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Impaired PRPP-synthesizing capacity compromises cell integrity signalling in Saccharomyces cerevisiae

School of Life Sciences, Heriot-Watt University, Edinburgh EH14 4AS, UK

Correspondence
Michael Schweizer
M.Schweizer{at}hw.ac.uk

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
In Saccharomyces cerevisiae, PRS genes comprise a family of five paralogous genes. Previously, it has been shown that in the cell the gene products are organized into two interacting complexes, one of which is a heterodimer and the other a heterotrimer. Here, it has been demonstrated that in addition to supplying the cell with the key metabolic intermediate PRPP [5-phospho-D-ribosyl-1()-pyrophosphate], the gene products contribute to the maintenance of cell integrity. Specifically, the phosphorylation of Rlm1, one of the end points of the cell integrity signalling pathway, is significantly impaired following deletion of any one of the PRS genes, in particular PRS1 and PRS3. This is reflected in changes in the expression of the alternative 1,3--glucan synthase catalytic subunit, Fks2, as measured by its promoter activity. Yeast two-hybrid analysis has shown that Prs1, specifically the non-homologous region, NHR1-1 and Prs3, and to a lesser extent Prs2 and Prs4, interact with the MAPK (mitogen-activated protein kinase) of the cell integrity pathway, Slt2. When PRS1 is lacking, the basal level of phosphorylation of Slt2 is increased. Furthermore, prs1 and prs3 strains have an increased chitin content under normal growth conditions. -Factor sensitivity and Calcofluor White resistance associated with the lack of Prs1 and Prs3 corroborate the involvement of these two gene products in cell integrity signalling. It is postulated that Prs polypeptides play a significant role in the remodelling of the cell wall and may have a direct involvement in cell integrity signalling.

*These authors contributed equally to the work.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
prpp [5-phospho-D-ribosyl-1()-pyrophosphate] is an essential biosynthetic precursor for the synthesis of purine, pyrimidine nucleotides and the pyridine nucleotides NAD⁺ and NADP⁺ (Becker, 2001). We have shown that the Saccharomyces cerevisiae genome contains five unlinked paralogous genes, each capable of encoding PRPP synthetase (Prs: EC 2.7.6.1; ATP : D-ribose-5-phosphate diphosphotransferase). A systematic phenotypic analysis has been carried out with our collection of strains representing all possible combinations of deletions of the five PRS genes. The results obtained define three phenotypes: (i) a synthetic lethal phenotype when PRS1 or PRS3 was deleted from a prs5 strain – simultaneous deletion of PRS2 and PRS4 in combination with loss of PRS1 or PRS3 also results in inviability; (ii) a second phenotype that is encountered in strains containing deletions of PRS1 and PRS3 together or in combination with lack of PRS2 or PRS4 manifests itself as a reduction in growth rate, enzyme activity and nucleotide content; and (iii) deletion of PRS2, PRS4 or PRS5 or combinations thereof has reduced enzyme activity, but are unimpaired in growth and nucleotide profiles (Carter et al., 1994; Hernando et al., 1998, 1999). Three viable triple deletion combinations, prs2 prs4 prs5, prs1 prs3 prs4 and prs1 prs2 prs3, define three minimal subunits, Prs1/Prs3, Prs2/Prs5, Prs4/Prs5, respectively An extensive two-hybrid (Y2H) analysis suggested the existence in the wild-type of two interacting functional entities which may have compensatory function since in the absence of one entity or one of its components the yeast cell can still survive (Hernando et al., 1998, 1999). In accordance with our data, interactions between Prs polypeptides have been identified in large-scale Y2H and high-throughput MS analyses (Bader et al., 2003; Ito et al., 2001; Uetz et al., 2000) (http://www.bind.ca/ or http://mips.gsf.de/proj/yeast/tables/interaction/).

Since none of the PRS deletant strains had an obvious phenotype with the exception of slow growth in prs1 and prs3 strains, it was significant that a prs3 mutation was uncovered in a colony-sectoring screen analysis for mutations which are co-lethal with whi2. Mutation of WHI2 results in caffeine sensitivity (Binley et al., 1999) and it is now known that Whi2 is a binding partner for Psr1 phosphatase and both proteins are necessary for a full activation of the general stress response (Kaida et al., 2002). We have shown that caffeine sensitivity is characteristic of all PRS deletant strains with those lacking PRS1 and/or PRS3 being the most sensitive. A possible link between Prs and the maintenance of cell integrity is provided by the observation that in prs1 and prs3 strains the caffeine-induced release of alkaline phosphatase can be counteracted by stabilizing the medium with 1 M sorbitol; this is a strong indication that the maintenance of cell integrity is compromised in these strains. Furthermore, electron microscopy indicated that in PRS deletant strains there is a higher incidence of plasma membrane invaginations and accumulation of cytoplasmic vesicles than in the wild-type (Schneiter et al., 2000).

