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
Yeast 15, 1459–1469 (1999)
The Yeast PRS3 Gene Is Required for Cell Integrity,
Cell Cycle Arrest upon Nutrient Deprivation, Ion
Homeostasis and the Proper Organization of the Actin
Cytoskeleton
K. M. BINLEY1†, P. A. RADCLIFFE1††, J. TREVETHICK1, K. A. DUFFY1 AND P. E. SUDBERY1*
1
Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK
PRS3 is one of a family of five genes encoding phosphoribosylpyrophosphate synthetase, an enzyme which catalyses
the first step in a variety of biosynthetic pathways, including purine and pyrimidine biosynthesis. We report here that
prs3Ä mutants have a number of phenotypes that suggest an unexpected role for PRS3 in linking nutrient availability
to cell cycle progression, cell integrity and the actin cytoskeleton. Upon nutrient limitation, prs3Ä mutants fail
to arrest in G1—cells remain budded and a significant fraction have a G2 DNA content. Furthermore, in such
conditions, prs3Ä mutants have a disorganized actin cytoskeleton: actin accumulates in one or two intensely staining
clumps per cell. Prs3Ä mutants also show defects in ion homeostasis and cell integrity. They fail to grow on medium
containing 1·0  NaCl, 5 m caffeine or when incubated at 37C. The caffeine and temperature sensitivity are
rescued by supplementing the growth medium with 1·0  sorbitol. These phenotypes resemble those of whi2Ä
mutations and indeed, a prs3 allele was recovered in a colony-sectoring screen for mutations that are co-lethal with
whi2Ä. However, further investigation showed that the prs3Ä whi2Ä double mutant was viable, with no obvious
growth defect compared to either single mutant. In the same colony-sectoring assay, an mpk1 allele was also
recovered. Multicopy PRS3 rescued the caffeine sensitivity of this mpk1 allele. Copyright 1999 John Wiley &
Sons, Ltd.
  — caffeine sensitivity; nutrient arrest; phosphoribosyl pyrophosphate synthetase; PRS3; S. cerevisiae;
WHI2
INTRODUCTION
When cells of the budding yeast Saccharomyces
cerevisiae are starved of nutrients they arrest in G1
of the cell cycle in an unbudded, phase-bright state
and undergo a series of changes that allow them
to survive the adverse environmental conditions
(Hartwell, 1974; Pringle and Hartwell, 1981; Saul
et al., 1985; Werner-Washburne et al., 1993). Whi2
mutants fail to show this response, so that in
*Correspondence to: P. E. Sudbery, Department of Molecular
Biology and Biotechnology, University of Sheffield, Western
Bank, Sheffield S10 2TN, UK. Tel: +44 114 2226186; fax: +44
114 2728697; e-mail: P.Sudbery@Sheffield.ac.uk
†Current address: Oxford Biomedica (UK) Ltd, Medawar
Centre, Robert Robinson Avenue, The Oxford Science Park,
Oxford OX4 4GA, UK.
††Current address: Laboratory of Cell Regulation,
Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields,
London WC2A 3PX, UK.
CCC 0749–503X/99/141459–11$17.50
Copyright 1999 John Wiley & Sons, Ltd.
stationary phase whi2 cells are abnormally small,
are arrested randomly in the cell cycle, retain the
phase-dark appearance of exponentially growing
cells, fail to accumulate glycogen and remain
sensitive to environmental stresses such as a 52C
heat shock (Saul et al., 1985; Sudbery et al., 1980).
Furthermore, in stationary phase, whi2 mutants
have a disturbed actin cytoskeleton—the actin
accumulates in one or two intensely staining
clumps per cell. The failure to show G1 arrest is
accompanied by deregulation of the G1 cyclins
CLN1 and CLN2, which may explain this aspect of
their phenotype (Radcliffe et al., 1997a). While a
deficiency of Whi2p results in the inappropriate
continuation of cell division, overproduction
from the GAL1 promoter on a multicopy plasmid
results in filamentous growth—cytokinesis is
inhibited, cell shape becomes elongated, the
budding pattern changes from axial to unipolar
Received 21 January 1999
Accepted 24 May 1999
1460
K. M. BINLEY ET AL.
Table 1.
Strains used in this study.
