Báo cáo khoa học: The yeast stress response Role of the Yap family of b-ZIP transcription factors The PABMB Lecture delivered on 30 June 2004 at the 29th FEBS Congress in Warsaw potx

REVIEW ARTICLE
The yeast stress response
Role of the Yap family of b-ZIP transcription factors
The PABMB Lecture delivered on 30 June 2004 at the 29th FEBS
Congress in Warsaw
Claudina Rodrigues-Pousada, Tracy Nevitt and Regina Menezes
Genomics and Stress Laboratory, Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Oeiras, Portugal
The capacity for adaptation to changes in intra- and
extracellular conditions is a universal prerequisite for
an organism’s survival and evolution. The existence of
molecular mechanisms of response, repair and adapta-
tion, many of which are greatly conserved across nat-
ure, endows the cell with the plasticity it requires to
adjust to its ever-changing environment, a homeostatic
event that is termed the stress response. Through the
sensing and transduction of the stress signal into the
nucleus, a genetic reprogramming occurs that leads, on
the one hand, to a decrease in the expression of house-
keeping genes and protein synthesis and, on the other
hand, to an enhancement of the expression of genes
encoding stress proteins. These include molecular
chaperones responsible for maintaining protein folding,
transcription factors that further modulate gene
expression and a diverse network of players including
membrane transporters and proteins involved in repair
and detoxification pathways, nutrient metabolism, and
osmolyte production, to name a few. Survival and
growth resumption imply successful cellular adaptation
to the new conditions as well as the repair of damage
incurred to the cell. Although specific stress conditions
elicit distinct cellular responses, underlying gene

Keywords
Saccharomyces cerevisae; stress response;
Yap
Correspondence
C. Rodrigues-Pousada, Genomics and
Stress Laboratory, Instituto de Tecnologia
Quimica e Biologica, Avenida da Republica,
EAN, Apt127, 2781-901 Oeiras, Portugal
Fax: +351 2144 11277
Tel: +351 2144 69624
E-mail:
Website: />Biological_Chemistry/Genomics_and_Stress/
(Received 2 February 2005, revised
22 March 2005, accepted 1 April 2005)
doi:10.1111/j.1742-4658.2005.04695.x
The budding yeast Saccharomyces cerevisiae possesses a very flexible and
complex programme of gene expression when exposed to a plethora of
environmental insults. Therefore, yeast cell homeostasis control is achieved
through a highly coordinated mechanism of transcription regulation invol-
ving several factors, each performing specific functions. Here, we present
our current knowledge of the function of the yeast activator protein family,
formed by eight basic-leucine zipper trans-activators, which have been
shown to play an important role in stress response.
Abbreviations
ACR, arsenic compounds resistance cluster; b-ZIP, basic leucine zipper; CRD, cysteine-rich domain; DBD, DNA-binding domain; ESR,
environmental stress response; HOG, high osmolarity glycerol; HSE, heat shock factor; HSR, heat shock element; MAP, mitogen-activated
protein; NEM, N-ethylmaleimide; NES, nuclear export signal; PC, phytochelatin; PKA, protein kinase A; ROS, reactive oxygen species; STRE,
stress responsive element; Ybp1, Yap 1 binding protein; YCF1, yeast cadmium factor gene.
FEBS Journal 272 (2005) 2639–2647 ª 2005 FEBS 2639
expression programmes common to all environmental

stress responses are at play [1]. Specific forms of stress
such as heat shock and some forms of oxidative stress
[2] demand the activation of the heat shock factor
(HSF), a modular protein consisting of a helix–turn–
helix class of DNA-binding domain (DBD), a leucine
zipper domain, required for trimerization, and a C-ter-
minal transcription activation domain [3]. Both the
HSFs of Saccharomyces cerevisiae and that of the clo-
sely related yeast Kluveromyces lactis contain a unique
transcription activation domain N-terminal to the
DBD [4,5]. The HSF is a pre-existing transcription
activator that binds to an array of a 5-bp heat shock
element (HSE; nGAAn) present upstream of all heat-
shock genes [3]. It has recently been shown that HSF
targets are activated not only upon heat shock, but
also by diamide, nitrogen depletion and stationary-
phase transition. However, this does not reflect a
general stress response because there is no significant
target gene induction upon treatment with hydrogen
peroxide [6]. HSF-independent mechanisms also exist,
namely the environmental stress response (ESR), lar-
gely mediated by the two transcription factors Msn2
and Msn4 [7]. Here, we review the response of the
budding yeast S. cerevisiae to several different forms
of stress highlighting, in particular, those in which the
Yap family of basic-leucine zipper (b-ZIP) transactiva-
tors play a role.
The Yap protein family
The Yap family of b-ZIP proteins comprises eight
members with a significant sequence similarity to the

true yeast AP-1 factor Gcn4 at the DNA-binding
domain [8]. However, in addition and common to all
family members, are several key residues that impart
distinct binding properties to these transcription fac-
tors. It has been determined that Yap1 through to
Yap5 preferentially bind to the consensus site
TTAG ⁄ CTAA, which differs from the true AP-1
recognition element bound by Gcn4 (TGAG ⁄ CTCA).
It is unclear how many YAP sites are required for tar-
get gene regulation. Work performed by Cohen et al.
[9] indicates that gene clusters enriched for Yap1- and
Yap2-depedent genes have, on average, 1.9 (P-value
8.0 · 10
)4
) and 1.8 (P-value 2.0 · 10
)3
) consensus Yap
sites, respectively. We cannot, however, exclude the
possibility that flanking bases around this core consen-
sus are also required. In the case of Yap8, it has been
shown that this protein binds the sequence TTAATAA
on target gene promoters [10] (and our own results).
The Yap family has been found to be implicated in a
variety of stress responses including oxidative, osmotic,
arsenic, drug and heat stress, among others [11].
Although much is currently understood about Yap1,
the major regulator of the oxidative stress response,
comparatively less is known about the remaining
family members.
Oxidative stress

