The disulfide relay system of mitochondria is connected to the respiratory chain

Abstract

All proteins of the intermembrane space of mitochondria are encoded by nuclear genes and synthesized in the cytosol. Many of these proteins lack presequences but are imported into mitochondria in an oxidation-driven process that relies on the activity of Mia40 and Erv1. Both factors form a disulfide relay system in which Mia40 functions as a receptor that transiently interacts with incoming polypeptides via disulfide bonds. Erv1 is a sulfhydryl oxidase that oxidizes and activates Mia40, but it has remained unclear how Erv1 itself is oxidized. Here, we show that Erv1 passes its electrons on to molecular oxygen via interaction with cytochrome c and cytochrome c oxidase. This connection to the respiratory chain increases the efficient oxidation of the relay system in mitochondria and prevents the formation of toxic hydrogen peroxide. Thus, analogous to the system in the bacterial periplasm, the disulfide relay in the intermembrane space is connected to the electron transport chain of the inner membrane.

Figure 1.

The redox state of Mia40 depends on the oxygen concentration. (A) Mitochondria were isolated from wild-type cells and GAL-ERV1 yeast mutants (Mesecke et al., 2005) in which Erv1 was down- or up-regulated by growth on a glucose- or galactose-containing medium. Thiol groups were trapped by incubation in 100 mM iodoacetamide at 25°C for 30 min. The samples were applied to nonreducing SDS-PAGE, and Mia40 was detected by Western blotting. Reduced (red.) and oxidized (ox.) states of Mia40 can be separated because of their different mobility in the gel. (B) Wild-type mitochondria were incubated for 30 min in the presence of the indicated glutathione (GSH) concentrations under oxygen-saturated or -depleted conditions. Thiol groups were trapped with iodoacetamide and the samples were analyzed by Western blotting. (C) Reduced and oxidized species of the Mia40 signals in B were quantified by densitometry. The percentage of oxidized Mia40 in the different samples is shown.

Figure 2.

The Mia40 redox status depends on complexes of the respiratory chain. (A) Mitochondria isolated from wild-type, Δcor1, Δcyt1, Δcyc1/Δcyc7, Δcox19, Δcox23, Δatp1, and Δatp10 yeast strains were incubated in 30 mM glutathione at 25°C for 30 min. Reduced thiol groups were trapped. After reisolation of the mitochondria, the samples were analyzed by SDS-PAGE and Western blotting. The fraction of oxidized Mia40 in the samples was quantified by densitometry. For comparison, the dotted line indicates the wild-type fraction of oxidized Mia40. (B) Mitochondria derived from wild-type, Δcyt1, and Δcox19 strains were incubated at different glutathione concentrations and treated with iodoacetamide. Oxidized and reduced fractions were separated by nonreducing SDS-PAGE and detected by immunoblotting. (C) Percentages of Mia40 oxidation of the experiment shown in B. (D) Wild-type mitochondria were incubated in the absence or presence of the respiratory chain inhibitors antimycin A (Ant A) or potassium cyanide (KCN). Reduced thiols were trapped and the samples were analyzed by SDS-PAGE, Western blotting, and densitometry. (E) Experiments described in D were performed three times in wild-type and cytochrome c–deletion mitochondria. The relative changes in the amounts of oxidized Mia40 were quantified. Error bars indicate SD.

Figure 3.

Erv1 transfers electrons to cytochrome c. (A) 40 μM of oxidized cytochrome c was incubated with 2 mM DTT and 8 μM of recombinant Erv1 in a cuvette, and the reduction of cytochrome c was monitored over time by spectroscopy at 550 nm. The amount of reduced cytochrome c was calculated and plotted against time (squares). For comparison, control samples lacking Erv1 (circles) or both Erv1 and DTT (triangles) are shown. (B) Wild-type and Δcyc1/Δcyc7 mitochondria were incubated in the presence of 7.5 mM glutathione under oxygen-saturated or -depleted conditions. All samples were treated with iodoacetamide. Oxidized and reduced fractions of Mia40 were separated by nonreducing SDS-PAGE and analyzed by Western blotting.

Figure 4.

Mia40-dependent protein import is influenced by the activity of the respiratory chain. (A) Radiolabeled Cox19 protein was incubated with wild-type, Δrip1, and Δcox18 mitochondria in the presence of different concentrations of DTT. Nonimported protein was removed by treatment with proteinase K on ice. Mitochondria were reisolated and dissolved in sample buffer. Proteins were analyzed by SDS-PAGE and autoradiography. Imported proteins were quantified by densitometry. Import efficiencies without DTT were set to 100% (control). (B) Radiolabeled Tim10 was imported into mitochondria from a wild-type and a Δcyc1/Δcyc7 mutant as described in A. (C) Wild-type mitochondria were incubated in the absence or presence of 100 μg/ml antimycin A and 10 mM potassium cyanide for 3 min at 25°C before radiolabeled Tim10 was imported. Mitochondria were treated with 65 mM iodoacetamide and reisolated, and proteins were analyzed by nonreducing SDS-PAGE. This allows the identification of monomeric Tim10 as well as of Mia40-associated Tim10 (Mia40 • Tim10).

Figure 5.

Cytochrome c prevents Erv1-dependent generation of hydrogen peroxide. (A) Production of hydrogen peroxide (H₂O₂) was assayed in a fluorescence-based assay using Amplex red. 2 μM of purified Erv1 was incubated with 50 mM Amplex red and 1 U/ml horseradish peroxidase in 600 μl of 100 mM potassium phosphate, pH 7.4. Upon addition of DTT, fluorescence emission at 610 nm was recorded at an excitation wavelength of 550 nm. Incubation with 150 and 300 nmol cytochrome c counteracted the production of hydrogen peroxide linearly with time (arrows). (top, inset) The generation of hydrogen peroxide represents the same measurement at a larger scale of the y axis. (B) 1 nmol hydrogen peroxide was preincubated with or without a twofold excess of oxidized cytochrome c for 1 min at 25°C before fluorescence was analyzed in the Amplex red assay. Note that the presence of cytochrome c did not quench the fluorescence signal. (C) Model for the interaction of the disulfide relay system and the mitochondrial respiratory chain. The electron flow from the imported proteins to the final electron acceptor oxygen is indicated. The cytochrome c–independent side reaction of Erv1 with oxygen is shown in light gray. Cytochrome c reductase and oxidase complexes are indicated as complexes III and IV, respectively. Q indicates the ubiquinone pool.