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PEX1 and PEX6 are two members of the A TPases a ssociated with diverse cellular a 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 in vitro 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 bona fide substrate of the PEX1-PEX6 complex., (© 2018 Pedrosa et al.)

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., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

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.

Peroxin 5 (PEX5), the peroxisomal protein shuttling receptor, binds newly synthesized peroxisomal matrix proteins in the cytosol and promotes their translocation across the organelle membrane. During the translocation step, PEX5 itself becomes inserted into the peroxisomal docking/translocation machinery. PEX5 is then monoubiquitinated at a conserved cysteine residue and extracted back into the cytosol in an ATP-dependent manner. We have previously shown that the ubiquitin-PEX5 thioester conjugate (Ub-PEX5) released into the cytosol can be efficiently disrupted by physiological concentrations of glutathione, raising the possibility that a fraction of Ub-PEX5 is nonenzymatically deubiquitinated in vivo. However, data suggesting that Ub-PEX5 is also a target of a deubiquitinase were also obtained in that work. Here, we used an unbiased biochemical approach to identify this enzyme. Our results suggest that ubiquitin-specific protease 9X (USP9X) is by far the most active deubiquitinase acting on Ub-PEX5, both in female rat liver and HeLa cells. We also show that USP9X is an elongated monomeric protein with the capacity to hydrolyze thioester, isopeptide, and peptide bonds. The strategy described here will be useful in identifying deubiquitinases acting on other ubiquitin conjugates.

Newly synthesized peroxisomal matrix proteins are targeted to the organelle by PEX5. PEX5 has a dual role in this process. First, it acts as a soluble receptor recognizing these proteins in the cytosol. Subsequently, at the peroxisomal docking/translocation machinery, PEX5 promotes their translocation across the organelle membrane. Despite significant advances made in recent years, several aspects of this pathway remain unclear. Two important ones regard the formation and disruption of the PEX5-cargo protein interaction in the cytosol and at the docking/translocation machinery, respectively. Here, we provide data on the interaction of PEX5 with catalase, a homotetrameric enzyme in its native state. We found that PEX5 interacts with monomeric catalase yielding a stable protein complex; no such complex was detected with tetrameric catalase. Binding of PEX5 to monomeric catalase potently inhibits its tetramerization, a property that depends on domains present in both the N- and C-terminal halves of PEX5. Interestingly, the PEX5-catalase interaction is disrupted by the N-terminal domain of PEX14, a component of the docking/translocation machinery. One or two of the seven PEX14-binding diaromatic motifs present in the N-terminal half of PEX5 are probably involved in this phenomenon. These results suggest the following: 1) catalase domain(s) involved in the interaction with PEX5 are no longer accessible upon tetramerization of the enzyme; 2) the catalase-binding interface in PEX5 is not restricted to its C-terminal peroxisomal targeting sequence type 1-binding domain and also involves PEX5 N-terminal domain(s); and 3) PEX14 participates in the cargo protein release step.

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 PEX5.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 ATP-dependent 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.

Pex5p, the peroxisomal protein cycling receptor, binds newly synthesized peroxisomal matrix proteins in the cytosol and promotes their translocation across the organelle membrane. During its transient passage through the membrane, Pex5p is monoubiquitinated at a conserved cysteine residue, a requisite for its subsequent ATP-dependent export back into the cytosol. Here we describe the properties of the soluble and membrane-bound monoubiquitinated Pex5p species (Ub-Pex5p). Our data suggest that 1) Ub-Pex5p is deubiquitinated by a combination of context-dependent enzymatic and nonenzymatic mechanisms; 2) soluble Ub-Pex5p retains the capacity to interact with the peroxisomal import machinery in a cargo-dependent manner; and 3) substitution of the conserved cysteine residue of Pex5p by a lysine results in a quite functional protein both in vitro and in vivo. Additionally, we show that MG132, a proteasome inhibitor, blocks the import of a peroxisomal reporter protein in vivo.

According to current models of peroxisomal biogenesis, newly synthesized peroxisomal matrix proteins are transported into the organelle by Pex5p. Pex5p recognizes these proteins in the cytosol, mediates their membrane translocation, and is exported back into the cytosol in an ATP-dependent manner. We have previously shown that export of Pex5p is preceded by (and requires) monoubiquitination of a conserved cysteine residue present at its N terminus. In yeasts, and probably also in plants, ubiquitination of Pex5p is mediated by a specialized ubiquitin-conjugating enzyme, Pex4p. In mammals, the identity of this enzyme has remained unknown for many years. Here, we provide evidence suggesting that E2D1/2/3 (UbcH5a/b/c) are the mammalian functional counterparts of yeast/plant Pex4p. The mechanistic implications of these findings are discussed.