The response to oxidative stress can be described as the
phenomenon by which the cell responds to alterations in
its redox state. As a consequence of aerobic growth, cells
are continuously exposed to reactive oxygen species
(ROS), potent oxidants capable of extensive cellular
damage at the level of DNA, protein and membrane
lipid content. As a result, organisms, from bacteria to
humans, have developed mechanisms of maintaining
cellular thiol redox homeostasis. This is achieved by lim-
iting the accumulation of O
2
-derived oxidants, control-
ling iron and copper metabolism, the activation of thiol
redox pathways and via damage repair [12].
Yap1 was initially characterized through the obser-
vation that the deletion mutant is hypersensitive to the
oxidants H
2
O
2
and t-BOOH, and to chemicals that
generate superoxide anions, including menadione,
plumbagine and methylviologen as well as to cad-
mium, methylglyoxal and cycloheximide. Recent gen-
ome-wide studies have focused on modulation of the
gene expression programmes that occur following
exposure to an oxidative insult in S. cerevisiae. Indeed,
Gasch et al. [7] and Causton et al. [13] have demon-
strated that the response to mild doses of H
2

O
2
leads
to the immediate and transient modulation of  24%
of the genome. Although approximately half of this
response can be attributed to the ESR, there is an
H
2
O
2
-specific response comprising genes encoding
most cellular antioxidants and components of thiol
redox pathways, heat shock proteins, drug transporters
and enzymes involved in carbohydrate metabolism.
Among these genes are TRX2 [14] and GSH1 [15], two
of the first Yap1 targets to be characterized and
induced under oxidative stress imposed by H
2
O
2
, di-
amide and t-BOOH. Since then, several Yap1 targets
involved in ROS detoxification have been identified,
including those involved in the thioredoxin and gluta-
thione systems, and other antioxidants such as catalase
and superoxide dismutase, among others. Yap1 is
therefore central to the adaptive response to oxidative
stress, regulating not only the response to H
2
O

2
-
induced stress, but also that to chemical oxidants
(redox cycling chemicals, thiol oxidants and alkylating
agents), cadmium and drug stress. Purified as a
90 kDa protein [16], Yap1 has a basal expression and,
in unstressed cells, shifts to and from the cytoplasm
Yap proteins and the yeast response to stress C. Rodrigues-Pousada et al.
2640 FEBS Journal 272 (2005) 2639–2647 ª 2005 FEBS
via interaction of the Crm1 nuclear exportin with the
Yap1 nuclear export signal (NES) [17,18]. Although
YAP1 mRNA basal levels are enhanced upon exposure
to an oxidative stimulus, the control of Yap1 activity
is primarily regulated through subcellular localization.
Indeed, Kuge et al. [14] demonstrated that Yap1 nuc-
lear retention is mediated by the cysteine-rich domain
(CRD) located at the C-terminus of the protein which
contains two cysteine-rich regions designated as the
n-CRD (C303, C310 and C315) and c-CRD (C598,
C620 and C629) (Fig. 1A). In response to diamide, the
c-CRD is sufficient to mediate a response. However, in
the case of H
2
O
2
, both n- and c-CRD regions are
required [17,19]. How does Yap1 sense oxidative
stress? It has been shown that the oxidant receptor
peroxidase Orp1 (also designated Hyr1 and Gpx3), is
the main signal sensor and that a third component of

this signal relay, Yap1 binding protein (Ybp1) is asso-
ciated with Yap1 [20]. Orp1 carries a conserved peroxi-
dase-active site cysteine residue (Cys36) of the Gpx
family, whose catalytic cycle is first oxidized to a sulf-
enic acid (Cys-SOH), and then reduced by GSH [18].
Orp1, however, contributes towards H
2
O
2
resistance
not as a peroxidase, but as a sensor of oxidative
stress. Orp1 activates Yap1 by forming an intermole-
cular disulfide bond between its Cys36 and the
Yap1 Cys598, which is then converted into the
Yap1 intramolecular Cys303-Cys598 disulfide bond
(Fig. 1B). Veal et al. [20] have shown that Ybp1 is
required for the signal transduction from Orp1 to
Yap1 because in its absence the intermolecular disul-
fide bond does not form. It has been suggested that
Ybp1 could act as chaperoning the formation of disul-
fide bonds through the guiding of Orp1 Cys36SOH to
Yap1 Cys598, and ⁄ or preventing the formation of the
competing Orp1 Cys36-Cys82 disulfide bond. Once
activated, the Yap1 NES that lies within the c-CRD is
masked leading to its retention in the nucleus and the
up-regulation of target genes. Ybp2 ⁄ Ybh1, a protein
homologous to Ybp1, was found in the genome of
S. cerevisiae and described as having an effect on
H
2

