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
´

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

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

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-

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

) 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

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

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],

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

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

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

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.

, 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

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

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

, 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.

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

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