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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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Tom13 and Tom38 are mitochondrial outer membrane proteins. Mitochondria were prepared from yeast strains expressing Tom13-HA or Tom38-FLAG. Mitochondria and mitoplasts generated by osmotic swelling (SW) were treated with 500 μg/ml PK for 30 min on ice. Mitochondria were treated with either 0.1 M Na2CO3 or 1% Triton X-100 with 500 mM NaCl (TX-100), and then pellets (ppt) and supernatants (sup) were separated by centrifugation. Proteins were detected by immunoblotting with the antibodies against the HA epitope (for Tom13-HA mitochondria), the FLAG epitope (for Tom38-FLAG mitochondria), or indicated proteins (for Tom38-FLAG mitochondria).
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fig1: Tom13 and Tom38 are mitochondrial outer membrane proteins. Mitochondria were prepared from yeast strains expressing Tom13-HA or Tom38-FLAG. Mitochondria and mitoplasts generated by osmotic swelling (SW) were treated with 500 μg/ml PK for 30 min on ice. Mitochondria were treated with either 0.1 M Na2CO3 or 1% Triton X-100 with 500 mM NaCl (TX-100), and then pellets (ppt) and supernatants (sup) were separated by centrifugation. Proteins were detected by immunoblotting with the antibodies against the HA epitope (for Tom13-HA mitochondria), the FLAG epitope (for Tom38-FLAG mitochondria), or indicated proteins (for Tom38-FLAG mitochondria).

Mentions: Tom13, the gene product of YOL026C, and Tom38, that of YHR083W, are deposited as essential proteins in the database of the yeast deletion project (Winzeler et al., 1999; http://www-deletion.stanford.edu/YDPM/YDPM_index.html). We analyzed their properties in mitochondrial association by protease treatment and alkaline extraction of isolated yeast mitochondria followed by immunoblotting for the HA or FLAG epitope tags attached to the COOH termini of Tom13 (Fig. 1, Tom13-HA) and Tom38 (Fig. 1, Tom38-FLAG). Both Tom13-HA and Tom38-FLAG were functional in vivo because they restored the growth defects of the mutants of their original genes (unpublished data). When mitochondria were treated with proteinase K (PK), both Tom13 and Tom38 disappeared, suggesting that their COOH termini are exposed to the cytosol (Fig. 1, lanes 1 and 2). Tom70, a surface-exposed outer membrane protein, was degraded; whereas Tim23, an inner membrane protein exposing a domain to the intermembrane space, and Mdj1p, a soluble matrix protein, remained intact after PK treatment. When the outer membrane of the mitochondria was selectively ruptured to make mitoplasts, treatment of the mitoplasts with PK led to degradation of Tom13, Tom38, Tom70, and Tim23, but not of Mdj1p (Fig. 1, lanes 3 and 4). This finding confirms that Tom13 and Tom38 reside outside the inner membrane. Tom13 was, like integral membrane proteins Tom70 and Tim23, not extracted by alkaline treatment of mitochondria, but was solubilized by treatment of mitochondria with Triton X-100 (Fig. 1, lanes 5–8). In contrast, Tom38 was extracted by both alkaline treatment of mitochondria or treatment of mitochondria with Triton X-100 (Fig. 1, lanes 5–8). These results indicate that Tom13 and Tom38 are an integral membrane protein and a peripheral membrane protein, respectively, of the mitochondrial outer membrane and expose at least their COOH termini to the cytosol.


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)

Tom13 and Tom38 are mitochondrial outer membrane proteins. Mitochondria were prepared from yeast strains expressing Tom13-HA or Tom38-FLAG. Mitochondria and mitoplasts generated by osmotic swelling (SW) were treated with 500 μg/ml PK for 30 min on ice. Mitochondria were treated with either 0.1 M Na2CO3 or 1% Triton X-100 with 500 mM NaCl (TX-100), and then pellets (ppt) and supernatants (sup) were separated by centrifugation. Proteins were detected by immunoblotting with the antibodies against the HA epitope (for Tom13-HA mitochondria), the FLAG epitope (for Tom38-FLAG mitochondria), or indicated proteins (for Tom38-FLAG mitochondria).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172422&req=5

fig1: Tom13 and Tom38 are mitochondrial outer membrane proteins. Mitochondria were prepared from yeast strains expressing Tom13-HA or Tom38-FLAG. Mitochondria and mitoplasts generated by osmotic swelling (SW) were treated with 500 μg/ml PK for 30 min on ice. Mitochondria were treated with either 0.1 M Na2CO3 or 1% Triton X-100 with 500 mM NaCl (TX-100), and then pellets (ppt) and supernatants (sup) were separated by centrifugation. Proteins were detected by immunoblotting with the antibodies against the HA epitope (for Tom13-HA mitochondria), the FLAG epitope (for Tom38-FLAG mitochondria), or indicated proteins (for Tom38-FLAG mitochondria).
Mentions: Tom13, the gene product of YOL026C, and Tom38, that of YHR083W, are deposited as essential proteins in the database of the yeast deletion project (Winzeler et al., 1999; http://www-deletion.stanford.edu/YDPM/YDPM_index.html). We analyzed their properties in mitochondrial association by protease treatment and alkaline extraction of isolated yeast mitochondria followed by immunoblotting for the HA or FLAG epitope tags attached to the COOH termini of Tom13 (Fig. 1, Tom13-HA) and Tom38 (Fig. 1, Tom38-FLAG). Both Tom13-HA and Tom38-FLAG were functional in vivo because they restored the growth defects of the mutants of their original genes (unpublished data). When mitochondria were treated with proteinase K (PK), both Tom13 and Tom38 disappeared, suggesting that their COOH termini are exposed to the cytosol (Fig. 1, lanes 1 and 2). Tom70, a surface-exposed outer membrane protein, was degraded; whereas Tim23, an inner membrane protein exposing a domain to the intermembrane space, and Mdj1p, a soluble matrix protein, remained intact after PK treatment. When the outer membrane of the mitochondria was selectively ruptured to make mitoplasts, treatment of the mitoplasts with PK led to degradation of Tom13, Tom38, Tom70, and Tim23, but not of Mdj1p (Fig. 1, lanes 3 and 4). This finding confirms that Tom13 and Tom38 reside outside the inner membrane. Tom13 was, like integral membrane proteins Tom70 and Tim23, not extracted by alkaline treatment of mitochondria, but was solubilized by treatment of mitochondria with Triton X-100 (Fig. 1, lanes 5–8). In contrast, Tom38 was extracted by both alkaline treatment of mitochondria or treatment of mitochondria with Triton X-100 (Fig. 1, lanes 5–8). These results indicate that Tom13 and Tom38 are an integral membrane protein and a peripheral membrane protein, respectively, of the mitochondrial outer membrane and expose at least their COOH termini to the cytosol.

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