essential for cell wall stability, cell growth, adaptation to salt stress, vacuolar morphogenesis and homologous DNA recombination (Luo et al., 2001; Dubois et al., 2002). However, the targets of these compounds remained to be identified. We have therefore used DNA microarray technology performed with strains deleted of Arg82p or Kcs1p, two inositol polyphosphate kinases, because these deletions led to a significant reduction of inositol pyrophosphate pools. We have compared the abundance of mRNAs between wild-type strain or strains overexpressing Arg82p or Kcs1p, to strains devoid of Arg82p or Kcs1p respectively. Among the genes identified by these experiments, two gene families clearly emerged: genes controlled in response to the quality of the nitrogen source (NCR) and genes regulated in response to phosphate availability (PHO). Expression of these genes was modified simultaneously in both arg82Δ and kcs1Δ strains. Microarray data and Northern blotting experiments showed that NCR genes were downregulated in arg82Δ or kcs1Δ cells, whereas PHO genes were upregulated in these strains growing on high phosphate medium. Analysis of expression of NCR and PHO genes under different physiological growth conditions indicates that Arg82p and Kcs1p are required for activation of NCR-regulated genes in response to nitrogen availability mainly through Nil1p, and for repression of PHO genes by phosphate. Mutations in Arg82p or Kcs1p, impairing their kinase activity but not their stability, and leading to a significant reduction of inositol pyrophosphates, affected expression of NCR and PHO genes to the same extent as deletion of each protein. Orthologous spArg82p and spKcs1p from S. pombe which contain the IP kinase domain but neither the polyaspartate stretch of Arg82p nor the leucine zippers of Kcs1p, could fulfill Arg82p and Kcs1p functions in the control of NCR and PHO genes in S. cerevisiae. Thus inositol pyrophosphates play a role in the transcriptional control of these genes. However, different inositol pyrophosphates could be required to control different cellular processes, as overexpression of Kcs1p in an arg82Δ strain restored vacuolar morphology, resistance to salt stress (shown previously) and PHO gene expression, but not NCR gene expression. The cellular growth and the cell wall integrity were only partially improved. It is worth stressing that although Kcs1p overexpression in an arg82Δ strain restored pyrophosphates, the pattern was different from the pyrophosphate pattern in the wild-type strain (Dubois et al., 2002). This may explain why NCR gene expression is not restored.

In contrast, inositol pyrophosphates are not involved in the control of Mcm1p-dependent genes, as Kcs1p and the kinase activity of Arg82p were not required for expression of these genes. Our data show clearly that Arg82p has two functions. One function requires the presence of the polyaspartate domain, essential for Mcm1p and Arg80p stabilization, suggesting a role of chaperone for Arg82p, and the other function which is to produce inositol polyphosphates, is independent of the polyaspartate domain.

Inositol pyrophosphates are required for numerous cellular processes regulated through transduction cascades involving protein phosphorylation. Cell wall integrity and resistance to salt stress imply two MAP kinase signalling pathways (reviewed in Banuett, 1998). The regulation of genes in response to phosphate availability requires activation of the cyclin-CDK (cyclin-dependent kinase) complex Pho80p-Pho85p (reviewed in Carroll and O’Shea, 2002). The TOR kinases have an essential role in preventing the expression of nitrogen-regulated genes in cells growing on an optimal nitrogen source (reviewed in Magasanik and Kaiser, 2002). The role of protein kinases in nitrogen and phosphate metabolism would be to prevent by phosphorylation the transcriptional activators (Gln3p, Nil1p and Pho4p) to translocate to the nucleus. We propose a role for inositol pyrophosphates in the transduction of the physiological signal in different regulatory pathways. However, full expression of PHO genes also requires inositol polyphosphates as Steger et al. (2003) have recently reported a role for Ins(1,3,4,5)P4, Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5, but not IP6 or pyrophosphates, in the SWI/SNF and INO80 chromatin remodelling allowing induction of transcription of some phosphate-responsive genes. This suggests that inositol polyphosphates and inositol pyrophosphates could control the expression of PHO genes at different levels and in opposite ways. InsP4 and InsP5 are required for activation of PHO gene expression at low phosphate concentration through chromatin remodelling, whereas inositol pyrophosphates are required to sustain repression of PHO genes at high phosphate concentration. Thus the inositol pyrophosphates could play a role in the phosphate signalling pathway.

Inositol pyrophosphates could contribute to the transduction of different physiological signals by controlling directly or indirectly the phosphorylation state of key signalling or regulatory proteins. It has been reported that the pyrophosphate groups can be donated to proteins, providing a novel means of protein phosphorylation. It was also proposed that the inositol pyrophosphates might regulate protein activation analogous to the way in which GTP regulates activity of G proteins (Luo et al., 2001; Dubois et al., 2002).

