Microbiology (2004), 150, 3327–3339
DOI 10.1099/mic.0.27373-0
Impaired PRPP-synthesizing capacity
compromises cell integrity signalling in
Saccharomyces cerevisiae
Ke Wang,* Stefano Vavassori,* Lilian M. Schweizer
and Michael Schweizer
Correspondence
School of Life Sciences, Heriot-Watt University, Edinburgh EH14 4AS, UK
Michael Schweizer
[email protected]
Received 3 June 2004
Revised
6 August 2004
Accepted 11 August 2004
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(a)-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-b-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, prs1D and prs3D strains have an increased chitin
content under normal growth conditions. a-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.
INTRODUCTION
PRPP [5-phospho-D-ribosyl-1(a)-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 prs5D 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
*These authors contributed equally to the work.
Abbreviations: CFW, Calcofluor White; MAPK, mitogen-activated
protein kinase; PRPP, 5-phospho-D-ribosyl-1(a)-pyrophosphate.
0002-7373 G 2004 SGM
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, prs2D prs4D prs5D, prs1D prs3D
prs4D and prs1D prs2D prs3D, 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 prs1D and
prs3D 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
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K. Wang and others
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 prs1D and prs3D 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-DeficiensSerum 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-b-glucan synthase, or GAS1, which encodes a GPIanchored cell surface protein with b-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
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(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-b-glucan synthase (Inoue et al., 1995).
METHODS
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 NHz
4 , 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 DH5a was used for plasmid
propagation. E. coli cells were cultured in LB-medium and transformed by standard methods (Ausubel et al., 1995).
Plasmids. Plasmid YEplac195-PKC1 (URA3/2 m) harbours a geno-
mic fragment containing PKC1 cloned into the HindIII and EcoRI
restriction sites of YEplac195 (Gietz & Sugino, 1988) and was kindly
provided by Dr M. J. R. Stark (University of Dundee). The pHPS100
series of plasmids was kindly provided by Dr J. J. Heinisch
(University of Osnabrück, Germany). This is a CEN/ARS vector
containing both a lexA-RLM1 fusion and a lacZ reporter gene preceded by five lexA binding sites. This lexA-Rlm1 fusion activates a
b-galactosidase reporter construct after phosphorylation of the Rlm1
moiety by Slt2. pHPS100-T, pHPS100-U and pHPS100-L carry the
selection markers TRP1, URA3 and LEU2, respectively (Kirchrath
et al., 2000). FKS2(2706/21)-lacZ containing the 2 m origin of
replication was generously provided by Dr P. de Groot (University
of Amsterdam, The Netherlands) and is described by de Nobel et al.
(2000) and Zhao et al. (1998). pGAD424-Slt2 (originally created by
Dr Maria Molina, Universidad Complutense, Madrid, Spain) was
kindly provided by Dr Hélène Martin-Yken (INSA, Toulouse,
France) after obtaining permission from Dr Molina. To obtain Slt2
in pGBT9 the 1?5 kb BamHI/BglII fragment from pGAD424-Slt2
containing the ORF of Slt2 was cloned into BamHI-restricted
pGBT9 and checked by restriction analysis.
Phenotypic growth assays. These were carried out under
appropriate conditions by spotting 3–5 ml 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 uC for
2–3 days.
