Revisiting the intraperoxisomal pathway of mammalian PEX7

Newly synthesized peroxisomal proteins containing a cleavable type 2 targeting signal (PTS2) are transported to the peroxisome by a cytosolic PEX5-PEX7 complex. There, the trimeric complex becomes inserted into the peroxisomal membrane docking/translocation machinery (DTM), a step that leads to the translocation of the cargo into the organelle matrix. Previous work suggests that PEX5 is retained at the DTM during all the steps occurring at the peroxisome but whether the same applies to PEX7 was unknown. By subjecting different pre-assembled trimeric PEX5-PEX7-PTS2 complexes to in vitro co-import/export assays we found that the export competence of peroxisomal PEX7 is largely determined by the PEX5 molecule that transported it to the peroxisome. This finding suggests that PEX7 is also retained at the DTM during the peroxisomal steps and implies that cargo proteins are released into the organelle matrix by DTM-embedded PEX7. The release step does not depend on PTS2 cleavage. Rather, our data suggest that insertion of the trimeric PEX5-PEX7-PTS2 protein complex into the DTM is probably accompanied by conformational alterations in PEX5 to allow release of the PTS2 protein into the organelle matrix.