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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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Tom38 forms a complex with Mas37 and Sam50. (A) Mitochondria containing Tom38-FLAG or Sam50-FLAG were solubilized as in Fig. 4 A and incubated with preimmune serum (Shift, preimmune) or anti-Mas37 antibodies (Shift, αMas37) for 1 h on ice. Solubilized protein complexes were analyzed by BN-PAGE and immunoblotting with the anti-FLAG antibody. The band marked with an asterisk is variable in different experiments. (B) Mitochondria containing Tom38-FLAG (top) or Tom13-FLAG (bottom) were solubilized with 0.8% digitonin (25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 50 mM 6-aminohexanoic acid, and 10% glycerol) in the presence (+Shift) or absence (−Shift) of the anti-FLAG antibody for 1 h on ice. Solubilized protein complexes were analyzed by glycerol density gradient centrifugation (10–30% glycerol and 0.2% digitonin in the same buffer for solubilization) at 200,000 g for 6 h at 4°C. After centrifugation, fractions were collected from the top and analyzed by immunoblotting using antibodies against the FLAG epitope, Tom38, Sam50, and Tim23. Numbers indicate fractions (from top to bottom). The asterisk indicates IgG.
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fig5: Tom38 forms a complex with Mas37 and Sam50. (A) Mitochondria containing Tom38-FLAG or Sam50-FLAG were solubilized as in Fig. 4 A and incubated with preimmune serum (Shift, preimmune) or anti-Mas37 antibodies (Shift, αMas37) for 1 h on ice. Solubilized protein complexes were analyzed by BN-PAGE and immunoblotting with the anti-FLAG antibody. The band marked with an asterisk is variable in different experiments. (B) Mitochondria containing Tom38-FLAG (top) or Tom13-FLAG (bottom) were solubilized with 0.8% digitonin (25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 50 mM 6-aminohexanoic acid, and 10% glycerol) in the presence (+Shift) or absence (−Shift) of the anti-FLAG antibody for 1 h on ice. Solubilized protein complexes were analyzed by glycerol density gradient centrifugation (10–30% glycerol and 0.2% digitonin in the same buffer for solubilization) at 200,000 g for 6 h at 4°C. After centrifugation, fractions were collected from the top and analyzed by immunoblotting using antibodies against the FLAG epitope, Tom38, Sam50, and Tim23. Numbers indicate fractions (from top to bottom). The asterisk indicates IgG.

Mentions: Because Tom38 plays a role in the Tom40 assembly similar to those by Mas37 and Sam50, Tom38 could be a subunit of the SAM complex. This is indeed the case (Fig. 5). When solubilized mitochondria with Tom38-FLAG were analyzed by BN-PAGE, the anti-FLAG antibody detected four bands with apparent molecular masses of 360, 290 (Fig. 5 A, lane 1, band a), 230 (Fig. 5 A, lane 1, band b), and 170 kD (Fig. 5 A, lane 1, band c). Bands a and b shifted to a higher molecular mass region upon addition of the anti-Mas37 antibodies (Fig. 5 A, lane 2). When solubilized mitochondria with Sam50 bearing the COOH-terminal FLAG epitope tag (Sam50-FLAG) were analyzed by BN-PAGE, the anti-FLAG antibody detected bands a, b, and c (Fig. 5 A, lane 3), and addition of the anti-Mas37 antibodies caused shifts of bands a and b, but not band c (Fig. 5 A, lane 4). These results suggest that bands a and b represent a complex consisting of Mas37, Tom38, and Sam50. Band c may represent a complex containing Tom38 and Sam50, but not Mas37. Evidently, Tom38 forms a complex with Mas37 and Sam50, although interactions among these subunits may be somehow dynamic. The interactions between Tom38 and Sam50 were also confirmed by coimmunoprecipitation (see online supplemental material).


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)

Tom38 forms a complex with Mas37 and Sam50. (A) Mitochondria containing Tom38-FLAG or Sam50-FLAG were solubilized as in Fig. 4 A and incubated with preimmune serum (Shift, preimmune) or anti-Mas37 antibodies (Shift, αMas37) for 1 h on ice. Solubilized protein complexes were analyzed by BN-PAGE and immunoblotting with the anti-FLAG antibody. The band marked with an asterisk is variable in different experiments. (B) Mitochondria containing Tom38-FLAG (top) or Tom13-FLAG (bottom) were solubilized with 0.8% digitonin (25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 50 mM 6-aminohexanoic acid, and 10% glycerol) in the presence (+Shift) or absence (−Shift) of the anti-FLAG antibody for 1 h on ice. Solubilized protein complexes were analyzed by glycerol density gradient centrifugation (10–30% glycerol and 0.2% digitonin in the same buffer for solubilization) at 200,000 g for 6 h at 4°C. After centrifugation, fractions were collected from the top and analyzed by immunoblotting using antibodies against the FLAG epitope, Tom38, Sam50, and Tim23. Numbers indicate fractions (from top to bottom). The asterisk indicates IgG.
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fig5: Tom38 forms a complex with Mas37 and Sam50. (A) Mitochondria containing Tom38-FLAG or Sam50-FLAG were solubilized as in Fig. 4 A and incubated with preimmune serum (Shift, preimmune) or anti-Mas37 antibodies (Shift, αMas37) for 1 h on ice. Solubilized protein complexes were analyzed by BN-PAGE and immunoblotting with the anti-FLAG antibody. The band marked with an asterisk is variable in different experiments. (B) Mitochondria containing Tom38-FLAG (top) or Tom13-FLAG (bottom) were solubilized with 0.8% digitonin (25 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 50 mM 6-aminohexanoic acid, and 10% glycerol) in the presence (+Shift) or absence (−Shift) of the anti-FLAG antibody for 1 h on ice. Solubilized protein complexes were analyzed by glycerol density gradient centrifugation (10–30% glycerol and 0.2% digitonin in the same buffer for solubilization) at 200,000 g for 6 h at 4°C. After centrifugation, fractions were collected from the top and analyzed by immunoblotting using antibodies against the FLAG epitope, Tom38, Sam50, and Tim23. Numbers indicate fractions (from top to bottom). The asterisk indicates IgG.
Mentions: Because Tom38 plays a role in the Tom40 assembly similar to those by Mas37 and Sam50, Tom38 could be a subunit of the SAM complex. This is indeed the case (Fig. 5). When solubilized mitochondria with Tom38-FLAG were analyzed by BN-PAGE, the anti-FLAG antibody detected four bands with apparent molecular masses of 360, 290 (Fig. 5 A, lane 1, band a), 230 (Fig. 5 A, lane 1, band b), and 170 kD (Fig. 5 A, lane 1, band c). Bands a and b shifted to a higher molecular mass region upon addition of the anti-Mas37 antibodies (Fig. 5 A, lane 2). When solubilized mitochondria with Sam50 bearing the COOH-terminal FLAG epitope tag (Sam50-FLAG) were analyzed by BN-PAGE, the anti-FLAG antibody detected bands a, b, and c (Fig. 5 A, lane 3), and addition of the anti-Mas37 antibodies caused shifts of bands a and b, but not band c (Fig. 5 A, lane 4). These results suggest that bands a and b represent a complex consisting of Mas37, Tom38, and Sam50. Band c may represent a complex containing Tom38 and Sam50, but not Mas37. Evidently, Tom38 forms a complex with Mas37 and Sam50, although interactions among these subunits may be somehow dynamic. The interactions between Tom38 and Sam50 were also confirmed by coimmunoprecipitation (see online supplemental material).

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