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Originally published In Press as doi:10.1074/jbc.M706325200 on August 28, 2007

J. Biol. Chem., Vol. 282, Issue 43, 31267-31272, October 26, 2007
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Ubiquitination of Mammalian Pex5p, the Peroxisomal Import Receptor*

Andreia F. Carvalho{ddagger}§1, Manuel P. Pinto{ddagger}§1, Cláudia P. Grou{ddagger}§1, Inês S. Alencastre{ddagger}§, Marc Fransen, Clara Sá-Miranda{ddagger}, and Jorge E. Azevedo{ddagger}§2

From the {ddagger}Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal, the §Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, Largo Professor Abel Salazar, 2, 4009-003 Porto, Portugal, and the Faculteit Geneeskunde, Departement Moleculaire Celbiologie, Katholieke Universiteit Leuven, Herestraat 49, B-3000 Leuven, Belgium

Received for publication, June 23, 2007 , and in revised form, August 27, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 Cys11 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein translocation across the peroxisomal membrane requires a complex machinery comprising many different peroxins. In yeasts, 15–16 peroxins have been implicated in this process, whereas in mammals, only 10 are known so far (reviewed in Refs. 1 and 2). For several of the extra peroxins found in yeasts, it is presently unclear if their apparent absence in mammals is related simply to a high rate of gene divergence during evolution, hampering the identification of the mammalian genes by homology searches, or if it reflects the existence of mechanistic differences in the protein import pathways of distantly related organisms. The fact that differences of this type do exist feeds this uncertainty. A good example is provided by the import pathways of matrix proteins possessing a type 2 peroxisomal targeting signal (PTS)3 (3). PTS2-containing proteins are recognized by Pex7p and transported to the peroxisome by Pex5p in many organisms. However, although yeasts also possess Pex5p, they do not rely on this peroxin for this purpose. Instead, Pex20p or Pex18p/Pex21p escorts the Pex7p-cargo protein complex to the organelle (reviewed in Ref. 4).

Despite these differences, all organisms characterized up to now use Pex5p to target PTS1-containing proteins to the peroxisomal matrix. These proteins are characterized by the existence of a conserved tripeptide at their extreme C terminus that is recognized by Pex5p (5). According to current models (1, 2), Pex5p interacts with newly synthesized matrix proteins in the cytosol and transports them to the peroxisomal membrane. Here, Pex5p participates in a complex network of protein-protein interactions involving several components of the importomer (6–9). These interactions ultimately result in the insertion of Pex5p into the organelle membrane with the concomitant translocation of the cargo protein across the membrane (10, 11). Remarkably, all available evidence suggests that energy in the form of ATP is not required at these steps. The need for ATP hydrolysis occurs only at the next step, when Pex5p is exported from the importomer by the action of Pex1p and Pex6p (12–14). Then, a new cycle of protein transportation can be initiated.

