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CELL BIOLOGY AND SIGNAL TRANSDUCTION

Import of the Peroxisomal Targeting Signal Type 2 Protein 3-Ketoacyl-Coenzyme A Thiolase into Glyoxysomes¹

Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Most peroxisomal matrix proteins possess a carboxy-terminal tripeptide targeting signal, termed peroxisomal targeting signal type 1 (PTS1), and follow a relatively well-characterized pathway of import into the organelle. The peroxisomal targeting signal type 2 (PTS2) pathway of peroxisomal matrix protein import is less well understood. In this study, we investigated the mechanisms of PTS2 protein binding and import using an optimized in vitro assay to reconstitute the transport events. The import of the PTS2 protein thiolase differed from PTS1 protein import in several ways. Thiolase import was slower than typical PTS1 protein import. Competition experiments with both PTS1 and PTS2 proteins revealed that PTS2 protein import was inhibited by addition of excess PTS2 protein, but it was enhanced by the addition of PTS1 proteins. Mature thiolase alone, lacking the PTS2 signal, was not imported into peroxisomes, confirming that the PTS2 signal is necessary for thiolase import. In competition experiments, mature thiolase did not affect the import of a PTS1 protein, but it did decrease the amount of radiolabeled full-length thiolase that was imported. This is consistent with a mechanism by which the mature protein competes with the full-length thiolase during assembly of an import complex at the surface of the membrane. Finally, the addition of zinc to PTS2 protein imports increased the level of thiolase bound and imported into the organelles.

Most peroxisomal matrix proteins use a carboxy-terminal tripeptide targeting signal, termed a peroxisomal targeting signal type 1 (PTS1; Gould et al., 1989). In contrast, thiolase is directed to the organelle by a distinct signal, the peroxisomal targeting signal type 2 (PTS2; Gietl, 1990; Osumi et al., 1991; Swinkels et al., 1991; Kato et al., 1996). The PTS2 is nonapeptide signal with the consensus sequence (R) X₆(H/Q)(L/A/F); it is located near the amino terminus of the peroxisomal protein (Flynn et al., 1998; Olsen, 1998). In addition to thiolase, other plant proteins that contain a PTS2 include citrate synthase (Kato et al., 1996), malate dehydrogenase (Gietl et al., 1994), amine oxidase (Faber et al., 1995), and at least one isozyme of Asp aminotransferase (Gebhardt et al., 1998). Plants appear to have more PTS2 proteins than other organisms.

Relatively few proteins follow the PTS2 pathway, but their import is necessary for the survival of the organism. Peroxisomal -oxidation is essential for seedling growth in Arabidopsis (Germain et al., 2001). In humans, the disease rhizomelic chondrodysplasia punctata is caused by the inability to import PTS2 proteins (Wiemer and Subramani, 1994); the import of PTS1 proteins into peroxisomes is normal in individuals afflicted with this disease. This observation and the discovery of classes of yeast mutants specifically defective in the import of PTS2 proteins (Erdmann et al., 1997), but not PTS1 proteins, provide strong evidence of separate import pathways for the two types of peroxisomal matrix proteins.

PTS1 and PTS2 peroxisomal matrix proteins are recognized in the cytosol and transported to peroxisomes by soluble receptor proteins (Olsen, 1998). It is now widely accepted that peroxin (Pex) 5p is the cytosolic receptor for PTS1 proteins and Pex7p is the PTS2 receptor. It has recently been shown that human Pex5p transports a cargo PTS1 protein to the peroxisomal membrane, where it appears to enter, at least partially, the matrix of the organelle along with the PTS1 protein, and then it is recycled back to the cytosol after releasing the cargo protein in the peroxisome matrix (Dammai and Subramani, 2001; Kunau, 2001). The use of in vitro systems to examine the specifics of PTS1 import has revealed additional mechanistic information (Horng et al., 1995; Brickner et al., 1997; Brickner and Olsen, 1998; Crookes and Olsen, 1998; Fransen et al., 1998; Pool et al., 1998). This approach was used to demonstrate the time, temperature, and energy dependence of PTS1 protein import (Behari and Baker, 1993; Brickner et al., 1997; Brickner and Olsen, 1998), as well as the role of cytosolic chaperones and membrane-associated proteins in the pathway (Crookes and Olsen, 1998; Fransen et al., 1998). Despite the number of reports using in vitro assays to study PTS1 protein import, there is only one account of reconstitution of PTS2 protein import using purified peroxisomes (Miura et al., 1994). Thus, the mechanistic details of the PTS2 pathway remain largely uncharacterized.

