Characterization of the Peroxisomal Cycling Receptor, Pex5p, Using a Cell-free in Vitro Import System

Alexandra M Gouveia,Carla P Guimarães,Márcia E Oliveira,Carlos Reguenga,Clara Sá-Miranda,Jorge E Azevedo

doi:10.1074/jbc.m209498200

Abstract

According to current models of peroxisomal biogenesis, Pex5p cycles between the cytosol and the peroxisome transporting newly synthesized proteins to the organelle matrix. However, little is known regarding the mechanism of this pathway. Here, we show that Pex5p enters and exits the peroxisomal compartment in a process that requires ATP. Insertion of Pex5p into the peroxisomal membrane is blocked by anti-Pex14p IgGs. At the peroxisomal level, two Pex14p-associated populations of Pex5p could be resolved, stage 2 and stage 3 Pex5p, both exposing the majority of their masses into the organelle lumen. Stage 3 Pex5p can be easily detected only under ATP-limiting conditions; in the presence of ATP it leaves the peroxisomal compartment rapidly. Our data suggest that translocation of PTS1-containing proteins across the peroxisomal membrane occurs concomitantly with formation of the Pex5p-Pex14p membrane complex and that this is probably the site from which Pex5p leaves the peroxisomal compartment.

Highlights

The vast majority of peroxisomal matrix proteins is targeted to the organelle via the so-called peroxisomal targeting signal 1 (PTS1)1 pathway
These data provide the basis for a model in which peroxins are classified into three functional groups: 1) docking proteins; 2) proteins involved in the translocation of matrix proteins across the peroxisomal membrane; and 3) proteins mediating the release of Pex5p from the peroxisomal compartment
Our results suggest, on one hand, that translocation of peroxisomal matrix proteins across the organelle membrane occurs through a Pex14p-containing structure via Pex5p, raising the possibility that no other peroxins other than Pex5p interact with the PTS1-containing proteins during the membrane translocation event and, on the other hand, that Pex5p is retained at the peroxisomal membrane during all steps of this process

Summary

Introduction

The vast majority of peroxisomal matrix proteins is targeted to the organelle via the so-called peroxisomal targeting signal 1 (PTS1)1 pathway (reviewed in Refs. 1 and 2). Immunoprecipitation of in Vitro Imported Pex5p—Two aliquots of a PNS fraction (1.3 mg of protein) were incubated in import buffer either in the presence or absence (mock-treated sample) of 5 ␮l of a reticulocyte lysate containing 35S-labeled Pex5p.

Results

Conclusion