Cell integrity is dependent upon a functional Pkc1-Slt2 signalling cascade (Gustin et al., 1998; Hohmann, 2002; Klis et al., 2002). The pathway is a MAPK (mitogen-activated protein kinase) cascade consisting of MAPKK kinase Bck1, the redundant MAPK kinases Mkk1 and Mkk2, and the MAP kinase Slt2 (Heinisch et al., 1999). Inputs into the pathway are mediated via sensors located in the plasma membrane, Mid2 and members of the Wsc family (Popolo et al., 2001; Rajavel et al., 1999; Verna et al., 1997). There are two known outputs: the transcription factors SBF, consisting of Swi4/Swi6 (Gray et al., 1997; Igual et al., 1996; Madden et al., 1988, 1990; Queralt & Igual, 2003) and Rlm1, a member of the MADS (Mcm1-Arg80-Deficiens-Serum response factor) box family (Dodou & Treisman, 1997; Jung & Levin, 1999; Watanabe et al., 1995, 1997). This pathway is induced when there is a requirement for polarized cell growth, e.g. during budding and formation of mating projections and also when cells are subjected to environmental stresses, such as increased temperature (Kamada et al., 1995; Zarzov et al., 1996). The cell integrity pathway is also activated when yeast are exposed to Calcofluor White (CFW), caffeine or Zymolyase, all of which interfere with cell wall synthesis (de Nobel et al., 2000; Martin et al., 2000). In addition, strains carrying mutations of FKS1, whose product is the major subunit of the 1,3--glucan synthase, or GAS1, which encodes a GPI-anchored cell surface protein with -1,3-glucanosyl transferase activity, also have an activated cell integrity pathway (de Groot et al., 2001; Lagorce et al., 2003; Popolo et al., 1997; Popolo & Vai, 1999). It has been shown that the Pkc1-Slt2 pathway is also required for viability on entry into stationary phase or following inactivation of TOR (target of rapamycin) function by rapamycin or nutrient exhaustion (Angeles de la Torre-Ruiz et al., 2002; Krause & Gray, 2002; Torres et al., 2002). With the increasing volume of results obtained from large-scale analyses, the interaction between various areas of metabolism is becoming more obvious. In recent publications genes of the functional category C-compound and carbohydrate utilization appear among the most responsive to CFW and Zymolyase when transcription profiles are measured, underlining the integration of metabolic networks (Boorsma et al., 2004; Garcia et al., 2004; Lagorce et al., 2003).

PRPP synthetase transfers the pyrophosphate moiety released from ATP to ribose-5-phosphate, thus linking carbon and nitrogen metabolism by directing energy from the pentose phosphate pathway to the biosynthetic intermediate PRPP, a precursor of purine, pyrimidine and pyridine nucleotides and the amino acids histidine and tryptophan (Hove-Jensen, 1988; Khorana et al., 1958). In light of our previous observations suggesting that an alteration of the cell's PRPP-synthesizing capacity impinges on cell integrity, we undertook to examine the impact of the PRPP status of the cell on various stages along the cell integrity pathway. Specifically, we investigated (i) input – raised temperature, exposure to CFW and mating pheromone; (ii) interaction with the MAPK Slt2; and (iii) output – activation of Rlm1 and Fks2, an alternative subunit of 1,3--glucan synthase (Inoue et al., 1995).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Strains and growth conditions.
S. cerevisiae strains used in this study are listed in Table 1. Yeast cultures were grown in YEPD (1 % yeast extract, 2 % Bacto peptone, 2 % glucose) or in synthetic complete (SC) medium (0·17 % yeast nitrogen base without amino acids and , 0·5 % (NH₄)₂SO₄, 2 % glucose) supplemented with the appropriate amino acids or nucleobases (Kaiser et al., 1994). Solid media contained 2 % agar. Yeast transformations were carried out using the high efficiency yeast transformation method (Gietz et al., 1992), the ‘lazy bones' method (Woods & Gietz, 2001) or the ‘Plate’ method (Elble, 1992). Escherichia coli DH5 was used for plasmid propagation. E. coli cells were cultured in LB-medium and transformed by standard methods (Ausubel et al., 1995).

