Abstract
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.
Highlights
To elucidate the hierarchy of the highly organized biogenesis of intracellular organelles, the peroxisome, a single membranebounded essential organelle (1), has been used as a model compartment in mammalian and yeast systems (2, 3)
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 peroxisome-targeting signal type 2 (PTS2) import defect
By functional phenotype complementation assay using Chinese hamster ovary (CHO) cell mutants, we isolated nine cDNAs encoding peroxisome biogenesis factors, termed peroxins, PEX1 (14), PEX2 (15), PEX3 (16), PEX5 (17), PEX6 (18), PEX12 (19, 20), PEX13 (21), PEX14 (22), and PEX19 (23). We showed that these PEXs, except for PEX14, are responsible for human fatal genetic disease, called peroxisome biogenesis disorders (PBD) such as Zellweger syndrome in which impaired peroxisome assembly is manifested (14, 17, 19, 20, 23–28)
Summary
To elucidate the hierarchy of the highly organized biogenesis of intracellular organelles, the peroxisome, a single membranebounded essential organelle (1), has been used as a model compartment in mammalian and yeast systems (2, 3). PEX7 encoding the PTS2 receptor is mutated in RCDP patients, where morphologically normal peroxisomes are present but defective in the import of PTS2 proteins (31–33). We more recently demonstrated, using CHO pex[5] cell mutants, that Pex5pL plays an exclusively pivotal role in PTS2 transport by interacting with Pex7p in mammals (38, 39). We have investigated the intracellular localization of Pex7p in CHO and human cells and found for the first time its bimodal distribution at the endogenous level, namely in the cytoplasm and peroxisomes. We have defined the function and dysfunction of Pex7p at molecular and cellular levels in PTS2 protein transport by making use of a CHO pex[7] mutant, ZPG207 (30). We report here that mutations of Pex7p identified in RCDP patients as well as ZPG207 cells affect the binding of Pex7p to both PTS2 protein and Pex5pL. Truncation analysis revealed that nearly the full length of Pex7p is required for the biological activity
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