Catarina Amaral

Yeast activator proteins and stress response: an overview

Yeast, and especially Saccharomyces cerevisiae, are continuously exposed to rapid and drastic changes in their external milieu. Therefore, cells must maintain their homeostasis, which is achieved through a highly coordinated gene expression involving a plethora of transcription factors, each of them performing specific functions. Here, we discuss recent advances in our understanding of the function of the yeast activator protein family of eight basic‐leucine zipper trans‐activators that have been implicated in various forms of stress response.

Contribution of Yap1 towards Saccharomyces cerevisiae adaptation to arsenic-mediated oxidative stress

In the budding yeast Saccharomyces cerevisiae, arsenic detoxification involves the activation of Yap8, a member of the Yap (yeast AP-1-like) family of transcription factors, which in turn regulates ACR2 and ACR3, genes encoding an arsenate reductase and a plasma-membrane arsenite-efflux protein respectively. In addition, Yap1 is involved in the arsenic adaptation process through regulation of the expression of the vacuolar pump encoded by YCF1 (yeast cadmium factor 1 gene) and also contributing to the regulation of ACR genes. Here we show that Yap1 is also involved in the removal of ROS (reactive oxygen species) generated by arsenic compounds. Data on lipid peroxidation and intracellular oxidation indicate that deletion of YAP1 and YAP8 triggers cellular oxidation mediated by inorganic arsenic. In spite of the increased amounts of As(III) absorbed by the yap8 mutant, the enhanced transcriptional activation of the antioxidant genes such as GSH1 (gamma- glutamylcysteine synthetase gene), SOD1 (superoxide dismutase 1 gene) and TRX2 (thioredoxin 2 gene) may prevent protein oxidation. In contrast, the yap1 mutant exhibits high contents of protein carbonyl groups and the GSSG/GSH ratio is severely disturbed on exposure to arsenic compounds in these cells. These results point to an additional level of Yap1 contribution to arsenic stress responses by preventing oxidative damage in cells exposed to these compounds. Transcriptional profiling revealed that genes of the functional categories related to sulphur and methionine metabolism and to the maintenance of cell redox homoeostasis are activated to mediate adaptation of the wild-type strain to 2 mM arsenate treatment.

Yap8p activation in Saccharomyces cerevisiae under arsenic conditions

Yap8p, a member of the Saccharomyces cerevisiae Yap family, is activated in response to arsenic. Both the mechanisms by which this activation takes place and its regulation have not yet been identified. In this report, we show that Yap8p is not activated at the transcriptional level but, rather, its nuclear transport is actively regulated and dependent on the exportin chromosome region maintenance protein. In addition, it is shown that Cys132, Cys137and Cys274 are essential for Yap8p localization and transactivation function both of which are required for its biological activity.

Repression of the Low Affinity Iron Transporter Gene FET4: a Novel Mechanism Against Cadmium Toxicity Orchestrated by Yap1 via Rox1

Background: Yap1 regulates cadmium accumulation in the vacuole and mitigates cadmium-induced ROS. Results: Yap1 induces the gene of the hypoxic repressor Rox1 that in turn represses FET4, avoiding cadmium uptake. Conclusion: Repression of FET4, via Rox1, is a novel line of defense mediated by Yap1 against cadmium toxicity. Significance: Evidence of cross-talk between oxidative and hypoxic regulators that results in increased tolerance to metal toxicity. Cadmium is a well known mutagenic metal that can enter cells via nonspecific metal transporters, causing several cellular damages and eventually leading to death. In the yeast Saccharomyces cerevisiae, the transcription factor Yap1 plays a key role in the regulation of several genes involved in metal stress response. We have previously shown that Yap1 represses the expression of FET4, a gene encoding a low affinity iron transporter able to transport metals other than iron. Here, we have studied the relevance of this repression in cell tolerance to cadmium. Our results indicate that genomic deletion of Yap1 increases FET4 transcript and protein levels. In addition, the cadmium toxicity exhibited by this strain is completely reversed by co-deletion of FET4 gene. These data correlate well with the increased intracellular levels of cadmium observed in the mutant yap1. Rox1, a well known aerobic repressor of hypoxic genes, conveys the Yap1-mediated repression of FET4. We further show that, in a scenario where the activity of Yap1 or Rox1 is compromised, cells activate post-transcriptional mechanisms, involving the exoribonuclease Xrn1, to compensate the derepression of FET4. Our data thus reveal a novel protection mechanism against cadmium toxicity mediated by Yap1 that relies on the aerobic repression of FET4 and results in the impairment of cadmium uptake.

