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1.
Mol Microbiol ; 35(4): 936-48, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10692169

ABSTRACT

Pos9 (Skn7) is an important transcription factor that, together with Yap1, induces the expression of oxidative stress target genes in Saccharomyces cerevisiae. The activation of Pos9 upon an oxidative stress signal occurs post-translationally. In a mutant screen for factors involved in the activation of a Pos9-dependent reporter gene upon oxidative stress, we identified the mutant fap7-1 (for factor activating Pos9). This point mutant failed to activate a Gal4-Pos9 hybrid transcription factor, assayed by hydrogen peroxide-induced GAL1-lacZ reporter gene activities. Additionally, the fap7-1 mutant strain was sensitive to oxidative stress and revealed slow growth on glucose compared with the wild type. The fap7-1 mutation also affected the induction of the Pos9 target gene TPX1 and of a synthetic promoter previously identified to be regulated in a Yap1- and Pos9-dependent manner. This lack of induction was specific as the fap7-1 mutant response to other stresses such as sodium chloride or co-application of both hydrogen peroxide and sodium chloride was not affected, as tested with the Pos9-independent expression pattern of a TPS2-lacZ reporter system. We identified the gene YDL166c to be allelic to the FAP7 gene and to be essential. Fluorescence microscopy of Fap7-GFP fusion proteins indicated a nuclear localization of the Fap7 protein. Our data suggest that Fap7 is a nuclear factor important for Pos9-dependent target gene transcription upon oxidative stress.


Subject(s)
Fungal Proteins/physiology , Nuclear Proteins/physiology , Oxidative Stress/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Adenylate Kinase , Amino Acid Sequence , Cell Nucleus/chemistry , DNA, Fungal/chemistry , DNA, Fungal/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Genes, Essential , Genetic Complementation Test , Green Fluorescent Proteins , Lac Operon/genetics , Luminescent Proteins/genetics , Microscopy, Fluorescence , Molecular Sequence Data , Mutation , Nuclear Proteins/genetics , Nucleoside-Triphosphatase , Osmotic Pressure , Phenotype , Phosphoric Monoester Hydrolases/genetics , Point Mutation , Recombinant Fusion Proteins/genetics , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Transcription Factors/genetics , Transcription Factors/physiology
2.
Mol Gen Genet ; 262(3): 437-47, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10589830

ABSTRACT

In Saccharomyces cerevisiae two transcription factors, Pos9 (Skn7) and Yap1, are involved in the response to oxidative stress. Fusion of the Pos9 response-regulator domain to the Gal4 DNA-binding domain results in a transcription factor which renders the expression of a GAL1-lacZ reporter gene dependent on oxidative stress. To identify genes which are involved in the oxygen-dependent activation of the Gal4-Pos9 hybrid protein we screened for mutants that failed to induce the heterologous test system upon oxidative stress (fap mutants for factors activating Pos9). We isolated several respiration-deficient and some respiration-competent mutants by this means. We selected for further characterization only those mutants which also displayed an oxidative-stress-sensitive phenotype. One of the respiration-deficient mutants (complementation groupfap6) could be complemented by the ISM1 gene, which encodes mitochondrial isoleucyl tRNA synthetase, suggesting that respiration competence was important for signalling of oxidative stress. In accordance with this notion a rho0 strain and a wild-type strain in which respiration had been blocked (by treatment with antimycin A or with cyanide) also failed to activate Gal4-Pos9 upon imposition of oxidative stress. Another mutant, fap24, which was respiration-competent, could be complemented by CCP1, which encodes the mitochondrial cytochrome c peroxidase. Mitochondrial cytochrome c peroxidase degrades reactive oxygen species within the mitochondria. This suggested a possible sensor function for the enzyme in the oxidative stress response. To test this we used the previously described point mutant ccp1 W191F, which is characterized by a 10(4)-fold decrease in electron flux between cytochrome c and cytochrome c peroxidase. The Ccp1W191F mutant was still capable of activating the Pos9 transcriptional activation domain, suggesting that the signalling function of Ccp1 is independent of electron flux rates.


Subject(s)
Cytochrome-c Peroxidase/metabolism , DNA-Binding Proteins/metabolism , Mitochondria/metabolism , Oxidative Stress/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Transcription Factors/metabolism , Gene Expression , Mitochondria/enzymology , Mutagenesis , Saccharomyces cerevisiae/enzymology , Signal Transduction , Transcriptional Activation
3.
Mol Gen Genet ; 261(4-5): 740-52, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10394911

