Arch Microbiol (2009) 191:721733

DOI 10.1007/s00203-009-0498-3
1 3
ORI GI NAL PAPER
Yeast protein phosphatases Ptp2p and Msg5p are involved
in G1S transition, CLN2 transcription, and vacuole
morphogenesis
Hermansyah Minetaka Sugiyama
Yoshinobu Kaneko Satoshi Harashima
Received: 25 March 2009 / Revised: 16 July 2009 / Accepted: 22 July 2009 / Published online: 14 August 2009
Springer-Verlag 2009
Abstract We previously reported that double disruption of
protein phosphatase (PPase) genes PTP2 (phosphotyrosine-
speciWc PPase) and MSG5 (phosphotyrosine and phosphothre-
onine/serine-PPase) causes Ca
2+
sensitive growth, whereas
the single disruptions do not. This Wnding suggests that Ptp2p
and Msg5p are involved in Ca
2+
-induced stress response in a
redundant manner. To gain insight into the molecular mecha-
nism causing calcium sensitivity of the ptp2 msg5 double
disruptant, we performed Xuorescence-activated cell sorting
analysis and found a delayed G1 phase. This delayed G1 was
consistent with the defect in bud emergence, and reduced
CLN2 transcription upon addition of CaCl
2
. We also found
that Slt2p is hyper-phosphorylated in the ptp2 msg5 double
disruptant and that the vacuole of the ptp2 msg5 double
disruptant is fragmented even in the absence of Ca
2+
. These
Wndings suggest that both Ptp2p and Msg5p are involved in
the G1 to S transition and vacuole morphogenesis possibly
through their regulation of Slt2 pathway.
Keywords Saccharomyces cerevisiae
Protein phosphatase PTP2 MSG5 Calcium sensitivity
Delayed G1
Abbreviations
PPase Protein phosphatase
MAPK Mitogen-activated protein kinase
Ca
s
Calcium sensitive
CWI Cell wall integrity
Introduction
In eukaryotic cells from humans to yeast, reversible phos-
phorylation and dephosphorylation of regulatory factors,
controls the majority of cellular pathways, including cell
signaling, cell cycle, and gene expression (Zolnierowicz
and Bollen 2000). Target proteins are phosphorylated at
speciWc site(s) by one or more protein kinases (PKases),
and speciWc protein phosphatases (PPases) remove those
phosphate residues. To understand the possible physiologi-
cal roles of PPases, we constructed 30 ppase disruptants
(all but two essential PPase genes) while to study the redun-
dant functions of these PPases, 435 double disruptants cov-
ering all possible combinations of the 30 viable gene
disruptions were constructed (Sakumoto et al. 1999, 2002).
We discovered that the ptp2 msg5 double disruptant had
a calcium sensitive (Ca
s
)-phenotype, whereas the single
disruptant of either PTP2 or MSG5 did not (Sakumoto et al.
2002). These phenotypes demonstrated that Ptp2p and
Msg5p are involved in cellular processes triggered by cal-
cium-stress response in a redundant manner. Both Ptp2p
and Msg5p are classiWed into the PTP family, but they are
classiWed into diVerent sub-families. Ptp2p belongs to the
protein tyrosine phosphatase sub-family which dephospho-
rylates tyrosine residues of a phosphorylated protein (Guan
et al. 1992). Msg5p belongs to dual-speciWcity protein
phosphatases (DSPs) sub-family which dephosphorylates
both threonine/serine and tyrosine residues (Doi et al.
1994). Ptp2p has been known to be a negative regulator of
Slt2p of the cell wall integrity (CWI) pathway, the Hog1p
of the high-osmolarity glycerol (HOG) MAPK sensing
pathway and the Fus3p of the MAPK pheromone pathway.
On the other hand, Msg5p inactivates both CWI MAPK
and the Fus3p MAPK pheromone pathways (Mattison et al.
1999; Gustin et al. 1998). Interestingly, Msg5p is inversely
Communicated by Axel Brakhage.
Hermansyah M. Sugiyama Y. Kaneko S. Harashima (&)
Department of Biotechnology, Graduate School of Engineering,
Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
e-mail: harashima@bio.eng.osaka-u.ac.jp
722 Arch Microbiol (2009) 191:721733
1 3
regulated by Slt2p in the CWI pathway (Flandez et al.
2004). The CWI MAPK cascade is comprised of Pkc1p and
three other PKases, i.e., Bck1p (MAPKKK), a pair of
redundant PKases Mkk1p/Mkk2p (MAPKK), and Slt2p
(MAPK). Activation of these MAPKs needs to be con-
trolled tightly and precisely because hyper-activation of
MAP kinases of the CWI pathway causes severe growth
defects (Martin et al. 2000).
Calcium-triggered signaling mechanisms regulate a wide
variety of cellular processes, such as the response to mating
pheromone (Muller et al. 2001) and the adaptation to envi-
ronmental stress (Batiza et al. 1996; Denis and Cyert 2002).
In an eVort to gain insight into the molecular mechanism by
which Ca
s
-phenotype is caused by the ptp2 msg5 double
disruption we characterized the ptp2 msg5 double dis-
ruptant in more detail. We found that the severe growth
defect of the ptp2 msg5 double disruptant in the pres-
ence of Ca
2+
occurs at the G1 phase and that CLN2 is
down-regulated and Slt2p is hyper-phosphorylated in this
strain. Furthermore, the ptp2 msg5 double disruption
caused fragmented vacuoles even when calcium levels
were not elevated. These observations suggest that, possi-
bly through the known regulation of Slt2p, both PTP2 and
MSG5 are involved in the G1 to S transition and in vacuole
morphogenesis.