Strain
Genotype
KTd2
Source
MAT a/á ade2-1/ade2-1, can1-100/can1-100,
his3-11/his3-11 leu2-3,112/leu2-3,112,
trp1-1/trp1-1, ura3-52 [psi + ]/ura3-52 [psi + ]
GAL1/GAL1 ssd1-2d/ssd1-2d
PAR3
As W303a whi2Ä::HIS3
PAR5
As Y763 whi2Ä::HIS3
KTd11
As W303a prs3Ä::URA3
KTd12
As W303a prs3Ä::URA3 whi2Ä::HIS3
W303a
MATa ade2-1, can1-100, his3-11,15 leu2-3,112
trp1-1 ura3[psi + ] GAL1 ssd1-d
Y763
Matá ura3-52 lys2-801 ade2-101 trp1-Ä1
his3-Ä200
YN94-18 MATa, prs3Ä::TRP1, ade2-1, his3-11,
leu2-3,ura3-1, can1-100
YC109
MATá ura3-52 lys2-801ade2-101 his3-Ä200
trp1-Ä1 slk1-Ä1::TRP1
mpk1siw9 As PAR5 mpk1siw9
and cell growth is hyperpolarized (Radcliffe et al.,
1997b). We have also observed that whi2 mutants
are sensitive to caffeine, 1·0  NaCl and calcofluor
white.
We carried out a colony-sectoring screen,
designed to isolate mutations that show colethality with whi2Ä. One of the mutations recovered affected the PRS3 gene. The S. cerevisiae
genome contains a family of five redundant genes
(PRS1–PRS5) encoding phosphoribosylpyrophosphate (PRPP) synthetase (Carter et al., 1994,
1995). This enzyme catalyses steps in a variety of
biosynthetic pathways, including those required
for the synthesis of purines, pyrimidines and
amino acids such as histidine and tryptophan
(Hove-Jenson, 1989). None of the genes are essential; however, disruption of PRS1 and PRS3 have
more drastic effects on growth, enzyme activity
and nucleotide pools than disruption of the other
PRS genes. Although further characterization
revealed that whi2Ä and prs3Ä are not strictly
co-lethal, we report here that a prs3Ä mutant has a
very similar phenotype to whi2Ä. Thus, surprisingly, a biosynthetic enzyme is also required for a
diverse array of cell functions including coordination of nutrient availability with division, cell
integrity and the organization of the actin
cytoskeleton.
Copyright 1999 John Wiley & Sons, Ltd.
Constructed by crossing
W303 MATa with W303
MATá
Radcliffe et al.
This work
This work
This work
M. Tyers
M. Snyder
M. Schweizer
M. Snyder and C. Costigan
MATERIALS AND METHODS
Strains and culture conditions
Strains used in this paper are described in
Table 1. They were routinely cultured at 30C in
YEPD (1% Difco Yeast Extract, 2% Difco
peptone, plus 2% glucose). Plates were solidified by
the addition of 2% Difco agar. Minimal medium
consists of 0·67% Difco Yeast Nitrogen Base plus
2% glucose. Synthetic complete medium consists of
minimal medium supplemented by all 20 amino
acids, adenine and uracil at standard concentrations. Plasmids used are described in Table 2.
Cell growth on plates was monitored by using a
standardized streaking pattern or by a dilution
drop test. For the latter, cells were harvested from
a freshly grown YEPD plate and resuspended in
YEPD to an OD600 of 8. This suspension was then
diluted 10 2, 10 3 and 10 4; 5 ìl of each dilution
was spotted onto the surface of the agar plate and
cultures were incubated for 3 days at 30C. Failure
to grow was regarded as absence of growth at the
intermediate cell concentration when wild-type
control cells were able to grow at the lowest cell
concentration. All growth tests were carried out
using YEPD media supplemented as indicated,
except for the 37C growth test in which minimal
medium was used.
Yeast 15, 1459–1469 (1999)
1461
PRS3 IS REQUIRED FOR CELL CYCLE ARREST AND CELL INTEGRITY
Table 2.
Plasmids used in this study.
