Báo cáo Y học: Cold induces stress-activated protein kinase-mediated response in the fission yeast Schizosaccharomyces pombe pot

Cold induces stress-activated protein kinase-mediated response
in the fission yeast
Schizosaccharomyces pombe
Teresa Soto
1
, Francisco F. Beltra
´
n
1
, Vanessa Paredes
1
, Marisa Madrid
1
, Jonathan B. A. Millar
2
,
Jero Vicente-Soler
1
, Jose
´
Cansado
1
and Mariano Gacto
1
1
Departamento de Gene
´
tica y Microbiologı
´
a, Facultad de Biologı
´

a, Universidad de Murcia, Spain;
2
Division of Yeast Genetics,
National Institute for Medical Research, London, UK
In the fission yeast Schizosaccharomyces pombe the Wak1p/
Win1p-Wis1p-Sty1p stress-activated protein kinase (SAPK)
pathway relays environmental signals to the transcriptional
machinery and modulates gene expression via a cascade of
protein phosphorylation. Cells of S. pombe subjected to cold
shock (transfer from 28 °Cto15°C) transiently activated
the Sty1p mitogen-activated protein kinase (MAPK) by
phosphorylation. Induction of this response was completely
abolished in cells disrupted in the upstream response regu-
lator Mcs4p. The cold-triggered Sty1p activation was par-
tially dependent on Wak1p MAPKKK and fully dependent
on Wis1p MAPKK suggesting that the signal transmission
follows a branched pathway, with the redundant MAPKKK
Win1p as alternative transducer to Wis1p, which subse-
quently activates the effector Sty1p MAPK. Also, the bZIP
transcription factor Atf1p became phosphorylated in a
Sty1p-dependent way during the cold shock and this phos-
phorylation was found responsible for the increased
expression of gpd1
+
, ctt1
+
, tps1
+
and ntp1
+

genes. Strains
deleted in transcription factors Atf1p or Pcr1p were unable
to grow upon incubation at low temperature whereas those
disrupted in any member of the SAPK pathway were able to
do so. These data reveal that S. pombe responds to cold by
inducing the SAPK pathway. However, such activation is
dispensable for yeast growth in cold conditions, supporting
that the presence of Atf1/Pcr1 heterodimers, rather than an
operative SAPK pathway, is critical to ensure yeast growth
at low temperature by an as yet undefined mechanism.
Keywords: cold; SAPK pathway; fission yeast.
Low temperature is an important environmental signal for
all living organisms. Adaptive response to cold stress
involves synthesis of several types of proteins. In bacteria,
thermal downshifts induce cold-shock proteins (Csp) that
function as RNA chaperones favouring efficient translation
of mRNAs at low temperature [1]. However, in eukaryotes
no proteins homologous to bacterial Csp’s have been
isolated and cold shock-inducible proteins range from
structural components involved in ribosomal biogenesis to
transcriptional regulation factors that activate gene expres-
sion in response to a drop in temperature [2,3].
The mitogen-activated protein kinase (MAPK) signalling
pathways are critical for the sensing and response of
eukaryotic cells to changes in the external environment [4].
These MAPK cascades are highly conserved through
evolution and serve to transduce signals to the nucleus,
which result in new patterns of gene expression [5,6]. Each
MAPK module comprises at least three protein kinases: a
MAP kinase is activated through phosphorylation on

specific threonine and tyrosine residues by a MAPK kinase
(MAPKK or MEK) which is in turn activated by
phosphorylation in one or several serine and threonine
residues by a MAPKK kinase (MAPKKK or MEKK).
Recently, different studies have revealed a key role for
MAPK cascades in the response of metazoan cells to
osmotic changes, heat shock, oxidative stress and UV
radiation, as well as to treatment with inflammatory
cytokines, DNA damaging agents and vasoactive neuro-
peptides [7–13]. In mammalian cells the c-Jun N-terminal
kinase (JNK) and p38/RK/CSBP kinases have been
characterized as stress-activated protein kinases (SAPKs)
[7,10–13] able to phosphorylate (and therefore activate)
transcription factors such as c-Jun [7,11], ATF2 [14–16] and
Elk-1 [17,18], which regulate gene expression in response to
various conditions.
The identification of a highly conserved SAPK pathway
in the fission yeast Schizosaccharomyces pombe allows to
analyse the precise mechanisms by which SAPKs are
activated in a system more amenable than higher eukaryotic
cells [19–22]. In this yeast, the central element of SAPK
cascade is the MAP kinase Sty1p (also known as Spc1p or
Phh1p), which is highly homologous to mammalian p38
kinase and becomes activated by a similar series of stresses
[20,21,23–25]. Deletion of sty1
+
brings about partially
sterile elongated cells that are sensitive to osmostress, heat
shock, oxidative treatment, and UV injury. Sty1p MAPK is
directly phosphorylated by Wis1p MAPKK in S. pombe

cells subjected to such stresses, and no Sty1p phosphory-
lation is detected in the absence of Wis1p under any stress
condition [20,21,23,24]. Activation of Sty1p is also con-
trolled by the action of two phosphatases, Pyp1p and Pyp2p
Correspondence to J. Cansado, Departamento de Gene
´
tica y
Microbiologı
´
a, Facultad de Biologı
´
a, University of Murcia,
30071 Murcia, Spain. Fax: + 34 68 363963, Tel.: + 34 68 364953,
E-mail:
Abbreviations: YES, yeast extract plus supplements; MEL, malt
extract liquid; EMM2, Edinburgh minimal medium; Ha6H,
hemagglutinin antigen epitope and six histidines; MAPK, mitogen-
activated protein kinase; SAPK, stress-activated protein kinase;
Csp, cold-shock proteins.
(Received 27 May 2002, revised 22 August 2002,
accepted 29 August 2002)
Eur. J. Biochem. 269, 5056–5065 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03214.x
[20]. The transmission pathway of the stress signal to Wis1p
is dual, and either the MAPKKKs Wak1p (also known as
Wis4p or Wik1p) or Win1p are responsible for Wis1p
phosphorylation [26–28]. A response regulator protein,
Mcs4p, associates with Wak1p, and probably with Win1p,
to regulate MAPKKK activity in response to several stimuli
[25,29]. In S. pombe different transcription factors function
downstream of the Sty1p MAP kinase cascade, among

