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...

Background: PEX5 binds newly synthesized peroxisomal proteins in the cytosol and releases them in the organelle matrix. Results: PEX5 binds monomeric catalase and releases it in the presence of PEX14. Conclusion: PEX14 participates in the cargo release step. Significance: Knowing how PEX5 interacts with cargo proteins and which factors disrupt this interaction are crucial for understanding this protein sorting pathway.

Background:The mammalian deubiquitinase that hydrolyzes the ubiquitin-PEX5 thioester conjugate was unknown. Results: USP9X was found to be the most active deubiquitinase acting on ubiquitin-PEX5. Conclusion:We propose that USP9X participates in the PEX5-mediated peroxisomal protein import pathway. Significance: The unbiased biochemical strategy described here will be useful to identify deubiquitinases acting on other substrates.

Background: How the soluble receptor PEX5 delivers its cargoes to the peroxisome remains largely unknown. Results: Cargo translocation occurs after docking of the receptor at the peroxisome and before any ATP-dependent step. Conclusion: Translocation is concomitant with PEX5 insertion into the docking/translocation machinery. Significance: These results support a model in which cargoes are pushed across the peroxisomal membrane by PEX5.

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and rapidly transported into the organelle by a complex machinery. The data gathered in recent years suggest that this machinery operates through a syringe-like mechanism, in which the shuttling receptor PEX5 - the "plunger" - pushes a newly synthesized protein all the way through a peroxisomal transmembrane protein complex - the "barrel" - into the matrix of the organelle. Notably, insertion of cargo-loaded receptor into the "barrel" is an ATP-independent process, whereas extraction of the receptor back into the cytosol requires its monoubiquitination and the action of ATP-dependent mechanoenzymes. Here, we review the main data behind this model.

dPeroxisomal matrix proteins are synthesized on cytosolic ribosomes and transported to the organelle by shuttling receptors. Matrix proteins containing a type 1 signal are carried to the peroxisome by PEX5, whereas those harboring a type 2 signal are transported by a PEX5-PEX7 complex. The pathway followed by PEX5 during the protein transport cycle has been characterized in detail. In contrast, not much is known regarding PEX7. In this work, we show that PEX7 is targeted to the peroxisome in a PEX5-and cargo-dependent manner, where it becomes resistant to exogenously added proteases. Entry of PEX7 and its cargo into the peroxisome occurs upstream of the first cytosolic ATP-dependent step of the PEX5-mediated import pathway, i.e., before monoubiquitination of PEX5. PEX7 passing through the peroxisome becomes partially, if not completely, exposed to the peroxisome matrix milieu, suggesting that cargo release occurs at the trans side of the peroxisomal membrane. Finally, we found that export of peroxisomal PEX7 back into the cytosol requires export of PEX5 but, strikingly, the two export events are not strictly coupled, indicating that the two proteins leave the peroxisome separately. P eroxisomal matrix proteins are synthesized on cytosolic ribosomes and posttranslationally targeted to the organelle via one of two peroxisomal targeting sequences (PTSs): (i) the PTS type 1 (PTS1), a small peptide frequently ending with the sequence SKL located at the C terminus of the vast majority of matrix proteins (1, 2), and (ii) the PTS2, a degenerated nonapeptide present at the amino terminus of a few matrix proteins (3-5). In contrast to the PTS1, the PTS2 is generally cleaved when the protein reaches the organelle matrix (5-7). In mammals and many other organisms, both PTS1 and PTS2 proteins are transported to the organelle by PEX5, the peroxisomal shuttling receptor (8-11). The interaction of PEX5 with PTS1 proteins is direct (12-16), whereas the interaction between PEX5 and PTS2 proteins requires the adaptor protein PEX7 (17-19). Interestingly, not all PEX5 proteins in a mammalian cell are capable of binding PEX7. This is due to alternative splicing of the PEX5 transcript, which yields two major isoforms of the receptor, PEX5S and PEX5L. In contrast to PEX5L, PEX5S is not able to bind PEX7 because it lacks an internal 37-amino-acid domain (8, 10). The situation in yeasts is different. While these organisms also use PEX5 to target PTS1 proteins to the peroxisome, import of PTS2 proteins is promoted by PEX7 and a species-specific member of the so-called PEX20 family (19-23), a group of proteins that have no mammalian counterpart but that display functional similarities with the N-terminal half of PEX5L (17,19,24).The pathway followed by PEX5 during the protein transport process is reasonably known (25-28). After binding a cargo protein in the cytosol, PEX5 interacts with the peroxisomal docking/ translocation machinery (DTM) (29), a peroxisomal membrane protein complex comprising PEX13, PEX14, and the RING peroxins PEX2, PE...

The peroxisomal protein import machinery displays remarkable properties. Be it its capacity to accept already folded proteins as substrates, its complex architecture or its energetics, almost every aspect of this machinery seems unique. The list of unusual properties is still growing as shown by the recent finding that one of its central components, Pex5p, is transiently monoubiquitinated at a cysteine residue. However, the data gathered in recent years also suggest that the peroxisomal import machinery is not that exclusive and similarities with p97/Cdc48-mediated processes and with multisubunit RING-E3 ligases are starting to emerge. Here, we discuss these data trying to distill the principles by which this complex machinery operates.

PEX1 and PEX6 are two members of the TPasesssociated with diverse cellular ctivities (AAA) family and the core components of the receptor export module of the peroxisomal matrix protein import machinery. Their role is to extract monoubiquitinated PEX5, the peroxisomal protein-shuttling receptor, from the peroxisomal membrane docking/translocation module (DTM), so that a new cycle of protein transportation can start. Recent data have shown that PEX1 and PEX6 form a heterohexameric complex that unfolds substrates by processive threading. However, whether the natural substrate of the PEX1-PEX6 complex is monoubiquitinated PEX5 (Ub-PEX5) itself or some Ub-PEX5-interacting component(s) of the DTM remains unknown. In this work, we used an established cell-free system coupled with photoaffinity cross-linking and protein PEGylation assays to address this problem. We provide evidence suggesting that DTM-embedded Ub-PEX5 interacts directly with both PEX1 and PEX6 through its ubiquitin moiety and that the PEX5 polypeptide chain is globally unfolded during the ATP-dependent extraction event. These findings strongly suggest that DTM-embedded Ub-PEX5 is a substrate of the PEX1-PEX6 complex.