Materials and methods
Yeast strains, plasmids, and culture conditions
Strains used in this study are described in Table 1. Yeast
strains SH5209 (=FY833) and SH5210 (=FY834) (Winston
et al. 1995) were used as a wild-type and parental strains.
The rich medium YPAD was prepared by supplementing
YPD broth (Sigma-Aldrich Co.) with 0.4 mg/ml adenine.
SC medium consisted of 0.67% yeast nitrogen base without
amino acids, 2% glucose and the required auxotrophic sup-
plements. SPM medium contained 0.30% potassium ace-
tate, 0.02% raYnose and was supplemented with 10 g/ml
of adenine, arginine, histidine, isoleucine, leucine, lysine,
methionine, phenylalanine, threonine, tryptophan, uracil,
and valine. Unless indicated otherwise, yeast strains were
grown at 30C. Plasmids were propagated in Escherichia
coli strain DH5 cultivated on LB-medium containing
100 g/ml ampicillin at 37C. Plasmid pCgHIS3 (Sakum-
oto et al. 1999; Kitada et al. 1995) carrying a 1.8-kbp frag-
ment of the Candida glabrata HIS3 (CgHIS3) gene and
p3005 constructed by subcloning a 1.7 kbp BamHIXhoI
fragment containing CgLEU2 (a kind gift from K. Kitada)
into pT7Blue-T vector (Novagen) were used as template for
the PCR-ampliWcation of PTP2 and MSG5 disruption cas-
settes.
Construction of yeast single and double disruptants
Single and double gene disruptants were constructed by tar-
geted gene replacement, crossing and tetrad analysis as
described previously (Sakumoto et al. 1999, 2002). Disrup-
tant ptp2::CgHIS3 (SH6790) was generated by transfor-
mation of SH5209 using a disruption cassette ampliWed
from plasmid pCgHIS3 with primers Kf-PTP2 (5-ATAA
CGGCAA TAGAATGGCT TCTTCCGCTA TATCGGA
AAA CACAGGAAAC AGCTATGACC-3) and Kr-PTP2
(5-GTAGCAATAT ACTTGAAATC AGGATTAATT
TGCGTGAGCT GTTGTAAAAC GACGGCCAGT-3).
Disruptant msg5::CgLEU2 (SH6791) was constructed
analogously from SH5210 using a disruption cassette gen-
erated with plasmid p3005 and primers Kf-MSG5 (5-A
CATCGATTT CAAGCCAAAC TCACCGCGTT CCTTA
CAAAA CACAGGAAAC AGCTAT GACC-3) and Kr-
MSG5 (5-TCGTTGTCCA CAGAAGCTTC CAGTGAA
TCT GCGGGTTGAG GTTGTAAAAC GACGGCCA
GT-3). Correct disruption of PTP2 and MSG5 was veriWed
by PCR using primers Kfc PTP2 (5-CTC AAGCTT
GGACACT CGTTTAATTTAGC CA-3) and Krc-PTP2
(5-CTC AAGCTT ATTCGGTATT GGCACAAACT TT-
3), and Kfc-MSG5 (5-CTC GGATCC GTAGTGATGG
ATAATGTGAT TT-3) and Krc-MSG5 (5-CTC
GGATCC GTGCCCATGG TAATTTTTGA CG-3),
respectively. The double disruptant ptp2 msg5 (SH6793)
was isolated by tetrad analysis of the diploid strain resulting
from the cross of ptp2::CgHIS3 (SH6790) with
msg5::CgLEU2 (SH6791) (Sherman and Hicks 1991).
Table 1 Strains used in this study
a
NBRP/YGRC, National BioResource Project/Yeast Genetic Research Center, Japan (http://yeast.lab.nig.ac.jp/nig/index_en.html)
Strains Genotype Source
SH5209 MATa ura3-52 his3200 leu21 lys2202 trp163 NBRP, YGRC
a
SH5210 MAT ura3-52 his3200 leu21 lys2202 trp163 NBRP, YGRC
a
SH6790 MATa ptp2::CgHIS3 ura3-52 his3200 leu21 lys2202 trp163 SH5209 disruptant
SH6791 MAT msg5::CgLEU2 ura3-52 his3200 leu21 lys2202 trp163 SH5210 disruptant
SH6793 MATa ptp2::CgHIS3 msg5::CgLEU2 ura3-52 his3200 leu21 lys2202 trp163 SH6790 SH6791
Arch Microbiol (2009) 191:721733 723
1 3
Spot assay of salt sensitivity
From yeast cells grown in YPAD medium to mid logarith-
mic phase, suspensions containing equal cell-numbers were
prepared on the basis of OD
660
and 10-fold serial dilutions
were spotted onto YPAD plates supplemented with 0.6 M
salt (either CaCl
2
, KCl, NaCl, MgCl
2
, or LiCl) that were
incubated for 12 days.
Synchronization of cell cycle
Cells grown in YPAD with or without 0.3 M CaCl
2
to mid
logarithmic phase were arrested in G1 phase by exposure to
20 g/ml -factor (Sigma) for 34 h, after which cells were
washed Wve times with YPAD and transferred into fresh
medium. At least 200 cells were counted to determine the
percentage of budded cells, which was repeated at 15, 30,
and 45 min after release from the -factor arrest.
