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The Omp85 family of proteins is essential for outer membrane biogenesis in mitochondria and bacteria.

Gentle I, Gabriel K, Beech P, Waller R, Lithgow T - J. Cell Biol. (2003)

Bottom Line: Integral proteins in the outer membrane of mitochondria control all aspects of organelle biogenesis, being required for protein import, mitochondrial fission, and, in metazoans, mitochondrial aspects of programmed cell death.How these integral proteins are assembled in the outer membrane had been unclear.In eukaryotes, Omp85 is present in the mitochondrial outer membrane.

View Article: PubMed Central - PubMed

Affiliation: Russell Grimwade School of Biochemistry and Molecular Biology, University of Melbourne, Melbourne 3010, Australia.

ABSTRACT
Integral proteins in the outer membrane of mitochondria control all aspects of organelle biogenesis, being required for protein import, mitochondrial fission, and, in metazoans, mitochondrial aspects of programmed cell death. How these integral proteins are assembled in the outer membrane had been unclear. In bacteria, Omp85 is an essential component of the protein insertion machinery, and we show that members of the Omp85 protein family are also found in eukaryotes ranging from plants to humans. In eukaryotes, Omp85 is present in the mitochondrial outer membrane. The gene encoding Omp85 is essential for cell viability in yeast, and conditional omp85 mutants have defects that arise from compromised insertion of integral proteins like voltage-dependent anion channel (VDAC) and components of the translocase in the outer membrane of mitochondria (TOM) complex into the mitochondrial outer membrane.

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Omp85 is a mitochondrial outer membrane protein in S. cerevisiae. (A) Thin sections of high-pressure frozen, wild-type yeast were labeled with affinity-purified Omp85 antiserum and 15-nm colloidal gold. The entire cell (left) shows that labeling, though weak, is specific to the mitochondrial periphery. Four, two, and three gold particles are present in the left, upper right, and lower right images, respectively. Bars, 500 nm. (B) Yeast cells fractionated into total extracts (T), cytosol (C), and purified mitochondria (M) were analyzed by SDS-PAGE and immunoblotting for the mitochondrial protein VDAC, Sec61 from the membrane of the endoplasmic reticulum, and the cytosolic protein hexokinase. (C) 35S-labeled Omp85 and VDAC were synthesized in vitro (10% of the total shown) and imported. Mitochondria were then extracted with 0.1 M Na2CO3 and purified by flotation through a sucrose cushion containing 0.1 M Na2CO3. After SDS-PAGE, 35S-labeled Omp85 and VDAC were detected by fluorography. (D) Omp85, Cyb2, and F1β were synthesized in vitro and incubated with yeast mitochondria. Where shown, p indicates the precursor form of the proteins. Mitochondria were then treated with 50 μg/ml proteinase K (+) in iso-osmotic buffer or in hypo-osmotic buffer.
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fig2: Omp85 is a mitochondrial outer membrane protein in S. cerevisiae. (A) Thin sections of high-pressure frozen, wild-type yeast were labeled with affinity-purified Omp85 antiserum and 15-nm colloidal gold. The entire cell (left) shows that labeling, though weak, is specific to the mitochondrial periphery. Four, two, and three gold particles are present in the left, upper right, and lower right images, respectively. Bars, 500 nm. (B) Yeast cells fractionated into total extracts (T), cytosol (C), and purified mitochondria (M) were analyzed by SDS-PAGE and immunoblotting for the mitochondrial protein VDAC, Sec61 from the membrane of the endoplasmic reticulum, and the cytosolic protein hexokinase. (C) 35S-labeled Omp85 and VDAC were synthesized in vitro (10% of the total shown) and imported. Mitochondria were then extracted with 0.1 M Na2CO3 and purified by flotation through a sucrose cushion containing 0.1 M Na2CO3. After SDS-PAGE, 35S-labeled Omp85 and VDAC were detected by fluorography. (D) Omp85, Cyb2, and F1β were synthesized in vitro and incubated with yeast mitochondria. Where shown, p indicates the precursor form of the proteins. Mitochondria were then treated with 50 μg/ml proteinase K (+) in iso-osmotic buffer or in hypo-osmotic buffer.

Mentions: Antibodies were raised to the yeast Omp85, affinity purified on the recombinant protein, and used to decorate thin sections of cryo-preserved yeast cells. Gold particles denote the presence of Omp85 on the surface of mitochondria (Fig. 2 A). Quantitation of gold labeling in 50 randomly selected cell sections showed 99 particles on or within ∼50 nm of the mitochondrial outer membrane, with a signal/noise ratio (West et al., 1998) of >66:1. Subcellular fractionation confirms that Omp85 is a mitochondrial protein; immunoblots decorated with specific antibodies show enrichment of both Omp85 and the outer membrane pore VDAC in purified mitochondria (Fig. 2 B).


