Limits...
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.

Show MeSH
Porin forms high molecular weight complexes in yeast. (A) Purified S. cerevisiae mitochondria of a por1Δ-strain and the corresponding wild-type (WT) (50 μg protein per lane) were lysed in SDS sample buffer and subjected to SDS-PAGE. The gel was stained with Coomassie brilliant blue G-250. (B) Mitochondria of por1Δ, por2Δ, and the wild-type (50 μg protein per lane) were lysed in digitonin buffer and subjected to BN-PAGE. After semidry blotting onto PVDF membranes, immunodecoration with antibodies directed against porin was performed and the signals were detected by chemiluminescence. (Ass. Porin) Complexes of assembled porin. (C) Wild-type mitochondria (50 μg protein) were treated with sodium carbonate, and the membrane pellet was subjected to BN-PAGE and immunodecoration with antibodies against porin (lane 2). Nontreated mitochondria are shown as control (lane 1).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199606&req=5

Figure 3: Porin forms high molecular weight complexes in yeast. (A) Purified S. cerevisiae mitochondria of a por1Δ-strain and the corresponding wild-type (WT) (50 μg protein per lane) were lysed in SDS sample buffer and subjected to SDS-PAGE. The gel was stained with Coomassie brilliant blue G-250. (B) Mitochondria of por1Δ, por2Δ, and the wild-type (50 μg protein per lane) were lysed in digitonin buffer and subjected to BN-PAGE. After semidry blotting onto PVDF membranes, immunodecoration with antibodies directed against porin was performed and the signals were detected by chemiluminescence. (Ass. Porin) Complexes of assembled porin. (C) Wild-type mitochondria (50 μg protein) were treated with sodium carbonate, and the membrane pellet was subjected to BN-PAGE and immunodecoration with antibodies against porin (lane 2). Nontreated mitochondria are shown as control (lane 1).

Mentions: To establish a specific assay for the import of porin, we characterized the properties of yeast mitochondrial porin (also termed porin1). Mitochondria from yeast wild-type and a porin-deficient strain (por1Δ) (Blachly-Dyson et al. 1990) were separated by SDS-PAGE and stained with Coomassie brilliant blue G-250. The mutant mitochondria selectively lacked a protein band at 30 kD, the expected size of porin (Fig. 3 A, lane 2); the identity as porin was confirmed by immunodecoration (not shown). A quantification revealed that porin is 1 of the 10 most abundant proteins of mitochondria.


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)

Porin forms high molecular weight complexes in yeast. (A) Purified S. cerevisiae mitochondria of a por1Δ-strain and the corresponding wild-type (WT) (50 μg protein per lane) were lysed in SDS sample buffer and subjected to SDS-PAGE. The gel was stained with Coomassie brilliant blue G-250. (B) Mitochondria of por1Δ, por2Δ, and the wild-type (50 μg protein per lane) were lysed in digitonin buffer and subjected to BN-PAGE. After semidry blotting onto PVDF membranes, immunodecoration with antibodies directed against porin was performed and the signals were detected by chemiluminescence. (Ass. Porin) Complexes of assembled porin. (C) Wild-type mitochondria (50 μg protein) were treated with sodium carbonate, and the membrane pellet was subjected to BN-PAGE and immunodecoration with antibodies against porin (lane 2). Nontreated mitochondria are shown as control (lane 1).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Porin forms high molecular weight complexes in yeast. (A) Purified S. cerevisiae mitochondria of a por1Δ-strain and the corresponding wild-type (WT) (50 μg protein per lane) were lysed in SDS sample buffer and subjected to SDS-PAGE. The gel was stained with Coomassie brilliant blue G-250. (B) Mitochondria of por1Δ, por2Δ, and the wild-type (50 μg protein per lane) were lysed in digitonin buffer and subjected to BN-PAGE. After semidry blotting onto PVDF membranes, immunodecoration with antibodies directed against porin was performed and the signals were detected by chemiluminescence. (Ass. Porin) Complexes of assembled porin. (C) Wild-type mitochondria (50 μg protein) were treated with sodium carbonate, and the membrane pellet was subjected to BN-PAGE and immunodecoration with antibodies against porin (lane 2). Nontreated mitochondria are shown as control (lane 1).
Mentions: To establish a specific assay for the import of porin, we characterized the properties of yeast mitochondrial porin (also termed porin1). Mitochondria from yeast wild-type and a porin-deficient strain (por1Δ) (Blachly-Dyson et al. 1990) were separated by SDS-PAGE and stained with Coomassie brilliant blue G-250. The mutant mitochondria selectively lacked a protein band at 30 kD, the expected size of porin (Fig. 3 A, lane 2); the identity as porin was confirmed by immunodecoration (not shown). A quantification revealed that porin is 1 of the 10 most abundant proteins of mitochondria.

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