1. Volume 121, Issue 7, Pages 1059-1069 (July 2005)
A Disulfide Relay System in the Intermembrane Space of Mitochondria that Mediates Protein Import 
Nikola Mesecke, Nadia Terziyska, Christian Kozany, Frank Baumann, Walter Neupert, Kai Hell, Johannes M. Herrmann 
Cell 
Volume 121, Issue 7, Pages (July 2005)
DOI: /j.cell
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2. Figure 1
Mia40 Forms Disulfide Bridges with Newly Imported Tim13 and Cox17
(A) Mitochondria were incubated with radiolabeled recombinant Tim13 protein for 20 min at 25°C in the presence (lanes 1 to 3) or absence (lanes 4 to 9) of 200 μM of the crosslinker DFDNB. The crosslinker was quenched with glycine, and nonimported material was removed by trypsin treatment. The mitochondria were washed and then lysed in SDS buffer. Five percent of the resulting extracts were directly applied to SDS-PAGE. The residual lysates were used for immunoprecipitation with antibodies against Mia40 (αMia40) or with preimmune (p.i.) serum. β-mercaptoethanol (β-ME) was added to the loading buffer of samples shown in lanes 1 to 6. Proteins were resolved by SDS-PAGE and visualized by autoradiography. Positions of molecular weight standards are depicted on the right. Arrowheads indicate bands corresponding to Mia40-Tim13 adducts in lanes 3 and 9, and to Tim13 in lane 6.
(B) Recombinant radiolabeled Cox17 was imported into mitochondria. The samples were treated with trypsin and loaded onto the gel either directly or following immunoprecipitation as described in (A). The arrowhead indicates the Mia40-Cox17 adduct.
(C) Wild-type mitochondria (100 μg per sample) were incubated with Cox17 (2 μg) or mock treated for 10 min at 25°C. The samples were incubated with proteinase K (PK) on ice, washed, and dissolved in sample buffer lacking (lanes 1 and 2) or containing (lanes 3 and 4) β-mercaptoethanol. Proteins were resolved by SDS-PAGE and transferred to nitrocellulose. Mia40 was detected by immunoblotting.
(D) Model showing the disulfide bridge between Mia40 and imported proteins.
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3. Figure 2 Import of Tim13 and Cox17 Is Sensitive to Reducing Agents
(A) Radiolabeled Tim13 was imported into isolated mitochondria for 10 min in the presence of 0, 5, or 10 mM DTT, as indicated. Nonimported material was removed by treatment with proteinase K. Imported Tim13 was visualized by autoradiography.
(B) Cox17 was imported in mitochondria in the presence of 0, 2.5, or 20 mM DTT; 2 mM NADH; 2 mM FAD; 2 mM EDTA; and ortho-phenanthroline (o-Phe) or 10 μM copper sulfate. Mitochondria were treated with proteinase K, and Cox17 was detected by autoradiography.
(C) Tim13, Cox17, the precursor form of Oxa1, and preSu9(1-69)-DHFR were imported into wild-type mitochondria in the presence of different concentrations of DTT. The imported proteins were detected as described in (A) and quantified by densitometry. Import efficiencies without DTT were set to 100% (control).
Cell  , DOI: ( /j.cell )
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4. Figure 3
Depletion of Erv1 Leads to the Reduction of Certain IMS Proteins and to an Increased DTT Sensitivity of Cell Growth
(A) Wild-type (wt) and GAL-Erv1 cells were grown on YPGal and shifted to glucose-containing medium for 48 hr. Mitochondria were isolated from these cells. Mitochondria (12, 25, 50, and 100 μg) were applied to the gel, and the levels of various proteins were detected by immunoblotting. These proteins are located in the IMS (Erv1, Cox17, Tim13, Sod1, cytochrome c heme lyase [CCHL], and cytochrome b2 [Cyt b2]), the outer membrane (Tom70), the inner membrane (ATP/ADP carrier [AAC], Mia40), and the matrix (aconitase [Aco1]).
