Arg82p is a bifunctional protein whose inositol polyphosphate kinase activity is essential for nitrogen and PHO gene expression but not for Mcm1p chaperoning in yeast

Molecular Microbiology (2003)

49

(2), 457–468 doi:10.1046/j.1365-2958.2003.03562.x

© 2003 Blackwell Publishing Ltd

Blackwell Science, LtdOxford, UKMMIMolecular Microbiology 1365-2958Blackwell Publishing Ltd, 200349

2457468

Original Article

Involvement of Arg82 IP kinase in gene expressionM. El Alami, F. Messenguy, B. Scherens and E. Dubois

Accepted 24 March, 2003. *For correspondence. [email protected]; Tel. (

+

32) 2526 72 77; Fax (

+

32) 2526 72 73.

Arg82p is a bifunctional protein whose inositol polyphosphate kinase activity is essential for nitrogen and

PHO

gene expression but not for Mcm1p chaperoning in yeast

Mohamed El Alami, Francine Messenguy,Bart Scherens and Evelyne Dubois*

Institut de Recherches Microbiologiques J-M Wiame, Laboratoire de Microbiologie de l’Université Libre de Bruxelles, 1 avenue Emile Gryzon, 1070 Bruxelles, Belgium.

Summary

In

Saccharomyces cerevisiae

, the synthesis of inosi-tol pyrophosphates is essential for vacuole biogene-sis and the cell’s response to certain environmentalstresses. The kinase activity of Arg82p and Kcs1p isrequired for the production of soluble inositol phos-phates. To define physiologically relevant targets ofthe catalytic products of Arg82p and Kcs1p, we usedDNA microarray technology. In

arg82

DDDD

or

kcs1

DDDD

cells,we observed a derepressed expression of genes reg-ulated by phosphate (PHO) on high phosphatemedium and a strong decrease in the expression ofgenes regulated by the quality of nitrogen source(NCR). Arg82p and Kcs1p are required for activationof NCR-regulated genes in response to nitrogen avail-ability, mainly through Nil1p, and for repression of

PHO

genes by phosphate. Only the catalytic activityof both kinases was required for

PHO

gene repres-sion by phosphate and for NCR gene activation inresponse to nitrogen availability, indicating a role forinositol pyrophosphates in these controls. Arg82palso controls expression of arginine-responsivegenes by interacting with Arg80p and Mcm1p, andexpression of Mcm1-dependent genes by interactingwith Mcm1p. We show here that Mcm1p and Arg80pchaperoning by Arg82p does not involve the inositolpolyphosphate kinase activity of Arg82p, but requiresits polyaspartate domain. Our results indicate thatArg82p is a bifunctional protein whose inositol kinaseactivity plays a role in multiple signalling cascades,and whose acidic domain protects two MADS-boxproteins against degradation.

Introduction

In yeast it is known that membrane-bound inositol lipidsplay important roles in adaptation to environmentalstresses. More recently it has been shown that solubleinositol polyphosphates, especially inositol pyrophos-phates play important roles in diverse cellular processes,such as cell wall maintenance, vacuolar morphogenesis,resistance to salt stress and also mediate homologousDNA recombination in yeast (Luo

et al

., 2001; Dubois

et al

., 2002). Arg82p and Kcs1p are two inositol poly-phosphate kinases (Saiardi

et al

., 1999; 2000; York

et al

.,1999). Arg82p converts Ins(1,4,5)P3 to Ins(1,3,4,5)P4and Ins(1,4,5,6)P4, which are converted toIns(1,3,4,5,6)P5. Kcs1p converts Ins(1,3,4,5,6)P5 andInsP6 to different inositol pyrophosphates, PPInsP4,PPInsP5 and (PP)

2

InsP3 (Saiardi

et al

., 1999; 2000)(Fig. 1). Cells deleted of

ARG82

or

KCS1

genes displaysevere growth defect at high temperature, vacuolar frag-mentation, increased leakiness of cellular phosphataseand hypersensitivity to salt stress (Dubois

et al

., 2002). Astrain impaired in Arg82p kinase activity not onlypresents 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, butalso a significant reduction of inositol pyrophosphates,whereas impairing Kcs1p kinase activity only decreasesthe synthesis of inositol pyrophosphates (Dubois

et al

.,2002). Consequently, the defects observed in bothdeleted strains would result from very low amounts ofinositol pyrophosphates.

Arg82p plays a second important role in the cell bystabilizing Mcm1p a protein essential for cell viability, andcontrolling 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), minichromo-some maintenance (Passmore

et al

., 1988) and argininemetabolism (Messenguy and Dubois, 1993). Arg82p sta-bilizes another MADS-box protein Arg80p, which is alsorequired for the formation of a regulatory complex with thearginine sensor Arg81p, at the ‘arginine boxes’ present inthe co-regulated arginine genes (Amar

et al

., 2000; ElBakkoury

et al

., 2000). Thus, for the arginine metabolism

458

M. El Alami, F. Messenguy, B. Scherens and E. Dubois

© 2003 Blackwell Publishing Ltd,

Molecular Microbiology

,

49

, 457–468

Arg82p acts as a protein chaperone, and Dubois

et al

.(2000; 2002) have shown that Arg82p kinase activity wasnot required for this function. However, it was not estab-lished whether the kinase activity of Arg82p was requiredfor Mcm1p-dependent gene expression.

