PEX5 protein binds monomeric catalase blocking its tetramerization and releases it upon binding the N-terminal domain of PEX14

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.     

A PEX7-Centered Perspective on the Peroxisomal Targeting Signal Type 2-Mediated Protein Import Pathway

Peroxisomal 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.     

Identification of ubiquitin-specific protease 9X (USP9X) as a deubiquitinase acting on ubiquitin-peroxin 5 (PEX5) thioester conjugate

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.     

The peroxisomal protein import machinery displays a preference for monomeric substrates

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and transported by the shuttling receptor PEX5 to the peroxisomal membrane docking/translocation machinery, where they are translocated into the organelle matrix. Under certain experimental conditions this protein import machinery has the remarkable capacity to accept already oligomerized proteins, a property that has heavily influenced current models on the mechanism of peroxisomal protein import. However, whether or not oligomeric proteins are really the best and most frequent clients of this machinery remain unclear. In this work, we present three lines of evidence suggesting that the peroxisomal import machinery displays a preference for monomeric proteins. First, in agreement with previous findings on catalase, we show that PEX5 binds newly synthesized (monomeric) acyl-CoA oxidase 1 (ACOX1) and urate oxidase (UOX), potently inhibiting their oligomerization. Second, in vitro import experiments suggest that monomeric ACOX1 and UOX are better peroxisomal import substrates than the corresponding oligomeric forms. Finally, we provide data strongly suggesting that although ACOX1 lacking a peroxisomal targeting signal can be imported into peroxisomes when co-expressed with ACOX1 containing its targeting signal, this import pathway is inefficient.     

Insertion of Pex5p into the peroxisomal membrane is cargo protein-dependent

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.     

PEX5 and Ubiquitin Dynamics on Mammalian Peroxisome Membranes

Peroxisomes are membrane-bound organelles within eukaryotic cells that post-translationally import folded proteins into their matrix. Matrix protein import requires a shuttle receptor protein, usually PEX5, that cycles through docking with the peroxisomal membrane, ubiquitination, and export back into the cytosol followed by deubiquitination. Matrix proteins associate with PEX5 in the cytosol and are translocated into the peroxisome lumen during the PEX5 cycle. This cargo translocation step is not well understood, and its energetics remain controversial. We use stochastic computational models to explore different ways the AAA ATPase driven removal of PEX5 may couple with cargo translocation in peroxisomal importers of mammalian cells. The first model considered is uncoupled, in which translocation is spontaneous, and does not immediately depend on PEX5 removal. The second is directly coupled, in which cargo translocation only occurs when its PEX5 is removed from the peroxisomal membrane. The third, novel, model is cooperatively coupled and requires two PEX5 on a given importomer for cargo translocation — one PEX5 with associated cargo and one with ubiquitin. We measure both the PEX5 and the ubiquitin levels on the peroxisomes as we vary the matrix protein cargo addition rate into the cytosol. We find that both uncoupled and directly coupled translocation behave identically with respect to PEX5 and ubiquitin, and the peroxisomal ubiquitin signal increases as the matrix protein traffic increases. In contrast, cooperatively coupled translocation behaves dramatically differently, with a ubiquitin signal that decreases with increasing matrix protein traffic. Recent work has shown that ubiquitin on mammalian peroxisome membranes can lead to selective degradation by autophagy, or ‘pexophagy.’ Therefore, the high ubiquitin level for low matrix cargo traffic with cooperatively coupled protein translocation could be used as a disuse signal to mediate pexophagy. This mechanism may be one way that cells could regulate peroxisome numbers.     

The energetics of Pex5p-mediated peroxisomal protein import

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.     

Pex14p, more than just a docking protein

After binding newly synthesized peroxisomal matrix proteins in the cytosol, the second task of Pex5p, the peroxisomal cycling receptor, is to carry these proteins to the peroxisomal membrane. Defining the nature of the events that occur at this membrane system and which ultimately result in the translocation of the cargo proteins into the matrix of the organelle and in the recycling of Pex5p back to the cytosol, is one of the major goals of the research in this field. Presently, it is generally accepted that all these steps are promoted by a large protein complex embedded in the peroxisomal membrane. This docking/translocation machinery or importomer, as it is often called, comprises many different peroxins of which one of the best characterized is Pex14p. Here, we review data regarding this membrane peroxin with emphasis on the interactions that it establishes with Pex5p. The available evidence suggests that the key to understand how folded proteins are capable of passing an apparently impermeable membrane may largely reside in this pair of peroxins.     

