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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.

According to current models of peroxisomal biogenesis, the Pex5p-mediated process of protein import can be divided into four steps. In the first step, newly synthesized PTS1-containing proteins interact with Pex5p in the cytosol. This protein-protein interaction involves the PTS1 signal on one side and the tetratricopeptide repeats domain of Pex5p on the other. The Pex5p-cargo protein complex is then recognized by the so-called docking machinery present in the peroxisomal membrane. Somewhere after this event, the PTS1-containing protein is released into the peroxisomal matrix. Finally, Pex5p is recycled back to the cytosol to catalyze additional rounds of transportation (reviewed in Refs. 10 -12).
One obvious property of such cyclic mechanism is that it needs some form of energy input to function, and indeed, basi-cally all the studies addressing this issue are unanimous in this respect: protein import into the peroxisomal matrix requires hydrolysis of ATP (13)(14)(15)(16)(17)(18)(19)(20)(21). However, the precise step(s) of this import pathway where energy input is necessary has not been firmly established. For instance, it is generally accepted that the step of protein translocation across the peroxisomal membrane requires ATP hydrolysis. Such conclusion derives from the fact that ATP depletion or the inclusion of non-hydrolysable ATP analogues in the several experimental systems used result in an inhibition of the peroxisomal import process. However, the possibility that recycling of Pex5p back to the cytosol is an ATP-dependent event and the rate-limiting step in all this process was never considered. In this scenario, inhibition of peroxisomal protein import by lack of ATP would result from the non-availability of peroxisomal docking/translocation sites for the Pex5p-cargo protein complex and not from the inhibition of some ATP-dependent protein catalyzing the translocation of the cargo proteins across the organelle membrane. Furthermore, many newly synthesized proteins are bound by chaperones while still in the cytosol (or in a cell-free in vitro translation system). It is known that disruption of this kind of protein interaction requires ATP hydrolysis (reviewed in Ref. 22). Thus, the observed ATP dependence of peroxisomal import may also be related, not to the translocation step per se (as assumed) but rather to the disruption of these chaperone-protein complexes with the concomitant production of import-competent cargo proteins. In fact, several data addressing the role of chaperones in peroxisomal protein import are already available and strongly suggest that this phenomenon does occur (23)(24)(25).
In this work, we have addressed the energetics of peroxisomal protein import by analyzing the effect of ATP on the insertion of Pex5p into the peroxisomal membrane. We provide data suggesting that insertion of Pex5p into the organelle membrane does not require ATP hydrolysis. This observation together with the fact that export of Pex5p from the organelle membrane is inhibited by ATP␥S (26) imply that, at the peroxisomal membrane, ATP is needed predominantly (if not exclusively) during the recycling step of the PTS1 receptor. It is proposed that protein translocation across the peroxisomal membrane is driven by protein-protein interactions involving the peroxisomal targeting domain of Pex5p, on one side, and the peroxisomal docking/translocation machinery on the other.

Preparation of Rat Liver Subcellular Fractions for in Vitro Import
Experiments-Rat liver post-nuclear supernatants (PNS) for in vitro import experiments were prepared as described before (26).
Isolation of highly pure peroxisomes from rat liver by differential centrifugation and Nycodenz gradient purification was done according to Hartl et al. (27), with minor modifications (28). Purified organelles were resuspended in 0.25 M sucrose, 20 mM 4-morpholinepropanesulfonic acid-KOH (pH 7.4), 1 mM EDTA-NaOH (pH 7.4) (protein concentration, 20 -40 mg/ml), frozen in liquid N 2 , and stored at Ϫ70°C. It is likely that this treatment results in some leakage of the peroxisomal matrix proteins, a factor that complicates the experiments in which the cargo dependence of Pex5p import is analyzed (see Fig. 2A). However, considering that: 1) the K d for the Pex5p-PTS1 protein complex is 190 nM (29); 2) this concentration of PTS1-containing proteins in our import assay would be easily reached assuming that 10% of the catalase alone (catalase corresponds to 15% of total peroxisomal from rat liver; Ref. 30) is released into the medium during the removal of the organelles from the Nycodenz gradient and additional pipetting; and 3) others have determined that the simple procedure of washing Nycodenz-purified rat liver peroxisomes with iso-osmotic isolation medium results in 30% leakage of catalase (13), no effort was made to obtain highly intact peroxisomal preparations (if at all possible).
