Domain Mapping of Human PEX5 Reveals Functional and Structural Similarities to Saccharomyces cerevisiae Pex18p and Pex21p

Abstract

PEX5 functions as an import receptor for proteins with the type-1 peroxisomal targeting signal (PTS1). Although PEX5 is not involved in the import of PTS2-targeted proteins in yeast, it is essential for PTS2 protein import in mammalian cells. Human cells generate two isoforms of PEX5 through alternative splicing, PEX5S and PEX5L, and PEX5L contains an additional insert 37 amino acids long. Only one isoform, PEX5L, is involved in PTS2 protein import, and PEX5L physically interacts with PEX7, the import receptor for PTS2-containing proteins. In this report we map the regions of human PEX5L involved in PTS2 protein import, PEX7 interaction, and targeting to peroxisomes. These studies revealed that amino acids 1-230 of PEX5L are required for PTS2 protein import, amino acids 191-222 are sufficient for PEX7 interaction, and amino acids 1-214 are sufficient for targeting to peroxisomes. We also identified a 21-amino acid-long peptide motif of PEX5L, amino acids 209 -229, that overlaps the regions sufficient for full PTS2 rescue activity and PEX7 interaction and is shared by Saccharomyces cerevisiae Pex18p and Pex21p, two yeast peroxins that act only in PTS2 protein import in yeast. A mutation in PEX5 that changes a conserved serine of this motif abrogates PTS2 protein import in mammalian cells and reduces the interaction of PEX5L and PEX7 in vitro. This peptide motif also lies within regions of Pex18p and Pex21p that interact with yeast PEX7. Based on these and other results, we propose that mammalian PEX5L may have acquired some of the functions that yeast Pex18p and/or Pex21p perform in PTS2 protein import. This hypothesis may explain the essential role of PEX5L in PTS2 protein import in mammalian cells and its lack of importance for PTS2 protein import in yeast.

Pex5p, the Peroxisomal Cycling Receptor, Is a Monomeric Non-globular Protein

“…The alternative would be to consider that binding of a PTS1 cargo protein to Pex5p also induces conformational alterations in regions of the Pex5p protein located more than 100 amino acid residues away as is the case for the Pex13p-binding domain of Pex5p (43). This possibility, however, is not supported by the observation that Pex5p-truncated versions lacking the TPR domains are still good substrates for the peroxisomal docking/translocation machinery and even for the ATPase catalyzing the export of Pex5p back into the cytosol (30,47,48). Thus, as suggested recently, it is possible that the peroxisomal targeting domain of Pex5p is a target of negative regulation exerted by the TPR domains (30).…”

The N Terminus of the Peroxisomal Cycling Receptor, Pex5p, Is Required for Redirecting the Peroxisome-associated Peroxin Back to the Cytosol

“…Presently, our knowledge on the structure/function of amino acid residues 118 -639 of mammalian Pex5p is quite vast. Indeed, a variety of biochemical and structural studies have shown that this region of the protein contains the binding site for PTS1-containing cargo proteins (7,(22)(23)(24)(25)(26)(27), seven Pex14p-binding domains (43)(44)(45), and binding sites for Pex7p (11)(12)(13)(14), Pex12p (46), and Pex13p (45). In sharp contrast, not much is known regarding the role of the first 117 amino acid residues of Pex5p.…”

“…This peroxin recognizes a degenerated nona-peptide complying to the consensus sequence (R/K)(L/I/V)X 5 (H/Q)(L/A/F), the socalled peroxisomal targeting signal type 2 (PTS2) (8 -10). In mammals and plants, but not in lower eukaryotes, targeting of these PTS2-containing proteins seems to require Pex5p, suggesting that the two import pathways actually converge at the level of the PTS1 receptor (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21).…”

Two Independent Pathways Traffic the Intraperoxisomal Peroxin PpPex8p into Peroxisomes: Mechanism and Evolutionary Implications

“…These two sets of genes appear to have coevolved. Mammals dispensed with the PEX20 gene by incorporating its PTS2-pathway function into an alternative exon in the PEX5 gene (Dodt et al, 2001). As a consequence, organisms such as plants and mammals that use the long (Pex5L) isoform of Pex5 to perform Pex20p-like functions, also lack a PEX8 gene.…”