Abstract
We describe the characterization of Ybp1, a novel protein, in Saccharomyces cerevisiae, that is required for the oxidative stress response to peroxides. Ybp1 is required for H2O2-induced expression of the antioxidant encoding gene TRX2. Our data indicate that the effects of Ybp1 are mediated through the Yap1 transcription factor. Indeed, Ybp1 forms a stress-induced complex with Yap1 in vivo and stimulates the nuclear accumulation of Yap1 in response to H2O2 but not in response to the thiol-oxidizing agent diamide. The H2O2-induced nuclear accumulation of Yap1 is regulated by the oxidation of specific cysteine residues and is dependent on the thiol peroxidase Gpx3. Our data suggest that Ybp1 is required for the H2O2-induced oxidation of Yap1 and acts in the same pathway as Gpx3. Consequently, Ybp1 represents a novel class of stress regulator of Yap1. These data have important implications for the regulation of protein oxidation and stress responses in eukaryotes.
Highlights
The cells of all aerobic organisms are exposed to reactive oxygen species (ROS)1 that include the superoxide (O2.) and hydroxyl (OH1⁄7) radicals and H2O2
We describe the characterization of Ybp1, a novel protein, in Saccharomyces cerevisiae, that is required for the oxidative stress response to peroxides
Identification of a Novel Protein Involved in Peroxide Resistance—The deletion of the TSA1 gene, which encodes thioredoxin peroxidase, in the W303-1a strain reduces the H2O2induced expression of the TRX2 promoter and increases peroxide sensitivity [19]
Summary
Yeast Strains and Growth Conditions—The S. cerevisiae strains used were as follows: W303–1a (MATa ade trp can100 leu112 his ura3); W303–1a diploid (MATa/MAT␣ ade trp can100 leu112 his ura3) [19]; skn7⌬ (MATa ade trp can100 leu112 his ura skn7::HIS3) [4]; yap1⌬ (MAT␣ ade trp can100 leu112 his ura yap1::TRP1) [4]; DY (MAT␣ ade trp can100 leu his ura3::(3xSV40AP1-lacZ)) [3]; DWYU (MAT␣ ade trp can100 leu his ura3::(3xSV40AP1-lacZ) yap1::URA3) [3]; SR1 (MAT␣ ade trp can100 leu112 his ura ybp1::HIS3); SR2 (MATa ade trp can100 leu112 his ura ybp1::HIS3); CTY10 –5d (MATa ade trp901 leu112 his 200 gal gal URA3::lexA op-lacZ) [20]. The YBP1 strain (SR6) was created by replacing the chromosomal ybp1::HIS3 in SR1 with the wild type YBP1 gene contained on a PCR fragment. YEplac181-YBP1-Pk was constructed by first ligating a 240-bp PstI/BamHI PCR fragment containing the C terminus of Ybp into the pRep42PkC plasmid [28] digested with the same enzymes to create pRep42-YBP1-PkC. To obtain the YCplac111-YBP1-His plasmid a 1.3-kb SalI/XhoI fragment of YBP1 was first ligated with SalI/XhoI-digested pET-Hnef-PFH [30]. Localization of GFP-tagged Yap1—Cells containing the GFP-Yap expression plasmid were concentrated into ϳ25 l of medium and 5 l spotted on to a glass slide. A coverslip was placed on top, and GFP-tagged Yap was detected by exciting cells with 450 – 490 nm using a Zeiss Axioscope fluorescence microscope, with a ϫ40 oil immersion objective and an Axiovision digital imaging system. DAPI-stained nuclei and anti-Pk immunofluorescence were visualized by excitation at 365 nm (DAPI) and 450 – 490 nm (Alexa 488) using a Zeiss Axioscope fluorescence microscope, with a ϫ63 oil immersion lens and an Axiovision digital imaging system
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