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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 affects assembly of Tom40 and porin in the mitochondrial outer membrane in vitro. (A) 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 yeast Tom40 was incubated with WT, Tom13↓, and Tom38↓ mitochondria at 23°C for the indicated times. The mitochondria were reisolated by centrifugation, solubilized with 1% digitonin (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.5 mM EDTA, and 10% glycerol) for 10 min on ice, and subjected to BN-PAGE analyses followed by radioimaging. Assembly I, the assembly intermediate I; Assembly II, the assembly intermediate II. (B) Radiolabeled Tom40 was imported into WT, Tom13↓, and Tom38↓ mitochondria for 60 min at 23°C. After reisolation, the mitochondria were solubilized with 1% digitonin; incubated with the antibodies against Tim23 (αTim23), Mas37 (αMas37), and Tom22 (αTom22) or without antibodies (none); and analyzed by BN-PAGE followed by radioimaging. (C) Radiolabeled Tom40 was imported into WT and Tom38↓ mitochondria for 90 min at 23°C and subjected to osmotic swelling (SW), trypsin treatment (100 μg/ml for 30 min on ice), or alkaline extraction (Na2CO3, as Fig. 1). As a control, translated Tom40 was treated with (lane 2) or without (lane 1) trypsin. Relative amounts of radiolabeled Tom40 imported into Tom38↓ mitochondria were compared with those into wild-type mitochondria (set to 100%): TOM40 complex (BN-PAGE), lane 15/lane 5 in A; Trypsin treatment, −SW, lane 4/lane 3 in C; Trypsin treatment, +SW, lane 6/lane 5 in C; Alkaline extraction, ppt, lane 8/lane 7 in C. Endogenous Tom40 was also subjected to similar trypsin treatment followed by immunoblotting with anti-Tom40 antibodies. Asterisks indicate a clipped form of Tom40. (D) In vitro import of porin into WT (closed circles), Tom13↓ (open circles), and Tom38↓ (open squares) mitochondria were performed at 23°C as described in Fig. 3. The amounts of imported, PK-protected protein were plotted.
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fig4: Depletion of Tom13 or Tom38 affects assembly of Tom40 and porin in the mitochondrial outer membrane in vitro. (A) 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 yeast Tom40 was incubated with WT, Tom13↓, and Tom38↓ mitochondria at 23°C for the indicated times. The mitochondria were reisolated by centrifugation, solubilized with 1% digitonin (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.5 mM EDTA, and 10% glycerol) for 10 min on ice, and subjected to BN-PAGE analyses followed by radioimaging. Assembly I, the assembly intermediate I; Assembly II, the assembly intermediate II. (B) Radiolabeled Tom40 was imported into WT, Tom13↓, and Tom38↓ mitochondria for 60 min at 23°C. After reisolation, the mitochondria were solubilized with 1% digitonin; incubated with the antibodies against Tim23 (αTim23), Mas37 (αMas37), and Tom22 (αTom22) or without antibodies (none); and analyzed by BN-PAGE followed by radioimaging. (C) Radiolabeled Tom40 was imported into WT and Tom38↓ mitochondria for 90 min at 23°C and subjected to osmotic swelling (SW), trypsin treatment (100 μg/ml for 30 min on ice), or alkaline extraction (Na2CO3, as Fig. 1). As a control, translated Tom40 was treated with (lane 2) or without (lane 1) trypsin. Relative amounts of radiolabeled Tom40 imported into Tom38↓ mitochondria were compared with those into wild-type mitochondria (set to 100%): TOM40 complex (BN-PAGE), lane 15/lane 5 in A; Trypsin treatment, −SW, lane 4/lane 3 in C; Trypsin treatment, +SW, lane 6/lane 5 in C; Alkaline extraction, ppt, lane 8/lane 7 in C. Endogenous Tom40 was also subjected to similar trypsin treatment followed by immunoblotting with anti-Tom40 antibodies. Asterisks indicate a clipped form of Tom40. (D) In vitro import of porin into WT (closed circles), Tom13↓ (open circles), and Tom38↓ (open squares) mitochondria were performed at 23°C as described in Fig. 3. The amounts of imported, PK-protected protein were plotted.

Mentions: Next, we analyzed the assembly of Tom40 in wild-type, Tom13↓, and Tom38↓ mitochondria. Model et al. (2001) reported that Tom40 was assembled into the 450-kD TOM40 complex via two successive intermediates of the 250-kD complex (the assembly intermediate I) and the 100-kD complex (the assembly intermediate II). Indeed, when we incubated radiolabeled Tom40 with isolated mitochondria from wild-type cells (14 or 10 h after shift to galactose-free medium) and analyzed the proteins by blue-native PAGE (BN-PAGE), we observed its accumulation in both the 250-kD double bands and the 100-kD band, and subsequently in the 450-kD band (Fig. 4 A, lanes 1–5). The 250-kD band represents the assembly intermediates I involving the SAM complex and the 450-kD band represents the final TOM40 complex because addition of the anti-Mas37 antibodies and the anti-Tom22 antibodies, respectively, shifted them to a higher molecular mass range on the BN-PAGE gel (Fig. 4 B, lane 3 and 4).