(B) Wild-type and GAL-Erv1 cells were grown to log phase in medium containing glucose or galactose as carbon sources. Equal amounts of cell culture were spread onto plates containing glucose or galactose. A filter disc was placed onto the cell lawn, which was soaked with 10 μl of 3 mM DTT. The plates were grown at 25°C for 2 (glucose) or 3 (galactose) days (upper panel). The graph shows the width of the halo (lower panel). The means and standard deviations were obtained from three plates per culture.
(C) The DTT sensitivity of the cells was assessed as in (B), but the plates were incubated in the absence of oxygen in an anaerobic chamber.
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5. Figure 4
Depletion of Functional Erv1 Increases the Sensitivity to DTT of the Import of Cox17 and Tim13
(A–C) Cox17, Tim13, and Oxa1 were imported into wild-type (wt) and Erv1-depleted (Erv1↓) mitochondria as described in Figure 2. (Upper panel) Autoradiography. (Lower panel) Quantification of autoradiography. Import efficiencies without DTT were set to 100% (control).
(D) Wild-type and erv1-ts mutant cells were grown in liquid culture to logarithmic phase, spotted onto YPD plates, and incubated for 48 hr at 25°C or 37°C as indicated.
(E and F) DTT sensitivity of the import of recombinant Cox17, and of in vitro synthesized Tim13, was analyzed as in (A).
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6. Figure 5
Depletion of Erv1 Diminishes the Import Rates of Cox17 and Tim13
(A) Cox17 (left and middle panels) and Tim13 (right panel) were incubated with wild-type and Erv1-depleted mitochondria in the presence of different concentrations of reduced glutathione (GSH). The amounts of imported proteins were assessed as described for Figure 2. The import reactions presented in the middle and the right panels were performed under microaerobic conditions in a nitrogen-filled glove bag. The broken line indicates the glutathione concentration determined for the cytosolic compartment of yeast cells (Ostergaard et al., 2004).
(B) The precursor forms of preSu9(1-69)-DHFR and Oxa1 were imported in the presence of increasing amounts of DTT under microaerobic conditions. The amounts of imported protein were quantified and expressed in comparison to the amount of protein imported without DTT.
(C) Cox17 was imported for various time periods in the presence of 15 mM glutathione under aerobic conditions. The amounts of Cox17 inaccessible to added proteinase K were quantified as described in Figure 2.
(D) Mitochondria (50 μg) from wild-type and Erv1-depleted cells were incubated with 5 μg recombinant radiolabeled Cox17 for 10 min in the absence or presence of 15 mM glutathione (GSH) as indicated. Nonimported Cox17 was removed by treatment with proteinase K. Mia40 and the Mia40-Cox17 complex were detected by immunoblotting with antibodies against Mia40 as described for Figure 1C.
(E and F) Wild-type and GAL-Erv1 cells were grown to logarithmic phase in glucose-containing medium. The cells were harvested, and total cell extracts (E) or mitochondrial (40 μg; Mito.) and cytosolic (200 μg; Cyto.) fractions (F) were prepared. The distribution of Cox17 was assessed by immunoblotting. Mitochondrial aconitase (Aco1), Erv1, and the cytosolic hexokinase (hexok.) served as controls.
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7. Figure 6 Erv1 Interacts with Mia40
(A) Mitochondria (500 μg) of wild-type or Mia40-His6 cells were lysed in 250 μl 1% Triton X-100, 300 mM NaCl, 20 mM imidazole, 2 mM phenylmethylsulfonyl fluoride, 50 mM sodium phosphate (pH 8.0). The extract was cleared by centrifugation for 20 min at 125,000 × g and incubated with NiNTA Sepharose for 60 min at 4°C. The beads were washed extensively with lysis buffer, and the bound material was eluted with sample buffer containing 300 mM imidazole. Proteins in the samples indicated were resolved by SDS-PAGE under reducing conditions and analyzed by immunoblotting with antibodies against Mia40 and Erv1. Mitochondria (50 μg) were directly dissolved in sample buffer and applied to SDS-PAGE (Mito.). The tagged and untagged versions of Mia40 present in the Mia40-His6 strain are indicated. Arrowheads depict the Erv1 protein copurified with Mia40-His6.
