A Cargo-centered Perspective on the PEX5 Receptor-mediated Peroxisomal Protein Import Pathway

Tânia Francisco,Tony A Rodrigues,Marta O Freitas,Cláudia P Grou,Andreia F Carvalho,Clara Sá-Miranda,Manuel P Pinto,Jorge E Azevedo

doi:10.1074/jbc.m113.487140

Abstract

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and post-translationally targeted to the organelle by PEX5, the peroxisomal shuttling receptor. The pathway followed by PEX5 during this process is known with reasonable detail. After recognizing cargo proteins in the cytosol, the receptor interacts with the peroxisomal docking/translocation machinery, where it gets inserted; PEX5 is then monoubiquitinated, extracted back to the cytosol and, finally, deubiquitinated. However, despite this information, the exact step of this pathway where cargo proteins are translocated across the organelle membrane is still ill-defined. In this work, we used an in vitro import system to characterize the translocation mechanism of a matrix protein possessing a type 1 targeting signal. Our results suggest that translocation of proteins across the organelle membrane occurs downstream of a reversible docking step and upstream of the first cytosolic ATP-dependent step (i.e. before ubiquitination of PEX5), concomitantly with the insertion of the receptor into the docking/translocation machinery.

Highlights

How the soluble receptor PEX5 delivers its cargoes to the peroxisome remains largely unknown
We have previously shown that cytosolic PEX5 becomes transiently inserted into the peroxisomal membrane docking/translocation module (DTM) in a process that is cargo protein-dependent but independent of cytosolic ATP [30, 31, 50]
Those findings together with data suggesting that DTM-embedded PEX5 adopts a transmembrane topology exposing a major fraction of its polypeptide chain into the lumen of the organelle [31, 49] led us to propose that the PEX5-mediated translocation of cargo proteins across the peroxisomal membrane is an ATP-independent event that occurs concomitantly with insertion of PEX5 into the DTM [50, 60]

Summary

Background

How the soluble receptor PEX5 delivers its cargoes to the peroxisome remains largely unknown. After recognizing cargo proteins in the cytosol, the receptor interacts with the peroxisomal docking/translocation machinery, where it gets inserted; PEX5 is monoubiquitinated, extracted back to the cytosol and, deubiquitinated Despite this information, the exact step of this pathway where cargo proteins are translocated across the organelle membrane is still ill-defined. The data suggest that translocation of pre-thiolase across the peroxisomal membrane occurs before ubiquitination of DTM-embedded PEX5, presumably during insertion of the receptor into the DTM [40] Whether this is a unique feature of PTS2 proteins emerging from the participation of PEX7 in this process (see “Discussion”), or a general property of the peroxisomal protein import machinery, remained unknown. Numbers to the left indicate the molecular masses of protein standards in kDa

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION