FIGURE 3.

Binding of cargo protein to PEX7 is required for its interaction with PEX5L. A, mammalian two-hybrid assay: COS7 cells were transfected with the luciferase and β-galactosidase reporter plasmids and either the empty plasmids or VP16^AD-HA-PEX5L and GAL4^DBD-PEX7 or mutated variants thereof that either severely interfere with cargo binding (E113R, E200R) or directly affect the interaction between PEX5L and PEX7 (E287R). B–E, immunofluorescence microscopy of COS7 cells transfected with mRFP-PEX7 (B) or variants thereof that harbor mutations interfering with cargo binding, mRFP-PEX7^E113R (C) or mRFP-PEX7^E200R (D), or interfering with PEX5L binding, mRFP-PEX7^E287R (E). mRFP autofluorescence (red) and α-PMP70 antibody staining (green) were used for detection of the cellular location of the protein. F, modified mammalian two-hybrid assay: COS7 cells were co-transfected with bait and prey plasmids encoding HA-PEX5L and PEX7 together with expression plasmids for EGFP (light gray), the PTS2-carrying cargo protein PTS2_thiolase-EGFP (dark gray), or a variant of the PTS2-carrying reporter protein harboring a mutation in the PTS2 (PTS2_thiolase^RS1E-EGFP) (gray). The ratio of luciferase activity and β-galactosidase activity is indicated.