The cell response to diverse stresses requires efficientMAP kinase signalling pathways (Banuett, 1998) whichcould be perturbed in strains lacking inositol pyrophos-phates. To identify the regulatory network of Arg82p andKcs1p, we conducted genome wide analysis. Among a setof genes whose expression was increased or decreasedin strains deleted of

ARG82

or

KCS1

genes compared towild-type strain, two families of genes emerged strikingly.In

arg82

D

or

kcs1

D

cells, genes controlled in response tothe quality of the nitrogen source (NCR) were downregu-lated, whereas genes controlled by phosphate availability(PHO) were upregulated in high phosphate medium. Inthis 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 inMcm1p stabilization.

Results

In

arg82

D

and

kcs1

D

strains, NCR genes are downregulated and

PHO

genes are upregulated

To determine the specific regulation network of Arg82pand Kcs1p, we used DNA microarrays allowing to analysechanges in transcript abundance in

arg82

D

strain com-pared to the same strain overexpressing

ARG82(

p

tetARG82

) or to the wild-type strain, and in

kcs1

D

straincompared to the same strain overexpressing

KCS1(

p

tetKCS1

) or to the wild-type strain. Expression of 14genes controlled in response to the quality of the nitrogensource was downregulated in strains deleted either of

ARG82

or

KCS1

genes. In contrast, expression of 15other genes controlled in response to phosphate availabil-ity was upregulated in the presence of high phosphateconcentration (Table 1). These microarrays did not identifyother gene families whose expression was dependent onArg82p or Kcs1p, not even genes involved in vacuolemorphogenesis, salt resistance or cell wall integrity asmight be expected from the phenotypes associated with

arg82

D

and

kcs1

D

strains.

In order to validate the results obtained for NCR and

PHO

genes, we performed Northern blotting analysisusing

DAL5, DAL7, ASP3, PHO5, PHO11, PHO84, VTC3

and

NRF1

probes. The results presented in Fig. 2 clearlyconfirmed that Arg82p and Kcs1p were required for NCRgene expression and for repression of phosphate-regu-lated genes. The mRNA levels of

PHO

and NCR geneswere comparable in a wild-type strain and in a strainoverexpressing

ARG82

or

KCS1

, indicating that high over-expression did not exaggerate the influence of Kcs1p or

Fig. 1.

Pathway for the synthesis of soluble inositol polyphosphates.

Table 1.

New gene categories regulated by Arg82p and Kcs1p.

ORFidentity Gene

a

Ratio

a

Ratio

tet-ARG82

/

arg82

D ±

SD

tet-KCS1

/

kcs1

D ±

SD

YLR158c

ASP3-3

4.90

±

1.12 5.10

±

1.74YLR160c

ASP3-4

4.89

±

0.97 3.92

±

1.65YLR157c

ASP3-2

4.76

±

0.99 4.67

±

1.78YLR155c

ASP3-1

3.40

±

0.69 3.78

±

1.06YJL172w

CPS1

2.70

±

0.54 1.34

±

0.15YKR039w

GAP1

2.66

±

0.29 1.52

±

0.32YKR033c 2.64

±

0.94 1.58

±

0.23YNL142w

MEP2

2.58

±

0.29 1.26

±

0.17YJR152w

DAL5

2.51

±

0.77 2.20

±

0.54YIR032c

DAL3

1.82

±

0.35 1.07

±

0.21YIR029w

DAL2

1.78

±

0.58 1.50

±

0.20YIR031c

DAL7

1.74

±

0.67 1.07

±

0.12YPR035w

GLN1

1.70

±

0.18 1.46

±

0.16YBR208c

DUR1,2

1.52

±

0.17 1.39

±

0.24YML123c

PHO84

0.06

±

0.02 0.12

±

0.02YAR071w

PHO11

0.07

±

0.01 0.40

±

0.05YHR215w

PHO12

0.11

±

0.04 0.47

±

0.09YBR296c

PHO89

0.18

±

0.09 0.58

±

0.11YPL019c VTC3 0.19 ± 0.04 0.27 ± 0.02YER072w VTC1 0.25 ± 0.05 0.24 ± 0.03YHR136c SPL2 0.26 ± 0.06 0.37 ± 0.03YER072w NRF1 0.30 ± 0.04 0.42 ± 0.04YJL117w PHO86 0.39 ± 0.01 0.61 ± 0.06YDR281c PHM6 0.40 ± 0.08 0.97 ± 0.12YDR481c PHO8 0.50 ± 0.05 0.68 ± 0.06YGR233c PHO81 0.56 ± 0.03 0.67 ± 0.07YFL004w VTC2 0.60 ± 0.13 0.67 ± 0.12YPL110c 0.61 ± 0.15 0.91 ± 0.03YJL012c VTC4 0.65 ± 0.08 0.54 ± 0.03

a. Expression of genes downregulated (ratios higher than 1), andupregulated (ratios lower than 1) in arg82D and kcs1D strains. Themean expression ratio was determined by microarray analysis com-paring a strain overexpressing Arg82p (tet-ARG82) to arg82D strain,and a strain overexpressing Kcs1p (tet-KCS1) to kcs1D strain. Eachvalue is the trimmed mean, excluding a single value from the top andbottom of the data set, for seven independent arrays.

Involvement of Arg82 IP kinase in gene expression 459

© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 49, 457–468

Arg82p on the expression of NCR and PHO genes(Fig. 2). Because PHO5 is strongly regulated by phos-phate (Toh-E et al., 1973), we included this gene in ourmRNA analysis, although PHO5 was not identified in themicroarray experiments indicating that some genes mayescape detection in such experiments. It is noteworthythat in many cases there are discrepancies between thegene expression ratios from microarray analysis (Table 1)and the Northern blots, comparing wild-type and mutantstrains. In our hands, the microarray technique seemsoften less sensitive than Northern blotting experiments.

To test the biological relevance of the effect of inositolpyrophosphates on the expression of PHO and NCRgenes, we fused the PHO5 promoter to the codingsequence of HIS3 (pFV309) and the DAL7 promoter tothe coding sequence of URA3 (pFV313). These plasmidswere used to transform the control diploid strain BY4743(ura3/ura3, his3/his3, leu2/leu2) and the kcs1D diploidstrain (ura3/ura3, his3/his3, leu2/leu2, kcs1::kanMX4/kcs1::kanMX4). The expression of HIS3 or URA3 wastested by growth of the transformed strains in the absenceof histidine or uracil respectively. At high phosphate con-centration (see Experimental procedures), a growth con-dition which prevents expression of PHO genes, theabsence of histidine led to a slower growth of the wild-type strain compared to the growth in the presence ofhistidine, and addition of 10 mM 3-amino triazole com-pletely abolished the growth (Fig. 3A). In contrast, thekcs1D strain transformed with the plasmid expressingPHO5-HIS3 grew in the presence of 10 mM 3-amino tria-zole without histidine as well as in the presence of histi-dine, despite the fact that a kcs1D strain grows morepoorly than the wild-type strain. These growth tests showthat the amount of the HIS3 gene product in a kcs1D strainis sufficient to ensure growth without addition of histidine,and confirm the derepression of PHO5 in this mutantstrain. The wild-type strain transformed with the plasmidexpressing DAL7-URA3 (pFV313) grew well on M. ammo-nia or glutamine media, two repressive nitrogen sources,even in the absence of uracil. In contrast in a kcs1Dbackground, the growth on M. ammonia was reduced andnearly abolished on glutamine as nitrogen source(Fig. 3B). This is consistent with the stronger repressionof NCR gene expression by glutamine than by ammonia.The weak expression of DAL7-URA3 in the kcs1D straingrown on glutamine medium was confirmed by the abilityof this strain to grow on a medium containing 5-fluoro-orotic acid + uracil (data not shown). Thus, when inositolpyrophosphates are reduced, expression of an NCR-reg-ulated gene such as DAL7 is sufficiently low to reduce theamount of URA3 product, leading to resistance on 5-FOAand to an auxotrophy on glutamine as nitrogen source.

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 tran-scriptional activators, the Pho80p-Pho85p cyclin-cyclindependent protein kinase (CDK) complex, and the Pho81CDK inhibitor. When the Pi concentration in the mediumis high, Pho85p kinase is active and hyperphosphorylatesPho4p, which is maintained in the cytoplasm (Kaffmanet al., 1994; Schneider et al., 1994; Oshima, 1997). At lowPi concentration Pho81p inhibits the Pho80p-Pho85p

Fig. 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 mg 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). arg82D strain was transformed with pCM262 (pURA3, lane 2) or pFV186 (pURA3, tet-ARG82, lane 3) plasmids. kcs1D strain was transformed with pCM262 (pURA3, lane 4) or pFV187 (pURA3, tet-KCS1, lane 5) plasmids (see Experimental procedures).

460 M. El Alami, F. Messenguy, B. Scherens and E. Dubois


kinase preventing the phosphorylation of Pho4p which isthen preferentially localized in the nucleus, where togetherwith Pho2p it activates target gene transcription (Kaffmanet al., 1994; Komeili and O’Shea, 1999). To determinewhether Arg82p and Kcs1p are required for repression ofPHO genes by phosphate, we have measured the mRNAlevels after growth of wild-type, arg82D and kcs1D strainson media containing high or low phosphate concentrations(Fig. 4). In a kcs1D strain, expression of PHO11 gene ishigh, independently of the phosphate concentration in thegrowth medium, and is comparable to its expression in awild-type strain at low phosphate concentration. This indi-cates that Kcs1p is necessary for repression of PHOgenes by phosphate. In an arg82D strain, the level ofPHO11 mRNA is high only at high phosphate concentra-tion. The weak amount of PHO11 mRNA at low phosphateconcentration in the arg82D strain is consistent with thedata presented recently by Steger et al. (2003) showing arole for Arg82p, but not for Kcs1p, in the regulation ofchromatin remodelling at PHO5 promoter.

