Abstract 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. The vast majority of peroxisomal matrix proteins is targeted to the organelle via the so-called peroxisomal targeting signal 1 (PTS1) 1 pathway (reviewed in Refs. Proteins belonging to this family contain at their extreme C terminus the sequence Ser-Lys-Leu (or a variant ()) that is recognized by Pex5p, the PTS1 receptor ().

In recent studies, miRNAs have been found to be extremely influential in many of the essential biological processes. They exhibit a self-regulatory mechanism through which they act as positive/negative regulators of expression of genes and other miRNAs. This has direct implications in the regulation of. Carlos Azevedo Biologia Celular Molecular Pdf Writer. Using other people’s research or ideas without giving them due credit is plagiarism.

The observation that Pex5p is predominantly found in the cytosol with only a small fraction associated with the peroxisome led several authors to propose the so-called cycling receptor mechanism (). According to this model, newly synthesized PTS1-containing peroxisomal proteins would be recognized by cytosolic Pex5p, transported to the surface of the peroxisome, and translocated across the membrane of the organelle. Experimental evidence compatible with this model was first provided by Dodt and Gould (), reversible accumulation of Pex5p at the peroxisomal compartment could be demonstrated in vivo by manipulating ATP levels and temperature in cultured human fibroblasts. More recently, Dammai and Subramani () were able to show that Pex5p goes through multiple rounds of cycling between the cytosol and the peroxisome.

Peroxisomal membrane docking, translocation of cargo proteins, and recycling of Pex5p requires a complex machinery. In the last years, more than 20 peroxins have been identified in several organisms, most of them involved in the Pex5p-mediated peroxisomal protein import (reviewed in Refs. However, our knowledge on the mechanism by which peroxisomal matrix proteins are translocated across the organelle membrane is still very limited. Much of what is known regarding this matter derives either from steady-state level analysis of peroxisomal Pex5p on several mutant cell lines ( e.g. ) or from studies aiming at the characterization of peroxin-peroxin interactions (see Ref. And references cited therein). Collectively, these data provide the basis for a model in which peroxins are classified into three functional groups: 1) docking proteins; 2) proteins involved in the translocation of matrix proteins across the peroxisomal membrane; and 3) proteins mediating the release (recycling) of Pex5p from the peroxisomal compartment (reviewed in Refs.

Although many aspects of this model may indeed be correct, the recent isolation of stable protein complexes comprising peroxins belonging to different functional groups raises some questions regarding this classification (, ). Here, we provide data suggesting that insertion of Pex5p into the peroxisomal membrane requires Pex14p, a proposed docking protein (). However, Pex14p-associated Pex5p exposes the majority of its mass into the peroxisomal matrix suggesting that translocation of cargo proteins into the matrix of the organelle occurs concomitantly with the formation of the Pex5p-Pex14p membrane complex. Furthermore, Pex14p-associated Pex5p is exported from the peroxisomal compartment in an ATP-dependent process. Thus, our results suggest, on one hand, that translocation of peroxisomal matrix proteins across the organelle membrane occurs through a Pex14p-containing structure via Pex5p, raising the possibility that no other peroxins other than Pex5p interact with the PTS1-containing proteins during the membrane translocation event and, on the other hand, that Pex5p is retained at the peroxisomal membrane during all steps of this process.

Synthesis of Radiolabeled Proteins The cDNA encoding full-length human Pex5p (the large isoform (, ) cloned in the pGEM TM-T Easy vector (pGEMT-Pex5) () was inserted into the SalI site of pGEM-4 (Promega) originating pGEM4-Pex5. This plasmid was transcribed in vitro using T7 RNA polymerase (Roche Molecular Biochemicals). 35S-Labeled Pex5p was synthesized using the translation kit Reticulocyte Type II (Roche Molecular Biochemicals) in the presence of [ 35S]methionine (specific activity >1000 Ci/mmol; ICN Biomedicals, Inc.) following the standard conditions of the manufacturer. Peroxisomal in Vitro Import Experiments Post-nuclear supernatants (PNS) from rat liver were prepared from male Wistar rats (1–2 months of age). Livers were rapidly removed and homogenized in SEM buffer (0.25 m sucrose, 5 m m MOPS-KOH, pH 7.2, and 1 m m EDTA-NaOH, pH 7.2). PNS fractions were obtained by centrifuging the homogenate two times at 600 × g for 10 min at 4 °C.

