Limits...
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.

Show MeSH
Erv1 promotes disulfide transfer to Mia40-bound precursors. (A) 35S-Tim9 precursors were treated with AMS (left) or imported into mitochondria at 15°C, and subjected to indirect thiol trapping (right). Samples were analyzed by Tricine–SDS-PAGE. (B) 35S-Tim9 was imported into mitochondria at 30°C and treated as described in A. (C) 35S-Tim9 precursors were imported into WT mitochondria. (D) 35S-Tim9 precursors were imported into Mia40His mitochondria and subjected to affinity purification and indirect thiol trapping. (E) 35S-Tim9C55S was imported into mitochondria for the indicated times or for 24 min in the presence of iodoacetamide (IA). Samples were treated as described in A. (F) 35S-Tim9C55S with one or three oxidized cysteines were quantified; error bars indicate SEM (n = 5). (G) 35S-Tim9C55S was imported into Mia40His mitochondria and analyzed as in D.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Erv1 promotes disulfide transfer to Mia40-bound precursors. (A) 35S-Tim9 precursors were treated with AMS (left) or imported into mitochondria at 15°C, and subjected to indirect thiol trapping (right). Samples were analyzed by Tricine–SDS-PAGE. (B) 35S-Tim9 was imported into mitochondria at 30°C and treated as described in A. (C) 35S-Tim9 precursors were imported into WT mitochondria. (D) 35S-Tim9 precursors were imported into Mia40His mitochondria and subjected to affinity purification and indirect thiol trapping. (E) 35S-Tim9C55S was imported into mitochondria for the indicated times or for 24 min in the presence of iodoacetamide (IA). Samples were treated as described in A. (F) 35S-Tim9C55S with one or three oxidized cysteines were quantified; error bars indicate SEM (n = 5). (G) 35S-Tim9C55S was imported into Mia40His mitochondria and analyzed as in D.

Mentions: Each small Tim protein contains two CysX3Cys motifs, forming two intramolecular disulfide bonds in the mature protein (Curran et al., 2002; Koehler, 2004; Webb et al., 2006). The fully oxidized proteins are released from Mia40 and assemble to hexameric complexes (Lu et al., 2004; Müller et al., 2008). To determine why Erv1 was needed for precursor release, we asked if it promoted disulfide formation on a Mia40-bound precursor. We monitored the oxidation of single cysteines of Tim9 by modification with the alkylating agent 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS; 500 D). Mutant precursors with a replacement of one to four cysteines were used as standards (Fig. 4 A, lanes 1–5). By indirect thiol trapping, the number of oxidized cysteines after import into mitochondria was determined: first, free thiol groups were blocked by iodoacetamide, then the mitochondria were lysed and all disulfides were reduced, followed by modification of the originally oxidized cysteines by AMS. At a low temperature (15°C), a sequential oxidation of the cysteines was observed (Fig. 4 A, lanes 6–10). At physiological temperature (30°C), three species were dissected: reduced Tim9, Tim9 with one oxidized cysteine, and fully oxidized Tim9 (Fig. 4 B). In erv1 mutant mitochondria, oxidation of the first cysteine was only mildly affected, but formation of the fully oxidized form was significantly inhibited (Fig. 4 B).


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)

Erv1 promotes disulfide transfer to Mia40-bound precursors. (A) 35S-Tim9 precursors were treated with AMS (left) or imported into mitochondria at 15°C, and subjected to indirect thiol trapping (right). Samples were analyzed by Tricine–SDS-PAGE. (B) 35S-Tim9 was imported into mitochondria at 30°C and treated as described in A. (C) 35S-Tim9 precursors were imported into WT mitochondria. (D) 35S-Tim9 precursors were imported into Mia40His mitochondria and subjected to affinity purification and indirect thiol trapping. (E) 35S-Tim9C55S was imported into mitochondria for the indicated times or for 24 min in the presence of iodoacetamide (IA). Samples were treated as described in A. (F) 35S-Tim9C55S with one or three oxidized cysteines were quantified; error bars indicate SEM (n = 5). (G) 35S-Tim9C55S was imported into Mia40His mitochondria and analyzed as in D.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Erv1 promotes disulfide transfer to Mia40-bound precursors. (A) 35S-Tim9 precursors were treated with AMS (left) or imported into mitochondria at 15°C, and subjected to indirect thiol trapping (right). Samples were analyzed by Tricine–SDS-PAGE. (B) 35S-Tim9 was imported into mitochondria at 30°C and treated as described in A. (C) 35S-Tim9 precursors were imported into WT mitochondria. (D) 35S-Tim9 precursors were imported into Mia40His mitochondria and subjected to affinity purification and indirect thiol trapping. (E) 35S-Tim9C55S was imported into mitochondria for the indicated times or for 24 min in the presence of iodoacetamide (IA). Samples were treated as described in A. (F) 35S-Tim9C55S with one or three oxidized cysteines were quantified; error bars indicate SEM (n = 5). (G) 35S-Tim9C55S was imported into Mia40His mitochondria and analyzed as in D.
Mentions: Each small Tim protein contains two CysX3Cys motifs, forming two intramolecular disulfide bonds in the mature protein (Curran et al., 2002; Koehler, 2004; Webb et al., 2006). The fully oxidized proteins are released from Mia40 and assemble to hexameric complexes (Lu et al., 2004; Müller et al., 2008). To determine why Erv1 was needed for precursor release, we asked if it promoted disulfide formation on a Mia40-bound precursor. We monitored the oxidation of single cysteines of Tim9 by modification with the alkylating agent 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS; 500 D). Mutant precursors with a replacement of one to four cysteines were used as standards (Fig. 4 A, lanes 1–5). By indirect thiol trapping, the number of oxidized cysteines after import into mitochondria was determined: first, free thiol groups were blocked by iodoacetamide, then the mitochondria were lysed and all disulfides were reduced, followed by modification of the originally oxidized cysteines by AMS. At a low temperature (15°C), a sequential oxidation of the cysteines was observed (Fig. 4 A, lanes 6–10). At physiological temperature (30°C), three species were dissected: reduced Tim9, Tim9 with one oxidized cysteine, and fully oxidized Tim9 (Fig. 4 B). In erv1 mutant mitochondria, oxidation of the first cysteine was only mildly affected, but formation of the fully oxidized form was significantly inhibited (Fig. 4 B).

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