Expression of nitrogen catabolic genes requires the twoGATA activators, Nil1p/Gat1p in response to a deficiencyof glutamate, and Gln3p in response to a deficiency ofglutamine. In the presence of a preferred nitrogen source,Gln3p is sequestered in the cytoplasm by Ure2p(reviewed in Magasanik and Kaiser, 2002). To testwhether Arg82p and Kcs1p also regulate the expressionof nitrogen-regulated genes dependently on nitrogenavailability, we have measured the mRNA levels of aseries of NCR genes, after growth on different nitrogen

sources and in different mutant strains. Figure 5 repre-sents the results for two such regulated genes, ASP3 andCPS1. We chose three growth conditions, ammoniamedium (am) on which only Nil1p is active, glutaminemedium (gln) on which Nil1p and Gln3p are inactive, andafter 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

Fig. 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 kcs1D strain (4743kcs1D) 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 kcs1D strain (4743kcs1D) transformed with the same plasmid, were plated and incubated at 30∞C for 4 days on the different media indicated.

Fig. 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 differ-ent strains grown on synthetic medium + 25 mg per ml uracil (see Experimental procedures) and 30 mg 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 4709arg82D, lanes 5 and 6 strain 4709kcs1D. These strains were grown on high phosphate (1.5 mg ml-1) (lanes 1, 3, 5) or low phos-phate (30 mg ml-1) (lanes 2, 4, 6).



ammonia medium than on glutamine, and are fully dere-pressed on proline (lanes 1, 2, 3). In an arg82D strain,there is a significant reduction of ASP3 and CPS1 expres-sion on ammonia medium, a slight reduction of expres-sion on glutamine, and almost no effect on proline (lanes10, 11, 12). It is noteworthy that in strains deprived ofGln3p or Nil1p, there is still a strong derepression ofASP3 and CPS1 on proline medium (lanes 6 and 9). Incontrast, the simultaneous deletion of gln3D and nil1D, ofgln3D and arg82D or of nil1D and arg82D, abolishedalmost entirely ASP3 and CPS1 expression on all threetested media (lanes 13–21). However, for CPS1 there isstill a slight derepression in the strain nil1D, arg82D aftergrowth on proline medium (lane 21). These results andthose obtained for other nitrogen regulated genes, DAL7,MEP2, GAP1 and GLN1 (data not shown) favour the ideathat Arg82p (and probably Kcs1p) is required to fully acti-vate NCR-regulated genes by Nil1p, and to a lesserextent by Gln3p.

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

Arg82p and Kcs1p contain several well conservedamino acid sequences among IP kinases, such as theIP binding site and the SSLL domain (Fig. 6). Mutationsin these regions abolished the IP kinase activity of eachprotein, without affecting the stability of the mutated pro-teins (Dubois et al., 2002). The polyaspartate stretch ofArg82p is required for the control of arginine metabo-lism, but not for the kinase function (Dubois et al.,

2000). Kcs1p presents two putative leucine zippermotifs, which could be involved in protein–protein inter-actions. To discriminate which domains of these proteinswere required for expression of nitrogen and phosphategenes, messenger RNA levels were determined instrains mutated in the different domains (IP binding siteSSLL and polyaspartate stretch for Arg82p, and SLLand leucine zipper motifs for Kcs1). Mutations affectingkinase 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 derepressedexpression of PHO11, VTC3, NRF1 genes. In contrast,mutations which did not impair Arg82p or Kcs1p kinaseactivity (arg82D282-303, lane 3; kcs1L794A,L801A-L857A,L864A, lane 9) did not affect expression of thesegenes.

Based on amino acid sequences comparison we haveidentified the S. pombe orthologues of Arg82p and Kcs1p.SpArg82p could be responsible for the inositol multikinaseactivity reported by Ongusaha et al. (1998). We clonedthese two genes and expressed them under the depen-dence of the tet promoter in S. cerevisiae arg82D andkcs1D strains respectively. It is noteworthy that spArg82pdoes not contain the aspartate-rich region and did notrestore the control of arginine metabolism impaired in anS. cerevisiae arg82D strain (unpubl. data). In contrast,overexpression of spArg82p restored nitrogen geneexpression and phosphate gene repression in an arg82Dstrain (Fig. 7, lane 6). Similarly, overexpression ofspKcs1p complemented the defect in gene expressionresulting from loss of Kcs1p (lane 3).

Fig. 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 mg per ml uracil (see Experimental procedures) and 30 mg were analysed by Northern blotting with 32P-labelled CPS1, ASP3 and ACT1 (control) DNA probes. Wild-type strain BY4709 (lanes 1, 2, 3), strain 4709gln3D (lanes 4–6), strain 4709nil1D (lanes 7–9), strain 4709arg82D (lanes 10–12), strain 03167b (gln3D, nil1D) (lanes 13–15), strain 4709gln3D, arg82D (lanes 16–18) and 4709nil1D, arg82D (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).



Taken together, these data indicate that the IP kinaseactivity of Arg82p and Kcs1p is essential for the expres-sion of nitrogen genes and for repression of phosphate-regulated genes by phosphate, suggesting a role for inos-itol pyrophosphates in these regulatory pathways.

