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

Wolfgang Schliebs,Kristina Kirchberg,Wolf-H. Kunau,Albrecht Wegner,Wolfgang Nastainczyk,Jürgen Saidowsky,Gabriele Dodt

doi:10.1074/jbc.m104647200

Wolfgang Schliebs, Kristina Kirchberg + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m104647200

Copy DOI

Abstract

PEX5 functions as a mobile import receptor for peroxisomal matrix proteins with a peroxisomal targeting signal 1 (PTS1). A critical step within the PTS1-import pathway is the interaction between PEX5 and the peroxisome membrane-associated protein PEX14. Based on two-hybrid analyses in mammalian cells and complementary in vitro binding assays, we demonstrate that the evolutionarily conserved pentapeptide repeat motifs, WX(E/D/Q/A/S)(E/D/Q)(F/Y), in PEX5 bind to PEX14 with high affinity. The results obtained indicate that each of the seven di-aromatic pentapeptides of human PEX5 interacts separately at the same binding site in the N terminus of PEX14 with equilibrium dissociation constants in the low nanomolar range. Mutational analysis of the PEX14-binding motifs reveals that the conserved aromatic amino acids at position 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 binding PEX5 to individual PEX14 molecules.

Highlights

Import of peroxisomal matrix proteins is a posttranslational multistep process which involves recognition of the peroxisomal proteins in the cytosol by specific soluble receptors, docking of the receptor-cargo complex at the surface of the organelle, and translocation of the matrix proteins with or without their receptors across the membrane
WXXX(F/Y) Motifs Are Required for PEX5-PEX14 Interaction in Mammalian Two-hybrid Assays—We previously demonstrated that human PEX5 possesses multiple binding sites for PEX14 and proposed that the various WXXX(F/Y) pentapeptide repeats might provide the structural basis for these interactions (19)
To map the PEX14-binding regions of PEX5 we expressed the long form of PEX5 and various truncated forms fused to the Gal4 DNA-binding domain in human fibroblast cells (Fig. 1)

Summary

Introduction

Import of peroxisomal matrix proteins is a posttranslational multistep process which involves recognition of the peroxisomal proteins in the cytosol by specific soluble receptors, docking of the receptor-cargo complex at the surface of the organelle, and translocation of the matrix proteins with or without their receptors across the membrane (for recent reviews, see Refs. 1– 4). There is not a strong correlation between the number of WXXX(F/Y) motifs and the expression level of CAT, these results indicate that the conserved pentapeptide repeats are essential for complex formation between PEX5 and PEX14. The binding affinities found by fluorescence spectroscopy for each of the peptides containing one WXXX(F/Y) motif are in the same range as that determined for full-length PEX5 toward PEX14(1–78) using surface plasmon resonance spectroscopy (19).

Results

Conclusion