which Atf1p, Pcr1p and Pap1p have been extensively
studied. Interestingly, Atf1p and Pcr1p were originally
reported as Mts1 and Mts2, respectively, and shown to be
involved in meiotic homologous recombination [30]. Atf1p
(also known as Gad7p) is a mammalian ATF-2 homologue
b-ZIP protein that associates to and is phosphorylated by
Sty1p following stress [31–33]. In fact, Sty1p is the only
known kinase involved in Atf1p phosphorylation so that
S. pombe mutants lacking atf1
+
gene show many of the
phenotypes previously described for sty1
–
cells [24]. Tran-
scription of several stress-response genes is controlled by
Atf1p [32,33]. On the other hand, strains lacking Pcr1p,
which forms heterodimers with Atf1p [30], display a
behaviour similar to atf1
–
cells [34]. Finally, Pap1p
transcription factor, with high homology to mammalian
c-Jun and similar DNA-binding properties [35], is a target
for Sty1p MAPK under oxidative stress [36]. However, in
contrast to Atf1p, Pap1p is neither phosphorylated nor a
substrate for Sty1p upon stress conditions. S. pombe cells
deleted in pap1
+
gene show high sensitivity to oxidative
stress but not to osmotic stress or nutrient deprivation [36].
Although different stimuli have been used in S. pombe to

reveal signalling routes that control cell adaptation, the
effect of low temperature has received no attention. In this
work we have dissected the SAPK cascade in cells of the
fission yeast subjected to a thermal downshift. We report
that cold activates the Wak1p/Win1p-Wis1p-Sty1p path-
way resulting in Atf1p phosphorylation and increased
expression of selected genes. Activation of the SAPK
cascade, however, is not essential for yeast growth in the
cold. Our data provide evidence for the existence of a novel
Atf1p/Pcr1p -mediated, SAPK-independent pathway that is
involved in growth determination at low temperature.
MATERIALS AND METHODS
Strains and culture media
The S. pombe strains employed in this study are listed in
Table 1. They were routinely grown with shaking at 28 °C
in yeast extract plus supplements (YES) [37] or Edinburgh
minimal medium (EMM2). Culture media were supple-
mented with adenine, leucine, histidine or uracil
(100 mgÆL
)1
, all obtained from Sigma Chemical Co.)
depending on the requirements for each particular strain.
Solid media were made by the addition of 2% (w/v) bacto-
agar (Difco Laboratories). Transformation of S. pombe
strains was performed by the lithium acetate method as
described elsewhere [37]. Escherichia coli DH5a was
employed as a host to propagate plasmids. It was grown
at 37 °C in Luria–Bertani medium plus 50 lgÆmL
)1
ampi-

cillin. Strains TS-1, TS-2, TS-3 and TS-4 were constructed
by mating the appropriate parental strains (see Table 1),
and selecting diploids in EMM2 medium with histidine plus
leucine (strains TS-1 and TS-2), or leucine (strains TS-3 and
TS-4). Sporulation was performed in malt extract liquid
(MEL) medium [37] and the spores purified by glusulase
treatment [38] were allowed to germinate in YES medium.
Strains with the desired genotype were identified by
Southern and immunoblot analysis with anti-Ha antibodies
(see below).
Stress treatments
Yeast cultures grown to mid-log phase (D
600
¼ 0.7–1) at
28 °C were subjected to heat (48 °C), osmotic (0.75
M
NaCl), oxidative (1 m
M
H
2
O
2
), or cold (15 °C) stresses.
At different times, the cells from 30 mL of culture were
collected in Falcon tubes containing ice (equivalent to
10 mL of distilled water) and harvested by centrifugation at
Table 1. S. pombe strains used in this study.
Strain Genotype Source/Reference
JM1059 h
–

ade6-M216 his7–366 leu 1–32 ura4-D18 J.B.A. Millar
JM1368 h
–
ade6-M216 his7–336 leu 1–32 ura4-D18 mcs4
+
::ura4
+
J.B.A. Millar
VB1700 h
–
ade6-M210 his7–336 leu 1–32 ura4-D18 sty1:Ha6H (ura4
+
)mcs4
+
(D412N) [29]
JM1478 h
–
ade6-M216 his7–366 leu 1–32 ura4-D18 wak1::ura4
+
J.B.A. Millar
JM1521 h
+
ade6-M210 his7–366 leu 1–32 ura4-D18 sty1:Ha6H (ura4
+
) J.B.A. Millar
JM1821 h
–
ade6-M216 leu 1–32 ura4-D18 atf1
+
:Ha6H (ura4

+
) J.B.A. Millar
TK003 h
–
leu 1–32 T. Kato
TK102 h
–
his1–102 leu 1–32 ura4-D18 wis1
+
:: his1
+
T. Kato
TK107 h
–
leu 1–32 ura4-D18 sty1
+
:: ura4
+
T. Kato
TK108 h
+
leu 1–32 ura4-D18 sty1
+
:: ura4
+
T. Kato
TS-1 h
–
ade6-M216 his7–336 leu 1–32 ura4-D18 sty1:Ha6H (ura4
+

) mcs4
+
::ura4
+
This work
TS-2 h
–
ade6-M216 his7–336 leu 1–32 ura4-D18 sty1:Ha6H (ura4
+
) wak1
+
::ura4
+
This work
TS-3 h
+
ade6-M210 his7–336 leu 1–32 ura4-D18 sty1:Ha6H (ura4
+
) wis1
+
::his1
+
This work
TS-4 h
–
ade6-M216 leu 1–32 ura4-D18 atf1:Ha6H (ura4
+
) sty1
+
::ura4

+
This work
WSP547 h
–
ade6-M210 his3-D1 leu 1–32 ura4-D18 W.P. Wahls
WSP643 h
–
ade6-M210 his3-D1 leu 1–32 ura4-D18 atf1
+
:: ura4
+
W.P. Wahls
WSP643 h
–
ade6-M210 his3-D1 leu 1–32 ura4-D18 pcr1
+
:: his3
+
W.P. Wahls
WSP672 h
–
ade6-M210 his3-D1 leu 1–32 ura4-D18 atf1
+
:: ura4
+
pcr1
+
::his3
+
W.P. Wahls

TP108–3c h
–
leu 1–32 ura4-D18 pap1
+
:: ura4
+
T. Toda
Ó FEBS 2002 Cold shock and MAP kinase activation in S. pombe (Eur. J. Biochem. 269) 5057
4 °C. Under these conditions, the previously described Sty1p
phosphorylation due to centrifugation [28] was not observed
in unstressed cells. After washing with NaCl/P
i
buffer, yeast
pellets were immediately frozen in liquid nitrogen.
Purification and detection of activated Sty1-hemagglu-
tinin antigen epitope and six histidines (Ha6H)
and Atf1-Ha6H proteins
To analyse Sty1p, total cell homogenates were prepared
under native conditions employing chilled acid-washed glass
beads and lysis buffer (10% glycerol, 50 m
M
Tris/HCl
pH 7.5, 150 m
M
NaCl, 0.1% Nonidet NP-40, plus specific
protease and phosphatase inhibitor cocktails for fungal and
yeast extracts obtained from Sigma Chemical Co.). The
lysates were removed and cleared by centrifugation at
10 000 g for 15 min. Ha6H-tagged Sty1p was purified by
using Ni