Fluorescent-activated cell sorting (FACS) analysis
A 0.25 ml aliquot of yeast cells (OD
660
= 1.0) grown in
YPAD with or without 0.3 M CaCl
2
media was harvested
by centrifugation, and, after being resuspended by gentle
vortexing, Wxed with cold ethanol (20C) for at least 12 h.
Cells were pelleted and resuspended in the dark with 300 l
of 0.2 M TrisHCl (pH 7.5) with 100 g/ml propidium
iodide, and then subjected to FACS analysis (Epix XL,
Beckman Coulter) as described (Haase and Lew 1997).
DNA microarray analysis
The yeast DNA microarray used was in Toray Industrial
Inc. (Kanagawa, Japan) and the experiment was carried out
according to the manufacturers instruction. BrieXy, RNA
was isolated by the hot phenol methods (Ausubel et al.
1989), cDNA was synthesized and puriWed with Amino
Allyl MessageAmp
TM
II aRNA ampliWcation kit (Ambion,
Applied Biosystems) and labeled with Cy3-dUTP or Cy5-
dUTP (Amersham Bioscience) for hybridization with the
DNA microarray. The Xuorescence intensities of Cy3 and
Cy5 were scanned with a microarray scanner (ScanArray
Lite; Perkin Elmer).
Transcriptional analysis
RNA isolated by the hot phenol method (Ausubel et al.
1989) was used for quantitative analysis of mRNA content
by real time (RT) PCR. First-strand cDNA was synthesized
using a High Capacity cDNA Archive kit (Applied Biosys-
tems) and used as the template for quantitative RT PCR
with a 7300 Real Time PCR system (Applied Biosystems)
and primers KfRT-CLN2 (5-TTACGGGACCAAGCC
AAATT-3) and KrRT-CLN2 (5-TTACAACCGCCCC
AAGTT TTA-3).
Vacuolar staining with FM4-64
Vacuoles were stained with FM4-64 as previously
described (Vida and Emr 1995) with minor modiWcations.
Cells grown in YPAD to an OD
660
= 1.0 were concentrated
20-fold in YPAD containing 40 M FM4-64 (Molecular
Probes Invitrogen) and incubated for 30 min at 4C with
shaking. The cells were harvested at 4C, resuspended into
fresh YPAD to an OD
660
= 10, and incubated at 30C with
vigorous shaking for 60 min. These cells were centrifuged,
resuspended into fresh YPAD and immediately analyzed
with a Xuorescence microscope (BX61-34-FL-I-D, Olym-
pus) using a MWIG2 (520550 nm) Wlter (Olympus), CCD
camera (CCD-Exi, Molecular Devices) and MetaMorph
version 6.1 software (Molecular Devices).
Immunoblot analysis of (phosphorylated) Slt2p
For Western blot analysis, protein extracts were prepared
using trichloroaceticacid (TCA) (An et al. 2006) and frac-
tionated on 10% SDS-PAGE. Proteins were transferred to
PVDF Immobilon membrane (Millipore corporation) and
probed overnight at 4C in the presence of 1% skim milk
(Difco) with either antiphospho-p44/42 MAPK (Thr
202
/
Tyr
204
) (Cell Signaling Technology) or anti-Mpk1(yC-
20):sc-6830 (Santa Cruz Biotechnology, Inc) antibodies at
1:1,000 dilution to detect phosphorylated or total Slt2p,
respectively. These primary antibodies were detected using
1:10,000 diluted horseradish peroxidase-conjugated anti-
rabbit or antigoat antibodies, respectively, and Western
Lightning Chemiluminescence Reagent Plus (PerkinEl-
mer LAS, Inc).
Results
ptp2 msg5 double disruptant displays delayed
G1 to S transition
In a previous study, we discovered that double disruption of
PTP2 and MSG5 caused a Ca
s
phenotype (Fig. 1a) (Sakum-
oto et al. 2002). The ptp2 msg5 double disruptant, how-
ever, is not sensitive to other cations (K
+
, Mg
2+
, Na
+
, and
Li
+
), as this strain grew as wild-type on media containing
0.6 M KCl, MgCl
2
, NaCl, or LiCl (Fig. 1b), suggesting that
Ptp2p and Msg5p are speciWcally involved in Ca
2+
-induced
stress response in a redundant manner.
To investigate whether the ptp2 msg5 double disrup-
tant exhibits growth arrest at any speciWc point of the cell
cycle, particularly in the presence of exogenous Ca
2+
we
724 Arch Microbiol (2009) 191:721733
1 3
conducted FACS analysis. We found that growth progres-
sion of the ptp2 msg5 double disruptant slowed down at
the G1 to S transition even in the absence of CaCl
2
, and this
delay was pronounced in the presence of CaCl
2
(Fig. 2a).
Delay in G1 to S transition in the ptp2 msg5 double dis-
ruptant was conWrmed by monitoring over time the percent-
age of budded cells in cultures synchronized with -factor,
a natural cell cycle inhibitor of S. cerevisiae (Zarzov et al.
1996) which arrests the cell cycle at G1. After release from
the -factor arrest, the percentages of budded cells in the
culture of the ptp2 msg5 double disruptant were 2- to
2.5-fold lower in the presence of 0.3 M CaCl
2
compared to
those in the wild type or in strains grown in the absence of
calcium (Fig. 2b). We conclude that the double disruption
of PTP2 and MSG5 causes signiWcant retardation of G1 to
S transition in the presence of a high external concentration
of CaCl
2
.