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Prs and cell integrity
Table 1. Yeast strains used in this study
Strain
PJ69-4A
YN94-1
YN94-2
YN94-4
YN94-18
YN94-19
YN94-20
YN94-23
YN95-25
YN95-36
YN96-1
YN96-50
YN96-52
YN96-72
YN96-77
YN97-3
YN97-4
YN97-12
YN97-68
YN97-70
YN97-86
YN97-88
YN97-90
Genotype
Reference
2
MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4D gal80D LYS2 : : GAL1UAS GAL1TATA-HIS3
GAL2UAS-GAL2TATA-ADE2 met2 : : GAL7-lacZ
MATa ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 GAL
MATa ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 ssd1-d2 GAL
YN94-1 prs1D : : HIS3
YN94-1 prs3D : : TRP1
YN94-1 prs2D : : URA3
YN94-1 prs4D : : LEU2
YN94-1 prs1D : : HIS3 prs4D : : LEU2
YN94-1 prs1D : : HIS3 prs3D : : TRP1 prs4D : : LEU2
YN94-1 prs1D : : HIS3 prs2D : : URA3 prs3D : : TRP1
YN94-1 prs5D : : KanMX4
YN94-1 prs1D : : loxP-KanMX4-loxP
YN94-1 prs3D : : loxP-KanMX4-loxP
YN94-1 prs2D : : URA3 prs4D : : LEU2 prs5D : : KanMX4
YN94-1 prs3D : : loxP
YN94-2 prs2D : : loxP-KanMX4-loxP
YN94-2 prs1D : : loxP prs3D : : loxP
YN94-2 prs2D : : loxP prs4D : : loxP-KanMX4-loxP
YN94-2 prs2D : : loxP prs5D : : loxP-KanMX4-loxP
YN94-2 prs2D : : loxP prs4D : : loxP prs5D : : loxP-KanMX4-loxP
YN94-2 prs1D loxP-KanMX4-loxP prs2D : : loxP
YN94-1 prs1D : : loxP prs3D : : loxP
YN94-2 prs4D : : loxP prs5D : : loxP
a-Factor sensitivity. When testing a-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 26105 cells ml21 in
buffered SC medium. Aliquots (50 ml) of each strain were removed
and spread on YEPD or selective medium. a-Factor (Sigma) was
then added to the cultures at a final concentration of 1 or 2 mM.
The cultures were incubated further at 30 uC over a period of 6–7 h.
Aliquots (50 ml) were removed at intervals of 100 min and plated
out on appropriate media. The plates were incubated for 48 h at
30 uC and the number of colonies formed on each plate was determined. a-Factor sensitivity was measured by the difference between
the number of colonies on pre- and post-a-factor plates for each
strain.
Measurement of Fks2 promoter activity. Individual trans-
formants containing the FKS2(2706/21)-lacZ reporter plasmid
were grown in duplicate in 10 ml selective medium to a density of
16108 cells ml21. One set of duplicates was grown at 37 uC for a
further 4 h and the second set were left to grow at 28 uC for the
same length of time. Cells were then harvested by centrifugation in
15 ml Falcon tubes (6000 r.p.m., 4 uC) and washed once in 1 ml
sterile ice-cold H2O, and, if not to be used immediately, the pellet
was stored at 280 uC until required. For cell disruption 300 ml Zbuffer (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 mm 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
http://mic.sgmjournals.org
James et al. (1996)
Hernando et al. (1999)
Hernando et al. (1999)
Carter et al. (1997)
Carter et al. (1997)
Carter et al. (1997)
Carter et al. (1997)
Laboratory collection
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according to the method of Bradford (1976) The b-galactosidase
assay was performed using O-nitrophenyl-b-D-galactopyranoside
(ONPG) as substrate. Depending on the protein concentration,
20–200 ml of crude extract in a final volume of 1 ml Z-buffer was
combined with 200 ml ONPG (4 mg ml21). After a pale yellow
colour developed, 0?5 ml 1 M Na2CO3 was added to stop the reaction. The specific activity of b-galactosidase [U (mg protein)21] 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, pGAD424Prs1(DNHR1-1) and pGBT9-(DNHR1-1), 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(D746-1281) and as a
BamHI/BglII fragment to give pGAD424-Prs1(D1-745) (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 uC. Crude extracts were prepared and assayed for
b-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 uC to OD600=0?5, harvested, rinsed once with an equal
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K. Wang and others
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 b-galactosidase activity was assayed by
using 20 ml crude extract with the chemiluminescent substrate
Galacto-Star, according to the manufacturer’s instructions (Applied
Biosystems). b-Galactosidase activity was expressed as relative light
units (RLU) (mg protein)21 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 a-factor on Rlm1 activation, the culture
was divided into two aliquots, one served as the control and the
other was incubated at 39 uC or treated with a-factor at a concentration of 2 mM for 4 h before determining b-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?06107 cells ml21 and harvested by centrifugation (6000 r.p.m.,
4 uC). 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)21; 1 ml 6 % KOH was added per 0?1 g cells and
the cell suspension was incubated at 80 uC 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 220 uC. Chitinase (Sigma #C-1650; 40 ml; 17 mg
per 400 ml 50 mM sodium phosphate buffer, pH 6?3) was added to
the pellet and the volume brought up to 600 ml with sodium phosphate buffer, pH 6?3. A control was set up containing 40 ml chitinase
and 540 ml 50 mM sodium phosphate buffer, pH 6?3. The suspensions were incubated at 37 uC for 2 h. After vortexing thoroughly, a
400 ml sample was transferred to a fresh 1?5 ml Eppendorf tube to
which 25 ml b-glucuronidase (Sigma #G0876) was added. The tubes
were incubated at 37 uC for 1 h, boiled for 1 min and centrifuged at
12 000 r.p.m. for 5 min. A sample of supernatant (50 ml) was transferred to a glass tube and the final volume was brought up to 250 ml
with H2O. K2B4O7.4H2O (250 ml 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 16 Reissig solution (106 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 uC for 40 min and the absorbance was measured at 585 nm.