Aliquots of the PNS fraction (at 40–60 mg/ml) were frozen in liquid nitrogen and stored at −70 °C. These fractions remain import-competent for at least 1 month. Import reactions (100 μl final volume) were performed in import buffer (0.25 m sucrose, 50 m m KCl, 5 m m MOPS-KOH, pH 7.2, 3 m m MgCl 2, 1 m m EDTA-NaOH, pH 7.2, 0.2% (w/v) lipid-free bovine serum albumin, and 20 μ m methionine) using 150 μg of rat liver PNS protein and 0.5 μl of the reticulocyte lysate containing 35S-labeled Pex5p. In some experiments the concentration of lipid-free bovine serum albumin in the import buffer was decreased to 0.002% (w/v) (import buffer B). Where indicated, nucleotides (100 m m stock solutions, pH 7.2, with NaOH; Sigma) were used at 1 m m final concentration.

The contributions of the reticulocyte lysate and rat liver PNS fraction to the levels of ATP in import reactions were not determined experimentally. Nevertheless, considering the total adenine nucleotide pool present in the reticulocyte lysate (Roche Molecular Biochemicals) and rat liver (), this value was estimated to be lower than 36 μ m. At the end of the import reaction, the samples were treated with proteinase K (300 μg/ml final concentration) for 30 min at 4 °C. The protease was inhibited with 500 μg/ml phenylmethysulfonyl fluoride (added from a 50 mg/ml stock solution in ethanol) for 2 min on ice.

The import reaction was diluted to 1 ml with SEM buffer and the organelles were isolated by centrifugation at 15,000 × g for 15 min at 4 °C. The samples were subjected to SDS-PAGE, transferred to nitrocellulose, and the radioactive proteins were detected by autoradiography. Under these conditions optimum import of Pex5p is observed at 26 °C (tested temperatures: 0, 8, 16, 26, and 37 °C) and occurs linearly during the first 45 min of incubation (data not shown). In antibody inhibition experiments, 150 μg of PNS protein, in 30 μl of import buffer, were preincubated with 20 μg of purified immunoglobins (5 μg/μl in PBS) for 20 min on ice before starting the import reaction.

GST-Pex5p and GST recombinant proteins were used in import reactions at a final concentration of 100 μg/ml. Immunoprecipitation of in Vitro Imported Pex5p Two aliquots of a PNS fraction (1.3 mg of protein) were incubated in import buffer either in the presence or absence (mock-treated sample) of 5 μl of a reticulocyte lysate containing 35S-labeled Pex5p. At the end of the incubation (30 min at 26 °C) both samples were treated with proteinase K as described above. The protease was inhibited and the organelles were diluted with 1 ml of SEM buffer containing 50 μg/ml phenylmethysulfonyl fluoride and 1:100 (v/v) mammalian protease inhibitors mixture (Sigma). After centrifugation (15,000 × g for 15 min at 4 °C) to sediment the organelles, 35S-labeled Pex5p (0.1 μl) was then added to the mock-treated sample. To avoid nonimport related Pex5p-Pex14p interactions, 10 μg of GST-Pex5p was added to each tube.

Both samples were then solubilized in 1.3 ml of immunoprecipitation buffer (1% (w/v) Nonidet P-40, 100 m m potassium acetate-acetic acid, pH 7.4, 2 m m EDTA-NaOH, pH 8.0, 100 μg/ml phenylmethysulfonyl fluoride, and 1:100 (v/v) mammalian protease inhibitors mixture) for 15 min on ice and subjected to centrifugation (60,000 × g for 15 min at 4 °C) to remove insoluble material. One aliquot (100 μl) of each supernatant was kept on ice during the rest of the procedure and subjected to trichloroacetic acid precipitation (10% (w/v) final concentration) before SDS-PAGE analysis. The remaining supernatants were divided into two halves. One-half received 5 mg of protein A-Sepharose beads previously incubated with 5 μl of anti-Pex14p serum; the other half received the same amount of beads previously incubated with 5 μl of preimmune serum. These samples were incubated for 60 min at 4 °C with gentle agitation, the beads were sedimented by centrifugation and washed three times with 1 ml of immunoprecipitation buffer. Immunoprecipitated proteins were eluted with 60 μl of Laemmli sample buffer and subjected to SDS-PAGE ().

Miscellaneous Centrifugation of PNS fractions on step Nycodenz gradients (1.5 ml of 45% (w/v), 6.5 ml of 30% (w/v), and 1 ml of 25% (w/v) Nycodenz in 5 m m MOPS-KOH, pH 7.2, and 1 m m EDTA-NaOH, pH 7.2) was performed in an angular rotor (65.13, Sorvall) at 60,000 × g for 45 min at 4 °C. Fractionation of the gradients, determination of the activities of marker enzymes, and trichloroacetic acid precipitation of gradient fractions were performed exactly as described (). Antibodies directed to human Pex14p () and Pex5p () were described before. Antibodies directed to rat PMP70 (Zymed Laboratories, Inc. (,)), catalase (Research Diagnostics, Inc.), and GST (AmershamBiosciences) were purchased. Rabbit and goat antibodies were detected on Western blots using alkaline phosphatase-conjugated anti-rabbit or anti-goat antibodies, respectively (Sigma). IgGs were purified from rabbit sera using protein A-Sepharose beads according to the manufacturer (Amersham Biosciences).

