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A discrete pathway for the transfer of intermembrane space proteins across the outer membrane of mitochondria.

Gornicka A, Bragoszewski P, Chroscicki P, Wenz LS, Schulz C, Rehling P, Chacinska A - Mol. Biol. Cell (2014)

Bottom Line: We identified a transient interaction between our model substrates and Tom40.Of interest, outer membrane translocation did not directly involve other core components of the TOM complex, including Tom22.Thus MIA-dependent proteins take another route across the outer mitochondrial membrane that involves Tom40 in a form that is different from the canonical TOM complex.

View Article: PubMed Central - PubMed

Affiliation: International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland.

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Competition experiments with recombinant TIM23-dependent precursor protein for import into mitochondria. (A) Radiolabeled presequence-containing precursor of b2167Δ-DHFR fusion protein was imported into mitochondria in the presence of increasing concentrations (up to 5 μg/100 μl import reaction) of b2167Δ-DHFR for 10 min. i, intermediate form. Quantitations of 35S-radiolabeled b2167Δ-DHFR import (bottom). Import into mitochondria without recombinant b2167Δ-DHFR was set to 100%. SEM of three independent experiments. (B) Radiolabeled presequence-containing precursor (b2167Δ-DHFR) was imported into mitochondria in the presence or absence of 5 μg of b2167Δ-DHFR/100 μl of import reaction. Quantitations of 35S-radiolabeled b2167Δ-DHFR import (bottom). Import into mitochondria without recombinant b2167Δ-DHFR after 15 min was set to 100%. SEM of three independent experiments. (C) Radiolabeled Tim9 was imported into mitochondria in the presence or absence of 5 μg/100 μl import reaction of b2167Δ-DHFR. Quantitations of 35S-radiolabeled Tim9 import (middle). Import into mitochondria without recombinant b2167Δ-DHFR after 15 min was set to 100%. SEM of three independent experiments. (D) Radiolabeled Cox19 was imported into mitochondria in the presence or absence of 5 μg/100 μl import reaction of b2167Δ-DHFR. Quantitations of 35S-radiolabeled Cox19 import (middle). Import into mitochondria without recombinant b2167Δ-DHFR after 27 min was set to 100%. SEM of three independent experiments. (A–D) The samples were treated with proteinase K as indicated and analyzed by nonreducing or reducing SDS–PAGE. Δψ, electrochemical potential; IA, iodoacetamide.
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Figure 1: Competition experiments with recombinant TIM23-dependent precursor protein for import into mitochondria. (A) Radiolabeled presequence-containing precursor of b2167Δ-DHFR fusion protein was imported into mitochondria in the presence of increasing concentrations (up to 5 μg/100 μl import reaction) of b2167Δ-DHFR for 10 min. i, intermediate form. Quantitations of 35S-radiolabeled b2167Δ-DHFR import (bottom). Import into mitochondria without recombinant b2167Δ-DHFR was set to 100%. SEM of three independent experiments. (B) Radiolabeled presequence-containing precursor (b2167Δ-DHFR) was imported into mitochondria in the presence or absence of 5 μg of b2167Δ-DHFR/100 μl of import reaction. Quantitations of 35S-radiolabeled b2167Δ-DHFR import (bottom). Import into mitochondria without recombinant b2167Δ-DHFR after 15 min was set to 100%. SEM of three independent experiments. (C) Radiolabeled Tim9 was imported into mitochondria in the presence or absence of 5 μg/100 μl import reaction of b2167Δ-DHFR. Quantitations of 35S-radiolabeled Tim9 import (middle). Import into mitochondria without recombinant b2167Δ-DHFR after 15 min was set to 100%. SEM of three independent experiments. (D) Radiolabeled Cox19 was imported into mitochondria in the presence or absence of 5 μg/100 μl import reaction of b2167Δ-DHFR. Quantitations of 35S-radiolabeled Cox19 import (middle). Import into mitochondria without recombinant b2167Δ-DHFR after 27 min was set to 100%. SEM of three independent experiments. (A–D) The samples were treated with proteinase K as indicated and analyzed by nonreducing or reducing SDS–PAGE. Δψ, electrochemical potential; IA, iodoacetamide.