In Vitro Import Experiments-Import reactions were performed at 26°C for 30 min (unless indicated otherwise) in 100 l of import buffer A (0.25 M sucrose, 50 mM KCl, 20 mM 4-morpholinepropanesulfonic acid-KOH, pH 7.4, 3 mM MgCl 2 , 0.2% (w/v) lipid-free bovine serum albumin, and 20 M methionine) using 0.5 l of a reticulocyte lysate containing 35 S-labeled Pex5p and 80 g (protein) of purified peroxisomes or 150 g of rat liver PNS. In some experiments, import buffer lacking MgCl 2 was used (import buffer B). Proteinase K treatment of import reactions, SDS-PAGE, and analysis by autoradiography were performed exactly as described before (26).
In antibody inhibition experiments, 80 g of rat liver peroxisomes were preincubated with 30 g of purified immunoglobulins in 100 l of import buffer A for 20 min on ice, before starting the import reaction by adding 35  Treatment of purified peroxisomes with EDTA-NaOH, pH 7.4 (final concentration, 12 mM), was made on ice for 10 min in import buffer B. Some samples were further incubated on ice for 5 min with 1 mM 1,10-phenanthroline (added from a 100-fold stock solution in ethanol). All the other samples received the same volume of ethanol. Reticulocyte lysates were treated in the same way after diluting the samples 1:10 with import buffer B.
ATP depletion of purified peroxisomes and reticulocyte lysates was performed according to a published protocol (32). Briefly, purified peroxisomes (80 g of protein) in 90 l of import buffer A were incubated for 10 min at 30°C in the presence of 10 units of hexokinase (Sigma H6380) and 1 mM 2-deoxy-D-glucose (Sigma). Reticulocyte lysates diluted 1:20 with import buffer A were treated with the same concentration of hexokinase but with 10 mM 2-deoxy-D-glucose. Import reactions were started by mixing the two components.
Miscellaneous-The synthesis of 35 S-labeled Pex5p (26) and antibodies directed to human Pex14p (33) and Pex5p (34) were described before. The antibody directed to rat PMP70 was purchased from Zymed Laboratories, Inc. The anti-catalase antibody is from Research Diagnostics, Inc. Immunoglobulins were purified from rabbit sera using Protein-A-Sepharose beads, according to the manufacturer's protocol (Amersham Biosciences). Rabbit antibodies were detected on Western blots using alkaline phosphatase-conjugated anti-rabbit antibodies (Sigma). Densitometric analysis of Western blots was performed using the UN-SCAN-IT automated digitizing system.

Import of Pex5p Is Influenced by the Levels of Endogenous
Peroxisomal Pex5p-Recently, we described a peroxisomal in vitro import system particularly suited to study Pex5p association with and release from the peroxisomal compartment (26). The system consists in incubating a PNS from rat liver with in vitro synthesized 35 S-labeled Pex5p. Import of Pex5p is monitored by protease treatment of import reactions; non-imported Pex5p is completely degraded, whereas the imported protein acquires resistance to proteinase K. By using this assay, two different populations of peroxisomal Pex5p were identified and characterized: the so-called stage 2 Pex5p, a population of the receptor exposing ϳ2 kDa of its N terminus into the cytosol; and stage 3 Pex5p, a membrane-associated molecule completely resistant to proteinase K. Data suggesting that stage 2 is the precursor of stage 3 Pex5p and that this population leaves the peroxisomal compartment in an ATP-dependent process were also provided (26).