ABSTRACT

Exposure of Saccharomyces cerevisiae to elevated concentrations of hydrogen peroxide induces transcription of several genes involved in the oxidative stress response. Two major transcription factors are involved in this induction, Pos9/Skn7 and Yap1. Fusions of either Yap1 or Pos9/Skn7 with the Gal4 DNA binding domain are active as transcription factors. Gal4-Yap1-dependent reporter gene activity is only weakly regulated by oxidative stress. In contrast, fusion of the Gal4 DNA binding domain to the Pos9/Skn7 protein results in a transcription factor that is independent of the YAP1 gene and is strictly regulated by oxidative stress, indicating that a signaling cascade impinges on the Pos9/Skn7 protein. We have observed that the Ras/PKA (cAMP-dependent protein kinase A) pathway affects this signaling. When PKA activity was low (in the presence of multicopy PDE2 or a cyr1(D822-->A) mutation) maximum reporter gene activity was observed even in the absence of oxidative stress. In contrast, high PKA activity (in strains mutant for either pde2 or bcy1, or expressing the dominant active Ras2Val19) resulted in a complete loss of activation following oxidative stress. The transcription of Pos9/Skn7 target genes was also affected in Ras/PKA pathway mutants. Furthermore, we demonstrated that activated Pos9/Skn7 is necessary for Yap1-dependent reporter gene induction.


Subject(s)
Cyclic AMP-Dependent Protein Kinases/metabolism , DNA-Binding Proteins/metabolism , Oxidative Stress , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/physiology , Transcription Factors/metabolism , ras Proteins/metabolism , Base Sequence , Fungal Proteins/metabolism , Genes, Reporter , Genotype , Hydrogen Peroxide/pharmacology , Kinetics , Molecular Sequence Data , Mutagenesis, Insertional , Oligodeoxyribonucleotides , Plasmids , Polymerase Chain Reaction , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Signal Transduction , beta-Galactosidase/biosynthesis , beta-Galactosidase/genetics
4.
Curr Genet ; 29(4): 327-34, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8598053

ABSTRACT

We have isolated mutants of Saccharomyces cerevisiae with an increased sensitivity to oxidative stress. All pos9 mutants (pos for peroxide sensitivity) were hypersensitive to methylviologene, hyperbaric oxygen or hydrogen peroxide, but grew similarly to the wild-type under all other conditions tested. Isolation and sequencing of the respective POS9 gene revealed that it was identical to SKN7. The predicted Skn7/Pos9 protein possesses a domain with high homology to prokaryotic response regulators. These regulatory proteins are part of a simple signalling cascade termed a "two-component system", where a phosphorylation signal of a histidine kinase is transferred to a conserved aspartate residue of the response regulator. To test the functional role of the respective aspartate residue of Skn7/Pos9 protein in oxidative stress, we mutagenized this residue in vitro to alanine, arginine and glutamate. Only the glutamate allele (D427 to E) was able to rescue the hydrogen peroxide-sensitivity of pos9 mutants. By fusion experiments with the Gal4 DNA-binding domain we identified the isolated response regulator-like domain as a novel eukaryotic domain sufficient for gene activation. Whereas this hybrid protein activated transcription of a lacZ reporter gene under aerobic conditions, no activation was observed under anaerobic conditions, indicating that the response regulator domain is involved in a signalling reaction. Two-hybrid investigations also suggest an oligomerization of the Pos9 protein. Our results indicate that a two-component system is involved in the oxidative-stress response of yeast.


Subject(s)
Fungal Proteins/genetics , Genes, Fungal , Oxidative Stress/drug effects , Oxidative Stress/genetics , Repressor Proteins/genetics , Saccharomyces cerevisiae/genetics , Anaerobiosis , Peroxides/pharmacology , Trans-Activators/physiology
5.
Curr Genet ; 27(5): 427-34, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7586028

ABSTRACT

Although oxidative stress is involved in many human diseases, little is known of its molecular basis in eukaryotes. In a genetic approach, S. cerevisiae was used to identify elements involved in oxidative stress. By using hydrogen peroxide as an agent for oxidative stress, 34 mutants were identified. All mutants were recessive and fell into 16 complementation groups (pos1 to pos16 for peroxide sensitivity). They corresponded to single mutations as shown by a 2:2 segregation pattern. Enzymes reportedly involved in oxidative stress, such as glucose-6-phosphate dehydrogenase, glutathione reductase, superoxide dismutase, as well as glutathione concentrations, were investigated in wild-type and mutant-cells. One complementation group lacked glucose-6-phosphate dehydrogenase and was shown to be allelic to the glucose-6-phosphate dehydrogenase structural gene ZWF1/MET19. In other mutants all enzymes supposedly involved in oxidative-stress resistance were still present. However, several mutants showed strongly elevated levels of glutathione reductase, gluconate-6-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase. One complementation group, pos9, was highly sensitive to oxidative stress and revealed the same growth phenotype as the previously described yap1/par1 mutant coding for the yeast homologue of mammalian transcriptional activator protein, c-Jun, of the proto-oncogenic AP-1 complex. However, unlike par1 mutants, which showed diminished activities of oxidative-stress enzymes and glutathion level, the pos9 mutants did not reveal any such changes. In contrast to other recombinants between pos mutations and par1, the sensitivity did not further increase in par1 pos9 recombinants, which may indicate that both mutations belong to the same regulating circuit.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Genes, Fungal , Mutation , Osmotic Pressure , Oxidative Stress , Saccharomyces cerevisiae/genetics , Cell Division , Hydrogen Peroxide/pharmacology , Mutagenesis , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/physiology
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