Related Experiment Videos
Intracellular localization, function, and dysfunction of the peroxisome-targeting signal type 2 receptor, Pex7p, in
Satoru Mukai1, Kamran Ghaedi, Yukio Fujiki
1Department of Biology, Faculty of Sciences, Kyushu University Graduate School, Fukuoka 812-8581, Japan.
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.
Insights
Researchers identified the Chinese hamster PEX7 gene, crucial for peroxisome-targeting signal type 2 (PTS2) protein import. This study elucidates Pex7p function and its role in peroxisomal disorders, highlighting the necessity of its full length for PTS2 import.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Peroxisome biogenesis relies on the import of specific proteins.
- Peroxisome-targeting signal type 2 (PTS2) proteins require the PEX7 receptor for import.
- A Chinese hamster ovary (CHO) cell mutant (ZPG207) exhibits a defect in PTS2 import.
Purpose of the Study:
- To clone and characterize the Chinese hamster PEX7 gene (ClPEX7).
- To investigate the molecular mechanisms underlying PTS2 import defects caused by PEX7 mutations.
- To determine the functional domains of Pex7p required for peroxisome import.
Main Methods:
- Gene cloning and sequencing of Chinese hamster PEX7.
- Complementation of the ZPG207 mutant with ClPEX7.
- Site-directed mutagenesis and analysis of Pex7p variants.
- Subcellular fractionation and protease protection assays.
- Analysis of Pex7p interaction with PTS2 cargo and Pex5p isoforms.
Main Results:
- Cloned Chinese hamster PEX7 (ClPEX7) and confirmed its role as the PTS2 receptor.
- Identified a mutation (W221ter) in the ZPG207 mutant's PEX7 gene.
- Mammalian Pex7p variants, including those found in rhizomelic chondrodysplasia punctata, impaired PTS2 import and binding to cargo and Pex5pL.
- Pex7p exhibits bimodal distribution between cytoplasm and peroxisomes.
- Pex5pL, but not Pex5pS, enhanced Pex7p-PTS2 translocation into peroxisomes.
- Specific N-terminal and C-terminal truncations of ClPex7p affected its activity, indicating the importance of WD motifs.
Conclusions:
- The cloned Chinese hamster PEX7 gene rescues PTS2 import defects.
- PEX7 mutations disrupt Pex7p binding to PTS2 cargo and Pex5pL, leading to import deficiency.
- Pex7p and its cargo shuttle between the cytoplasm and peroxisomes, facilitated by Pex5pL.
- The full length of Pex7p, including all WD motifs, is essential for its function in PTS2 protein import.