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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 involved in mitochondrial protein import in vivo. (A) Yeast strains in which Tom13 or Tom38 is down-regulated slowed their growth on galactose-free medium. Wild-type cells (WT) and those carrying the TOM13 (GAL-TOM13) or TOM38 gene (GAL-TOM38) under control of the GAL7 promoter were first grown on galactose-containing medium and then transferred to galactose-free medium. (B) One of the two chromosomal TOM13 (top) or TOM38 (bottom) genes in a yeast diploid strain, W303-AB, was disrupted, the diploid cells were sporulated, and six different asci were dissected. The four spores recovered from each asci were allowed to germinate and to grow on YPD for 45 h at 30°C. (C) Total lysates were prepared from WT, GAL-TOM13, and GAL-TOM38 cells, which were grown at 23°C for12 h in lactate medium (+0.1% galactose), diluted, and then grown at 23°C for the indicated hours in lactate medium (+0.1% glucose). Total proteins were isolated and analyzed by SDS-PAGE and immunoblotting with antibodies against the indicated proteins. The arrowheads indicate the accumulated precursor forms of mtHsp60 and Mdj1p.
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fig2: Tom13 and Tom38 are involved in mitochondrial protein import in vivo. (A) Yeast strains in which Tom13 or Tom38 is down-regulated slowed their growth on galactose-free medium. Wild-type cells (WT) and those carrying the TOM13 (GAL-TOM13) or TOM38 gene (GAL-TOM38) under control of the GAL7 promoter were first grown on galactose-containing medium and then transferred to galactose-free medium. (B) One of the two chromosomal TOM13 (top) or TOM38 (bottom) genes in a yeast diploid strain, W303-AB, was disrupted, the diploid cells were sporulated, and six different asci were dissected. The four spores recovered from each asci were allowed to germinate and to grow on YPD for 45 h at 30°C. (C) Total lysates were prepared from WT, GAL-TOM13, and GAL-TOM38 cells, which were grown at 23°C for12 h in lactate medium (+0.1% galactose), diluted, and then grown at 23°C for the indicated hours in lactate medium (+0.1% glucose). Total proteins were isolated and analyzed by SDS-PAGE and immunoblotting with antibodies against the indicated proteins. The arrowheads indicate the accumulated precursor forms of mtHsp60 and Mdj1p.

Mentions: To assess the functions of Tom13 and Tom38, we constructed yeast strains GAL-TOM13 and GAL-TOM38 in which the galactose-inducible GAL7 promoter was integrated into the chromosome in front of YOL026C and YHR083W, respectively, to achieve regulated expression of Tom13 and Tom38 by galactose. Tom38 is essential for viability of yeast cells as confirmed by tetrad analysis (Fig. 2 B), and the GAL-TOM38 cell growth slowed down significantly 20 h after shift to galactose-free medium (Fig. 2 A).


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 involved in mitochondrial protein import in vivo. (A) Yeast strains in which Tom13 or Tom38 is down-regulated slowed their growth on galactose-free medium. Wild-type cells (WT) and those carrying the TOM13 (GAL-TOM13) or TOM38 gene (GAL-TOM38) under control of the GAL7 promoter were first grown on galactose-containing medium and then transferred to galactose-free medium. (B) One of the two chromosomal TOM13 (top) or TOM38 (bottom) genes in a yeast diploid strain, W303-AB, was disrupted, the diploid cells were sporulated, and six different asci were dissected. The four spores recovered from each asci were allowed to germinate and to grow on YPD for 45 h at 30°C. (C) Total lysates were prepared from WT, GAL-TOM13, and GAL-TOM38 cells, which were grown at 23°C for12 h in lactate medium (+0.1% galactose), diluted, and then grown at 23°C for the indicated hours in lactate medium (+0.1% glucose). Total proteins were isolated and analyzed by SDS-PAGE and immunoblotting with antibodies against the indicated proteins. The arrowheads indicate the accumulated precursor forms of mtHsp60 and Mdj1p.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172422&req=5

fig2: Tom13 and Tom38 are involved in mitochondrial protein import in vivo. (A) Yeast strains in which Tom13 or Tom38 is down-regulated slowed their growth on galactose-free medium. Wild-type cells (WT) and those carrying the TOM13 (GAL-TOM13) or TOM38 gene (GAL-TOM38) under control of the GAL7 promoter were first grown on galactose-containing medium and then transferred to galactose-free medium. (B) One of the two chromosomal TOM13 (top) or TOM38 (bottom) genes in a yeast diploid strain, W303-AB, was disrupted, the diploid cells were sporulated, and six different asci were dissected. The four spores recovered from each asci were allowed to germinate and to grow on YPD for 45 h at 30°C. (C) Total lysates were prepared from WT, GAL-TOM13, and GAL-TOM38 cells, which were grown at 23°C for12 h in lactate medium (+0.1% galactose), diluted, and then grown at 23°C for the indicated hours in lactate medium (+0.1% glucose). Total proteins were isolated and analyzed by SDS-PAGE and immunoblotting with antibodies against the indicated proteins. The arrowheads indicate the accumulated precursor forms of mtHsp60 and Mdj1p.
Mentions: To assess the functions of Tom13 and Tom38, we constructed yeast strains GAL-TOM13 and GAL-TOM38 in which the galactose-inducible GAL7 promoter was integrated into the chromosome in front of YOL026C and YHR083W, respectively, to achieve regulated expression of Tom13 and Tom38 by galactose. Tom38 is essential for viability of yeast cells as confirmed by tetrad analysis (Fig. 2 B), and the GAL-TOM38 cell growth slowed down significantly 20 h after shift to galactose-free medium (Fig. 2 A).

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