We have also shown that insertion of 35 S-labeled Pex5p into the peroxisomal membrane is strongly inhibited in the presence of ATP␥S (a non-hydrolysable ATP analogue) or by enzymatic ATP depletion of the import reactions (26). This observation could suggest that the peroxisomal import of Pex5p requires ATP. However, as emphasized previously (26), the possibility that the majority of the docking/translocation sites of the peroxisomal membrane were already occupied by endogenous rat liver Pex5p could not be excluded. The fact that peroxisomal Pex5p is exported from the organelle in an ATPdependent process (26) precluded any conclusion on the role of ATP (if any) on the Pex5p import step.
As an attempt to determine whether the rate of import of 35 S-labeled Pex5p is influenced by peroxisomal export of endogenous Pex5p, we tried to separate in time the two processes. For this purpose, a PNS fraction was incubated in the presence of 1 mM ATP at 26°C for 15 min, conditions that promote peroxisomal export of endogenous Pex5p (see below; Ref. 26). The import reaction was then supplemented with 10 mM ATP␥S, and 2 min later, 35 S-labeled Pex5p was added. Two control import reactions were performed in parallel. In one reaction, a PNS fraction was first incubated with 1 mM ATP␥S for 15 min (endogenous Pex5p is not exported from the peroxisome under these conditions), and only then a mixture of ATP and ATP␥S was added. The final concentrations of ATP and ATP␥S in this reaction were 1 and 10 mM, respectively, as above. Import was initiated 2 min later by adding 35 S-labeled Pex5p. In the second control reaction, ATP alone was used both in the pre-incubation step (1 mM) and in the import reaction (final concentration, 11 mM). The rates of peroxisomal import of 35 S-labeled Pex5p observed under these three conditions were then compared by analyzing protease-treated aliquots of the import reactions removed 5 and 15 min after adding the 35 Slabeled protein. As shown in Fig. 1A, when ATP is the only exogenous nucleotide present, import of 35 S-labeled Pex5p can be easily detected 5 min after starting the import reaction (upper panel, lane 7). Under these conditions, stage 2 Pex5p is the most prominent peroxisomal population detected, as described before (26). In the sample where the PNS fraction was preincubated with ATP␥S and afterward subjected to an import reaction in the presence of 1 mM ATP and 10 mM ATP␥S, no import of Pex5p could be detected during the complete time of the incubation (lanes 6 and 9). In sharp contrast, under the same concentrations of ATP and ATP␥S, a robust amount of protease-protected Pex5p (corresponding to stage 2 and stage 3 Pex5p) is observed in the sample preincubated with ATP ( lanes  5 and 8).
The behavior of the endogenous peroxisomal pool of Pex5p was also monitored in this experiment. The amounts of protease-protected Pex5p as well as the ratios of stage 2 to stage 3 Pex5p obtained under different incubation conditions (Fig. 1A, middle panel, lanes 2-10) were compared with those present in a PNS fraction that was subjected to no incubation (lane 1). Treatment of a PNS fraction with 1 mM ATP␥S for 15 min followed by a 17-min incubation in the presence of 1 mM ATP plus 10 mM ATP␥S does not change significantly the total amount of peroxisomal protease-protected Pex5p (compare lanes 3, 6, and 9 with lane 1 in Fig. 1A, middle panel; see also there was no radiolabeled stage 2 Pex5p when ATP␥S was added to the reaction mixture). Thus, 35 S-labeled Pex5p has first to be imported into peroxisomes originating in stage 2 Pex5p, and only then can stage 3 Pex5p be formed. Considering that this stage 2 to stage 3 transition requires some minutes to occur (see Fig. 3 in Ref. 26), a low stage 3:stage 2 Pex5p ratio would be expected in this case. Second, in contrast to the results obtained with 35 S-labeled Pex5p, there is a small amount of protease-resistant endogenous Pex5p resembling stage 3 Pex5p that remains detectable in PNS fractions incubated with ATP (Fig. 1A, middle panel, lanes 4, 7, and 10). We think that this population represents inactive Pex5p (i.e. Pex5p present in docking/translocation sites that are no longer active in the export of the peroxin) and/or inaccessible Pex5p (i.e. Pex5p entrapped into membrane vesicles of non-peroxisomal origin). When monitoring the behavior of in vitro synthesized Pex5p, neither insertion into inactive, already occupied, docking/translocation sites nor entrapment into membrane vesicles should occur, explaining why the results obtained with 35 Slabeled Pex5p are always much more clear than the ones obtained with the endogenous peroxin. Although the existence of this ATP-unresponsive Pex5p population inflates the stage 3:stage 2 Pex5p ratios calculated for the endogenous peroxin, it does not change the conclusions of this experiment.
