Figure 5. Roles of Tim23–Tim50 interactions in the IMS in the early stage of protein translocation across the inner membrane. (A, Binding) Radiolabeled pSu9-DHFR was incubated with CCCP-treated mitochondria isolated from wild-type control (WT), tim23 mutant, and tim50 mutant cells for 15 min at 30°C. Mitochondria were then washed with buffer containing indicated concentration of KCl, and bound proteins were analyzed by SDS-PAGE and radioimaging. (Chase) Mitochondria after binding of pSu9-DHFR in the presence of 100 mM KCl were incubated in chase buffer for indicated times (without following PK treatment). Proteins were analyzed by SDS-PAGE and radioimaging, and the chased, mature form was quantified. C, bound pSu9-DHFR (100%); p, precursor form; m, mature form. (B) Radiolabeled pSu9-DHFR containing BPA at position 21 was bound to wild-type control (WT), tim23 mutant, and tim50 mutant mitochondria in SM buffer with 10 mM KCl, 5 mM MgCl2, and 2 mM methionine with 10 mg/ml valinomycin at 30°C for 15 min. Mitochondria were washed with SM buffer containing 150 mM KCl and kept on ice with (UV+) or without (UV−) UV irradiation in the same buffer for 5 min. The UV-irradiated mitochondria were solubilized and subjected to immunoprecipitation with anti-Tim50 antibodies (+IP), and proteins were analyzed by SDS-PAGE and radioimaging. Closed and open triangles indicate cross-linked products with Tim50 and Tom22, respectively. (C) Mitochondria isolated from wild-type, tim23 mutant, tim21Δ, and tim50 mutant cells were treated with 500 μg/ml PK for 20 min at 4 or 16°C. After stopping the reaction with 1 mM PMSF, the mitochondria were reisolated and proteins were analyzed by SDS-PAGE and immunoblotting with antibodies against Tim54 (with a domain exposed to the IMS), Tom20 (with a domain exposed to the cytosol), and Tim23. Total amounts of Tim23 and Tim23*, a protease-resistant fragment, are set to 100%. (D) Mitochondria isolated from indicated cells expressing Tim23FLAG with BPA at position 41 after UV irradiation (UV+) or mock treatment (UV−) were analyzed by SDS-PAGE and immunoblotting with the anti-FLAG antibody (left) or were subjected to immunoprecipitation with the anti-FLAG antibody, and Tom22 was detected by anti-Tom22 antibodies (right). (E) C-terminally (His)10-tagged Tom22 with BPA at position 132, 134, or 136 was expressed in cells with either wild-type (W) or C-terminally FLAG-tagged (F) Tim50 and purified with Ni-NTA resin after UV irradiation (UV+) or mock treatment (UV−). The purified proteins were analyzed by SDS-PAGE and immunoblotting with anti-Tom22 antibodies. The weak bands above the cross-linked products (denoted with a closed triangle) are nonspecific bands. (F) Cell extracts were prepared from TIM50FLAG/TOM22-BPA-X (X = 132, 134, and 136) and were subjected to immunoprecipitation with the antibody against the FLAG epitope tag attached to the C terminus of Tim50. The purified proteins were analyzed by SDS-PAGE and immunoblotting with anti-Tim50 antibodies (left) and anti-Tom22 antibodies (right). (G) Tom22-(His)10 with BPA at position 132 or 136 was expressed in tim23-71, tim50-279,282,286, or their corresponding wild-type cells and purified with Ni-NTA resin after UV irradiation (UV+) or mock treatment (UV−). Cross-linked products between Tom22 and Tim50 were detected by immunoblotting with anti-Tom22 antibodies and quantified with normalization by the total amount of authentic Tom22 plus Tom22-(His)10. Amounts of the cross-linked products in corresponding wild-type cells were set to 100%. Error bars represent SDs from three independent experiments.
Figure 5.

Roles of Tim23–Tim50 interactions in the IMS in the early stage of protein translocation across the inner membrane. (A, Binding) Radiolabeled pSu9-DHFR was incubated with CCCP-treated mitochondria isolated from wild-type control (WT), tim23 mutant, and tim50 mutant cells for 15 min at 30°C. Mitochondria were then washed with buffer containing indicated concentration of KCl, and bound proteins were analyzed by SDS-PAGE and radioimaging. (Chase) Mitochondria after binding of pSu9-DHFR in the presence of 100 mM KCl were incubated in chase buffer for indicated times (without following PK treatment). Proteins were analyzed by SDS-PAGE and radioimaging, and the chased, mature form was quantified. C, bound pSu9-DHFR (100%); p, precursor form; m, mature form. (B) Radiolabeled pSu9-DHFR containing BPA at position 21 was bound to wild-type control (WT), tim23 mutant, and tim50 mutant mitochondria in SM buffer with 10 mM KCl, 5 mM MgCl2, and 2 mM methionine with 10 mg/ml valinomycin at 30°C for 15 min. Mitochondria were washed with SM buffer containing 150 mM KCl and kept on ice with (UV+) or without (UV−) UV irradiation in the same buffer for 5 min. The UV-irradiated mitochondria were solubilized and subjected to immunoprecipitation with anti-Tim50 antibodies (+IP), and proteins were analyzed by SDS-PAGE and radioimaging. Closed and open triangles indicate cross-linked products with Tim50 and Tom22, respectively. (C) Mitochondria isolated from wild-type, tim23 mutant, tim21Δ, and tim50 mutant cells were treated with 500 μg/ml PK for 20 min at 4 or 16°C. After stopping the reaction with 1 mM PMSF, the mitochondria were reisolated and proteins were analyzed by SDS-PAGE and immunoblotting with antibodies against Tim54 (with a domain exposed to the IMS), Tom20 (with a domain exposed to the cytosol), and Tim23. Total amounts of Tim23 and Tim23*, a protease-resistant fragment, are set to 100%. (D) Mitochondria isolated from indicated cells expressing Tim23FLAG with BPA at position 41 after UV irradiation (UV+) or mock treatment (UV−) were analyzed by SDS-PAGE and immunoblotting with the anti-FLAG antibody (left) or were subjected to immunoprecipitation with the anti-FLAG antibody, and Tom22 was detected by anti-Tom22 antibodies (right). (E) C-terminally (His)10-tagged Tom22 with BPA at position 132, 134, or 136 was expressed in cells with either wild-type (W) or C-terminally FLAG-tagged (F) Tim50 and purified with Ni-NTA resin after UV irradiation (UV+) or mock treatment (UV−). The purified proteins were analyzed by SDS-PAGE and immunoblotting with anti-Tom22 antibodies. The weak bands above the cross-linked products (denoted with a closed triangle) are nonspecific bands. (F) Cell extracts were prepared from TIM50FLAG/TOM22-BPA-X (X = 132, 134, and 136) and were subjected to immunoprecipitation with the antibody against the FLAG epitope tag attached to the C terminus of Tim50. The purified proteins were analyzed by SDS-PAGE and immunoblotting with anti-Tim50 antibodies (left) and anti-Tom22 antibodies (right). (G) Tom22-(His)10 with BPA at position 132 or 136 was expressed in tim23-71, tim50-279,282,286, or their corresponding wild-type cells and purified with Ni-NTA resin after UV irradiation (UV+) or mock treatment (UV−). Cross-linked products between Tom22 and Tim50 were detected by immunoblotting with anti-Tom22 antibodies and quantified with normalization by the total amount of authentic Tom22 plus Tom22-(His)10. Amounts of the cross-linked products in corresponding wild-type cells were set to 100%. Error bars represent SDs from three independent experiments.

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