Functional characterization of two missense mutations in Pex5p - C11S and N526K

Most newly synthesized peroxisomal proteins are targeted to the organelle by Pex5p, the peroxisomal cycling receptor. Pex5p interacts with these proteins in the cytosol, transports them to the peroxisomal docking/translocation machinery and promotes their translocation across the organelle membrane. Finally, Pex5p is recycled back to the cytosol in order to catalyse additional rounds of transportation. Although several properties of this protein sorting pathway have been recently uncovered, we are still far from comprehending many of its basic principles. Here, we describe the mechanistic implications of two single-amino acid substitutions in Pex5p. The first mutation characterized, Cys11Ser, blocks the recycling of Pex5p back into the cytosol at the step in which stage 2 Pex5p is converted into stage 3 Pex5p. The mutation Asn526Lys, previously described in a child with neonatal adrenoleukodystrophy and shown to abolish the PTS1-binding capacity of Pex5p, results in a Pex5p protein exhibiting import capacity. Protease assays suggest that the Asn526Lys mutation causes conformational alterations at the N-terminal half of Pex5p mimicking the ones induced by binding of a PTS1-containing peptide to the normal peroxin. The implications of these findings on the mechanism of protein translocation across the peroxisomal membrane are discussed.     

Functional role of the AAA peroxins in dislocation of the cycling PTS1 receptor back to the cytosol

Peroxisomal import receptors bind their cargo proteins in the cytosol and target them to docking and translocation machinery at the peroxisomal membrane (reviewed in ref. 1). The receptors release the cargo proteins into the peroxisomal lumen and, according to the model of cycling receptors, they are supposed to shuttle back to the cytosol. This shuttling of the receptors has been assigned to peroxins including the AAA peroxins Pex1p and Pex6p, as well as the ubiquitin-conjugating enzyme Pex4p (reviewed in ref. 2). One possible target for Pex4p is the PTS1 receptor Pex5p, which has recently been shown to be ubiquitinated. Pex1p and Pex6p are both cytosolic and membrane-associated AAA ATPases of the peroxisomal protein import machinery, the exact function of which is still unknown. Here we demonstrate that the AAA peroxins mediate the ATP-dependent dislocation of the peroxisomal targeting signal-1 (PTS1) receptor from the peroxisomal membrane to the cytosol.     

Peroxisome biogenesis

Peroxisome biogenesis conceptually consists of the (a) formation of the peroxisomal membrane, (b) import of proteins into the peroxisomal matrix and (c) proliferation of the organelles. Combined genetic and biochemical approaches led to the identification of 25 PEX genes-encoding proteins required for the biogenesis of peroxisomes, so-called peroxins. Peroxisomal matrix and membrane proteins are synthesized on free ribosomes in the cytosol and posttranslationally imported into the organelle in an unknown fashion. The protein import into the peroxisomal matrix and the targeting and insertion of peroxisomal membrane proteins is performed by distinct machineries. At least three peroxins have been shown to be involved in the topogenesis of peroxisomal membrane proteins. Elaborate peroxin complexes form the machinery which in a concerted action of the components transports folded, even oligomeric matrix proteins across the peroxisomal membrane. The past decade has significantly improved our knowledge of the involvement of certain peroxins in the distinct steps of the import process, like cargo recognition, docking of cargo-receptor complexes to the peroxisomal membrane, translocation, and receptor recycling. This review summarizes our knowledge of the functional role the known peroxins play in the biogenesis and maintenance of peroxisomes. Ideas on the involvement of preperoxisomal structures in the biogenesis of the peroxisomal membrane are highlighted and special attention is paid to the concept of cargo protein aggregation as a presupposition for peroxisomal matrix protein import. Electronic Publication     

Novel proteins interacting with peroxisomal protein receptor PEX7 in Arabidopsis thaliana

Peroxisomal matrix protein transport relies on 2 cytosolic receptors, PEX5 and PEX7, which import peroxisomal targeting signal type 1 (PTS1) and PTS2-containing proteins, respectively. To better understand the transport mechanism of PEX7, we isolated PEX7 complexes using proteomics. We identified PEX5 as well as PTS1- and PTS2-containing proteins within the complex, thereby confirming the interaction between PEX5 and PEX7 during cargo transport that had been previously characterized by biochemical approaches. In addition, a chaperone T-complex and 2 small Rab GTPases were identified. We recently reported that the RabE1c is involved in the degradation of the PEX7 when abnormal PEX7 is accumulated on the peroxisomal membrane. This study expands our knowledge on the transport machinery via PEX7 by identifying both known and novel PEX7-interacting proteins and thus is helpful for further investigation of the regulation of the peroxisomal protein receptor during its translocation.     

PEX5, the Shuttling Import Receptor for Peroxisomal Matrix Proteins,Is a Redox‐Sensitive Protein

Peroxisome maintenance depends on the import of nuclear‐encoded proteins from the cytosol. The vast majority of these proteins is destined for the peroxisomal lumen and contains a C‐terminal peroxisomal targeting signal, called PTS1. This targeting signal is recognized in the cytosol by the receptor PEX5. After docking at the peroxisomal membrane and release of the cargo into the organelle matrix, PEX5 is recycled to the cytosol through a process requiring monoubiquitination of an N‐terminal, cytosolically exposed cysteine residue (Cys11 in the human protein). At present, the reason why a cysteine, and not a lysine residue, is the target of ubiquitination remains unclear. Here, we provide evidence that PTS1 protein import into human fibroblasts is a redox‐sensitive process. We also demonstrate that Cys11 in human PEX5 functions as a redox switch that regulates PEX5 activity in response to intracellular oxidative stress. Finally, we show that exposure of human PEX5 to oxidized glutathione results in a ubiquitination‐deficient PEX5 molecule, and that substitution of Cys11 by a lysine can counteract this effect. In summary, these findings reveal that the activity of PEX5, and hence PTS1 import, is controlled by the redox state of the cytosol. The potential physiological implications of these findings are discussed.      