We noted that in the presence of 0.3 M Ca
2+
, the transcrip-
tion of CLN2 was down-regulated signiWcantly in the ptp2
msg5 double disruptant compared to that in the wild-type
strain. Out of more than 300 genes in which the transcription
was altered (Tables 2, 3) signiWcantly (transcription ratio of
gene in the ptp2 msg5/wild type is 2.0 or 0.6), about
228 ORFs had signiWcant increase or decrease in transcrip-
tion in the ptp2 msg5 double disruptant, but not in single
ptp2 and msg5 disruptant (Tables 2, 3).
Real-time RT-PCR analysis revealed that CLN2 was
down-regulated 2-fold in the presence of 0.3 M Ca
2+
in the
ptp2 msg5 double disruptant. An increase in the concen-
tration of Ca
2+
to 0.6 M resulted in an approximately 4-fold
decrease of CLN2 levels (Fig. 2c), suggesting that higher
exogenous Ca
2+
concentrations cause a more severe down-
regulation of CLN2 in the absence of Ptp2p and Msg5p.
ptp2 msg5 double disruption increases expression
of Slt2p and also leads to hyper-activation
of the Slt2 pathway
Over-expression of PTP2 and MSG5 is known to suppress
the growth defect caused by hyper-activation of the Slt2
pathway (Mattison et al. 1999; Watanabe et al. 1995). This
is expected because Ptp2p and Msg5p are negative regula-
tors for the Slt2 pathway (Flandez et al. 2004; Martin et al.
2000). These reports motivated us to investigate the
involvement of the Slt2 pathway in the defective growth of
the ptp2 msg5 double disruptant in the presence of exog-
enous Ca
2+
although DNA microarray analysis revealed
that transcriptional level of SLT2 had no signiWcant alter-
ation in the ptp2 msg5 double disruptant. This result
suggested that Ptp2p and Msg5p may regulate the Slt2
pathway at the post-transcriptional level. However, its
molecular mechanism still remains to be elucidated.
Therefore, we determined the level of Slt2p by perform-
ing immunoblot analysis using two kinds of antibodies one
of which distinctively detects the phosphorylated form of
Slt2p and the other detects Slt2p irrespective of its phos-
phorylation status. Results showed that phosphorylated
Slt2p was induced in the wild-type, the single ptp2 and
msg5 disruptants, and the ptp2 msg5 double disruptant
by addition of Ca
2+
(Fig. 2d), indicating that 0.3 M Ca
2+
Fig. 1 Calcium sensitivity of
the ptp2 msg5 double disrup-
tant. a Streak test. Cells of the
wild-type strain (SH5209),
ptp2 (SH6790), msg5
(SH6791), and the ptp2 msg5
double disruptant (SH6793)
were streaked on a YPAD plate
containing 0.6 M CaCl
2
and
incubated at 30C for 2 days.
b Spot test for sensitivity to
various cations. Ten-fold serial
dilutions of wild type (SH5209),
ptp2 (SH6790), msg5
(SH6791), and ptp2 msg5
(SH6793) were spotted on
YPAD plates containing 0.6 M
CaCl
2
, KCl, MgCl
2
, NaCl, or
LiCl, and incubated at 30C for
2 days
(A)
Wild type ptp2
ptp2 msg5
msg5
YPAD + 0.6M CaCl
2
(B)
Wild type
ptp2
msg5
ptp2 msg5
+ 0.6 M KCl
+ 0.6 M MgCl
2
+ 0.6 M NaCl + 0.6 M LiCl
+ 0.6 M CaCl
2
YPAD
10
6
cells

Arch Microbiol (2009) 191:721733 725
1 3
Fig. 2 EVect of high, exogenous concentration of Ca
2+
on ptp2
msg5 cell cycle. a FACS proWle of propidium iodide stained cells of
the ptp2 msg5 double disruptant (SH6793) and wild-type strain
(SH5209). Cells were cultivated in YPAD medium containing 0.3 M
CaCl
2
at 30C to OD
660
= 1.0 and subjected to FACS analysis. b Per-
centage of bud emergence. Cells were grown to reach mid-log phase
and treated to arrest in G1 phase by addition of 20 g/ml -factor for
34 h, then released into either YPAD with or without 0.3 M CaCl
2
. At
least 200 cells were counted to estimate the percentage of budded cells.
c Transcript levels of CLN2 in strains, wild type (SH5209), and ptp2
msg5 (SH6793) cultivated in YPAD medium containing 0, 0.3, and
0.6 M CaCl
2
as determined by real-time PCR analysis. d Detection of
Slt2p phosphorylation and expression levels. Top panel anti-phospho-
Slt2p immunoblot analysis of wild type (SH5209), ptp2 msg5
(SH6793), ptp2 (SH6790), msg5 (SH6791), and slt2. Proteins ex-
tracted from cells grown in media with or without 0.3 M CaCl
2
to an
OD
660
= 1.0 at 30C were separated on SDS-PAGE, and immunoblot-
ted with anti-phospho-p44/42 MAPK (Thr
202
/Tyr
204
) antibody. Mid
panel identical samples were used to detect total Slt2p by immunoblot
with anti-Mpk1p/Slt2p. Bottom lane panel identical samples were used
to detect actin by immunoblot with anti-actin antibody
(A) (B)
30
40
50
60
70
Wild type (0 M CaCl
2
)
Wild type (0.3 M CaCl
2
)
t 2 5 (0 MC Cl )
-CaCl
2
+CaCl
2
Wild type
0
10
20
30
0 10 20 30 40 50
Minutes
%

b
u
d
d
e
d

c
e
l
l
s
p p2 msg a
2
ptp2msg5 (0.3 M CaCl
2
)
1C 2C 1C 2C
(C)
ptp2 msg5
0 8
1
1.2
1.4
t
i
v
e
t
o
A
C
T
1
1C 2C 1C 2C
0
0.2
0.4
0.6
.
