Additional file 5: of Using in vivo oxidation status of one- and two-component redox relays to determine H2O2 levels linked to signaling and toxicity

Figure S5. OxyR as a sensor of intracellular H2O2 concentrations. The relation between intracellular H2O2 concentration and OxyR oxidation levels observed at steady state after addition of H2O2 to the WT (filled squares), Δtrx1 (filled diamonds), or Δtrx3 (filled circles) strains is plotted according to the data shown in Table 1 in the main text. (PDF 17 kb

Additional file 2: of Using in vivo oxidation status of one- and two-component redox relays to determine H2O2 levels linked to signaling and toxicity

Figure S2. Kinetics of OxyR and Tpx1 oxidation. a In cells deficient in Tpx1 or Trx1, HA-OxyR oxidizes at low concentrations of peroxides. Aerobic or anaerobic cultures of strains AD29 (WT), AD61 (Δtrx1), and AD36 (Δtpx1) carrying an integrative sty1 promoter-driven HA-oxyR gene were treated or not with the indicated concentrations of H2O2 for the times indicated. TCA extracts were analyzed as in Fig. 2a. b Cultures of strain AD29 (WT) were treated or not with 20, 50, or 100 μM H2O2 for the times indicated. TCA extracts were analyzed as in Fig. 2a, using antibodies against Tpx1 (ox. Tpx1 dimer is the upper band in the panels; red. Tpx1 monomer is the lower band in the panels). (PDF 476 kb

Additional file 8: of Using in vivo oxidation status of one- and two-component redox relays to determine H2O2 levels linked to signaling and toxicity

Figure S7. Biological replicates of main figures. (PDF 1383 kb

The glutaredoxin Grx4 is a Fe-S cluster-containing protein.

<p>(A) Cells pellets of <i>E</i>. <i>coli</i> over-expressing GST-HA-Grx4 are brown. Cells transformed with plasmids pGEX-2T-TEV (GST) or p400 (pGEX-2T-TEV-GST-<i>HA-grx4</i>; GST-Grx4) were grown into LB, and protein expression was induced by IPTG. Cells were pelleted on Elisa plates and photographed. (B and C) Reconstitution of the Fe-S cluster of Grx4. UV-visible absorption spectra of reconstituted Fe-HA-Grx4 (Fe-Grx4). The red dashed line indicates the spectrum of the apo-protein, obtained in the absence of added Fe. (D) The reconstituted Fe-S cluster of Grx4 is sensitive to oxygen. UV-visible absorption spectra of reconstituted Fe-HA-Grx4 (Fe-Grx4) protein before (solid line) and after (red dashed line) 15 minutes of oxygen exposure. (E) GSH is required for reconstitution of the Grx4 Fe-S cluster. UV-visible absorption spectra of Grx4 reconstitution reactions performed in the presence (solid line) or absence (red dashed line) of GSH. (F) Cell lacking Gcs1, auxotrophic for GSH, display constitutive activation of the Fe starvation response after 8 h of GSH withdrawal. Strains 972 (WT), NG81 (<i>Δgrx4</i>) and NG77 (<i>Δgcs1</i>) were grown in YE media, and shifted to MM without GSH, when DIP was added. When indicated for strain NG77, growth proceeded for 8 h prior to DIP addition. Total RNA was analyzed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005106#pgen.1005106.g001" target="_blank">Fig. 1E</a>. (G) Same as in F, with strains 972 (WT), NG81 (<i>Δgrx4</i>) and JE7 (<i>Δgcs1 Δgrx4</i>).</p

The Fep1 repressor is also a Fe-containing protein.