Plasmids Description
Source
pAD12
C. Costigan and M. Snyder
TRP1 ADE2 CEN6/ARSH4 engineered for
unstable replication
pPR9
2 ìm URA3-based plasmid containing WHI2
pPR14
WHI2 ORF cloned into pAD12
YCp50
Centromeric URA3-based plasmid used to
construct S. cerevisiae genomic library
pYES2.0 URA3 based 2 ìm vector containing GAL1
promoter
Yep352 URA3 based 2 ìm shuttle vector with blue–white
selection for bacterial cloning
Colony sectoring assay
The WHI2 gene was deleted using the one-step
gene disruption method (Rothstein, 1983) as follows. Strain Y763 was transformed with a linear
DNA fragment consisting of the WHI2 region in
which 1420 bp was replaced with the HIS3 gene,
that is from a BamHI site 258 bp upstream of the
start of the WHI2 ORF to a XhoI site 328 bp
before the end of the ORF. The deletion was
confirmed by Southern hybridization. This red
strain, PAR5, was transformed with pPR14
(Table 2) which consists of the WHI2 sequence
cloned into pAD12, a CEN6/ARSH4-based plasmid which shows unstable replication and carries
the ADE2 and TRP1 genes. The resulting white
strain, PAR5-pPR14, produced either red colonies
or white colonies with red sectors at high frequency. It was mutagenized with EMS to a survival of 25%. The survivors were plated onto
synthetic medium, supplemented with appropriate
amino acids and 10 ìg/ml adenine and incubated
for 7 days at 27C, followed by 3 days at 4C to aid
development of the red colour. 40 000 colonies
were screened initially and 2250 white nonsectoring colonies were retained, of which 420
remained white upon re-testing. These colonies
were then tested for growth on glycerol to eliminate petite mutations which are known to prevent
the red colour developing, and plates lacking
adenine to eliminate mutations which block the
adenine biosynthetic pathway upstream of ADE2,
and would thus also prevent the red colour developing. Twenty putative mutants remained after
these tests. Each of these was transformed with
pPR9 which contains the WHI2 gene on a multicopy URA3 plasmid. This allows the segregation
Copyright 1999 John Wiley & Sons, Ltd.
This work
(Rose et al., 1987)
obtained from ATTC
Invitrogen
Invitrogen
of pPR14 and the reappearance of red sectors if
the non-sectoring phenotype was due to a requirement for WHI2 for viability. Strains which showed
such sectoring were finally tested as follows to
demonstrate that their ability to form colonies was
dependent on either pPR14 or pPR9. Cells from a
white sector containing both pPR9 and pPR14
were allowed to grow to stationary phase on
YEPD medium, diluted and plated either on medium containing 5-fluoro-orotic acid (5-FOA),
which selects for cells which have lost the URA3based pPR9, or medium without 5-FOA (Boeke
et al., 1984). Both red and white colonies formed
on the medium lacking 5-FOA, but only white
colonies formed on medium containing 5-FOA.
This test demonstrated that, in the absence of
pPR9, cells were forced to retain pPR14.
Cloning and characterization of the PRS3 gene
Mutants were transformed with a genomic
library based on YCp50, a centromeric URA3
vector (Rose et al., 1987). Ura + transformants,
representing approximately 5-genome equivalents,
were replica-plated to YEPD plates containing
5 m caffeine, and transformants which grew were
retained for further study. Many of the colonies
that grew no longer required pPR14, as judged
by the appearance of red sectors, indicating that
there was no longer any selection that prevents
the loss of plasmid pPR14 (ADE2 WHI2) used
in the colony-sectoring screen. The dependency of
caffeine-resistant growth on the presence of a
plasmid from the library was examined by growing
transformants on media containing 5-FOA, which
selects for cells which have lost the YCp50-based
plasmid containing the URA3 gene (Boeke et al.,
Yeast 15, 1459–1469 (1999)
1462
Figure 1. PRS3 complements the siw17 mutation. The complementing plasmid contained a 13 kb genomic insert spanning
bases 78 000–91 000 on chromosome VIII. This fragment contained PRS3 (YHL011c), an open reading frame (YHL1010c)
encoding a hypothetical protein of with no obvious homologies,
and YAP3 (YHL009c). The fragment also contained TY4, a
transposable element. The double-headed arrows indicate the
extent of the insert carried in subclones A–C, which were
generated as follows. Subclones A and B were generated by
digesting the complementing plasmid with HindIII and subcloning the resulting fragments into Yep352. Subclone C was
generated by digesting the complementing plasmid with SalI
and religating (the right hand SalI site is located in the YCp50
vector close to the site of insertion of the genomic fragment).