A standard curve was prepared using appropriately diluted Nacetylglucosamine (10 mg ml21 stock). For chitin measurements
following exposure to CFW, cells were cultured in the presence of
100 mg CFW ml21 at 28 uC for 4 h prior to harvesting.
Western blotting. Yeast cells were grown overnight at 28 uC to
mid-exponential phase and diluted to OD600=0?3 (96106 cells ml21)
in a volume of 200 ml YEPD. The cultures were allowed to grow
for one generation before removing a 10 ml (16108–26108 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 uC. At hourly intervals thereafter the contents of a
single universal were processed as indicated above. Each individual
pellet was resuspended in 100 ml 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 % b-mercaptoethanol and
10 % 1 M Tris-base. For the sample taken at time zero a volume of
the suspension, equivalent to approximately 46107 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 46107 cells. Cell extracts were separated in SDS-8 % polyacrylamide gels and transferred to Hybond-P PVDF membrane (0?2 mm;
Amersham Bioscience) using the Mini Trans-Blot Electrophoretic
3330
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 antirabbit antibody (1 : 5000 dilution; Sigma) with the ECL+Plus detection
system from Amersham Bioscience. Slt2 was detected by anti-GSTSlt2 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
The requirement for Prs in the yeast cell
response to stress
Various prsD 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 uC prs1D and prs3D are impaired in their
growth and the double deletant prs1D prs3D is inviable.
The temperature-sensitive phenotype of prs1D prs3D can
be reversed by the addition of 1 M sorbitol to the
medium (data not shown). No thermosensitive phenotype
was observed in a prs4D or prs5D strain or in the triple
deletant strain prs2D prs4D prs5D.
The ability of the PRS deletants to grow in the presence of
CFW is also shown in Fig. 1; prs1D, prs3D and prs1D prs3D
prs4D strains have acquired resistance to CFW, whereas
the prs1D prs3D and the prs1D prs2D prs3D strains are, in
contrast, sensitive to this compound. Deletion of PRS2,
PRS4 or PRS5 singly or in combination confers a slight,
albeit variable, degree of resistance to CFW.
It is known that exposure to a-factor induces morphological
changes in the cell wall which activate the cell integrity
pathway (Buehrer & Errede, 1997). Treatment with 1 mM
a-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 prs3D strain,
77 %, after 6 h (Fig. 2a). Surprisingly, the a-factor sensitivity of prs1D prs3D can be overcome by the additional
deletion of either PRS2 or PRS4; these triple deletants
retained their wild-type response even when the a-factor
concentration was increased to 2 mM. Deletion of PRS2
PRS4 PRS5 had no effect on the response of a MATa cell
to a-factor (Fig. 2b).