<p>(A) Scheme of the 564 aa-long Fep1 protein, showing the four Cys residues between the zinc fingers (ZF1 and ZF2) which were mutated to serine residues (Fep1.C4S). (B) Cells pellets of <i>E</i>. <i>coli</i> over-expressing GST-Fep1, but not GST-Fep1.C4S, are brown. Cells transformed with plasmids pGEX-2T-TEV (GST), p514 (pGEX-2T-TEV-<i>fep1;</i> GST-Fep1) or p514.C4S (pGEX2T-TEV-<i>fep1</i>.<i>C4S;</i> GST-Fep1.C4S) were grown and cell color analyzed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005106#pgen.1005106.g002" target="_blank">Fig. 2A</a>. (C) UV-Visible spectra of GST-Fep1 (solid line) or GST-Fep1.C4S (dashed line). (D) The Fep1-dependent gene expression program is compromised in cells expressing HA-Fep1.C4S. Total RNA from strains 972 (WT), JE16 (<i>Δfep1</i>) alone or transformed with episomal plasmids p516.81x or p516.81x.C4S (allowing the expression of HA-Fep1 or HA-Fep1.C4S under the control of the weak <i>nmt</i> promoter) was obtained from MM cultures after 18 h thiamine withdrawal, and analyzed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005106#pgen.1005106.g001" target="_blank">Fig. 1E</a>. (E) Cells pellets of <i>E</i>. <i>coli</i> over-expressing GST-Fep1^1–245, but not GST-Fep1^1–245.C4S, are brown. Cells containing p514.NTD (pGEX-2T-TEV-<i>fep1</i>^{<i>1–245</i>}<i>;</i> GST-Fep1^1–245) or p514.NTD.C4S (pGEX-2T-TEV-<i>fep1</i>^{<i>1–245</i>}.<i>C4S;</i> GST-Fep1^1–245.C4S) were grown and cell color analyzed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005106#pgen.1005106.g002" target="_blank">Fig. 2A</a>. (F) UV-Visible spectra of GST-Fep1^1–245 (solid line) or GST-Fep1^1–245.C4S (red dashed line) proteins. (G) A sulfur donor (L-Cys) is not required for reconstitution of the GST-Fep1^1–245 cluster. UV-visible absorption spectra after reconstitution reactions of apo GST-Fep1^1–245 (solid black line) in the presence of Fe (Fe; solid grey line), Fe and DTT (solid red line), Fe, DTT and L-Cys (dashed blue line) or the standard reconstitution reaction with Fe, DTT, L-Cys and GSH (solid green line).</p

The Fe-S cluster of Grx4 is essential for both Fe delivery and Fe sensing.

<p>(A) Scheme of the 244 aa-long Grx4 protein, showing the conserved Cys-containing thioredoxin (Trx) and glutaredoxin (Grx) domains. (B and C) Cells expressing Grx4.C172S display growth defects under aerobic conditions. (B) Survival spots of cultures from strains 972 (WT), NG81 (<i>Δgrx4</i>), NG86.C35S (<i>grx4</i>.<i>C35S</i>), NG86.C172S (<i>grx4</i>.<i>C172S</i>), NG2 (<i>Δfep1</i>) and NG40 (<i>Δphp4</i>), as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005106#pgen.1005106.g001" target="_blank">Fig. 1B</a>. (C) Growth curves of 972 (WT), NG81 (<i>Δgrx4</i>), NG86.C35S (<i>grx4</i>.<i>C35S</i>) and NG86.C172S (<i>grx4</i>.<i>C172S</i>) strains were recorded as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005106#pgen.1005106.g001" target="_blank">Fig. 1C</a>. (D) Total RNA from strains 972 (WT), NG81 (<i>Δgrx4</i>), NG86.C35S (<i>grx4</i>.<i>C35S</i>) and NG86.C172S (<i>grx4</i>.<i>C172S</i>) was processed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005106#pgen.1005106.g001" target="_blank">Fig. 1E</a>. (E) Cells pellets of <i>E</i>. <i>coli</i> over-expressing GST-Grx4.C172S are not brown. Cells transformed with plasmids pGEX-2T-TEV (GST), p400 (pGEX-2T-TEV-<i>HA-grx4</i>; GST-Grx4), p400.C35S (pGEX-2T-TEV-<i>HA-grx4</i>.<i>C35S</i>; GST-Grx4.C35S) or p400.C172S (pGEX-2T-TEV-<i>HA-grx4</i>.<i>C172S</i>; GST-Grx4.C172S) were grown and cell color analyzed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005106#pgen.1005106.g002" target="_blank">Fig. 2A</a>. (F) UV-visible absorption spectra of wild-type and mutant Grx4 after Fe-S cluster reconstitution.</p

Reverse Fe transfer between Grx4-apo-Fra2 and Fe-containing Fep1 followed by UV/visible spectroscopy.