‘(+)’ or ‘()’ indicates whether the clone complemented the
caffeine sensitivity of the original prs3siw17 mutation.
1984). Colonies that grew on 5-FOA were no
longer able to grow on a YEPD plate containing
5 m caffeine. Plasmids were recovered from five
independent colonies by transformation of E. coli
with total yeast DNA preparations. Each of these
plasmids had the same HindIII restriction map.
One of the plasmids was re-transformed into the
prs3 mutant and was found to complement the
caffeine sensitivity.
Two oligonucleotides, 5-GCCACTATCGACT
ACGCGATCA-3 and 5-CACGATGCGTCCG
GCGTAGA-3, which anneal to YCp50 either
side of the BamHI site used to construct the
library, were used to determine the sequence of
400 bp at either end of the genomic insert in the
plasmid. These sequences were compared to the
S. cerevisiae genome database (http://genomewww.stanford.edu/Saccharomyces/) and the intervening sequence retrieved from the database. The
genomic insert was a 13 kb fragment from chromosome VIII containing three ORFs (Figure 1):
YAP3, a bZIP DNA binding protein, PRS3,
encoding phosphoribosylpyrophosphate synthetase, and YHL1010c, a hypothetical reading frame
with no obvious homologies to any other protein.
In addition, the insert contained TY4. Appropriate
subcloning experiments identified PRS3 as the
complementing ORF (Figure 1).
Copyright 1999 John Wiley & Sons, Ltd.
K. M. BINLEY ET AL.
A prs3Ä strain was generated by one-step gene
disruption of a trp1/trp1 diploid strain, using a
construct kindly provided by M. Schweizer in
which the PRS3 ORF had been replaced by the
TRP1 gene. All putative prs3Ä alleles were verified
by appropriate Southern blots. The heterozygous
diploid was then sporulated, the resulting tetrads
showed 2:2 segregation of the Trp + phenotype,
showing that the prs3Ä allele is viable. Sensitivity
to caffeine co-segregated with the Trp + phenotype.
Actin staining
Cultures were stained with phalloidin conjugated to tetramethylrhodaminyl-isothiocyanate
(TRITC) (Sigma) (Adams and Pringle, 1983).
Flow cytometry
1 ml of stationary phase culture was harvested
by centrifugation, the cell pellet resuspended in
1 ml 70% ethanol and incubated with shaking for
1 h at room temperature. The cells were harvested
by centrifugation, washed twice in 1 ml 50 m
TRIS, pH 7.0, and resuspended in 0·5 ml 50 m
TRIS, pH 7·0, to which 20 ìl of 10 mg . ml 1
preboiled RNase A was added. The suspension
was then incubated with shaking for 4 h at 37C.
The cells were harvested by centrifugation and
resuspended in 0·5 mg ml 1 pepsin (Sigma),
freshly dissolved in 55 m HCl and incubated
with shaking for 30 min at 37C. The cells were
harvested by centrifugation, washed once in 1 ml
FACS buffer (200 m TRIS, pH 7·5, 211 m
NaCl, 78 m MgCl2) and resuspended in FACS
buffer, to which was added 55 ìl of 0·5 mg . ml 1
propidium iodide (Sigma). The suspension was
sonicated for 3 s to break up cell clumps. DNA
distribution in the suspension was determined
using a Becton-Dickinson FACSort Desk top
flow cytometer with a 488 nm air cooled laser.
Propidium iodide fluorescence was channelled with
a 650 nm long pass filter.
RESULTS
A colony sectoring assay identifies an interaction
between PRS3 and WHI2
In order to identify genes that may interact with
WHI2, designated SIW genes (synthetic interaction with whi2Ä), we carried out a colony sectoring assay. For this screen we applied the system
used by Costigan et al. (1992) to isolate slk1/ bck1
as a synthetic lethal mutation with spa2. The
Yeast 15, 1459–1469 (1999)
PRS3 IS REQUIRED FOR CELL CYCLE ARREST AND CELL INTEGRITY
parent strain for this screen was PAR5 (ade2 ura3
whi2Ä pPR14-WHI2 ADE2; for full genotype, see
Table 1; plasmid pPR14 is described in Table 2).
The screen identifies mutants that require WHI2
for viability because they form colonies that are
homogeneously white, in contrast to colonies of
wild-type cells which have red sectors (for details,
see Materials and Methods.) We report here a
characterization of one of the mutants recovered
from this screen, designated prs3siw17, because it
was subsequently shown to be allelic to prs3 (see
below).
Prs3siw17 was sensitive to caffeine (see below).
The caffeine sensitivity (and other phenotypes
described below) segregated 2:2 in crosses to a
wild-type parent, indicating that the strain harboured a single mutation in a nuclear gene. We
made use of the caffeine sensitivity to clone, by
complementation, the wild-type copy from a lowcopy chromosomal library (see Materials and
Methods). The complementing ORF was demonstrated to be PRS3 (Figure 1). To confirm that the
mutation recovered from the colony sectoring
assay was allelic to prs3, the mutant was crossed to
YN94-18 containing a prs3Ä allele. The resulting
diploid was caffeine-sensitive and the caffeine sensitivity phenotype segregated 4:0 in the meiotic
progeny upon sporulation.
In order to ascertain whether prs3Ä and whi2Ä
are co-lethal, a prs3Ä allele was generated in
KTd2, a diploid strain which is heterozygous for
a whi2Ä mutation (WHI2/whi2Ä::HIS3). Upon
sporulation it was found that TRP1 HIS3
(prs3Äwhi2Ä) progeny were viable and showed no
obvious growth defect compared to either single
parent. This indicates that whi2Ä and prs3Ä are
not co-lethal and, with respect to growth on
YEPD, neither mutation enhances the phenotype
of the other, despite the isolation of prs3siw17 in the
original screen.
The prs3Ä allele results in cell integrity and ion
homeostasis defects
Since prs3 was recovered in a colony-sectoring
screen with whi2Ä, we examined whether prs3Ä
mutants have the same or similar phenotypes as a
whi2Ä mutant. We examined mutations in the
original strain background (Y763) and in the
W303a background. We also compared the strain
harbouring the original prs3siw17 mutation and
strains harbouring a prs3Ä allele. We found that
neither the strain background nor the nature of the
Copyright 1999 John Wiley & Sons, Ltd.
1463
prs3 mutation affected the phenotype examined.
We report here the properties of a prs3Ä in the
W303a background.
A prs3Ä mutant was unable to grow on YEPD
plates containing 5 m caffeine (Figure 2). This
defect was rescued by the addition of 1·0  sorbitol
to the growth medium suggesting it arises from a
defect in cell integrity (Figure 2). Growth of a
prs3Ä strain was also impaired on YEPD plates
containing 1·0  NaCl (Figure 2). Although
growth was evident at high plating densities, no
growth was apparent at low plating density when
cells were plated in isolation. A whi2Ä strain was
more severely inhibited by 1·0  NaCl compared
to prs3Ä (Figure 2). The whi2Ä prs3Ä double
mutant strain was not more severely affected than
the whi2Ä single mutant, indicating that prs3Ä did
not enhance the salt sensitivity of whi2Ä. This
defect was rescued in both types of mutant by
inclusion of 5 m CaCl2, stimulating the calcineurin dependent Na + PMR2/ENA1 ion pump
(Danielsson et al., 1996). Sensitivity to low concentrations of Mn2+ ions has been shown to be a
characteristic of mutations that cause sensitivity
to caffeine and NaCl, such as those affecting
the calcineurin pathway (Farcasanu et al., 1995).
Curiously, the growth of a prs3Ä strain is more
resistant to 6 m MnCl2 than either a wild-type
parent or a parent harbouring a whi2Ä allele
(Figure 2). The increased sensitivity to 1·0  NaCl
and increased resistance to 6 m MnCl2 suggest
abnormalities in ion homeostasis. Growth at 37C
on minimal medium was also impaired at low
plating densities; this was rescued by the inclusion
of 1·0  sorbitol in the growth medium, suggesting
a defect in cell integrity (Figure 2). The whi2Ä
prs3Ä double mutant showed similar sensitivity to
temperature, caffeine and 1·0  NaCl, but was less
resistant to 6 m MnCl2 (Figure 2).
