The Peroxisomal Targeting Signal 1 in sterol carrier protein 2 is autonomous and essential for receptor recognition

Chris P Williams,Ralf Erdmann,Wolfgang Schliebs,Matthias Wilmanns,Colin A Thompson,Charles S Bond,Will A Stanley,Marlene Van Den Berg,Nicole Schueller,Ben Distel,Simon D Van Haren

doi:10.1186/1471-2091-12-12

Abstract

BackgroundThe majority of peroxisomal matrix proteins destined for translocation into the peroxisomal lumen are recognised via a C-terminal Peroxisomal Target Signal type 1 by the cycling receptor Pex5p. The only structure to date of Pex5p in complex with a cargo protein is that of the C-terminal cargo-binding domain of the receptor with sterol carrier protein 2, a small, model peroxisomal protein. In this study, we have tested the contribution of a second, ancillary receptor-cargo binding site, which was found in addition to the characterised Peroxisomal Target Signal type 1.ResultsTo investigate the function of this secondary interface we have mutated two key residues from the ancillary binding site and analyzed the level of binding first by a yeast-two-hybrid assay, followed by quantitative measurement of the binding affinity and kinetics of purified protein components and finally, by in vivo measurements, to determine translocation capability. While a moderate but significant reduction of the interaction was found in binding assays, we were not able to measure any significant defects in vivo.ConclusionsOur data therefore suggest that at least in the case of sterol carrier protein 2 the contribution of the second binding site is not essential for peroxisomal import. At this stage, however, we cannot rule out that other cargo proteins may require this ancillary binding site.

Highlights

The majority of peroxisomal matrix proteins destined for translocation into the peroxisomal lumen are recognised via a C-terminal Peroxisomal Target Signal type 1 by the cycling receptor Pex5p
The PTS1 sequence binds to Pex5p in an extended conformation, which is accommodated in a deep cavity in the tetratricopeptide repeat (TPR) domain of Pex5p [3,4] (Figure 1A)
Remote from the PTS1 binding site, of about 500 Å2 has been found to form between Pex5p and the model cargo protein sterol carrier protein 2 (SCP2)

Summary

Introduction

The majority of peroxisomal matrix proteins destined for translocation into the peroxisomal lumen are recognised via a C-terminal Peroxisomal Target Signal type 1 by the cycling receptor Pex5p. It has been found to recognise the type 1 Peroxisomal Targeting Signal (PTS1) - a C-terminal tripeptide of consensus sequence -[S/A/C]-[K/H/R]-[L/M]CO2-, carried by some 40 human proteins destined for the peroxisomal lumen. Remote from the PTS1 binding site, of about 500 Å2 has been found to form between Pex5p and the model cargo protein sterol carrier protein 2 (SCP2), Inspection of the secondary interface of the Pex5p(C)SCP2 complex structure reveals several intermolecular hydrogen bonds (Figure 1C), in particular: the carboxylate group of Glu from SCP2 interacts with the side chains of two Pex5p residues, Arg608 and Tyr620; Lys from SCP2 forms a salt bridge with Asp624 from Pex5p; Lys from SCP2 interacts with the main chain carbonyl group of Gly615 from Pex5p(C). An alignment of a set of Pex5p sequences (Figure 1B) indicates that while Tyr620 and Asp624 are only partly conserved, a basic residue at the position equivalent to Arg608 in human Pex5p is invariant

Methods

Results

Conclusion