In summary, these results show that in this in vitro system peroxisomal import of Pex5p is highly dependent on the process of export of endogenous Pex5p; efficient import of 35 S-labeled Pex5p can be obtained in the presence of 1 mM ATP plus 10 mM ATP␥S but only when peroxisomal docking/translocation sites were freed previously from endogenous Pex5p by preincubating PNS fractions in the presence of ATP. Thus, the observation that import of Pex5p is inhibited by ATP␥S when this nucleotide is added at time zero of import cannot be taken as evidence to suggest that import of Pex5p requires ATP hydrolysis. In fact, the finding that the amount of imported 35 S-labeled Pex5p obtained in an import reaction containing 1 mM ATP and 10 mM ATP␥S is not smaller than the one obtained in the presence of 11 mM ATP suggests the opposite.
Import of Pex5p into Purified Peroxisomes Is Cargo Proteinand Pex14p-dependent-The best approach to determine whether a given process requires ATP or not is, of course, to quantify its efficiency both in the presence and in the absence of ATP. In the present situation, this is not an easy task because of the fact that export of endogenous peroxisomal Pex5p (an ATP-dependent process) influences the rate of import of Pex5p. It could be considered that one strategy to address this problem could consist of incubating PNS fractions in the presence of ATP (to promote export of endogenous Pex5p) and afterward to degrade this ATP enzymatically. Import of 35 S-labeled Pex5p would then be quantified in the presence and absence of ATP. However, some incorrect assumptions are made in this rationale, the major one being that the initial ATP treatment would result in export of endogenous peroxisomal Pex5p with 100% efficiency. As shown above, this is clearly not the case. Thus, some stimulation of the peroxisomal import rate of Pex5p by ATP would always be obtained in this kind of experiment, regardless of the need for ATP in this process. Clearly, the energetics of Pex5p import should be addressed in a system where the behavior of endogenous pool of the PTS1 receptor is not a variable.
As described recently (26), protease-protection assays using PNS fractions from rat liver revealed the existence of two peroxisomal membrane populations of Pex5p, stage 2 and stage 3 Pex5p. When purified rat liver peroxisomes were subjected to a protease-protection assay and analyzed by Western blotting using a Pex5p antibody, a different result was obtained; only stage 2 Pex5p could be detected (26,34). The absence of stage 3 Pex5p in these peroxisomal preparations led us to hypothesize that export of Pex5p from the membrane of isolated organelles is, for some reason, blocked, which prompted us to test this material in in vitro import experiments. As described below, purified peroxisomes from rat liver do import 35 S-labeled Pex5p when subjected to an in vitro import reaction. However, as expected, the import efficiency of these organelles is quite low when compared with the efficiency of a PNS fraction. Indeed, we need to use in these import reactions 80 g of purified peroxisomes to detect the amount of imported 35 Slabeled Pex5p obtained with 150 g of a PNS fraction. Considering that peroxisomal proteins correspond to 2% of total rat liver protein (27), this observation implies that, upon isolation, the import efficiency of rat liver peroxisomes decreases at least 26-fold. In fact, this factor is probably higher because import of the in vitro synthesized Pex5p is not competed by endogenous (cytosolic) Pex5p when purified organelles are used in these assays. Nevertheless, as shown below, this residual peroxisomal import of Pex5p presents two important properties: 1) it is cargo protein-dependent; and 2) it requires available Pex14p on the peroxisomal membrane.