Phenotypic growth assays.
These were carried out under appropriate conditions by spotting 3–5 µl serial tenfold dilutions of a culture of OD₆₀₀=1·0 onto solid medium. Growth was recorded after 48–72 h. CFW was added to YEPD plates at the concentrations indicated. Incubation was carried out at either 28 or 37 °C for 2–3 days.

-Factor sensitivity.
When testing -factor sensitivity appropriately supplemented liquid SC medium was buffered by the addition of succinic acid to a final concentration of 85 mM and NaOH to a final concentration of 19 mM (Ketela et al., 1999). Cultures of MATa strains containing either control plasmids or plasmids bearing genes of interest were diluted to 2x10⁵ cells ml^–1 in buffered SC medium. Aliquots (50 µl) of each strain were removed and spread on YEPD or selective medium. -Factor (Sigma) was then added to the cultures at a final concentration of 1 or 2 µM. The cultures were incubated further at 30 °C over a period of 6–7 h. Aliquots (50 µl) were removed at intervals of 100 min and plated out on appropriate media. The plates were incubated for 48 h at 30 °C and the number of colonies formed on each plate was determined. -Factor sensitivity was measured by the difference between the number of colonies on pre- and post--factor plates for each strain.

Measurement of Fks2 promoter activity.
Individual transformants containing the FKS2(–706/–1)-lacZ reporter plasmid were grown in duplicate in 10 ml selective medium to a density of 1x10⁸ cells ml^–1. One set of duplicates was grown at 37 °C for a further 4 h and the second set were left to grow at 28 °C for the same length of time. Cells were then harvested by centrifugation in 15 ml Falcon tubes (6000 r.p.m., 4 °C) and washed once in 1 ml sterile ice-cold H₂O, and, if not to be used immediately, the pellet was stored at –80 °C until required. For cell disruption 300 µl Z-buffer (60 mM Na₂HPO₄.12 H₂O, 40 mM NaH₂PO₄.2H₂O, 10 mM KCl, 1 mM MgSO₄.7H₂O) was added to the cells followed by 0·5 g acid-washed glass beads (425–600 µm diam.) and the whole was vortexed at maximum speed for 1 min followed by 1 min incubation on ice. This step was repeated three times before centrifugation as described above (Guarente, 1983). The supernatant was transferred to a fresh tube and protein concentration was determined according to the method of Bradford (1976) The -galactosidase assay was performed using O-nitrophenyl--D-galactopyranoside (ONPG) as substrate. Depending on the protein concentration, 20–200 µl of crude extract in a final volume of 1 ml Z-buffer was combined with 200 µl ONPG (4 mg ml^–1). After a pale yellow colour developed, 0·5 ml 1 M Na₂CO₃ was added to stop the reaction. The specific activity of -galactosidase [U (mg protein)^–1] was calculated as described by Miller (1972).

Yeast two-hybrid analysis (Y2H).
The Gal4pDBD-Prs1-5 and Gal4pAd-Prs1-5 fusion plasmids (Hernando et al., 1999), and pGBT9-Snf1 and pGAD424-Snf4 (Roder et al., 1999) have been described previously. The truncated versions of Prs1, pGAD424-Prs1(NHR1-1) and pGBT9-(NHR1-1), were created by PCR to remove NHR1-1 which extends from nt 604 to 925 in the ORF of Prs1. For the other two constructs PRS1 in pGAD424 was bisected using the BamHI restriction site located at nt 746–751 of the PRS1 ORF; the two halves of the ORF were cloned into pGAD424 as a EcoRI/BamHI fragment to give pGAD424-Prs1(746-1281) and as a BamHI/BglII fragment to give pGAD424-Prs1(1-745) (A. Carter, unpublished data). The host strain PJ69-4A (James et al., 1996) was transformed with the two plasmids whose interaction was to be tested. At least three independent transformants were inoculated singly into 10 ml SC minus leucine and tryptophan and incubated overnight at 30 °C. Crude extracts were prepared and assayed for -galactosidase activity as described above with ONPG as substrate. The transformants were also tested for their ability to grow on selective media lacking adenine or histidine plus 150 mM 3-aminotriazole.