Using an in vitro import/export system, Erdmann and co-workers (15) have shown recently that export of yeast Pex5p from the importomer requires Pex4p and Pex22p. They also demonstrated that a small fraction of membrane-bound yeast Pex5p can be monoubiquitinated by recombinant Pex4p, although the lability of this ubiquitinated Pex5p population precluded its detection in the in vitro import/export experiments. These data led the authors to propose that yeast Pex5p has to be monoubiquitinated to be recognized by the export machinery. Whether or not such a step also applies to the mechanism of protein translocation in mammals is not clear at present. In fact, many independent observations could suggest otherwise. For instance, although several Pex4p-like proteins can be found in the human proteome, the protein displaying the highest similarity to yeast/plant Pex4p has been reported to be localized in the nucleus (16). In the case of Pex22p, no evidence for the existence of the corresponding gene in mammals could be found, despite several attempts (17, 18). Proteomic approaches aiming at defining the protein repertoire of mammalian peroxisomes also failed in identifying these peroxins (19, 20). Furthermore, the genes involved in the so-called peroxisomal biogenesis disorders have been identified for all complementation groups presently known, and none corresponds to a PEX4- or PEX22-like gene (21). Finally, despite the numerous experiments that have been described by several groups over the years aiming at characterizing the Pex5p-mediated protein import cycle in mammals, no evidence for a ubiquitinated Pex5p population was ever found. Obviously, all these facts represent negative observations that may lose significance in face of positive results. This is, we believe, what happens in this work.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
In Vitro Import Reactions—Rat liver post-nuclear supernatants were prepared in SE buffer (0.25 M sucrose, 5 mM MOPS-KOH, pH 7.2, and 1 mM EDTA-NaOH, pH 7.2) exactly as described previously (22). In vitro import reactions (100 µl) contained 400 µg of post-nuclear supernatant protein and 0.5–1.0 µl of the relevant reticulocyte lysates in import buffer (0.25 M sucrose, 50 mM KCl, 20 mM MOPS-KOH, pH 7.2, 3 mM MgCl2, 20 µM methionine, and 2 µg/ml N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide). Unless specified otherwise, import reactions were incubated for 30 min at 37 °C. ATP and ATP{gamma}S were used at a 10 mM final concentration. Where indicated, 25 µg of glutathione S-transferase (GST)-ubiquitin (Ub) (23) or negative control GST protein (GST-Control; referred to as GST-LKS in Ref. 24) was used per import reaction. The synthesis of 35S-labeled proteins using a rabbit reticulocyte lysate was described previously (22). The cDNA encoding Pex5p(C11S) was obtained with the QuikChange® site-directed mutagenesis kit (Stratagene) using primer pair 5'-GGAGCTGGTGGAGGCCGAAAGCGGGGGTGCCAACCCGC-3' and 5'-GCGGGTTGGCACCCCCGCTTTCGGCCTCCACCAGCTCC-3' and pGEM4-Pex5p (22) as the template. The plasmid pGEM4-Pex5p encodes the large iso-form of human Pex5p (25, 26). Proteinase K treatment of import reactions, processing of protein samples for SDS-PAGE (performed at 4 °C), and autoradiography were done as described (22).

GST Pulldown Assays and Immunoprecipitations—Organelle pellets were resuspended in solubilization buffer containing 1% (w/v) Triton X-100, 150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA-NaOH, pH 8.0, 5 mM N-ethylmaleimide (NEM), 1:100 (v/v) mammalian protease inhibitor mixture (Sigma), and 50 µg/ml phenylmethylsulfonyl fluoride. After 30 min on ice, the insoluble material was removed by centrifugation, and the supernatants were incubated at 4 °C for 2 h with either glutathione-Sepharose beads (50-µl bed volume) or 5 mg of protein G-Sepharose previously incubated with 10 µg of a monoclonal antibody directed against ubiquitin (clone FK2, BIOMOL International). An anti-c-Myc antibody (10 µg; clone 9E10, Roche Applied Science) was used as a negative control in the immunoprecipitation experiments. The Sepharose beads were then washed three times with 1 ml of solubilization buffer and once with the same buffer lacking detergent and incubated with Laemmli gel loading buffer (lacking reducing agents) supplemented with 10 mM NEM at 45 °C for 30 min. Where indicated, samples received 100 mM dithiothreitol (DTT) and were then heated at 60 °C for 15 min.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
In the experiments presented below, we will make frequent reference to two peroxisomal membrane-associated Pex5p populations. Their properties have been described (see Ref. 27 and references cited therein). However, for the sake of clarity, a summary is presented here. Both populations represent Pex5p molecules passing through the importomer during the protein transport cycle and are operationally defined by a protease protection assay and SDS-PAGE analysis under reducing conditions. The so-called stage 2 Pex5p exposes the majority of its mass to the matrix of the organelle and ~2 kDa of its N terminus to the cytosol as assessed with proteinase K; stage 3 Pex5p is completely resistant to this protease as long as the organelles are kept intact. The conversion of stage 2 into stage 3 Pex5p is a relatively slow process. In contrast, the subsequent export of stage 3 Pex5p into the cytosol is very rapid at 37 °C, provided that ATP is not limiting. In fact, under these conditions, stage 3 Pex5p is a minor species. In the absence of exogenous ATP (i.e. in the presence of the small amounts of ATP derived from the post-nuclear supernatant and the reticulocyte lysates used in these experiments), stage 3 Pex5p is easily detectable; in the presence of ATP{gamma}S, it becomes the most abundant species. It is also important to note that ATP{gamma}S blocks the export step of Pex5p from the peroxisomal membrane. Because of the fact that a significant fraction of the peroxisomal importomer present in our reactions is already occupied by endogenous rat liver Pex5p (13), the addition of ATP{gamma}S at the beginning of the reactions results in a low import rate of the 35S-labeled peroxin.