O
2
tolerance, through different mechanisms [21].
However, these data should be regarded with caution
because most of the conclusions are derived from
indirect results. These sensing mechanisms appear con-
served in Schizosaccharomyces pombe in which a two-
cysteine-based peroxidase functions in a similar way to
Orp1 in the activation of Pap1, the Yap1 orthologue
[22]. In addition, a second Yap1 redox centre involved
in the direct binding of N-ethylmaleimide (NEM), the
quinone menadione, both an electrophile and super-
oxide anion generator, was shown to operate. Under
conditions favouring superoxide anion generation, Yap1
is activated by H
2
O
2
formed by the dismutation of the
A
B
Fig. 1. (A) Comparison of the CRD of Yap1,
Yap2 and Yap8, NES is underlined. (B) The
two Yap1 redox centres. Under nonoxidizing
conditions, Yap1 is cytoplasmic owing to
Crm1-dependent nuclear export. Upon H
2
O
2
exposure, the formation of an intermolecular

bond occurs between the Orp1 Cys36 and
the Cys598 of Yap1 leading to its activation.
The subsequent formation of the Yap1
Cys303-Cys598 disulfide bond masks the
NES retaining it in the nucleus where it
activates target genes. Under thiol-reactive
agents, and possibly the metalloids, a
second redox centre operates involving the
Cys598, Cys620 and Cys629 of Yap1, to
which the drug binds directly.
C. Rodrigues-Pousada et al. Yap proteins and the yeast response to stress
FEBS Journal 272 (2005) 2639–2647 ª 2005 FEBS 2641
superoxide. In contrast, menadione acts as an electro-
phile in the absence of oxygen and in this case binds
directly to the c-CRD Cys598, Cys620 and Cys629 in a
manner independent of the Orp1 pathway [23].
Metalloid and metal stress
The widespread distribution of the toxic metalloid
arsenic in nature leads to the acquisition of its resist-
ance in almost all living organisms [24,25]. In S. cere-
visiae, resistance to arsenic is achieved through the
activation of the arsenic compounds-resistance (ACR)
cluster [26], which is composed by the positive regula-
tor Acr1 (Yap8), the arsenate-reductase Acr2 and the
plasma membrane arsenite efflux protein Acr3 [27].
The yeast cadmium factor (YCF1) gene encodes an
independent detoxification system that operates by
sequestering As(GS)
3
into the vacuole [28–30]. Induc-

tion of the expression of ACR2, ACR3 and also YCF1
by the transcription factor Yap8 is essential to arsenic
stress response. Like Yap1, Yap8 is constitutively
expressed, and under physiological conditions shuttles
to and from the nucleus [31]. This is in contrast to the
results obtained by Wysocki et al. [10] and may be due
to the fact that the latter use a multicopy vector,
whereas the former look at the green fluorescent pro-
tein construct within a normal chromosomal context.
Under arsenic stress conditions, Yap8 is activated at
the level of its transactivation potential as well as its
nuclear accumulation, which is triggered by the loss of
interaction with Crm1 [31]. Yap8 cysteine residues
Cys132, Cys137 and Cys274 are essential to both pro-
cesses (Fig. 1A). Work by Haugen et al. [32] on the
integration of phenotypic and expression profiles
involved in arsenic response has revealed the array of
genes whose transcription is enriched, including those
involved in methionine metabolism and sulfur assimil-
ation, protein degradation and transcriptional regula-
tion, and by proteins that form a stress response
network, including Fhl1, Msn2 Msn4, Yap1, Cad1
(Yap2), Hsf1 and Rpn4 among others. Furthermore,
results obtained in microarray analyses point towards
the existence of further Yap8-mediated arsenic detoxifi-
cation pathways (C Amaral, F Devause, R Menezes,
C Facq & C Rodrigues-Pousada, unpublished observa-
tions), highlighting the relevance of multiple mecha-
nisms of arsenic management. A distinct detoxification
strategy employed by S. pombe, nematodes and plants

makes use of phytochelatins (PCs) for metalloid che-
lation. The observation that overexpression of the
S. cerevisiae ACR3-encoded arsenite transporter not
only complements the lack of phytochelatins in
S. pombe, but also confers hyper-resistance to arsenic
compounds to the levels observed in the budding yeast
and prokaryotes [33] further accentuates the effective-
ness of this pathway in arsenic detoxification. Yap1
activation by arsenic compounds is similar to its acti-
vation by thiol-reactive chemicals [23] because it is
unaffected by the absence of the sensor Orp1 ⁄ Gpx3
and does not depend on the n-CRD cysteines
(Fig. 1B). In contrast to Yap8, under arsenic stress
conditions YAP1 basal expression is slightly enhanced
and the presence of this metalloid does not signifi-
cantly modulate Yap1 transactivation function [11].
Heavy metals including copper, zinc, iron and man-
ganese play an important role in cellular biochemistry
and physiology [34]. However, when the concentration
of these metals is elevated, toxicity arises for the organ-
isms. Although cadmium and mercury are not essential
metals they cause severe damage even in low amounts.
Organisms therefore possess cellular detoxification
mechanisms that maintain homeostasis through the con-
trol of intracellular ion levels. One of these involves the
activation of Yap1 and Yap2 (Cad1) [9,35,36]. Yap2
overexpression confers resistance to a plethora of stress
agents such as cadmium, cerulenin and 1,10-phenanthro-
line among others, suggesting a role in the response to
drug stress. Indeed, several target genes encoding a set of

proteins involved in the stabilization and folding of pro-
teins in an oxidative environment have been identified by
microarray analyses [9]. Induced upon exposure to cad-
mium stress [8], Yap2 re-localizes to the nucleus via a
Crm1-dependent mechanism, where it activates the tran-
scription of its target gene FRM2, encoding a protein
homologous to nitroreductase, whose precise role in the
metal stress response remains unclear. The strong
sequence homology between Yap2 and Yap1 in the
C-terminal CRD (residues 570–650 in Yap1 and 330–409
in Yap2) was used to further provide an insight into the
function Yap2. Domain swapping of the Yap1 c-CRD
by that of Yap2 has shown that the fusion protein is
regulated by cadmium but not by H
2
O
2
(D Azevedo
& C Rodrigues-Pousada, unpublished data). Nuclear
localization of the fusion protein correlates not only with
activation of FRM2 transcription, but also with growth
in increasing concentrations of cadmium but not of
H
2
O
2
. Because of the high degree of homology to Yap1,
the role of the Yap2 cysteine residues may prove relevant
for its activation. Furthermore, it has been shown that
Yap2 interacts with the cytoplasmic kinase Rck1 under

