Figure 4(A) Experimental layout of the 'acute stress-washout' assay performed in (B) and in Appendix Fig S4A. Cells were pre-treated with either 0, 1, 5, 10, 25 or 50 mM H2O2 for 10 mins after which H2O2 was removed and cells were resuspended in fresh buffer. The response of a mitochondrial matrix-localized Grx1-roGFP2 probe to an exogenous bolus of 1 mM H2O2 was then measured. (B) 'Acute stress-washout' assay. Δprx1 cells containing a Prx1-WT plasmid and expressing a mitochondrial matrix-localized Grx1-roGFP2 probe were grown to exponential phase in SGal (-Leu, -Ura) medium harvested and treated with either 0, 1, 5, 10, 25 or 50 mM H2O2 for 10 mins. After removal of H2O2 and resuspension in fresh buffer the response of a mitochondrial matrix-localized Grx1-roGFP2 probe to the addition of 1 mM H2O2 was measured. The middle panel is an enlargement of the left panel. The graph in the right panel shows the steady state Grx1-roGFP2 oxidation following H2O2 pre-treatment as well as the maximum Grx1-roGFP2 oxidation in response to the subsequent second H2O2 treatment. 'Acute stress-washout' assay. Δprx1 cells expressing a mitochondrial matrix-localized Grx1-roGFP2 probe were grown to exponential phase in SGal (-Leu, -Ura) medium harvested and treated with either 0, 1, 5, 10, 25 or 50 mM H2O2 for 10 mins. (C) The same experiment as in (B) performed with Δprx1 cells transformed with a Prx1-C91A plasmid. (D) ∆prx1 cells transformed with a vector encoding wild-type Prx1, a vector encoding the matrix-targeted D-aminooxidase (su9-DAO) and expressing a mitochondrial matrix-localized Grx1-roGFP2 were grown to exponential phase in SGal (-Leu, -Ura, -His) and then pre-treated with 0.15 M D-alanine for 0, 0.5, 1.5 hours. Afterwards, the cells were washed and the response of Grx1-roGFP2 to the addition of 1 mM H2O2 was measured. (E) Left panel: the response of mitochondrial matrix-localized roGFP2-Prx1 and roGFP2 probes expressed in wild-type cells to exogenous H2O2 at the indicated concentrations. Cells were grown in SGal (-Leu) medium and harvested at early exponential phase. Right panel: model illustrating that hyperoxidation of the Prx1 moiety in the roGFP2-Prx1 sensor leads to a roGFP2-like behavior. (F) Scheme illustrating the work-flow of the experiment preformed in (G, H). Dependent on the initial state of the single cysteine of Prx1 different outcomes of the modification approach can consequently different behaviour on SDS-PAGE can be expected. In sample (1) "Unmodified", no shift expected, since no mmPEG24 modification occurs. In sample (2) "TCEP reduced", treatment with TCEP reduces only disulfide bonds and sulfenic acids to thiols and these are modified with mmPEG24 - hyperoxidized cysteine residues are not modifiable. In lane 3 "Steady state", only reduced thiols are directly modified with mmPEG24 - disulfide bonds, sulfenic acids and hyperoxidized cysteine residues are not modifiable. (G) Left panel: a scheme illustrating the principle of the thiol modification-based 'redox shift' assay, for which reduced thiols (but not thiols oxidized to a disulphide bond or a sulfinic or sulfonic acid) can be irreversibly modified by the alkylating agent mmPEG24 leading to a ~2.4kDa increase in mass and thus decreased mobility on an SDS-PAGE gel. Right panel: redox shift assay to assess the oxidation state of the Prx1 catalytic cysteine residue performed in Δprx1+Prx1-WT, wild-type + empty vector and Δprx1+Prx1-C91A. (H) Redox shift assays of Prx1. Δprx1 cells transformed with a plasmid encoding wild-type Prx1 were grown to early exponential phase in SGal (-Ura) medium. Cells were subsequently treated with the indicated concentrations of H2O2 for 10 mins. Exposure to H2O2 oxidizes the single cysteine of Prx1 to a state that cannot be reduced with TCEP. (I) Experimental layout of the 'acute stress-washout' assay in the presence or absence of the ribosome inhibitor cycloheximide (CHX) as performed in (J). Wild-type cells expressing a mitochondrial matrix-localized Grx1-roGFP2 probe were grown to early exponential phase in SGal (-Leu). Cells were pre-treated either with water or 50 mM H2O2 for 10 mins and afterwards washed and resuspended in fresh medium. During the next 2 hour recovery, cells were either treated with the translation inhibitor cycloheximide (CHX) or with DMSO as a vehicle control. The response of a mitochondrial matrix-localized Grx1-roGFP2 probe to the subsequent addition of 1 mM H2O2 was then measured. (J) The response of a mitochondrial matrix-localized Grx1-roGFP2, expressed in wild-type cells grown to the addition of exogenous 1 mM H2O2. Cells were grown in SGal (-Leu) and harvested at early exponential phase. Left panel, the pre-treatment efficiently inhibits the ability of Prx1 to transfer oxidizing equivalents to EGSH. Right panel, newly synthesized Prx1 is required to recover the transfer of oxidizing equivalents to EGSH in the mitochondrial matrix. (K) Hyperoxidation of Prx1 can prevent H2O2-induced oxidation of the matrix glutathione pool and de novo synthesis is required to replace hyperoxidized Prx1.