Ubiquitination of Pex5p in yeasts is well documented and serves two different purposes (14, 15, 28–31). Polyubiquitination of Pex5p is observed in several peroxisomal deficient mutant strains and reflects the existence of a quality control system aiming at removing arrested/entangled Pex5p molecules from the importomer; monoubiquitination of Pex5p was recently linked to the export of yeast Pex5p from the importomer.

In an attempt to determine whether mammalian Pex5p is also poly/monoubiquitinated, we initiated a series of in vitro import experiments using 35S-labeled Pex5p and Pex5p(C11S). The substitution of Cys11 with serine blocks the ATP-dependent export step of Pex5p from the peroxisomal membrane by specifically impeding the stage 2-to-stage 3 transition (27). Thus, high amounts of this protein can be accumulated at the peroxisomal membrane in in vitro import reactions containing ATP, a property that should increase our chances to detect ubiquitination of Pex5p. To facilitate the identification of putative ubiquitinated Pex5p or Pex5p(C11S) molecules, a recombinant GST-Ub fusion protein was included in some of the reactions. GST-Ub is 28 kDa larger than ubiquitin alone, and thus, a similar increase in the molecular mass of in vitro ubiquitinated molecules should be observed in the presence of this ubiquitin analog. Finally, considering that any putative ubiquitinated molecules should be, in principle, exposed to the cytosol (and thus protease accessible), both protease-treated and -untreated reactions were analyzed.


Figure 1
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  		FIGURE 1.
GST-Ub interferes with the export step of Pex5p from the peroxisomal membrane. 35S-Labeled Pex5p or Pex5p(C11S) was subjected to import reactions in the absence of exogenous nucleotides (–lanes) or in the presence of ATP or ATP{gamma}S as indicated. One set of reactions received GST-Ub, and the other received GST-Control (negative control GST fusion protein). The import reactions were halved, and proteinase K (PK; 400 µg/ml final concentration; lower panel) or NEM (20 mM final concentration; upper panel) was added to each half. After 30 min on ice, the samples were diluted with SE buffer containing 20 mM NEM, and the organelles were isolated by centrifugation and analyzed by SDS-PAGE under reducing conditions. Arrows a and b mark the 100- and 130-kDa species (see "Results" for details). The migrations of stage 2 and 3 Pex5p are also indicated. The asterisks indicate truncated proteins arising from internal initiation during the in vitro translation reactions. These truncated molecules are export-incompetent and thus become concentrated in the peroxisomes in the presence of ATP (see Ref. 27 for details). IA and IB lanes, 10% of the reticulocyte lysates containing 35S-labeled Pex5p or Pex5p(C11S), respectively, used in each lane. Numbers to the left indicate the molecular masses of protein standards in kilodaltons. Note that the exposure time in the autoradiography shown in the upper panel was 3-fold longer than that used in the lower panel to allow detection of the low abundance 100- and 130-kDa species.