The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by alpha- and beta-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate approximately 50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter. Although this research has greatly improved our understanding of peroxisomes and their role in mammalian metabolism, deeper insight into biochemistry and functions of peroxisomes is required to expand our knowledge of this low abundance but vital organelle. In this work, we used classical subcellular fractionation in combination with MS-based proteomics methodologies to characterize the proteome of mouse kidney peroxisomes. We could identify virtually all known components involved in peroxisomal metabolism and biogenesis. Moreover through protein localization studies by using a quantitative MS screen combined with statistical analyses, we identified 15 new peroxisomal candidates. Of these, we further investigated five candidates by immunocytochemistry, which confirmed their localization in peroxisomes. As a result of this joint effort, we believe to have compiled the so far most comprehensive protein catalogue of mammalian peroxisomes.

Protein translocation across the peroxisomal membrane requires the concerted action of numerous peroxins. One central component of this machinery is Pex5p, the cycling receptor for matrix proteins. Pex5p recognizes newly synthesized proteins in the cytosol and promotes their translocation across the peroxisomal membrane. After this translocation step, Pex5p is recycled back into the cytosol to start a new protein transport cycle. Here, we show that mammalian Pex5p is ubiquitinated at the peroxisomal membrane. Two different types of ubiquitination were detected, one of which is thiol-sensitive, involves Cys(11) of Pex5p, and is necessary for the export of the receptor back into the cytosol. Together with mechanistic data recently described for yeast Pex5p, these findings provide strong evidence for the existence of Pex4p- and Pex22p-like proteins in mammals.

HFE C282Y, the mutant protein associated with hereditary hemochromatosis (HH), fails to acquire the correct conformation in the endoplasmic reticulum (ER) and is targeted for degradation. We have recently shown that an active unfolded protein response (UPR) is present in the cells of patients with HH. Now, by using HEK 293T cells, we demonstrate that the stability of HFE C282Y is influenced by the UPR signaling pathway that promotes its degradation. Treatment of HFE C282Y-expressing cells with tauroursodeoxycholic acid (TUDCA), a bile acid derivative with chaperone properties, or with the chemical chaperone sodium 4-phenylbutyrate (4PBA) impeded the UPR activation. However, although TUDCA led to an increased stability of the mutant protein, 4PBA contributed to a more efficient disposal of HFE C282Y to the degradation route. Fluorescence microscopy and biochemical analysis of the subcellular localization of HFE revealed that a major portion of the C282Y mutant protein forms intracellular aggregates. Although neither TUDCA nor 4PBA restored the correct folding and intracellular trafficking of HFE C282Y, 4PBA prevented its aggregation. These data suggest that TUDCA hampers the UPR activation by acting directly on its signal transduction pathway, whereas 4PBA suppresses ER stress by chemically enhancing the ER capacity to cope with the expression of misfolded HFE, facilitating its degradation. Together, these data shed light on the molecular mechanisms involved in HFE C282Y-related HH and open new perspectives on the use of orally active chemical chaperones as a therapeutic approach for HH.

Biogenesis of the mammalian peroxisomal membrane requires the action of Pex3p and Pex16p, two proteins present in the organelle membrane, and Pex19p, a protein that displays a dual subcellular distribution (peroxisomal and cytosolic). Pex19p interacts with most peroxisomal intrinsic membrane proteins, but whether this property reflects its role as an import receptor for this class of proteins or a chaperone-like function in the assembly/disassembly of peroxisomal membrane proteins has been the subject of much controversy. Here, we describe an in vitro system particularly suited to address this issue. It is shown that insertion of a reporter protein into the peroxisomal membrane is a Pex3p-dependent process that does not require ATP/GTP hydrolysis. The system can be programmed with recombinant versions of Pex19p, allowing us to demonstrate that Pex19p-cargo protein complexes formed in the absence of peroxisomes are the substrates for the peroxisomal docking/insertion machinery. Data suggesting that cargo-loaded Pex19p displays a much higher affinity for Pex3p than Pex19p alone are also provided. These results suggest that soluble Pex19p participates in the targeting of newly synthesized peroxisomal membrane proteins to the organelle membrane and support the existence of a cargo-induced peroxisomal targeting mechanism for Pex19p.

In mammals, targeting of newly synthesized peroxisomal matrix proteins to the organelle requires Pex5p, the peroxisomal cycling receptor. Pex5p is a multidomain protein involved in a complex network of transient protein-protein interactions. Besides interacting directly with most peroxisomal proteins en route to the organelle, Pex5p has also binding domains for several components of the peroxisomal docking/translocation machinery. However, our knowledge of how binding of a cargo protein to Pex5p influences its properties is still rather limited. Here, we describe a protease assay particularly useful for identifying and characterizing protein-protein interactions involving human Pex5p. Binding of a PTS1-containing peptide/protein to Pex5p as well as the interaction of this peroxin with the Src homology domain 3 of Pex13p could be easily demonstrated using this assay. To address the possible effects of these Pex5p-interacting peptides/proteins on the assumed quaternary structure of Pex5p, we have analyzed the hydrodynamic properties of human Pex5p using size exclusion chromatography, sucrose gradient centrifugation, and sedimentation equilibrium centrifugation. Our results show that Pex5p is a monomeric protein with an abnormal shape. The implications of these findings on current models of protein translocation across the peroxisomal membrane are discussed.

