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.
Highlights
PEX5 functions as an import receptor for proteins with the type-1 peroxisomal targeting signal (PTS1)
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
Additional studies have established that mammalian cells express two forms of PEX5 that differ by 37 amino acids and that these forms are generated by alternative splicing of a single exon of the PEX5 gene [27, 64, 65]
Summary
The human skin fibroblast cell lines were kindly provided by A. The plasmid encoding full-length PEX5L, an N-terminal hexahistidinyl (His6) tag and a tobacco etch virus protease cleavage side in a derivative of pET9d (Novagen) has been described before [70]. This plasmid was transformed into Escherichia coli BL21(DE3), and recombinant PEX5L protein was purified under native conditions according to Schliebs et al [70]. Yeast Two-Hybrid System—The full-length PEX5L and PEX5S coding regions were excised from pGD106 and pGD100 with NcoI (blunted) and BglII and cloned into the SmaI and BglII sites of the activating domain vector pPC86 [71]. Mammalian Two-Hybrid System—HsPEX7 in pM (pEB6.4) was generated from pEB6.6 by excision of the full-length DNA with EcoRI
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