 
The results of one of these experiments are presented in Fig. 1. When the protease treatment step was omitted, a faint radioactive protein band migrating above Pex5p(C11S) (apparent molecular mass of 100 kDa) could be detected upon SDS-PAGE under reducing conditions (Fig. 1, upper panel, lane 5). This abnormally migrating protein band was no longer visible when the import reaction was supplemented with GST-Ub. Instead, a new species displaying an apparent molecular mass of 130 kDa was observed (lane 11), suggesting that it represents a ubiquitinated Pex5p(C11S) population (see also below). Occasionally, a similar 100-kDa protein band was also detected when Pex5p was used in these experiments, although the fraction of the in vitro imported protein displaying such a behavior was always much smaller than that detected with Pex5p(C11S) (compare lanes 2 and 5). This observation could suggest that this ubiquitination event is linked to the existence of a quality control system acting on defective Pex5p molecules, although further data are necessary to clarify this issue.

To our surprise, inclusion of GST-Ub in the import reactions revealed a phenomenon of a completely different magnitude. Indeed, in the protease-treated aliquots, stage 2 Pex5p was no longer the predominant species in the ATP-supplemented reaction (Fig. 1, lower panel, compare lanes 2 and 8). A fully protease-protected species corresponding to (or resembling) stage 3 Pex5p was the most abundant species. Notably, the total amount of protease-protected Pex5p in this sample was similar to that detected for Pex5p(C11S) under the same conditions (compare lanes 8 and 11). Clearly, GST-Ub strongly inhibited the export of Pex5p from the importomer, an observation that provides, for the first time, evidence that ubiquitin is involved in the Pex5p-mediated import pathway also in mammals.

The finding that GST-Ub blocks peroxisomal Pex5p at the stage 3 level together with the fact that Pex5p(C11S) is unable to reach this stage led us to address the possibility that Cys11 might be the residue modified by ubiquitin. In fact, this hypothesis was formulated before for both Pex5p and Pex20p, a Pex5p-like peroxin from Pichia pastoris (27, 32). This type of ubiquitin-conjugated molecule cannot be detected under reducing conditions because the bond linking the carboxyl group of the C-terminal glycine of ubiquitin to the thiol group of a cysteine is thiol-sensitive (33). Thus, we subjected 35S-labeled Pex5p and Pex5p(C11S) to import reactions containing ATP and GST-Ub, but this time, the reactions were analyzed by SDS-PAGE under both reducing and nonreducing conditions. Two additional questions were addressed in this experiment. 1) How fast is this ubiquitination event? 2) Does it occur in the soluble (cytosolic) phase of the reactions, in the organelle fraction, or in both? As shown in Fig. 2A, a prominent thiol-sensitive 130-kDa protein band was visible in the Pex5p-containing organelles (compare upper and lower panels). Data indicating that this protein band corresponds to GST-Ub-Pex5p are presented in Fig. 2B. In agreement with the results described above, Pex5p(C11S) was also modified by this ubiquitin analog. However, in this case, treatment with DTT did not destroy the GST-Ub-conjugated species, as expected (see also Fig. 2B). Another important property differentiates these two GST-Ub-conjugated populations. In the case of Pex5p, this species was easily detectable already after 5 min of import, whereas with Pex5p(C11S), it was necessary to incubate the reactions for 45 min to generate similar amounts of the 130-kDa band protein (Fig. 2A).

There are six cysteines in the primary structure of the large isoform of human Pex5p (25, 26). Although the results described above already suggest that the thiol-sensitive bond involves Cys11 of Pex5p, we decided to address this issue in a direct way. For this purpose, we explored the properties of a truncated version of Pex5p comprising its first 324 amino acid residues ({Delta}C1Pex5p) and containing a single cysteine residue, the one at position 11. Although it lacks the PTS1-binding domain, this C-terminally truncated molecule is still a substrate for the peroxisomal import and export machineries (24, 34). As shown in Fig. 2C, inclusion of GST-Ub in import reactions containing ATP also led to an arrest of {Delta}C1Pex5p at the importomer (right panel, compare GST-Control and GST-Ub lanes). Again, a species corresponding to (or resembling) stage 3 was observed. Notably, under these conditions, a thiol-sensitive protein band displaying an apparent molecular mass of ~80 kDa (the expected value for a GST-Ub-conjugated {Delta}C1Pex5p species) was detected (left and middle panels, compare GST-Ub lanes). Taken together, these data suggest that Cys11 of Pex5p is the residue involved in the thiol ester bond.