(B) Extracts of wild-type and Mia40-His6 mitochondria were prepared and incubated with NiNTA Sepharose as in (A). Proteins present in the bound fractions were resolved under nonreducing conditions by SDS-PAGE, transferred to nitrocellulose, and immunodecorated with Erv1-specific antibodies.
(C) Mia40-His6 cells were lysed with 1% digitonin (Dig.) or 1% Triton X-100 (TX) in the absence or presence of 10 mM DTT as indicated. Mia40-His6 was isolated on NiNTA Sepharose. Mia40 and Erv1 in the mitochondrial extract and the eluate were analyzed by immunoblotting.
(D and E) Wild-type mitochondria (50 μg) were incubated in the presence or absence of 50 mM DTT. The mitochondria were washed and incubated with 50 mM iodoacetamide for 20 min to modify reduced thiol groups. Mitochondria were reisolated and lysed in sample buffer. The proteins were resolved by SDS-PAGE under nonreducing conditions, and Mia40 (D) or Erv1 (E) were visualized by immunoblotting.
(F and G) Mitochondria were isolated from wild-type and GAL-Erv1 cells grown on glucose (F) or galactose (G) and incubated with reduced glutathione (GSH) of the concentrations indicated. The samples were treated with iodoacetamide, and the redox state of Mia40 was analyzed as in (D). Reduced and oxidized species of Mia40 were quantified by densitometry, and the proportion of oxidized to total Mia40 was calculated.
Cell  , DOI: ( /j.cell )
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8. Figure 7
The Presence of Disulfide Bridges in Mia40 Is Critical for Import of Cox17 and Tim13
(A) To determine the stage in the import pathway of Cox17 and Tim13 at which disulfide bonds are critical, mitochondria were exposed either before (A) or during (B) the import reaction to 50 mM DTT (cf. upper panel). Mitochondria were preincubated in import buffer for 10 min at 25°C in the absence or the presence (sample “A”) of DTT. Mitochondria were reisolated and resuspended again in import buffer. DTT was added to sample “B,” and 0.2 μg radiolabeled Cox17 was added to both samples. After 10 min at 25°C, mitochondria were again reisolated and then resuspended in 600 mM sorbitol, HEPES/KOH (pH 7.4) containing 50 μg/ml proteinase K. After incubation for 20 min, mitochondria were isolated and washed. The amounts of imported Cox17 were assessed by SDS-PAGE, autoradiography, and densitometry.
(B) A mitochondrial suspension was split into four aliquots, two of which were incubated in the absence and two of which were incubated in the presence of 10 mM DTT for 10 min. In order to modify reduced thiol groups, 50 mM NEM was added to one of each type of aliquots. After 10 min, mitochondria were reisolated and split into two halves, which were incubated in import buffer with either recombinant Cox17 or Tim13. Nonimported material was removed by protease treatment. The mitochondria were washed, reisolated, and dissolved in sample buffer lacking β-mercaptoethanol. One-half of the sample buffer was directly loaded on a gel to detect reduced and oxidized species of Mia40. The other half was reduced by addition of β-mercaptoethanol, resolved by SDS-PAGE, and autoradiographed to detect imported Cox17 and Tim13.
(C) Left part (“Import”): Recombinant Cox17 was imported into wild-type mitochondria in the presence or absence of 50 mM DTT or 2 mM EDTA and 2 mM o-phenanthroline as indicated. The mitochondria were reisolated, washed, and treated with proteinase K. Right part (“Postincubation”): Cox17 was imported in the absence of DTT and chelators. DTT and chelators were then added as indicated during the postincubation of the samples with protease. The amounts of imported Cox17 were quantified and expressed in relation to the amounts of protein imported in the absence of DTT and chelators.
(D) Model for Erv1-dependent formation of oxidized Mia40, which has the capacity to bind newly imported proteins via disulfide bridges. See text for details.
Cell  , DOI: ( /j.cell )
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