FIGURE 1.

Ybp1 is limiting for H₂O₂ tolerance. A, a low-copy number vector plasmid (pRS315) or this same plasmid containing the strong promoter from the glycolytic gene PGK1 driving YBP1, YBH1 (YBP1 homologue involved in centromere function (11, 12, 32)), or GPX3 were introduced into wild-type (SEY6210) S. cerevisiae cells. Addtionally, a high-copy number vector plasmid (YEp352) or this same plasmid containing the YAP1 gene were similarly introduced into SEY6210 cells. Transformants were grown to mid-log phase, placed on medium containing H₂O₂, and photographed after incubation at 30 °C. B, whole cell protein extracts were prepared from transformants described above containing the indicated expression plasmids. Equal aliquots of extracts were separated on SDS-PAGE and analyzed by Western blotting with an anti-HA antibody. Each expression plasmid contains two HA epitopes fused to the N terminus of the PGK1-driven protein. Molecular mass of the recombinant proteins is indicated in kDa on the left side of the panel. Ponceau S staining of total protein is shown at the bottom of panels B and C to confirm equal loading. C, either wild-type (WT:SEY6210) or isogenic yap1Δ cells transformed with the indicated high-copy number plasmids were grown to mid-log phase, protein extracts were prepared and analyzed by Western blotting using anti-Yap1 antibody (20).