As shown before, insertion of Pex5p into the peroxisomal membrane requires the presence of PTS1-containing proteins in the import medium (31). This is not a problem when using PNS fractions in in vitro import experiments because the amount of peroxisomal PTS1-containing proteins released into the soluble phase during the homogenization procedure of rat liver is sufficient to support an efficient import of Pex5p. A similar phenomenon is observed when purified peroxisomes are used in in vitro import experiments (see "Experimental Procedures"); insertion of Pex5p into the peroxisomal membrane can be obtained easily (Fig. 2, lane 1), although no exogenous PTS1containing proteins were added to the import reaction. However, an almost complete inhibition on the import of Pex5p is observed when the import reaction is performed in the presence of a GST-fusion protein comprising amino acid residues 312-639 of Pex5p (Fig. 2, lane 5). This portion of the PTS1 receptor contains the complete PTS1-binding domain of Pex5p (and thus, should sequester PTS1-containing proteins present in the import medium) but lacks its peroxisomal targeting information (35,36). A partial reversion of this inhibitory action can be obtained by including a PTS1-containing protein in the import reaction (Fig. 2, lane 4). Thus, under these experimental conditions, insertion of Pex5p into the peroxisomal membrane is cargo protein-dependent.
Pex14p is one of the components of the docking/translocation complex (reviewed in Refs. 10 -12), and antibodies directed to this peroxin inhibit peroxisomal import of Pex5p (26). When purified peroxisomes are incubated with anti-Pex14p antibodies and then subjected to an import reaction, a strong inhibition on the import process of Pex5p is observed (see Fig. 2B).
Purified Peroxisomes Do Not Export Pex5p-The data presented above strongly suggest that, for the initial steps in the Pex5p-mediated import pathway, purified rat liver peroxisomes display the same qualitative properties of a PNS fraction in our in vitro import assay. However, the similarities between the two systems end here. Indeed, purified peroxisomes do not support the stage 2 to stage 3 Pex5p transition. Furthermore, no in vitro imported Pex5p can be exported back to the soluble fraction of the import reaction. These conclusions derive from in vitro pulse-chase experiments in which purified peroxisomes are subjected to a three-step import reaction. In the first step, 35 S-labeled Pex5p is imported into peroxisomes for a short period of time ("pulse"); in the second step, further import of the PTS1 receptor is blocked by adding a vast excess of recombinant GST-Pex5p to the reaction tube; finally, the behavior of the imported 35 S-labeled Pex5p as a function of time is monitored ("chase"). As shown in Fig. 3, when 35 S-labeled Pex5p is imported into purified peroxisomes in the presence of ATP and then subjected to a chase incubation in the presence of the same nucleotide, no decrease in the amount of stage 2 Pex5p can be detected. This result is in contrast with our previous observations showing that when a PNS fraction is used in this type of experiment, in vitro imported 35 S-labeled Pex5p is exported from the peroxisomal compartment in an ATP-dependent process (26). It is also noteworthy that, in the absence of exogenous ATP, no stage 3 Pex5p can be detected in these experiments, even when ATP␥S is added to an import reaction just before the chase incubation. Clearly, purified peroxisomes are not able to promote export of Pex5p, suggesting that some component necessary for the export of Pex5p was either lost or inactivated during the purification procedure of the organelles. At present, the second possibility seems more likely because various attempts to reconstitute the export activity of these  5) and GST-SKL (lanes 2 and 4) or GST-LKS (lanes 3 and 5) for 30 min at 26°C in import buffer A containing 10 mM ATP. B, rat liver peroxisomes were preincubated in import buffer A in the absence (lane Ϫ) or presence of anti-PMP70, anti-Pex14p, or pre-immune immunoglobulins. Import reactions (containing 10 mM ATP) were started by adding 35 S-labeled Pex5p. Lane I, 35 S-labeled Pex5p reticulocyte lysate (5% of input in each lane).
