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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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Porin is in the vicinity of Tom20, Tom22, and Tom40 on its insertion pathway. Radiolabeled porin precursor was incubated with isolated OMVs for 2 min at 0°C. One aliquot was left on ice (−DSG) while the chemical cross-linker DSG was added to the others (+DSG) and incubated for 40 min on ice. The cross-linking reagent was quenched with 80 mM glycine (pH 8.0), the OMVs were pelleted, and the first two aliquots (25% of material subjected to immunoprecipitation) were loaded onto SDS-PAGE. The pellets from the other aliquots were first solubilized with SDS- and Triton X-100–containing buffer and then diluted to buffer lacking SDS. Aliquots were subjected to immunoprecipitation with antibodies against either Tom20, Tom22, Tom40, or with preimmune serum (PIS). The immunoprecipitates were solubilized in sample buffer and analyzed by SDS-PAGE and digital autoradiography. *Cross-links of porin to Tom proteins.
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Figure 9: Porin is in the vicinity of Tom20, Tom22, and Tom40 on its insertion pathway. Radiolabeled porin precursor was incubated with isolated OMVs for 2 min at 0°C. One aliquot was left on ice (−DSG) while the chemical cross-linker DSG was added to the others (+DSG) and incubated for 40 min on ice. The cross-linking reagent was quenched with 80 mM glycine (pH 8.0), the OMVs were pelleted, and the first two aliquots (25% of material subjected to immunoprecipitation) were loaded onto SDS-PAGE. The pellets from the other aliquots were first solubilized with SDS- and Triton X-100–containing buffer and then diluted to buffer lacking SDS. Aliquots were subjected to immunoprecipitation with antibodies against either Tom20, Tom22, Tom40, or with preimmune serum (PIS). The immunoprecipitates were solubilized in sample buffer and analyzed by SDS-PAGE and digital autoradiography. *Cross-links of porin to Tom proteins.

Mentions: To obtain independent evidence for the involvement of components of the GIP complex in porin import, we performed cross-linking of porin precursor molecules during the import process. Radiolabeled porin precursor was accumulated as an import intermediate in Neurospora OMVs by incubation at 0°C. Addition of the homobifunctional cross-linking reagent, DSG, led to cross-linking of porin to each of Tom20, Tom22, and Tom40 (Fig. 9). Cross-linked Tom40 products tend to appear as multiple bands on SDS-PAGE, probably caused by the existence of different conformations of Tom40 molecules in the membrane (Rapaport et al. 1997, Rapaport et al. 1998; Kanamori et al. 1999). The abundance of the adduct with Tom20 is at least partly due to the fact that the experiments are performed at 0°C, which insures that cross-linking occurs before the porin precursor becomes fully inserted. These conditions also favor association of porin with the receptor rather than with pore components. Nonetheless, the data clearly show that both Tom22 and Tom40 are in the vicinity of the porin precursor during import. No cross-linking of endogenous porin, the most abundant outer membrane protein, to Tom40 was observed (not shown), demonstrating the specificity of the cross-linking approach and excluding that the observed cross-linking of porin precursor to Tom40 could be explained as random interactions between assembled porin with Tom40. Although the levels of cross-linking of the porin precursor to the GIP complex components are low, they are comparable to those observed previously for the matrix-destined precursor pSu9-DHFR imported under similar conditions (Rapaport et al. 1997) and suggest that the initial stages of import for the two precursors occur via a similar pathway. As observed with matrix-targeted preproteins (Neupert 1997; Pfanner et al. 1997), Tom20 may thus interact first with the porin precursor, followed by Tom22 and Tom40.


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 is in the vicinity of Tom20, Tom22, and Tom40 on its insertion pathway. Radiolabeled porin precursor was incubated with isolated OMVs for 2 min at 0°C. One aliquot was left on ice (−DSG) while the chemical cross-linker DSG was added to the others (+DSG) and incubated for 40 min on ice. The cross-linking reagent was quenched with 80 mM glycine (pH 8.0), the OMVs were pelleted, and the first two aliquots (25% of material subjected to immunoprecipitation) were loaded onto SDS-PAGE. The pellets from the other aliquots were first solubilized with SDS- and Triton X-100–containing buffer and then diluted to buffer lacking SDS. Aliquots were subjected to immunoprecipitation with antibodies against either Tom20, Tom22, Tom40, or with preimmune serum (PIS). The immunoprecipitates were solubilized in sample buffer and analyzed by SDS-PAGE and digital autoradiography. *Cross-links of porin to Tom proteins.
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Related In: Results  -  Collection

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Figure 9: Porin is in the vicinity of Tom20, Tom22, and Tom40 on its insertion pathway. Radiolabeled porin precursor was incubated with isolated OMVs for 2 min at 0°C. One aliquot was left on ice (−DSG) while the chemical cross-linker DSG was added to the others (+DSG) and incubated for 40 min on ice. The cross-linking reagent was quenched with 80 mM glycine (pH 8.0), the OMVs were pelleted, and the first two aliquots (25% of material subjected to immunoprecipitation) were loaded onto SDS-PAGE. The pellets from the other aliquots were first solubilized with SDS- and Triton X-100–containing buffer and then diluted to buffer lacking SDS. Aliquots were subjected to immunoprecipitation with antibodies against either Tom20, Tom22, Tom40, or with preimmune serum (PIS). The immunoprecipitates were solubilized in sample buffer and analyzed by SDS-PAGE and digital autoradiography. *Cross-links of porin to Tom proteins.
Mentions: To obtain independent evidence for the involvement of components of the GIP complex in porin import, we performed cross-linking of porin precursor molecules during the import process. Radiolabeled porin precursor was accumulated as an import intermediate in Neurospora OMVs by incubation at 0°C. Addition of the homobifunctional cross-linking reagent, DSG, led to cross-linking of porin to each of Tom20, Tom22, and Tom40 (Fig. 9). Cross-linked Tom40 products tend to appear as multiple bands on SDS-PAGE, probably caused by the existence of different conformations of Tom40 molecules in the membrane (Rapaport et al. 1997, Rapaport et al. 1998; Kanamori et al. 1999). The abundance of the adduct with Tom20 is at least partly due to the fact that the experiments are performed at 0°C, which insures that cross-linking occurs before the porin precursor becomes fully inserted. These conditions also favor association of porin with the receptor rather than with pore components. Nonetheless, the data clearly show that both Tom22 and Tom40 are in the vicinity of the porin precursor during import. No cross-linking of endogenous porin, the most abundant outer membrane protein, to Tom40 was observed (not shown), demonstrating the specificity of the cross-linking approach and excluding that the observed cross-linking of porin precursor to Tom40 could be explained as random interactions between assembled porin with Tom40. Although the levels of cross-linking of the porin precursor to the GIP complex components are low, they are comparable to those observed previously for the matrix-destined precursor pSu9-DHFR imported under similar conditions (Rapaport et al. 1997) and suggest that the initial stages of import for the two precursors occur via a similar pathway. As observed with matrix-targeted preproteins (Neupert 1997; Pfanner et al. 1997), Tom20 may thus interact first with the porin precursor, followed by Tom22 and Tom40.

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