Using semipermeabilized mammalian cells, thiolase import was recently shown to be temperature and time dependent and to require energy and cytosol (Legakis and Terlecky, 2001). PTS2 protein import in mammalian cells requires the presence of the soluble receptors Pex5p and Pex7p, as well as the membrane protein Pex14p and the chaperones Hsp70 and Hsp40. Most of these requirements have not yet been demonstrated in other organisms, including plants.

In this study, we investigated the mechanisms of PTS2 protein binding and import into glyoxysomes using an optimized in vitro assay to reconstitute the transport events. This assay permits independent manipulation of each of the biochemical parameters of the import reaction. First, we characterized PTS2 protein import using time courses of import and competition experiments with both PTS1 and PTS2 proteins. Next, we examined the effect of a PTS2 protein lacking its targeting signal on the import of full-length PTS1 and PTS2 proteins. Finally, we examined the role of zinc in the binding and import of PTS2 proteins.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Import of Thiolase into Isolated Pumpkin (Cucurbita pepo) Glyoxysomes

To study the PTS2 pathway of protein import, we performed in vitro peroxisomal protein import assays using glyoxysomes isolated from pumpkin cotyledons (Brickner et al., 1997). Protease resistance defined proteins imported into glyoxysomes; proteins protected by the organelle membrane were not degraded. In each import experiment, all protease-protected protein was considered as imported; there was no distinction made between precursor and processed forms of imported thiolase, because processing of the protein could occur at any time after import. In vitro studies with rat peroxisomal thiolase have provided evidence that processing of the thiolase precursor is not necessarily directly coupled with import (Miura et al., 1994).

Additional Pex7p (the PTS2 protein receptor) was not routinely added to standard in vitro import assays because the addition of in vitro synthesized Pex7p did not significantly increase the import of thiolase (T. Johnson and L. Olsen, unpublished data). This is most likely because sufficient Pex7p is included in the purified glyoxysome preparations, either loosely associated with the outside of the peroxisome or present inside the organelle (Dodt and Gould, 1996), such that it is not a limiting factor for the in vitro import reactions. It is also possible that a small pool of soluble receptors is supplied by the cell-free extract in which the radiolabeled matrix proteins were synthesized (rabbit reticulocyte lysate or wheat germ lysate).

The sequence of thiolase from Arabidopsis (GenBank Accession no. T20943) is shown in Figure 1. Arabidopsis thiolase is 96% identical and 98% similar to canola (Brassica napus) thiolase (Olesen and Brandt, 1996). To determine whether the putative PTS2 signal is functional, we constructed a plasmid encoding the predicted mature form of 3-ketoacyl-CoA thiolase (pMTL). The Leu indicated in Figure 1 is predicted, by analogy to mammalian thiolase, to be the first residue of the mature plant thiolase, assuming that cleavage occurred in vivo at the position between the carboxy-terminal Cys of the presequence and the amino-terminal Leu of the mature plant thiolase (Gietl et al., 1997). This truncation removes the PTS2 signal and a Ser-rich region that is found in many PTS2 plant proteins. It is important to note that the predicted mature form of canola thiolase has been shown to be enzymatically active (Olesen et al., 1997). For technical reasons, the mature form of thiolase engineered for the experiments presented here has Met as the first residue, rather than Leu.

View larger version (56K): [in this window] [in a new window]   Figure 1. Alignment of 3-ketoacyl-CoA thiolase from Arabidopsis and canola. The full-length amino acid sequences of 3-ketoacyl-CoA thiolase from Arabidopsis (At; accession no. T20943) and canola (Bn; accession no. CAA63598) are shown. These two proteins differ in only 17/462 residues. The PTS2 import signal is underlined. The Ser-rich region found in many PTS2 proteins is indicated in bold. The Leu in the full-length protein at the predicted cleavage site of the PTS2 processing protease is indicated by a black box; this Leu was changed to a Met as the first residue of the engineered mature thiolase protein.

The results of typical import experiments are shown in Figure 2. The pattern of thiolase import into peroxisomes was similar to that of the control peroxisomal protein glycolate oxidase (GLO), a PTS1 protein whose import has been studied extensively (Brickner et al., 1997; Brickner and Olsen, 1998). The proteins detected in samples shown in lane 1 represent all protein bound to the membrane or imported into the organelle. Protease was added to samples shown in lane 2 to remove bound proteins (not protected by the peroxisome membrane), leaving only imported proteins. The import reaction was also performed at 4°C to demonstrate that thiolase import is temperature dependent. In the absence of ATP, neither thiolase nor GLO was imported (data not shown), as expected (Brickner et al., 1997). Import reactions containing purified glyoxysomes, radiolabeled mature thiolase translation products, and ATP were incubated at room temperature (25°C). Mature thiolase, which lacks the targeting signal, was not imported into glyoxysomes (Fig. 2), confirming the necessity of the PTS2 for thiolase import.