Preparation of Fab fragments using immobilized papain was carried out using the ImmunoPure Fab Preparation Kit (Pierce). For expression of the fusion protein GST-PEX5p, the plasmid pGEMT-Pex5 was used as template in a PCR reaction using the upper primer 5′-GGCGTCGACTGGAGATCTATGGCAATGCGGGAGCTGGTGGA-3′ and the lower primer 5′-GGGTCTAGAGCGGCCGCGTCGACCTGTCACTGGGGCAGGCC-3′.

The amplified DNA fragment was digested with the enzymes BglII and NotI and cloned into the BamHI and NotI sites of pGEX-4T-3 (Amersham Biosciences). GST-Pex5p and GST were expressed in the XL1-Blue strain of Escherichia coli, purified by affinity chromatography using glutathione-Sepharose 4B (Amersham Biosciences), and dialyzed against SEM buffer. Digestion of proteins with Genenase I (2 units/digestion; New England BioLabs) was performed in a buffer containing 0.25 m sucrose, 20 m m Tris-HCl, pH 8.0, 1 m m EDTA-NaOH, pH 8.0, 1 m m dithiothreitol, 100 m m NaCl, 50 μg/ml phenylmethysulfonyl fluoride, and 1% (w/v) Triton X-100 for 30 min at 23 °C. Digestions were stopped with trichloroacetic acid (10% (w/v) final concentration) and the samples were subjected to SDS-PAGE. Protease protection assays were performed as described (). In Vitro Synthesized Pex5p Is Specifically Imported into Peroxisomes Using a Cell-free System Transport of PTS1-containing proteins across the peroxisomal membrane requires the concerted action of many different peroxins. Although a significant fraction of this machinery resides and functions in the peroxisomal compartment, there are several peroxins (Pex5p included) that seem to exist in a dynamic equilibrium between the cytosol and the peroxisome.

Furthermore, presently, the involvement of other subcellular compartments in the process of peroxisomal biogenesis cannot be formally excluded (reviewed in Ref. Thus, we just decided to use a PNS to set up a peroxisomal in vitro import system. To test the validity of this strategy, in vitro synthesized 35S-labeled Pex5p (the large isoform (, )) was incubated with a rat liver PNS in import buffer (see “Experimental Procedures”) containing 1 m m ATP, 1 m mATPγS or no exogenous nucleotides.

After 30 min at 26 °C, the samples were put on ice and treated with proteinase K to degrade nonimported Pex5p. The organelles were then isolated by centrifugation and analyzed by SDS-PAGE. As shown in Fig. A, in the presence of exogenous ATP, a small fraction of the 35S-labeled Pex5p used in these experiments (1–5%) resisted the protease treatment ( lane 2). Most importantly, the vast majority of this protease-resistant Pex5p population (hereafter referred to as stage 2 Pex5p; see “Discussion”) does not represent the full-length peroxin; a 2-kDa fragment was clipped from Pex5p by the action of proteinase K.

As shown before, this is exactly the proteolytic profile of Pex5p present in highly pure rat liver peroxisomes (), suggesting that the in vitro synthesized Pex5p entered the peroxisomal compartment. In the absence of exogenous nucleotides, two protease-resistant Pex5p populations were observed ( lane 1): full-length Pex5p (hereafter referred to as stage 3 Pex5p) and a 2-kDa truncated form (stage 2 Pex5p; see later).

All attempts to degrade stage 3 Pex5p, leaving stage 2 Pex5p intact, have failed (data not shown); stage 3 Pex5p can only be degraded under conditions that disrupt biological membranes (see below). The fact that 35S-labeled Pex5p acquires a protease-resistant location in the absence of exogenous nucleotides could imply that the observed import does not require ATP. Alternatively, the low levels of ATP present under these conditions (derived from both the PNS fraction and the reticulocyte lysate; see “Experimental Procedures”) may be sufficient to promote this process. The result obtained when ATPγS is used in an import assay favors this last possibility, the amount of protease-resistant Pex5p is highly decreased ( lane 3). A similar inhibition on the import of 35S-labeled Pex5p was observed in the presence of AMP-PNP or when the reaction components were pretreated with apyrase (data not shown). Figure 1 35 S-Labeled Pex5p is in vitro imported into peroxisomes.

