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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 not affected in tom40-3 mutant mitochondria. (A) The experiment was performed as described in the legend to Fig. 4 A except that mitochondria isolated from the yeast mutant strain tom40-3 and the corresponding wild-type strain (WT) were used, and import was performed for the indicated periods. (Non-ass. porin) Porin precursor. The assembly of porin in tom40-3 mitochondria was comparable to that of wild-type mitochondria, independently whether the import was directly performed at 25°C (as in A), or whether the mitochondria were preincubated at 37°C. (B) Quantification of assembled porin. The amount of assembled radiolabeled porin in wild-type mitochondria after 30 min was set to 100% (control).
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Figure 6: Import of porin is not affected in tom40-3 mutant mitochondria. (A) The experiment was performed as described in the legend to Fig. 4 A except that mitochondria isolated from the yeast mutant strain tom40-3 and the corresponding wild-type strain (WT) were used, and import was performed for the indicated periods. (Non-ass. porin) Porin precursor. The assembly of porin in tom40-3 mitochondria was comparable to that of wild-type mitochondria, independently whether the import was directly performed at 25°C (as in A), or whether the mitochondria were preincubated at 37°C. (B) Quantification of assembled porin. The amount of assembled radiolabeled porin in wild-type mitochondria after 30 min was set to 100% (control).

Mentions: Tom40 is an essential yeast protein; therefore, deletion mutants are not viable (Baker et al. 1990). A conditional mutant allele of Tom40 has been characterized, termed tom40-3 (isp42-3 in the old nomenclature) (Kassenbrock et al. 1993; Pfanner et al. 1996; Schleiff et al. 1999). We used the BN-PAGE assay to analyze assembly of porin in tom40-3 mitochondria. For comparison, radiolabeled porin was incubated with isolated mitochondria of the corresponding wild-type strain. After lysis in digitonin and BN-PAGE, assembled porin was mainly found in the 200-kD complex and to a smaller degree in the 440-kD complex (Fig. 6 A, lanes 1–3). Immunodecoration of preexisting porin revealed an indistinguishable behavior on BN-PAGE, confirming the specificity of the assay (not shown). (A higher molecular weight species [∼500 kD] was a mitochondrial complex not related to porin that became nonspecifically radiolabeled.) The two major assembly complexes (200 and 440 kD) of porin are thus observed in different strain backgrounds (with variations in the relative amounts). When radiolabeled porin was incubated with tom40-3 mitochondria, we did not observe any significant defect in import and assembly of porin (Fig. 6 A, lanes 4–6, and B), demonstrating that tom40-3 mitochondria indeed are not deficient in porin biogenesis. This is in agreement with results reported by Schleiff et al. 1999.


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 not affected in tom40-3 mutant mitochondria. (A) The experiment was performed as described in the legend to Fig. 4 A except that mitochondria isolated from the yeast mutant strain tom40-3 and the corresponding wild-type strain (WT) were used, and import was performed for the indicated periods. (Non-ass. porin) Porin precursor. The assembly of porin in tom40-3 mitochondria was comparable to that of wild-type mitochondria, independently whether the import was directly performed at 25°C (as in A), or whether the mitochondria were preincubated at 37°C. (B) Quantification of assembled porin. The amount of assembled radiolabeled porin in wild-type mitochondria after 30 min was set to 100% (control).
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Related In: Results  -  Collection

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Figure 6: Import of porin is not affected in tom40-3 mutant mitochondria. (A) The experiment was performed as described in the legend to Fig. 4 A except that mitochondria isolated from the yeast mutant strain tom40-3 and the corresponding wild-type strain (WT) were used, and import was performed for the indicated periods. (Non-ass. porin) Porin precursor. The assembly of porin in tom40-3 mitochondria was comparable to that of wild-type mitochondria, independently whether the import was directly performed at 25°C (as in A), or whether the mitochondria were preincubated at 37°C. (B) Quantification of assembled porin. The amount of assembled radiolabeled porin in wild-type mitochondria after 30 min was set to 100% (control).
Mentions: Tom40 is an essential yeast protein; therefore, deletion mutants are not viable (Baker et al. 1990). A conditional mutant allele of Tom40 has been characterized, termed tom40-3 (isp42-3 in the old nomenclature) (Kassenbrock et al. 1993; Pfanner et al. 1996; Schleiff et al. 1999). We used the BN-PAGE assay to analyze assembly of porin in tom40-3 mitochondria. For comparison, radiolabeled porin was incubated with isolated mitochondria of the corresponding wild-type strain. After lysis in digitonin and BN-PAGE, assembled porin was mainly found in the 200-kD complex and to a smaller degree in the 440-kD complex (Fig. 6 A, lanes 1–3). Immunodecoration of preexisting porin revealed an indistinguishable behavior on BN-PAGE, confirming the specificity of the assay (not shown). (A higher molecular weight species [∼500 kD] was a mitochondrial complex not related to porin that became nonspecifically radiolabeled.) The two major assembly complexes (200 and 440 kD) of porin are thus observed in different strain backgrounds (with variations in the relative amounts). When radiolabeled porin was incubated with tom40-3 mitochondria, we did not observe any significant defect in import and assembly of porin (Fig. 6 A, lanes 4–6, and B), demonstrating that tom40-3 mitochondria indeed are not deficient in porin biogenesis. This is in agreement with results reported by Schleiff et al. 1999.

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.

Show MeSH
Related in: MedlinePlus