N
A
C
L
N
2
r
e
l
a
t
ptp2msg5
(D)
- + - + - + - + - +
WT ptp2msg5 ptp2 msg5 slt2
0.3 M CaCl
2
0 M CaCl
2
0.3 M CaCl
2
0.6 M CaCl
2
m
R
Phospho-Slt2p
Slt2p Slt2p
Actin
Wild type
Table 2 The transcription of
genes which increased signiW-
cantly (transcriptional level
2.0) in the ptp2 msg5
double disruptant in the presence
of 0.3 M Ca
2+
ORF Gene Molecular function Expression
level
YJL052W TDH1 Glyceraldehyde-3-phosphate
dehydrogenase (phosphorylating) activity
2.0
YMR015C ERG5 C-22 sterol desaturase activity 2.0
YKR024C DBP7 ATP-dependent RNA helicase activity 2.0
YMR223W UBP8 Ubiquitin-speciWc protease activity 2.0
YDR096W GIS1 Transcription factor activity 2.0
YOR279C RFM1 Unfolded protein binding 2.0
YOL101C IZH4 Metal ion binding 2.0
YEL039C CYC7 Electron carrier activity 2.0
YKL025C PAN3 Poly(A)-speciWc ribonuclease activity 2.0
YGR060W ERG25 C-4 methylsterol oxidase activity 2.1
YDL131W LYS21 Homocitrate synthase activity 2.1
YHR011W DIA4 Serine-tRNA ligase activity 2.1
YPL254W HFI1 Transcription coactivator activity 2.1
YJR066W TOR1 Protein binding 2.1
726 Arch Microbiol (2009) 191:721733
1 3
Table 2 continued
ORF Gene Molecular function Expression
level
YOL115W PAP2 Polynucleotide adenylyltransferase activity 2.1
YBR041W FAT1 Long-chain-fatty-acid-CoA ligase activity 2.1
YOL091W SPO21 Structural molecule activity 2.1
YJL157C FAR1 Cyclin-dependent protein kinase inhibitor activity 2.1
YDL181W INH1 Enzyme inhibitor activity 2.1
YOR208W PTP2 Protein tyrosine phosphatase activity 2.1
YNL126W SPC98 Structural constituent of cytoskeleton 2.1
YML065W ORC1 ATPase activity 2.1
YJL127C SPT10 Histone acetyltransferase activity 2.1
YDR508C GNP1 Amino acid transporter activity 2.1
YBR006W UGA2 Succinate-semialdehyde dehydrogenase [NAD(P) +] activity 2.1
YHR052W CIC1 Protein binding, bridging 2.1
YJL125C GCD14 tRNA (adenine-N1-)-methyltransferase activity 2.1
YBR021W FUR4 Uracil permease activity 2.1
YKL005C BYE1 Transcriptional elongation regulator activity 2.2
YLR300W EXG1 Glucan 1,3-beta-glucosidase activity 2.2
YOL145C CTR9 RNA polymerase II transcription
elongation factor activity
2.2
YPL088W Aryl-alcohol dehydrogenase activity 2.2
YMR294W JNM1 Structural constituent of cytoskeleton 2.2
YKL148C SDH1 Succinate dehydrogenase (ubiquinone) activity 2.2
YCR093W CDC39 35-exoribonuclease activity 2.2
YJR043C POL32 Delta DNA polymerase activity 2.2
YPL095C EEB1 Hydrolase activity, acting on ester bonds 2.2
YPL280W HSP32 Unfolded protein binding 2.2
YGR129W SYF2 RNA splicing factor activity,
transesteriWcation mechanism
2.2
YJR156C THI11 Protein binding 2.2
YBR180W DTR1 Multidrug transporter activity 2.2
YBR034C HMT1 Protein-arginine N-methyltransferase activity 2.3
YKL194C MST1 Threonine-tRNA ligase activity 2.3
YNL275W BOR1 Anion transporter activity 2.3
YNL192W CHS1 Chitin synthase activity 2.3
YER075C PTP3 Protein tyrosine phosphatase activity 2.3
Q0080 ATP8 Hydrogen-transporting ATP synthase activity,
rotational mechanism
2.3
YPL146C NOP53 rRNA binding 2.3
YJL071W ARG2 Amino-acid N-acetyltransferase activity 2.3
YEL004W YEA4 UDP-N-acetylglucosamine transporter activity 2.4
YOR180C DCI1 Dodecenoyl-CoA delta-isomerase activity 2.4
YJL098W SAP185 Protein serine/threonine phosphatase activity 2.4
YBR106W PHO88 Phosphate transporter activity 2.4
YDR297W SUR2 Sphingosine hydroxylase activity 2.4
YOR348C PUT4 L-proline permease activity 2.5
YML130C ERO1 Electron carrier activity 2.5
YLR222C UTP13 snoRNA binding 2.5
YOR330C MIP1 Gamma DNA-directed DNA polymerase activity 2.5
YOR100C CRC1 Carnitine:acyl carnitine antiporter activity 2.5
Arch Microbiol (2009) 191:721733 727
1 3
Table 2 continued
ORF Gene Molecular function Expression
level
YEL003W GIM4 Tubulin binding 2.5
YGR184C UBR1 Ubiquitin-protein ligase activity 2.5
YNL180C RHO5 GTPase activity 2.5
YOR032C HMS1 Transcription factor activity 2.5
Q0250 COX2 Cytochrome-c oxidase activity 2.6
YBR158W AMN1 Protein binding 2.7
YNL065W AQR1 Monocarboxylic acid transporter activity 2.7
YMR192W GYL1 Protein binding 2.7
YDR218C SPR28 Structural constituent of cytoskeleton 2.7
YKL216W URA1 Dihydroorotate dehydrogenase activity 2.9
YNL117W MLS1 Malate synthase activity 2.9
YOL136C PFK27 6-Phosphofructo-2-kinase activity 2.9
YOL126C MDH2 L-malate dehydrogenase activity 3.0
Q0050 AI1 Endonuclease activity 3.1
YPR186C PZF1 RNA polymerase III transcription factor activity 3.1
YJR159W SOR1 L-iditol 2-dehydrogenase activity 3.4
YDR243C PRP28 RNA splicing factor activity,
transesteriWcation mechanism
3.6
YMR028W TAP42 Protein binding 3.7
YGL028C SCW11 Glucan 1,3-beta-glucosidase activity 3.8
YGL209W MIG2 SpeciWc RNA polymerase II transcription factor activity 3.9
