Fig. 2
Expression of E. coli’s OxyR in yeast cells. a Tpx1-independent oxidation of OxyR in fission yeast. MM cultures of strains AD29 (WT) and AD36 (Δtpx1), carrying an integrative sty1 promoter-driven HA-oxyR gene, were treated or not with 0.1 mM or 0.5 mM H2O2 for the times indicated. TCA/AMS protein extracts were obtained, processed by SDS-PAGE, and analyzed by Western blot using antibodies against HA. Reduced (red.) and oxidized (ox.) HA-OxyR are indicated with arrows. b The Trx system participates in OxyR reduction. MM cultures of strains AD29 (WT), AD47 (Δtrx3), and AD61 (Δtrx1), all constitutively expressing HA-OxyR, were treated or not with 0.2 mM H2O2 for the times indicated. TCA/AMS extracts were prepared and analyzed as described in (b) c H2O2 scavenging by Tpx1, and the role of Trx1 and Trx3 in the recycling of disulfide-link Tpx1. d Cells lacking Tpx1, or both Trx1 and Trx3, display constitutive levels of oxidized OxyR. Overexpression of Ctt1 suppresses basal OxyR oxidation in cells lacking Tpx1 or cytosolic Trxs. TCA extracts of MM cultures of the HA-OxyR-expressing strains AD62 (Δtrx1 Δtrx3), AD98 + p464 (Δtrx1 Δtrx3 pctt1; it overexpresses catalase from plasmid p464) and AD36 (Δtpx1), AD94 + p464 (Δtpx1 pctt1; it overexpresses catalase from plasmid p464) treated or not with 0.2 mM H2O2 for the times indicated, were processed and analyzed as in (b) AMS 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid, E. coli Escherichia coli, MM minimal medium, ox. oxidized, red. reduced, SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis, TCA trichloroacetic acid, WT wild type