The Omp85 family of proteins is essential for outer membrane biogenesis in mitochondria and bacteria.

Gentle I, Gabriel K, Beech P, Waller R, Lithgow T - J. Cell Biol. (2003)

Omp85 is a mitochondrial outer membrane protein in S. cerevisiae. (A) Thin sections of high-pressure frozen, wild-type yeast were labeled with affinity-purified Omp85 antiserum and 15-nm colloidal gold. The entire cell (left) shows that labeling, though weak, is specific to the mitochondrial periphery. Four, two, and three gold particles are present in the left, upper right, and lower right images, respectively. Bars, 500 nm. (B) Yeast cells fractionated into total extracts (T), cytosol (C), and purified mitochondria (M) were analyzed by SDS-PAGE and immunoblotting for the mitochondrial protein VDAC, Sec61 from the membrane of the endoplasmic reticulum, and the cytosolic protein hexokinase. (C) 35S-labeled Omp85 and VDAC were synthesized in vitro (10% of the total shown) and imported. Mitochondria were then extracted with 0.1 M Na2CO3 and purified by flotation through a sucrose cushion containing 0.1 M Na2CO3. After SDS-PAGE, 35S-labeled Omp85 and VDAC were detected by fluorography. (D) Omp85, Cyb2, and F1β were synthesized in vitro and incubated with yeast mitochondria. Where shown, p indicates the precursor form of the proteins. Mitochondria were then treated with 50 μg/ml proteinase K (+) in iso-osmotic buffer or in hypo-osmotic buffer.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171957&req=5

fig2: Omp85 is a mitochondrial outer membrane protein in S. cerevisiae. (A) Thin sections of high-pressure frozen, wild-type yeast were labeled with affinity-purified Omp85 antiserum and 15-nm colloidal gold. The entire cell (left) shows that labeling, though weak, is specific to the mitochondrial periphery. Four, two, and three gold particles are present in the left, upper right, and lower right images, respectively. Bars, 500 nm. (B) Yeast cells fractionated into total extracts (T), cytosol (C), and purified mitochondria (M) were analyzed by SDS-PAGE and immunoblotting for the mitochondrial protein VDAC, Sec61 from the membrane of the endoplasmic reticulum, and the cytosolic protein hexokinase. (C) 35S-labeled Omp85 and VDAC were synthesized in vitro (10% of the total shown) and imported. Mitochondria were then extracted with 0.1 M Na2CO3 and purified by flotation through a sucrose cushion containing 0.1 M Na2CO3. After SDS-PAGE, 35S-labeled Omp85 and VDAC were detected by fluorography. (D) Omp85, Cyb2, and F1β were synthesized in vitro and incubated with yeast mitochondria. Where shown, p indicates the precursor form of the proteins. Mitochondria were then treated with 50 μg/ml proteinase K (+) in iso-osmotic buffer or in hypo-osmotic buffer.
Mentions: Antibodies were raised to the yeast Omp85, affinity purified on the recombinant protein, and used to decorate thin sections of cryo-preserved yeast cells. Gold particles denote the presence of Omp85 on the surface of mitochondria (Fig. 2 A). Quantitation of gold labeling in 50 randomly selected cell sections showed 99 particles on or within ∼50 nm of the mitochondrial outer membrane, with a signal/noise ratio (West et al., 1998) of >66:1. Subcellular fractionation confirms that Omp85 is a mitochondrial protein; immunoblots decorated with specific antibodies show enrichment of both Omp85 and the outer membrane pore VDAC in purified mitochondria (Fig. 2 B).

Bottom Line: Integral proteins in the outer membrane of mitochondria control all aspects of organelle biogenesis, being required for protein import, mitochondrial fission, and, in metazoans, mitochondrial aspects of programmed cell death.How these integral proteins are assembled in the outer membrane had been unclear.In eukaryotes, Omp85 is present in the mitochondrial outer membrane.

View Article: PubMed Central - PubMed

Affiliation: Russell Grimwade School of Biochemistry and Molecular Biology, University of Melbourne, Melbourne 3010, Australia.

ABSTRACT
Integral proteins in the outer membrane of mitochondria control all aspects of organelle biogenesis, being required for protein import, mitochondrial fission, and, in metazoans, mitochondrial aspects of programmed cell death. How these integral proteins are assembled in the outer membrane had been unclear. In bacteria, Omp85 is an essential component of the protein insertion machinery, and we show that members of the Omp85 protein family are also found in eukaryotes ranging from plants to humans. In eukaryotes, Omp85 is present in the mitochondrial outer membrane. The gene encoding Omp85 is essential for cell viability in yeast, and conditional omp85 mutants have defects that arise from compromised insertion of integral proteins like voltage-dependent anion channel (VDAC) and components of the translocase in the outer membrane of mitochondria (TOM) complex into the mitochondrial outer membrane.

Show MeSH