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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 F1β and porin into N. crassa mitochondria with damaged outer membranes. (A) Mitochondria were isolated from mutant strain 861 (tom22861) and the control strain 76-26 (WT) using standard procedures. Import of radiolabeled precursor proteins was conducted at 20°C for the indicated times. After the import reactions, mitochondria were reisolated and subjected to SDS-PAGE. The gels were blotted to nitrocellulose and subjected to autoradiography. The leftmost lane for each precursor contained 33% of the input lysate used in each import reaction. The precursor (p) and mature (m) forms of F1β are indicated. (B) After phosphorimaging of the imported proteins, the blot was decorated with antiserum to porin. (C) Effect of proteinase K on proteins from different submitochondrial compartments. Isolated mitochondria were either digested with proteinase K as described in Materials and Methods or were incubated at 0°C for 15 min without proteinase K. After reisolation, electrophoresis, and blotting, the membranes were decorated with antiserum to the indicated proteins. Tom20′, proteolytic fragment of Tom20. PK, proteinase K.
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Figure 1: Import of F1β and porin into N. crassa mitochondria with damaged outer membranes. (A) Mitochondria were isolated from mutant strain 861 (tom22861) and the control strain 76-26 (WT) using standard procedures. Import of radiolabeled precursor proteins was conducted at 20°C for the indicated times. After the import reactions, mitochondria were reisolated and subjected to SDS-PAGE. The gels were blotted to nitrocellulose and subjected to autoradiography. The leftmost lane for each precursor contained 33% of the input lysate used in each import reaction. The precursor (p) and mature (m) forms of F1β are indicated. (B) After phosphorimaging of the imported proteins, the blot was decorated with antiserum to porin. (C) Effect of proteinase K on proteins from different submitochondrial compartments. Isolated mitochondria were either digested with proteinase K as described in Materials and Methods or were incubated at 0°C for 15 min without proteinase K. After reisolation, electrophoresis, and blotting, the membranes were decorated with antiserum to the indicated proteins. Tom20′, proteolytic fragment of Tom20. PK, proteinase K.

Mentions: The mitochondrial outer membrane of certain mutant strains of N. crassa is easily broken during standard mitochondrial isolation procedures (Nargang et al. 1998; Grad et al. 1999). In such mitochondria, we found that most precursor proteins were imported with the same efficiency as in mitochondria with intact outer membranes. An exception was the precursor of porin, which was imported at reduced levels. This is shown in Fig. 1 A where mitochondria, isolated by standard procedures from a control strain (WT) and a tom22 mutant strain susceptible to opening of the outer membrane during isolation under standard conditions (tom22861) (Nargang et al. 1998), were used to assess the import of the precursors of the β subunit of the mitochondrial ATPase (F1β) and porin. The import of F1β was virtually indistinguishable in the two strains, whereas porin import was significantly reduced in the mutant. Quantification of the levels of import in tom22861 relative to the control strain in three separate experiments gave averages of 91% for F1β and 34% for 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 F1β and porin into N. crassa mitochondria with damaged outer membranes. (A) Mitochondria were isolated from mutant strain 861 (tom22861) and the control strain 76-26 (WT) using standard procedures. Import of radiolabeled precursor proteins was conducted at 20°C for the indicated times. After the import reactions, mitochondria were reisolated and subjected to SDS-PAGE. The gels were blotted to nitrocellulose and subjected to autoradiography. The leftmost lane for each precursor contained 33% of the input lysate used in each import reaction. The precursor (p) and mature (m) forms of F1β are indicated. (B) After phosphorimaging of the imported proteins, the blot was decorated with antiserum to porin. (C) Effect of proteinase K on proteins from different submitochondrial compartments. Isolated mitochondria were either digested with proteinase K as described in Materials and Methods or were incubated at 0°C for 15 min without proteinase K. After reisolation, electrophoresis, and blotting, the membranes were decorated with antiserum to the indicated proteins. Tom20′, proteolytic fragment of Tom20. PK, proteinase K.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199606&req=5

Figure 1: Import of F1β and porin into N. crassa mitochondria with damaged outer membranes. (A) Mitochondria were isolated from mutant strain 861 (tom22861) and the control strain 76-26 (WT) using standard procedures. Import of radiolabeled precursor proteins was conducted at 20°C for the indicated times. After the import reactions, mitochondria were reisolated and subjected to SDS-PAGE. The gels were blotted to nitrocellulose and subjected to autoradiography. The leftmost lane for each precursor contained 33% of the input lysate used in each import reaction. The precursor (p) and mature (m) forms of F1β are indicated. (B) After phosphorimaging of the imported proteins, the blot was decorated with antiserum to porin. (C) Effect of proteinase K on proteins from different submitochondrial compartments. Isolated mitochondria were either digested with proteinase K as described in Materials and Methods or were incubated at 0°C for 15 min without proteinase K. After reisolation, electrophoresis, and blotting, the membranes were decorated with antiserum to the indicated proteins. Tom20′, proteolytic fragment of Tom20. PK, proteinase K.
Mentions: The mitochondrial outer membrane of certain mutant strains of N. crassa is easily broken during standard mitochondrial isolation procedures (Nargang et al. 1998; Grad et al. 1999). In such mitochondria, we found that most precursor proteins were imported with the same efficiency as in mitochondria with intact outer membranes. An exception was the precursor of porin, which was imported at reduced levels. This is shown in Fig. 1 A where mitochondria, isolated by standard procedures from a control strain (WT) and a tom22 mutant strain susceptible to opening of the outer membrane during isolation under standard conditions (tom22861) (Nargang et al. 1998), were used to assess the import of the precursors of the β subunit of the mitochondrial ATPase (F1β) and porin. The import of F1β was virtually indistinguishable in the two strains, whereas porin import was significantly reduced in the mutant. Quantification of the levels of import in tom22861 relative to the control strain in three separate experiments gave averages of 91% for F1β and 34% for 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.

Show MeSH
Related in: MedlinePlus