Rlm1 transcriptional activation.
Transformants containing one of the pHPS100 series of plasmids were grown in selective medium at 28 °C to OD₆₀₀=0·5, harvested, rinsed once with an equal volume of Z-buffer (see above) and centrifuged, and the pellet was taken up in 1/5 volume Z-buffer. The cells were broken by two cycles of freezing and thawing and -galactosidase activity was assayed by using 20 µl crude extract with the chemiluminescent substrate Galacto-Star, according to the manufacturer's instructions (Applied Biosystems). -Galactosidase activity was expressed as relative light units (RLU) (µg protein)^–1 using a tube luminometer, Lumat LB 9507 (Berthold Technologies). Protein concentration was determined according to the method of Bradford (1976). When measuring the influence of temperature or -factor on Rlm1 activation, the culture was divided into two aliquots, one served as the control and the other was incubated at 39 °C or treated with -factor at a concentration of 2 µM for 4 h before determining -galactosidase activity.

Measurement of chitin content.
This was performed according to the protocol kindly provided by Dr Laura Popolo (University of Milan, Italy). A 50 ml culture was grown overnight to a density of 1·0x10⁷ cells ml^–1 and harvested by centrifugation (6000 r.p.m., 4 °C). The cells were washed twice with 1·5 ml H₂O, the wet weight determined and the cells resuspended at a concentration of 1 g wet weight (ml H₂O)^–1; 1 ml 6 % KOH was added per 0·1 g cells and the cell suspension was incubated at 80 °C for 90 min. After cooling to room temperature, 0·1 ml glacial acetic acid was added and centrifuged at 13 000 r.p.m. for 15 min. The pellet was washed twice in 50 mM sodium phosphate buffer, pH 6·3. At this stage the pellet could be stored at –20 °C. Chitinase (Sigma #C-1650; 40 µl; 17 mg per 400 µl 50 mM sodium phosphate buffer, pH 6·3) was added to the pellet and the volume brought up to 600 µl with sodium phosphate buffer, pH 6·3. A control was set up containing 40 µl chitinase and 540 µl 50 mM sodium phosphate buffer, pH 6·3. The suspensions were incubated at 37 °C for 2 h. After vortexing thoroughly, a 400 µl sample was transferred to a fresh 1·5 ml Eppendorf tube to which 25 µl -glucuronidase (Sigma #G0876) was added. The tubes were incubated at 37 °C for 1 h, boiled for 1 min and centrifuged at 12 000 r.p.m. for 5 min. A sample of supernatant (50 µl) was transferred to a glass tube and the final volume was brought up to 250 µl with H₂O. K₂B₄O₇.4H₂O (250 µl 0·27 M, pH 9·0) was added to the tube and the whole was boiled for 8 min. The sample was cooled to room temperature and 3 ml 1x Reissig solution (10x stock: 10 g dimethylaminobenzaldehyde, 12·5 ml 10 M HCl, 87·5 ml glacial acetic acid) was added. After vortexing the tubes were incubated at 37 °C for 40 min and the absorbance was measured at 585 nm. A standard curve was prepared using appropriately diluted N-acetylglucosamine (10 mg ml^–1 stock). For chitin measurements following exposure to CFW, cells were cultured in the presence of 100 µg CFW ml^–1 at 28 °C for 4 h prior to harvesting.

Western blotting.
Yeast cells were grown overnight at 28 °C to mid-exponential phase and diluted to OD₆₀₀=0·3 (9x10⁶ cells ml^–1) in a volume of 200 ml YEPD. The cultures were allowed to grow for one generation before removing a 10 ml (1x10⁸–2x10⁸ cells) aliquot which was processed as described by Reinoso-Martin et al. (2003) and Schuetzer-Muehlbauer et al. (2003). Four further 10 ml aliquots were dispensed into individual pre-warmed universals and incubated at 37 °C. At hourly intervals thereafter the contents of a single universal were processed as indicated above. Each individual pellet was resuspended in 100 µl loading buffer (40 mM Tris/HCl, pH 6·8, 8·0 M urea, 5 % SDS, 0·1 mM EDTA, 10 mg bromophenol blue) which was supplemented with 1 % -mercaptoethanol and 10 % 1 M Tris-base. For the sample taken at time zero a volume of the suspension, equivalent to approximately 4x10⁷ cells, was loaded onto the gel. To compensate for cell growth during the 4 h incubation period, the remaining samples were diluted with an appropriate amount of loading buffer to a concentration which corresponded to 4x10⁷ cells. Cell extracts were separated in SDS-8 % polyacrylamide gels and transferred to Hybond-P PVDF membrane (0·2 µm; Amersham Bioscience) using the Mini Trans-Blot Electrophoretic Transfer system (Bio-Rad) according to the manufacturer's instructions. Phosphorylated Slt2 was detected using anti-phospho-p44/p42 MAPK (Thr²⁰²/Tyr²⁰⁴) as the primary antibody (1 : 2000 dilution; New England Biolabs) (Martin et al., 2000) and detected by goat anti-rabbit antibody (1 : 5000 dilution; Sigma) with the ECL+Plus detection system from Amersham Bioscience. Slt2 was detected by anti-GST-Slt2 antibody (1 : 1000 dilution) (Martin et al., 1993) generously provided by Drs Maria Molina and Humberto Martin (Universidad Complutense de Madrid, Spain) and detected as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
The requirement for Prs in the yeast cell response to stress
Various prs strains were examined for their growth characteristics following exposure to elevated temperature (Kamada et al., 1995) and CFW (Ketela et al., 1999). Fig. 1 shows that at 37 °C prs1 and prs3 are impaired in their growth and the double deletant prs1 prs3 is inviable. The temperature-sensitive phenotype of prs1 prs3 can be reversed by the addition of 1 M sorbitol to the medium (data not shown). No thermosensitive phenotype was observed in a prs4 or prs5 strain or in the triple deletant strain prs2 prs4 prs5.