Ubiquitination of mammalian Pex5p, the peroxisomal import receptor

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.     

Characterization of Trypanosoma brucei PEX14 and its role in the import of glycosomal matrix proteins.

It has been shown previously in various organisms that the peroxin PEX14 is a component of a docking complex at the peroxisomal membrane, where it is involved in the import of matrix proteins into the organelle after their synthesis in the cytosol and recognition by a receptor. Here we present a characterization of the Trypanosoma brucei homologue of PEX14. It is shown that the protein is associated with glycosomes, the peroxisome-like organelles of trypanosomatids in which most glycolytic enzymes are compartmentalized. The N-terminal part of the protein binds specifically to TbPEX5, the cytosolic receptor for glycosomal matrix proteins with a peroxisome-targeting signal type 1 (PTS-1). TbPEX14 mRNA depletion by RNA interference results, in both bloodstream-form and procyclic, insect-stage T. brucei, in mislocalization of glycosomal proteins to the cytosol. The mislocalization was observed for different classes of matrix proteins: proteins with a C-terminal PTS-1, a N-terminal PTS-2 and a polypeptide internal I-PTS. The RNA interference experiments also showed that TbPEX14 is essential for the survival of bloodstream-form and procyclic trypanosomes. These data indicate the protein's great potential as a target for selective trypanocidal drugs.     

The AAA-type ATPases Pex1p and Pex6p and their role in peroxisomal matrix protein import in Saccharomyces cerevisiae

The recognition of the conserved ATP-binding domains of Pex1p, p97 and NSF led to the discovery of the family of AAA-type ATPases. The biogenesis of peroxisomes critically depends on the function of two AAA-type ATPases, namely Pex1p and Pex6p, which provide the energy for import of peroxisomal matrix proteins. Peroxisomal matrix proteins are synthesized on free ribosomes in the cytosol and guided to the peroxisomal membrane by specific soluble receptors. At the membrane, the cargo-loaded receptors bind to a docking complex and the receptor-docking complex assembly is thought to form a dynamic pore which enables the transition of the cargo into the organellar lumen. The import cycle is completed by ubiquitination- and ATP-dependent dislocation of the receptor from the membrane to the cytosol, which is performed by the AAA-peroxins. Receptor ubiquitination and dislocation are the only energy-dependent steps in peroxisomal protein import. The export-driven import model suggests that the AAA-peroxins might function as motor proteins in peroxisomal import by coupling ATP-dependent removal of the peroxisomal import receptor and cargo translocation into the organelle.     

Covalent Label Transfer between Peroxisomal Importomer Components Reveals Export-driven Import Interactions

Peroxisomes are vital metabolic organelles found in almost all eukaryotic organisms, and they rely exclusively on import of their matrix protein content from the cytosol. In vitro import of proteins into isolated peroxisomal fractions has provided a wealth of knowledge on the import process. However, the common method of protease protection garnered no information on the import of an N-terminally truncated PEX5 (PEX5C) receptor construct or peroxisomal malate dehydrogenase 1 (pMDH1) cargo protein into sunflower peroxisomes because of high degrees of protease susceptibility or resistance, respectively. Here we present a means for analysis of in vitro import through a covalent biotin label transfer and employ this method to the import of PEX5C. Label transfer demonstrates that the PEX5C construct is monomeric under the conditions of the import assay. This technique was capable of identifying the PEX5-PEX14 interaction as the first interaction of the import process through competition experiments. Labeling of the peroxisomal protein import machinery by PEX5C demonstrated that this interaction was independent of added cargo protein, and, strikingly, the interaction between PEX5C and the import machinery was shown to be ATP-dependent. These important mechanistic insights highlight the power of label transfer in studying interactions, rather than proteins, of interest and demonstrate that this technique should be applied to future studies of peroxisomal in vitro import.     

Insights into Peroxisome Function from the Structure of PEX3 in Complex with a Soluble Fragment of PEX19

The human peroxins PEX3 and PEX19 play a central role in peroxisomal membrane biogenesis. The membrane-anchored PEX3 serves as the receptor for cytosolic PEX19, which in turn recognizes newly synthesized peroxisomal membrane proteins. After delivering these proteins to the peroxisomal membrane, PEX19 is recycled to the cytosol. The molecular mechanisms underlying these processes are not well understood. Here, we report the crystal structure of the cytosolic domain of PEX3 in complex with a PEX19-derived peptide. PEX3 adopts a novel fold that is best described as a large helical bundle. A hydrophobic groove at the membrane-distal end of PEX3 engages the PEX19 peptide with nanomolar affinity. Mutagenesis experiments identify phenylalanine 29 in PEX19 as critical for this interaction. Because key PEX3 residues involved in complex formation are highly conserved across species, the observed binding mechanism is of general biological relevance.     

Pex5p, the peroxisomal cycling receptor, is a monomeric non-globular protein

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.