A, PNS fractions were incubated with 35S-labeled Pex5p in import buffer containing 1 m m ATP ( lane 2), no exogenous nucleotides ( lane 1), or 1 m m ATPγS ( lane 3). At the end of the incubation, samples were treated with proteinase K to remove nonimported Pex5p and subjected to SDS-PAGE. Lane I, 5% of the 35S-labeled Pex5p used in each import reaction. The numbers at the left indicate the molecular masses of the applied standards in kDa. B, PNS fractions (1.5 mg of protein) subjected to an import reaction with 35S-labeled Pex5p either in the presence ( panels A– D) or absence ( panel E) of 1 m m ATP were subjected to Nycodenz gradient centrifugation. Samples shown in panels D and Ewere treated with proteinase K before loading onto the gradients. After centrifugation, the gradients were fractionated from the bottom ( lane 1) to the top ( lane 12) of the tubes and the distributions of 35S-labeled Pex5p ( panels C– E), rat liver Pex14p ( panel B), and several marker enzymes ( panel A) were analyzed.

♦, cytochrome c oxidase (mitochondria); ▪, esterase (endoplasmic reticulum); ▴, β-glucocerebrosidase (lysosomes); ■, catalase (peroxisomes). Lane I, reticulocyte lysate containing 35S-labeled Pex5p. To provide evidence regarding the peroxisomal location of in vitro imported Pex5p, import experiments in the presence or absence of exogenous ATP were performed.

After import, one-half of each import reaction was treated with proteinase K; the other halves received buffer only. The samples were then loaded onto the top of discontinuous Nycodenz gradients and centrifuged.

The gradients were fractionated, and the distributions of in vitro synthesized Pex5p, endogenous Pex14p, and several marker enzymes were determined. As shown in Fig. B ( panels A and B), both catalase and endogenous Pex14p display a dual behavior under these conditions: one fraction is recovered in fractions 2 and 3 and corresponds to highly pure peroxisomes; the other fraction remains at the top of the gradient and probably reflects the existence of peroxisomes of lower density () together with some disrupted organelles. When the behavior of 35S-labeled Pex5p in the samples not treated with protease is analyzed, a quite asymmetric distribution is observed (Fig. B, panel C; shown only for the sample subjected to import in the presence of exogenous ATP). Most of the radiolabeled protein remains at the top of the gradient and only a small fraction is found in fractions 2 and 3. The majority (but not all) of the 35S-labeled Pex5p staying at the top of gradient represents nonimported peroxin, as expected.

Indeed, when the behaviors of stage 2 Pex5p ( panel D) and stage 2 plus stage 3 Pex5p ( panel E) are analyzed a more symmetric distribution, paralleling the one displayed by endogenous Pex14p, is observed. Clearly, both peroxisomal populations detected in these kinds of gradients are import-competent for Pex5p. Taken together, these data indicate that in vitrosynthesized Pex5p can be specifically imported into peroxisomes. Characterization of the Pex5p Membrane Insertion Step Previously, we provided data suggesting that endogenous Pex5p from rat liver peroxisomes is a subunit of a Pex14p-containing complex (, ). On the other hand, several authors have proposed that Pex14p is the docking protein for Pex5p (). Thus, the possible involvement of Pex14p on the in vitro import of Pex5p was assessed on both functional and structural levels.

We first asked whether antibodies directed to Pex14p block in vitro import of Pex5p. For this purpose, rat liver PNS fractions were preincubated with IgGs directed to either Pex14p or PMP70 (the most abundant peroxisomal membrane protein ()) and subjected to an import reaction. The relative amounts of Pex5p in stage 2 and stage 3 in the two samples were compared. As shown in Fig. A, IgGs directed to Pex14p strongly inhibit the appearance of protease-protected Pex5p.

Virtually the same inhibition was observed when the import reaction was performed in the presence of ATP (data not shown). These experiments were also performed using Fab fragments directed to Pex14p.

Although these Fab fragments were as efficient as the precursor IgGs in recognizing Pex14p in Western blots, we were unable to see an inhibitory effect of these fragments on the in vitro import rate of Pex5p (data not shown). It is likely that the observed import inhibition with anti-Pex14p IgGs results from sterical hindrance effects and not by blocking of the Pex5p-binding site on Pex14p. The Pex5p-binding domain of Pex14p (comprising the first 78 amino acid residues of Pex14p ()) is probably not exposed into the cytosol ().