2
-nitrilotriacetic acid agarose beads (Qiagen Inc.),
as reported previously [39]. The purified proteins were
resolved in 10% SDS/PAGE gels, transferred to nitrocel-
lulose filters (Amersham Pharmacia), and incubated with
either a mouse anti-Ha (Roche Molecular Biochemicals,
clone 12CA5) or mouse anti-(phospho-p38) (New England
Biolabs) antibodies. The immunoreactive bands were
revealed with an HRP-conjugated anti-(mouse Ig) Ig
secondary antibody (Sigma Chemical Co.) and the ECL
system (Amersham-Pharmacia). For Atf1p-Ha6H purifica-
tion, the pelleted cells were lysed into denaturing lysis buffer
(6
M
Guanidine HCl, 0.1
M
sodium phosphate, 50 m
M
Tris
HCl, pH 8.0) and the Atf1p protein isolated by affinity
precipitation on Ni
2
-nitrilotriacetic acid agarose beads as
previously described [40]. The purified proteins were
resolved in 6% SDS/PAGE gels, transferred to nitrocellu-
lose filters (Amersham Pharmacia), and incubated with a
mouse anti-Ha antibody (12CA5). The immunoreactive
bands were detected as described above.
Plate assay of cold sensitivity for growth
S. pombe mutants and wild-type strains were grown in YES

liquid medium to mid-log phase and, after appropriate
dilutions, different number of cells were spotted per
duplicate on YES agar plates and incubated either at
28 °C for 3 days, or 15 °C for 10 days.
RNA isolation and hybridization
Total RNA preparations from cold-shocked strains were
obtained essentially as described in [39] and resolved
through 1.5% agarose-formaldehyde gels. Northern
(RNA)-hybridization analyses were performed as described
by Sambrook et al. [41]. A 1.2Kb fragment of the gpd1
+
gene [42] was amplified by PCR with the 5¢ oligonucleotide
TGGATATGGTCAACAAGG and the 3¢ oligonucleotide
GTTTCAGTACCGCCCTCG, and used to probe for
gpd1
+
mRNA, while a 1 Kb fragment of the ctt1
+
gene
[43] was amplified with the 5¢ oligonucleotide CGTCCCTG
TTTACAC and the 3¢ oligonucleotide GCTTCCTTGGA
ACAT. Probes for tps1
+
and ntp1
+
were prepared as
previously reported [44,45]. An approximately 900 bp
fragment of the leu1
+
gene was amplified by PCR [46],

andusedtoprobeforleu1
+
mRNA as an internal standard
for the RNA amount loaded in each lane. To establish
quantitative conclusions, the level of mRNAs was quanti-
fied in a Phosphorimager (Molecular Dynamics) and
compared with the internal control (leu1
+
mRNA).
RESULTS
Sty1p activation following a cold stress
The effects of low temperature in the fission yeast have been
scarcely investigated [34]. Because in S. pombe arangeof
environmental stresses activates Sty1 MAP kinase through
phosphorylation [28] we examined if Sty1p was also
activated following a cold stress. To this end, we used
strain JM1521 which harbors a genomic copy of sty1
+
tagged with two copies of the Ha epitope and six histidine
residues. Exponentially growing cultures of this strain were
subjected to either cold, heat shock, osmotic or oxidative
stresses. Samples were collected from 0 to 360 min, and
Sty1p-Ha6H protein was purified by affinity chromatogra-
phy employing Ni
2
-nitrilotriacetic acid agarose beads. The
activation status of Sty1p was analysed by Western
immunoblotting, using antiphospho-p38 antibodies,
whereas duplicated samples were probed with a monoclonal
antibody to the Ha-tag in order to normalize the protein

level during the course of the experiment. As shown in
Fig. 1, a thermal downshift from 28 to 15 °C provoked a
clear and largely maintained activation of Sty1p, with
significant phosphorylation level within 1 h of treatment, a
maximum at 2–3 h, and a rather slight decrease afterwards.
The kinetics of cold-induced Sty1p activation was markedly
different from the quick and transient activation achieved
under heat shock [28] (Fig. 1). Osmotic stress also produced
a relatively quick activation of Sty1 kinase followed by a
slow decrease in Sty1p phosphorylation (Fig. 1). Oxidative
stress promoted a similar Sty1p rapid activation pattern
which, however, was maintained for even longer time than
during cold shock (Fig. 1). These results show that phos-
phorylation of Sty1 kinase of S. pombe can be fully induced
by low temperature at 15 °C, although with a delayed
kinetics as compared to the effect of other previously
characterized stimuli. Cold shocks performed in a range
from 10 to 25 °C indicated that the kinetics of cold-induced
phosphorylation of Sty1p is rather sensitive and dependent
on the particular temperature chosen for stress. We
observed a rather rapid response at 25 °C, which is relatively
close to optimal temperature for growth (28–30 °C), and
significant longer lags as the temperature of exposure
dropped to lower values (results not shown).
Wis1p is the MAPK kinase that activates Sty1p during
cold stress
Earlier studies have demonstrated that the presence of
Wis1p is critical to ensure Sty1p phosphorylation during
heat shock and osmotic or oxidative stresses [19–22]. To
asses whether cold-induced activation of Sty1p takes place

in a Wis1p-dependent manner we constructed strain TS-3,
which expresses Ha6H-tagged Sty1 protein in a wis1
–
background. In contrast to wis1
+
control strain JM1521, no
signal of phosphorylated Sty1p was detected when strain
TS-3 was subjected to a cold shock at 15 °C(Fig.2A).
These analyses revealed that Wis1 MAPK kinase is essential
for Sty1p activation during a cold stress in S. pombe.
5058 T. Soto et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Wis1p-Sty1p activation during cold stress is largely
dependent on Wak1 MAPKKK and Mcs4p
The MAPKKK homologue Wak1p/Wis4p/Wik1p is an
essential regulator of the Wis1p-Sty1p cascade in S. pombe.
Shiozaki et al. [28] demonstrated that Sty1p activation is
greatly reduced in wak1
–
cells during an osmotic stress,
whereas oxidative stress or heat shock still induced a strong
activation of Sty1p kinase. In this context, we tried to
estimate the levels of Sty1p phosphorylation in strain TS-2
(wak1
–
, Sty1p-Ha6H) to determine the role of Wak1p in the
cold-induced activation process. As shown in Fig. 2A, only
a slight increase in Sty1p activation was observed at 15 °Cin
strain TS-2 as compared to control strain JM1521. In fact,
we detected Sty1p phosphorylation in strain TS-2 only at
180 min, which corresponds to the maximum of Sty1p

