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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 mediates protein insertion and assembly into the mitochondrial outer membrane. (A) Mitochondria (50 mg) isolated from wild-type or mutant yeast cells were incubated with 35S-labeled Tom40 for the indicated times and then isolated for BN-PAGE and phosphorimage analysis. The identity of each TOM complex intermediate was estimated from the size of marker proteins (Model et al., 2001). (B) Mitochondria (50 mg) isolated from wild-type or mutant yeast cells were incubated with 35S-labeled VDAC. Duplicate assays were committed either to BN-PAGE or, after treatment with proteinase K, analyzed by SDS-PAGE and fluorography (Krimmer et al., 2001). Quantitation of the VDAC complexes is graphed beside the fluorogram. (C) 35S-labeled Su9-DHFR was incubated with mitochondria for the indicated times, and then the mitochondria were treated with proteinase K. Imported proteins were analyzed by SDS-PAGE and fluorography. p indicates the precursor form of Su9-DHFR. (D) Mitochondria were isolated from wild-type or omp85↓ cells and assayed by immunoblotting for levels of Omp85 and the TOM complex subunits Tom20 and Tom40. (E) Mitochondria (50 mg) isolated from wild-type or omp85↓ cells were incubated with 35S-labeled Tom40 for the indicated times and then isolated for BN-PAGE and phosphorimage analysis.
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fig3: Omp85 mediates protein insertion and assembly into the mitochondrial outer membrane. (A) Mitochondria (50 mg) isolated from wild-type or mutant yeast cells were incubated with 35S-labeled Tom40 for the indicated times and then isolated for BN-PAGE and phosphorimage analysis. The identity of each TOM complex intermediate was estimated from the size of marker proteins (Model et al., 2001). (B) Mitochondria (50 mg) isolated from wild-type or mutant yeast cells were incubated with 35S-labeled VDAC. Duplicate assays were committed either to BN-PAGE or, after treatment with proteinase K, analyzed by SDS-PAGE and fluorography (Krimmer et al., 2001). Quantitation of the VDAC complexes is graphed beside the fluorogram. (C) 35S-labeled Su9-DHFR was incubated with mitochondria for the indicated times, and then the mitochondria were treated with proteinase K. Imported proteins were analyzed by SDS-PAGE and fluorography. p indicates the precursor form of Su9-DHFR. (D) Mitochondria were isolated from wild-type or omp85↓ cells and assayed by immunoblotting for levels of Omp85 and the TOM complex subunits Tom20 and Tom40. (E) Mitochondria (50 mg) isolated from wild-type or omp85↓ cells were incubated with 35S-labeled Tom40 for the indicated times and then isolated for BN-PAGE and phosphorimage analysis.

Mentions: To assay the kinetics of mitochondrial protein insertion and assembly, mitochondria were isolated from the omp85-94 mutant and incubated with radiolabeled Tom40 or VDAC (Fig. 3). Tom40 is assembled into the hetero-oligomeric TOM complex through a series of transient assembly intermediates, visualized in time course experiments analyzed by BN-PAGE (Model et al., 2001; Wiedemann et al., 2003). The omp85-94 mutant shows a twofold defect in the kinetics of Tom40 assembly into the TOM complex (Fig. 3 A). Similarly, assembly of VDAC into the trimeric native state can be analyzed by BN-PAGE (Krimmer et al., 2001) and is achieved at 50% the wild-type rate in the omp85-94 mutant (Fig. 3 B). Insertion of VDAC into the outer membrane, as judged by accumulation of a protease-resistant form, is only marginally compromised in the mutants (Fig. 3 B, SDS-PAGE). The protein translocation defect in the omp85-94 mutants is selective for outer membrane proteins, with import of the matrix-located protein Su9-DHFR proceeding at wild-type rates in the omp85-94 mitochondria (Fig. 3 C).


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 mediates protein insertion and assembly into the mitochondrial outer membrane. (A) Mitochondria (50 mg) isolated from wild-type or mutant yeast cells were incubated with 35S-labeled Tom40 for the indicated times and then isolated for BN-PAGE and phosphorimage analysis. The identity of each TOM complex intermediate was estimated from the size of marker proteins (Model et al., 2001). (B) Mitochondria (50 mg) isolated from wild-type or mutant yeast cells were incubated with 35S-labeled VDAC. Duplicate assays were committed either to BN-PAGE or, after treatment with proteinase K, analyzed by SDS-PAGE and fluorography (Krimmer et al., 2001). Quantitation of the VDAC complexes is graphed beside the fluorogram. (C) 35S-labeled Su9-DHFR was incubated with mitochondria for the indicated times, and then the mitochondria were treated with proteinase K. Imported proteins were analyzed by SDS-PAGE and fluorography. p indicates the precursor form of Su9-DHFR. (D) Mitochondria were isolated from wild-type or omp85↓ cells and assayed by immunoblotting for levels of Omp85 and the TOM complex subunits Tom20 and Tom40. (E) Mitochondria (50 mg) isolated from wild-type or omp85↓ cells were incubated with 35S-labeled Tom40 for the indicated times and then isolated for BN-PAGE and phosphorimage analysis.
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Related In: Results  -  Collection

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fig3: Omp85 mediates protein insertion and assembly into the mitochondrial outer membrane. (A) Mitochondria (50 mg) isolated from wild-type or mutant yeast cells were incubated with 35S-labeled Tom40 for the indicated times and then isolated for BN-PAGE and phosphorimage analysis. The identity of each TOM complex intermediate was estimated from the size of marker proteins (Model et al., 2001). (B) Mitochondria (50 mg) isolated from wild-type or mutant yeast cells were incubated with 35S-labeled VDAC. Duplicate assays were committed either to BN-PAGE or, after treatment with proteinase K, analyzed by SDS-PAGE and fluorography (Krimmer et al., 2001). Quantitation of the VDAC complexes is graphed beside the fluorogram. (C) 35S-labeled Su9-DHFR was incubated with mitochondria for the indicated times, and then the mitochondria were treated with proteinase K. Imported proteins were analyzed by SDS-PAGE and fluorography. p indicates the precursor form of Su9-DHFR. (D) Mitochondria were isolated from wild-type or omp85↓ cells and assayed by immunoblotting for levels of Omp85 and the TOM complex subunits Tom20 and Tom40. (E) Mitochondria (50 mg) isolated from wild-type or omp85↓ cells were incubated with 35S-labeled Tom40 for the indicated times and then isolated for BN-PAGE and phosphorimage analysis.
Mentions: To assay the kinetics of mitochondrial protein insertion and assembly, mitochondria were isolated from the omp85-94 mutant and incubated with radiolabeled Tom40 or VDAC (Fig. 3). Tom40 is assembled into the hetero-oligomeric TOM complex through a series of transient assembly intermediates, visualized in time course experiments analyzed by BN-PAGE (Model et al., 2001; Wiedemann et al., 2003). The omp85-94 mutant shows a twofold defect in the kinetics of Tom40 assembly into the TOM complex (Fig. 3 A). Similarly, assembly of VDAC into the trimeric native state can be analyzed by BN-PAGE (Krimmer et al., 2001) and is achieved at 50% the wild-type rate in the omp85-94 mutant (Fig. 3 B). Insertion of VDAC into the outer membrane, as judged by accumulation of a protease-resistant form, is only marginally compromised in the mutants (Fig. 3 B, SDS-PAGE). The protein translocation defect in the omp85-94 mutants is selective for outer membrane proteins, with import of the matrix-located protein Su9-DHFR proceeding at wild-type rates in the omp85-94 mitochondria (Fig. 3 C).

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