1. Volume 37, Issue 4, Pages 516-528 (February 2010)
Mitochondrial Disulfide Bond Formation Is Driven by Intersubunit Electron Transfer in Erv1 and Proofread by Glutathione 
Melanie Bien, Sebastian Longen, Nikola Wagener, Ilona Chwalla, Johannes M. Herrmann, Jan Riemer 
Molecular Cell 
Volume 37, Issue 4, Pages (February 2010)
DOI: /j.molcel
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2. Molecular Cell 2010 37, 516-528DOI: (10.1016/j.molcel.2010.01.017)
Copyright © 2010 Elsevier Inc. Terms and Conditions

3. Figure 1 The FAD Domain Facilitates Noncovalent Dimerization of Erv1
(A) Scheme of Erv1 displaying the N (Erv1(N)) and FAD (Erv1(C)) domain of the protein and the respective redox-active cysteine residues.
(B) Conservation of residues in the dimerization interface of Erv1, RnALR, Erv2, and AtErv1. Residues contributing to subunit interactions in the published structures are indicated by asterisks. Arrows indicate cysteine residues of the CxxC motif of the FAD domain.
(C) Mitochondria were isolated from ERV1 deletion strains carrying plasmids expressing wild-type, expressing His- or HA-tagged Erv1, or coexpressing the HA- and His-tagged Erv1 variants. Isolated mitochondria were incubated with 10 mM TCEP for 15 min at 25°C, and free thiol groups were blocked by NEM treatment. Then, mitochondria were lysed and His-tagged Erv1 was precipitated. Precipitates were analyzed by western blotting (WB) against His and HA. T, total; B, bound material.
(D) Isolated wild-type mitochondria were treated with 10 mM TCEP for 15 min at 25°C. Free thiol groups were blocked by treatment with 50 mM NEM for 30 min. Mitochondria were treated with either 40 or 200 μM of the indicated crosslinkers. Samples were analyzed by western blotting (WB) using an Erv1 antibody.
(E) Purified Erv1 or Erv1(C) was subjected to gel filtration chromatography on a Superdex 200 column. Fractions were analyzed by absorption spectroscopy. Sizes of proteins used for calibration are indicated.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Electrons Are Shuttled from the N onto the FAD Domain by Intersubunit Electron Transfer
(A) An ERV1 deletion strain encoding wild-type Erv1 on a URA3-containing plasmid was transformed with LEU1 plasmids carrying wild-type or cysteine mutant ERV1. Empty plasmid (pYX142) was transformed as control. Resulting transformants were grown on plates containing 5-FOA to counterselect against the URA3 plasmid.
(B) As in (A), except that LEU- and TRP-containing plasmids encoding Erv1 cysteine mutants (C33S/C133S and C33S/C130S) were cotransformed into the ERV1 shuffle strain. Growth was tested on plates lacking leucine (Leu) or tryptophan (Trp).
(C) LEU- and TRP-containing plasmids encoding His- and HA-tagged versions of Erv1 cysteine mutants were cotransformed into the ERV1 shuffle strain. Resulting transformants were grown on plates containing 5-FOA to counterselect against the URA3 plasmid. These strains and a strain carrying the ERV1 gene under the control of the GalL promoter (GalL-ERV1) were used. In the GalL-ERV1 strain, the expression of the GalL promoter was repressed by growth on glucose (Erv1↓). Mitochondria were isolated and incubated with 10 mM GSH for 10 min at 4°C to counteract air oxidation of Mia40. Samples were TCA-precipitated, and free thiol groups were modified using 20 mM NEM. Proteins were separated by nonreducing SDS-PAGE, and Mia40 was detected by western blotting (WB). Reduced and oxidized states of Mia40 are indicated.
(D) As in (A), except that LEU- and TRP-containing plasmids for expression of either the N- or the C-terminal part of Erv1 were transformed into the ERV1 shuffle strain.
(E) A strain carrying the ERV1 gene under the control of the GalL promoter (GalL-ERV1) was transformed with plasmids encoding wild-type Erv1, Erv1(N), or Erv1(C). Expression from the GalL promoter was repressed by growth on glucose (Erv1↓). Subsequently, mitochondria were isolated, and the experiment was performed as described in (C).