FIG. 3. Purified peroxisomes do not export Pex5p. 35 S-labeled Pex5p was incubated with rat liver-purified peroxisomes in import buffer A containing 10 mM ATP (Pulse ϩ ATP) or no exogenous ATP (Pulse Ϫ ATP). After 20 min at 26°C, GST-Pex5p was added to the import reactions to block further import. An aliquot was removed 7 min later (lanes 27Ј), and 10 mM ATP (Chase ϩ ATP) or 10 mM ATP␥S (Chase ϩ ATP␥S) were added to two of the import reactions. Eight and 23 min later, aliquots of the import reactions were removed (lanes 35Ј and 50Ј, respectively). All the samples were digested with proteinase K, subjected to SDS-PAGE, and analyzed by autoradiography (upper panel) and Western blotting using the anti-Pex5p antibody (lower panel). Lane I, 35 S-labeled Pex5p reticulocyte lysate (5% of input in each lane).
organelles by supplementing the import reactions with cytosolic fractions have failed (data not shown).
Insertion of Pex5p into the Membrane of Purified Organelles Does Not Require Hydrolysis of ATP-Having established that purified peroxisomes are not functional in exporting Pex5p from the organelle membrane but are still able to import the PTS1 receptor, we addressed the energetics of this process. For this purpose, purified rat liver peroxisomes and the Pex5pcontaining reticulocyte lysate were separately subjected to a standard ATP depletion protocol (see "Experimental Procedures"). Three import reactions were performed using these ATP-depleted components. One reaction received no further additions; the two other reactions received either ATP or ATP␥S to a final concentration of 10 mM each. As shown in Fig.  4A, no differences on the peroxisomal import of 35 S-labeled Pex5p can be detected under these experimental conditions.
Most ATP-utilizing enzymes use Mg 2ϩ ⅐ATP as their true substrate. Thus, the requirement for Mg 2ϩ on the in vitro import of Pex5p was also assessed. Removal of this ion from the import buffer or even the inclusion of the chelating agents EDTA and 1,10-phenanthroline in the import reaction have no effect on the peroxisomal import of 35 S-labeled Pex5p (see Fig. 4B).
These results strongly suggest that insertion of Pex5p into the peroxisomal membrane does not require ATP hydrolysis. DISCUSSION The energetics of peroxisomal protein import has attracted much attention from the very beginning of this research field. Different experimental systems have been used by several groups to address this issue. The unanimous conclusion that emerged from these studies is that energy provided by ATP hydrolysis is necessary for peroxisomal protein import into the organelle matrix (13)(14)(15)(16)(17)(18)(19)(20)(21). However, the exact steps in the Pex5p-mediated import pathway where energy input is necessary have remained unclear.
Here, we show that insertion of Pex5p into the peroxisomal membrane does not require ATP hydrolysis. This observation, together with previous data indicating that peroxisomal export of the PTS1 receptor is abolished by ATP␥S (Ref. 26; see also Fig. 1) strongly suggest that, at the peroxisomal membrane level, ATP is predominantly (if not exclusively) used to reset the Pex5p-mediated transport system, i.e. to recycle Pex5p back into the cytosol. This conclusion does not exclude the need for ATP at other steps of the peroxisomal protein import mech-anism. In fact, if we consider that many peroxisomal proteins (if not all) are imported into the organelle in their folded state (37)(38)(39)(40)(41)(42)(43) and that protein folding is a chaperone-assisted process that requires ATP hydrolysis (reviewed in Ref. 22), then ATP is certainly used upstream of the translocation step. It should be emphasized that in the experimental system used here, import of Pex5p into the peroxisomal membrane occurs in the presence of already folded PTS1-containing proteins that have leaked from the organelles during the preparation of the rat liver fractions used in these assays. Thus, no conclusions regarding this segment of the Pex5p-mediated import pathway can be withdrawn from these experiments.
Steady-state level analysis of peroxisomal Pex5p present in several mutant cell lines deficient in PTS1 protein import led to the idea that Pex1p and Pex6p (the only two known peroxins having ATP-binding domains) could be involved in the export of Pex5p back to the cytosol (44). Recently, however, these data were reinterpreted; it was proposed that Pex1p and Pex6p act upstream of the translocation step and/or in the vectorial transport of proteins across the peroxisomal membrane and/or in the assembly of the docking/translocation machinery (45). Although determining the identity of the ATPase(s) involved in the export of Pex5p from the peroxisomal compartment was beyond the scope of this work, our data strongly argue against a role of Pex1p and Pex6p in any of these processes. Thus, if Pex1p and Pex6p are indeed mechanistically involved in the process of protein translocation across the peroxisomal membrane, then the hypothesis that these peroxins are involved in the export of peroxisomal Pex5p seems more likely.