Mentions: To determine whether the translocation of MIA-dependent precursor proteins across the OM uses the same pathway as the import of presequence-containing precursors, we applied an import competition assay with purified precursor proteins. A recombinant cytosolic dihydrofolate reductase protein that was fused to the presequence of cytochrome b2 (b2167Δ-DHFR) was applied as a model presequence-containing substrate. It was arrested in translocation complexes in the presence of a substrate analogue, methotrexate (Dekker et al., 1997; Chacinska et al., 2003; Schulz and Rehling, 2014). On import into mitochondria isolated from wild-type yeast Saccharomyces cerevisiae, the b2167Δ-DHFR precursor protein is cleaved by the matrix-processing peptidase into an intermediate form that is protected against the exogenously added protease. We reasoned that this purified precursor protein, when used in large amounts and even without arrest by methotrexate, should saturate the import sites and compete with a radiolabeled protein if both precursor proteins share a translocation pathway. As expected, we observed that the presence of increasing amounts of recombinant b2167Δ-DHFR inhibited the import of radiolabeled b2167Δ-DHFR into the protease-protected location inside mitochondria (Figure 1A). We chose a concentration of 5 μg of b2167Δ-DHFR per 100 μl of import reaction for the kinetic experiments. The import efficiency of radiolabeled b2167Δ-DHFR was decreased to ∼50% compared with the control reaction without the addition of a recombinant precursor (Figure 1B). We examined the import of the MIA-dependent precursor proteins Tim9 and Cox19 under the same conditions. Of interest, the presence of saturating b2167Δ-DHFR did not inhibit the import of the MIA-dependent precursor proteins Tim9 (Figure 1C, lanes 1–8 and graph) and Cox19 (Figure 1D, lanes 1–8 and graph). On their transfer across the OM, these proteins form a disulfide-bonded intermediate with Mia40 that is stably maintained in nonreducing denaturing electrophoresis (Milenkovic et al., 2007). The lack of competition between Tim9 or Cox19 and b2167Δ-DHFR was also reflected by the equal formation of the covalent intermediate complex with Mia40 (Figure 1, C, lanes 9–16 and D, lanes 9–16).


A discrete pathway for the transfer of intermembrane space proteins across the outer membrane of mitochondria.

Gornicka A, Bragoszewski P, Chroscicki P, Wenz LS, Schulz C, Rehling P, Chacinska A - Mol. Biol. Cell (2014)

