Limits...
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.

Show MeSH
Competition experiments with recombinant MIA-dependent precursor proteins for import into mitochondria. (A) Radiolabeled Mix17, Tim9, Cox19, F1β, Su9-DHFR, or b2167Δ-DHFR was imported into mitochondria in the presence of increasing concentrations (up to 3 μg/100 μl import reaction) of Tim10HIS for 10 min (top). Quantitations of 35S-radiolabeled precursor import (bottom). Import into mitochondria without recombinant Tim10HIS was set to 100%. SEM of three independent experiments. (B) Radiolabeled Mix17, Tim9, Su9-DHFR, or b2167Δ-DHFR was imported into mitochondria in the presence of increasing concentrations (up to 4 μg/100 μl import reaction) of Tim12HIS for 10 min. (A, B) The samples were treated with proteinase K and analyzed by reducing SDS–PAGE. Δψ, electrochemical potential; m, mature form.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4263444&req=5

Figure 2: Competition experiments with recombinant MIA-dependent precursor proteins for import into mitochondria. (A) Radiolabeled Mix17, Tim9, Cox19, F1β, Su9-DHFR, or b2167Δ-DHFR was imported into mitochondria in the presence of increasing concentrations (up to 3 μg/100 μl import reaction) of Tim10HIS for 10 min (top). Quantitations of 35S-radiolabeled precursor import (bottom). Import into mitochondria without recombinant Tim10HIS was set to 100%. SEM of three independent experiments. (B) Radiolabeled Mix17, Tim9, Su9-DHFR, or b2167Δ-DHFR was imported into mitochondria in the presence of increasing concentrations (up to 4 μg/100 μl import reaction) of Tim12HIS for 10 min. (A, B) The samples were treated with proteinase K and analyzed by reducing SDS–PAGE. Δψ, electrochemical potential; m, mature form.

Mentions: We also performed experiments with a reverse experimental setup in which we used purified MIA-dependent proteins (Böttinger et al., 2012) to compete with radiolabeled presequence-containing proteins. We observed that the increasing amounts of recombinant Tim10HIS gradually inhibited the transport of radiolabeled MIA substrates such as Mix17, Tim9, and Cox19 (Figure 2A, lanes 1–5 and graph). A similar result was obtained when Tim12HIS was imported in increasing concentrations (Figure 2B, lanes 1–5). The import of the presequence-containing precursors was unaffected by the presence of increasing amounts of Tim10HIS (Figure 2A, lanes 6–10 and graph) and Tim12HIS (Figure 2B, lanes 6–11). Large amounts of MIA substrates significantly inhibited the import of radiolabeled MIA-dependent precursors but did not influence TIM23-dependent preproteins. These results suggest the possibility that an alternative translocation route for MIA-dependent proteins exists that differs from the pathway for presequence-containing proteins.


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 MIA-dependent precursor proteins for import into mitochondria. (A) Radiolabeled Mix17, Tim9, Cox19, F1β, Su9-DHFR, or b2167Δ-DHFR was imported into mitochondria in the presence of increasing concentrations (up to 3 μg/100 μl import reaction) of Tim10HIS for 10 min (top). Quantitations of 35S-radiolabeled precursor import (bottom). Import into mitochondria without recombinant Tim10HIS was set to 100%. SEM of three independent experiments. (B) Radiolabeled Mix17, Tim9, Su9-DHFR, or b2167Δ-DHFR was imported into mitochondria in the presence of increasing concentrations (up to 4 μg/100 μl import reaction) of Tim12HIS for 10 min. (A, B) The samples were treated with proteinase K and analyzed by reducing SDS–PAGE. Δψ, electrochemical potential; m, mature form.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4263444&req=5

Figure 2: Competition experiments with recombinant MIA-dependent precursor proteins for import into mitochondria. (A) Radiolabeled Mix17, Tim9, Cox19, F1β, Su9-DHFR, or b2167Δ-DHFR was imported into mitochondria in the presence of increasing concentrations (up to 3 μg/100 μl import reaction) of Tim10HIS for 10 min (top). Quantitations of 35S-radiolabeled precursor import (bottom). Import into mitochondria without recombinant Tim10HIS was set to 100%. SEM of three independent experiments. (B) Radiolabeled Mix17, Tim9, Su9-DHFR, or b2167Δ-DHFR was imported into mitochondria in the presence of increasing concentrations (up to 4 μg/100 μl import reaction) of Tim12HIS for 10 min. (A, B) The samples were treated with proteinase K and analyzed by reducing SDS–PAGE. Δψ, electrochemical potential; m, mature form.
Mentions: We also performed experiments with a reverse experimental setup in which we used purified MIA-dependent proteins (Böttinger et al., 2012) to compete with radiolabeled presequence-containing proteins. We observed that the increasing amounts of recombinant Tim10HIS gradually inhibited the transport of radiolabeled MIA substrates such as Mix17, Tim9, and Cox19 (Figure 2A, lanes 1–5 and graph). A similar result was obtained when Tim12HIS was imported in increasing concentrations (Figure 2B, lanes 1–5). The import of the presequence-containing precursors was unaffected by the presence of increasing amounts of Tim10HIS (Figure 2A, lanes 6–10 and graph) and Tim12HIS (Figure 2B, lanes 6–11). Large amounts of MIA substrates significantly inhibited the import of radiolabeled MIA-dependent precursors but did not influence TIM23-dependent preproteins. These results suggest the possibility that an alternative translocation route for MIA-dependent proteins exists that differs from the pathway for presequence-containing proteins.

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