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.