conditions of oxidative stress [37], although the nature
and relevance of this interaction remain elusive. Given
that overexpression phenotypes do not necessarily reflect
a true biological function and that no phenotype has yet
been associated to the yap2 mutant, the precise role for
Yap2 remains to be deciphered.
Yap proteins and the yeast response to stress C. Rodrigues-Pousada et al.
2642 FEBS Journal 272 (2005) 2639–2647 ª 2005 FEBS
Osmotic stress
Hyperosmotic stress leads to the passive efflux of water
from the cell to the exterior, resulting in a decrease in
cell volume, loss of cell turgor pressure and increased
concentration of cellular solutes. Conversely, an aque-
ous hypo-osmolar environment allows the movement
of water into the cell, leading to cell swelling, high tur-
gor pressure and diluted intracellular milieu [38,39].
To counteract these effects, the cell makes use of
osmolytes, small compatible solutes such as the sugar
alcohol glycerol and trehalose, which, via active accu-
mulation or extrusion, protect the cell against the
effects of an osmotic challenge by altering the intracel-
lular osmotic pressure [40,41]. Many of the changes to
gene expression upon an osmotic challenge, therefore,
are dedicated to altering the metabolism and cell per-
meability to these compounds.
Upon an upshift in extracellular osmolarity, the high
osmolarity glycerol (HOG) mitogen-activated protein
(MAP) kinase pathway is activated via the action of
two membrane-bound receptors, Sho1 and Sln1 that
form two independent signal input branches conver-

ging on the MAP kinase kinase (MAPKK) Pbs2. The
increased sensitivity of the Sln1 branch, as well as its
graded response [42], disregards the notion of pathway
input redundancy suggesting a capacity of the cell for
finely sensing and adjusting to external changes. Upon
Pbs2 phosphorylation, the Hog1 kinase is activated
through dual Thr ⁄ Tyr phosphorylation, promoting its
rapid translocation into the nucleus and increasing its
kinase activity [43]. Hog1 nuclear residence is regulated
by the two tyrosine phosphatases Ptp2 and Ptp3 [44],
three phosphoserine ⁄ threonine phosphatases Ptc1–3
and by several of the transcription factors it interacts
with, namely, Msn2 ⁄ 4 [45], Hot1 and Msn1 [46], which
subsequently mediate signal amplification via the tran-
sient modulation of global gene expression, often with
overlapping functions [47]. In all, the expression of
 10% of the yeast the genome is affected by an osmo-
tic upshift. This includes most of the genes typically
induced by the ESR, many of which show Hog1-
dependent gene expression [47] and a reduced number
of osmo-specific gene responses, comprising genes of
unknown function. Altogether, Hog1 has been shown
to regulate not only genes required for the immediate
response to increased osmolarity, but also for the res-
toration of gene expression upon osmo-adaptation,
controlling the extent of gene expression as well as its
duration [48]. Recently, Hog1 has been found to be
located at several gene target promoters through
association with the transcription factors it interacts
with [49,50]. Deletion of the HOG1 gene gives rise to a

severe cellular sensitivity to increased external osmo-
tica [51], whereas HOG1 pathway hyperactivation is
lethal [42,52] highlighting not only the importance of
this pathway to the yeast response to increased osmo-
larity, but also the absolute requirement for cellular
mechanisms that accurately measure and grade the
response without compromising cell viability.
Msn2 and Msn4 are two zinc-finger transcription
factors initially described as mediators of the yeast
general stress response [53] because of their capacity to
jointly modulate the expression of a large battery of
unrelated genes in response to a shift to suboptimal
growth conditions. Regulation is mediated by the bind-
ing of these factors to the stress response element
(STRE) (C4T) [54,55] present on the promoter of tar-
get genes. Cytosolic, under normal growth conditions,
Msn2 ⁄ 4 rapidly accumulate in the nucleus under stress
conditions in a manner that can be inversely correlated
to protein kinase A (PKA) activity [56,57]. The Msn5
exportin contributes towards Msn2 nuclear retention
through recognition of its phosphorylation state [58].
Furthermore, work by Bose et al. [59] revealed that
the initial burst of stress-induced STRE-driven gene
expression is quickly converted into the observed tran-
sient response through Msn2 nuclear-dependent degra-
dation and target gene transcriptional repression by
Srb10 kinase, a member of the mediator complex. The
magnitude of target gene induction varies greatly from
gene to gene, primarily due to promoter context,
whereby STRE-driven regulation can be jointly modu-

lated by other transcription factors including Yap1
and Hot1 [7,47]. Induction of the Msn2 ⁄ 4 target genes
in response to one form of stress gives rise to the phe-
nomenon of cross-protection against an aggravated
form of the same stress or to a different type of envi-
ronmental insult altogether.
That Msn2 ⁄ 4 form a downstream branch of the
HOG MAP kinase pathway under conditions of hyper-
osmolarity can be inferred from the fact that, although
many Hog1-dependent genes do not show Msn2 ⁄ 4
dependence, virtually all genes affected by the absence
of these factors are also affected by the deletion of
HOG1 [47]. Indeed, it has been shown that YAP4,
induced under hyperosmotic stress, is regulated by
Msn2 in a Hog1-dependent way via STRE located
within the upstream promoter region (Fig. 2) [60]. The
observation that, under these conditions, YAP4 is not
regulated by Msn4, further supports growing evidence
that the two zinc-finger transcription factors are not
entirely redundant in function [59]. Yap4 and Yap6
are constitutively located in the nucleus [61] and are
the Yap family members that share the greatest simi-
larity at the protein level with almost 33% identity
C. Rodrigues-Pousada et al. Yap proteins and the yeast response to stress
FEBS Journal 272 (2005) 2639–2647 ª 2005 FEBS 2643
between them [11]. Although no significant sensitivity
can be observed in the yap6 null mutant, the yap4-
deleted strain displays impaired growth at moderate
concentrations of hyperosmolarity [60]. Furthermore,
YAP4 overexpression can significantly relieve the