PRS3 is required for cell cycle arrest upon nutrient
deprivation
A prs3Ä strain was allowed to grow to
stationary phase in YEPD medium and examined
under the phase contrast microscope (Figure 3).
Under these conditions, the W303a parent strain
arrests with phase-bright, unbudded cells in G1. By
contrast, cells of a prs3Ä strain were budded and
phase-dark, suggesting that they have failed to
show the normal response to nutrient deprivation.
Unlike whi2Ä cells, however, they did not appear
to become smaller. This was confirmed by
Yeast 15, 1459–1469 (1999)
1464
K. M. BINLEY ET AL.
Figure 2. The growth of prs3Ä mutants is sensitive to 5 m caffeine,
37C, and 1·0  NaCl and resistant to 5 m MnCl2. Serial dilutions of
W303a (wild-type), KTd12 (W303a prs3Ä whi2Ä), KTd11 (pres3Ä), PAR3
(W303a whi2Ä) were incubated for 3 days on YEPD, MM at 37C,
YEPD+5 m Caffeine, YEPD+1·0  NaCl and YEPD+6 m MnCl2
and incubated for 3 days.
measurements of size distribution: in stationary
phase the median and modal cell volumes were
respectively 48 ìm3 and 29 ìm3 for strain W303a
and 45 ìm3 and 43 ìm3 for prs3Ä. The budded
appearance of the cells suggested that they had
not arrested in G1. This was confirmed by FACS
Copyright 1999 John Wiley & Sons, Ltd.
analysis which showed that in a stationary phase
prs3Ä culture, approximately 50% of the cells
arrested with a G2 DNA content (Figure 4).
Besides a failure to show cell cycle arrest, whi2Ä
cells in stationary phase fail to become resistant
to environmental stresses, such as 53C heat
Yeast 15, 1459–1469 (1999)
1465
PRS3 IS REQUIRED FOR CELL CYCLE ARREST AND CELL INTEGRITY
Figure 3. A prs3Ä strain remains budded in stationary phase. Strains W303a (wildtype), KTd12 (W303a prs3Ä whi2Ä), KTd11 (W303a prs3Ä), and PAR3 (W303a whi2Ä)
were grown in YEPD liquid culture until the cell number had stopped increasing and
photographed using a phase contrast microscope.
treatment (Radcliffe et al., 1997a; Saul et al.,
1985). However, the fraction of prs3Ä cells which
survived a 53C heat shock was not distinguishable
from an isogenic wild-type strain (data not shown).
So, although prs3Ä prevents cell cycle arrest
upon nutrient limitation, it does not prevent the
acquisition of stress resistance.
The actin cytoskeleton is disturbed in prs3Ä
strains
When wild-type cells enter stationary phase,
staining with TRITC-conjugated phalloidin
reveals a small number of cortical actin patches
(Figure 5). All cells in a whi2Ä strain show an
abnormal distribution of actin in stationary phase,
displaying one or two clumps of actin per cell
which stain much more intensely than the cortical
actin patches of wild-type cells (Figure 5). We refer
to these as ‘actin clumps’. Cells containing the
Copyright 1999 John Wiley & Sons, Ltd.
prs3Ä mutation showed an intermediate phenotype: a proportion of cells in a stationary phase
culture showed the actin clumps evident in whi2Ä
cells (Figure 5). This proportion was both straindependent and varied between different cultures of
the same strain. In W303a prs3Ä, the proportion
varied (15% and 30%). It was much higher (50–
70%) in stationary phase cells of strain YN94-18 (a
prs3Ä strain supplied by M. Schweizer). The disturbance to the actin phenotype was not additive,
the phenotype of a whi2Ä prs3Ä double mutant
was identical to a whi2Ä single mutant (data not
shown).
Multicopy PRS3 suppresses the caffeine sensitivity
of an mpk1 allele
Since whi2Ä and prs3Ä have similar phenotypes, we investigated whether overexpression of
the wild-type allele of one rescued any of the
Yeast 15, 1459–1469 (1999)
1466
K. M. BINLEY ET AL.
shown). PRS3 on a multicopy plasmid had little
effect on the phenotypes of a whi2Ä strain,
although there was some very limited rescue of the
37C growth defect (data not shown).
Another mutation, siw9, isolated in the colonysectoring assay, proved to be allelic to mpk1 (slt2).