YML091C RPM2 Ribonuclease P activity 4.0
YNL181W Oxidoreductase activity 4.0
YKR034W DAL80 Transcription factor activity 4.6
YKR103W NFT1 ATPase activity, coupled to transmembrane
movement of substances
5.3
YKR039W GAP1 L-proline permease activity 5.4
YOL152W FRE7 Ferric-chelate reductase activity 6.9
YFR012W Molecular function unknown 2.0
YDR482C CWC21 Molecular function unknown 2.0
YGL057C Molecular function unknown 2.0
YDR381C-A Molecular function unknown 2.0
YER044C-A MEI4 Molecular function unknown 2.0
YJR122W CAF17 Molecular function unknown 2.1
YHL039W Molecular function unknown 2.1
YMR018W Molecular function unknown 2.1
YNL334C SNO2 Molecular function unknown 2.1
YPR030W CSR2 Molecular function unknown 2.1
YGR168C Molecular function unknown 2.1
YJR082C EAF6 Molecular function unknown 2.1
YOR287C Molecular function unknown 2.1
YBR272C HSM3 Molecular function unknown 2.1
YJR119C Molecular function unknown 2.1
YGR174C CBP4 Molecular function unknown 2.1
YOR152C Molecular function unknown 2.1
YOL019W Molecular function unknown 2.1
YGL236C MTO1 Molecular function unknown 2.2
YJR083C ACF4 Molecular function unknown 2.2
728 Arch Microbiol (2009) 191:721733
1 3
activates the Slt2 pathway, by enhancing Slt2p translation
and phosphorylation, which has not been reported before.
These observations suggest that the Ca
s
delay of the G1 to S
transition of the ptp2 msg5 double disruptant, presum-
ably caused by a decrease in CLN2 expression, is related to
hyper-activation of the Slt2 pathway (Fig. 2a, d).
Double disruption of PTP2 and MSG5 causes a defect
in vacuole morphology
The vacuole serves as a storage organelle for excess cal-
cium and its defect leads to sensitivity to Ca
2+
(HoVman-
Sommer et al. 2005). We examined vacuole morphology of
Table 2 continued
ORF Gene Molecular function Expression
level
YBL054W Molecular function unknown 2.2
YNL146C-A Molecular function unknown 2.2
YGL262W Molecular function unknown 2.3
YMR075W RCO1 Molecular function unknown 2.3
YOR062C Molecular function unknown 2.3
YBR105C VID24 Molecular function unknown 2.3
YGL080W FMP37 Molecular function unknown 2.3
YJL052C-A Molecular function unknown 2.4
YMR147W Molecular function unknown 2.4
YOL166W-A Molecular function unknown 2.4
YNL146W Molecular function unknown 2.4
YBR296C-A Molecular function unknown 2.4
YOR020W-A Molecular function unknown 2.5
YKL124W SSH4 Molecular function unknown 2.5
YPL192C PRM3 Molecular function unknown 2.5
YHR033W Molecular function unknown 2.5
YHR175W-A Molecular function unknown 2.6
YLR456W Molecular function unknown 2.6
YML066C SMA2 Molecular function unknown 2.6
YMR052W FAR3 Molecular function unknown 2.6
YOR381W-A Molecular function unknown 2.7
YDR532C Molecular function unknown 2.8
YKR075C Molecular function unknown 2.9
YHR140W Molecular function unknown 2.9
YIL070C MAM33 Molecular function unknown 2.9
YDR055W PST1 Molecular function unknown 3.1
YNL182C IPI3 Molecular function unknown 3.1
YAL063C-A Molecular function unknown 3.2
YPL201C YIG1 Molecular function unknown 3.3
YNR071C Molecular function unknown 3.3
YJL193W Molecular function unknown 3.3
YHR214C-E Molecular function unknown 3.4
YBR184W Molecular function unknown 3.7
YJL049W Molecular function unknown 3.7
YGL169W SUA5 Molecular function unknown 4.2
YGL188C-A Molecular function unknown 5.0
YOL019W-A Molecular function unknown 5.6
YGR035C Molecular function unknown 6.7
YAR061W Molecular function unknown 6.8
YDR034C-D Molecular function unknown 4.7
YOR068C VAM10 Molecular function unknown 8.1
Expression level is ratio of gene
expression in the ptp2 msg5/
in wild-type strain
Arch Microbiol (2009) 191:721733 729
1 3
Table 3 The transcription of
genes which decreased signiW-
cantly (transcriptional
level 0.6) in the ptp2 msg5
double disruptant in the presence
of 0.3 M Ca
2+
ORF Gene Molecular function Expression level
YDR210W-B Molecular function unknown 0.2
YLR157C-B Molecular function unknown 0.2
YDR261C-D Molecular function unknown 0.2
YDR380W ARO10 Pyruvate decarboxylase activity 0.3
YDR365W-B Molecular function unknown 0.3
YLR234W TOP3 DNA topoisomerase type I activity 0.3
YAR009C Molecular function unknown 0.3
YML117W NAB6 Molecular function unknown 0.3
YDR210C-D Molecular function unknown 0.3
YBL101W-A Molecular function unknown 0.3
YOR083W WHI5 Transcriptional repressor activity 0.4
YMR084W Molecular function unknown 0.4
YJL144W Molecular function unknown 0.4
YEL075C Molecular function unknown 0.4
YFR015C GSY1 Glycogen (starch) synthase activity 0.4
YOL104C NDJ1 Telomeric DNA binding 0.4
YCR021C HSP30 Molecular function unknown 0.4
YLR154W-E Molecular function unknown 0.4
YBR012W-B Molecular function unknown 0.4