severe hog1 salt-sensitive phenotype, indicative of a
role in the yeast response to increased osmolarity.
Although its precise function remains unclear, micro-
array analyses revealed that this transcription factor
contributes towards the regulation of several osmo-
induced genes. Of significance, genes involved in
glycerol metabolism, GCY1 encoding a putative
glycerol dehydrogenase and GPP2, encoding a NAD-
dependent glycerol-3-phosphate phosphatase, show
decreased expression in the YAP4-deleted strain
(Fig. 2). Crucial to osmo-tolerance, glycerol metabo-
lism and accumulation form a relevant part of the
yeast response to hyperosmolarity [39]. Indeed, a heat-
shock-stimulated increase in the level of intracellular
glycerol is sufficient to completely abolish hog1 sensi-
tivity to hyperosmotic stress [62]. HXT5, encoding a
hexose transporter also partially regulated by Yap4,
shows a further decrease in gene expression in the dou-
ble yap4yap6 mutant strain, suggesting cooperation
between these two transcription factors in mediating
the stress response. This is further substantiated by
computational interactome data that predict their
interaction [63]. Interestingly, the observation that
Yap4 and Yap6 are induced by a variety of unrelated
forms of environmental stress [11,64] has hinted
towards a more fundamentally universal role for Yap4
and Yap6 in the yeast response to stress which is in
contrast to what is currently understood for the
remaining family members.
Perspectives

A particularity of the yeast S. cerevisiae is that is pos-
sesses an extended family of Yap transcription factors.
S. pombe Pap1 shares a high degree of similarity to
Yap1. However, multiple environmental insults in
S. pombe activate the Sty1-mediated MAP kinase path-
way, itself strongly homologous to Hog1 and to mam-
malian p38, making this pathway more analogous to
higher eukaryotes [39,65,66]. Although Yap1 and
Yap8 orthologues exist in the genomes of several other
Saccharomyces species [67], the remaining Yap mem-
bers appear to be exclusive to this microorganism,
hinting towards the possibility that this extended fam-
ily arose through gene duplication events to fulfil a
wider genetic programme required for its environmen-
tal adaptation. Experimental data support both a func-
tional overlap as well as distinct biological roles for
this protein family [11] endowing S. cerevisiae with an
added flexibility with regards to sensing and grading
its stress response. Furthermore, data are beginning to
emerge on the cross-talk between several members of
this family. In particular, the double mutant yap1yap2
is more sensitive to cadmium as well as the double
mutant yap1yap8 to metalloid than either single
mutant, respectively. Indeed, several studies are emer-
ging with data supporting the condition-specific
cooperation between distinct sets of transcriptional
modulators in target gene regulation [32,68]. As was
recently shown by studies using benomyl [69], it is
possible that other chemical stresses also affect the
early expression of genes dependent on different tran-

scription factors. Also, it cannot be neglected that cells
respond to different stresses, for instance, those produ-
cing different oxygen species [70] using distinct mecha-
nisms, pointing to the selective use of different
transcription factors, different combinations or differ-
ent mechanisms of their activation. The fact that
YAP4 is responsive to a plethora of environmental
insults, allied to the richness of cis-elements in its pro-
moter, suggests an important role in response to stress.
Given that Yap4 overexpression gives resistance to cis-
platin, a chemotherapeutic drug that binds the TATA
box [71], it is plausible to hypothesize that Yap4 may
Fig. 2. Yap4 is under the HOG pathway. Upon exposure to osmotic
stress the nuclear accumulation of Hog1 activates downstream
transcription factors. YAP4 is activated via Msn2 and subsequently
the encoded factor elicits the transcription of its target genes.
Yap proteins and the yeast response to stress C. Rodrigues-Pousada et al.
2644 FEBS Journal 272 (2005) 2639–2647 ª 2005 FEBS
play a role in the realm of the basic transcriptional
machinery. The construction of a strain deleted for all
Yaps in well-defined background may prove an invalu-
able tool for the functional study of each family mem-
ber. Indeed, it is being shown that the various strains
not only have different sensitivities to the stress
imposed, but also that significant differences occur at
the level of gene regulation.
Acknowledgements
This study was supported by grants from Fundac¸ a
˜
o

para a Cieˆ ncia e Tecnologia (FCT) to CR-P (POCTI ⁄
BME34967 ⁄ 99), fellowships to RAM (SFR ⁄ BPD ⁄
11438 ⁄ 2002) and to TN (SSRH ⁄ BD ⁄ 1162 ⁄ 2000).
References
1 Estruch F (2000) Stress-controlled transcription factors,
stress-induced genes and stress tolerance in budding
yeast. FEMS Microbiol Rev 24, 469–486.
2 Grably MR, Stanhill A, Tell O & Engelberg D (2002)
HSF and Msn2 ⁄ 4p can exclusively or cooperatively acti-
vate the yeast HSP104 gene. Mol Microbiol 44, 21–35.
3 Trott A & Morano KA (2003) The yeast response to
heat shock. In Yeast Stress Responses (Hohmann S &
Mager PWH, eds), pp. 71–119. Springer-Verlag, Heidle-
berg.
4 Nieto-Sotelo J, Wiederrecht G, Okuda A & Parker CS
(1990) The yeast heat shock transcription factor contains
a transcriptional activation domain whose activity is
repressed under nonshock conditions. Cell 62, 807–817.
5 Sorger PK & Pelham HR (1988) Yeast heat shock factor
is an essential DNA-binding protein that exhibits tem-
perature-dependent phosphorylation. Cell 54, 855–864.
6 Hahn JS, Hu Z, Thiele DJ & Iyer VR (2004) Genome-
wide analysis of the biology of stress responses through
heat shock transcription factor. Mol Cell Biol 24, 5249–
5256.
7 Gasch AP, Spellman PT, Kao CM, Carmel-Harel O,
Eisen MB, Storz G, Botstein D & Brown PO (2000)
Genomic expression programs in the response of yeast
cells to environmental changes. Mol Biol Cell 11, 4241–
4257.