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Strains and media

BY4709 (MATα, ura3) and BY4719 (MATa, trp1, ura3) yeast strains (Brachmann et al., 1998) were used to construct deletion of ARG82 and KCS1 genes as described in Dubois et al. (2002), yielding strains 03127c (arg82::kanMX4, ura3), 4719arg82Δ (arg82::kanMX4, trp1, ura3), and 4709kcs1Δ (kcs1::kanMX4, ura3). BY4709 was also used to create deletion of GLN3 or NIL1 genes using the long flanking homology strategy (Wach, 1996). The kanMX4 cassettes flanked by about 500 bp corresponding to the promoter and terminator regions of the target genes were synthesized by a two-step PCR. The DNA fragments containing the different cassettes were used to transform strain BY4709 on rich media plates containing 200 µg ml−1 of geneticin. The correct targeting of the deletions in G418r transformants was verified by PCR using whole cells as a source of DNA and appropriate primers. The following strains were obtained: gln3::kanMX4 (4709gln3Δ) and nil1::kanMX4 (4709nil1Δ and 4700nil1Δ). To construct strains with multiple deletions (gln3Δ, arg82Δ and nil1Δ, arg82Δ) we have used the gene disruption cassette loxP-kanMX4-loxP (Guldener et al., 1996). To eliminate the kanMX4 marker from the disrupted gene, the mutated strain was transformed with the cre expression plasmid pSH47, which carries the URA3 marker gene and the cre gene under the control of the inducible GAL1 promoter. Expression of the cre recombinase was induced by shifting cells from YPD (glucose) to YPG (galactose) medium for 2 h. The loss of the kanMX4 cassette was detected by plating cells on YPD and replica plating the colonies onto YPD-G418. The cre expression plasmid was removed from the strain by streaking cells on plates containing 5-fluoroorotic acid to counterselect for the loss of the plasmid. Strain 03167b (gln3Δ, nil1Δ) was obtained by crossing strain 4709gln3Δ and 4700nil1Δ. BY4743 (Brachmann et al., 1998), a diploid strain (ura3/ura3, his3/his3, leu2/leu2) and 4743kcs1Δ (ura3/ura3, his3/his3, leu2/leu2, kcs1::kanMX4/kcs1::kanMX4) were used as recipient strains for plasmids expressing PHO5-HIS3 or DAL7-URA3. Strain HY (Louvet et al., 1997) was used for transformation by plasmids pAS1, pACTII and their derivatives (two-hybrid analysis) (Durfee et al., 1993). E. coli strain XL1B was used for plasmid amplification and for in vitro mutagenesis. For RNA preparations and for the two-hybrid experiments, yeast strains were grown on synthetic medium containing 0.7% yeast nitrogen base with ammonia and without amino acids and 3% glucose, or on 0.7% yeast nitrogen base without ammonia and without amino acids + 1 mg ml−1 glutamine and 3% glucose. Growth on proline was achieved by filtering the cells grown on glutamine medium and cultivating them on fresh minimal medium with 1 mg ml−1 proline as a nitrogen source for 2 h. The phosphate amount was 1.5 mg KH2PO4 per ml (high phosphate medium) or 30 µg KH2PO4 per ml (low phosphate medium). For the two-hybrid experiments the yeast nitrogen base medium with ammonia was supplemented with all the amino acids, except those whose omission was required for plasmid selection. For Western blot experiments, yeast strains were grown on minimal medium (pH 6.5) which contained 1% galactose, vitamins and mineral traces (Messenguy, 1976).

Genetic manipulations

The low copy pFL38 plasmid was used in this work to bear wild-type and mutated arg82 (Dubois et al., 2000) and kcs1 genes (Dubois et al., 2002). Plasmid pFV145 (pFL38-ARG82) was used to create the following amino acid changes, S257A-S258A-L259A-L269A, by in vitro mutagenesis leading to plasmid pFV215.

The plasmids pFV186 and pFV187 overexpressing ARG82 and KCS1 genes, respectively, under the dependence of the tet promoter were described in Dubois et al. (2002).

To overexpress ARG82 and KCS1 genes from S. pombe, we have fused their coding sequence to the tet promoter present in plasmid pCM262 (gift from E. Herrero). For ARG82sp (locus CAB63791), a 810 bp BamHI-BamHI DNA fragment was synthesized by PCR using appropriate oligonucleotides as primers with BamHI restriction sites, and genomic wild-type DNA from S. pombe as template, blunt-ended with T4 DNA polymerase and inserted in the PmeI restriction site of plasmid pCM262, yielding plasmid pFV234. For KCS1sp (locus CAA20701), a 2.9 kb EcoRI-EcoRI DNA fragment was synthesized by PCR using appropriate oligonucleotides as primers with EcoRI restriction sites, and genomic wild-type DNA from S. pombe as template, blunt-ended with T4 DNA polymerase and inserted in the PmeI restriction site of plasmid pCM262, yielding plasmid pFV236.