View larger version (107K): [in this window] [in a new window]   Fig. 1. Loss of PRS1 and PRS3 impairs growth at 37 °C and confers resistance to CFW. Wild-type (YN94-1, WT) and congenic prs strains (YN94-4 prs1; YN94-18 prs3; YN94-19 prs2; YN94-20 prs4; YN96-1 prs5; YN97-88 prs1 prs3; YN96-72 prs2 prs4 prs5; YN95-25 prs1 prs3 prs4; YN95-36 prs1 prs2 prs3 were grown to early stationary phase (1x108 cells ml–1) in YEPD and serial tenfold dilutions prepared. Aliquots (3 µl) were spotted onto YEPD and YEPD+CFW (500 µg ml–1) plates and incubated for 3 days at the temperatures indicated. The experiment was repeated at least three times and a representative illustration is shown.

It is known that exposure to -factor induces morphological changes in the cell wall which activate the cell integrity pathway (Buehrer & Errede, 1997). Treatment with 1 µM -factor over a period of 6 h revealed that in comparison to the wild-type, strains lacking PRS1 displayed a 46 % reduction and strains lacking PRS3 a 73 % reduction, in viability. Simultaneous deletion of PRS1 and PRS3 has an additive effect since viability decreased more rapidly within 2 h and reached a similar level to the prs3 strain, 77 %, after 6 h (Fig. 2a). Surprisingly, the -factor sensitivity of prs1 prs3 can be overcome by the additional deletion of either PRS2 or PRS4; these triple deletants retained their wild-type response even when the -factor concentration was increased to 2 µM. Deletion of PRS2 PRS4 PRS5 had no effect on the response of a MATa cell to -factor (Fig. 2b).

View larger version (23K): [in this window] [in a new window]   Fig. 2. Response of PRS deletant strains to -factor and influence of PKC1 overexpression on prs-induced phenotypes. Cells were grown at 30 °C in appropriately supplemented buffered SC medium containing -factor over a period of 6 h and samples were withdrawn at the times indicated to determine the number of viable cells represented as a percentage of viable cells at time zero. (a) Response curve of strains lacking PRS1 and/or PRS3. Strains were identical to those listed in the legend to Fig. 1 with the exception of prs3 which was YN96-77. -Factor was used at a concentration of 1 µM. Filled squares, wild-type; open squares, prs1; filled triangles, prs1 prs3; open triangles, prs3. (b) Response curve of PRS triple deletants. The concentration of -factor was 2 µM. Filled squares, wild-type; open squares, prs2 prs4 prs5; filled triangles, prs1 prs3 prs4; open triangles, prs1 prs2 prs3. (c) -Factor sensitivity of prs1 and prs3 strains with or without YEplac195-PKC1. The concentration of -factor was 2 µM. Filled squares, prs1[PKC1]; open squares, prs1; filled triangles, prs3[PKC1]; open triangles, prs3. For all experiments values are means±SD (n=4).