We refer to this allele as mpk1siw9. The MPK1 gene
encodes the MAP kinase of the PKC1–MPK1
pathway (Irie et al., 1993; Lee et al., 1993; Torres
et al., 1991). Mutants of this pathway have a
similar phenotype to whi2Ä and prs3Ä mutants
(Costigan et al., 1992; Irie et al., 1993; Lee et al.,
1993; Lee and Levin, 1992; Levin and BartlettHeubusch, 1992; Levin et al., 1994; Levin et al.,
1990; Martin et al., 1993; Mazzoni et al., 1993;
Paravicini et al., 1992; Torres et al., 1991; Zarzov
et al., 1996). First, they fail to show cell cycle arrest
upon nutrient deprivation. Secondly, they show a
growth defect at 37C which is rescued by 1·0 
sorbitol, or in the case of pkc1 mutants they
require 1·0  sorbitol at all temperatures for
growth. Thirdly, growth is sensitive to caffeine and
1·0  NaCl. The mpk1siw9 mutant showed all of
these phenotypes. However, the minimum inhibitory concentration of caffeine was 4 m compared
to 2 m for an mpk1Ä strain. It is possible, therefore, that it is not a complete null allele. PRS3 on
a multicopy plasmid completely rescued the
caffeine sensitivity but not the temperature sensitivity of mpk1siw9 (Figure 6). Interestingly, multicopy PRS3 was unable to rescue an mpk1Ä allele
(data not shown), suggesting that PRS3 can only
rescue an mpk1 allele when it retains some residual
activity. Figure 6 also shows that multicopy
SIW14 (YNL032w), another gene identified in the
colony-sectoring assay, also suppresses the caffeine
sensitivity of mpk1siw9. We shall describe the characterization of SIW14 elsewhere (Binley et al., in
preparation).
DISCUSSION
Figure 4. A prs3Ä strain fails to show G1 arrest in stationary
phase. Strains W303a (wild-type haploid), L3566 (wild-type
diploid), KTd11 (W303a prs3Ä) and PAR3 (W303a whi2Ä)
were grown in YEPD liquid culture until the cell number had
stopped increasing. DNA distributions were determined in
stationary phase by FACS analysis.
phenotypes of the mutant allele of the other.
WHI2 on a multicopy plasmid did not rescue any
of the phenotypes of a prs3Ä strain (data not
Copyright 1999 John Wiley & Sons, Ltd.
PRS3 is one of a family of five genes that encodes
PRPP synthetase, an enzyme that catalyses the first
step in a variety of biosynthetic pathways, including amino acid and nucleotide biosynthesis. We
show here that prs3Ä mutants are disturbed in a
wide variety of cell functions, including cell cycle
arrest upon nutrient limitation, cell integrity, ion
homeostasis and the organization of the actin
cytoskeleton in stationary phase. So either the
metabolic disturbances caused by loss of PRS3
Yeast 15, 1459–1469 (1999)
1467
PRS3 IS REQUIRED FOR CELL CYCLE ARREST AND CELL INTEGRITY
Figure 5. Formation of actin bars in a stationary phase prs3Ä strain.
Strains W303a, KTd11 (W303a prs3Ä) and PAR3 (W303a whi2Ä) were
grown in YEPD liquid culture until the cell number had stopped increasing
and actin was visualized by staining with TRITC-phalloidin, as described
in Materials and Methods. The three sub-panels to the bottom right show,
in each case, an enlarged view of a cell selected from the population shown
in the other three panels.
Figure 6. Multicopy PRS3 rescues the caffeine sensitivity of mpk1siw9. The
mpk1siw9 mutant recovered in the colony sectoring assay was transformed with
pYES2 (a 2 ìm-based vector) and pYES2 containing cloned copies of genes,
identified in the colony-sectoring assay, that interact with whi2. Although
pYES2 carries the GAL1 promoter, the genes were cloned with at least 1·5 kb
of upstream sequence, so that the expression of the coding regions was under
the control of the native promoters.
have far-reaching consequences in cell biology,
despite the presence of four other genes which
encode an apparently functionally identical
enzyme, or PRS3 has other functions besides its
role in biosynthetic pathways.
Copyright 1999 John Wiley & Sons, Ltd.
