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Mitochondrial protein import: precursor oxidation in a ternary complex with disulfide carrier and sulfhydryl oxidase.

Stojanovski D, Milenkovic D, Müller JM, Gabriel K, Schulze-Specking A, Baker MJ, Ryan MT, Guiard B, Pfanner N, Chacinska A - J. Cell Biol. (2008)

Bottom Line: The biogenesis of mitochondrial intermembrane space proteins depends on specific machinery that transfers disulfide bonds to precursor proteins.The machinery shares features with protein relays for disulfide bond formation in the bacterial periplasm and endoplasmic reticulum.We have analyzed the cooperation of the disulfide relay components during import of precursors into mitochondria and identified a ternary complex of all three components.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung, Universität Freiburg, 79104 Freiburg, Germany.

ABSTRACT
The biogenesis of mitochondrial intermembrane space proteins depends on specific machinery that transfers disulfide bonds to precursor proteins. The machinery shares features with protein relays for disulfide bond formation in the bacterial periplasm and endoplasmic reticulum. A disulfide-generating enzyme/sulfhydryl oxidase oxidizes a disulfide carrier protein, which in turn transfers a disulfide to the substrate protein. Current views suggest that the disulfide carrier alternates between binding to the oxidase and the substrate. We have analyzed the cooperation of the disulfide relay components during import of precursors into mitochondria and identified a ternary complex of all three components. The ternary complex represents a transient and intermediate step in the oxidation of intermembrane space precursors, where the oxidase Erv1 promotes disulfide transfer to the precursor while both oxidase and precursor are associated with the disulfide carrier Mia40.

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Influence of Erv1 on the ternary complex. (A) 35S-Tim9 was incubated with mitochondria and mitoplasts for 30 min and analyzed by native electrophoresis. (B) Mitoplasts were incubated with 35S-Tim9. (C) Growth of mia40-4 cells overproducing Erv1. (D) 35S-Tim9 was incubated with mitochondria and analyzed by nonreducing SDS-PAGE. (E–G) Mitoplasts were incubated with recombinant Erv1 forms. Pellets (E and G) and supernatants (F) were analyzed by nonreducing SDS-PAGE.
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fig3: Influence of Erv1 on the ternary complex. (A) 35S-Tim9 was incubated with mitochondria and mitoplasts for 30 min and analyzed by native electrophoresis. (B) Mitoplasts were incubated with 35S-Tim9. (C) Growth of mia40-4 cells overproducing Erv1. (D) 35S-Tim9 was incubated with mitochondria and analyzed by nonreducing SDS-PAGE. (E–G) Mitoplasts were incubated with recombinant Erv1 forms. Pellets (E and G) and supernatants (F) were analyzed by nonreducing SDS-PAGE.

Mentions: In erv1 mutant mitochondria, the Mia40–intermediate II formed with an efficiency close to that of wild-type mitochondria (Fig. 3 A). Although the original erv1-1 mutant showed only a minor effect on assembly of small Tim proteins, as described previously, the other erv1 mutants were strongly impaired in the formation of the mature Tim9–Tim10 and TIM22 complexes (Figs. 1 A and 3 A; Allen et al., 2005; Rissler et al., 2005; Milenkovic et al., 2007; Müller et al., 2008). We reasoned that Erv1 may be rate-limiting for the turnover of intermediate II. When Erv1 was overexpressed, the amount of intermediate II was indeed decreased (Fig. 3 B). Moreover, overexpression of Erv1 in mia40-4 cells restored growth at elevated temperatures (Fig. 3 C) and decreased the amount of intermediate II (Fig. 3 D). To directly test if Erv1 was needed for dissociation of intermediate II, we generated the intermediate in mitoplasts and added recombinant Erv1. Thereby, the amount of intermediate II was significantly decreased (Fig. 3 E), and released precursor was found in the supernatant fraction (Fig. 3 F). Mutant forms of Erv1 (Hofhaus et al., 2003) did not change the amount of intermediate II (Fig. 3 G). We conclude that Erv1 promotes a dissociation of the ternary complex.


