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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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Insertion of porin is inhibited by blocking the translocation pore with import intermediates. N. crassa mitochondria (30 μg protein per import reaction) in import buffer containing 2 mM NADH, 2 mM ATP, and 37 μM carbonyl cyanide m-chlorophenylhydrazone were incubated for 15 min at 0°C in the absence (A) or presence (B) of 20 μg/ml trypsin. After inactivation of the trypsin by soybean trypsin inhibitor, each sample was split into two, and one half was incubated for 10 min at 0°C with 3.2 μM pSu9(1–69)-DHFR. Radiolabeled porin precursor was then added and further incubated at 15°C (A) or 25°C (B) for the indicated periods. At the end of the import reactions proteinase K (100 μg/ml) was added, proteins were analyzed by SDS-PAGE, and imported porin was quantified.
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Figure 2: Insertion of porin is inhibited by blocking the translocation pore with import intermediates. N. crassa mitochondria (30 μg protein per import reaction) in import buffer containing 2 mM NADH, 2 mM ATP, and 37 μM carbonyl cyanide m-chlorophenylhydrazone were incubated for 15 min at 0°C in the absence (A) or presence (B) of 20 μg/ml trypsin. After inactivation of the trypsin by soybean trypsin inhibitor, each sample was split into two, and one half was incubated for 10 min at 0°C with 3.2 μM pSu9(1–69)-DHFR. Radiolabeled porin precursor was then added and further incubated at 15°C (A) or 25°C (B) for the indicated periods. At the end of the import reactions proteinase K (100 μg/ml) was added, proteins were analyzed by SDS-PAGE, and imported porin was quantified.

Mentions: To investigate the role of TOM complex components in porin insertion, we added excess amounts of a matrix-destined precursor protein to saturate import sites and then evaluated the effect on the import of porin into mitochondria in vitro. Chemical amounts of a fusion protein consisting of the presequence of Fo-ATPase subunit 9 and pSu9-DHFR were added to N. crassa mitochondria under conditions where completion of import to the matrix was prevented by dissipation of the inner membrane potential with the protonophore carbonyl cyanide m-chlorophenylhydrazone. Subsequent import of radiochemical amounts of porin precursor was reduced about fourfold relative to import with no competitor present (Fig. 2 A). This indicates that the pSu9-DHFR and porin compete for similar sites required for insertion. To determine if the inhibitory effect was due solely to competition for binding sites on the receptors of the outer membrane (Tom20/Tom22), or if it also involved the actual import pore, the experiment was repeated using mitochondria pretreated with trypsin to remove the surface receptors. Under these conditions, receptor-dependent precursors enter mitochondria at a lower rate due to “bypass import,” which occurs via direct interaction with the GIP (Pfaller et al. 1989). As expected for trypsinized mitochondria, the import of porin was reduced relative to untreated mitochondria even without the addition of excess amounts of competing pSu9-DHFR precursor (Fig. 2 B). However, the saturation of the pore with excess precursor substantially decreased the level of porin import even via the bypass route. Since the binding of the competing precursor was also reduced in the bypass route, lower levels of competition were expected. As the matrix-destined pSu9-DHFR precursor was found to be in the vicinity of the Tom40 pore component under similar “bypass” conditions (Rapaport et al. 1997), the data suggested that the import of porin is also dependent on components of the TOM complex that make up the translocation pore. Therefore, we decided to perform a detailed analysis for the requirement of individual Tom proteins in import 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)

Insertion of porin is inhibited by blocking the translocation pore with import intermediates. N. crassa mitochondria (30 μg protein per import reaction) in import buffer containing 2 mM NADH, 2 mM ATP, and 37 μM carbonyl cyanide m-chlorophenylhydrazone were incubated for 15 min at 0°C in the absence (A) or presence (B) of 20 μg/ml trypsin. After inactivation of the trypsin by soybean trypsin inhibitor, each sample was split into two, and one half was incubated for 10 min at 0°C with 3.2 μM pSu9(1–69)-DHFR. Radiolabeled porin precursor was then added and further incubated at 15°C (A) or 25°C (B) for the indicated periods. At the end of the import reactions proteinase K (100 μg/ml) was added, proteins were analyzed by SDS-PAGE, and imported porin was quantified.
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Related In: Results  -  Collection

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Figure 2: Insertion of porin is inhibited by blocking the translocation pore with import intermediates. N. crassa mitochondria (30 μg protein per import reaction) in import buffer containing 2 mM NADH, 2 mM ATP, and 37 μM carbonyl cyanide m-chlorophenylhydrazone were incubated for 15 min at 0°C in the absence (A) or presence (B) of 20 μg/ml trypsin. After inactivation of the trypsin by soybean trypsin inhibitor, each sample was split into two, and one half was incubated for 10 min at 0°C with 3.2 μM pSu9(1–69)-DHFR. Radiolabeled porin precursor was then added and further incubated at 15°C (A) or 25°C (B) for the indicated periods. At the end of the import reactions proteinase K (100 μg/ml) was added, proteins were analyzed by SDS-PAGE, and imported porin was quantified.
Mentions: To investigate the role of TOM complex components in porin insertion, we added excess amounts of a matrix-destined precursor protein to saturate import sites and then evaluated the effect on the import of porin into mitochondria in vitro. Chemical amounts of a fusion protein consisting of the presequence of Fo-ATPase subunit 9 and pSu9-DHFR were added to N. crassa mitochondria under conditions where completion of import to the matrix was prevented by dissipation of the inner membrane potential with the protonophore carbonyl cyanide m-chlorophenylhydrazone. Subsequent import of radiochemical amounts of porin precursor was reduced about fourfold relative to import with no competitor present (Fig. 2 A). This indicates that the pSu9-DHFR and porin compete for similar sites required for insertion. To determine if the inhibitory effect was due solely to competition for binding sites on the receptors of the outer membrane (Tom20/Tom22), or if it also involved the actual import pore, the experiment was repeated using mitochondria pretreated with trypsin to remove the surface receptors. Under these conditions, receptor-dependent precursors enter mitochondria at a lower rate due to “bypass import,” which occurs via direct interaction with the GIP (Pfaller et al. 1989). As expected for trypsinized mitochondria, the import of porin was reduced relative to untreated mitochondria even without the addition of excess amounts of competing pSu9-DHFR precursor (Fig. 2 B). However, the saturation of the pore with excess precursor substantially decreased the level of porin import even via the bypass route. Since the binding of the competing precursor was also reduced in the bypass route, lower levels of competition were expected. As the matrix-destined pSu9-DHFR precursor was found to be in the vicinity of the Tom40 pore component under similar “bypass” conditions (Rapaport et al. 1997), the data suggested that the import of porin is also dependent on components of the TOM complex that make up the translocation pore. Therefore, we decided to perform a detailed analysis for the requirement of individual Tom proteins in import 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.

Show MeSH
Related in: MedlinePlus