Mutation of putative zinc finger of KREPB4 prevents normal cleavage activities in TAP-isolated complexes. Asterisks in the schematics denote location of radiolabel. Cleavage product (arrows) is only observed in the presence of guide RNA (+g), not in its absence (−g). Positive control activity (20S+) was obtained using a ∼20S fraction from gradient fractionated mitochondrial or whole cell lysate. The reference ladder was produced by RNase T1 (T1). (A) Insertion cleavage assay of complexes isolated via KREPB4 wild type (WT), C51A/C54A, and E284A (left panel) or via KREPB5 wild type, C14A, E236A, V5-tagged wild type (WT-V5), F114S, and E122A (right panel). (B) Deletion cleavage assay of complexes described above. Note the lack of cleavage product by complexes with the C51A/C54A mutation in KREPB4.