Yap4 PKA- and GSK3-dependent phosphorylation affects its stability but not its nuclear localization.

Yap4 is a nuclear‐resident transcription factor induced in Saccharomyces cerevisiae when exposed to several stress conditions, which include mild hyperosmotic and oxidative stress, temperature shift or metal exposure. This protein is also phosphorylated. Here we report that this modification is driven by PKA and GSK3. In order to ascertain whether Yap4 is directly or indirectly phosphorylated by PKA, we searched for stress and PKA‐related kinases that could phosphorylate Yap4. We show that phosphorylation is independent of the kinases Rim15, Yak1, Sch9, Slt2, Ste20 and Ptk2. In addition, we showed that Yap4 phosphorylation is also abrogated in the triple GSK3 mutant mck1 rim11 yol128c. Furthermore, our data reveal that Yap4 nuclear localization is independent of its phosphorylation state. This protein has several putative phosphorylation sites, but only the mutation of residues T192 and S196 impairs its phosphorylation under different stress conditions. The ability of the non‐phosphorylated forms of Yap4 to partially rescue the hog1 severe sensitivity phenotype is not affected, suggesting that Yap4 activity is maintained in the absence of phosphorylation. However, this modification seems to be required for stability of the protein, as the non‐phosphorylated form has a shorter half‐life than the phosphorylated one. Copyright

Two Residues in the Basic Region of the Yeast Transcription Factor Yap8 Are Crucial for Its DNA-Binding Specificity

In Saccharomyces cerevisiae, the transcription factor Yap8 is a key determinant in arsenic stress response. Contrary to Yap1, another basic region-leucine zipper (bZIP) yeast regulator, Yap8 has a very restricted DNA-binding specificity and only orchestrates the expression of ACR2 and ACR3 genes. In the DNA-binding basic region, Yap8 has three distinct amino acids residues, Leu26, Ser29 and Asn31, at sites of highly conserved positions in the other Yap family of transcriptional regulators and Pap1 of Schizosaccharomyces pombe. To evaluate whether these residues are relevant to Yap8 specificity, we first built a homology model of the complex Yap8bZIP-DNA based on Pap1-DNA crystal structure. Several Yap8 mutants were then generated in order to confirm the contribution of the residues predicted to interact with DNA. Using bioinformatics analysis together with in vivo and in vitro approaches, we have identified several conserved residues critical for Yap8-DNA binding. Moreover, our data suggest that Leu26 is required for Yap8 binding to DNA and that this residue together with Asn31, hinder Yap1 response element recognition by Yap8, thus narrowing its DNA-binding specificity. Furthermore our results point to a role of these two amino acids in the stability of the Yap8-DNA complex.

Inhibition of Yap2 activity by MAPKAP kinase Rck1 affects yeast tolerance to cadmium

Yap2 is a cadmium responsive transcription factor that interacts with MAPK‐activated protein (MAPKAP) kinase Rck1. We show that Rck1 deletion confers protection against cadmium toxicity and that the mechanism underlying this observation relies on Yap2. Rck1 removal from the yeast genome potentiates Yap2 activity by increasing protein half‐life and delaying its nuclear export. As a consequence, several Yap2 antioxidant targets are over‐activated by a mechanism that also requires Yap1. Several genes of the cell wall integrity (CWI) pathway are upregulated under cadmium stress in a Yap2 dependent way. We showed that deletion of CWI genes renders yeast cells more sensitive to cadmium. These findings led us to suggest that in response to cadmium stress Yap2 may serve a dual purpose: oxidative stress attenuation and cell wall maintenance.