YDL059C RAD59 Protein binding 0.4
YBL005W-B Molecular function unknown 0.4
YFL064C Molecular function unknown 0.4
YJL213W Molecular function unknown 0.4
YDR540C IRC4 Molecular function unknown 0.5
YJR153W PGU1 Polygalacturonase activity 0.5
YCR105W ADH7 Alcohol dehydrogenase (NADP+) activity 0.5
YLR045C STU2 Structural constituent of cytoskeleton 0.5
YIL166C Transporter activity 0.5
YNL029C KTR5 Mannosyltransferase activity 0.5
YLR154W-B Molecular function unknown 0.5
YBL005W-A Molecular function unknown 0.5
YBL111C Molecular function unknown 0.5
YDL012C Molecular function unknown 0.5
YCL064C CHA1 L-serine ammonia-lyase activity 0.5
YGR126W Molecular function unknown 0.5
YHL049C Molecular function unknown 0.5
YHL021C FMP12 Molecular function unknown 0.5
YJL165C HAL5 Protein kinase activity 0.5
YOR394C-A Molecular function unknown 0.5
YHR214W Molecular function unknown 0.5
YBL113C Helicase activity 0.5
YHR105W YPT35 Molecular function unknown 0.5
YIL152W Molecular function unknown 0.5
YDR098C-B Molecular function unknown 0.5
YER180C-A SLO1 Small GTPase regulator activity 0.5
YAL016C-B Molecular function unknown 0.5
YCR068W ATG15 Lipase activity 0.5
730 Arch Microbiol (2009) 191:721733
1 3
the ptp2 msg5 double disruptant by staining the vacuolar
membrane with FM4-64, a lypophilic styryl dye. Micro-
scopic observation revealed that even in the absence of
Ca
2+
the vacuole of the ptp2 msg5 double disruptant was
fragmented, whereas that of either ptp2 or msg5 was not
(Fig. 3). Consistent with this observation, single ptp2 and
msg5 disruptants were not among the genes with a vacuo-
lar fragmentation phenotype listed in a previous systematic
study of a S. cerevisiae single-gene knock-out collection
(Seeley et al. 2002). These observations suggest that there
is a relationship between redundant activities of Ptp2p and
Msg5p and vacuole fragmentation. A fragmented vacuole
was also observed in wild-type cells in the presence of
0.2 M Ca
2+
(Kellermayer et al. 2003) and strains with muta-
tions causing fragmented vacuoles under normal growth
conditions became sensitive to high extracellular calcium
concentrations (Kellermayer et al. 2003; Kane 2006).
Therefore, the abnormal morphology of the vacuole of the
ptp2 msg5 double disruptant contributes to its Ca
s
-phe-
notype.
Discussion
Ptp2p and Msg5p are known to negatively regulate the
MAPK Slt2 pathway (Mattison et al. 1999; Flandez et al.
Table 3 continued
ORF Gene Molecular function Expression level
YMR153W NUP53 Structural molecule activity 0.6
YOR216C RUD3 Molecular function unknown 0.6
YEL019C MMS21 SUMO ligase activity 0.6
YFL047W RGD2 Rho GTPase activator activity 0.6
YKL187C Molecular function unknown 0.6
YML010W SPT5 RNA polymerase II transcription elongation factor activity 0.6
YOL016C CMK2 Calcium- and calmodulin-dependent protein kinase activity 0.6
YOL067C RTG1 Transcription coactivator activity 0.6
YER153C PET122 Translation regulator activity 0.6
YDL220C CDC13 Single-stranded dna binding 0.6
YGR136W LSB1 Molecular function unknown 0.6
YPL233W NSL1 Molecular function unknown 0.6
YLL052C AQY2 Water channel activity 0.6
YLR132C Molecular function unknown 0.6
YLR106C MDN1 ATPase activity 0.6
YGL137W SEC27 Molecular function unknown 0.6
YMR273C ZDS1 Protein binding 0.6
YGL061C DUO1 Structural constituent of cytoskeleton 0.6
YIL149C MLP2 Ribonucleoprotein binding 0.6
YIR041W PAU15 Molecular function unknown 0.6
YIL137C TMA108 Metalloendopeptidase activity 0.6
YPL256C CLN2 Cyclin-dependent protein kinase regulator activity 0.6
YBL029C-A Molecular function unknown 0.6
YIL078W THS1 Threonine-tRNA ligase activity 0.6
YIR039C YPS6 Aspartic-type endopeptidase activity 0.6
YFR034C PHO4 Transcription factor activity 0.6
YJL059W YHC3 Basic amino acid transporter activity 0.6
YPR013C Molecular function unknown 0.6
YKR011C Molecular function unknown 0.6
YNR003C RPC34 DNA-directed RNA polymerase activity 0.6
YOR385W Molecular function unknown 0.6
YCR104W PAU3 Molecular function unknown 0.6
YHR129C ARP1 Structural constituent of cytoskeleton 0.6
YNL088W TOP2 DNA topoisomerase (ATP-hydrolyzing) activity 0.6
YLL053C Molecular function unknown 0.6
YGR013W SNU71 RNA binding 0.6
Expression level is ratio of gene
expression in the ptp2 msg5/
in wild-type strain
Arch Microbiol (2009) 191:721733 731
1 3
2004), which is activated during G1 to S transition concom-
itant with bud emergence (Zarzov et al. 1996). In
S. cerevisiae, G1 cyclins (CLN1, CLN2, and CLN3) play an
essential role to activate Cdc28p, the primary cyclin-depen-
dent kinase (CDK) that controls cell cycle progression and
is required for G1 to S progression (Wittenberg et al. 1990).