activation in wild-type strains (see Fig. 1). Also, we studied
the level of Sty1p activation in strain TS-2 subjected to an
osmotic shock to confirm that, as previously described [28],
only a weak increase in Sty1p phosphorylation was likewise
occurring (compare Fig. 2B with Fig. 1). Taken together,
these results indicate that, similar to osmostress, Wak1p is a
key element in the regulation of Sty1p activation by cold in
S. pombe. However, the existence of some Sty1p activation
in the absence of Wak1p reveals that to some extent other
MEKK, likely Win1p, can transmit the cold stress signal to
the Wis1p-Sty1p cascade as an alternative branch of the
pathway.
Sensing of multiple stresses through the SAPK pathway
leads to Mcs4p phosphorylation, that alters the activity of
Wak1p, and probably also the Win1p MAPKKK, to
promote sequential phosphorylation of Wis1p MAPKK
and Sty1p MAPK [29]. We examined Sty1p phosphory-
lation in strain TS-1 which shows mcs4
+
disrupted. Upon
a cold shock Sty1p was not phosphorylated in strain TS-1
(Fig. 2A). Thus, the lack of interaction between Mcs4p
and either Wak1p or Win1p totally blocks the transmis-
sion of the signal induced by low temperature that results
in phosphorylation of MAPK Sty1p. Recently, it has been
reported that during oxidative stress Mcs4p acts in a
conserved phospho-relay system initiated by two PAS/
PAC domain-containing histidine kinases, Mak2p ad
Mak3p [29]. Mcs4p phosphorylation at aspartate 412
appears critical for Sty1p activation in response to

hydrogen peroxide, but not to other environmental
stresses [29]. Hence, we performed a time-course study
of Sty1p phosphorylation during cold stress in strain
VB1700, where mcs4
+
is replaced by a mutant allele
bearing a nonphosphorylable asparagine at residue 412.
As shown in Fig. 2C, mcs4(D412N) cells displayed a
pattern of Sty1p phosphorylation similar to wild-type cells
(Fig. 2A), suggesting that phosphorylation of Mcs4p
D412 is not required for activation of Sty1p by cold
stress.
Atf1p is phosphorylated in a Sty1p-dependent way
and regulates gene expression during cold stress
Among the several bZIP transcription factors that appear to
function downstream of the Sty1 MAP kinase cascade,
Atf1p has been investigated in some detail during the last
years. Atf1p is phosphorylated by Sty1p both in vivo and
in vitro under different stress conditions and induces the
expression of different stress-response genes [33]. The
existence of a Wis1p-Sty1p-mediated response to cold stress
in S. pombe, led us to explore the phosphorylation status of
Atf1p during a thermal downshift. To this purpose we used
strain JM1821, which carries a genomic copy of the atf1
+
gene tagged with two copies of the Ha epitope and six
histidine residues, and took advantage of previous findings
demonstrating that Atf1p of unstressed cells migrates in gel
as a single protein band of approximately 85 kDa that
undergoes a Sty1p-dependent band shift due to phosphory-

lation under different stresses [29,32]. Cold treatment of
wild-type strain JM1821 of S. pombe induced Atf1p phos-
phorylation in vivo (Fig. 3). This response was evident upon
90 min of treatment at 15 °C and was maintained for at
least 3 h. Besides, the kinetics of Atf1p phosphorylation
matched closely with Sty1p activation (see Fig. 1). Also, the
level of Atf1p increased at longer treatment times, an effect
that has been interpreted by others as due to Atf1p
stimulation of its own expression under stress [24,47].
Contrary to these results, Atf1p purified from sty1
–
strain
TS-4 subjected to the low temperature treatment migrated
Fig. 1. Kinetics of cold-induced activation of Sty1p in S. pombe. Wild-
type strain JM1521 carrying a Ha6H-tagged chromosomal version of
the sty1
+
gene was grown in YES medium to mid-log phase and
subjected to a cold stress (15 °C), heat shock (40 °C), osmotic shock
(0.5
M
Na Cl) or oxidative stress (1 m
M
H
2
O
2
) for the times indicated.
Aliquots were harvested and Sty1p was purified by affinity chroma-
tography. Activated Sty1p was detected by inmunoblotting with

anti(phospho p38) antibodies. Total Sty1p was determined by inmu-
noblotting with anti-Ha antibody as loading control.
Ó FEBS 2002 Cold shock and MAP kinase activation in S. pombe (Eur. J. Biochem. 269) 5059
always with the same apparent size, corresponding to the
unphosphorylated form (Fig. 3). These results clearly indi-
cate that Sty1p is the only MAP kinase that phosphorylates
Atf1p in vivo following a cold stress in S. pombe.
A number of stress-responsive genes have been shown
to be targets of the Atf1p transcription factor. One of
these, gpd1
+
, encoding glycerol-3 phosphate dehydroge-
nase, is involved in the synthesis of glycerol, whose
intracellular accumulation is important in response to high
osmolarity conditions. The expression of gpd1
+
is induced
in S. pombe via the Wis1p-Sty1p-Atf1p pathway
[23,25,32,33]. With these precedents, we studied the level
of expression of gpd1
+
gene in wild-type, sty1
–
,andatf1
–
strains of S. pombe during a cold stress. As shown in
Fig. 4, a modest but reproducible increase of gpd1
+
expression was evident after 4–5 h of treatment in wild-
type strain induced by cold stress. However, this increase

was absent in sty1
–
and atf1
–
strains under the same
conditions (Fig. 4). Also, the expression of the cytoplas-
mic catalase gene ctt1
+
, which is characteristically
Fig. 2. Functional SAPK pathway is required for cold stress activation of Sty1p. (A) Sty1p phosphorylation in S. pombe cellssubjectedtoacold
stress is dependent on Wis1p, Wak1p and Mcs4p. Wild-type (JM1521), Dwis1 mutant (TS-3), Dwak1 mutant (TS-2) and Dmcs4 mutant (TS-1)
strains carrying a Ha6H-tagged chromosomal version of the sty1
+
gene were subjected to a cold stress at 15 °C for the times indicated. Aliquots
were harvested and Sty1p was purified by affinity chromatography. Activated Sty1p was detected by inmunoblotting with anti-(phospho p38)
antibodies and anti-Ha antibody inmunoblotting was used as a control to determine loaded Sty1p. Wis1p and Mcs4p function is critical in the cold-
induced activation of Sty1p while Wak1p plays an important role. (B) Osmostress-induced Sty1p activation is largely dependent on Wak1p. The
Dwak1 mutant strain TS-2 was subjected to an osmotic stress with 0.5
M
NaCl in YES medium. Aliquots were processed and analyzed as described
in (A). (C) D412 of Mcs4p is not required for the activation of Sty1p MAP kinase in response to cold stress. The mcs4 (D412N) strain VB1700 was
subjected to a cold stress at 15 °C in YES medium, and aliquots were processed as described in (A).
Fig. 3. Sty1p-dependent phosphorylation of Atf1p in vivo followingacoldstress.Wild-type (JM1821) and Dsty1 (TS-4) strains carrying a chro-
mosomal copy of Ha-tagged atf1
+
gene were grown at 28 °C(0time)andshiftedto15°C for the times indicated. Aliquots were harvested and the
Atf1p-Ha6H tagged protein was purified with Ni
2
-nitrilotriacetic acid beads and analyzed by SDS/PAGE followed by inmunoblotting with anti-Ha
antibodies. Samples marked with asterisk (*) show a typical Atf1p shift due to phosphorylation.