Mitochondrial protein import: precursor oxidation in a ternary complex with disulfide carrier and sulfhydryl oxidase.

Stojanovski D, Milenkovic D, Müller JM, Gabriel K, Schulze-Specking A, Baker MJ, Ryan MT, Guiard B, Pfanner N, Chacinska A - J. Cell Biol. (2008)

Influence of Erv1 on the ternary complex. (A) 35S-Tim9 was incubated with mitochondria and mitoplasts for 30 min and analyzed by native electrophoresis. (B) Mitoplasts were incubated with 35S-Tim9. (C) Growth of mia40-4 cells overproducing Erv1. (D) 35S-Tim9 was incubated with mitochondria and analyzed by nonreducing SDS-PAGE. (E–G) Mitoplasts were incubated with recombinant Erv1 forms. Pellets (E and G) and supernatants (F) were analyzed by nonreducing SDS-PAGE.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2568017&req=5

fig3: Influence of Erv1 on the ternary complex. (A) 35S-Tim9 was incubated with mitochondria and mitoplasts for 30 min and analyzed by native electrophoresis. (B) Mitoplasts were incubated with 35S-Tim9. (C) Growth of mia40-4 cells overproducing Erv1. (D) 35S-Tim9 was incubated with mitochondria and analyzed by nonreducing SDS-PAGE. (E–G) Mitoplasts were incubated with recombinant Erv1 forms. Pellets (E and G) and supernatants (F) were analyzed by nonreducing SDS-PAGE.
Mentions: In erv1 mutant mitochondria, the Mia40–intermediate II formed with an efficiency close to that of wild-type mitochondria (Fig. 3 A). Although the original erv1-1 mutant showed only a minor effect on assembly of small Tim proteins, as described previously, the other erv1 mutants were strongly impaired in the formation of the mature Tim9–Tim10 and TIM22 complexes (Figs. 1 A and 3 A; Allen et al., 2005; Rissler et al., 2005; Milenkovic et al., 2007; Müller et al., 2008). We reasoned that Erv1 may be rate-limiting for the turnover of intermediate II. When Erv1 was overexpressed, the amount of intermediate II was indeed decreased (Fig. 3 B). Moreover, overexpression of Erv1 in mia40-4 cells restored growth at elevated temperatures (Fig. 3 C) and decreased the amount of intermediate II (Fig. 3 D). To directly test if Erv1 was needed for dissociation of intermediate II, we generated the intermediate in mitoplasts and added recombinant Erv1. Thereby, the amount of intermediate II was significantly decreased (Fig. 3 E), and released precursor was found in the supernatant fraction (Fig. 3 F). Mutant forms of Erv1 (Hofhaus et al., 2003) did not change the amount of intermediate II (Fig. 3 G). We conclude that Erv1 promotes a dissociation of the ternary complex.

Bottom Line: The biogenesis of mitochondrial intermembrane space proteins depends on specific machinery that transfers disulfide bonds to precursor proteins.The machinery shares features with protein relays for disulfide bond formation in the bacterial periplasm and endoplasmic reticulum.We have analyzed the cooperation of the disulfide relay components during import of precursors into mitochondria and identified a ternary complex of all three components.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung, Universität Freiburg, 79104 Freiburg, Germany.

ABSTRACT
The biogenesis of mitochondrial intermembrane space proteins depends on specific machinery that transfers disulfide bonds to precursor proteins. The machinery shares features with protein relays for disulfide bond formation in the bacterial periplasm and endoplasmic reticulum. A disulfide-generating enzyme/sulfhydryl oxidase oxidizes a disulfide carrier protein, which in turn transfers a disulfide to the substrate protein. Current views suggest that the disulfide carrier alternates between binding to the oxidase and the substrate. We have analyzed the cooperation of the disulfide relay components during import of precursors into mitochondria and identified a ternary complex of all three components. The ternary complex represents a transient and intermediate step in the oxidation of intermembrane space precursors, where the oxidase Erv1 promotes disulfide transfer to the precursor while both oxidase and precursor are associated with the disulfide carrier Mia40.

Show MeSH