To overexpress MCM1, a 760 bp BamHI-BamHI DNA fragment was synthesized by PCR using appropriate oligonucleotides as primers with BamHI restriction sites, and genomic wild-type DNA from S. cerevisiae as template, blunt-ended with T4 DNA polymerase and inserted in the PmeI restriction site of plasmid pCM262, yielding plasmid pFV188.

Plasmid pFV260 (pGAL10-ARG80-V5, 2µ, URA3) was obtained by insertion of a 530 bp EcoRI-EcoRI DNA fragment in the EcoRI site of plasmid pYES2 (pGAL10,V5, 2µ, URA3, Invitrogen).

To express the HIS3 gene under the dependence of the PHO5 promoter, we synthesized by PCR a BamHI-BamHI 540 bp DNA fragment, covering the PHO5 region from − 541 to − 1 containing the Pho4p and Pho2p binding sites, and a 2.3 kb BamHI–BamHI DNA fragment covering the HIS3 region from + 1 to + 2298, containing the coding sequence and the terminator of the gene. The 3′ end of the PHO5 fragment contained an extended sequence complementary to an extended sequence at the 5′ end of HIS3. The products of the two previous PCRs were used as templates to synthesize by PCR with appropriate primers, a DNA fragment containing the PHO5 promoter fused to the coding sequence of HIS3. This 2.85 kb BamHI–BamHI DNA fragment was inserted in the BamHI site of pFL38 vector (URA3, CEN6, ARS4) yielding plasmid pFV309.

To express the URA3 gene under the dependence of the DAL7 promoter, we synthesized by PCR a BamHI–BamHI 450 bp DNA fragment, covering the DAL7 region from − 451 to − 1 containing the GATAA sequences, Gln3p and Gat1p binding sites, and a 1.3 kb BamHI–BamHI DNA fragment covering the URA3 region from + 1 to + 1300, containing the coding sequence and the terminator of the gene. The 3′ end of the DAL7 DNA fragment contained an extended sequence complementary to an extended sequence at the 5′ end of URA3. The products of the two previous PCRs were used as templates to synthesize by PCR with appropriate primers, a fragment containing the DAL7 promoter fused to the coding sequence of URA3. This 1.75 kb BamHI–BamHI DNA fragment was inserted in the BamHI site of pFL36 vector (LEU2, CEN6, ARS4) yielding plasmid pFV313.

RNA preparation

Total RNAs were extracted following Schmitt et al. (1990), and purified using the RNeasy kit (Qiagen).

Microarray procedures

The yeast DNA chips were manufactured by Eurogentec (Sart Tilman, Belgium). Fluorescent cDNA synthesis for microarray hybridizations was performed according to Foury and Talibi (2001), using Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech). Hybridizations were performed according to Foury and Talibi (2001). The hybridization signal was detected by scanning microarrays using GenePix 4000 laser scanner and genepix 3.01 software (Axon Instruments). The normalization factors calculated by this program were used to set optimal PMT settings (photomultiplier tube) on the scanner to ensure maximum data integrity upon acquisition. Artefactual, saturated and low signal spots were excluded. The ratio's represented in Table 1 were obtained by calculating the mean ratio eliminating the top and bottom values of seven independent microarray experiments with independent RNA preparations, for tetARG82/arg82Δ and tetKCS1/kcs1Δ.

Northern blot analysis

Northern blot analysis was performed as described by Foury and Talibi (2001). DNA probes of about 500 bp were generated by PCR using appropriate oligonucleotides, and labelled using the ‘prime a gene® labelling system’ from Promega, with [α-32P]-dCTP from ICN. Hybridizations were carried out using standard procedures.

Western blot analysis

Western blot analysis was performed according to El Bakkoury et al. (2000). Mcm1p was detected with antibodies raised against GST-Mcm1p, and Arg80p was detected with antibodies raised against V5 epitope (Invitrogen).

Two-hybrid assays

Two-hybrid assays to examine the interactions between wild-type and mutated Arg82p and cofactors were performed with plasmids pME18 (GAD-Arg82), pNA14 (GAD-arg82Δasp), pFV174 (GAD-arg82D131 A), pME46 (GBD-Arg80) and pNA51 (GBD-Mcm1), as described previously (El Bakkoury et al., 2000). β-galactosidase activity was assayed as described by Miller (1972). Protein contents were determined by the Folin method.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We would like to thank F. Vierendeels and L. Delys for their assistance in the achievement of some experiments, and J-P ten Have for the figures. We are grateful to Eurogentec for the use of their microarray facilities, and to E. Herrero for the gift of plasmids and strains. We also thank S.B. Shears and C. Erneux for helpful comments about the manuscript.

This work was supported by EEC grant GARNISH (contract number QLK3-CT-2000–00174) and by a grant from Bruxelles-Capitale.

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  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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