<p>(A) Upon long DIP treatments, Grx4 and Fra2 are dispensable for activation of Fep1-dependent genes. Total RNA from YE cultures of strains 972 (WT), NG81 (<i>Δgrx4</i>) and NG101 (<i>Δfra2</i>), before and after the indicated time in hours with DIP, was processed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005106#pgen.1005106.g001" target="_blank">Fig. 1E</a>. (B) Scheme of the metal transfer reaction between GST beads-bound GST-Fep1^1–245 and Grx4-apo-Fra2. (C and D) <i>In vitro</i> Fe transfer from Fe-Fep1 to apo-Grx4-Fra2. UV/visible spectra of GST-tagged Fep1 (C) and Grx4-Fra2 (D) were recorded before (dashed line) and after (solid line) incubation in a 1:1 protein ratio and protein separation through GSH-affinity chromatography. (E) Model proposed for the reverse metal transfer reaction between Fe (solid circles)-containing Fep1 and Fe-depleted Grx4-Fra2. See text for details.</p

The BolA protein Fra2 participates in the Grx4-dependent signaling cascade.

<p>(A) Scheme of the 84 aa-long Fra2 protein, showing the conserved Cys and histidine residues important in the <i>S</i>. <i>cerevisiae</i> homolog. (B to D) Reconstitution of a Fe-S cluster bridging Grx4 and Fra2. (B) UV-visible absorption spectra of reconstituted Fe-Grx4 (dashed line) or Grx4-Fe-Fra2 (solid line) proteins. (C) UV-visible absorption spectra of reconstituted Grx4-Fe-Fra2 protein before (solid line) and after (dashed line) 15 minutes of oxygen exposure. (D) UV-visible absorption spectra reconstitution of Grx4-Fe-Fra2 in presence (solid line) or absence (dashed line) of GSH. (E) Cells lacking Fra2 display growth defects under aerobic conditions. Strains 972 (WT), NG101 (<i>Δfra2</i>) and NG81 (<i>Δgrx4</i>) were spotted and grown on YE and MM plates under aerobic or anaerobic conditions. (F) The Fep1-dependent gene expression program is compromised in cells lacking Fra2 in response to DIP. Total RNA from strains 972 (WT), NG101 (<i>Δfra2</i>) and NG81 (<i>Δgrx4</i>) was processed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005106#pgen.1005106.g001" target="_blank">Fig. 1E</a>. (G) Fra2 is not fully dispensable in the Php4-dependent Fe starvation response in response to BPS. Total RNA was obtained from YE cultures of strains 972 (WT), NG101 (<i>Δfra2</i>) and NG81 (<i>Δgrx4</i>), treated or not with 25 μM BPS for the indicated times in minutes, and analyzed by Northern blot with the probes indicated. <i>rRNA</i> was used as a loading control. (H) Fra2 displays both cytosolic and nuclear localization. Strain JE3 (<i>fra2-GFP</i>) was analyzed by fluorescence microscopy before and after DIP treatment. (I) Fra2 interacts with Grx4 <i>in vivo</i>. Strains JE5 (<i>fra2-myc</i>), NG115 (<i>grx4-GFP</i>) and JE17 (<i>grx4-GFP fra2-myc</i>) were analyzed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005106#pgen.1005106.g001" target="_blank">Fig. 1F</a>.</p

The glutaredoxin Grx4 functions in both Fe signaling and Fe traffic.