5060 T. Soto et al. (Eur. J. Biochem. 269) Ó FEBS 2002
regulated by Atf1p [24,26], was induced in a Sty1p-Atf1p-
dependent manner during exposure of S. pombe to a cold
shock (Fig. 4). Similarly, we observed a retarded cold-
induced increase in the expression of tps1
+
and ntp1
+
,
which code for trehalose-6-phosphate synthase and neut-
ral trehalase, respectively (Fig. 4). These results show that
S. pombe is able to transduce a cold-induced stress signal
through the Wis1-Sty1 MAP kinase pathway, to promote
Atf1p phosphorylation and to trigger the subsequent
expression of stress-responsive genes.
Sensitivity of mutants in SAPK pathway to cold stress
The experiments described above demonstrate that the
SAPK pathway of S. pombe can be fully activated in
response to low temperatures. We next addressed the
question of the biological significance of such a response by
studying the ability of different mutants affected in this
pathway to grow at low temperatures in YES solid medium.
As shown in Fig. 5, all the strains assayed displayed normal
growth when incubated at 28 °C for 3 days. However,
significant differences were observed in cell viability among
different mutants after incubation at 15 °C for 10 days.
S. pombe strains lacking Mcs4p, Wak1p, Wis1p or Sty1p
kinases were slightly more sensitive to cold stress than their
wild-type counterparts (Fig. 5), whereas atf1
–

, pcr1
–
,and
atf1
–
pcr1
–
strains were unable to grow at these conditions.
On the contrary, the strain TP108–3C, which lacks
transcription factor pap1
–
, did not exhibit the cold tem-
perature defective phenotype and exhibited a growth similar
to wild-type cells. The failure of atf1
–
and pcr1
–
mutants to
grow at low temperature is not related to a marked decrease
in the viability of these strains because a shift of cold-
stressed cultures back to 28 °Cresultedingrowthresump-
tion (not shown). Taken together, these results indicate that
the SAPK pathway plays a discrete role in the survival of
S. pombe to low temperatures and that cell viability
(measured as growth) appears to be fully dependent on
the existence of Atf1p-Pcr1p heterodimers. As Atf1p is not
phosphorylated during cold stress in the absence of the Sty1
kinase that channels the stress signal (Fig. 4), the role of
Atf1p/Pcr1pingrowthatlowtemperatureislikely
independent on the existence of an operative SAPK

pathway.
Fig. 4. Sty1p MAP kinase regulates the induction of gpd1
+
, ctt1
+
,tps1
+
and ntp1
+
genes during cold stress through the Atf1p transcription factor.
Strains TK003 (WT), TK107 (Dsty1) and WSP 643 (Datf1)weregrowninYESmediumtomid-logphaseandshiftedto15°Cforthetimes
indicated. Total RNA was extracted from each sample and 20 lg were resolved in 1.5% agarose-formaldehyde gels. The denatured RNAs were
transferred to nylon membranes and hybridized with
32
P-labelled probes of gpd
+
,ctt1
+
, tps1
+
, ntp1
+
and leu1
+
(loading control). Numbers below
each frame indicate normalized relative values of expression.
Ó FEBS 2002 Cold shock and MAP kinase activation in S. pombe (Eur. J. Biochem. 269) 5061
DISCUSSION
The main aim of this study was to analyse the cold shock
response in S. pombe,ayeastthatdisplaysacascadeof

stress activated protein kinases homologous to the SAPK
pathway present in higher eukaryotes [20–25]. In rich
medium, wild-type S. pombe strains are able to growth in a
range from 15 to 37 °C, although the optimum temperature
forgrowthis28–30°C [48]. The key element of the SAPK
pathway in this yeast is the Sty1p MAP kinase, that
becomes phosphorylated by different stressful conditions.
We have demonstrated that Sty1p from S. pombe is
specifically phosphorylated at threonine and tyrosine resi-
dues during a thermal downshift to 15 °C, with maximal
level within 2–3 h of exposure. This cold-induced activation
is unnoticed unless the span of sampling to detect signal of
Sty1p phosphorylation extends for longer times than those
usually considered for other stress stimuli. Our data provide
the first evidence that low temperature is an activator of the
SAPK pathway and that this signal transduction system
controls gene transcription in response to cold environ-
ments.
An interesting observation concerns the delay in the
kinetics of phosphorylation and dephosphorylation of
Sty1p during cold stress as compared to other stimuli.
Unexpectedly, we detected Sty1 activation even at temper-
atures well below 15 °C, for example, immediately prior to
the freezing of cell samples at )20 °C (data not shown).
Indeed, this pattern of activation is slower than the
described for heat shock, osmostress, treatment with
reactive oxygen species or UV radiation [23,24], but similar
to the kinetics observed in response to nitrogen starvation
[32]. It thus appears that the cold stress signal is transduced
through the SAPK cascade in S. pombe with a rate specific

of this type of stress and relatively dependent of the severity
of the thermal downshift, with longer delays at lower tem-
peratures. It has been proposed that different environmental
stresses may be sensed by specific membrane-bound cellular
receptors that trigger activation of Sty1p but the nature of
the upstream components involved in this signalling remain
for most part unknown [25]. A possible reason for the slow
response in Sty1p phosphorylation during sensing and
transduction of the cold shock signal could be that
membrane fluidity decreases greatly upon temperature
downshifts, thereby slowing down membrane associated
cellular functions. Also, a decreased rate in general meta-
bolism would limit the generation of second messengers.
The level of Sty1p phosphorylation increased markedly in
response to cold shock and was maintained for at least 6 h
(Fig. 1). Contrariwise, a severe heat shock induced a quick
and transient phosphorylation of Sty1p that became
dephosphorylated within 60–90 min. This is likely due to
the activity of threonine-phosphatases Ptc1p and Ptc3p and
tyrosine-phosphatase Pyp1 [49]. Other treatments, such as
osmotic stress, induced an early burst of Sty1p phosphory-
lation which, even under continuous stress, returned sub-
sequently close to the basal level only after 60 min (Fig. 1).
Pyp1p and Pyp2p phosphatases appear to play a key role in
the dephosphorylation of the osmotic stress-activated Sty1p
[20]. Interestingly, however, the Sty1p activation induced by
oxidative stress, although rather rapid, was followed by a
very slow decrease in Sty1p phosphorylation, as during a
cold shock (see Fig. 1). This differential behaviour may
reflect that one or several of the protein phosphatases are