8 Fernandes L, Rodrigues-Pousada C & Struhl K (1997)
Yap, a novel family of eight bZIP proteins in Saccharo-
myces cerevisiae with distinct biological functions. Mol
Cell Biol 17, 6982–6993.
9 Cohen BA, Pilpel Y, Mitra RD & Church GM (2002)
Discrimination between paralogs using microarray ana-
lysis: application to the Yap1p and Yap2p transcrip-
tional networks. Mol Biol Cell 13, 1608–1614.
10 Wysocki R, Fortier PK, Maciaszczyk E, Thorsen M,
Leduc A, Odhagen A, Owsianik G, Ulaszewski S,
Ramotar D & Tamas MJ (2004) Transcriptional activa-
tion of metalloid tolerance genes in Saccharomyces cere-
visiae requires the AP-1-like proteins Yap1p and Yap8p.
Mol Biol Cell 15, 2049–2060.
11 Rodrigues-Pousada CA, Nevitt T, Menezes R, Azevedo
D, Pereira J & Amaral C (2004) Yeast activator pro-
teins and stress response: an overview. FEBS Lett 567,
80–85.
12 Toledano M, Delaunay A, Biteau B, Spector D &
Azevedo D (2003) Oxidative stress responses in yeast. In
Yeast Stress Responses (Hohmann S & Mager PWH,
eds), pp. 241–303. Springer-Verlag, Heidleberg.
13 Causton HC, Ren B, Koh SS, Harbison CT, Kanin E,
Jennings EG, Lee TI, True HL, Lander ES & Young
RA (2001) Remodeling of yeast genome expression in
response to environmental changes. Mol Biol Cell 12,
323–337.
14 Kuge S, Jones N & Nomoto A (1997) Regulation of
yAP-1 nuclear localization in response to oxidative
stress. EMBO J 16, 1710–1720.

15 Wu AL & Moye-Rowley WS (1994) GSH1, which
encodes gamma-glutamylcysteine synthetase, is a target
gene for yAP-1 transcriptional regulation. Mol Cell Biol
14, 5832–5839.
16 Harshman KD, Moye-Rowley WS & Parker CS (1988)
Transcriptional activation by the SV40 AP-1 recognition
element in yeast is mediated by a factor similar to AP-1
that is distinct from GCN4. Cell 53, 321–330.
17 Delaunay A, Isnard AD & Toledano MB (2000) H
2
O
2
sensing through oxidation of the Yap1 transcription fac-
tor. EMBO J 19, 5157–5166.
18 Delaunay A, Pflieger D, Barrault MB, Vinh J & Tole-
dano MB (2002) A thiol peroxidase is an H
2
O
2
receptor
and redox-transducer in gene activation. Cell 111, 471–
481.
19 Kuge S, Arita M, Murayama A, Maeta K, Izawa S,
Inoue Y & Nomoto A (2001) Regulation of the yeast
Yap1p nuclear export signal is mediated by redox sig-
nal-induced reversible disulfide bond formation. Mol
Cell Biol 21, 6139–6150.
20 Veal EA, Ross SJ, Malakasi P, Peacock E & Morgan
BA (2003) Ybp1 is required for the hydrogen peroxide-
induced oxidation of the Yap1 transcription factor.

J Biol Chem 278, 30896–30904.
21 Gulshan K, Rovinsky SA & Moye-Rowley WS (2004)
YBP1 and its homologue YBP2 ⁄ YBH1 influence oxida-
tive-stress tolerance by nonidentical mechanisms in Sac-
charomyces cerevisiae. Eukaryot Cell 3, 318–330.
22 Toledano MB, Delaunay A, Monceau L & Tacnet F
(2004) Microbial H
2
O
2
sensors as archetypical redox
signaling modules. Trends Biochem Sci. 29, 351–357.
23 Azevedo D, Tacnet F, Delaunay A, Rodrigues-Pousada
C & Toledano MB (2003) Two redox centers within
Yap1 for H
2
O
2
and thiol-reactive chemicals signaling.
Free Radical Biol Med 35, 889–900.
C. Rodrigues-Pousada et al. Yap proteins and the yeast response to stress
FEBS Journal 272 (2005) 2639–2647 ª 2005 FEBS 2645
24 Liu J & Rosen BP (1997) Ligand interactions of the
ArsC arsenate reductase. J Biol Chem 272, 21084–
21089.
25 Rosen BP (1999) Families of arsenic transporters.
Trends Microbiol 7, 207–212.
26 Bobrowicz P, Wysocki R, Owsianik G, Goffeau A &
Ulaszewski S (1997) Isolation of three contiguous genes,
ACR1, ACR2 and ACR3 involved in resistance to