View larger version (45K): [in this window] [in a new window]   Figure 2. In vitro import of GLO, full-length thiolase (THL), and mature thiolase (mTHL) into isolated pumpkin glyoxysomes. Isolated glyoxysomes were incubated with radiolabeled GLO (top) for 30 min, full-length thiolase (middle) for 60 min, or mature thiolase (bottom) for 60 min under standard import conditions (see "Materials and Methods") at either 25°C (lanes 1-3) or 4°C (lane 4). After import, thermolysin was added to samples in lanes 2 to 4. The detergent Triton X-100 was added to samples in lane 3 to lyse the organelles. All subsequent treatments were as described in "Materials and Methods." The results shown are from representative experiments that were repeated three times for GLO and mature thiolase and four times for full-length thiolase. TR, Translation product.

Although the PTS2 protein appeared to import similarly to PTS1 proteins, one difference was observed. In standard time-course experiments, the amount of thiolase imported increased over time to a maximum level after about 180 min. Interestingly, this is slower than the import time course observed for the PTS1 protein GLO (Fig. 3; see also Brickner et al., 1997). The amount of GLO imported into glyoxysomes also increased over time, but it reached a maximum import level much sooner than thiolase. Eventually, the levels of import of thiolase and GLO appeared to reach the same maximum, i.e. 10% of the amount of protein presented to the glyoxysomes (quantified by phosphor-imaging analysis; see "Materials and Methods"), although thiolase reached this level later than GLO.

View larger version (13K): [in this window] [in a new window]   Figure 3. Import of thiolase (THL) into glyoxysomes is slower than import of GLO. Standard in vitro import reactions were performed for 0 to 180 min for thiolase (solid line) and for 0 to 120 min for GLO (dotted line), as described in "Materials and Methods." At the indicated time points, samples were placed on ice to stop the import reactions and treated with thermolysin to remove nonimported proteins. The level of GLO import at 40 min and the amount of thiolase imported at 180 min were set at 100% relative import for comparison with the other time points from the same experiment. Each protein reached a maximum import level of approximately 10% of the radiolabeled protein presented to the organelles. The experiment was repeated six times with thiolase and four times with GLO; the results are presented as the average ± SE.

PTS1 protein import is saturable, and PTS1 proteins compete for import (Brickner et al., 1997). To determine whether the PTS2 pathway was also saturable and competitive, in vitro import reactions were performed with radiolabeled thiolase in the presence of increasing amounts of unlabeled thiolase (Fig. 4A). An excess of unlabeled thiolase should saturate receptor and membrane docking sites, thus competitively blocking the import of radiolabeled PTS2 proteins into glyoxysomes. As expected, incubating glyoxysomes with increasing levels of nonradioactive thiolase in the presence of a constant amount of radiolabeled protein decreased the amount of protease-resistant (imported) radiolabeled thiolase observed (Fig. 4A). In contrast, when lysate alone (without radiolabeled protein) was used as a nonspecific protein control, there was no effect on the import of thiolase (data not shown). Interestingly, the PTS1 protein GLO did not inhibit import of thiolase (Fig. 4B). In fact, addition of GLO to import reactions actually increased the amount of thiolase imported into the peroxisomes. This stimulation of PTS2 import was also observed in experiments in which either of two additional PTS1 proteins, isocitrate lyase or Ala:glyoxylate aminotransferase (data not shown), was present. Thus, PTS2 protein import was stimulated by all three of the PTS1 proteins tested.

View larger version (13K): [in this window] [in a new window]   Figure 4. Import of thiolase is decreased by excess thiolase but not by the PTS1 protein GLO. Isolated glyoxysomes were incubated with increasing amounts of non-radiolabeled full-length thiolase (A) or GLO (B) in the presence of a constant amount of radiolabeled protein. The amount of trichloroacetic acid-precipitable counts obtained from a standard translation reaction was used to calculate the equivalent amount of non-radiolabeled protein for import reactions. For example, twice as much non-radiolabeled as radiolabeled protein is "2 equivalents," and so on. The amount of thiolase imported in the absence of non-radiolabeled protein was set at 100% for comparison with the other data points from this experiment. The experiments were repeated three times; the results are presented as the average ± SE.

In vitro competition reactions were also performed with radiolabeled full-length thiolase in the presence of increasing amounts of mature thiolase, which lacks the PTS2. Incubating glyoxysomes with increasing levels of mature thiolase in the presence of a constant amount of full-length thiolase decreased the amount of protease-resistant (imported) full-length thiolase observed (Fig. 5A). Conversely, when mature thiolase was radiolabeled and incubated in the presence of non-labeled full-length thiolase, protease-protected mature thiolase could be detected (data not shown), suggesting that mature thiolase "piggy-backed" onto full-length thiolase and used its PTS2 signal to achieve import. Notably, the addition of mature thiolase had no effect on the import of the PTS1 protein GLO (Fig. 5B).

