Engineering presequence probes for receptor screening. (A) Primary structure of presequence probes and rat ALDH presequence (pALDH). (B) Presequence probes were imported into isolated mitochondria in the presence or absence of a Δψ for the indicated times at 25°C. After import, mitochondria were treated with proteinase K and samples analyzed by Western blotting using streptavidin-HRP (horseradish peroxidase). (C) Radiolabeled precursor was imported for 15 min at 25°C into isolated mitochondria in the presence of indicated amounts of peptides. After proteinase K treatment, import reactions were analyzed by SDS-PAGE and digital autoradiography. i, intermediate. (D) Mitochondria were incubated with and without presequence peptide in the presence or absence of Δψ. After re-isolation of mitochondria, protein import of a radiolabeled precursor protein was assessed in a second incubation. Therefore, radiolabeled precursor was added to treated or untreated mitochondria and import performed for 15 min. A control import was performed in the presence of peptide. Samples were analyzed by SDS-PAGE and digital autoradiography as in C. i, intermediate. (E) Isolated mitochondria were incubated with 2 µM of the respective photo-peptide, equilibrated for 10 min, and subjected to UV irradiation for 30 min. PA, photo-adduct. (F) After in organello photo cross-linking for 30 min, photo-adducts (PA) were purified from mitochondria by streptavidin agarose chromatography. Samples were analyzed by Western blotting with indicated antibodies. Total, 5%; Eluate, 100%. PA, photo-adduct.
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