1. Circadian Oscillation of Sulfiredoxin in the Mitochondria
In Sup Kil, Keun Woo Ryu, Se Kyoung Lee, Jeong Yeon Kim, Sei Yoon Chu, Ju Hee Kim, Sunjoo Park, Sue Goo Rhee 
Molecular Cell 
Volume 59, Issue 4, Pages (August 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 59, 651-663DOI: (10.1016/j.molcel.2015.06.031)
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3. Figure 1
Hyperoxidation of PrxIII and Daily Oscillation of Mitochondrial PrxIII-SO2 and Srx Abundance
(A) Immunoblot analysis of tissue homogenates from Srx–/– (KO) or wild-type (WT) mice with antibodies to Prx-SO2, to PrxIII, to Srx, and to GAPDH (loading control). Lysates of Y-1 cells that had been exposed to H2O2 were examined as a positive control (H2O2) for PrxI-SO2/PrxII-SO2 and PrxIII-SO2 (rightmost lane). Two different blot exposures are shown for Prx-SO2. WAT, white adipose tissue; BAT, brown adipose tissue.
(B) C57BL/6J mice were housed under a 12 hr light, 12 hr dark cycle and killed at 4 hr intervals, with zeitgeber time (ZT) 0 indicating 7 a.m. Cytosolic and mitochondrial (Mito) fractions of AG, BAT, and heart were then prepared and subjected to immunoblot analysis with antibodies to Srx, to p-p38, to p38, to α-tubulin (loading control), to Prx-SO2, and to PrxIII (upper panels). The blot intensities of p-p38 in cytosol and of PrxIII-SO2 and Srx in mitochondria relative to those of total p38 and PrxIII, respectively, were determined as means ± SD from three independent experiments (lower panels). See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 Import of Srx into Mitochondria in Cultured Cells and AG
(A–D) Y-1 cells (A and B) and A549 cells (C and D) were exposed to 250 μM H2O2 for 10 min (0.16 hr), washed with PBS, and cultured for the indicated times in fresh medium. Cytosolic and mitochondrial fractions of the cells were then subjected to immunoblot analysis (A and C). Blot intensities of PrxI-SO2/PrxII-SO2 in cytosol and of PrxIII-SO2 in mitochondria relative to those of PrxII and PrxIII, respectively, were determined (B and D, upper panels), as were those of Srx in cytosol relative to that of PrxII and of Srx in mitochondria relative to that of PrxIII (B and D, lower panels). Quantitative data are means ± SD from three independent experiments.
(E and F) Mouse AGs were incubated in the absence (Con) or presence of 500 nM ACTH, 50 μM rotenone (Rot), or 50 μM antimycin A (Anti) for the indicated times, after which cytosolic and mitochondrial fractions were prepared and subjected to immunoblot analysis (E). The immunoblot intensity of mitochondrial (Mito) Srx relative to that of PrxIII was determined as the mean ± SD (n = 6–8 individual glands) from two independent experiments (F). ∗p < 0.05 versus corresponding value for Con.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 Mitochondrial Import of Srx In Vitro
(A) Purified recombinant mouse Srx (10 μg) was incubated for 3 hr at 30°C with mitochondrial (500 μg) and cytosolic (1 mg) fractions prepared from the liver of Srx–/– mice, after which mitochondrial lysates were subjected to immunoblot analysis with antibodies to Srx and to PrxIII.
(B) Srx import was assayed as in (A) but in the presence of the indicated amounts of liver cytosol (Native) or of liver cytosol boiled for 1 min (Denatured).
(C) Srx import was assayed as in (A) but in the presence of the indicated concentrations of DTT.
(D) Mitochondria incubated with Srx and cytosol as in (A) were washed and then further incubated for 10 min at 30°C either with 0.1% digitonin (Dig) or 1% Triton X-100 (Tri) in the presence of proteinase K (P K) or without detergent and proteinase K (Con). Mitochondrial lysates were then subjected to immunoblot analysis. See also Figure S2.
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6. Figure 4 The N Terminus of Srx Is Required for Mitochondrial Import
(A and B) Purified WT, ΔN10, and ΔN20 (A) as well as R4.11A (B) forms of mouse Srx were assayed for mitochondrial import for the indicated times as in Figure 3A.
(C) HeLa cells transfected with an expression vector for FLAG-WT, FLAG-ΔN20, or FLAG-R4.11A forms of human Srx were incubated in the absence or presence of 250 μM H2O2 for 10 min, washed with PBS, and cultured for 8 hr in fresh medium, after which cytosolic and mitochondrial fractions were prepared and subjected to immunoblot analysis with antibodies to FLAG, to PrxII, and to PrxIII.