Figure 2
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  		FIGURE 2.
Cys11 of in vitro imported Pex5p forms a thiol ester conjugate with GST-Ub. A, 35S-labeled Pex5p or Pex5p(C11S) was subjected to import reactions in the presence of ATP and GST-Ub. At time 0, one aliquot of the import reactions was withdrawn, supplemented with NEM (20 mM), and kept on ice (T lanes). Three other aliquots were removed from the import reactions at the indicated time periods. These aliquots were supplemented with NEM and separated into an organelle fraction (Pellet) and a soluble phase (Supernatant) by centrifugation. The samples were then analyzed by SDS-PAGE under reducing (lower panel) or nonreducing (upper panel) conditions. To avoid protein overloading problems, only one-third of the Supernatant and T samples were analyzed in these gels. The arrowheads indicate the 130-kDa species. Note that the 130-kDa bands detected with Pex5p and Pex5p(C11S) in the upper panel seem to display different migrations. This is due to a small distortion of this gel because the two bands display exactly the same migration when analyzed side-by-side. B, organelles from import reactions containing Pex5p or Pex5p(C11S) performed in the presence of GST-Ub and ATP were isolated by centrifugation, resuspended in solubilization buffer, and subjected to a pulldown assay using glutathione-Sepharose beads. The total (T), unbound (Unb), and bound (in this case, treated (B+) or not (B–) with DTT) fractions were subjected to SDS-PAGE. Three equivalents of the bound fractions in relation to the total and unbound samples were loaded onto the gel. C, 35S-labeled {Delta}C1Pex5p was subjected to import reactions in the presence of ATP and GST-Ub or GST-Control. At the end of the 37 °C incubation, each reaction was divided into three equal aliquots that were treated (+PK) or not (–PK) with proteinase K, as specified in the legend to Fig. 1. The organelles were then isolated by centrifugation and analyzed by SDS-PAGE under reducing (+DTT) or nonreducing (–DTT) conditions. The thiol-sensitive GST-Ub-{Delta}C1Pex5p species and stage 2 and 3 {Delta}C1Pex5p are indicated. I lanes, 10% of the reticulocyte lysate containing {Delta}C1Pex5p used in each lane. Numbers to the left indicate the molecular masses of protein standards in kilodaltons.

 
The experiments described above unveil the existence of a ubiquitin-conjugating system acting on mammalian Pex5p during its transient passage through the importomer. However, because GST-Ub interferes with the normal transit of Pex5p, these data do not clearly reveal the relationship between ubiquitinated Pex5p and the stage 2 and 3 Pex5p populations. In an attempt to clarify this issue, Pex5p was subjected to standard import reactions in the absence of exogenous nucleotides or in the presence of ATP or ATP{gamma}S. Each reaction was then divided into four aliquots that were treated or not with proteinase K and analyzed by SDS-PAGE under reducing or nonreducing conditions. The results obtained for each of these four sets of samples are shown in Fig. 3A. A thiol-sensitive 100-kDa protein band could be easily detected in these assays (compare–PK/–DTT and–PK/+DTT panels), suggesting that 35S-labeled Pex5p forms a conjugate with endogenous rat liver ubiquitin. Indeed, the 100-kDa species was recognized by an anti-ubiquitin antibody (Fig. 3B). In addition, inclusion of GST-Ub in the ATP{gamma}S-containing import reactions led to the loss of this species with the concomitant appearance of a 130-kDa protein band (Fig. 3C). Notably, the ubiquitinated Pex5p species detected in these experiments corresponds to stage 3 Pex5p. This relationship was particularly evident when the protease-treated samples were analyzed under reducing or nonreducing conditions (Fig. 3A, +PK/+DTT and +PK/–DTT panels, respectively). Whereas stage 2 Pex5p behaved in a similar way under both conditions (ATP lanes), nonreduced stage 3 Pex5p (ATP{gamma}S lanes) was detected as a group of protein bands derived from partial proteolysis of the 100-kDa species. We note that this proteolysis does not affect Pex5p itself but rather the conjugated ubiquitin molecule because, as expected, all these bands disappeared upon reduction with DTT, yielding the intact peroxin (i.e. the stage 3 Pex5p population, as defined at the beginning of "Results"). (See Ref. 10 for a topological model of stage 3 Pex5p.)

