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: claudina@itqb.unl.pt Website: http://www.itqb.unl.pt/Research/ 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 . 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,. regulated by Yap4 , shows a further decrease in gene expression in the dou- ble yap4 yap6 mutant strain, suggesting cooperation between these two transcription factors in mediating the stress response. . 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