Overexpression of PKC1 partially counteracts
PRS deletant-induced cell integrity pathway
malfunction
The slightly reduced growth of prs1D and prs3D observed at
37 uC (Fig. 1) and the a-factor sensitivity of prs1D and prs3D
strains (Fig. 2a) are alleviated by overexpression of Pkc1, a
well-documented upstream regulator of the cell integrity
pathway (Heinisch et al., 1999). The a-factor sensitivities
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Prs and cell integrity
YEPD + CFW
_
(500 mg m 1)
YEPD
Strain
30 ºC
37 ºC
30 ºC
WT
prs2D
prs4D
Fig. 1. Loss of PRS1 and PRS3 impairs
growth at 37 6C and confers resistance to
CFW. Wild-type (YN94-1, WT) and congenic prsD strains (YN94-4 prs1D; YN9418 prs3D; YN94-19 prs2D; YN94-20
prs4D; YN96-1 prs5D; YN97-88 prs1D
prs3D; YN96-72 prs2D prs4D prs5D;
YN95-25 prs1D prs3D prs4D; YN95-36
prs1D prs2D prs3D were grown to early
stationary phase (16108 cells ml”1) in
YEPD and serial tenfold dilutions prepared.
Aliquots (3 ml) were spotted onto YEPD and
YEPD+CFW (500 mg 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.
prs5D
prs2D prs4D prs5D
prs1D
prs3D
prs1D prs3D
prs1D prs2D prs3D
prs1D prs3D prs4D
of prs1D, prs3D strains (Fig. 2c) and the wild-type (data
not shown) each containing Pkc1 are in agreement with
each other when exposed to a-factor at a concentration
of 2 mM. However, overexpression of Pkc1 could correct
neither the thermosensitivity nor the a-factor sensitivity
of the double deletant prs1D prs3D (data not shown).
With respect to growth on CFW at a concentration of
300 mg ml21, overexpression of Pkc1 in a prs1D prs3D
strain has a growth-promoting effect which is, however,
not sufficient to confer CFW resistance. Conversely, overexpression of Pkc1 reduces growth in the wild-type and
has a sensitizing effect on the growth of prs1D and prs3D
strains under the above conditions (data not shown).
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 prs1D prs3D there is a 50 % increase in chitin
content at 28 uC, whereas in the presence of CFW the chitin
content is virtually doubled.
Sensitivity to a-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 a-factor
sensitivity of a mid2D strain. The independent nature of the
apparently similar prs1D- and/or prs3D- and mid2D-induced
phenotypes is underlined by the observation that overexpression of either PRS1 or PRS3 does not correct the
mid2D-associated a-factor-sensitive phenotype (data not
shown).
Rlm1 activation in prsD strains
Chitin levels in PRS deletant strains
CFW resistance goes hand in hand with altered chitin
synthesis (Roncero & Duran, 1985; Roncero et al., 1988).
http://mic.sgmjournals.org
The observations described above suggest an involvement
of Prs1 and Prs3 polypeptides with the cell integrity pathway. Therefore, it seemed a logical step to examine one of
the end points of the cell integrity pathway, the transcription factor Rlm1 (Heinisch et al., 1999), in strains lacking
one or other of the Prs polypeptides. To this end, a plasmid
carrying a lexA-Rlm1 fusion, which when phosphorylated
activates a b-galactosidase reporter on the same plasmid
(Kirchrath et al., 2000), thus providing a measure of Rlm1
activation by the cell integrity pathway, was introduced
into various prsD strains. The loss of any of the Prs polypeptides had a dramatic effect on Rlm1 activation under
normal growth conditions: deletion of PRS5 reduced Rlm1
activation by 30 %; however, lack of PRS2 or PRS4 alone
or in combination with PRS5 reduced Rlm1 transcriptional
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3331
Glucosamine_
1
[mg (mg wet wt) ]
K. Wang and others
2.5
2
1.5
1
.
05
0
WT
prs2D prs4D prs5D prs2D prs1D prs3D prs1D
prs4D
prs3D
prs5D
Fig. 3. Chitin content of prsD strains. Strains as in Fig. 1 were
grown in the presence or absence of 100 mg 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.
