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Two novel proteins in the mitochondrial outer membrane mediate beta-barrel protein assembly.

Ishikawa D, Yamamoto H, Tamura Y, Moritoh K, Endo T - J. Cell Biol. (2004)

Bottom Line: Recent evidence has shown that mitochondrial beta-barrel protein assembly in the outer membrane requires specific translocator proteins in addition to the components of the general translocator complex in the outer membrane, the TOM40 complex.Here we report two novel mitochondrial outer membrane proteins in yeast, Tom13 and Tom38/Sam35, that mediate assembly of mitochondrial beta-barrel proteins, Tom40, and/or porin in the outer membrane.Depletion of Tom13 or Tom38/Sam35 affects assembly pathways of the beta-barrel proteins differently, suggesting that they mediate different steps of the complex assembly processes of beta-barrel proteins in the outer membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.

ABSTRACT
Mitochondrial outer and inner membranes contain translocators that achieve protein translocation across and/or insertion into the membranes. Recent evidence has shown that mitochondrial beta-barrel protein assembly in the outer membrane requires specific translocator proteins in addition to the components of the general translocator complex in the outer membrane, the TOM40 complex. Here we report two novel mitochondrial outer membrane proteins in yeast, Tom13 and Tom38/Sam35, that mediate assembly of mitochondrial beta-barrel proteins, Tom40, and/or porin in the outer membrane. Depletion of Tom13 or Tom38/Sam35 affects assembly pathways of the beta-barrel proteins differently, suggesting that they mediate different steps of the complex assembly processes of beta-barrel proteins in the outer membrane.

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Depletion of Tom13 or Tom38 does not affect import of mtHsp60 or AAC into mitochondria in vitro. Mitochondria were isolated from yeast strains W303-1A (WT), GAL-TOM13 (Tom13↓), and GAL-TOM38 (Tom38↓) after cultivation in lactate medium (+0.1% glucose) for 14, 14, and 10 h, respectively, at 23°C. Radiolabeled mtHsp60 precursor and AAC were incubated with Tom13↓ (lanes 4–6 and open circles), Tom38↓ (lanes 7–9 and open squares), and WT mitochondria (lanes 1–3 and closed circles) at 23°C for the indicated times. The mitochondria were treated with PK, and the imported proteins were analyzed by SDS-PAGE and radioimaging. The amounts of radiolabeled proteins added to each reaction are set to 100%.
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fig3: Depletion of Tom13 or Tom38 does not affect import of mtHsp60 or AAC into mitochondria in vitro. Mitochondria were isolated from yeast strains W303-1A (WT), GAL-TOM13 (Tom13↓), and GAL-TOM38 (Tom38↓) after cultivation in lactate medium (+0.1% glucose) for 14, 14, and 10 h, respectively, at 23°C. Radiolabeled mtHsp60 precursor and AAC were incubated with Tom13↓ (lanes 4–6 and open circles), Tom38↓ (lanes 7–9 and open squares), and WT mitochondria (lanes 1–3 and closed circles) at 23°C for the indicated times. The mitochondria were treated with PK, and the imported proteins were analyzed by SDS-PAGE and radioimaging. The amounts of radiolabeled proteins added to each reaction are set to 100%.

Mentions: We tested the in vitro import abilities of mitochondria isolated from Tom13-depleted (Tom13↓) cells and from Tom38-depleted (Tom38↓) cells after 14 and 10 h cultivation, respectively, in the absence of galactose for various radiolabeled precursor proteins. Mitochondria isolated from Tom13↓ and Tom38↓ cells did not exhibit decrease in ΔΨ, which is essential for protein import via the TIM23 or TIM22 complexes (unpublished data). We analyzed the import of radiolabeled matrix and inner-membrane proteins: the precursor of mtHsp60, a presequence-containing precursor protein that uses the TOM40 complex and the TIM23 complex to move across the outer and inner membranes, respectively, and ADP/ATP carrier (AAC), a presequence-less polytopic inner membrane protein, which uses the TOM40 complex to move across the outer membrane and the TIM22 complex to be inserted into the inner membrane. The import rates of the precursor of mtHsp60 (Fig. 3 A) and of AAC (Fig. 3 B) were not affected by depletion of Tom13 or Tom38 at all.