Sensitivity to -factor is also encountered in strains carrying a deletion of MID2, a gene which encodes a putative cell surface sensor known to activate the cell integrity pathway via Pkc1 (Ketela et al., 1999; Philip & Levin, 2001; Rajavel et al., 1999). Deletion of PRS1 and/or PRS3 in a strain lacking MID2 does not exacerbate the inherent -factor sensitivity of a mid2 strain. The independent nature of the apparently similar prs1- and/or prs3- and mid2-induced phenotypes is underlined by the observation that overexpression of either PRS1 or PRS3 does not correct the mid2-associated -factor-sensitive phenotype (data not shown).

Chitin levels in PRS deletant strains
CFW resistance goes hand in hand with altered chitin synthesis (Roncero & Duran, 1985; Roncero et al., 1988). Therefore, we decided to measure the chitin content of certain PRS deletant strains in the presence or absence of CFW. As is shown in Fig. 3, strains deleted for PRS2, PRS4 or PRS5, singly or combined, respond in the same way as the wild-type, i.e. they increase their chitin content at least fourfold upon exposure to CFW. On the other hand, deletion of PRS1 or PRS3 causes a threefold increase in chitin content and impairs the ability of the cell to increase its chitin content upon exposure to CFW. For the double deletant prs1 prs3 there is a 50 % increase in chitin content at 28 °C, whereas in the presence of CFW the chitin content is virtually doubled.

View larger version (21K): [in this window] [in a new window]   Fig. 3. Chitin content of prs strains. Strains as in Fig. 1 were grown in the presence or absence of 100 µg CFW ml–1 and then processed for chitin determination as described in Methods. Values are means±SD (n=5). White bars, YEPD; grey bars, CFW.

Rlm1 activation in prs strains

View larger version (30K): [in this window] [in a new window]   Fig. 4. Rlm1 activation is distorted by a lack of Prs. Strains as in Fig. 1, harbouring plasmids of the pHPS100 series, were grown in appropriately supplemented SC medium at 28 °C to mid-exponential phase (white bars). Incubation at 39 °C for 4 h (grey bars) and treatment with 2 µM -factor (black bars) were carried out as described in the legend to Fig. 2. -Galactosidase activity was determined luminometrically. Values are means±SD (n=3). Please note that different scales are used to represent -galactosidase activity in (a) and (b).

Next, we examined the effect of overexpression of Pkc1 on the impaired Rlm1 response of strains lacking PRS1 and/or PRS3 under normal growth conditions. Introduction of Pkc1 in a prs1 strain carrying the Rlm1 reporter plasmid had a positive effect on the transcriptional activation of Rlm1, increasing it by approximately twofold at 28 °C, 39 °C and following exposure to -factor (Fig. 5a). When PRS3 was lacking, overexpression of Pkc1 increased Rlm1 activation threefold at 28 and 39 °C, but only 1·7-fold after exposure to -factor (Fig. 5b). Overexpression of Pkc1 was also able to correct the defective activation of Rlm1 in a prs1 prs3 strain at 28 °C by effecting a sixfold increase; when -factor was tested in the same strain the response was improved almost fourfold to just slightly above the Rlm1 activation observed in the prs1 prs3 strain grown at 28 °C. However, Pkc1 overexpression does not improve Rlm1 activation sufficiently to allow the temperature-sensitive phenotype of the prs1 prs3 strain to be overcome (Fig. 5c).

View larger version (30K): [in this window] [in a new window]   Fig. 5. Influence of PKC1 on Rlm1 transcriptional activation in prs1 and/or prs3 strains. Rlm1 transcriptional activation was measured in YN94-4 prs1 (a), YN94-18 prs3 (b) and in YN97-88 prs1 prs3 (c) containing the appropriate plasmid of the pHPS100 series with or without the YEplac195-PKC1 plasmid. Values are means±SD (n=3). White bars, prs1 and prs3; grey bars, prs1 or prs3[PKC1].

View larger version (35K): [in this window] [in a new window]   Fig. 6. FKS2 activation in response to heat shock in wild-type and PRS deletant strains. Strains YN94-1 (wild-type, WT), YN96-50 (prs1), YN95-23 (prs2), YN96-52 (prs3), YN94-20 (prs4), YN96-1 (prs5) and YN97-88 (prs1 prs3), each containing the FKS2(–706/–1)-lacZ plasmid, were grown in SC medium minus uracil to a density of 1x108 cells ml–1 at 28 °C and aliquots were then grown further for 4 h at either 28 (light grey bars) or 37 °C (dark grey bars). Crude extracts were prepared and -galactosidase activity was measured as described in Methods. Values are means±SD (n=6).