Thus, taken together, these data do not prove that Pex14p is the initial peroxisomal docking protein for Pex5p, but they do indicate that Pex14p and/or some Pex14p-bound component(s) is functionally involved in the formation of stage 2 and/or stage 3 Pex5p. Figure 2 Functional/structural involvement of Pex14p on the in vitro import of Pex5p. A, PNS fractions (150 μg of protein) were preincubated with preimmune IgGs ( lane 1), anti-PMP70 IgGs ( lane 2), or anti-Pex14p IgGs ( lane 3), and subjected to an import reaction with 35S-labeled Pex5p in the absence of exogenous nucleotides. After proteinase K treatment, the organelles were isolated by centrifugation and subjected to SDS-PAGE. Lane I, 35S-labeled Pex5p (5% of the input in each lane). B, two PNS fractions were incubated in import buffer either in the presence ( sample A) or absence ( sample B) of 35S-labeled Pex5p.

After protease treatment, 35S-labeled Pex5p (2% of the amount used in sample A) was added to sample B. After solubilization, aliquots corresponding to 600 μg of the initial PNS protein were subjected to immunoprecipitation ( IPP) using either anti-Pex14p IgGs (Pex14p, lanes A and B) or preimmune IgGs ( Preimmune; lanes A and B). Lanes Total, aliquots of samples A and B (corresponding to 100 μg of PNS protein) after the solubilization step. IPP lanes were subjected to autoradiography 6 times longer than lanes Total.

Data demonstrating a structural involvement of Pex14p on the import pathway of Pex5p was obtained as follows. 35S-Labeled Pex5p was subjected to an import reaction in the absence of exogenous ATP. After proteinase K treatment, the organelles were solubilized using a mild detergent and subjected to immunoprecipitation using the anti-Pex14p antibody. As shown in Fig.

B, both stage 2 and stage 3 Pex5p can be co-immunoprecipitated with the anti-Pex14p antibody. Control experiments (see legend to Fig. ) clearly indicate that the observed immunoprecipitation is specific and occurs only when 35S-labeled Pex5p was subjected to an import reaction. Based on these data, we conclude that both stage 2 and stage 3 Pex5p are components of a Pex14p-containing complex. To clarify this point, we tried to explore the fact that recombinant GST-Pex5p inhibits the import of in vitro synthesized Pex5p into peroxisomes (see Fig. Furthermore, in the absence of exogenous ATP, GST-Pex5p is unable to displace stage 2 and stage 3 Pex5p from the peroxisomal compartment (Fig.

In the presence of ATP a different result is obtained. After addition of the recombinant protein to the import reaction, the amount of stage 2 Pex5p decreases slowly over time (Fig. There are two possibilities to explain this result: either GST-Pex5p is displacing 35S-labeled Pex5p from the peroxisomal compartment in an ATP-dependent process; or, more likely, in vitrosynthesized Pex5p simply exits the peroxisome under these conditions (as observed for the endogenous peroxin, see below), in a process that is not directly related to the presence of GST-Pex5p but that can only be detected when import de novo is blocked. Independently of the reasons behind this event, we reasoned that the properties displayed by GST-Pex5p could still be explored to establish a relationship between stage 2 and stage 3 Pex5p.

Figure 4 GST-Pex5p blocks in vitroimport of Pex5p. A, GST-Pex5p ( lanes 2, 4, and 6) or GST ( lanes 1, 3, and 5) were added to import reactions in the presence (+ ATP) or absence of exogenous ATP (− ATP) at the indicated times. The total import time was 30 min. The import reactions were treated with proteinase K and the organelles were analyzed by SDS-PAGE.

B, 35S-labeled Pex5p was imported into peroxisomes either in the presence (+ ATP) or absence (− ATP) of exogenous ATP for 18 min. GST-Pex5p or GST were then added to the import reactions as indicated.

Aliquots of the import reactions were taken at the indicated time points, treated with proteinase K, and analyzed by SDS-PAGE. Lane I, 35S-labeled Pex5p (1% of the input). For this purpose, two in vitro pulse-chase import experiments were performed. Ds150e New Vci Keygen Generator. In the first experiment, Pex5p was imported into peroxisomes for 20 min in the presence of ATP. GST-Pex5p was then added to stop de novo import of Pex5p and, 7 min later, ATPγS (1 m m final concentration) was added to the import reaction (import of Pex5p in the presence of equimolar amounts of ATP and ATPγS results in the formation of stage 2 and stage 3 Pex5p; data not shown). Aliquots of the import reaction were removed at several time points and analyzed. As shown in Fig., stage 2 Pex5p can be transformed into stage 3 Pex5p upon addition of ATPγS.

In the second experiment, in vitro synthesized Pex5p was subjected to an import reaction in the absence of exogenous ATP. After 20 min, import of Pex5p was stopped by adding GST-Pex5p and a chase in the presence of ATP (1 m m final concentration) was done. Stage 3 Pex5p disappears rapidly from the peroxisomal compartment; the amount of stage 2 Pex5p also decreases but in a slower way.

