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Tim50 contains two separate functional domains for presequence and Tim23 binding. (A) Ni-NTA agarose with purified Tim23IMS or without (control) was incubated with mitochondrial detergent extracts, bound proteins eluted, and analyzed by Western blotting with the indicated antibodies. Total, 5%; Eluate, 100%. (B) Immunoprecipitation from solubilized mitochondria containing reduced levels of Tim50 and Tim50HA1 or Tim50ΔPBDHA1 using anti-HA or -6xHis (control) antibodies. Samples were analyzed as in A. Total, 5%; Eluate, 100%. (C) Isolated mitochondria from indicated strains were incubated with presequence probes and subjected to photo cross-linking for 30 min. Samples were analyzed as in A. PA, photo-adduct. Asterisk denotes cross-reactive protein. (D) Radiolabeled F1β was imported into wild-type and Tim50↑ mitochondria for the indicated times at 25°C. Subsequent to proteinase K digestion samples were analyzed by SDS-PAGE and digital autoradiography. Amounts of processed and protease-protected protein were quantified. The amount after import for 16 min in wild-type mitochondria was set to 100%. m, mature protein. (E) Steady-state protein analysis of isolated wild-type and Tim23 down-regulated mitochondria analyzed by Western blotting with the indicated antibodies. (F) Isolated mitochondria were incubated with 2 µM of the indicated presequence probes and subjected UV irradiation for 30 min. Samples were analyzed as in A. PA, photo-adduct.