Figure 4. Tim23 and Tim50 IMS domain... | Rockefeller University Press

$Figure 4. Tim23 and Tim50 IMS domain mutants are defective in Tim23–Tim50 interactions. (A) Wild-type control (WT) and tim23 mutant mitochondria with FLAG-tagged Tim50 were solubilized with 1% digitonin and subjected to immunoprecipitation with the anti-FLAG antibody. 5%, 5% of the loaded proteins; F, 10% of unbound proteins; E, 100% of proteins eluted with 100 mM glycine-HCl, pH 2.5. (B) Immunoprecipitation was performed as in A (Tim50 is not FLAG tagged) using anti-Tim23 antibodies. (C) wild-type, tim23-64, tim23-71, tim23-78, tim23-71,78, and tim23-64,71,78 mitochondria were solubilized with 0.5% Triton X-100 and subjected to incubation with Ni-NTA agarose loaded with purified recombinant Tim50 IMS. Bound proteins were eluted with 500 mM imidazole and analyzed by SDS-PAGE and immunoblotting with anti-Tim23 antibodies. 5%, 5% of the loaded proteins; FT, 10% of unbound proteins; W2-W3, 100% of washed fractions; E, 100% of eluted proteins. (D) Wild-type and tim50 mutant mitochondria with FLAG-tagged Tim23 were solubilized with 1% digitonin and subjected to immunoprecipitation with the anti-FLAG antibody as in A. 15%, 15% of the loaded proteins; F, 10% of unbound proteins; E, 100% of eluted proteins. (E) Radiolabeled Tim23 with BPA incorporated at position 64, 71, or 78 was imported into mitochondria (W303-1A) at 25°C for 1 h and subjected to UV irradiation. Identified cross-linked partners are indicated. (F) Mitochondria with the FLAG-tagged version of Tim23 (WT) or L64,71,78S Tim23 (tim23-64,71,78) were solubilized with 1% digitonin and subjected to immunoprecipitation as in A. Error bars represent SDs from three independent experiments.$

Figure 4.

Tim23 and Tim50 IMS domain mutants are defective in Tim23–Tim50 interactions. (A) Wild-type control (WT) and tim23 mutant mitochondria with FLAG-tagged Tim50 were solubilized with 1% digitonin and subjected to immunoprecipitation with the anti-FLAG antibody. 5%, 5% of the loaded proteins; F, 10% of unbound proteins; E, 100% of proteins eluted with 100 mM glycine-HCl, pH 2.5. (B) Immunoprecipitation was performed as in A (Tim50 is not FLAG tagged) using anti-Tim23 antibodies. (C) wild-type, tim23-64, tim23-71, tim23-78, tim23-71,78, and tim23-64,71,78 mitochondria were solubilized with 0.5% Triton X-100 and subjected to incubation with Ni-NTA agarose loaded with purified recombinant Tim50 IMS. Bound proteins were eluted with 500 mM imidazole and analyzed by SDS-PAGE and immunoblotting with anti-Tim23 antibodies. 5%, 5% of the loaded proteins; FT, 10% of unbound proteins; W2-W3, 100% of washed fractions; E, 100% of eluted proteins. (D) Wild-type and tim50 mutant mitochondria with FLAG-tagged Tim23 were solubilized with 1% digitonin and subjected to immunoprecipitation with the anti-FLAG antibody as in A. 15%, 15% of the loaded proteins; F, 10% of unbound proteins; E, 100% of eluted proteins. (E) Radiolabeled Tim23 with BPA incorporated at position 64, 71, or 78 was imported into mitochondria (W303-1A) at 25°C for 1 h and subjected to UV irradiation. Identified cross-linked partners are indicated. (F) Mitochondria with the FLAG-tagged version of Tim23 (WT) or L64,71,78S Tim23 (tim23-64,71,78) were solubilized with 1% digitonin and subjected to immunoprecipitation as in A. Error bars represent SDs from three independent experiments.

This Feature Is Available To Subscribers Only