We have shown previously that overexpression ofKcs1p in the arg82D background, rescued vacuolarmorphology and resistance to salt stress, whereas thegrowth rate and cell wall integrity were improved butnot fully restored (Dubois et al., 2002). Our Northernblot analysis with RNAs extracted from the arg82Dstrain overexpressing Kcs1p, showed that expression ofphosphate regulated genes (PHO11, VTC3) wasrepressed but that nitrogen regulated genes (ASP3,DAL7) remained downregulated (Fig. 7, lane 7). arg82Dstrain overexpressing Kcs1p contains substantial levelsof higher inositol polyphosphates such as (PP)2InsP4and PP-InsP5 (Dubois et al., 2002). Synthesis of suchproducts restored fully the repression of PHO genes,but was not sufficient to restore expression of NCRgenes.

Fig. 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 mg 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). arg82D strain was transformed with pFV145 (pURA3, ARG82, lane 1), pFL38 (pURA3, lane 2), pFV160 (pURA3, arg82Dasp, lane 3), pFV148 (pURA3, arg82D131 A, lane4), pFV215 (pURA3, arg82SLL, lane 5) plasmids. kcs1D 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).

Fig. 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 mg were analysed by Northern blotting with 32P-labelled DAL7, ASP3, PHO11, VTC3 and ACT1 (con-trol) DNA probes. Strains were deleted of KCS1 (lanes 1–3) or ARG82 genes (lanes 4–8). kcs1D strain was transformed with pCM262 (pURA3, lane 1), pFV187 (pURA3, tetKCS1sc, lane 2), pFV236 (pURA3, tetKCS1sp, lane 3) plasmids (see Experimental procedures). arg82D 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).



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 Arg82pin the control of the metabolism of arginine was to recruitand stabilize in the nucleus the two MADS-box proteinsMcm1p and Arg80p (Dubois et al., 2000; El Bakkouryet al., 2000). Overexpression of Mcm1p in an arg82Dstrain partially restored Mcm1-dependent functions, suchas mating and control of cell cycle genes (Dubois andMessenguy, 1994; El Bakkoury et al., 2000). To testwhether the IP kinase activities of Arg82p and Kcs1p wererequired for expression of Mcm1p-dependent genes,RNAs were extracted from strains arg82D, arg82D trans-formed with pARG82, parg82D131 A, parg82Dasp,ptetspARG82, ptetKCS1, or ptetMCM1, and from strainskcs1D and kcs1D transformed with pKCS1. These RNAswere probed with MFa1, an a-specific gene, whoseexpression 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 theexpression of MFa1 (lane 6), whereas the deletion of thepolyaspartate residues (lane 5) impaired its expression.Overexpresssion of Mcm1p, but not of Kcs1p norspArg82p, restored MFa1 RNA levels (lanes 8, 9, 10). It

is noteworthy that overexpression of Mcm1p in an arg82Dstrain had no effect on NCR or phosphate-regulatedgenes (Fig. 7, lane 8).

All these data show that expression of Mcm1p-depen-dent genes does not necessitate the production of inositolpyrophosphates in contrast to NCR and phosphate-regu-lated genes. Thus the role of Arg82p in Mcm1-mediatedgene expression is to interact with and stabilize Mcm1p,as overexpression of this protein can compensate for thelack of Arg82p.

The polyaspartate domain of Arg82p was shown to becrucial for its role in the arginine regulation (Qiu et al.,1990; Dubois et al., 2000) indicating that this region couldbe important for the interaction between Arg82p and theMADS-box proteins Mcm1p and Arg80p. To address thispoint, two-hybrid assays were used to examine the inter-action between Arg80p or Mcm1p and arg82p mutatedproteins (arg82D131 A, arg82D282-303). Deletion of thepolyaspartate domain and not mutation impairing the IPkinase activity caused a significant decrease in the inter-action with Mcm1p or Arg80p (Table 2). In addition weexamined the stability of Mcm1p and Arg80p in the wild-type and mutated arg82 strains by Western blot. Mcm1pwas produced under the control of tet promoter and wasdetected using antibodies raised against purified GST-Mcm1p, whereas Arg80p tagged at its C-terminal endwith V5 epitope, was produced under the control ofGAL10 promoter, and detected with anti-V5 antibodies.Protein stability was measured after addition of cyclohex-imide to the culture (20 mg per ml). The stability of Mcm1pand Arg80p was impaired in the arg82Dasp strain to thesame extent as in a arg82D strain. In contrast, impairingthe IP kinase activity of Arg82p (mutation D131A) had noeffect on the stability of the two MADS-box proteins(Fig. 9).

Fig. 8. Northern blotting of MFa1 gene in different kcs1 or arg82 mutant strains, and in arg82D strain overexpressing different proteins. Total RNAs were extracted from different strains grown on synthetic medium (see Experimental procedures) and 30 mg were analysed by Northern blotting with 32P-labelled MFa1 and ACT1 DNA probes. Strains were deleted of KCS1 (lanes 1 and 2) or ARG82 genes (lanes 3–10). kcs1D strain was transformed with pFV241 (pURA3, KCS1, lane 1), pCM262 (pURA3, lane 2), plasmids (see Experimental pro-cedures). arg82D strain was transformed with pFV145 (pURA3, ARG82, lane 3), pCM262 (pURA3, lane 4), pFV160 (pURA3, arg82Dasp, 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).