Two novel proteins in the mitochondrial outer membrane mediate beta-barrel protein assembly.

Ishikawa D, Yamamoto H, Tamura Y, Moritoh K, Endo T - J. Cell Biol. (2004)

Depletion of Tom13 or Tom38 does not affect import of mtHsp60 or AAC into mitochondria in vitro. Mitochondria were isolated from yeast strains W303-1A (WT), GAL-TOM13 (Tom13↓), and GAL-TOM38 (Tom38↓) after cultivation in lactate medium (+0.1% glucose) for 14, 14, and 10 h, respectively, at 23°C. Radiolabeled mtHsp60 precursor and AAC were incubated with Tom13↓ (lanes 4–6 and open circles), Tom38↓ (lanes 7–9 and open squares), and WT mitochondria (lanes 1–3 and closed circles) at 23°C for the indicated times. The mitochondria were treated with PK, and the imported proteins were analyzed by SDS-PAGE and radioimaging. The amounts of radiolabeled proteins added to each reaction are set to 100%.
© Copyright Policy
Related In: Results  -  Collection

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fig3: Depletion of Tom13 or Tom38 does not affect import of mtHsp60 or AAC into mitochondria in vitro. Mitochondria were isolated from yeast strains W303-1A (WT), GAL-TOM13 (Tom13↓), and GAL-TOM38 (Tom38↓) after cultivation in lactate medium (+0.1% glucose) for 14, 14, and 10 h, respectively, at 23°C. Radiolabeled mtHsp60 precursor and AAC were incubated with Tom13↓ (lanes 4–6 and open circles), Tom38↓ (lanes 7–9 and open squares), and WT mitochondria (lanes 1–3 and closed circles) at 23°C for the indicated times. The mitochondria were treated with PK, and the imported proteins were analyzed by SDS-PAGE and radioimaging. The amounts of radiolabeled proteins added to each reaction are set to 100%.
Mentions: We tested the in vitro import abilities of mitochondria isolated from Tom13-depleted (Tom13↓) cells and from Tom38-depleted (Tom38↓) cells after 14 and 10 h cultivation, respectively, in the absence of galactose for various radiolabeled precursor proteins. Mitochondria isolated from Tom13↓ and Tom38↓ cells did not exhibit decrease in ΔΨ, which is essential for protein import via the TIM23 or TIM22 complexes (unpublished data). We analyzed the import of radiolabeled matrix and inner-membrane proteins: the precursor of mtHsp60, a presequence-containing precursor protein that uses the TOM40 complex and the TIM23 complex to move across the outer and inner membranes, respectively, and ADP/ATP carrier (AAC), a presequence-less polytopic inner membrane protein, which uses the TOM40 complex to move across the outer membrane and the TIM22 complex to be inserted into the inner membrane. The import rates of the precursor of mtHsp60 (Fig. 3 A) and of AAC (Fig. 3 B) were not affected by depletion of Tom13 or Tom38 at all.

Bottom Line: Recent evidence has shown that mitochondrial beta-barrel protein assembly in the outer membrane requires specific translocator proteins in addition to the components of the general translocator complex in the outer membrane, the TOM40 complex.Here we report two novel mitochondrial outer membrane proteins in yeast, Tom13 and Tom38/Sam35, that mediate assembly of mitochondrial beta-barrel proteins, Tom40, and/or porin in the outer membrane.Depletion of Tom13 or Tom38/Sam35 affects assembly pathways of the beta-barrel proteins differently, suggesting that they mediate different steps of the complex assembly processes of beta-barrel proteins in the outer membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.

ABSTRACT
Mitochondrial outer and inner membranes contain translocators that achieve protein translocation across and/or insertion into the membranes. Recent evidence has shown that mitochondrial beta-barrel protein assembly in the outer membrane requires specific translocator proteins in addition to the components of the general translocator complex in the outer membrane, the TOM40 complex. Here we report two novel mitochondrial outer membrane proteins in yeast, Tom13 and Tom38/Sam35, that mediate assembly of mitochondrial beta-barrel proteins, Tom40, and/or porin in the outer membrane. Depletion of Tom13 or Tom38/Sam35 affects assembly pathways of the beta-barrel proteins differently, suggesting that they mediate different steps of the complex assembly processes of beta-barrel proteins in the outer membrane.

Show MeSH