Our proposal that insertion of Pex5p into the peroxisomal membrane does not require hydrolysis of ATP raises an important question regarding the energetics of protein translocation across the peroxisomal membrane. If no ATP is involved in this step and if a membrane potential is irrelevant for this process (13,17), 2 then what form of energy is used to transport proteins against a concentration gradient? Perhaps the answer to this question resides on the protein-protein interactions that the PTS1 receptor establishes with the docking/translocation machinery of the peroxisomal membrane. Although our knowledge on the architecture of this protein assembly is still limited, data indicating the presence of Pex12p, Pex13p, and Pex14p on the docking/translocation complex are now irrefutable (33,46,47). All of these membrane peroxins are capable of binding directly to the PTS1 receptor (36, 48 -52), and in principle, the driving force for protein translocation across the peroxisomal membrane could reside in any of these interactions. Presently, not much is known regarding the energetics of the Pex5p interaction with Pex12p or Pex13p. The Pex14p-Pex5p interaction, however, is well documented, and several properties of this interaction may be pertinent here. First, the N-terminal half of mammalian Pex5p possesses seven binding sites for Pex14p; each of these sites binds Pex14p with high affinity (K d values in the low nanomolar ratio) (53,54). Second, there is good evidence to suggest that, in vivo at least, some of these binding sites are occupied by Pex14p (34). Finally, Pex14p interacts with itself via the C-terminal coiled-coil domain (55,56). Thus, in vivo, the Pex14p-Pex5p interaction is most likely mutually multivalent. The energy involved in the Pex5p-Pex14p complex formation is not known at the moment. Nevertheless, on a simple thermodynamic basis the energy released during the interaction of just one Pex14p molecule with Pex5p would be more than sufficient to move one protein from the cytosol to the peroxisomal matrix against a high (e.g. 1:10,000) concentration FIG. 4. Insertion of Pex5p into purified peroxisomes does not require ATP hydrolysis. A, ATP-depleted purified peroxisomes and 35 S-labeled Pex5p reticulocyte lysate were subjected to import reactions in import buffer A containing 10 mM ATP (lane ATP), 10 mM ATP␥S (lane ATP␥S), or no exogenous nucleotides (laneϪ). A standard import reaction (in import buffer A containing 10 mM ATP) was also performed as a control (lane C). B, rat liver-purified peroxisomes were subjected to import reactions in import buffer B (lane 1), import buffer B containing 12 mM EDTA (lane 2), or 12 mM EDTA plus 1 mM 1,10-phenanthroline (lane 3) or import buffer A (lane 4). Lane I, 35 S-labeled Pex5p reticulocyte lysate (5% of input in each lane).
gradient. Although definite proof supporting such hypothesis will probably require membrane reconstitution experiments using purified peroxins, such an ATP-independent mechanism of protein translocation across a biological membrane would not be novel. Indeed, the translocation step of many receptorcargo protein complexes across the nuclear pore has been found to be energy-independent (reviewed in Ref. 57).
The data presented here, suggesting that no ATP is required for protein translocation across the peroxisomal membrane, may appear in contradiction with the observation that Pex5p (58) and newly synthesized PTS1-containing proteins (59) accumulate in vivo at the peroxisomal surface under energylimiting conditions. However, all of these observations can be easily conciliated by assuming that each docking/translocation complex has at least two binding sites for Pex5p (e.g. one for docking and one for the translocation itself). Binding of Pex5p to each of these two sites would be, according to our data, ATP-independent. However, in the absence of energy the number of available translocation sites would decrease rapidly (export of Pex5p is blocked) leading to an accumulation of Pex5pcargo protein complexes at the docking sites. Defining the protein domain(s) involved in the peroxisomal docking step of the Pex5p-cargo protein complex is currently one of our aims.