Mediator, SWI/SNF and SAGA complexes regulate Yap8-dependent transcriptional activation of ACR2 in response to arsenate

Response to arsenic stress in Saccharomyces cerevisiae is orchestrated by the regulatory protein Yap8, which mediates transcriptional activation of ACR2 and ACR3. This study contributes to the state of art knowledge of the molecular mechanisms underlying yeast stress response to arsenate as it provides the genetic and biochemical evidences that Yap8, through cysteine residues 132, 137, and 274, is the sensor of presence of arsenate in the cytosol. Moreover, it is here reported for the first time the essential role of the Mediator complex in the transcriptional activation of ACR2 by Yap8. Based on our data, we propose an order-of-function map to recapitulate the sequence of events taking place in cells injured with arsenate. Modification of the sulfhydryl state of these cysteines converts Yap8 in its activated form, triggering the recruitment of the Mediator complex to the ACR2/ACR3 promoter, through the interaction with the tail subunit Med2. The Mediator complex then transfers the regulatory signals conveyed by Yap8 to the core transcriptional machinery, which culminates with TBP occupancy, ACR2 upregulation and cell adaptation to arsenate stress. Additional co-factors are required for the transcriptional activation of ACR2 by Yap8, particularly the nucleosome remodeling activity of SWI/SNF and SAGA complexes.

An HcpR paralog of Desulfovibrio gigas provides protection against nitrosative stress.

Desulfovibrio gigas belongs to the group of sulfate reducing bacteria (SRB). These ubiquitous and metabolically versatile microorganisms are often exposed to reactive nitrogen species (RNS). Nonetheless, the mechanisms and regulatory elements involved in nitrosative stress protection are still poorly understood. The transcription factor HcpR has emerged as a putative regulator of nitrosative stress response among anaerobic bacteria. HcpR is known to orchestrate the expression of the hybrid cluster protein gene,hcp, proposed to be involved in cellular defense against RNS. According to phylogenetic analyses, the occurrence ofhcpR paralog genes is a common feature among severalDesulfovibrio species. Within theD. gigas genome we have identified two HcpR‐related sequences. One of these sequences,hcpR1, was found in the close vicinity of thehcp gene and this finding prompted us to proceed with its functional characterization. We observed that the growth of aD. gigas strain lackinghcpR1 is severely impaired under nitrosative stress. Anin silico search revealed several putative targets of HcpR1 that were experimentally validated. The fact that HcpR1 regulates several genes encoding proteins involved in nitrite and nitrate metabolism, together with the sensitive growth phenotype to NO displayed by anhcpR1 mutant strain, strongly supports a relevant role of this factor under nitrosative stress. Moreover, the finding that severalDesulfovibrio species possess HcpR paralogs, which have been transmitted vertically in the evolution and diversification of the genus, suggests that these sequences may confer adaptive or survival advantage to these organisms, possibly by increasing their tolerance to nitrosative stress.

Ace1 prevents intracellular copper accumulation by regulating Fet3 expression and thereby restricting Aft1 activity

In the yeast Saccharomyces cerevisiae Aft1, the low iron‐sensing transcription factor is known to regulate the expression of the FET3 gene. However, we found that a strain‐lacking FET3 is more sensitive to copper excess than a strain‐lacking AFT1, and accordingly, FET3 expression is not fully compromised in the latter. These findings suggest that, under such conditions, another regulator comes into play and controls FET3 expression. In this work, we identify Ace1, the regulator of copper detoxification genes, as a regulator of FET3. We suggest that the activation of FET3 by Ace1 prevents the hyper activation of Aft1, possibly by assuring the adequate functioning of mitochondrial iron–sulfur cluster biogenesis. While reinforcing the link between iron and copper homeostasis, this work unveils a novel protection mechanism against copper toxicity mediated by Ace1, which relies in the activation of FET3 and results in the restriction of Aft1 activity as a means to prevent excessive copper accumulation.