Competition experiments with recombinant TIM23-dependent precursor protein for import into mitochondria. (A) Radiolabeled presequence-containing precursor of b2167Δ-DHFR fusion protein was imported into mitochondria in the presence of increasing concentrations (up to 5 μg/100 μl import reaction) of b2167Δ-DHFR for 10 min. i, intermediate form. Quantitations of 35S-radiolabeled b2167Δ-DHFR import (bottom). Import into mitochondria without recombinant b2167Δ-DHFR was set to 100%. SEM of three independent experiments. (B) Radiolabeled presequence-containing precursor (b2167Δ-DHFR) was imported into mitochondria in the presence or absence of 5 μg of b2167Δ-DHFR/100 μl of import reaction. Quantitations of 35S-radiolabeled b2167Δ-DHFR import (bottom). Import into mitochondria without recombinant b2167Δ-DHFR after 15 min was set to 100%. SEM of three independent experiments. (C) Radiolabeled Tim9 was imported into mitochondria in the presence or absence of 5 μg/100 μl import reaction of b2167Δ-DHFR. Quantitations of 35S-radiolabeled Tim9 import (middle). Import into mitochondria without recombinant b2167Δ-DHFR after 15 min was set to 100%. SEM of three independent experiments. (D) Radiolabeled Cox19 was imported into mitochondria in the presence or absence of 5 μg/100 μl import reaction of b2167Δ-DHFR. Quantitations of 35S-radiolabeled Cox19 import (middle). Import into mitochondria without recombinant b2167Δ-DHFR after 27 min was set to 100%. SEM of three independent experiments. (A–D) The samples were treated with proteinase K as indicated and analyzed by nonreducing or reducing SDS–PAGE. Δψ, electrochemical potential; IA, iodoacetamide.
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Figure 1: Competition experiments with recombinant TIM23-dependent precursor protein for import into mitochondria. (A) Radiolabeled presequence-containing precursor of b2167Δ-DHFR fusion protein was imported into mitochondria in the presence of increasing concentrations (up to 5 μg/100 μl import reaction) of b2167Δ-DHFR for 10 min. i, intermediate form. Quantitations of 35S-radiolabeled b2167Δ-DHFR import (bottom). Import into mitochondria without recombinant b2167Δ-DHFR was set to 100%. SEM of three independent experiments. (B) Radiolabeled presequence-containing precursor (b2167Δ-DHFR) was imported into mitochondria in the presence or absence of 5 μg of b2167Δ-DHFR/100 μl of import reaction. Quantitations of 35S-radiolabeled b2167Δ-DHFR import (bottom). Import into mitochondria without recombinant b2167Δ-DHFR after 15 min was set to 100%. SEM of three independent experiments. (C) Radiolabeled Tim9 was imported into mitochondria in the presence or absence of 5 μg/100 μl import reaction of b2167Δ-DHFR. Quantitations of 35S-radiolabeled Tim9 import (middle). Import into mitochondria without recombinant b2167Δ-DHFR after 15 min was set to 100%. SEM of three independent experiments. (D) Radiolabeled Cox19 was imported into mitochondria in the presence or absence of 5 μg/100 μl import reaction of b2167Δ-DHFR. Quantitations of 35S-radiolabeled Cox19 import (middle). Import into mitochondria without recombinant b2167Δ-DHFR after 27 min was set to 100%. SEM of three independent experiments. (A–D) The samples were treated with proteinase K as indicated and analyzed by nonreducing or reducing SDS–PAGE. Δψ, electrochemical potential; IA, iodoacetamide.
Mentions: To determine whether the translocation of MIA-dependent precursor proteins across the OM uses the same pathway as the import of presequence-containing precursors, we applied an import competition assay with purified precursor proteins. A recombinant cytosolic dihydrofolate reductase protein that was fused to the presequence of cytochrome b2 (b2167Δ-DHFR) was applied as a model presequence-containing substrate. It was arrested in translocation complexes in the presence of a substrate analogue, methotrexate (Dekker et al., 1997; Chacinska et al., 2003; Schulz and Rehling, 2014). On import into mitochondria isolated from wild-type yeast Saccharomyces cerevisiae, the b2167Δ-DHFR precursor protein is cleaved by the matrix-processing peptidase into an intermediate form that is protected against the exogenously added protease. We reasoned that this purified precursor protein, when used in large amounts and even without arrest by methotrexate, should saturate the import sites and compete with a radiolabeled protein if both precursor proteins share a translocation pathway. As expected, we observed that the presence of increasing amounts of recombinant b2167Δ-DHFR inhibited the import of radiolabeled b2167Δ-DHFR into the protease-protected location inside mitochondria (Figure 1A). We chose a concentration of 5 μg of b2167Δ-DHFR per 100 μl of import reaction for the kinetic experiments. The import efficiency of radiolabeled b2167Δ-DHFR was decreased to ∼50% compared with the control reaction without the addition of a recombinant precursor (Figure 1B). We examined the import of the MIA-dependent precursor proteins Tim9 and Cox19 under the same conditions. Of interest, the presence of saturating b2167Δ-DHFR did not inhibit the import of the MIA-dependent precursor proteins Tim9 (Figure 1C, lanes 1–8 and graph) and Cox19 (Figure 1D, lanes 1–8 and graph). On their transfer across the OM, these proteins form a disulfide-bonded intermediate with Mia40 that is stably maintained in nonreducing denaturing electrophoresis (Milenkovic et al., 2007). The lack of competition between Tim9 or Cox19 and b2167Δ-DHFR was also reflected by the equal formation of the covalent intermediate complex with Mia40 (Figure 1, C, lanes 9–16 and D, lanes 9–16).

Bottom Line: We identified a transient interaction between our model substrates and Tom40.Of interest, outer membrane translocation did not directly involve other core components of the TOM complex, including Tom22.Thus MIA-dependent proteins take another route across the outer mitochondrial membrane that involves Tom40 in a form that is different from the canonical TOM complex.

View Article: PubMed Central - PubMed

Affiliation: International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland.

Show MeSH
Related in: MedlinePlus