Precursor oxidation by Mia40 and Erv1 promotes vectorial transport of proteins into the mitochondrial intermembrane space

Abstract

The mitochondrial intermembrane space contains chaperone complexes that guide hydrophobic precursor proteins through this aqueous compartment. The chaperones consist of hetero-oligomeric complexes of small Tim proteins with conserved cysteine residues. The precursors of small Tim proteins are synthesized in the cytosol. Import of the precursors requires the essential intermembrane space proteins Mia40 and Erv1 that were proposed to form a relay for disulfide formation in the precursor proteins. However, experimental evidence for a role of Mia40 and Erv1 in the oxidation of intermembrane space precursors has been lacking. We have established a system to directly monitor the oxidation of precursors during import into mitochondria and dissected distinct steps of the import process. Reduced precursors bind to Mia40 during translocation into mitochondria. Both Mia40 and Erv1 are required for formation of oxidized monomers of the precursors that subsequently assemble into oligomeric complexes. Whereas the reduced precursors can diffuse back into the cytosol, the oxidized precursors are retained in the intermembrane space. Thus, oxidation driven by Mia40 and Erv1 determines vectorial transport of the precursors into the mitochondrial intermembrane space.

Figure 1.

Tim13 is oxidized upon import into mitochondria. (A) Isolated yeast wild-type mitochondria were incubated with ³⁵S-labeled precursor of Tim13 for the indicated times. Mitochondria were treated with proteinase K (Prot. K), reisolated, and subjected to denaturation in the presence of 50 mM IA or 15 mM AMS as indicated. Lanes 1 and 2, the Tim13 precursor was directly treated with 50 mM IA or 15 mM AMS as indicated to serve as a migration standard. Red., reduced. (B) ³⁵S-labeled wild-type Tim13 and single cysteine mutants of Tim13 were incubated with wild-type mitochondria for the indicated times and analyzed without proteinase K treatment. When the mitochondria were treated with proteinase K after the import reaction, the single cysteine mutants were accessible to the protease to the same extent as the reduced wild-type precursor of Tim13. (C) ³⁵S-labeled Tim13 was imported for the indicated time periods. Proteinase K treatment was performed in SEM buffer containing 1% Triton X-100. Proteins were precipitated before IA or AMS treatment. Samples were analyzed by nonreducing Tricine-SDS-PAGE. Protein bands were visualized by digital autoradiography.

Figure 2.

Tim8 is oxidized upon import into mitochondria. (A) Isolated wild-type mitochondria were incubated with ³⁵S-labeled precursor of Tim8 for the indicated times. Mitochondria were treated with proteinase K (Prot. K), reisolated, and subjected to denaturation in the presence of 50 mM IA or 15 mM AMS. The samples were analyzed by nonreducing Tricine-SDS-PAGE. Lanes 1 and 2, the Tim8 precursor was directly treated with 50 mM IA or 15 mM AMS. Oxid. + Red., oxidized and reduced. (B and C) ³⁵S-labeled precursors of Tim8 were imported into isolated yeast wild-type mitochondria. In B, mitochondria were reisolated and treated with 50 mM DTT for 15 min at 20°C as indicated; lane 1, import was performed for 30 min, and mitochondria were solubilized in the presence of 0.5% SDS and heated at 75°C. All samples of B and C, after solubilization in the presence of 50 mM IA, assembly reactions were separated by blue native electrophoresis and analyzed by digital autoradiography.

Figure 3.

The oxidized monomer of Tim8 is located inside mitochondria and released after reduction. ³⁵S-labeled precursors of Tim8 were imported into isolated yeast wild-type mitochondria. (A) Mitochondria were reisolated and treated with proteinase K (Prot. K) as indicated. (B) Mitochondria were spun down, and supernatants were collected. Half of the samples were treated with 50 mM DTT at 20°C for 15 min. Mitochondrial pellets and supernatants were analyzed by blue native electrophoresis and autoradiography.

Figure 4.

Mia40 is required to oxidize small Tim proteins. (A and C) ³⁵S-labeled precursors of Tim13 and Tim8 were imported into wild-type (WT) and mia40 mutant mitochondria. Where indicated, the mitochondria were treated with proteinase K (Prot. K.) followed by denaturation in the presence of 15 mM AMS. Oxid., oxidized; Red., reduced. Lanes 1 and 2, precursors were directly treated with 50 mM IA or 15 mM AMS. Proteins were separated by nonreducing Tricine-SDS-PAGE and analyzed by autoradiography. (B and D) Import of ³⁵S-labeled Tim8 into WT and mia40 mutant mitochondria was performed for the indicated times and analyzed by blue native electrophoresis.

Figure 5.

Influence of erv1 mutants on oxidation of small Tim proteins. (A and B) ³⁵S-labeled precursors of Tim13 and Tim8 were imported in wild-type (WT) and erv1 mutant mitochondria. Where indicated, the mitochondria were treated with proteinase K (Prot. K.), followed by denaturation in the presence of 15 mM AMS. Lanes 1 and 2, precursors were directly treated with 50 mM IA or 15 mM AMS. Proteins were separated by nonreducing Tricine-SDS-PAGE and analyzed by autoradiography. (C) Import of ³⁵S-labeled Tim8 into WT and erv1-2 mutant mitochondria for the indicated time periods, followed by blue native electrophoresis.

Figure 6.

Cysteine 159 mutant of Erv1 (erv1-5) is defective in formation of disulfide bonds in small Tim proteins. (A) Schematic representation of Erv1. The cysteine residues and FAD binding site in the primary structure of S. cerevisiae Erv1 are indicated previously (Hofhaus et al., 2003). Erv1-5 contains the amino acid substitution C159S. (B) Wild-type (WT) and erv1-5 cells were subjected to serial dilutions, plated on YPD medium and incubated at the indicated temperatures. (C) Mitochondria (15 and 30 μg of protein) from WT and erv1-5 strains were analyzed by SDS-PAGE and Western blotting. (D and E) ³⁵S-labeled precursors of Tim9 and Tim13, respectively, were incubated with WT and erv1-5 mitochondria. The mitochondria were solubilized with digitonin and analyzed by blue native electrophoresis and autoradiography. (F and G) ³⁵S-labeled precursors of Tim13 and Tim8 were imported into WT and erv1-5 mutant mitochondria. Where indicated, the mitochondria were treated with proteinase K (Prot. K.), followed by denaturation in the presence of 15 mM AMS. Lanes 1 and 2, precursors were directly treated with 50 mM IA or 15 mM AMS. Proteins were separated by nonreducing Tricine-SDS-PAGE and analyzed by autoradiography. (H) Import of ³⁵S-labeled Tim8 was analyzed as described for D and E.

Figure 7.

The oxidized monomer of Tim8 is a productive intermediate in formation of the Tim8–Tim13 complex. (A) Mitochondria from single deletion mutant strains (tim8Δ, tim13Δ), the double deletion strain tim8Δ tim13Δ, and wild-type (WT) were analyzed by SDS-PAGE and Western blotting. The level of Tim8 in tim13Δ mitochondria and the level of Tim13 in tim8Δ mitochondria was <5–10% of the level in wild-type mitochondria, respectively. (B and C) ³⁵S-labeled precursors of Tim13 and Tim8, respectively, were imported into WT, tim8Δ, tim13Δ, and tim8Δ tim13Δ mitochondria. In B, mitochondria were treated with proteinase K before nonreducing Tricine-SDS-PAGE. In C, mitochondria were solubilized in the presence of 50 mM IA, and proteins were separated by blue native electrophoresis. Protein bands were visualized by digital autoradiography.