A Cell-Free In Vitro Import System for Peroxisomal Proteins Containing a Type 2 Targeting Signal (PTS2)

Cell-free in vitro systems are invaluable tools to study the molecular mechanisms of protein translocation across biological membranes. We have been using such a strategy to dissect the mechanism of the mammalian peroxisomal matrix protein import machinery. Here, we provide a detailed protocol to import proteins containing a peroxisomal targeting signal type 2 (PTS2) into the organelle. The in vitro system consists of incubating a ³⁵S-labeled reporter protein with a post-nuclear supernatant from rat/mouse liver. At the end of the incubation, the organelle suspensions are generally treated with an aggressive protease to degrade reporter proteins that did not enter peroxisomes, and the organelles are isolated by centrifugation and analyzed by SDS-PAGE and autoradiography. This in vitro system is particularly suited to characterize the functional consequences of PEX5 and PEX7 mutations found in patients affected with a peroxisomal biogenesis disorder.