Exposure of the wild-type to a-factor causes a 20 % decrease
in Rlm1 activation. However, deletion of PRS2 increases
Rlm1 activation fivefold and deletion of PRS4 causes a
more modest activation of twofold, similar to the increase
observed in the prs2D prs4D prs5D strain (Fig. 4a). In a
prs5D background exposure to a-factor results in a 50 %
reduction of Rlm1 activation. In contrast to the effect of
increased temperature, loss of PRS1, in spite of causing a
activation further by 80 % (Fig. 4a). The most pronounced
effect on Rlm1 activation is observed when PRS1 and/or
PRS3 is removed; in a prs1D prs3D strain Rlm1 activation is
virtually abolished (Fig. 4b). However, when the growth
temperature is increased to 39 uC, strains which lack PRS2
or PRS4 or PRS2 PRS4 PRS5 are capable of increasing
Rlm1 activation. Interestingly, the loss of PRS5 or the loss
of PRS1 and/or PRS3 alone results in Rlm1 activation
remaining virtually unaltered at the higher temperature.
3332
250 000 (a)
200 000
150 000
100 000
b-Galactosidase activity
_1
[RLU (mg protein) ]
Fig. 2. Response of PRS deletant strains to a-factor and influence of PKC1 overexpression on prsD-induced phenotypes.
Cells were grown at 30 6C in appropriately supplemented buffered SC medium containing a-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 prs3D which was
YN96-77. a-Factor was used at a concentration of 1 mM. Filled
squares, wild-type; open squares, prs1D; filled triangles, prs1D
prs3D; open triangles, prs3D. (b) Response curve of PRS triple
deletants. The concentration of a-factor was 2 mM. Filled
squares, wild-type; open squares, prs2D prs4D prs5D; filled
triangles, prs1D prs3D prs4D; open triangles, prs1D prs2D
prs3D. (c) a-Factor sensitivity of prs1D and prs3D strains with
or without YEplac195-PKC1. The concentration of a-factor was
2 mM. Filled squares, prs1D[PKC1]; open squares, prs1D; filled
triangles, prs3D[PKC1]; open triangles, prs3D. For all experiments values are means±SD (n=4).
50 000
0
2500
WT
prs2D
prs4D
prs5D
prs2D
prs4D
prs5D
prs1D
prs3D
prs1D
prs3D
prs1D
prs3D
prs4D
prs1D
prs2D
prs3D
(b)
2000
1500
1000
500
0
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 6C to
mid-exponential phase (white bars). Incubation at 39 6C for
4 h (grey bars) and treatment with 2 mM a-factor (black bars)
were carried out as described in the legend to Fig. 2. bGalactosidase activity was determined luminometrically. Values
are means±SD (n=3). Please note that different scales are
used to represent b-galactosidase activity in (a) and (b).
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Microbiology 150
Prs and cell integrity
b-Galactosidase (U mg-1)
35
30
25
20
15
10
5
0
WT
prs1D prs2D
prs3D
prs4D
prs5D prs1D
prs3D
Fig. 6. FKS2 activation in response to heat shock in wild-type
and PRS deletant strains. Strains YN94-1 (wild-type, WT),
YN96-50 (prs1D), YN95-23 (prs2D), YN96-52 (prs3D), YN9420 (prs4D), YN96-1 (prs5D) and YN97-88 (prs1D prs3D),
each containing the FKS2(”706/”1)-lacZ plasmid, were grown
in SC medium minus uracil to a density of 16108 cells ml”1 at
28 6C and aliquots were then grown further for 4 h at either 28
(light grey bars) or 37 6C (dark grey bars). Crude extracts were
prepared and b-galactosidase activity was measured as
described in Methods. Values are means±SD (n=6).
Fig. 5. Influence of PKC1 on Rlm1 transcriptional activation in
prs1D and/or prs3D strains. Rlm1 transcriptional activation was
measured in YN94-4 prs1D (a), YN94-18 prs3D (b) and in
YN97-88 prs1D prs3D (c) containing the appropriate plasmid
of the pHPS100 series with or without the YEplac195-PKC1
plasmid. Values are means±SD (n=3). White bars, prs1D and
prs3D; grey bars, prs1D or prs3D[PKC1].
considerable loss of Rlm1 activation, does not alter the
a-factor-induced reduction in Rlm1 response. However,
deletion of PRS3 results in a twofold increase, whereas a
prs1D prs3D strain is severely impaired in its Rlm1 response
following exposure to a-factor. The same holds true for the
triple deletant strains prs1D prs2D prs3D and prs1D prs3D
prs4D (Fig. 4b).