maintained partially inactive when S. pombe is subjected to
any of these two stresses.
The fact that Wis1p is apparently the only MAPKK that
activates Sty1p during cold stress (Fig. 2) is not surprising,
as this is the case for other stresses [20,21,23,24]. Wis1p is
thus able to integrate the transmission of different stress
signals to Sty1p MAP kinase, including thermal downshifts.
From the two MAPKKKs that are known to activate
Wis1p (i.e.Win1p or Wak1p), Wak1p appears to be the
main responsible for Sty1p activation during cold shock
Fig. 5. The requirement of Atf1p and Pcr1p
for S. pombe growth at low temperature is
not dependent on the existence of an operative
SAPK pathway. The indicated number of
cells from wild-type and mutants in the SAPK
pathway were spotted onto YES plates and
incubated at 28 °Cor15 °C for 3 and 10 days,
respectively, prior to being photographed.
5062 T. Soto et al. (Eur. J. Biochem. 269) Ó FEBS 2002
(Fig. 2A), similar to what happens during osmostress [28].
The Win1p mitotic regulator, which controls the activation
of Sty1p kinase under multiple stressful conditions [27], is
likely responsible for the slight cold-induced activation of
Sty1p observed in the absence of Wak1p (Fig. 2B). Thus,
the signal transmission for cold appears to follow a
branched pathway, with either Wak1p or the redundant
MAPKKKWin1pasanalternativeviatoactivatetosome
extent the Sty1p kinase through Wis1p. On the other hand,
our results with cells disrupted in the upstream response
regulator Mcs4p indicate that the signal for cold does not

reach in such cells the level of MAPKKs, which strongly
supports the suggestion that Mcs4p likely interacts with
both Wak1p and Win1p [27,29]. Also, the occurrence of
Sty1p phosphorylation in S. pombe cells expressing a
D412N version of Mcs4p indicated that the Mak2p and
Mak3p phosphorelay system is not involved in Sty1p
phosphorylation following a cold stress, which is similar to
what happens during other environmental stresses except
during oxidative stress. In any case, the control of SAPK
pathways by two-component systems appears exclusive of
lower eukaryotes under specific stresses, as no structural
homologues of phospho-relay proteins have been identified
in mammals [29]. Thus, because such a system is not
operative in S. pombe during cold induced activation of the
SAPK pathway, this model may be relevant to study and
characterize the transduction of temperature downshifts
signals in cells from higher eukayotes, including mammals.
Following a shift to low temperature, the bZIP trancrip-
tion factor Atf1p becomes phosphorylated in vivo in a
Sty1p-dependent manner (Fig. 3). In coincidence with
previous studies, our data confirm that Sty1p is the only
kinase able to phosphorylate Atf1p at any stress condition
[32,33]. As Atf1p becomes phosphorylated under cold
temperature, one might anticipate changes in the expression
of Atf1p-regulated genes upon incubation of S. pombe at
low temperatures. Indeed, this happens for several Atf1-
regulated genes studied in this work, gpd1
+
, ctt1
+

, tps1
+
and ntp1
+
, whose expression rises significantly by a Sty1p-
Atf1p-dependent mechanism after a thermal downshift
(Fig. 4). The physiological significance of the cold-triggered
expression of gpd1
+
might be interpreted in terms of
synthesis of a cryoprotectant metabolite [50]. Catalase may
also act as a protectant as it has been shown in plants and
yeast cells that cold involves oxidative stress [51,52].
Additionally, we have observed a retarded cold-induced
increase in the expression of tps1
+
and ntp1
+
, which code
for enzymes involved in trehalose metabolism. This is
congruent with accumulation and turnover of the low
molecular mass carbohydrate trehalose, a well known
stabilizer of macromolecular components [53]. As a whole,
it appears that the induction of compatible solutes and
defences against oxidant species forms part of the response
to low temperature and that the expression of a conserved
set of stress-responsive genes is the basis of the general stress
response underlying crossed stress tolerance. In this respect,
pretreatment with low temperature induces a significant
adaptive response to osmostress in S. pombe cells (F.F.

Beltran and J. Cansado, unpublished results). It is worth to
mention that transcription factor Pap1p is neither activated
nor translocated from the cytoplasm to the cell nucleus at
low temperatures and that ctt1
+
expression was induced at
low temperatures in a pap1-deficient strain (data not
shown). Thus, in analogy to what happens during osmotic
stress [33], the expression of ctt1
+
at cold temperature
appears to be mostly dependent on Atf1p/Pcr1p.
Although the MAP kinase pathway confers a slight, but
consistent, growth advantage at 15 °C (Fig. 5), this path-
way is not essential for growth at such temperature.
Instead, the transcription factors Atf1p and Pcr1p, which
are key effectors of the SAPK phosphorylation cascade
required for a variety of developmental decisions [31–34]
and dispensable for growth at 28 °C, were needed for
growth in the cold. Surprisingly, however, the cold sensitive
phenotype in atf1
–
or pcr1
–
strains is not shared by mutants
disrupted in either sty1
+
or wis1
+
(Fig. 5), indicating that

the role of these factors at low temperature is independent
of their function as SAPK-driven multifunctional switches
that activate specific responses against extracellular condi-
tions. Hence, although cold stress in S. pombe induces the
SAPK pathway, the function of this cascade does not
guarantee far more than a slight better adjustment of cell
growth to cold conditions. On the contrary, the presence of
Atf1p and Pcr1p (presumably acting as a heterodimer) is
vital for growth al low temperature by a mechanism
unrelated to the SAPK pathway. Preliminary observations
indicate that there is not specific cell cycle block in Datf1
cells arrested at 15 °C. Altogether, these data are consistent
with a new role for these factors in transcriptional events
sustaining specific development programs in the cold.
ACKNOWLEDGEMENTS
We are indebted to W.P. Wahls, T. Kato and T. Toda for kind
supply of yeast strains, and to F. Garro for technical assistance. V.
Paredes and M. Madrid are predoctoral fellows from the Fundacio
´
n
Se
´
neca (Regio
´
n de Murcia) and the Fundacio
´
nRamo
´
n Areces,
respectively. This work was supported in part by grant BMC

