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
Import of porin requires components of the GIP. (A) Levels of Tom proteins in mitochondria isolated from yeast strains lacking individual TOM genes (Table ). Isolated mitochondria (25 μg protein per lane) were subjected to SDS-PAGE and immunodecoration with antibodies directed against the indicated proteins. Tim44, translocase of inner membrane subunit of 44 kD. (B) Import and assembly of radiolabeled porin. The experiment was performed as described in the legend to Fig. 4 A, except tom mutant mitochondria were used, and import was performed for the indicated periods. (Non-ass. Porin) Porin precursor in the low molecular weight range that is associated with mitochondria but not assembled. (C) Quantification of assembled porin. Hatched bars, high molecular weight complexes of assembled porin (440 and 400 kD); white bars, total assembled porin (440, 400, and 200 kD complexes together). The total amount of assembled radiolabeled porin in wild-type mitochondria after 15 min was set to 100% (control).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199606&req=5

Figure 5: Import of porin requires components of the GIP. (A) Levels of Tom proteins in mitochondria isolated from yeast strains lacking individual TOM genes (Table ). Isolated mitochondria (25 μg protein per lane) were subjected to SDS-PAGE and immunodecoration with antibodies directed against the indicated proteins. Tim44, translocase of inner membrane subunit of 44 kD. (B) Import and assembly of radiolabeled porin. The experiment was performed as described in the legend to Fig. 4 A, except tom mutant mitochondria were used, and import was performed for the indicated periods. (Non-ass. Porin) Porin precursor in the low molecular weight range that is associated with mitochondria but not assembled. (C) Quantification of assembled porin. Hatched bars, high molecular weight complexes of assembled porin (440 and 400 kD); white bars, total assembled porin (440, 400, and 200 kD complexes together). The total amount of assembled radiolabeled porin in wild-type mitochondria after 15 min was set to 100% (control).

Mentions: Mitochondria were isolated from yeast strains with single deletions of TOM22, TOM20, TOM7, TOM6, or TOM5. The steady-state levels of most mitochondrial marker proteins, including the respective other components of the TOM complex and the TIM machinery (shown here with Tim44), were found to be similar to those of wild-type mitochondria (Fig. 5 A) (Alconada et al. 1995; Hönlinger et al. 1996; Dietmeier et al. 1997; Dekker et al. 1998; van Wilpe et al. 1999). This was explained in the following way: mutations impairing the function of the TOM complex result in slower mitochondrial protein import that consequently becomes rate-limiting for mitochondrial and cellular growth. The strain lacking Tom20, however, is an exception since tom20Δ mitochondria are known to have a strongly reduced level of Tom22 (Lithgow et al. 1994). Therefore, we used a tom20Δ strain that additionally expressed TOM22 from a high copy number plasmid (Table ) (Dekker et al. 1998), restoring the mitochondrial content of Tom22 to a level comparable to that of wild-type (Fig. 5 A, lower left panel). Thus, the mutant mitochondria used are selectively defective in only one Tom protein.


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)

Import of porin requires components of the GIP. (A) Levels of Tom proteins in mitochondria isolated from yeast strains lacking individual TOM genes (Table ). Isolated mitochondria (25 μg protein per lane) were subjected to SDS-PAGE and immunodecoration with antibodies directed against the indicated proteins. Tim44, translocase of inner membrane subunit of 44 kD. (B) Import and assembly of radiolabeled porin. The experiment was performed as described in the legend to Fig. 4 A, except tom mutant mitochondria were used, and import was performed for the indicated periods. (Non-ass. Porin) Porin precursor in the low molecular weight range that is associated with mitochondria but not assembled. (C) Quantification of assembled porin. Hatched bars, high molecular weight complexes of assembled porin (440 and 400 kD); white bars, total assembled porin (440, 400, and 200 kD complexes together). The total amount of assembled radiolabeled porin in wild-type mitochondria after 15 min was set to 100% (control).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Import of porin requires components of the GIP. (A) Levels of Tom proteins in mitochondria isolated from yeast strains lacking individual TOM genes (Table ). Isolated mitochondria (25 μg protein per lane) were subjected to SDS-PAGE and immunodecoration with antibodies directed against the indicated proteins. Tim44, translocase of inner membrane subunit of 44 kD. (B) Import and assembly of radiolabeled porin. The experiment was performed as described in the legend to Fig. 4 A, except tom mutant mitochondria were used, and import was performed for the indicated periods. (Non-ass. Porin) Porin precursor in the low molecular weight range that is associated with mitochondria but not assembled. (C) Quantification of assembled porin. Hatched bars, high molecular weight complexes of assembled porin (440 and 400 kD); white bars, total assembled porin (440, 400, and 200 kD complexes together). The total amount of assembled radiolabeled porin in wild-type mitochondria after 15 min was set to 100% (control).
Mentions: Mitochondria were isolated from yeast strains with single deletions of TOM22, TOM20, TOM7, TOM6, or TOM5. The steady-state levels of most mitochondrial marker proteins, including the respective other components of the TOM complex and the TIM machinery (shown here with Tim44), were found to be similar to those of wild-type mitochondria (Fig. 5 A) (Alconada et al. 1995; Hönlinger et al. 1996; Dietmeier et al. 1997; Dekker et al. 1998; van Wilpe et al. 1999). This was explained in the following way: mutations impairing the function of the TOM complex result in slower mitochondrial protein import that consequently becomes rate-limiting for mitochondrial and cellular growth. The strain lacking Tom20, however, is an exception since tom20Δ mitochondria are known to have a strongly reduced level of Tom22 (Lithgow et al. 1994). Therefore, we used a tom20Δ strain that additionally expressed TOM22 from a high copy number plasmid (Table ) (Dekker et al. 1998), restoring the mitochondrial content of Tom22 to a level comparable to that of wild-type (Fig. 5 A, lower left panel). Thus, the mutant mitochondria used are selectively defective in only one Tom protein.

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