PEX5 Binds the PTS1 Independently of Hsp70 and the Peroxin PEX12

Courtney C Harper,Jeremy M Berg,Stephen J Gould

doi:10.1074/jbc.m206651200

Abstract

Most peroxisomal enzymes are targeted to peroxisomes by virtue of a type-1 peroxisomal targeting signal (PTS1) at their extreme C terminus. PEX5 binds the PTS1 through its C-terminal 40-kDa tetratricopeptide repeat domain and is essential for import of PTS1-contining proteins into peroxisomes. Here we examined the PTS1-binding activity of purified, recombinant, full-length PEX5 using a fluorescence anisotropy-based assay. Like its C-terminal fragment, full-length tetrameric PEX5 exhibits high intrinsic affinity for the PTS1, with a K(d) of 35 nm for the peptide lissamine-Tyr-Gln-Ser-Lys-Leu-COO(-). The specificity of this interaction was demonstrated by the fact that PEX5 had no detectable affinity for a peptide in which the Lys was replaced with Glu, a substitution that inactivates PTS1 signals in vivo. Hsp70 has been found to regulate the affinity of PEX5 for a PTS1-containing protein, but we found that the kinetics of PEX5-PTS1 binding was unaffected by Hsp70, Hsp70 plus ATP, or Hsp70 plus ADP. In addition, we found that another protein known to interact with the PTS1-binding domain of PEX5, the PEX12 zinc RING domain, also had no discernable effect on PEX5-PTS1 binding kinetics. Taken together, these results suggest that the initial step in peroxisomal protein import, the recognition of enzymes by PEX5, is a relatively simple process and that Hsp70 most probably stimulates this process by catalyzing the folding of newly synthesized peroxisomal enzymes and/or enhancing the accessibility of their PTS1.

Highlights

Peroxisomes lack nucleic acids and import all of their protein content [1]
Plotting the amount of peptide bound against PEX5 protein concentration reveals a rectangular hyperbola showing that PEX5 displayed an apparent affinity (Kd) for the PTS1 of 35 nM, which is similar to the Kd reported for the interaction between the same peptide and the C-terminal ligand-binding domain of PEX5 [10]
When purified PEX5L is separated by gel filtration chromatography, nearly half of the protein migrates with a size of ϳ300 kDa, as expected for a homotetramer of 70 kDa subunits, whereas the other half of the protein migrates in the void volume and probably represents aggregated forms of PEX5L that may not contribute to PTS1 binding (Fig. 1B)

Summary

Introduction

Peroxisomes lack nucleic acids and import all of their protein content [1]. Peroxisomal matrix protein import is initiated when newly synthesized peroxisomal enzymes are bound by the peroxisomal matrix protein import receptors, PEX5 or PEX7 [2]. PEX5 binds the PTS1 through its C-terminal 40-kDa tetratricopeptide repeat domain and is essential for import of PTS1-contining proteins into peroxisomes. Data Analysis—Determination of the binding affinity of PEX5 for the PTS1 requires that we first determine the fraction of peptide bound (fB) for any given anisotropy value (r).

Results

Conclusion