2001–0135 from MCYT, Spain.
REFERENCES
1. Phadtare,S.,Alsina,J.&Inouye,M.(1999)Cold-shockresponse
and cold-shock proteins. Curr. Opin. Microbiol. 2, 175–180.
2. Kondo, K., Kowalski, L. & Inouye, M. (1992) Cold-shock
induction of yeast NSR-1 protein and its role in pre-RNA pro-
cessing. J. Biol. Chem. 267, 16259–16265.
3. Shinwari, Z.K., Nakashima, K., Miura, S., Kasuga, M., Seki, M.,
Yamaguchi-Shinozaki, K. & Shinozaki, K. (1998) An Arabidopsis
gene family encoding DRE/CRT binding proteins involved in low-
temperature-responsive gene expression. Biochem. Biophys. Res.
Commun. 250, 161–170.
4. Treisman, R. (1996) Regulation of transcription by MAP kinase
cascades. Curr. Opin. Cell Biol. 8, 205–215.
5. Marshall, C.J. (1995) Specificity of receptor tyrosine kinase sig-
nalling: transient versus sustained extracellular signal-regulated
kinase activation. Cell 80, 179–185.
6. Waskiewicz, A.J. & Cooper, J.A. (1995) Mitogen and stress
response pathways: MAP kinase cascades and phosphatase
regulation in mammals and yeast. Curr. Opin. Cell Biol. 7, 98–805.
7. Derijard, B., Hibi, M., Wu, I.H., Barrett, T., Su, B., Deng, T.,
Karin, M. & Davis, R.J. (1994) JNK1: a protein kinase stimulated
by UV light and Ha-Ras that binds and phosphorylates the c-Jun
activation domain. Cell 76, 1025–1037.
8. Freshney, N.W., Rawlison, L., Gusdon, F., Jones, E., Cowley, S.,
Hsuan, J. & Saklatvala, J. (1994) Interleukin-1 activates a novel
protein kinase cascade that results in the phosphorylation of
Hsp27. Cell 78, 1039–1049.
Ó FEBS 2002 Cold shock and MAP kinase activation in S. pombe (Eur. J. Biochem. 269) 5063
9. Galcheva-Gargova, Z., Derijard, B., Wu, I.H. & Davis, R.J.

(1994) An osmosensing signal transduction pathway in mamma-
lian cells. Science 265, 806–808.
10.Han,J.,Lee,J.D.,Bibbs,L.&Ulevitch,R.J.(1994)AMAP
kinase targeted by endotoxin and hyperosmolarity in mammalian
cells. Science 265, 808–811.
11. Kyriakis, J.M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E.A.,
Ahmad, M.F., Avruch, J. & Woodgett, J.R. (1994) The stress-
activated protein kinase subfamily of c-Jun kinases. Nature 369,
156–160.
12. Lee, J.C., Laydon, J.T., McDonell, P.C., Gallagher, T.F., Kumar,
S., Green, D., McNulty, D., Blumenthal, M.J., Heys, J.R.,
Landwatter, S.W., Strickler, J.A., McLaughlin, M.M., Siemens,
I.V., Fisher, S.M., Livi, G.P., White, J.R., Adams, J.L. & Young,
P.R. (1994) A protein kinase involved in the regulation of
inflammatory cytokine biosynthesis. Nature 372, 739–746.
13. Rouse, J., Cohen, P., Trigon, S., Morange, M., Alonso-Llamaz-
ares, A., Zamanillo, D., Hunt, T. & Nebreda, A. (1994) A novel
kinase cascade triggered by stress and heat shock that stimulates
MAPKAP kinase-2 and phosphorylation of the small heat shock
proteins. Cell 78, 1027–1037.
14. Gupta, S., Campbell, D., Derijard, B. & Davis, R.J. (1995)
Independent human MAP- kinase signal transduction pathways
defined by MEK and MKK isoforms. Science 267, 389–393.
15. Livingstone, C., Patel, G. & Jones, N. (1995) ATF-2 contains a
phosphorylation- dependent transcriptional activation domain.
EMBO J. 14, 1785–1797.
16. Raingeaud, J., Whitmarsh, A.J., Barrett, T., Derijard, B. & Davis,
R.J. (1996) MKK3- and MKK6-regulated gene expression is
mediated by the p38 mitogen-activated protein kinase signal
transduction pathway. Mol. Cell. Biol. 16, 1247–1255.

17. Cavigelli, M., Dolfi, F., Claret, F.X. & Karin, M. (1995) Induction
of c-fos expression through JNK-mediated TCF/Elk-1 phos-
phorylation. EMBO J. 14, 6269–6279.
18. Price, M.A., Cruzalegui, F.H. & Treisman, R. (1996) The p38 and
ERK MAP kinase pathways cooperate to activate Ternary
Complex Factors and c-fos transcription in response to UV light.
EMBO J. 15, 6552–6563.
19. Warbrick,E.&Fantes,P.(1991)Thewis1proteinkinaseisa
dosage-dependent regulator of mitosis in Schizosaccharomyces
pombe. EMBO J. 10, 4291–4299.
20. Millar, J.B.A., Buck, V. & Wilkinson, M.G. (1995) Pyp1 and Pyp2
PTPases dephosphorylate an osmosensing MAP kinase control-
ling cell size at division in fission yeast. Genes Dev. 9, 2117–2130.
21. Shiozaki, K. & Russell, P. (1995) Cell-cycle control linked to
extracellular environment by MAP kinase pathway in fission
yeast. Nature 378, 739–743.
22. Kato, T., Okazaki, K., Murakami, H., Stettler, S., Fantes, P. &
Okayama, H. (1996) Stress signal, mediated by a Hog1-like MAP
kinase, controls sexual development in fission yeast. FEBS Lett.
378, 207–212.
23. Degols, G., Shiozaki, K. & Russell, P. (1996) Activation and
regulation of the Spc1 stress-activated protein kinase in Schizo-
saccharomyces pombe. Mol. Cell. Biol. 16, 2870–2877.
24. Degols, G. & Russell, P. (1997) Discrete roles of the Spc1 kinase
and the Atf1 transcription factor in the UV response of Schizo-
saccharomyces pombe. Mol. Cell. Biol. 17, 3356–3363.
25. Shieh, J.C., Wilkinson, M.G., Buck, V., Morgan, B., Makino, K.
& Millar, J.B.A. (1997) The Mcs4 response regulator coordinately
controls the stress-activated Wak1-Wis1-Sty1 MAP kinase path-
way and fission yeast cell cycle. Genes Dev. 11, 1008–1022.

