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.

PEX1 and PEX6 are two members of the ATPases Associated with diverse cellular Activities (AAA) family and the core components of the receptor export module (REM) 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 which 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 in vitro system coupled with photoaffinity crosslinking 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 bona fide substrate of the PEX1.PEX6 complex.

PEX13 and PEX14 are two core components of the so‐called peroxisomal docking/translocation module, the transmembrane hydrophilic channel through which newly synthesized peroxisomal proteins are translocated into the organelle matrix. The two proteins interact with each other and with PEX5, the peroxisomal matrix protein shuttling receptor, through relatively well characterized domains. However, the topologies of these membrane proteins are still poorly defined. Here, we subjected proteoliposomes containing PEX13 or PEX14 and purified rat liver peroxisomes to protease‐protection assays and analyzed the protected protein fragments by mass spectrometry, Edman degradation and western blotting using antibodies directed to specific domains of the proteins. Our results indicate that PEX14 is a bona fide intrinsic membrane protein with a N_in‐C_out topology, and that PEX13 adopts a N_out‐C_in topology, thus exposing its carboxy‐terminal Src homology 3 [SH3] domain into the organelle matrix. These results reconcile several enigmatic findings previously reported on PEX13 and PEX14 and provide new insights into the organization of the peroxisomal protein import machinery.Enzymes: Trypsin, EC3.4.21.4; Proteinase K, EC3.4.21.64; Tobacco etch virus protease, EC3.4.22.44.

A remarkable property of the machinery for import of peroxisomal matrix proteins is that it can accept already folded proteins as substrates. This import involves binding of newly synthesized proteins by cytosolic peroxisomal biogenesis factor 5 (PEX5) followed by insertion of the PEX5–cargo complex into the peroxisomal membrane at the docking/translocation module (DTM). However, how these processes occur remains largely unknown. Here, we used truncated PEX5 molecules to probe the DTM architecture. We found that the DTM can accommodate a larger number of truncated PEX5 molecules comprising amino acid residues 1–197 than full-length PEX5 molecules. A shorter PEX5 version (PEX5(1–125)) still interacted correctly with the DTM; however, this species was largely accessible to exogenously added proteinase K, suggesting that this protease can access the DTM occupied by a small PEX5 protein. Interestingly, the PEX5(1–125)–DTM interaction was inhibited by a polypeptide comprising PEX5 residues 138–639. Apparently, the DTM can recruit soluble PEX5 through interactions with different PEX5 domains, suggesting that the PEX5–DTM interactions are to some degree fuzzy. Finally, we found that the interaction between PEX5 and PEX14, a major DTM component, is stable at pH 11.5. Thus, there is no reason to assume that the hitherto intriguing resistance of DTM-bound PEX5 to alkaline extraction reflects its direct contact with the peroxisomal lipid bilayer. Collectively, these results suggest that the DTM is best described as a large cavity-forming protein assembly into which cytosolic PEX5 can enter to release its cargo.