Table 2. Ability of mutated GAD-arg82 proteins to interact with GBD-Arg80p and GBD-Mcm1p.

Hybrid

b-Galactosidase specific activity (nmol of o-nitrophenyl-b-D-

galactopyranoside hydrolysed min-1 mg-1 of protein)

GBD-Arg80p(pME46)

GBD-Mcm1p(pNA51)

GAD (pACTII) 2 2GAD-Arg82p (pME18) 23 26GAD-arg82Dasp (pNA14) 2 2GAD-arg82D131A (pFV174) 17 20

Transcription activation of the lacZ gene was estimated by determi-nation of b-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-arg82pfusions. Values are the mean of three independent measurementswith variations less than 15%.



Discussion

Recent studies have shown that inositol pyrophosphatesare essential for cell wall stability, cell growth, adaptationto salt stress, vacuolar morphogenesis and homologousDNA recombination (Luo et al., 2001; Dubois et al., 2002).However, the targets of these compounds remained to beidentified. We have therefore used DNA microarray tech-nology performed with strains deleted of Arg82p or Kcs1p,two inositol polyphosphate kinases, because these dele-tions led to a significant reduction of inositol pyrophos-phate pools. We have compared the abundance ofmRNAs between wild-type strain or strains overexpress-ing Arg82p or Kcs1p, to strains devoid of Arg82p or Kcs1prespectively. Among the genes identified by these exper-iments, two gene families clearly emerged: genes con-

trolled in response to the quality of the nitrogen source(NCR) and genes regulated in response to phosphateavailability (PHO). Expression of these genes was modi-fied simultaneously in both arg82D and kcs1D strains.Microarray data and Northern blotting experimentsshowed that NCR genes were downregulated in arg82Dor kcs1D cells, whereas PHO genes were upregulated inthese strains growing on high phosphate medium. Analy-sis of expression of NCR and PHO genes under differentphysiological growth conditions indicates that Arg82p andKcs1p are required for activation of NCR-regulated genesin response to nitrogen availability mainly through Nil1p,and for repression of PHO genes by phosphate. Mutationsin Arg82p or Kcs1p, impairing their kinase activity but nottheir stability, and leading to a significant reduction ofinositol pyrophosphates, affected expression of NCR andPHO genes to the same extent as deletion of each protein.Orthologous spArg82p and spKcs1p from S. pombe whichcontain the IP kinase domain but neither the polyaspartatestretch of Arg82p nor the leucine zippers of Kcs1p, couldfulfill Arg82p and Kcs1p functions in the control of NCRand PHO genes in S. cerevisiae. Thus inositol pyrophos-phates play a role in the transcriptional control of thesegenes. However, different inositol pyrophosphates couldbe required to control different cellular processes, as over-expression of Kcs1p in an arg82D strain restored vacuolarmorphology, resistance to salt stress (shown previously)and PHO gene expression, but not NCR gene expression.The cellular growth and the cell wall integrity were onlypartially improved. It is worth stressing that althoughKcs1p overexpression in an arg82D strain restored pyro-phosphates, the pattern was different from the pyrophos-phate pattern in the wild-type strain (Dubois et al., 2002).This may explain why NCR gene expression is notrestored.

In contrast, inositol pyrophosphates are not involved inthe control of Mcm1p-dependent genes, as Kcs1p and thekinase activity of Arg82p were not required for expressionof these genes. Our data show clearly that Arg82p hastwo functions. One function requires the presence of thepolyaspartate domain, essential for Mcm1p and Arg80pstabilization, suggesting a role of chaperone for Arg82p,and the other function which is to produce inositol poly-phosphates, is independent of the polyaspartate domain.

Inositol pyrophosphates are required for numerous cel-lular processes regulated through transduction cascadesinvolving protein phosphorylation. Cell wall integrity andresistance to salt stress imply two MAP kinase signallingpathways (reviewed in Banuett, 1998). The regulation ofgenes in response to phosphate availability requires acti-vation of the cyclin-CDK (cyclin-dependent kinase) com-plex Pho80p-Pho85p (reviewed in Carroll and O’Shea,2002). The TOR kinases have an essential role in prevent-ing the expression of nitrogen-regulated genes in cells

Fig. 9. Determination of Arg80p or Mcm1p stability in wild-type and mutated arg82 strains, using Western blotting. Aliquots of exponen-tially growing cells were taken at different times (indicated in the figure) after the addition of 20 mg ml-1 cycloheximide at time 0. Pro-teins were extracted, electrophoresed on 10% SDS-polyacrylamide gel and electrotransferred to Hybond membrane. Each lane contains about 25 mg protein.Strain 4719arg82D (ura3, trp1, arg82::kanMX4) was transformed with pFL39 (TRP1), pFV194 (TRP1, ARG82), pFV196 (TRP1, arg82D131 A) and pFV197 (TRP1, arg82Dasp) plas-mids.A. Arg80p protein tagged with V5 epitope (see Experimental proce-dures) was visualized with anti-V5 antibodies. To detect Arg80p, the above strains were also transformed with plasmid pFV260 (pGAL10-ARG80-V5, 2m, 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, 2m, URA3).



