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Presequence recognition by Tim50 is required for inner membrane transport. (A) Radiolabeled precursor was imported at 25°C into isolated mitochondria in the presence or absence of Δψ. Mitochondria were treated with proteinase K and analyzed by SDS-PAGE and digital autoradiography. Amounts of processed protein were quantified (right). The amount after import for 16 min in wild-type mitochondria was set to 100% (n = 3, SEM). p, precursor; m, mature. (B) Radiolabeled AAC was imported into indicated mitochondria at 25°C. After import and proteinase K treatment, mitochondria were solubilized and analyzed by BN-PAGE and digital autoradiography. (C) [35S]Su9-DHFR precursor was imported into mitochondria in the absence of Δψ, and subsequently diluted in buffer containing methotrexate and NADPH. After re-isolation, mitochondria were treated with proteinase K or left untreated as indicated. Samples were analyzed as in A. P, pellet; S, supernatant; p, precursor; f, protease-resistant DHFR fragment. (D) Radiolabeled precursor proteins were imported into mitochondria or mitoplasts for 15 min at 25°C in the presence of affinity-purified anti-Tim50PBD or control antibodies. Samples were analyzed as in A. 100%, import in the absence of antibody. Import into mitoplasts was calculated as percentage of the corresponding import into mitochondria. (E) Radiolabeled precursor was imported into mitoplasts in the presence or absence of Δψ. After import, mitoplasts were treated with proteinase K and analyzed and quantified as in A. p, precursor; m, mature.