It is interesting that when the protease treatment step was omitted, a significant amount of ubiquitinated Pex5p could be detected in the supernatants of import reactions performed in the presence of exogenous ATP (Fig. 3D, lane 2). In contrast, although ATP{gamma}S is as efficient as ATP in supporting the energetics of the ubiquitin-conjugating cascade (35, 36), the vast majority of ubiquitinated Pex5p was detected in the organelle pellet when this ATP analog was used in the import reactions (Fig. 3D, lanes 3 and 4). This observation is in perfect agreement with our earlier finding that ATP{gamma}S blocks the export of peroxisomal Pex5p at the stage 3 level (22). Taken together, these data strongly suggest that ubiquitinated Pex5p (unlike GST-Ub-Pex5p; see Fig. 2A) is a substrate for the peroxisomal export machinery.


Figure 3
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  		FIGURE 3.
Stage 3 Pex5p represents ubiquitinated Pex5p and is export-competent. A, 35S-labeled Pex5p was subjected to import reactions containing no exogenous nucleotides (–lane) or ATP or ATP{gamma}S as indicated. At the end of the 37 °C incubation, each reaction was divided into four equal aliquots. The aliquots were treated (+PK) or not (–PK) with proteinase K and analyzed by SDS-PAGE under reducing (+DTT) or nonreducing (–DTT) conditions as indicated. Pex5p, the 100-kDa species (arrow a), and stage 2 and 3 Pex5p are indicated. B, an organelle pellet from an import reaction performed in the presence of ATP{gamma}S was solubilized with detergent and subjected to immunoprecipitation using an anti-ubiquitin antibody (upper panel) or a control antibody (lower panel) (see "Experimental Procedures" for details). The total (T), unbound (Unb), and immunoprecipitated (in this case, treated (B+) or not (B–) with DTT) fractions were subjected to SDS-PAGE. Note that although the yield in this immunoprecipitation experiment was quite low, the anti-ubiquitin antibody recognized the 100-kDa component, but not the unmodified Pex5p species. C, 35S-labeled Pex5p was subjected to import reactions containing 10 mM ATP{gamma}S and GST-Control or GST-Ub for 10 min at 37 °C. The reactions were diluted with SE buffer supplemented with 20 mM NEM, and the organelles were isolated by centrifugation and analyzed by SDS-PAGE under nonreducing conditions. The 100-kDa (ubiquitinated Pex5p) and 130-kDa (GST-Ub-Pex5p) protein bands are indicated by arrows a and b, respectively. D, 35S-labeled Pex5p was subjected to import reactions containing ATP or ATP{gamma}S as indicated. The reactions received NEM (20 mM) and were then separated into an organelle fraction (P) and a soluble phase (S) by centrifugation. The samples were analyzed by SDS-PAGE under nonreducing conditions. Arrow a indicates ubiquitinated Pex5p. Numbers to the left indicate the molecular masses of protein standards in kilodaltons. I lane, 10% of the Pex5p reticulocyte lysate used in each lane.