growing on an optimal nitrogen source (reviewed inMagasanik and Kaiser, 2002). The role of protein kinasesin nitrogen and phosphate metabolism would be to pre-vent by phosphorylation the transcriptional activators(Gln3p, Nil1p and Pho4p) to translocate to the nucleus.We propose a role for inositol pyrophosphates in the trans-duction of the physiological signal in different regulatorypathways. However, full expression of PHO genes alsorequires 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 pyro-phosphates, in the SWI/SNF and INO80 chromatinremodelling allowing induction of transcription of somephosphate-responsive genes. This suggests that inositolpolyphosphates and inositol pyrophosphates could controlthe expression of PHO genes at different levels and inopposite ways. InsP4 and InsP5 are required for activationof PHO gene expression at low phosphate concentrationthrough chromatin remodelling, whereas inositol pyro-phosphates are required to sustain repression of PHOgenes at high phosphate concentration. Thus the inositolpyrophosphates could play a role in the phosphate signal-ling pathway.

Inositol pyrophosphates could contribute to the trans-duction of different physiological signals by controllingdirectly or indirectly the phosphorylation state of key sig-nalling or regulatory proteins. It has been reported thatthe pyrophosphate groups can be donated to proteins,providing a novel means of protein phosphorylation. It wasalso proposed that the inositol pyrophosphates might reg-ulate protein activation analogous to the way in whichGTP regulates activity of G proteins (Luo et al., 2001;Dubois et al., 2002).

Experimental procedures

Strains and media

BY4709 (MATa, ura3) and BY4719 (MATa, trp1, ura3) yeaststrains (Brachmann et al., 1998) were used to construct dele-tion of ARG82 and KCS1 genes as described in Dubois et al.(2002), yielding strains 03127c (arg82::kanMX4, ura3),4719arg82D (arg82::kanMX4, trp1, ura3), and 4709kcs1D(kcs1::kanMX4, ura3). BY4709 was also used to create dele-tion of GLN3 or NIL1 genes using the long flanking homologystrategy (Wach, 1996). The kanMX4 cassettes flanked byabout 500 bp corresponding to the promoter and terminatorregions of the target genes were synthesized by a two-stepPCR. The DNA fragments containing the different cassetteswere used to transform strain BY4709 on rich media platescontaining 200 mg ml-1 of geneticin. The correct targeting ofthe deletions in G418r transformants was verified by PCRusing whole cells as a source of DNA and appropriate prim-ers. The following strains were obtained: gln3::kanMX4(4709gln3D) and nil1::kanMX4 (4709nil1D and 4700nil1D). Toconstruct strains with multiple deletions (gln3D, arg82D andnil1D, arg82D) we have used the gene disruption cassette

loxP-kanMX4-loxP (Guldener et al., 1996). To eliminate thekanMX4 marker from the disrupted gene, the mutated strainwas transformed with the cre expression plasmid pSH47,which carries the URA3 marker gene and the cre gene underthe control of the inducible GAL1 promoter. Expression of thecre recombinase was induced by shifting cells from YPD(glucose) to YPG (galactose) medium for 2 h. The loss of thekanMX4 cassette was detected by plating cells on YPD andreplica plating the colonies onto YPD-G418. The cre expres-sion plasmid was removed from the strain by streaking cellson plates containing 5-fluoroorotic acid to counterselect forthe loss of the plasmid. Strain 03167b (gln3D, nil1D) wasobtained by crossing strain 4709gln3D and 4700nil1D.BY4743 (Brachmann et al., 1998), a diploid strain (ura3/ura3,his3/his3, leu2/leu2) and 4743kcs1D (ura3/ura3, his3/his3,leu2/leu2, kcs1::kanMX4/kcs1::kanMX4) were used as recip-ient strains for plasmids expressing PHO5-HIS3 or DAL7-URA3. Strain HY (Louvet et al., 1997) was used for transfor-mation by plasmids pAS1, pACTII and their derivatives (two-hybrid analysis) (Durfee et al., 1993). E. coli strain XL1B wasused for plasmid amplification and for in vitro mutagenesis.For RNA preparations and for the two-hybrid experiments,yeast strains were grown on synthetic medium containing0.7% yeast nitrogen base with ammonia and without aminoacids and 3% glucose, or on 0.7% yeast nitrogen base with-out ammonia and without amino acids + 1 mg ml-1 glutamineand 3% glucose. Growth on proline was achieved by filteringthe cells grown on glutamine medium and cultivating themon fresh minimal medium with 1 mg ml-1 proline as a nitrogensource for 2 h. The phosphate amount was 1.5 mg KH2PO4

per ml (high phosphate medium) or 30 mg KH2PO4 per ml (lowphosphate medium). For the two-hybrid experiments theyeast nitrogen base medium with ammonia was supple-mented with all the amino acids, except those whose omis-sion was required for plasmid selection. For Western blotexperiments, yeast strains were grown on minimal medium(pH 6.5) which contained 1% galactose, vitamins and mineraltraces (Messenguy, 1976).