arsenic compounds in the yeast Saccharomyces cerevi-
siae. Yeast 13, 819–823.
27 Wysocki R, Bobrowicz P & Ulaszewski S (1997) The
Saccharomyces cerevisiae ACR3 gene encodes a putative
membrane protein involved in arsenite transport. J Biol
Chem 272, 30061–30066.
28 Szczypka MS, Wemmie JA, Moye-Rowley WS & Thiele
DJ (1994) A yeast metal resistance protein similar to
human cystic fibrosis transmembrane conductance regu-
lator (CFTR) and multidrug resistance-associated pro-
tein. J Biol Chem 269, 22853–22857.
29 Li ZS, Szczypka M, Lu YP, Thile DJ & Rea PA (1996)
The yeast cadmiun factor protein (YCF1 ) is a vacuolar
glutathione S-conjugate pump. J Biol Chem 271, 6509–
6517.
30 Ghosh M, Shen J & Rosen BP (1999) Pathway of As
(III) detoxification in Saccharomyces cerevisiae. Proc
Natl Acad Sci USA 96, 5001–5006.
31 Menezes RA, Amaral C, Delaunay A, Toledano M &
Rodrigues-Pousada C (2004) Yap8p activation in Sac-
charomyces cerevisiae under arsenic conditions. FEBS
Lett 566, 141–146.
32 Haugen AC, Kelley R, Collins JB, Tucker CJ, Deng C,
Afshari CA, Brown JM, Ideker T & Van Houten B
(2004) Integrating phenotypic and expression profiles to
map arsenic-response networks. Genome Biol 5, R95.
33 Wysocki R, Clemens S, Augustyniak D, Golik P,
Maciaszczyk E, Tamas MJ & Dziadkowiec D (2003)
Metalloid tolerance based on phytochelatins is not
functionally equivalent to the arsenite transporter

Acr3p. Biochem Biophys Res Commun 304 , 293–300.
34 Shiraishi E, Inouhe M, Joho M & Tohoyama H (2000)
The cadmium-resistant gene, CAD2, which is a mutated
putative copper-transporter gene (PCA1), controls the
intracellular cadmium-level in the yeast S. cerevisiae.
Curr Genet 37, 79–86.
35 Wemmie JA, Szczypka MS, Thiele DJ & Moye-Rowley
WS (1994) Cadmium tolerance mediated by the yeast
AP-1 protein requires the presence of an ATP-binding
cassette transporter-encoding gene, YCF1. J Biol Chem
269, 32592–32597.
36 Vido K, Spector D, Lagniel G, Lopez S, Toledano MB
& Labarre J (2001) A proteome analysis of the cad-
mium response in Saccharomyces cerevisiae. J Biol Chem
276, 8469–8474.
37 Bilsland E, Molin C, Swaminathan S, Ramne A & Sun-
nerhagen P (2004) Rck1 and Rck2 MAPKAP kinases
and the HOG pathway are required for oxidative stress
resistance. Mol Microbiol 53, 1743–1756.
38 Blomberg A & Adler L (1992) Physiology of osmotoler-
ance in fungi. Adv Microb Physiol 33, 145–212.
39 Hohmann S (2002) Osmotic stress signaling and osmo-
adaptation in yeasts. Microbiol Mol Biol Rev 66,
300–372.
40 Yancey PH, Clark ME, Hand SC, Bowlus RD & Som-
ero GN (1982) Living with water stress: evolution of
osmolyte systems. Science 217, 1214–1222.
41 Reed RH, Chudek JA, Foster R & Gadd GM (1987)
Osmotic significance of glycerol accumulation in expo-
nentially growing yeasts. Appl Environ Microbiol 53,

2119–2123.
42 Maeda T, Takekawa M & Saito H (1995) Activation of
yeast PBS2 MAPKK by MAPKKKs or by binding of
an SH3-containing osmosensor. Science 269, 554–558.
43 Proft M, Pascual-Ahuir A, de Nadal E, Arino J, Serr-
ano R & Posas F (2001) Regulation of the Sko1 tran-
scriptional repressor by the Hog1 MAP kinase in
response to osmotic stress. EMBO J 20, 1123–1133.
44 Mattison CP & Ota IM (2000) Two protein tyrosine
phosphatases, Ptp2 and Ptp3, modulate the subcellular
localization of the Hog1 MAP kinase in yeast. Genes
Dev 14, 1229–1235.
45 Reiser V, Ruis H & Ammerer G (1999) Kinase activity-
dependent nuclear export opposes stress-induced nuclear
accumulation and retention of Hog1 mitogen-activated
protein kinase in the budding yeast Saccharomyces cere-
visiae. Mol Biol Cell 10, 1147–1161.
46 Rep M, Reiser V, Gartner U, Thevelein JM, Hohmann
S, Ammerer G & Ruis H (1999) Osmotic stress-induced
gene expression in Saccharomyces cerevisiae requires
Msn1p and the novel nuclear factor Hot1p. Mol Cell
Biol 19, 5474–5485.
47 Rep M, Krantz M, Thevelein JM & Hohmann S (2000)
The transcriptional response of Saccharomyces cerevisiae
to osmotic shock. Hot1p and Msn2p ⁄ Msn4p are
required for the induction of subsets of high osmolarity
glycerol pathway-dependent genes. J Biol Chem 275,
8290–8300.
48 O’Rourke SM & Herskowitz I (2004) Unique and
redundant roles for HOG MAPK pathway components