Taken together, these experiments suggest that stage 2 Pex5p is the precursor of stage 3 Pex5p and that this population of peroxisomal Pex5p is capable of exiting the organelle in an ATP-dependent process. Figure 5 Relationship between stage 2 and stage 3 Pex5p. In vitro synthesized Pex5p was imported into peroxisomes either in the absence ( Pulse −ATP) or presence ( Pulse +ATP) of exogenous ATP for 20 min.

GST-Pex5p was then added to both import reactions and, 7 min later, an aliquot was removed ( lanes 27′). ATP ( Chase +ATP) or ATPγS ( Chase +ATPγ S) were then added to the import reactions, as indicated. Aliquots of the import reactions were taken 8 and 23 min later ( lanes 35′ and 50′, respectively).

All the samples were treated with proteinase K and analyzed by SDS-PAGE. Lane I, 35S-labeled Pex5p (1% of the input). Export of Endogenous Rat Liver Pex5p from the Peroxisome Requires ATP Hydrolysis To obtain independent evidence supporting the existence and properties of stage 2 and stage 3 Pex5p, we have repeated some of the experiments described above but this time the behavior of the endogenous (rat liver) Pex5p was monitored. PNS fractions were incubated in import buffer under several conditions (see Fig.). The samples were then treated with proteinase K, the organelles were isolated by centrifugation and analyzed by Western blotting. As shown in Fig.

( panel A), both stage 2 and stage 3 Pex5p can be detected in organelles incubated at 4 °C in the absence of exogenous ATP ( lane 1). Interestingly, the ratio of endogenous stage 2 to stage 3 Pex5p is much smaller than observed in the in vitro import experiments performed in the absence of exogenous ATP, suggesting that the majority of peroxisome-associated rat liver Pex5p is just on its way to the cytosol. Similar ratios of stage 2 to stage 3 Pex5p can be observed when PNS fractions are incubated in the presence of 1 m mATPγS ( lane 4) or in the absence of exogenous nucleotides at 26 °C ( lane 2). In sharp contrast, when a PNS fraction is incubated at 26 °C in the presence of 1 m m ATP the stage 2/stage 3 Pex5p ratio is largely increased ( lane 3), suggesting that a significant amount of stage 3 Pex5p was exported from the peroxisomal compartment. Figure 6 Export of endogenous Pex5p from the peroxisomal compartment requires ATP hydrolysis. PNS fractions (300 μg of protein) were incubated for 30 min at 0 ( lane 1) or 26 °C ( lanes 2–4) in import buffer B (see “Experimental Procedures”) containing no exogenous nucleotides ( lanes 1 and 2), 1 m m ATP ( lane 3), or 1 m m ATPγS ( lane 4). Samples were then divided into two equal aliquots.

One-half was treated with proteinase K, the organelles were isolated and subjected to Western blotting analysis using anti-Pex5p ( panel A) or anti-catalase antibodies ( panel B). The other half was centrifuged and the organelle pellets ( panel C) and the supernatants ( panel D) were analyzed by Western blotting using an anti-Pex5p antibody.

Note that in the ATP-treated sample, the decrease in organelle-associated Pex5p ( panel C, lane 3) is associated with a slight increase in the amount of soluble Pex5p ( panel D, lane 3). Membrane Topology of Stage 2 and Stage 3 Pex5p In a previous work (), it was shown that Pex5p from purified rat liver peroxisomes is quite resistant to exogenous proteases as long as the peroxisomal compartment is left intact (). When this is not the case ( e.g. If Triton X-100 is used in the protease assays) Pex5p is completely degraded by the proteases. These observations were based on Western blot analysis using a polyclonal antibody directed to Pex5p.

Because the Pex5p epitopes recognized by this antibody are not known, no conclusions regarding the extent of exposure of Pex5p to the peroxisomal matrix could be withdrawn from those experiments. In this work, the availability of radiolabeled peroxisomal Pex5p prompted us to repeat those protease assays. Thus, an in vitro import experiment in the absence of exogenous ATP was performed to produce stage 2 and stage 3 Pex5p. The organelles were isolated by centrifugation, resuspended gently, and incubated with proteinase K under several conditions. As shown in Fig., proteinase K treatment of intact organelles resulted in two bands (stage 2 and stage 3 Pex5p), as expected. However, when detergent-solubilized or sonicated organelles are subjected to the same treatment only small radiolabeled peptides can be detected.

