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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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Assembly of in vitro–imported porin into preexisting complexes. (A) Radiolabeled precursor of yeast porin (10 μl reticulocyte lysate per lane) was incubated with por1Δ, por2Δ, and wild-type mitochondria (50 μg protein) at 25°C for the indicated periods. The mitochondria were reisolated, lysed in digitonin-containing buffer, and subjected to BN-PAGE and digital autoradiography. (Ass. Porin) Complexes of assembled porin. Asterisk indicates a putative assembly intermediate of porin. Non-assembled (monomeric) porin is shown in the low molecular weight range. (B) Quantification of the amounts of assembled porin by digital autoradiography. The amount of assembled radiolabeled porin in wild-type mitochondria after 15 min was set to 100% (control).
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Figure 4: Assembly of in vitro–imported porin into preexisting complexes. (A) Radiolabeled precursor of yeast porin (10 μl reticulocyte lysate per lane) was incubated with por1Δ, por2Δ, and wild-type mitochondria (50 μg protein) at 25°C for the indicated periods. The mitochondria were reisolated, lysed in digitonin-containing buffer, and subjected to BN-PAGE and digital autoradiography. (Ass. Porin) Complexes of assembled porin. Asterisk indicates a putative assembly intermediate of porin. Non-assembled (monomeric) porin is shown in the low molecular weight range. (B) Quantification of the amounts of assembled porin by digital autoradiography. The amount of assembled radiolabeled porin in wild-type mitochondria after 15 min was set to 100% (control).

Mentions: We used the characteristic behavior of mature endogenous porin on BN-PAGE to establish a specific assay for the correct import and assembly of in vitro–synthesized porin precursors. Porin was synthesized in rabbit reticulocyte lysate in the presence of [35S]methionine/cysteine and imported into isolated wild-type mitochondria. The mitochondria were reisolated, lysed in digitonin-containing buffer, and subjected to BN-PAGE and digital autoradiography. The radiolabeled porin was found in three high molecular weight complexes with a mobility indistinguishable from that of endogenous porin (Fig. 4 A, lanes 1–3). When por1Δ mitochondria were employed, however, none of the three high molecular weight complexes was observed (Fig. 4 A, lanes 4–6, and B), indicating that preexisting porin was required for the assembly of the radiolabeled porin. por2Δ mitochondria behaved like wild-type mitochondria (Fig. 4 A, lanes 7–9 vs. 1–3, and B). With all three types of mitochondria, a low molecular weight form of porin was found, likely representing the monomeric form of the porin precursor that was associated with mitochondria yet not assembled (Fig. 4 A). Additionally, radiolabeled porin was found at an ∼100-kD position (Fig. 4 A, asterisk). The formation of this species required the presence of preexisting porin (Fig. 4 A, lanes 4–6), raising the possibility that it represents an assembly intermediate of porin. Since the 100-kD band does not represent a major form of mature porin (Fig. 3 B) and is observed to variable extent (depending on the yeast strain used), we only used the mature 200–440-kD complexes to determine the efficiency of assembly of radiolabeled porin. We conclude that BN-PAGE of digitonin-lysed mitochondria represents a specific assay to determine correct import and assembly of porin in yeast.


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)

Assembly of in vitro–imported porin into preexisting complexes. (A) Radiolabeled precursor of yeast porin (10 μl reticulocyte lysate per lane) was incubated with por1Δ, por2Δ, and wild-type mitochondria (50 μg protein) at 25°C for the indicated periods. The mitochondria were reisolated, lysed in digitonin-containing buffer, and subjected to BN-PAGE and digital autoradiography. (Ass. Porin) Complexes of assembled porin. Asterisk indicates a putative assembly intermediate of porin. Non-assembled (monomeric) porin is shown in the low molecular weight range. (B) Quantification of the amounts of assembled porin by digital autoradiography. The amount of assembled radiolabeled porin in wild-type mitochondria after 15 min was set to 100% (control).
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Related In: Results  -  Collection

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Figure 4: Assembly of in vitro–imported porin into preexisting complexes. (A) Radiolabeled precursor of yeast porin (10 μl reticulocyte lysate per lane) was incubated with por1Δ, por2Δ, and wild-type mitochondria (50 μg protein) at 25°C for the indicated periods. The mitochondria were reisolated, lysed in digitonin-containing buffer, and subjected to BN-PAGE and digital autoradiography. (Ass. Porin) Complexes of assembled porin. Asterisk indicates a putative assembly intermediate of porin. Non-assembled (monomeric) porin is shown in the low molecular weight range. (B) Quantification of the amounts of assembled porin by digital autoradiography. The amount of assembled radiolabeled porin in wild-type mitochondria after 15 min was set to 100% (control).
Mentions: We used the characteristic behavior of mature endogenous porin on BN-PAGE to establish a specific assay for the correct import and assembly of in vitro–synthesized porin precursors. Porin was synthesized in rabbit reticulocyte lysate in the presence of [35S]methionine/cysteine and imported into isolated wild-type mitochondria. The mitochondria were reisolated, lysed in digitonin-containing buffer, and subjected to BN-PAGE and digital autoradiography. The radiolabeled porin was found in three high molecular weight complexes with a mobility indistinguishable from that of endogenous porin (Fig. 4 A, lanes 1–3). When por1Δ mitochondria were employed, however, none of the three high molecular weight complexes was observed (Fig. 4 A, lanes 4–6, and B), indicating that preexisting porin was required for the assembly of the radiolabeled porin. por2Δ mitochondria behaved like wild-type mitochondria (Fig. 4 A, lanes 7–9 vs. 1–3, and B). With all three types of mitochondria, a low molecular weight form of porin was found, likely representing the monomeric form of the porin precursor that was associated with mitochondria yet not assembled (Fig. 4 A). Additionally, radiolabeled porin was found at an ∼100-kD position (Fig. 4 A, asterisk). The formation of this species required the presence of preexisting porin (Fig. 4 A, lanes 4–6), raising the possibility that it represents an assembly intermediate of porin. Since the 100-kD band does not represent a major form of mature porin (Fig. 3 B) and is observed to variable extent (depending on the yeast strain used), we only used the mature 200–440-kD complexes to determine the efficiency of assembly of radiolabeled porin. We conclude that BN-PAGE of digitonin-lysed mitochondria represents a specific assay to determine correct import and assembly of porin in yeast.

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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