Most newly synthesized peroxisomal matrix proteins are transported to the organelle by Pex5p, a remarkable multidomain protein involved in an intricate network of transient protein-protein interactions. Presently, our knowledge regarding the structure/function of amino acid residues 118 to the very last residue of mammalian Pex5p is quite vast. Indeed, the cargo-protein receptor domain as well as the binding sites for several peroxins have all been mapped to this region of Pex5p. In contrast, structural/functional data regarding the first 117 amino acid residues of Pex5p are still scarce. Here we show that a truncated Pex5p lacking the first 110 amino acid residues (DeltaN110-Pex5p) displays exactly the peroxisomal import properties of the full-length peroxin implying that this N-terminal domain is involved neither in cargo-protein binding nor in the docking/translocation step of the Pex5p-cargo protein complex at the peroxisomal membrane. However, the ATP-dependent export step of DeltaN110-Pex5p from the peroxisomal membrane is completely blocked, a phenomenon that was also observed for a Pex5p version lacking just the first 17 amino acid residues but not for a truncated protein comprising amino acid residues 1-324 of Pex5p. By exploring the unique properties of DeltaN110-Pex5p, the effect of temperature on the import/export kinetics of Pex5p was characterized. Our data indicate that the export step of Pex5p from the peroxisomal compartment (in contrast with its insertion into the organelle membrane) is highly dependent on the temperature.

Most newly synthesized peroxisomal matrix proteins are targeted to the organelle by Pex5p, the peroxisomal cycling receptor. According to current models of peroxisomal biogenesis, Pex5p interacts with cargo proteins in the cytosol and transports them to the peroxisomal membrane. After delivering the passenger protein into the peroxisomal matrix, Pex5p returns to the cytosol to catalyze additional rounds of transportation. Obviously, such cyclic pathway must require energy, and indeed, data confirming this need are already available. However, the exact step(s) of this cycle where energy input is necessary remains unclear. Here, we present data suggesting that insertion of Pex5p into the peroxisomal membrane does not require ATP hydrolysis. This observation raises the possibility that at the peroxisomal membrane ATP is needed predominantly (if not exclusively) downstream of the protein translocation step to reset the Pex5p-mediated transport system.

It is now generally accepted that Pex5p, the receptor for most peroxisomal matrix proteins, cycles between the cytosol and the peroxisomal compartment. According to current models of peroxisomal biogenesis, this intracellular trafficking of Pex5p is coupled to the transport of newly synthesized peroxisomal proteins into the organelle matrix. However, direct evidence supporting this hypothesis was never provided. Here, using an in vitro peroxisomal import system, we show that insertion of Pex5p into the peroxisomal membrane requires the presence of cargo proteins. Strikingly the peroxisomal docking/translocation machinery is also able to catalyze the membrane insertion of a Pex5p truncated molecule lacking any known cargo-binding domain. These results suggest that the cytosol/peroxisomal cycle in which Pex5p is involved is directly or indirectly regulated by Pex5p itself and not by the peroxisomal docking/translocation machinery.

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.

Although many of the proteins involved in the biogenesis of the mammalian peroxisome have already been identified, our knowledge of the architecture of all this machinery is still very limited. In this work we used native gel electrophoresis and sucrose gradient sedimentation analysis in combination with immunoprecipitation experiments to address this issue. After solubilization of rat liver peroxisomes with the mild detergent digitonin, comigration of Pex5p, Pex14p, and a fraction of Pex12p was observed upon native electrophoresis and sucrose gradient sedimentation. The existence of a complex comprising Pex2p, Pex5p, Pex12p, and Pex14p was demonstrated by preparative coimmunoprecipitation experiments using an antibody directed to Pex14p. No stoichiometric amounts of Pex13p were detected in the Pex2p-Pex5p-Pex12p-Pex14p complex, although the presence of a small fraction of Pex13p in this complex could be demonstrated by Western blot analysis. Pex13p is also a component of a high molecular mass complex. Strikingly, partial purification of this Pex13p-containing complex revealed Pex13p as the major (if not the only) component. Taken together, our data indicate that Pex2p, Pex5p, Pex12p, and Pex14p, on one side, and Pex13p, on the other, are subunits of two stable protein complexes that probably interact with each other in the peroxisomal membrane.

Pex5p is the receptor for the vast majority of peroxisomal matrix proteins. Here, we show that about 15% of rat liver Pex5p is found in the peroxisomal fraction representing 0.06% of total peroxisomal protein. This population of Pex5p displays all the characteristics of an intrinsic membrane protein. Protease protection assays indicate that this pool of Pex5p has domains exposed on both sides of the peroxisomal membrane. The strong interaction of Pex5p with the membrane of the organelle is not affected by mild protease treatment of intact organelles, conditions that result in the partial degradation of Pex13p. Cytosolic Pex5p is a monomeric protein. In contrast, virtually all peroxisomal Pex5p was found to be part of a stable 250-kDa protein assembly. This complex was isolated and shown to comprise just two subunits, Pex5p and Pex14p.