Domain mapping of human PEX5 reveals functional and structural similarities to Saccharomyces cerevisiae Pex18p and Pex21p

PEX5 functions as an import receptor for proteins with the type-1 peroxisom al 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 alterna tive splicing, PEX5S and PEX5L, and PEX5L contains an additional insert 37 amino acids long. Only one isoform, PEX5L, is involved in PTS2 protein impo rt, and PEX5L physically interacts with PEX7, the import receptor for PTS2- containing proteins. In this report we map the regions of human PEX5L invol ved in PTS2 protein import, PEX7 interaction, and targeting to peroxisomes. These studies revealed that amino acids 1-230 of PEX5L are required for PT S2 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 209-22 9, that overlaps the regions sufficient for fall PTS2 rescue activity and P EX7 interaction and is shared by Saccharomyces cerevisiae Pex18p and Pex21p , two yeast peroxins that act only in PTS2 protein import in yeast. A mutat ion in PEX5 that changes a conserved serine of this motif abrogates PTS2 pr otein import in mammalian cells and reduces the interaction of PEX5L and PE X7 in vitro. This peptide motif also lies within regions of Pex18p and Pex2 1p that interact with yeast PEX7. Based on these and other results, we prop ose that mammalian PEX5L may have acquired some of the functions that yeast Pex18p and/or Pex21p perform in PTS2 protein import. This hypothesis may e xplain the essential role of PEX51, in PTS2 protein import in mammalian cel ls and its lack of importance for PTS2 protein import in yeast.