Cln2p disruption has been suggested to cause a delay of the
G1S transition (Queralt and Igual 2004); Mizunuma et al.
2005) and although all three cyclins function in a redundant
manner (Huang et al. 1997), it seems that the strong G1S
transition delay observed in the ptp2 msg5 double dis-
ruptant in the presence of exogenous Ca
2+
is caused by
down-regulation of CLN2.
Although higher Ca
2+
concentration causes more severe
delay in G1/S of the ptp2 msg5 double disruptant, slight
G1/S delay is exhibited in the ptp2 msg5 double disrup-
tant in the absence of Ca
2+
also (Fig. 2a). On other hand, we
observed that the CLN2 expression was down-regulated
only in the presence of Ca
2+
. This result indicates that Ca
2+
-
induced CLN2 down-regulation was involved only in the
pronounced delay in G1/S of the ptp2 msg5 double dis-
ruptant. Furthermore, microarray analysis suggested that
other genes, such as FAR1 and FAR3 may be implicated in
G1/S delay of the ptp2 msg5 double disruptant in the
presence of calcium. However, further study to elucidate
the detailed mechanism including identiWcation of other
genes that are involved in Ca
s
phenotype of the ptp2
msg5 double disruptant is currently underway.
On other hand, we found that disruption of PPases PTP2
and MSG5 genes, either by themselves or in combination,
caused induced phosphorylation of Slt2p compared to the
wild-type strain in the presence of Ca
2+
. In the case of the
double disruptant this phosphorylation was remarkably
enhanced and was detectable even in the absence of Ca
2+
,
suggesting a synthetic eVect of both disruptions (Fig. 2d).
We suppose that double disruption of the PTP2 and MSG5
genes in combination with high concentration of Ca
2+
strongly activated Slt2 pathway, leading to calcium sensi-
tivity. It is known that Slt2p and Mlp1p positively regulates
a transcription factor SBF (Swi4p/Swi6p) which in turn
activates G1 cyclin genes including CLN2 (Madden et al.
1997; Kim et al. 2008). However, we found in this study
that hyper-activation of Slt2p did not result in up- (Fig. 2c,
d) but rather down-regulation of CLN2 in the ptp2 msg5
double disruptant, suggesting a diVerent pathway by which
CLN2 expression was regulated in these circumstances. The
hyper-activation of Slt2p might reXect a situation in which
its initial function of dealing with high exogenous calcium
levels is not properly regulated by lack of the PPase activi-
ties of Ptp2p and Msg5p, possibly establishing an constitu-
tive cell-wall stress response that results in reduced bud
formation and growth retardation.
Fig. 3 Vacuole fragmentation
in the ptp2 msg5 double dis-
ruptant. Wild type (SH5209),
ptp2 (SH6790), msg5
(SH6791), and ptp2 msg5
(SH6793) were stained with
FM4-64 and photographed as
described in the Materials and
methods. Bar 5 m
DIC FM4-64
Wild type
ptp2 msg5
ptp2
msg5
732 Arch Microbiol (2009) 191:721733
1 3
In conclusion, we propose that the Ca
s
-phenotpe of the
ptp2 msg5 double disruptant is caused by three diVerent
eVects (Fig. 4) that could either reXect independent path-
ways or be part of a single cascade; (a) In the presence of
Ca
2+
insuYcient activation of Cln2-Cdc28p complex medi-
ated by down-regulation of Cln2p (Fig. 2c) results in a
defect in G1 to S progression, (b) Ca
2+
-mediated and ptp2
msg5 double disruption-mediated hyper-activation of
Slt2p (Fig. 2d) leads to a growth defect, and (c) fragmented
vacuoles (Fig. 3) disable the ptp2 msg5 double disrup-
tant to tolerate high concentrations of exogenous Ca
2+
.
How hyper-activation of Slt2p due to absence of PTP2 and
MSG5 is linked to down-regulation of CLN2 and vacuole
fragmentation awaits further analysis.
Acknowledgment This work was supported by a Grant-in-Aid for
ScientiWc Research B, 2007 to 2009, to S.H. from the Ministry of Edu-
cation, Science, Sports and Culture of Japan.
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