In vitro import of peroxisome-targeting signal type 2 (PTS2) receptor Pex7p into peroxisomes

Pex7p, the peroxisome-targeting signal type 2 (PTS2) receptor, transports PTS2 proteins to peroxisomes from the cytosol. We here established a cell-free Pex7p translocation system. In assays using post-nuclear supernatant fractions each from wild-type CHO-K1 and pex7 ZPG207 cells, 35S-labeled Pex7p was imported into peroxisomes. 35S-Pex7p import was also evident using rat liver peroxisomes. 35S-Pex7p was not imported into peroxisomal remnants from a pex5 ZPG231 defective in PTS2 import and pex2 Z65. When the import of 35S-Pex5pL was inhibited with an excess amount of recombinant Pex5pS, 35S-Pex7p import was concomitantly abrogated, suggesting that Pex5pL was a transporter for Pex7p, unlike a yeast cochaperone, Pex18p. 35S-Pex7p as well as 35S-Pex5p was imported in an ATP-independent manner, whilst the import of PTS1 and PTS2 cargo-proteins was ATP-dependent. Thereby, ATP-independent import of Pex7p implicated that Pex5p export requiring ATP hydrolysis is not a limiting step for its cargo recruitment to peroxisomes. PTS1 protein import was indeed insensitive to N-ethylmaleimide, whereas Pex5p export was N-ethylmaleimide-sensitive. Taken together, the cargo-protein translocation through peroxisomal membrane more likely involves another ATP-requiring step in addition to the Pex5p export. Moreover, upon concurrent import into peroxisomes, 35S-Pex5pL and 35S-Pex7p were detected at mutually distinct ratios in the immunoprecipitates each of the import machinery peroxins including Pex14p, Pex13p, and Pex2p, hence suggesting that Pex7p as well as Pex5p translocated from the initial docking complex to RING complex on peroxisomes.