These results strongly suggest that the bulky part of Pex5p is exposed into the peroxisomal matrix. Figure 7 The bulky portion of peroxisomal Pex5p is exposed into the lumen of the organelle. 35S-Labeled Pex5p was incubated with a PNS fraction (600 μg of protein) in import buffer in the absence of exogenous ATP for 30 min. The organelles were isolated by centrifugation and carefully resuspended in SEM buffer (see “Experimental Procedures”). The organelles were divided into four equal aliquots and proteinase K was added to three of these samples, as indicated. One of these samples received Triton X-100 ( TX-100) ( lane 3); another was sonicated immediately after addition of the protease ( lane 4).

After trichloroacetic acid precipitation, all the samples were analyzed by SDS-PAGE. Lane I, 35S-labeled Pex5p (1% of the input). The numbers at the right indicate the molecular masses of the applied standards in kDa. In an attempt to define more precisely the membrane topology of stage 2 Pex5p, we tried to determine which terminus of this protein was exposed into the cytosol. This was accomplished by using Genenase I.

This engineered protease cleaves only once to GST-Pex5p (Fig. Analysis of the cleavage products obtained with GST-Pex5p clearly indicate that Genenase I cleaves at the N terminus of Pex5p. Thus, we used this protease to map the Pex5p domain that is cleaved when intact organelles are subjected to proteinase K treatment. As shown in Fig. B, both in vitro imported stage 2 Pex5p and Pex5p from purified rat liver peroxisomes are no longer cleaved by Genenase I after proteinase K treatment.

We conclude that the N terminus (and not the C-terminal receptor domain) of stage 2 Pex5p is exposed into the cytosol. Figure 8 The N terminus of stage 2 Pex5p is exposed into the cytosol. A, GST-Pex5p (2 μg/lane) and 35S-labeled Pex5p (1 μl of reticulocyte lysate/lane) in 40 μl of Genenase I buffer (see “Experimental Procedures”) were incubated in the presence ( lanes 2) or absence ( lanes 1) of Genenase I. After trichloroacetic acid precipitation the samples were analyzed by SDS-PAGE.

The digestion of GST-Pex5p was monitored by both Coomassie Blue staining ( Coomassie) and Western blotting using an anti-GST antibody ( Anti-GST). Δ N- Pex5p, Pex5p cleaved at its N terminus; GST′, GST containing at its C terminus a few Pex5p amino acid residues. The numbers at the left indicate the molecular masses of the applied standards in kDa. B, 35S-labeled Pex5p was incubated with a PNS fraction in import buffer in the presence of ATP. After import, the organelles were isolated by centrifugation and carefully resuspended in SEM buffer.

One-half of this sample was treated with proteinase K ( lanes 3 and 4); the other half received buffer only ( lanes 1 and 2). Phenylmethylsulfonyl fluoride was then added to both samples, the organelles were reisolated by centrifugation and resuspended in Genenase I buffer.

Genenase I was added as indicated. Protease treatment of purified rat liver peroxisomes ( Purified peroxisomes) was done in the same way. After trichloroacetic acid precipitation, proteins were resolved by SDS-PAGE and blotted onto a nitrocellulose membrane. 35S-Labeled Pex5p was detected by autoradiography; peroxisomal rat liver Pex5p was detected using the anti-Pex5p antibody. DISCUSSION In this work, a characterization of Pex5p entry into and exit from the peroxisomal compartment is presented. Using a postnuclear supernatant from rat liver, specific import of in vitrosynthesized Pex5p into peroxisomes was demonstrated by three independent lines of evidence: first, in vitro imported Pex5p acquires the membrane topology of the endogenous peroxin; second, Nycodenz gradient centrifugation of PNS fractions subjected to import reactions show a typical peroxisomal distribution for the in vitro synthesized peroxin; finally, insertion of Pex5p into a protease-resistant location is strongly inhibited by anti-Pex14p IgGs.

Import of Pex5p in the absence of exogenous ATP revealed the existence of two different protease-resistant populations. The existence of these two populations was also confirmed with the endogenous (rat liver) Pex5p. The first population (stage 2 Pex5p) exposes ∼2 kDa of its N terminus into the cytosol. The second population (stage 3 Pex5p) is completely inaccessible to proteinase K. Thus, stage 3 Pex5p could represent an intraperoxisomal population of the PTS1 receptor, a hypothesis that would be in agreement with the extended version of the cycling receptor model ().

However, it should be noted that stage 3 Pex5p can be co-immunoprecipitated with Pex14p, a peroxisomal membrane protein (). Furthermore, neither stage 2 nor stage 3 Pex5p can be extracted from the peroxisomal membrane by sonication in the presence of low or high ionic strength buffers. 2 Thus, stage 3 Pex5p is still a membrane-associated protein. Finally, both stage 2 and stage 3 Pex5p can be completely degraded by proteinase K when the peroxisomal membrane is disrupted prior to protease treatment.