Genetic manipulations

The low copy pFL38 plasmid was used in this work to bearwild-type and mutated arg82 (Dubois et al., 2000) and kcs1genes (Dubois et al., 2002). Plasmid pFV145 (pFL38-ARG82) was used to create the following amino acidchanges, S257A-S258A-L259A-L269A, by in vitro mutagen-esis leading to plasmid pFV215.

The plasmids pFV186 and pFV187 overexpressing ARG82and KCS1 genes, respectively, under the dependence of thetet 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 promoterpresent in plasmid pCM262 (gift from E. Herrero). ForARG82sp (locus CAB63791), a 810 bp BamHI-BamHI DNAfragment was synthesized by PCR using appropriate oligo-nucleotides as primers with BamHI restriction sites, andgenomic wild-type DNA from S. pombe as template, blunt-ended with T4 DNA polymerase and inserted in the PmeIrestriction site of plasmid pCM262, yielding plasmid pFV234.For KCS1sp (locus CAA20701), a 2.9 kb EcoRI-EcoRI DNAfragment was synthesized by PCR using appropriate oligo-



nucleotides as primers with EcoRI restriction sites, andgenomic wild-type DNA from S. pombe as template, blunt-ended with T4 DNA polymerase and inserted in the PmeIrestriction site of plasmid pCM262, yielding plasmid pFV236.

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

Plasmid pFV260 (pGAL10-ARG80-V5, 2m, URA3) wasobtained by insertion of a 530 bp EcoRI-EcoRI DNA fragmentin the EcoRI site of plasmid pYES2 (pGAL10,V5, 2m, URA3,Invitrogen).

To express the HIS3 gene under the dependence of thePHO5 promoter, we synthesized by PCR a BamHI-BamHI540 bp DNA fragment, covering the PHO5 region from -541to -1 containing the Pho4p and Pho2p binding sites, and a2.3 kb BamHI–BamHI DNA fragment covering the HIS3region from +1 to +2298, containing the coding sequenceand the terminator of the gene. The 3¢ end of the PHO5fragment contained an extended sequence complementaryto an extended sequence at the 5¢ end of HIS3. The productsof the two previous PCRs were used as templates to synthe-size by PCR with appropriate primers, a DNA fragment con-taining the PHO5 promoter fused to the coding sequence ofHIS3. This 2.85 kb BamHI–BamHI DNA fragment wasinserted in the BamHI site of pFL38 vector (URA3, CEN6,ARS4) yielding plasmid pFV309.

To express the URA3 gene under the dependence of theDAL7 promoter, we synthesized by PCR a BamHI–BamHI450 bp DNA fragment, covering the DAL7 region from -451to -1 containing the GATAA sequences, Gln3p and Gat1pbinding sites, and a 1.3 kb BamHI–BamHI DNA fragmentcovering the URA3 region from +1 to +1300, containing thecoding sequence and the terminator of the gene. The 3¢ endof the DAL7 DNA fragment contained an extended sequencecomplementary to an extended sequence at the 5¢ end ofURA3. The products of the two previous PCRs were used astemplates to synthesize by PCR with appropriate primers, afragment containing the DAL7 promoter fused to the codingsequence of URA3. This 1.75 kb BamHI–BamHI DNA frag-ment 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 formicroarray hybridizations was performed according to Fouryand Talibi (2001), using Cy3-dCTP or Cy5-dCTP (AmershamPharmacia Biotech). Hybridizations were performed accord-ing to Foury and Talibi (2001). The hybridization signal wasdetected by scanning microarrays using GenePix 4000 laserscanner and GENEPIX 3.01 software (Axon Instruments). The

normalization factors calculated by this program were usedto set optimal PMT settings (photomultiplier tube) on thescanner 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 calculat-ing the mean ratio eliminating the top and bottom values ofseven independent microarray experiments with independentRNA preparations, for tetARG82/arg82D and tetKCS1/kcs1D.

Northern blot analysis

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

Western blot analysis

Western blot analysis was performed according to El Bakk-oury et al. (2000). Mcm1p was detected with antibodiesraised against GST-Mcm1p, and Arg80p was detected withantibodies 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 withplasmids pME18 (GAD-Arg82), pNA14 (GAD-arg82Dasp),pFV174 (GAD-arg82D131 A), pME46 (GBD-Arg80) andpNA51 (GBD-Mcm1), as described previously (El Bakkouryet al., 2000). b-galactosidase activity was assayed asdescribed by Miller (1972). Protein contents were determinedby the Folin method.

Acknowledgements

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

This work was supported by EEC grant GARNISH (con-tract number QLK3-CT-2000–00174) and by a grant fromBruxelles-Capitale.

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Documents

Arg82p is a bifunctional protein whose inositol polyphosphate kinase activity is essential for nitrogen and PHO gene expression but not for Mcm1p chaperoning in yeast