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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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Two new mutant alleles of TOM40. (A) Predicted amino acid sequences (single letter code) of wild-type Tom40 and the two mutant alleles tom40-2 and tom40-4. Alterations to the wild-type sequence are indicated in bold. (B) Steady state levels of mitochondrial proteins. Isolated mitochondria (25 μg protein per lane) from wild-type or tom40 mutant yeast strains were separated by SDS-PAGE using the Tris-tricine buffer system (Schägger and von Jagow 1987), followed by immunodecoration with antibodies directed against the indicated yeast proteins. Tom40′, truncated form of Tom40. AAC, ADP/ATP carrier; Ssc1, matrix Hsp70; Mge1, matrix cochaperone of the GrpE-type. (C) Presence of the 400-kD GIP complex in the tom40 mutant mitochondria. Isolated mitochondria (50 μg protein per lane) were lysed in digitonin-containing buffer and subjected to BN-PAGE, followed by immunodecoration with antibodies directed against Tom5. (D) Import of a matrix-targeted preprotein is inhibited in the tom40 mutant mitochondria. The fusion protein b2-DHFR (1 μg protein per lane) was imported into isolated mitochondria (50 μg protein per lane) for the indicated time points. The processed form of the protein was detected by immunodecoration.
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Figure 7: Two new mutant alleles of TOM40. (A) Predicted amino acid sequences (single letter code) of wild-type Tom40 and the two mutant alleles tom40-2 and tom40-4. Alterations to the wild-type sequence are indicated in bold. (B) Steady state levels of mitochondrial proteins. Isolated mitochondria (25 μg protein per lane) from wild-type or tom40 mutant yeast strains were separated by SDS-PAGE using the Tris-tricine buffer system (Schägger and von Jagow 1987), followed by immunodecoration with antibodies directed against the indicated yeast proteins. Tom40′, truncated form of Tom40. AAC, ADP/ATP carrier; Ssc1, matrix Hsp70; Mge1, matrix cochaperone of the GrpE-type. (C) Presence of the 400-kD GIP complex in the tom40 mutant mitochondria. Isolated mitochondria (50 μg protein per lane) were lysed in digitonin-containing buffer and subjected to BN-PAGE, followed by immunodecoration with antibodies directed against Tom5. (D) Import of a matrix-targeted preprotein is inhibited in the tom40 mutant mitochondria. The fusion protein b2-DHFR (1 μg protein per lane) was imported into isolated mitochondria (50 μg protein per lane) for the indicated time points. The processed form of the protein was detected by immunodecoration.

Mentions: Two possibilities are conceivable to explain this result. Tom40 is not involved in import of porin at all, or the competence of Tom40 with regard to porin assembly is not compromised in cells harboring the allele tom40-3. The PCR mutagenesis of TOM40 (Kassenbrock et al. 1993) had yielded several further uncharacterized mutant alleles that confer temperature-sensitive growth. Like tom40-3 and the corresponding wild-type strain, all these strains harbor a chromosomal deletion of TOM40 and express Tom40 from a plasmid. We performed an initial screen of the mutant strains in order to select strains where mitochondrial marker proteins, including Tom proteins, are present in normal amounts to minimize indirect effects of the mutations. We then selected two mutant strains, termed tom40-2 and tom40-4. The TOM40 ORFs of the two new alleles were sequenced, demonstrating their independence (Fig. 7 A). Each allele led to several amino acid alterations; additionally, a premature stop in tom40-4 led to a Tom40 lacking 17 amino acid residues at the COOH terminus (Fig. 7 A).


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)

Two new mutant alleles of TOM40. (A) Predicted amino acid sequences (single letter code) of wild-type Tom40 and the two mutant alleles tom40-2 and tom40-4. Alterations to the wild-type sequence are indicated in bold. (B) Steady state levels of mitochondrial proteins. Isolated mitochondria (25 μg protein per lane) from wild-type or tom40 mutant yeast strains were separated by SDS-PAGE using the Tris-tricine buffer system (Schägger and von Jagow 1987), followed by immunodecoration with antibodies directed against the indicated yeast proteins. Tom40′, truncated form of Tom40. AAC, ADP/ATP carrier; Ssc1, matrix Hsp70; Mge1, matrix cochaperone of the GrpE-type. (C) Presence of the 400-kD GIP complex in the tom40 mutant mitochondria. Isolated mitochondria (50 μg protein per lane) were lysed in digitonin-containing buffer and subjected to BN-PAGE, followed by immunodecoration with antibodies directed against Tom5. (D) Import of a matrix-targeted preprotein is inhibited in the tom40 mutant mitochondria. The fusion protein b2-DHFR (1 μg protein per lane) was imported into isolated mitochondria (50 μg protein per lane) for the indicated time points. The processed form of the protein was detected by immunodecoration.
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Figure 7: Two new mutant alleles of TOM40. (A) Predicted amino acid sequences (single letter code) of wild-type Tom40 and the two mutant alleles tom40-2 and tom40-4. Alterations to the wild-type sequence are indicated in bold. (B) Steady state levels of mitochondrial proteins. Isolated mitochondria (25 μg protein per lane) from wild-type or tom40 mutant yeast strains were separated by SDS-PAGE using the Tris-tricine buffer system (Schägger and von Jagow 1987), followed by immunodecoration with antibodies directed against the indicated yeast proteins. Tom40′, truncated form of Tom40. AAC, ADP/ATP carrier; Ssc1, matrix Hsp70; Mge1, matrix cochaperone of the GrpE-type. (C) Presence of the 400-kD GIP complex in the tom40 mutant mitochondria. Isolated mitochondria (50 μg protein per lane) were lysed in digitonin-containing buffer and subjected to BN-PAGE, followed by immunodecoration with antibodies directed against Tom5. (D) Import of a matrix-targeted preprotein is inhibited in the tom40 mutant mitochondria. The fusion protein b2-DHFR (1 μg protein per lane) was imported into isolated mitochondria (50 μg protein per lane) for the indicated time points. The processed form of the protein was detected by immunodecoration.
Mentions: Two possibilities are conceivable to explain this result. Tom40 is not involved in import of porin at all, or the competence of Tom40 with regard to porin assembly is not compromised in cells harboring the allele tom40-3. The PCR mutagenesis of TOM40 (Kassenbrock et al. 1993) had yielded several further uncharacterized mutant alleles that confer temperature-sensitive growth. Like tom40-3 and the corresponding wild-type strain, all these strains harbor a chromosomal deletion of TOM40 and express Tom40 from a plasmid. We performed an initial screen of the mutant strains in order to select strains where mitochondrial marker proteins, including Tom proteins, are present in normal amounts to minimize indirect effects of the mutations. We then selected two mutant strains, termed tom40-2 and tom40-4. The TOM40 ORFs of the two new alleles were sequenced, demonstrating their independence (Fig. 7 A). Each allele led to several amino acid alterations; additionally, a premature stop in tom40-4 led to a Tom40 lacking 17 amino acid residues at the COOH terminus (Fig. 7 A).

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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