as revealed by whole-genome expression analysis. Mol
Biol Cell 15, 532–542.
49 Alepuz PM, Jovanovic A, Reiser V & Ammerer G
(2001) Stress-induced map kinase Hog1 is part of tran-
scription activation complexes. Mol Cell 7, 767–777.
50 Proft M & Struhl K (2002) Hog1 kinase converts the
Sko1–Cyc8–Tup1 repressor complex into an activator
that recruits SAGA and SWI ⁄ SNF in response to osmo-
tic stress. Mol Cell 9, 1307–1317.
51 Siderius M, Van Wuytswinkel O, Reijenga KA, Kelders
M & Mager WH (2000) The control of intracellular gly-
cerol in Saccharomyces cerevisiae influences osmotic
Yap proteins and the yeast response to stress C. Rodrigues-Pousada et al.
2646 FEBS Journal 272 (2005) 2639–2647 ª 2005 FEBS
stress response and resistance to increased temperature.
Mol Microbiol 36, 1381–1390.
52 Maeda T, Tsai AY & Saito H (1993) Mutations in a
protein tyrosine phosphatase gene (PTP2) and a protein
serine ⁄ threonine phosphatase gene (PTC1) cause a syn-
thetic growth defect in Saccharomyces cerevisiae. Mol
Cell Biol 13, 5408–5417.
53 Mager WH & De Kruijff AJ (1995) Stress-induced tran-
scriptional activation. Microbiol Rev 59, 506–531.
54 Martinez-Pastor MT, Marchler G, Schuller C, Marchler-
Bauer A, Ruis H & Estruch F (1996) The Saccharomyces
cerevisiae zinc finger proteins Msn2p and Msn4p are
required for transcriptional induction through the stress
response element (STRE). EMBO J 15, 2227–2235.
55 Schmitt AP & McEntee K (1996) Msn2p, a zinc finger
DNA-binding protein, is the transcriptional activator of

the multistress response in Saccharomyces cerevisiae.
Proc Natl Acad Sci USA 93, 5777–5782.
56 Gorner W, Durchschlag E, Martinez-Pastor MT, Est-
ruch F, Ammerer G, Hamilton B, Ruis H & Schuller C
(1998) Nuclear localization of the C
2
H
2
zinc finger pro-
tein Msn2p is regulated by stress and protein kinase A
activity. Genes Dev 12, 586–597.
57 Kobayashi N & McEntee K (1990) Evidence for a heat
shock transcription factor-independent mechanism for
heat shock induction of transcription in Saccharomyces
cerevisiae. Proc Natl Acad Sci USA 87, 6550–6554.
58 Gorner W, Durchschlag E, Wolf J, Brown EL,
Ammerer G, Ruis H & Schuller C (2002) Acute glucose
starvation activates the nuclear localization signal of a
stress-specific yeast transcription factor. EMBO J 21,
135–144.
59 Bose S, Dutko JA & Zitomer RS (2005) Genetic factors
that regulate the attenuation of the general stress
response of yeast. Genetics 169, 1215–1226.
60 Nevitt T, Pereira J, Azevedo D, Guerreiro P & Rodri-
gues-Pousada C (2004) Expression of YAP4. Saccharo-
myces cerevisiae under osmotic stress. Biochem J 379,
367–374.
61 Furuchi T, Ishikawa H, Miura N, Ishizuka M, Kajiya
K, Kuge S & Naganuma A (2001) Two nuclear pro-
teins, Cin5 and Ydr259c, confer resistance to cisplatin

in Saccharomyces cerevisiae. Mol Pharmacol 59, 470–
474.
62 Wojda I, Alonso-Monge R, Bebelman JP, Mager WH
& Siderius M (2003) Response to high osmotic condi-
tions and elevated temperature in Saccharomyces cerevi-
siae is controlled by intracellular glycerol and involves
coordinate activity of MAP kinase pathways. Microbiol-
ogy 149, 1193–1204.
63 Lee TI, Rinaldi NJ, Robert F, Odom DT, Bar-Joseph Z,
Gerber GK, Hannett NM, Harbison CT, Thompson
CM, Simon I et al. (2002) Transcriptional regulatory net-
works in Saccharomyces cerevisiae. Science 298, 799–804.
64 Nevitt T, Pereira J & Rodrigues-Pousada C (2004)
YAP4 gene expression is induced in response to several
forms of stress in Saccharomyces cerevisiae. Yeast 21,
1365–1374.
65 Millar JB (1999) Stress-activated MAP kinase (mitogen-
activated protein kinase) pathways of budding and fis-
sion yeasts. Biochem Soc Symp 64, 49–62.
66 Degols G & Russell P (1997) Discrete roles of the Spc1
kinase and the Atf1 transcription factor in the UV
response of Schizosaccharomyces pombe . Mol Cell Biol
17, 3356–3363.
67 Maciaszczyk E, Wysocki R, Golik P, Lazowska J &
Ulaszewski S (2004) Arsenical resistance genes in Sac-
charomyces douglasii and other yeast species undergo
rapid evolution involving genomic rearrangements and
duplications. FEMS Yeast Res 4, 821–832.
68 Amoros M & Estruch F (2001) Hsf1p and Msn2 ⁄ 4p
cooperate in the expression of Saccharomyces cerevisiae

genes HSP26 and HSP104 in a gene- and stress type-
dependent manner. Mol Microbiol 39, 1523–1532.
69 Lucau-Danila A, Lelandais G, Kozovska Z, Tanty V,
Delaveau T, Devaux F & Jacq C (2005) Early expres-
sion of yeast genes affected by chemical stress. Mol Cell
Biol 25, 1860–1868.
70 Thorpe GW, Fong CS, Alic N, Higgins VJ & Dawes
IW (2004) Cells have distinct mechanisms to maintain
protection against different reactive oxygen species: oxi-
dative-stress-response genes. Proc Natl Acad Sci USA
101, 6564–6569.
71 Vichi P, Coin F, Renaud JP, Vermeulen W, Hoeijmak-
ers JH, Moras D & Egly JM (1997) Cisplatin- and
UV-damaged DNA lure the basal transcription factor
TFIID ⁄ TBP. EMBO J 16, 7444–7456.
C. Rodrigues-Pousada et al. Yap proteins and the yeast response to stress
FEBS Journal 272 (2005) 2639–2647 ª 2005 FEBS 2647