(D and E) HeLa cells transfected with an expression vector for FLAG-WT, FLAG-ΔN20, or FLAG-R4.11A forms of Srx (or with the corresponding empty vector) were exposed to 250 μM H2O2 for 10 min, washed with PBS, and cultured for the indicated times in fresh medium. The cells were then subjected to immunoblot analysis (D). The blot intensities of PrxI-SO2/PrxII-SO2 and PrxIII-SO2 relative to that of β-actin were determined as means ± SD from three independent experiments (E).
(F) Liver lysate prepared from Srx–/– mice was incubated without (−) or with recombinant WT or R4.11A forms of Srx and then subjected to immunoprecipitation (IP) with antibodies to Srx. The resulting precipitates as well as the liver lysate were subjected to immunoblot analysis with antibodies to TOM70, to TOM20, and to Srx. See also Figure S3.
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7. Figure 5 Requirement for HSP90 and FKBP in Mitochondrial Import of Srx
(A) Purified GST or GST-Srx was subjected to a pull-down assay by incubation overnight at 4°C with a liver lysate of Srx–/– mice. The precipitated proteins, as well as the liver lysate, were subjected to immunoblot analysis with antibodies to HSP90, to HSC70, or to GST.
(B) Purified GST-Srx was subjected to a pull-down assay by incubation overnight at 4°C with liver lysate (1 mg) or with pool #1 (HSC70) or pool #2 (HSP90) obtained by chromatography of liver lysate (Figure S4A). The precipitated proteins as well as input samples were then subjected to immunoblot analysis as in (A).
(C) Purified GST or GST-WT or C99S mutant forms of Srx were subjected to a pull-down assay by incubation overnight at 4°C with liver lysate of Srx–/– mice. The precipitated proteins as well as the liver lysate were then subjected to immunoblot analysis with antibodies to HSP90 or to GST.
(D) Purified GST-M domain of HSP90α was subjected to a pull-down assay by incubation overnight at 4°C with purified His6-WT or C99S forms of Srx. The resulting precipitates were then subjected to SDS-PAGE in the absence (−) or presence (+) of DTT followed by immunoblot analysis with antibodies to HSP90 and to Srx.
(E) HeLa cells transfected with expression vectors for HA-HSP90α and FLAG-Srx were exposed to 250 μM H2O2 for 10 min, washed with PBS, and cultured for the indicated times in fresh medium. Cell lysates were then subjected to immunoprecipitation with antibodies to HA, and the resulting precipitates (IP) as well as the whole-cell lysates (WCL) were subjected to immunoblot analysis with antibodies to FLAG and to HA.
(F) HeLa cells expressing HA-HSP90α and FLAG-WT or C99S forms of Srx were exposed (or not) to 250 μM H2O2 for 10 min, washed with PBS, and cultured for 2 hr in fresh medium. Cell lysates were then subjected to immunoprecipitation and immunoblot analysis as in (E).
(G) HeLa cells expressing FLAG-WT or C99S forms of Srx were exposed (or not) to 250 μM H2O2 for 10 min, washed with PBS, and cultured for 8 hr in fresh medium. Cytosolic and mitochondrial fractions of the cells were then prepared and subjected to immunoblot analysis with antibodies to FLAG, to PrxII, and to PrxIII.
(H) HeLa cells expressing FLAG-Srx were incubated in the absence (−) or presence (+) of 6 μM geldanamycin (GA) for 1 hr and then in the additional absence or presence of 250 μM H2O2 for 10 min. The cells were then washed with PBS, cultured for 8 hr in fresh medium without (−) or with (+) 6 μM geldanamycin, and fractionated for immunoblot analysis of cytosolic and mitochondrial fractions as in (G).
(I) Purified Srx was incubated for 3 hr at 30°C with mitochondrial and cytosolic fractions prepared from the liver of Srx–/– mice as well as in the absence (−) or presence of 10 μM geldanamycin, 10 μM 17-AAG, FK506 (1 μg/ml), or cyclosporine A (1 μg/ml). Lysates of the mitochondria were then subjected to immunoblot analysis.
(J) HeLa cells expressing FLAG-Srx were incubated in the absence or presence of 20 μM FK506 for 1 hr and then in the additional absence or presence of 250 μM H2O2 for 10 min. They were then washed with PBS, cultured for 8 hr in fresh medium with or without FK506, and fractionated for immunoblot analysis of cytosolic (Cyto) and mitochondrial (Mito) fractions. See also Figure S4.
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8. Figure 6
Effects of PrxIII and PrxIII-SO2 on Mitochondrial Srx Degradation
(A) HeLa cells transfected with an expression vector for FLAG-WT, R4.11A, or mito forms of Srx were exposed to cycloheximide (0.1 mg/ml) for the indicated times and then subjected to immunoblot analysis with antibodies to FLAG.
(B) Whole homogenates as well as cytosolic and mitochondrial fractions of the indicated tissues (AG, adrenal gland) from WT, PrxI–/– (I), PrxII–/– (II), or PrxIII–/– (III) mice were subjected to immunoblot analysis with antibodies to Srx, to α-tubulin (cytosolic marker), and to SOD2 (mitochondrial marker). Equal protein amounts were loaded for each sample.
(C and D) A breast tumor cell line (MP3N2) derived from PrxIII–/– mice was infected with a retrovirus encoding WT or ΔC mutant forms of PrxIII (to yield WTPrxIII and ΔCPrxIII cells, respectively), and the infected cells were then transfected with an expression vector for FLAG-mito Srx and exposed to cycloheximide (0.1 mg/ml) for the indicated times. Cell lysates were then subjected to immunoblot analysis with antibodies to FLAG and to β-actin (C). The immunoblot intensity of FLAG-mito Srx relative to that of β-actin was determined as the mean ± SD from three independent experiments (D). ∗p < 0.05, ∗∗p < 0.01 versus the corresponding value for ΔCPrxIII cells.
(E and F) Purified GST-Srx was subjected to a pull-down assay by incubation for 3 hr at 4°C with AG lysate from Srx−/− mice in which ∼30% of PrxIII is hyperoxidized. The indicated amounts of AG lysate and pull-down fraction were subjected to immunoblot analysis of PrxIII and PrxIII-SO2 (E). The average value (x1) of PrxIII band intensity per microgram of tissue lysate was determined from the first three lanes of the PrxIII blot, and the average value (y1) of PrxIII band intensity per microliter of the pull-down fraction was determined from the last three lanes of the PrxIII blot. The corresponding values (x2 and y2) were determined for PrxIII-SO2 band intensity. The ratio (y2/x2) for PrxIII-SO2 is presented as relative interaction, with the ratio (y1/x1) for PrxIII taken as 1 (F). Data are means ± SD from three independent experiments. ∗p < 0.05.
(G) The kinetic constants for binding of purified PrxIII-SH or PrxIII-SO2 to GST-Srx were evaluated by surface plasmon resonance analysis performed with 7.9 μM GST-Srx and the indicated concentrations of PrxIII-SH or PrxIII-SO2 and with a sensor chip coated with antibodies to GST. RU, relative units. See also Figure S5.
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9. Figure 7 Mitochondrial Srx Is Degraded by Lon Protease
(A and B) A549 cells were exposed for 1 hr to 10 μM MG132 or DMSO vehicle, incubated for 10 min in the additional presence of 250 μM H2O2, and then cultured for the indicated times in fresh medium with DMSO or MG132. Cytosolic (Cyto) and mitochondrial (Mito) fractions were then prepared and subjected to immunoblot analysis (A). The blot intensity of mitochondrial Srx relative to that of PrxIII was determined as the mean ± SD from three independent experiments (B). ∗p < 0.05 versus corresponding value for DMSO.
(C) HeLa cells transfected with an expression vector for FLAG-WT or L117R forms of mito Srx were incubated in the absence or presence of 10 μM MG132 for 1 hr and then in the additional absence or presence of cycloheximide (CHX, 0.1 mg/ml) for 4 hr. Cell lysates were then subjected to immunoblot analysis with antibodies to FLAG and to β-actin.
(D and E) LonRNAi cells were cultured in the absence (−) or presence (+) of doxycyclin (Dox, 1 μg/ml) for 15 days, transfected with an expression vector for FLAG-mito Srx, exposed to cycloheximide (0.1 mg/ml) for the indicated times, and subjected to immunoblot analysis with antibodies to Lon, to FLAG, and to β-actin (D). The immunoblot intensity of FLAG-mito Srx relative to that of β-actin was determined as the mean ± SD from three independent experiments (E). ∗p < 0.05, ∗∗p < 0.01 versus corresponding value for the absence of Dox.
(F) Model for the mechanism underlying daily oscillation of PrxIII-SO2 and Srx levels in mitochondria of AG, BAT, and heart. See Discussion for details. See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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