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 affects assembly of Tom40 and porin in the mitochondrial outer membrane in vitro. (A) 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 yeast Tom40 was incubated with WT, Tom13↓, and Tom38↓ mitochondria at 23°C for the indicated times. The mitochondria were reisolated by centrifugation, solubilized with 1% digitonin (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.5 mM EDTA, and 10% glycerol) for 10 min on ice, and subjected to BN-PAGE analyses followed by radioimaging. Assembly I, the assembly intermediate I; Assembly II, the assembly intermediate II. (B) Radiolabeled Tom40 was imported into WT, Tom13↓, and Tom38↓ mitochondria for 60 min at 23°C. After reisolation, the mitochondria were solubilized with 1% digitonin; incubated with the antibodies against Tim23 (αTim23), Mas37 (αMas37), and Tom22 (αTom22) or without antibodies (none); and analyzed by BN-PAGE followed by radioimaging. (C) Radiolabeled Tom40 was imported into WT and Tom38↓ mitochondria for 90 min at 23°C and subjected to osmotic swelling (SW), trypsin treatment (100 μg/ml for 30 min on ice), or alkaline extraction (Na2CO3, as Fig. 1). As a control, translated Tom40 was treated with (lane 2) or without (lane 1) trypsin. Relative amounts of radiolabeled Tom40 imported into Tom38↓ mitochondria were compared with those into wild-type mitochondria (set to 100%): TOM40 complex (BN-PAGE), lane 15/lane 5 in A; Trypsin treatment, −SW, lane 4/lane 3 in C; Trypsin treatment, +SW, lane 6/lane 5 in C; Alkaline extraction, ppt, lane 8/lane 7 in C. Endogenous Tom40 was also subjected to similar trypsin treatment followed by immunoblotting with anti-Tom40 antibodies. Asterisks indicate a clipped form of Tom40. (D) In vitro import of porin into WT (closed circles), Tom13↓ (open circles), and Tom38↓ (open squares) mitochondria were performed at 23°C as described in Fig. 3. The amounts of imported, PK-protected protein were plotted.
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fig4: Depletion of Tom13 or Tom38 affects assembly of Tom40 and porin in the mitochondrial outer membrane in vitro. (A) 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 yeast Tom40 was incubated with WT, Tom13↓, and Tom38↓ mitochondria at 23°C for the indicated times. The mitochondria were reisolated by centrifugation, solubilized with 1% digitonin (20 mM Tris-HCl, pH 7.4, 50 mM NaCl, 0.5 mM EDTA, and 10% glycerol) for 10 min on ice, and subjected to BN-PAGE analyses followed by radioimaging. Assembly I, the assembly intermediate I; Assembly II, the assembly intermediate II. (B) Radiolabeled Tom40 was imported into WT, Tom13↓, and Tom38↓ mitochondria for 60 min at 23°C. After reisolation, the mitochondria were solubilized with 1% digitonin; incubated with the antibodies against Tim23 (αTim23), Mas37 (αMas37), and Tom22 (αTom22) or without antibodies (none); and analyzed by BN-PAGE followed by radioimaging. (C) Radiolabeled Tom40 was imported into WT and Tom38↓ mitochondria for 90 min at 23°C and subjected to osmotic swelling (SW), trypsin treatment (100 μg/ml for 30 min on ice), or alkaline extraction (Na2CO3, as Fig. 1). As a control, translated Tom40 was treated with (lane 2) or without (lane 1) trypsin. Relative amounts of radiolabeled Tom40 imported into Tom38↓ mitochondria were compared with those into wild-type mitochondria (set to 100%): TOM40 complex (BN-PAGE), lane 15/lane 5 in A; Trypsin treatment, −SW, lane 4/lane 3 in C; Trypsin treatment, +SW, lane 6/lane 5 in C; Alkaline extraction, ppt, lane 8/lane 7 in C. Endogenous Tom40 was also subjected to similar trypsin treatment followed by immunoblotting with anti-Tom40 antibodies. Asterisks indicate a clipped form of Tom40. (D) In vitro import of porin into WT (closed circles), Tom13↓ (open circles), and Tom38↓ (open squares) mitochondria were performed at 23°C as described in Fig. 3. The amounts of imported, PK-protected protein were plotted.
Mentions: Next, we analyzed the assembly of Tom40 in wild-type, Tom13↓, and Tom38↓ mitochondria. Model et al. (2001) reported that Tom40 was assembled into the 450-kD TOM40 complex via two successive intermediates of the 250-kD complex (the assembly intermediate I) and the 100-kD complex (the assembly intermediate II). Indeed, when we incubated radiolabeled Tom40 with isolated mitochondria from wild-type cells (14 or 10 h after shift to galactose-free medium) and analyzed the proteins by blue-native PAGE (BN-PAGE), we observed its accumulation in both the 250-kD double bands and the 100-kD band, and subsequently in the 450-kD band (Fig. 4 A, lanes 1–5). The 250-kD band represents the assembly intermediates I involving the SAM complex and the 450-kD band represents the final TOM40 complex because addition of the anti-Mas37 antibodies and the anti-Tom22 antibodies, respectively, shifted them to a higher molecular mass range on the BN-PAGE gel (Fig. 4 B, lane 3 and 4).

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
Related in: MedlinePlus