 
Besides linking stage 3 Pex5p to ubiquitinated Pex5p, the results described above have another implication. Indeed, they suggest that Pex5p molecules at the stage 2 and 3 levels may adopt similar membrane topologies. The observation that the polypeptide chain of Pex5p at the stage 3 level is not cleaved by proteinase K may now be explained by a protection effect exerted by the cytosol-exposed conjugated ubiquitin molecule and/or by some still unknown ubiquitin-binding protein. The fact that no significant stage 3 population can be detected with Pex5p(C11S) (it lacks the cysteine residue necessary for ubiquitin conjugation) and the observation that import conditions leading to an accumulation of GST-Ub-Pex5p at the importomer result in such high amounts of stage 3 species corroborate this conclusion.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The data presented here provide, for the first time, an indication that mammalian Pex5p is ubiquitinated at the peroxisomal membrane. Two different types of ubiquitination were demonstrated in our experiments. The first involves conjugation of ubiquitin molecules to Pex5p via thiol-resistant bonds in a process that was quite slow in our experimental system. The physiological relevance of this process remains to be determined. The second type of ubiquitination involves Cys11 of Pex5p. This finding and our previous observations that alkylation or mutation of this residue results in a Pex5p protein unable to exit the importomer (27) strongly suggest that this type of ubiquitination is not only involved in, but is also absolutely necessary for the normal mechanism of protein translocation across the peroxisomal membrane. One additional observation reported here supports this conclusion. This regards the potent inhibitory effect of GST-Ub on the export step of Pex5p. Although our in vitro system contained endogenous ubiquitin (~1 µM assuming that ubiquitin comprises 0.2% of total protein) (37, 38), the total levels of protease-protected Pex5p obtained in import reactions containing ATP and GST-Ub (6.7 µM) were similar to those obtained under the same conditions with Pex5p(C11S). This implies that the degree of inhibition induced by GST-Ub is also similar to that associated with the C11S mutation, which in turn suggests that most, if not all, Pex5p molecules passing through the importomer are ubiquitinated. The reason why GST-Ub-Pex5p is not a good substrate for the export machinery is presently unknown, although it could be related to a failure of this machinery to recognize such a bulky ubiquitin analog. (Note that GST alone is a homodimeric protein of 52 kDa (39).)

While this manuscript was in preparation, Williams and co-workers (40) reported the Pex4p-dependent ubiquitination of the conserved cysteine present at the N terminus of yeast Pex5p. Because mutation of this cysteine did not result in the peroxisomal accumulation of the mutant Pex5p species, the true mechanistic meaning of this modification could not be defined. The authors nevertheless hypothesized that such ubiquitination could be involved in the recycling of Pex5p. The data presented here strongly suggest that this is indeed the case. To our surprise, they also reveal the remarkably conserved nature of the Pex5p-mediated protein transport pathway in so distantly related organisms. The search for the mammalian functional counterparts of Pex4p and Pex22p should be the next step.


   FOOTNOTES
 
* This work was supported in part by the POCTI Program of the Fundação para a Ciência e Tecnologia; the Fundo Europeu de Desenvolvimento Regional, Portugal; and European Union VI Framework Program Grant LSHG-CT-2004-512018 (Peroxisomes in Health and Disease). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by the Fundação para a Ciência e Tecnologia. Back

2 To whom correspondence should be addressed: Inst. de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal. Tel.: 351-226-074-900; Fax: 351–226-099-157; E-mail: jazevedo{at}ibmc.up.pt.

3 The abbreviations used are: PTS, peroxisomal targeting signal; MOPS, 3-(N-morpholino) propanesulfonic acid; ATP{gamma}S, adenosine 5'-O-(thiotriphosphate); GST, glutathione S-transferase; Ub, ubiquitin; NEM, N-ethylmaleimide; DTT, dithiothreitol. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Anabela Ferro and Patrícia Maciel (Universidade do Minho, Braga, Portugal) for the kind gift of the plasmid encoding GST-Ub.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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