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Biogenesis of porin of the outer mitochondrial membrane involves an import pathway via receptors and the general import pore of the TOM complex.

Krimmer T, Rapaport D, Ryan MT, Meisinger C, Kassenbrock CK, Blachly-Dyson E, Forte M, Douglas MG, Neupert W, Nargang FE, Pfanner N - J. Cell Biol. (2001)

Bottom Line: The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40.Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway.We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biochemistry and Molecular Biology, University of Freiburg, D-79104 Freiburg, Germany.

ABSTRACT
Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro-imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.

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Import of porin is inhibited into tom40 mutant mitochondria. (A) Radiolabeled porin precursor (10 μl reticulocyte lysate per lane) was incubated with isolated yeast mitochondria as indicated (50 μg protein per lane) at 25°C. The mitochondria were reisolated, lysed in digitonin-containing buffer, and subjected to BN-PAGE and digital autoradiography. (B) Quantification of assembled porin by digital autoradiography. The total amount of assembled radiolabeled porin after 30 min in wild-type mitochondria was set to 100% (control). When the mitochondria were preincubated at 37°C, the assembly of porin in the tom40 mutant mitochondria was similarly reduced compared with wild-type mitochondria.
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Figure 8: Import of porin is inhibited into tom40 mutant mitochondria. (A) Radiolabeled porin precursor (10 μl reticulocyte lysate per lane) was incubated with isolated yeast mitochondria as indicated (50 μg protein per lane) at 25°C. The mitochondria were reisolated, lysed in digitonin-containing buffer, and subjected to BN-PAGE and digital autoradiography. (B) Quantification of assembled porin by digital autoradiography. The total amount of assembled radiolabeled porin after 30 min in wild-type mitochondria was set to 100% (control). When the mitochondria were preincubated at 37°C, the assembly of porin in the tom40 mutant mitochondria was similarly reduced compared with wild-type mitochondria.

Mentions: We then incubated radiolabeled porin with isolated wild-type, tom40-2, and tom40-4 mitochondria and analyzed them by BN-PAGE. The assembly of porin was inhibited in the mutant mitochondria (Fig. 8 A, lanes 4–9). A quantification showed a 60–70% reduction of porin assembly compared with the wild-type mitochondria (Fig. 8 B). Since the levels of all marker proteins analyzed along with the GIP complex are not disturbed in the mutant mitochondria, we conclude that a functional Tom40 is required for the biogenesis of porin.


Biogenesis of porin of the outer mitochondrial membrane involves an import pathway via receptors and the general import pore of the TOM complex.

Krimmer T, Rapaport D, Ryan MT, Meisinger C, Kassenbrock CK, Blachly-Dyson E, Forte M, Douglas MG, Neupert W, Nargang FE, Pfanner N - J. Cell Biol. (2001)

Import of porin is inhibited into tom40 mutant mitochondria. (A) Radiolabeled porin precursor (10 μl reticulocyte lysate per lane) was incubated with isolated yeast mitochondria as indicated (50 μg protein per lane) at 25°C. The mitochondria were reisolated, lysed in digitonin-containing buffer, and subjected to BN-PAGE and digital autoradiography. (B) Quantification of assembled porin by digital autoradiography. The total amount of assembled radiolabeled porin after 30 min in wild-type mitochondria was set to 100% (control). When the mitochondria were preincubated at 37°C, the assembly of porin in the tom40 mutant mitochondria was similarly reduced compared with wild-type mitochondria.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199606&req=5

Figure 8: Import of porin is inhibited into tom40 mutant mitochondria. (A) Radiolabeled porin precursor (10 μl reticulocyte lysate per lane) was incubated with isolated yeast mitochondria as indicated (50 μg protein per lane) at 25°C. The mitochondria were reisolated, lysed in digitonin-containing buffer, and subjected to BN-PAGE and digital autoradiography. (B) Quantification of assembled porin by digital autoradiography. The total amount of assembled radiolabeled porin after 30 min in wild-type mitochondria was set to 100% (control). When the mitochondria were preincubated at 37°C, the assembly of porin in the tom40 mutant mitochondria was similarly reduced compared with wild-type mitochondria.
Mentions: We then incubated radiolabeled porin with isolated wild-type, tom40-2, and tom40-4 mitochondria and analyzed them by BN-PAGE. The assembly of porin was inhibited in the mutant mitochondria (Fig. 8 A, lanes 4–9). A quantification showed a 60–70% reduction of porin assembly compared with the wild-type mitochondria (Fig. 8 B). Since the levels of all marker proteins analyzed along with the GIP complex are not disturbed in the mutant mitochondria, we conclude that a functional Tom40 is required for the biogenesis of porin.

Bottom Line: The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40.Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway.We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biochemistry and Molecular Biology, University of Freiburg, D-79104 Freiburg, Germany.

ABSTRACT
Porin, also termed the voltage-dependent anion channel, is the most abundant protein of the mitochondrial outer membrane. The process of import and assembly of the protein is known to be dependent on the surface receptor Tom20, but the requirement for other mitochondrial proteins remains controversial. We have used mitochondria from Neurospora crassa and Saccharomyces cerevisiae to analyze the import pathway of porin. Import of porin into isolated mitochondria in which the outer membrane has been opened is inhibited despite similar levels of Tom20 as in intact mitochondria. A matrix-destined precursor and the porin precursor compete for the same translocation sites in both normal mitochondria and mitochondria whose surface receptors have been removed, suggesting that both precursors utilize the general import pore. Using an assay established to monitor the assembly of in vitro-imported porin into preexisting porin complexes we have shown that besides Tom20, the biogenesis of porin depends on the central receptor Tom22, as well as Tom5 and Tom7 of the general import pore complex (translocase of the outer mitochondrial membrane [TOM] core complex). The characterization of two new mutant alleles of the essential pore protein Tom40 demonstrates that the import of porin also requires a functional Tom40. Moreover, the porin precursor can be cross-linked to Tom20, Tom22, and Tom40 on its import pathway. We conclude that import of porin does not proceed through the action of Tom20 alone, but requires an intact outer membrane and involves at least four more subunits of the TOM machinery, including the general import pore.

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