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 prs1D strain carrying the Rlm1 reporter plasmid
had a positive effect on the transcriptional activation of
Rlm1, increasing it by approximately twofold at 28 uC,
39 uC and following exposure to a-factor (Fig. 5a). When
PRS3 was lacking, overexpression of Pkc1 increased Rlm1
activation threefold at 28 and 39 uC, but only 1?7-fold after
exposure to a-factor (Fig. 5b). Overexpression of Pkc1 was
also able to correct the defective activation of Rlm1 in a
http://mic.sgmjournals.org
prs1D prs3D strain at 28 uC by effecting a sixfold increase;
when a-factor was tested in the same strain the response
was improved almost fourfold to just slightly above the
Rlm1 activation observed in the prs1D prs3D strain grown
at 28 uC. However, Pkc1 overexpression does not improve
Rlm1 activation sufficiently to allow the temperaturesensitive phenotype of the prs1D prs3D strain to be overcome (Fig. 5c).
FKS2 activity in PRS deletant strains
Introduction of the FKS2-lacZ reporter which contains
the FKS2 promoter from 2706 to 21 linked to the bgalactosidase reporter module into the wild-type and PRS
deletant strains allowed us to determine the effect of the
loss of Prs polypeptides on the expression of the FKS2
gene, which encodes an alternative catalytic subunit of
1,3-b-glucan synthase (Inoue et al., 1995) and is expressed
under conditions of cellular stress and Rlm1 activation.
As shown in Fig. 6, in the wild-type FKS2 promoter activity is almost three times higher following incubation at
37 uC than at 28 uC. Deletion of either PRS2 or PRS4 has
no effect on this response. Deletion of PRS1, however,
although it reduces FKS2 promoter activity at 28 uC, apparently does not affect the heat-inducibility. Deletion of
PRS3 reduces the basal FKS2 promoter activity slightly,
but completely abolishes the heat-inducibility. In a prs5D
strain the basal activity is hardly altered, but there is
a significant reduction in b-galactosidase activity at 37 uC.
A strain lacking PRS1 and PRS3 exhibited a reduced
basal FKS2 promoter activity which was further reduced
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3333
K. Wang and others
following growth at 37 uC. This is in accordance with the
Rlm1 activation in the same strain under temperature
stress (Fig. 4b).
Interactions of Prs polypeptides with
components of the cell integrity pathway
To determine whether or not the effects of the Prsdependent cell integrity-related phenotypes are caused by
direct interaction between Prs polypeptides and elements
of the cell wall integrity pathway, we undertook a Y2H
analysis. The yeast reporter strain PJ69-4A was transformed with appropriate plasmids to give pairwise combinations of PRS1–PRS5 with SLT2 fused to either the
activation or the binding domain of GAL4. The transformants were tested for b-galactosidase activity and their
ability to grow on selective media for histidine and adenine
prototrophy. In addition to several negative controls, two
positive controls were used: one measuring an interaction
between Prs4 and Prs5 and the other, which is Prsindependent, measuring the interaction between Snf1 and
Snf4. The results are summarized in Table 2. It can be seen
that each of the Prs polypeptides is capable of interacting
with Slt2 in either orientation with the strongest interaction occurring with Prs3. All pairwise combinations
grow in the absence of histidine, and adenine prototrophy
is encountered in all combinations with the exception of
Slt2 and Prs5 in either orientation, although the interaction
of Prs5 and Slt2 supports a b-galactosidase activity above
that of the background.
As a way of confirming these data we used three truncations
of Prs1 to investigate the interaction between Prs1 and Slt2.
As is obvious from Table 2, deletion of NHR1-1 (Carter
et al., 1994) completely abolishes the Prs1/Slt2 interaction.
Interaction of Prs1 with Prs2 or Prs3 is not destroyed by
deletion of NHR1-1 (Hernando et al., 1999). On the other
hand, when the PRS1 ORF was split into approximately
equal parts there was no loss of interaction between Prs1
and Slt2. In contrast, neither Prs2 nor Prs3 was capable of
interacting with either Prs1(D1-745) or Prs1(D746-1281)
(data not shown).