Phosphorylation status of Slt2 in PRS deletant strains
An antibody which recognizes the dually phosphorylated MAPKs containing a TEY motif in the activation domain (Rodriguez-Pachon et al., 2002) was used to detect the phosphorylation status of Slt2 in the wild-type and strains deleted for individual PRS genes. Crude extracts of PRS deletant strains, cultivated as described in Methods, were separated on 8 % SDS-PAGE and transferred to a PVDF membrane followed by probing with anti-phospho-p44/42 MAPK (Thr²⁰²/Tyr²⁰⁴) antibodies. In the wild-type strain there was a faint signal corresponding to Slt2-P at time zero which increases in intensity with respect to time of incubation at 37 °C. In contrast in the prs1 strain there was a strong signal at time zero which was maintained over a period of 4 h incubation at 37 °C. A strain lacking PRS3 was also phosphorylated at time zero and this signal increased steadily over a period of 4 h. For control purposes the same membranes were stripped and reprobed with anti-GST-Slt2 antibody (Fig. 7).

View larger version (16K): [in this window] [in a new window]   Fig. 7. Slt2 phosphorylation in crude extracts of wild-type (WT) YN94-1, prs1 (YN96-50) and prs3 (YN96-52) strains. The strains were grown in YEPD to mid-exponential phase at 28 °C (time point zero) and then heat-shocked at 37 °C for the times indicated (h). The level of phospho-Slt2 was detected immunologically and the filter was stripped and reprobed with GST-Slt2 antibodies as described in Methods.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

We also examined the possibility of the involvement of Mid2 which is a putative cell wall stress sensor and upstream activator of the cell integrity pathway and whose deletion invokes phenotypes similar to those of prs1 and/or prs3 strains, e.g. -factor and caffeine sensitivity, and CFW resistance (Ketela et al., 1999; Rajavel et al., 1999). The viability of a mid2 prs1 prs3 strain suggests that the impact of the metabolic disturbance caused by altering the PRPP-synthesizing capacity of the cell impinges on the cell integrity pathway via Wsc sensors (Zu et al., 2001) or a cell internal signalling pathway parallel to the MID2-dependent signal transduction pathway.

It has been shown that when global transcript profiles of yeast exposed to CFW and Zymolyase are analysed with the REDUCE and Quontology algorithms there is an upregulation of Rlm1-regulated genes (Boorsma et al., 2004). The transcription factor Rlm1, one of the end points of the cell integrity pathway, is directly phosphorylated by Slt2 (Dodou & Treisman, 1997; Kirchrath et al., 2000; Watanabe et al., 1995, 1997). Induction of FKS2 expression is regarded as a measure of cell wall damage (de Nobel et al., 2000) and is regulated by both the cell wall integrity pathway and independently thereof (Jung & Levin, 1999). We have demonstrated that activation of Rlm1, as measured with the pHSP-100 series of plasmids, is reduced in each of the five prs strains, with the possible exception of prs5, in comparison to the wild-type. However, prs2, prs4 and prs2 prs4 prs5 strains are able to increase Rlm1 phosphorylation at 39 °C (Fig. 4a). This heat-induced response of Rlm1 may be sufficient to mediate the wild-type response of the FKS2 promoter observed in prs2 and prs4 strains (Fig. 6). Rlm1 activation and FKS2 promoter activity are also correlated in a prs5 strain since a temperature-dependent response was not observed for either reporter plasmid. In prs1 and prs3 strains Rlm1 activation is reduced by 90 % and is no longer responsive to increased temperature; this is reflected in the decreased levels of FKS2 promoter activity measured in the same strains (Figs 4 and 6).