(F) 40 μM oxidized cytochrome c was incubated with purified Erv1 (4 μM) or Erv1(C) (4 μM) and DTT (100 μM) as indicated. The reduction of cytochrome c was monitored by following the absorption at 550 nm. The amount of reduced cytochrome c was calculated and plotted against time. Control measurements were performed with cytochrome c and buffer, DTT, or Erv1.
(G) As in (F), except that 100 μM reduced Erv1(N) was incubated with 40 μM cytochrome c and 4 μM Erv1(C).
(H) 30 μM purified reduced Erv1(N) was incubated with 60 μM purified Erv1(C) for the indicated time under oxygen-depleted conditions. Samples were TCA-precipitated and treated with 15 mM AMS for 1 hr. Proteins were separated by nonreducing SDS-PAGE and analyzed by Ponceau staining after blotting.
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5. Figure 3
Electrons Are Transferred from Mia40 via the N to the FAD Domain of Erv1
(A) The cytochrome c reduction assay was performed as in Figure 2F. Purified reduced Mia40 (100 μM) was incubated with 40 μM oxidized cytochrome c and 4 μM purified Erv1. Control measurements were performed with cytochrome c and DTT (100 μM), Erv1 and DTT, or reduced Mia40.
(B) As in (A), except that reduced Mia40 (100 μM) was incubated with cytochrome c (40 μM) and either 4 μM Erv1 or a combination of 4 μM Erv1(N) and 4 μM Erv1(C). Control measurements were performed with cytochrome c and either Erv1(C) and Erv1(N) or Erv1(C) and reduced Mia40.
(C) Purified, reduced Mia40 (30 μM) was either incubated with Erv1, Erv1(N), or Erv1(C) (all at concentrations of 60 μM) for the indicated times or left untreated. Samples were TCA-precipitated and treated with 20 mM NEM for 1 hr, subjected to nonreducing SDS-PAGE, and analyzed by Ponceau staining after blotting.
(D) Mitoplasts were generated from 100 μg wild-type mitochondria and treated with 3 mM DTT for 10 min at 25°C. DTT was removed by centrifugation, and mitoplasts were then incubated with purified Erv1, Erv1(N), or Erv1(C) as indicated. Samples were TCA-precipitated and treated with 20 mM NEM. Proteins were separated by nonreducing SDS-PAGE and analyzed by western blotting (WB) against Mia40.
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6. Figure 4
Complete Reconstitution of the Mitochondrial Disulfide Relay with Purified Cox19 as Substrate
(A) Scheme of Cox19 displaying the reduced (unfolded) and the oxidized (mature) forms of the protein. The cysteine residues contributing to the inner and outer disulfide bond are indicated.
(B) The cytochrome c reduction assay was performed as described in Figure 2F. Reduced Cox19 (50 μM) was incubated with 40 μM oxidized cytochrome c and 4 μM of Erv1, Mia40, or a combination of both. A control measurement was performed with cytochrome c and reduced Cox19.
(C) Purified, reduced Cox19 was incubated with cytochrome c and either both Mia40 and Erv1 (top panel) or Erv1, Mia40, or buffer for the indicated times. Samples were TCA-precipitated and treated with 15 mM AMS for 1 hr, subjected to nonreducing SDS-PAGE, and analyzed by western blotting (WB) against Cox19 (0, 2, 4 AMS; number of AMS modifications added to Cox19 with 4 AMS representing fully reduced Cox19 and 0 AMS fully oxidized Cox19).
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7. Figure 5 Mia40 Introduces Both Disulfide Bonds into Cox19
(A) Lanes 1–3: Small amounts of in vitro translated radioactive Cox19 or Cox19C40,52S were left untreated or TCA-precipitated and incubated with 15 mM AMS for 1 hr at 25°C. Proteins were analyzed by nonreducing SDS-PAGE and autoradiography. In vitro translated radioactive Cox19 was incubated with buffer (lanes 4–9) or 30 μM purified Mia40 (lanes 10–14) for the indicated times. Subsequently, samples were TCA-precipitated, treated with 15 mM AMS for 1 hr at 25°C, and then subjected to nonreducing SDS-PAGE and analyzed by autoradiography (4/2/0 AMS, different redox states of monomeric Cox19; 3/1 AMS, different redox states of Cox19-Mia40 disulfide-linked intermediates).
