Mapping the Cargo Protein Membrane Translocation Step into the PEX5 Cycling Pathway

Abstract

Newly synthesized peroxisomal matrix proteins are targeted to the organelle by PEX5, the peroxisomal cycling receptor. Over the last few years, valuable data on the mechanism of this process have been obtained using a PEX5-centered in vitro system. The data gathered until now suggest that cytosolic PEX5⅐ cargo protein complexes dock at the peroxisomal docking/ translocation machinery, where PEX5 becomes subsequently inserted in an ATP-independent manner. This PEX5 species is then monoubiquitinated at a conserved cysteine residue, a mandatory modification for the next step of the pathway, the ATPdependent dislocation of the ubiquitin-PEX5 conjugate back into the cytosol. Finally, the ubiquitin moiety is removed, yielding free PEX5. Despite its usefulness, there are many unsolved mechanistic aspects that cannot be addressed with this in vitro system and that call for a cargo protein-centered perspective instead. Here we describe a robust peroxisomal in vitro import system that provides this perspective. The data obtained with it suggest that translocation of a cargo protein across the peroxisomal membrane, including its release into the organelle matrix, occurs prior to PEX5 ubiquitination.Peroxisomal matrix proteins are synthesized in cytosolic ribosomes and post-translationally targeted to the organelle (1, 2). The vast majority of proteins destined to this compartment possess the so-called peroxisomal targeting sequence type 1 (PTS1), 3 a short domain present at their extreme C termini and frequently ending with the sequence SKL (3, 4). A small number of matrix proteins lack this domain and contain instead a PTS2, a degenerated nonapeptide with the sequence (R/K)(L/V/I)X 5 (H/Q)(L/A) present at their N termini (5, 6). In contrast to the PTS1, which is not cleaved upon peroxisomal import, the PTS2 signal is proteolytically removed in the peroxisomal matrix of many organisms by a peroxisomal processing peptidase (1,7,8).In mammals and many other organisms, both PTS1-containing and PTS2-containing proteins are targeted to the organelle by PEX5, the peroxisomal cycling receptor (9 -12). PTS1 proteins interact directly with the C-terminal half of PEX5, a region comprising seven tetratricopeptide repeats arranged into a ring-like structure, whereas the PEX5-PTS2 interaction is bridged by the adaptor protein PEX7 (13-19). This adaptor protein interacts with a small region within the largely unfolded N-terminal half of PEX5 (17,18,20). Interestingly, not all proteins derived from the mammalian PEX5 gene have the capacity to bind PEX7. This is due to alternative splicing of the PEX5 transcript yielding two major mRNAs, one encoding the so-called large isoform of PEX5 (PEX5L) and the other coding for the small PEX5 isoform (PEX5S). PEX5S lacks a 37-amino-acid region that is involved in the PEX7 interaction, and so it is incompetent in the peroxisomal targeting of PTS2 proteins (16 -18).In recent years, valuable data on the mechanistic details of the PEX5-mediated protein import pathway in mammals have been obtained using...

Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes

“…The nature of the interactions between the peroxins has been examined in a qualitative fashion, but with a few exceptions there is still rather little quantitative or structural data for these interactions and a paucity of kinetic data to allow rigorous testing of models. An important step forward is the development of a cargo-dependent cell-free import system which has provided evidence that cargo unloading precedes ubiquitination of PEX5 (Alencastre et al, 2009).…”

“…Also, Pex8p is only found in yeast cells (Rehling et al, 2000). Cargo release occurs prior to ubiquitination of Pex5p, which regulates its recycling (see below) (Alencastre et al, 2009), and receptor ubiquitination does not affect its cargo-binding properties . Thus the mechanism of receptor unloading remains enigmatic.…”

Identification of Ubiquitin-specific Protease 9X (USP9X) as a Deubiquitinase Acting on Ubiquitin-Peroxin 5 (PEX5) Thioester Conjugate

“…Here, the PEX5-cargo protein complex becomes inserted into the DTM with the concomitant translocation of the cargo protein across the organelle membrane (27,28). In at least one case, that of the PTS2-containing protein thiolase, it is also at this step that PEX5 releases its cargo into the peroxisomal matrix (29). Notably, in vitro import experiments suggest that none of these steps requires hydrolysis of ATP, suggesting that the driving force for peroxisomal protein import relies on the strong protein-protein interactions that are established between PEX5 on one side and members of the DTM on the other side (29 -32).…”

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

“…The DNA fragment was digested with XbaI and KpnI and cloned into the XbaI/KpnI-digested pGEM4 vector (Promega). 35 S-labeled proteins were synthesized in vitro as described before (40).…”

“…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). However, whether this is a unique feature of PTS2 proteins emerging from the participation of PEX7 in this process (see also "Discussion"), or a general property of the peroxisomal protein import machinery, remained unknown.…”