26. Wilkinson, M.G. & Millar, J.B.A. (1998) SAPKs and transcrip-
tion factors do the nucleocytoplasmic tango. Genes Dev. 12, 1391–
1397.
27. Shieh, J.C., Wilkinson, M.G. & Millar, J.B.A. (1998) The Win1
mitotic regulator is a component of the fission yeast stress-acti-
vated Sty1 MAPK pathway. Mol. Biol. Cell 9, 311–322.
28. Shiozaki, K., Shiozaki, M. & Russell, P. (1998) Heat stress acti-
vates fission yeast Spc1/StyI MAPK by a MEKK-independent
mechanism. Mol. Biol. Cell 9, 1339–1349.
29. Buck, V., Quinn, J., Soto, T., Martin, H., Saldanha, J., Makino,
K., Morgan, B.A. & Millar, J.B.A. (2001) Peroxide sensors for the
fission yeast stress-activated mitogen-activated protein kinase
pathway. Mol. Biol. Cell 12, 407–419.
30. Wahls, W.P. & Smith, G.R. (1994) A heteromeric protein that
binds to a meiotic homologous recombination hot spot: correla-
tion of binding and hot spot activity. Genes Dev. 8, 1693–1702.
31. Takeda, T., Toda, T., Kominami, K., Kohnosu, A., Yanagida, M.
& Jones, N. (1995) Schizosaccharomyces pombe atf1+ encodes a
transcription factor required for sexual development and entry
into stationary phase. EMBO J. 14, 6193–6208.
32. Shiozaki, K. & Russell, P. (1996) Conjugation, meiosis, and the
osmotic stress response are regulated by Spc1 kinase through Atf1
transcription factor in fission yeast. Genes Dev. 10, 2276–2288.
33. Wilkinson, M.G., Samuels, M., Takeda, T., Toone, M.W., Shieh,
J., Toda, T., Millar, J.B.A. & Jones, N. (1996) The Atf1 tran-
scription factor is a target for the Sty1 stress- activated MAP
kinase pathway in fission yeast. Genes Dev. 10, 2289–2301.
34.Watanabe,Y.&Yamomoto,M.(1996)Schizosaccharomyces
pombe pcr1+ encodes a CREB/ATF protein involved in regula-
tion of gene expression for sexual development. Mol. Cell. Biol. 16,

704–711.
35. Toda, T., Shimanuki, Y. & Yanagida, M. (1991) Fission yeast
genes that confer resistance to staurosporine encode an AP-1-like
transcription factor and a protein kinase related to the mammalian
ERK1/MAP2 and budding yeast FUS3 and KSS1 kinases. Genes
Dev. 5, 60–73.
36. Toone, W.M., Kuge, S., Samuels, S., Morgan, B.A., Toda, T. &
Jones, N. (1998) Regulation of the fission yeast transcription
factor Pap1 by oxidative stress: requirement for the nuclear export
factor Crm1 (Exportin) and the stress-activated MAP kinase Sty1/
Spc1. Genes Dev. 12, 1453–1463.
37. Moreno, S., Klar, A. & Nurse, P. (1991) Molecular genetic ana-
lysis of fission yeast Schizosaccharomyces pombe. Methods
Enzymol. 194, 795–823.
38. Kelly, T.J., Martin, G.S., Forsburg, S.L., Stephen, R.J., Russo, A.
& Nurse, P. (1993) The fission yeast cdc18+ gene product couples
S phase to START and mitosis. Cell 74, 371–382.
39. Franco, A., Soto, T., Vicente-Soler, J., Valero Guillen, P.,
Cansado, J. & Gacto, M. (2000) Characterization of tpp1+ as
encoding a main trehalose-6P phosphatase in the fission yeast
Schizosaccharomyces pombe. J. Bacteriol. 182, 5880–5884.
40. Shiozaki, K. & Russell, P. (1997) Stress-activated protein kinase
pathway in cell cycle control of fission yeast. Methods Enzymol.
283, 503–520.
41. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular
Cloning: a Laboratory Manual, 2nd edn. Cold Spring. Harbor
Laboratory Press, Cold Spring Harbor, N.Y.
42. Pidoux, A.L., Fawell, E.H. & Armstrong, J. (1990) Glycerol-
3-phosphate dehydrogenase homologue from Schizosacchar-
omyces pombe. Nucleic Acids Res. 18, 7145.

43. Nakagawa, C.W., Mutoh, N. & Hayashi, Y. (1995) Transcriptional
regulation of catalase gene in the fission yeast Schizosacchar-
omyces pombe: molecular cloning of the catalase gene and north-
ern blot analyses of the transcript. J. Biochem. 118, 109–116.
44. Fernandez, J., Soto, T., Vicente-Soler, J., Cansado, J. & Gacto, M.
(1997) Heat-shock response in Schizosaccharomyces pombe cells
lacking cyclic AMP-dependent phosphorylation. Curr. Genet. 31,
112–118.
45. Cansado, J., Soto, T., Fernandez, J., Vicente-Soler, J. & Gacto, M.
(1998) Characterization of mutants devoid of neutral trehalase
activity in the fission yeast Schizosaccharomyces pombe: partial
protection from heat shock and high-salt stress. J. Bacteriol. 180,
1342–1345.
5064 T. Soto et al. (Eur. J. Biochem. 269) Ó FEBS 2002
46. Kikuchi, Y., Kitazawa, Y., Shimatake, H. & Yamamoto, M.
(1988) The primary structure of the leu1+ gene of Schizosaccha-
romyces pombe. Curr. Genet. 14, 375–379.
47. Wilkinson, M.G., Soto, T., Tournier, S., Buck, V., Martin, H.,
Christiansen, J., Wilkinson, D.G. & Millar, J.B.A. (1999) Sin1: an
evolutionarily conserved component of the eukaryotic SAPK
pathway. EMBO J. 18, 4210–4221.
48. Yarrow, D. (1984) The Yeasts, a Taxonomic Study (Kreger-Van
Rij, N.J.W., eds), pp. 414–422. Elsevier Science, Amsterdam
49. Nguyen, A.N. & Shiozaki, K. (1999) Heat-shock-induced activa-
tion of stress MAP kinase is regulated by threonine- and tyrosine-
specific phosphatases. Genes Dev. 13, 1653–1663.
50.Beney,L.,Marechal,P.A.&Gervais,P.(2001)Coupling
effects of osmotic pressure and temperature on the viability of
Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 56,
513–516.

51. Xiong, L., Schumaker, K.S. & Zhu, J.K. (2002) Cell signaling
during cold, drought and salt stress. Plant Cell 14(Suppl.), S165–
S183.
52. Park, J.L., Grant, C.M., Davies, M.J. & Dawes, I.W. (1998) The
cytoplasmic Cu,Zn superoxide dismutase of Saccharomyces cere-
visiae is required for resistance to freeze- thaw stress. Generation
of free radicals during freezing and thawing. J. Biol. Chem. 273,
22921–22928.
53. Singer, M.A. & Lindquist, S. (1998) Multiple effects of trehalose
on protein folding in vitro and in vivo. Mol. Cell 1, 639–648.
Ó FEBS 2002 Cold shock and MAP kinase activation in S. pombe (Eur. J. Biochem. 269) 5065