(B) Cox19 signals from three independent experiments as performed in (A) were quantified. The sum of the signals in each lane was set to 100%. Data are represented as average ±SD.
(C) As in (A), except that radiolabeled Cox19C30,62S or Cox19C40,52S were used instead of Cox19. 0/2 AMS; different redox states of Cox19 mutants with 2 AMS representing fully reduced and 0 AMS oxidized Cox19 mutants. 1 AMS; Cox19 mutant-Mia40 mixed disulfide intermediate with one free cysteine.
(D) As in (A), lanes 10–14, except that small amounts of radiolabeled Cox19 were incubated with Erv1 (lanes 1–5), Erv1, and Mia40 (lanes 6–10) or with Mia40 after 20 min preincubation with Erv1 (lanes 12–16) (all purified proteins at a concentration of 30 μM). For quantification of all gels in Figure 5, see Figure S5. All experiments were performed under oxygen-depleted conditions. Experiments performed under aerobic conditions yielded similar results (data not shown).
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8. Figure 6
Glutathione Supports the Correct and Efficient Protein Folding of Substrates of the Erv1-Mia40 Import Pathway
(A) The cytochrome c reduction assay was performed as detailed in Figure 2F. Oxidized cytochrome c (40 μM) was incubated with 10 mM GSH in the presence or absence of purified Erv1, Erv1(C), or a combination of Erv1 and Mia40 (all at concentrations of 4 μM). Control measurements were performed with cytochrome c and either GSH or Erv1(C) and DTT.
(B) As in (A), except that 40 μM cytochrome c, 4 μM Erv1, and 4 μM Mia40 were incubated with 50 μM purified reduced Cox19 in the presence of 10 mM GSH. Control measurements were performed with cytochrome c, Erv1, and Mia40 with GSH.
(C) The experiments were performed as in Figure 4C in the presence and absence of 10 mM GSH.
(D) As in (C), except that the experiment was blotted against Mia40.
(E) Experiments with in vitro translated radioactive Cox19 lysate were performed as in Figures 5A and 5D, but in the presence of 10 mM GSH.
(F) Quantification of the oxidized and reduced forms of Cox19 in the experiments depicted in Figures 5A (lanes 10–14), 5D (lanes 1–5), and 6E (lanes 4–13). Quantifications of three independent experiments were performed as described in Figure 5B, and a Student's t test was performed. ∗∗∗p < 0.005, ∗∗p < 0.05 and ∗p < 0.5. Data are represented as average ±SD.
(G) As in (E), except that Cox19-Mia40 intermediates were preformed by incubating radioactive Cox19 lysate with Mia40 for 20 min before following the reaction for additional 20 min in the presence or absence of GSH. For quantification, see Figure S5.
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9. Figure 7 Glutathione Increases the Efficiency of Mitochondrial Import
(A) Isolated mitochondria from the indicated strains were incubated with different amounts of GSH. Thiol-disulfide exchange was prevented by NEM addition, and the samples were analyzed by nonreducing SDS-PAGE and western blotting (WB) against Mia40. Substrate-Mia40 disulfide-linked intermediates are indicated.
(B) In vitro translated radioactive Cox19 or Tim9 was incubated with mitochondria in the presence of different amounts of GSH. Nonimported protein was removed by treatment with Proteinase K. Imported protein was analyzed by reducing SDS-PAGE and autoradiography. Signals were quantified and the amount of imported protein was blotted. Control; amount of imported protein in the absence of GSH was set to 100%.
(C) As in (B), except that the import was performed for different times in the presence or absence of 10 mM GSH. Total; amount of protein in the lysate used for the import study was set to 100%.
(D) As in (B), except that the import was performed in the presence of DTT.
Molecular Cell  , DOI: ( /j.molcel )
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