The di-aromatic pentapeptide repeats of the human peroxisome import receptor PEX5 are separate high affinity binding sites for the peroxisomal membrane protein PEX14

PEX5 functions as a mobile import receptor for peroxisomal matrix proteins with a peroxisomal targeting signal 1 (PTS1). A critical step within the PT S1-import pathway is the interaction between PEX5 and the peroxisome membra ne-associated protein PEX14. Based on two-hybrid analyses in mammalian cell s and complementary in vitro binding assays, we demonstrate that the evolut ionarily conserved pentapeptide repeat motifs, WX(E/D/Q/A/S)(E/D/Q)(F/Y), i n PEX5 bind to PEX14 with high affinity. The results obtained indicate that each of the seven di-aromatic,pentapeptides of human PEX5 interacts separa tely at the same binding site in the N terminus of PEX14 with equilibrium d issociation constants in the low nanomolar range. Mutational analysis of th e PEX14-binding motifs reveals that the conserved aromatic amino acids at p osition 1 or 5 are essential for high affinity binding. We propose that the side chains of the aromatic amino acids are in close proximity as part of an amphipathic alpha -helix and together form hydrophobic anchors for bindi ng PEX5 to individual PEX14 molecules.