Table 2. Y2H interactions of Prs with MAPKs of the cell integrity pathway
PJ69-4A transformants were tested for b-galactosidase activity and growth on adenine-free (ADE auxotrophy) and histidine-free plus 150 mM 3-aminotriazole (HIS auxotrophy) SC media. pGBT9-Snf1/
pGAD424-Snf4 and pGBT9-Prs5/pGAD424-Prs4 were used as positive controls and the combinations
pGBT9/pGAD424, pGBT9-Slt2/pGAD424 and pGBT9/pGAD424-Slt2 served as negative controls. Values
are means±SD (n=6 or n=3) for Prs1/Slt2 and Prs1-truncated/Slt2 combinations, respectively.
Plasmid combinations
pGBT9/pGAD424
pGBT9-Slt2/pGAD424
pGBT9-Slt2/pGAD424-Prs1
pGBT9-Slt2/pGAD424-Prs2
pGBT9-Slt2/pGAD424-Prs3
pGBT9-Slt2/pGAD424-Prs4
pGBT9-Slt2/pGAD424-Prs5
pGBT9/pGAD424- Prs1(DNHR1-1)
pGBT9-Slt2/pGAD424-Prs1(DNHR1-1)
pGBT9/pGAD424-Prs1(D746-1281)
pGBT9-Slt2/pGAD424-Prs1(D746-1281)
pGBT9/pGAD424-Prs1(D1-745)
pGBT9-Slt2/pGAD424-Prs1(D1-745)
pGBT9/pGAD424-Slt2
pGBT9-Prs1/pGAD424-Slt2
pGBT9-Prs2/pGAD424-Slt2
pGBT9-Prs3/pGAD424-Slt2
pGBT9-Prs4/pGAD424-Slt2
pGBT9-Prs5/pGAD424-Slt2
pGBT9-Prs1(DNHR1-1)/pGAD424
pGBT9-Prs1(DNHR1-1)/pGAD424-Slt2
pGBT9-Snf1/pGAD424-Snf4
pGBT9-Prs5/pGAD424-Prs4
3334
Relative b-galactosidase
activity (U mg”1±SD)
ADE
auxotrophy
HIS
auxotrophy
3?2±0?1
1?6±0?3
19?3±2?3
13?9±1?1
22?9±0?1
17?2±1?0
6?1±0?7
2?9±0?79
2?8±0?076
2?9±0?38
13?1±1?56
3?1±0?43
12?4±0?88
2?6±1?0
13?3±0?1
19?1±0?3
32?5±1?0
10?7±0?2
5?1±0?6
2?7±0?05
3?3±0?78
11?1±0?08
12?8±0?5
2
2
+
+
+
+
2
2
2
2
+
2
+
2
+
+
+
+
2
2
2
+
+
2
2
+
+
+
+
2
2
2
2
+
2
+
2
+
+
+
+
+
2
2
+
+
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Microbiology 150
Prs and cell integrity
Fig. 7. Slt2 phosphorylation in crude extracts of wild-type (WT) YN94-1, prs1D (YN96-50) and prs3D (YN96-52) strains.
The strains were grown in YEPD to mid-exponential phase at 28 6C (time point zero) and then heat-shocked at 37 6C 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.
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 uC. In contrast in the prs1D strain there
was a strong signal at time zero which was maintained over
a period of 4 h incubation at 37 uC. 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).
