Adaptation of Saccharomyces cerevisiae cells to arsenic stress is mediated through the activation... more Adaptation of Saccharomyces cerevisiae cells to arsenic stress is mediated through the activation of arsenic detoxification machinery by the Yap8 transcription factor. Yap8 is targeted by the ubiquitin proteasome system for degradation under physiological conditions, yet it escapes proteolysis in arsenic-injured cells by a mechanism that remains to be elucidated. Here, we show that Ufd2, an E4-Ubiquitin (Ub) ligase, is upregulated by arsenic compounds both at mRNA and protein levels. Under these conditions, Ufd2 interacts with Yap8 mediating its stabilization, thereby controlling expression of ACR3 and capacity of cells to adapt to arsenic injury. We also show that Ufd2 U-box domain, which is associated to the ubiquitination activity of specific ubiquitin ligases, is dispensable for Yap8 stability and has no role in cell tolerance to arsenic stress. Thus, our data disclose a novel Ufd2 role beyond degradation. This finding is further supported by genetic analyses showing that protei...
Yap2 is a cadmium responsive transcription factor that interacts with MAPK-activated protein (MAP... more 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.
Gal1p carries out two functions in the galactose pathway of yeast. It activates Gal4p by interact... more Gal1p carries out two functions in the galactose pathway of yeast. It activates Gal4p by interacting with Gal80p--a function that can also served by Gal3p--and it catalyzes the formation of galactose-1-phosphate. Recently, we and others have presented biochemical evidence for complex formation between Gal1p and Gal80p. Here, we extend these data and present genetic evidence for an interaction between Gal1p and Gal80p in vivo, using a two-hybrid assay. Interaction between Gal1p and Gal80p depends on the presence of galactose, but not on the catalytic activity of Gal1p. A new class of Kluyveromyces lactis mutants was isolated, designated Klgal1-m, which have lost the derepressing activity but retain galactokinase activity, indicating that the two Gal1p activities are functionally independent. The KlGal1-m proteins are defective in their ability to interact with Gal80p in a two-hybrid assay. The locations of gall-m mutations identify putative interaction sites in Gal1p and Gal80p. A dominant mutation, KlGAL1-d, leads to a high level of constitutive expression of genes of the galactose pathway. The behavior of chimeric proteins consisting of Gal3p and KlGal1p sequences indicates that both the N-terminal and C-terminal halves of KlGal1p are involved in specific interaction with KlGal80p.
Cobalt has a rare occurrence in nature, but yet may accumulate in cells to toxic levels. In the p... more Cobalt has a rare occurrence in nature, but yet may accumulate in cells to toxic levels. In the present study, we have investigated how the transcription factor Yap1mediates tolerance to cobalt toxicity. Fluorescence microscopy was used to address how cobalt activates Yap1. Using microarray analysis, we compared the transcriptional profile of a strain lacking Yap1 to that of its parental strain. To evaluate the extent of the oxidative damage caused by cobalt, GSH was quantified by HPLC and protein carbonylation levels were assessed. Cobalt activates Yap1 under aerobiosis and anaeobiosis growth conditions. This metal generates a severe oxidative damage in the absence of Yap1. However, when challenged with high concentrations of cobalt, yap1 mutant cells accumulate lower levels of this metal. Accordingly, microarray analysis revealed that the expression of the high affinity phosphate transporter, PHO84, a well-known cobalt transporter, is compromised in the yap1 mutant. Moreover, we s...
Cadmium is a well known mutagenic metal that can enter cells via nonspecific metal transporters, ... more 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.
The budding yeast Saccharomyces cerevisiae possesses a very flexible and complex programme of gen... more 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 involving 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.
Adaptation of Saccharomyces cerevisiae cells to arsenic stress is mediated through the activation... more Adaptation of Saccharomyces cerevisiae cells to arsenic stress is mediated through the activation of arsenic detoxification machinery by the Yap8 transcription factor. Yap8 is targeted by the ubiquitin proteasome system for degradation under physiological conditions, yet it escapes proteolysis in arsenic-injured cells by a mechanism that remains to be elucidated. Here, we show that Ufd2, an E4-Ubiquitin (Ub) ligase, is upregulated by arsenic compounds both at mRNA and protein levels. Under these conditions, Ufd2 interacts with Yap8 mediating its stabilization, thereby controlling expression of ACR3 and capacity of cells to adapt to arsenic injury. We also show that Ufd2 U-box domain, which is associated to the ubiquitination activity of specific ubiquitin ligases, is dispensable for Yap8 stability and has no role in cell tolerance to arsenic stress. Thus, our data disclose a novel Ufd2 role beyond degradation. This finding is further supported by genetic analyses showing that protei...
Yap2 is a cadmium responsive transcription factor that interacts with MAPK-activated protein (MAP... more 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.
Gal1p carries out two functions in the galactose pathway of yeast. It activates Gal4p by interact... more Gal1p carries out two functions in the galactose pathway of yeast. It activates Gal4p by interacting with Gal80p--a function that can also served by Gal3p--and it catalyzes the formation of galactose-1-phosphate. Recently, we and others have presented biochemical evidence for complex formation between Gal1p and Gal80p. Here, we extend these data and present genetic evidence for an interaction between Gal1p and Gal80p in vivo, using a two-hybrid assay. Interaction between Gal1p and Gal80p depends on the presence of galactose, but not on the catalytic activity of Gal1p. A new class of Kluyveromyces lactis mutants was isolated, designated Klgal1-m, which have lost the derepressing activity but retain galactokinase activity, indicating that the two Gal1p activities are functionally independent. The KlGal1-m proteins are defective in their ability to interact with Gal80p in a two-hybrid assay. The locations of gall-m mutations identify putative interaction sites in Gal1p and Gal80p. A dominant mutation, KlGAL1-d, leads to a high level of constitutive expression of genes of the galactose pathway. The behavior of chimeric proteins consisting of Gal3p and KlGal1p sequences indicates that both the N-terminal and C-terminal halves of KlGal1p are involved in specific interaction with KlGal80p.
Cobalt has a rare occurrence in nature, but yet may accumulate in cells to toxic levels. In the p... more Cobalt has a rare occurrence in nature, but yet may accumulate in cells to toxic levels. In the present study, we have investigated how the transcription factor Yap1mediates tolerance to cobalt toxicity. Fluorescence microscopy was used to address how cobalt activates Yap1. Using microarray analysis, we compared the transcriptional profile of a strain lacking Yap1 to that of its parental strain. To evaluate the extent of the oxidative damage caused by cobalt, GSH was quantified by HPLC and protein carbonylation levels were assessed. Cobalt activates Yap1 under aerobiosis and anaeobiosis growth conditions. This metal generates a severe oxidative damage in the absence of Yap1. However, when challenged with high concentrations of cobalt, yap1 mutant cells accumulate lower levels of this metal. Accordingly, microarray analysis revealed that the expression of the high affinity phosphate transporter, PHO84, a well-known cobalt transporter, is compromised in the yap1 mutant. Moreover, we s...
Cadmium is a well known mutagenic metal that can enter cells via nonspecific metal transporters, ... more 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.
The budding yeast Saccharomyces cerevisiae possesses a very flexible and complex programme of gen... more 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 involving 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.
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Papers by Regina Menezes