The prs3 mutation was recovered in a colonysectoring screen with whi2Ä but genetic analysis
showed that prs3Ä and whi2Ä mutations are not
co-lethal. It is not clear why the screen yielded a
prs3 mutant. The lack of red sectors in the original
Yeast 15, 1459–1469 (1999)
1468
mutant (prs3 ade2 whi2Ä pPR14 WHI2 ADE2)
indicates that loss of pPR14 (Table 2) is selected
against. Since PRS3 catalyses the first step in
the adenine biosynthetic pathway, we considered
the possibility that the absence of red sectors in the
original prs3 mutant might be due to a reduction in
metabolic flux through the adenine biosynthetic
pathway. We think this is unlikely, however,
because prs3Ä mutants formed red colonies in two
different ade2 backgrounds. Furthermore, the
original prs3siw17 mutant that contained pPR14
was able to grow on medium lacking adenine. This
indicates that the phosphoribosylpyrophosphate
synthetase encoded by one or more of the redundant PRS genes provides sufficient flux through
this pathway to support growth on adeninedeficient medium. Since the product of Prs3p,
phosphoribosylpyrophosphate, is the substrate for
Trp1p, we also considered the possibility that loss
of pPR14 was selected against because of an
interaction between the TRP1 gene carried by
pPR14 and the prs3siw17 mutation. Again, we think
that this is unlikely, since any such effect would
cause tryptophan auxotrophy and there was no
selection against tryptophan auxotrophs in the
conditions used in the screen. Indeed, the parent
Y763 strain is trp1, and colony sectors lacking
pPR14, which would thus be tryptophan auxotrophs, were apparent in most of the colonies
examined in the screen. Even if there was some
form of interaction, it was not responsible for the
phenotypes documented here, since a wild-type
TRP1 gene was used to generate the prs3Ä allele
in W303a.
In general, the phenotypes of prs3Ä and whi2Ä
resemble each other, although the severity of the
phenotypes examined differs. Cells containing the
prs3Ä mutation are more sensitive to caffeine than
whi2Ä cells, but less sensitive to 37C and 1 
NaCl. Both prs3Ä and whi2Ä cells fail to arrest in
G1 upon nutrient starvation, arresting as budded
cells with a significant fraction containing a G2
content of DNA. However, the cell cycle defect of
prs3Ä is not as severe as whi2Ä, since prs3Ä cells do
not become smaller in stationary phase and they
also acquire resistance to environmental stresses,
as judged by their ability to survive 53C heat
treatment. Acquisition of environmental stress
resistance does not depend upon entry into G1
(Elliott and Futcher, 1993), so failure to show G1
arrest does not inevitably have to be linked to the
acquisition of stress resistance. Both prs3Ä and
whi2Ä mutants show disturbances to the actin
Copyright 1999 John Wiley & Sons, Ltd.
K. M. BINLEY ET AL.
cytoskeleton in stationary phase cultures, but
whereas essentially all whi2Ä cells in a stationary
phase culture display actin clumps, actin clumps
are only evident in a proportion of prs3Ä cells. In
all phenotypes examined, the phenotype of the
double mutant was identical to the phenotype of
the more severely affected single mutant. This lack
of additivity may indicate that the mutations act
in the same pathway, although this conclusion
must remain tentative in the absence of further
supporting data.
Similar defects to those documented here are
displayed by mutants of the PKC1–MPK1 pathway. A number of observations have implicated
the PKC1–MPK1 pathway in a complex network
that links regulation of enzymes required for cell
wall synthesis with the cell cycle engine, probably
in a way that involves the SBF transcription
factor, a dimer consisting of Swi4p and Swi6p
(Gray et al., 1997; Madden et al., 1997). It is
possible that Prs3p forms part of this network. In
support of this speculation, we also recovered a
mpk1 allele in our colony-sectoring screen affecting
the MAP kinase of this pathway. The caffeine
sensitivity of this allele was suppressed by multicopy PRS3. While the precise role of PRS3 is
currently unclear, it is not surprising that an
enzyme that catalyses the first step in a variety of
biosynthetic pathways, including that of nucleic
acid precursors, thus playing a key role in growth
processes, should have inputs into a regulatory
network that links bud formation, cell wall synthesis, the actin cytoskeleton and progress through
the cell cycle.
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