Intracellular localization, function, and dysfunction of the peroxisome-targeting signal type 2 receptor, Pex7p, in mammalian cells.

We previously isolated and characterized a Chinese hamster ovary (CHO) cell mutant, ZPG207, that is defective in import of proteins carrying a peroxisome-targeting signal type 2 (PTS2) nonapeptide. Herein we have cloned Chinese hamster (Cl) PEX7 encoding the PTS2 receptor. ClPex7p consists of 318 amino acids, shorter than human Pex7p by 5 residues, showing 91 and 30% identity with Pex7p from humans and the yeast Saccharomyces cerevisiae, respectively. Expression of ClPEX7 rescued the impaired PTS2 import in pex7 ZPG207. Mutation in ZPG207 PEX7 was determined by reverse transcription PCR; a G-to-A transition caused a 1-amino acid substitution, W221ter. We investigated the molecular dysfunction of Pex7p variants in mammals, including Pex7p-W221ter and Pex7p with one site mutation at G217R, A218V, or L292ter, which frequently occurs in the human fatal genetic peroxisomal disease rhizomelic chondrodysplasia punctata, showing a cell phenotype of PTS2 import defect. All types of the mutations affected Pex7p in binding to both PTS2 cargo protein and the longer isoform of PTS1 receptor Pex5pL that is responsible for transport of the Pex7p-PTS2 complex. Subcellular fractionation and protease protection studies demonstrated bimodal distribution of Pex7p between the cytoplasm and peroxisomes in CHO and human cells. Moreover, expression of Pex5pL, but not of the shorter isoform Pex5pS, enhanced translocation of Pex7p-PTS2 proteins into peroxisomes, thereby implying that both PTS receptors shuttle between peroxisomes and the cytosol. Furthermore, a ClPex7p mutant with a deletion of 7 amino acids from the N terminus retained peroxisome-restoring activity, whereas an 11-amino acid truncation abrogated the activity. ClPex7p with a C-terminal 9- amino acid truncation, comprising residues 1--309, maintained the activity, whereas a 14-amino acid shorter form lacking several amino acids of the sixth WD motif lost the activity. Therefore, nearly the full length of Pex7p, including all WD motifs, is required for its function.