Taken together, these data indicate that the bulky part of these two Pex5p populations (the tetratricopeptide repeat-containing domain included) is exposed into the matrix of the peroxisome. Considering that import of Pex5p into peroxisomes is strongly inhibited by anti-Pex14p IgGs and that both stage 2 and stage 3 Pex5p can be immunoprecipitated with anti-Pex14p antibodies, our Pex5p topology results have a major implication: translocation of newly synthesized peroxisomal proteins across the peroxisomal membrane probably occurs concomitantly with the formation of the Pex5p-Pex14p complex. Thus, Pex14p is not just part of the docking site for cargo-loaded Pex5p but is, most likely, a member of the translocation machinery itself. Recent data from our and other laboratories corroborate this interpretation: using mild solubilization conditions both mammalian () and yeast Pex14p () can be isolated in a complex containing several peroxins presumably involved in the translocation step of peroxisomal proteins across the organelle membrane. Using in vitro import pulse-chase experiments we provide data suggesting that stage 2 is the precursor of stage 3 Pex5p and that stage 3 Pex5p leaves the peroxisomal compartment rapidly in the presence of ATP.

An observation compatible with these data was obtained when the behavior of endogenous rat liver Pex5p was monitored: ATP (but not ATPγS) induced the release of a major fraction of endogenous stage 3 Pex5p from peroxisomes. Thus, export of Pex5p from the peroxisomal compartment requires ATP hydrolysis. Obviously, this observation raises some questions concerning the need for ATP in Pex5p import: if all the Pex5p-docking sites present in our PNS fractions were occupied by the endogenous peroxin then import of 35S-labeled Pex5p would only be possible after peroxisomal export of at least a fraction of the endogenous Pex5p.

In the absence of experimental data addressing this possibility, our results do not allow us to infer whether or not import of Pex5p requires ATP. However, even if this proves to be the case, our data indicate that export of Pex5p from the peroxisome is much more demanding on the ATP concentration than its import into the organelle. The fact that stage 3 Pex5p exits the peroxisomal compartment in a ATP-dependent process, together with the observation that this Pex5p population can be immunoprecipitated by an anti-Pex14p antibody provide the basis for the second major conclusion of this work: Pex14p-associated Pex5p is a target for a ATP-utilizing component mediating its release from the peroxisomal compartment. We do not know the identity of this ATPase but it is obvious that Pex1p and/or Pex6p (the only known peroxins containing ATP-binding domains ()) are good candidates. These two peroxins have been implicated in the recycling of Pex5p back to the cytosol ().

The results presented here allow us to propose a hypothetical model for the Pex5p-mediated import of peroxisomal matrix proteins. Free Pex5p (defined as stage 0 Pex5p) would bind newly synthesized PTS1-containing proteins in the cytosol. This Pex5p-cargo protein complex (stage 1 Pex5p) would then diffuse to the peroxisomal membrane. We do not know whether stage 1 Pex5p enters directly to the Pex14p-containing docking-translocation complex at this stage or if it interacts first with some other peroxisomal protein at the surface of the organelle ( e.g. Pex13p ()): in this work only protease-protected Pex5p species were characterized.

Insertion of stage 1 Pex5p into the Pex14p-containing complex results in stage 2 Pex5p. The conformation/environment of stage 2 Pex5p is then changed, generating stage 3 Pex5p. We do not know at what stage cargo proteins are released from Pex5p. In principle, the conformation of the receptor domain of Pex5p has to be modified to ensure that not only PTS1-containing proteins are released but also prevented from re-binding. The stage 2/stage 3 transition could represent such a conformational modification. The reason why stage 3 Pex5p is completely proteinase K-resistant remains speculative. It is possible that the N terminus of Pex5p moves toward the matrix side of the membrane as the PTS1 receptor adopts the conformation required for its export from the peroxisomal compartment.

Alternatively, the N terminus of stage 3 Pex5p is not accessible to proteinase K as the result of a shielding effect performed by some other protein ( e.g. The ATPase itself). The fact that in purified rat liver peroxisomes only stage 2 Pex5p is detected (see Fig. B ()) together with the observation that in vitro import of Pex5p using purified organelles is not inhibited by ATPγS and results in detection of stage 2 Pex5p only, 2 could support this interpretation. Finally, stage 3 Pex5p would be pulled from the peroxisomal membrane by an ATP-utilizing component regenerating stage 0 Pex5p. It is obvious that this hypothetical model raises more questions than it can answer. However, we believe that the results and the experimental strategy presented in this work provide a good starting point for an extensive mechanistic analysis of the Pex5p-mediated protein import pathway.

Similar studies using additional anti-peroxin antibodies and mutant cell lines deficient in peroxisomal biogenesis will surely provide valuable data.

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