DISCUSSION
Metabolic disturbances in any organism have far-reaching
and unexpected consequences. In this report we have
demonstrated that altering the capacity of the yeast cell
for synthesizing PRPP, a key biosynthetic intermediate
linking carbon and nitrogen metabolism (Khorana et al.,
1958), causes the cell to display cell wall damage-related
phenotypes and interferes with signalling in the cell integrity pathway. Stress conditions, such as elevated temperature and mating pheromone treatment, trigger the Slt2
MAPK pathway which is essential for maintenance of the
cell wall (Heinisch et al., 1999). Our finding that 1 M
sorbitol prevented prs1D and prs3D strains from releasing
alkaline phosphatase and reversed their caffeine sensitivity
(Schneiter et al., 2000) suggested that the PRS gene products may in some way be connected with the maintenance
of cell integrity. To extend these results we performed a
number of experiments aimed at investigating the influence of the five gene products responsible for the production of PRPP on the cell integrity pathway. The data
http://mic.sgmjournals.org
presented in Fig. 1 show that strains lacking PRS1 or PRS3
are resistant to CFW. These two genes encode one of the
Prs complexes postulated on the basis of the Y2H analysis
and is also one of the three minimal functional units
identified by genetic analysis (Hernando et al., 1999). The
existence of the Prs1/Prs3 complex has also been confirmed
immunologically. Using our collection of PRS deletant
strains in which PRS1 has been replaced by a functional
GFP-PRS1 fusion (Schneiter et al., 2000), we were able to
demonstrate that in a strain carrying a deletion of PRS3
the GFP signal is lost, whereas in all other strains bearing
the GFP-PRS1 fusion the GFP signal is clearly visible
(M. Oufir, unpublished data). Therefore, we postulate that
the phenotypes of temperature-sensitivity, CFW resistance
(Fig. 1) and caffeine sensitivity (Schneiter et al., 2000) are
associated with loss of the Prs1/Prs3 complex. Such phenotypes have not been observed in strains lacking PRS2, PRS4
or PRS5 whose products form the second complex defined
by Y2H and the other two minimal functional units, Prs2/
Prs5 and Prs4/Prs5 (Hernando et al., 1999). The triple
deletant YN96-72 (prs2D prs4D prs5D) which relies on the
Prs1/Prs3 minimal functional unit for provision of PRPP
has a phenotype which with respect to temperature sensitivity is indistinguishable from the wild-type. The other two
triple deletant strains YN95-36 (prs1D prs2D prs3D) and
YN95-25 (prs1D prs3D prs4D) are both temperaturesensitive and the latter is also CFW-resistant (Fig. 1),
suggesting that there is an inherent difference in the minimal functional units Prs2/Prs5 and Prs4/Prs5. However,
with regard to a-factor sensitivity all three triple deletants
behave as wild-type (Fig. 2b). It would appear that the
a-factor sensitivity observed in prs1D and prs3D strains
is a result of the dysfunction of the cell integrity pathway,
since overexpression of PKC1 in these strains corrects the
phenotype (Fig. 2c).
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 prs1D and/or prs3D
strains, e.g. a-factor and caffeine sensitivity, and CFW
resistance (Ketela et al., 1999; Rajavel et al., 1999). The
viability of a mid2D prs1D prs3D strain suggests that the
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3335
K. Wang and others
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 prsD strains, with the possible exception of prs5D, in
comparison to the wild-type. However, prs2D, prs4D and
prs2D prs4D prs5D strains are able to increase Rlm1 phosphorylation at 39 uC (Fig. 4a). This heat-induced response
of Rlm1 may be sufficient to mediate the wild-type response
of the FKS2 promoter observed in prs2D and prs4D strains
(Fig. 6). Rlm1 activation and FKS2 promoter activity are
also correlated in a prs5D strain since a temperaturedependent response was not observed for either reporter
plasmid. In prs1D and prs3D 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 prs1D, prs3D and prs1D
prs3D 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
3336
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 prs1D prs2D prs3D strain. A prs1D prs3D prs4D strain
contains only about 10 % of the UTP+UDP+UMP level of
the wild-type, whereas in the triple deletant prs2D prs4D
prs5D 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 prsD 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 b-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 b-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 uC leads to an increase in
the signal for phosphorylated Slt2 from that obtained at
28 uC 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 wildtype, in a prs1D strain the signal corresponding to phosphoSlt2 is stronger at 28 uC and is maintained over a period
of 4 h during incubation at 37 uC (Fig. 7). Interestingly,
incubation at 37 uC 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 prs3D 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 uC (data
not shown).
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Prs and cell integrity
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
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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.
Msg5 dual-specificity protein phosphatase modulates the yeast cell
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Francois, J., Nombela, C. & Arroyo, J. (2004). The global trans-
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