<p>(A) Scheme of the putative roles of Grx4 in Fe delivery and Fe signaling. Grx4 as a Fe sensor regulates the repressors Fep1 and Php4. (B) Only cells lacking Grx4 display growth defects under aerobic conditions. Strains 972 (WT), NG2 (<i>Δfep1</i>), NG40 (<i>Δphp4</i>) and NG81 (<i>Δgrx4</i>) were spotted and grown on YE plates under aerobic or anaerobic conditions. (C) Cells lacking Fep1, Php4 or Grx4 display growth defects in the presence of Fe chelators or Fe excess. Strains as in B were spotted and grown under anaerobic conditions on plates containing or not DIP or ammonium ferrous sulfate (Fe). (D) The Fe chelators BPS and DIP trigger the activation of the Fe starvation response with different kinetics. Total RNA was obtained from YE cultures of wild-type strain 972 treated or not with 25 μM BPS, 0.1 mM DIP or 0.25 mM DIP for the indicated times in minutes, and analyzed by Northern blot with the probes indicated. <i>rRNA</i> was used as a loading control. (E) Php4, Fep1 and Grx4 are essential for the induction of the Fe starvation response. Total RNA from strains as in B was obtained from YE cultures treated or not with 0.25 mM DIP for 90 min, and analyzed by Northern blot with the probes indicated. <i>rRNA</i> was used as a loading control. (F and G) The interaction of Grx4 with Php4, but not with Fep1, is disturbed upon Fe starvation. (F) Strains NG108 (<i>fep1-myc</i>), NG115 (<i>grx4-GFP</i>) and NG109 (<i>grx4-GFP fep1-myc</i>) were treated or not with 0.25 mM DIP for the indicated times. Total native protein extracts were immuno-precipitated with GFP-trap beads. Immuno-precipitates were analyzed by SDS–PAGE and blotted with anti-Myc or anti-GFP antibodies. As a loading control, whole-cell extracts were loaded (WCE). (G) Strains NG107 (<i>php4-myc</i>), NG115 (<i>grx4-GFP</i>) and NG120 (<i>grx4-GFP php4-myc</i>) were treated as described in F.</p

Loss-of-function of Tpx1 suppresses the lethal phenotype of a <i>Δtrr1 Δgrx1</i> double deletion.

<p>(A) Sequence comparison of the wild-type and mutant <i>tpx1</i> loci. The suppressor mutation is a nucleotide deletion at codon 26 (indicated in red in both loci) causing a frame shift leading to a truncated polypeptide of 70 amino acids (stop codon indicated in blue in the mutant allele). (B) Deletion of <i>tpx1</i> is a suppressor of the synthetic lethality of <i>Δtrr1 Δgrx1</i>. Strains SG169 (<i>trr1</i>::<i>kan)</i> and MC138 (<i>grx1</i>::<i>hph tpx1</i>::<i>nat</i>) were crossed and analyzed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006858#pgen.1006858.g005" target="_blank">Fig 5E</a>. Four very small colonies were isolated from four tetrads and recovered in GSH-containing plates grown under semi-anaerobic conditions. Again, no growth was detected for the double deletion (<i>Δ1 Δ2</i>: <i>Δtrr1 Δgrx1</i> †), which was inferred after subtracting all the markers accumulated in the remaining spores of the second tetrad. (C) TCA extracts from YE cultures of strains SB104 (WT), SB112 (<i>Δtpx1</i>), SB106 (<i>Δtrx1</i>), AD175 (<i>Δtrx1 Δtpx1</i>), SB121 (<i>Δtrx1 Δtrx3</i>), AD176 (<i>Δtrx1 Δtrx3 Δtpx1</i>), SB308 (<i>Δtrx1 Δtrx3 Δgrx1</i>) and AD172 (<i>Δtrx1 Δtrx3 Δgrx1 Δtpx1</i>), all of them carrying an <i>HA</i>-tagged version of <i>cdc22</i> at the endogenous locus, were analyzed as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006858#pgen.1006858.g001" target="_blank">Fig 1B</a>. Reduced/active (red. Cdc22-HA) and oxidized/inactive Cdc22 (ox. Cdc22-HA) are indicated. (D) Graph shows the average percentage of oxidized Cdc22-HA from three independent experiments as in C. Error bars (SEM) are shown.</p