The chitin measurements further emphasize the involvement of the Prs1/Prs3 complex in cell wall construction. First, the elevated chitin content in prs1, prs3 and prs1 prs3 strains points to a response on the part of the cell to a disturbance in the cell wall synthesis machinery. Second, in these strains the CFW-induced increase in chitin synthesis observed in the wild-type and strains compromised for, or lacking, the Prs2/Prs4/Prs5 complex is impaired (Fig. 3). This chitin-associated strengthening of the cell wall would suggest that the Prs1/Prs3 complex plays some role in the compensatory mechanism induced by perturbations of the yeast cell wall. This role is likely to be subtle since in none of the recent genome-wide analyses of the response to cell wall mutations has any of the PRS genes been identified. However, clustering of the data obtained following treatment with cell-wall-damaging agents (Garcia et al., 2004) or from strains carrying mutations affecting different aspects of cell wall construction (Lagorce et al., 2003) has indicated an enrichment of gene categories whose products are involved in metabolism and energy generation, and PRS genes can be included in these categories since their products are responsible for the synthesis of PRPP which is essential for nucleotide production. For instance UTP is required in the penultimate step of chitin synthesis to produce UDP-N-acetylglucosamine (Orlean, 1997; Roncero, 2002; Ruiz-Herrera et al., 2002). We have shown previously that the UTP+UDP+UMP content of strains lacking PRS1 and/or PRS3 is reduced by 75 and 80 %, respectively, in a prs1 prs2 prs3 strain. A prs1 prs3 prs4 strain contains only about 10 % of the UTP+UDP+UMP level of the wild-type, whereas in the triple deletant prs2 prs4 prs5 the level is indistinguishable from the wild-type. The levels of adenine and guanine tri-, di- and mononucleotides are also reduced in line with the levels of UTP+UDP+UMP (Hernando et al., 1998, 1999). These reduced nucleotide levels may be the primary signal which causes the cell wall defects in prs strains. However, this does not appear to be the whole story.

The Y2H analysis, which has been performed between the Prs polypeptides and the MAPK Slt2, would suggest that the Prs polypeptides do in fact interact with this element of the cell integrity pathway. In particular, there is a strong interaction between Slt2 and Prs1 or Prs3 in both orientations which gives rise to -galactosidase activities two- to threefold higher than the positive controls, Prs4/Prs5 and Snf1/Snf4 (Table 2). Furthermore, interaction between the Prs polypeptides and Slt2 can, with the exception of Slt2/Prs5, confer adenine and histidine prototrophy on the strain. The interaction Slt2/Prs5, which appears to be the weakest, is able to confer only histidine prototrophy in one orientation. Any interaction between Mkk1 or Mkk2 and Prs1–Prs4, although generating -galactosidase activity in excess of the background, is not sufficiently strong to switch on the adenine or histidine reporters (data not shown).

Removal of NHR1-1, which is a characteristic of the PRS1 ORF and is also found in the Prs-associated proteins of rat and man (Becker, 2001; Hernando et al., 1999), completely abolishes the Prs1/Slt2 interaction (Table 2). Other work has shown that deletion of NHR1-1 does not impair the interaction of Prs1 with the other Prs polypeptides (A. Carter, unpublished data). In contrast, there is still a Prs1/Slt2 interaction when Slt2 is co-transformed independently with the two halves of Prs1. It remains for these Prs1/Slt2 interactions to be validated by co-immunoprecipitation.

In the wild-type, incubation at 37 °C leads to an increase in the signal for phosphorylated Slt2 from that obtained at 28 °C in agreement with published data (Lagorce et al., 2003; Martin et al., 2000; Rodriguez-Pachon et al., 2002). Western blot analysis shows that, in contrast to the wild-type, in a prs1 strain the signal corresponding to phospho-Slt2 is stronger at 28 °C and is maintained over a period of 4 h during incubation at 37 °C (Fig. 7). Interestingly, incubation at 37 °C for a further 2 h resulted in a drastic reduction in the strength of the signal (data not shown). The phosphorylated form of Slt2 in a prs3 strain shows the same profile in a Western blot as that of the wild-type, albeit with a stronger signal at time zero. However, this signal is not lost upon further incubation at 37 °C (data not shown).

The loss of the phospho-Slt2 signal at elevated temperature in the absence of PRS1 together with the Y2H interaction of Slt2 with both Prs1 and Prs3 would suggest that the Prs1/Prs3 complex may have a supporting role in the functioning of the cell integrity pathway. Intriguing possibilities are that it may provide a dedicated source of PRPP for ATP production for Slt2 phosphorylation or that the complex functions as a scaffold or regulates a phosphatase, such as Msg5 (Collister et al., 2002; Flandez et al., 2004; Hahn & Thiele, 2002; Huang & Symington, 1995; Martin et al., 2000) known to be essential for maintaining a low level of signalling through the cell integrity pathway.

	ACKNOWLEDGEMENTS

 
This work was supported by Heriot-Watt University, Eda Lady Jardine Trust (S. V.) and the Henry Lester Trust Ltd (K. W.). We would also like to thank Piet de Groot, Jürgen J. Heinisch, Humberto Martin, Hélène Martin-Yken and Maria Molina for supplying plasmids and antibodies (HM, MM) and A. Bensmail for help with preliminary experiments.

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