Figure 6. Roles of Tim23–Tim50 interactions in the IMS in the late step of protein translocation across the inner membrane. (A) Mitochondria isolated from wild-type control (WT), in which Tim23 is supplied from the plasmid, and tim23 mutant cells were incubated in import buffer containing 0.05% BSA and 75 mM disuccinimidylglutarate on ice for 30 min. After quenching the reaction with 50 mM Tris-HCl, pH 7.4, Tim23 dimer formation was analyzed by SDS-PAGE followed by immunoblotting with anti-Tim23 antibodies. The amount of the Tim23 dimer in wild-type mitochondria was set to 100%. (B) Radiolabeled Tim23 was imported into wild-type mitochondria (WT) with or without Tim23FLAG and into tim50-279,282,286 mitochondria (T) with Tim23FLAG for 20 min at 25°C and PK treated. The mitochondria was solubilized with 1.0% digitonin and subjected to coimmunoprecipitation with the anti-FLAG antibody. Proteins were analyzed by SDS-PAGE followed by radioimaging and immunoblotting with indicated antibodies before and after import. (C) Radiolabeled pb2(220)Δ19-DHFR was incubated with mitochondria for 20 min at 30°C in the presence of 10 mM methotrexate and 100 mM NADPH. The mitochondria were treated with 20 mg/ml PK for 20 min on ice. PK treatment was stopped by addition of 1 mM PMSF, and the mitochondria were reisolated by centrifugation. Proteins were analyzed by SDS-PAGE and radioimaging (top). The amounts of the PK-resistant processed intermediate (i) were quantified (bottom). The amounts of the processed intermediate without PK treatment are set to 100%. p, precursor form. (D) Serial dilutions of wild-type, tim23, and tim50 mutant strains carrying yeast 2μ plasmid, pYO326-TIM23, and pYO325-SSC1 were plated on SD (−Ura, −Trp, −Leu; left) and SD (−Trp, −Leu, and +1 mg/ml 5′-fluoroorotic acid; right) and grown for 4 d at 23°C. (E) Serial dilutions of tim23-64,71,78 carrying yeast 2μ plasmid (pYO326) encoding indicated genes were plated on YPD plates and grown for 2 d at 30 or 35°C. (F) Serial dilutions of tim23 and tim50 mutant strains lacking the PAM17 gene with plasmid, pRS316-Tim23Δ50, and pRS316-Tim50, respectively, were plated on a SCD (−Trp and +FOA) plate. Error bars represent SDs from three independent experiments.
Figure 6.

Roles of Tim23–Tim50 interactions in the IMS in the late step of protein translocation across the inner membrane. (A) Mitochondria isolated from wild-type control (WT), in which Tim23 is supplied from the plasmid, and tim23 mutant cells were incubated in import buffer containing 0.05% BSA and 75 mM disuccinimidylglutarate on ice for 30 min. After quenching the reaction with 50 mM Tris-HCl, pH 7.4, Tim23 dimer formation was analyzed by SDS-PAGE followed by immunoblotting with anti-Tim23 antibodies. The amount of the Tim23 dimer in wild-type mitochondria was set to 100%. (B) Radiolabeled Tim23 was imported into wild-type mitochondria (WT) with or without Tim23FLAG and into tim50-279,282,286 mitochondria (T) with Tim23FLAG for 20 min at 25°C and PK treated. The mitochondria was solubilized with 1.0% digitonin and subjected to coimmunoprecipitation with the anti-FLAG antibody. Proteins were analyzed by SDS-PAGE followed by radioimaging and immunoblotting with indicated antibodies before and after import. (C) Radiolabeled pb2(220)Δ19-DHFR was incubated with mitochondria for 20 min at 30°C in the presence of 10 mM methotrexate and 100 mM NADPH. The mitochondria were treated with 20 mg/ml PK for 20 min on ice. PK treatment was stopped by addition of 1 mM PMSF, and the mitochondria were reisolated by centrifugation. Proteins were analyzed by SDS-PAGE and radioimaging (top). The amounts of the PK-resistant processed intermediate (i) were quantified (bottom). The amounts of the processed intermediate without PK treatment are set to 100%. p, precursor form. (D) Serial dilutions of wild-type, tim23, and tim50 mutant strains carrying yeast 2μ plasmid, pYO326-TIM23, and pYO325-SSC1 were plated on SD (−Ura, −Trp, −Leu; left) and SD (−Trp, −Leu, and +1 mg/ml 5′-fluoroorotic acid; right) and grown for 4 d at 23°C. (E) Serial dilutions of tim23-64,71,78 carrying yeast 2μ plasmid (pYO326) encoding indicated genes were plated on YPD plates and grown for 2 d at 30 or 35°C. (F) Serial dilutions of tim23 and tim50 mutant strains lacking the PAM17 gene with plasmid, pRS316-Tim23Δ50, and pRS316-Tim50, respectively, were plated on a SCD (−Trp and +FOA) plate. Error bars represent SDs from three independent experiments.

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