View larger version (16K): [in this window] [in a new window]   Figure 5. Import of full-length thiolase, but not GLO, is inhibited by mature thiolase. A, Isolated glyoxysomes were incubated with increasing amounts of radiolabeled mature thiolase in the presence of a constant amount of radiolabeled full-length thiolase protein. The amount of protein imported in the absence of radiolabeled mature thiolase was set at 100% for comparison with the other data points from this experiment. B, Isolated glyoxysomes were incubated with increasing amounts of radiolabeled mature thiolase in the presence of a constant amount of radiolabeled GLO. The amount of GLO imported in the absence of mature thiolase was set at 100% for comparison with the other data points from this experiment. The experiments were repeated three times; the results are presented as the average ± SE.

Effect of Zinc on PTS2 Protein Import

Evidence from Terlecky and colleagues (2001) indicates that zinc stimulates PTS1 import. As shown in Figure 6, the addition of zinc to import reactions greatly increased the level of thiolase imported into the peroxisome. This enhanced import was observed with all zinc salts (zinc chloride, zinc acetate, and zinc sulfate) tested. The effect appears to be specific for zinc, because the respective potassium salts (potassium chloride, potassium acetate, and potassium sulfate) had no effect on import (Fig. 7). Addition of the ion chelator 1-10-phenanthroline completely abolished the import stimulation provided by exogenous zinc (Fig. 6B, last bar). Similar stimulation of import by the addition of zinc was also observed with three PTS1 proteins (isocitrate lyase, GLO, and Ala:glyoxylate aminotransferasae; data not shown), as well as with a second PTS2 protein, Asp aminotransferase 3 (data not shown). Other divalent cations were previously shown to have no effect on PTS1 protein import (Brickner and Olsen, 1998).

View larger version (15K): [in this window] [in a new window]   Figure 6. Import of thiolase (THL) is stimulated by the addition of zinc chloride. Isolated glyoxysomes were incubated with increasing amounts of zinc chloride or the zinc chelator, 1-10-phenanthroline, in the presence of a constant amount of radiolabeled thiolase. The amount of thiolase imported in the absence of zinc chloride was set at 100% for comparison with the other data points from the experiment. The experiments were repeated three times; the results presented represent the average ± SE.

View larger version (20K): [in this window] [in a new window]   Figure 7. Zinc stimulates PTS2 protein import. Isolated glyoxysomes were incubated with different concentrations of zinc or potassium salts in the presence of a constant amount of radiolabeled thiolase. The amount of thiolase imported in the absence of additional salt (black bar) was set at 100% for comparison with the other data points from each experiment. The salts added were zinc chloride and potassium chloride (light gray bars), zinc acetate (ZnAc2) and potassium acetate (KAc2; dark gray bars), and zinc sulfate and potassium sulfate (white bars). The experiments were repeated three times; the results presented represent the average ± SE.

The import of matrix proteins involves three steps: receptor/cargo complex assembly in the cytosol, binding of the receptor/cargo complex to the peroxisome membrane, and translocation of the complex into the peroxisomal matrix with the subsequent release of the cargo from the complex. To examine where in the import pathway zinc is acting, we compared the amount of receptor/cargo complex bound to the peroxisomal membrane with and without the addition of zinc. None of the samples shown in Figure 8 was treated with protease. Radioactive proteins present in samples that were incubated at room temperature (Fig. 8, lanes 1 and 2) include all proteins bound and imported into the glyoxysomes. Proteins present in samples that were incubated at 4°C include only bound proteins (Fig. 8, lanes 3 and 4) because proteins are not imported into the organelle at this temperature (Brickner et al., 1997). Proteins bound to the membrane at 4°C are protease sensitive. Full-length thiolase (3-ketoacyl-CoA-thiolase [THL]) associated with peroxisomal membranes at 25°C (Fig. 8, lane 1), and also at 4°C (Fig. 8, lane 3), demonstrating that thiolase binding is not temperature dependent. When zinc was added to reactions in which thiolase was bound to peroxisomes, the amount of full-length thiolase associated with the membrane increased (Fig. 8, compare lanes 1 and 3 with 2 and 4). Thus, zinc may act, at least partially, at the membrane-binding step in the import pathway. Mature thiolase (mTHL) did not significantly bind to the peroxisome membrane (Fig. 8, lower panel), confirming the necessity of the PTS2 for typical thiolase import. The addition of zinc had no effect on the binding or import of mature thiolase.

View larger version (21K): [in this window] [in a new window]   Figure 8. In vitro binding of thiolase (THL) is increased in the presence of zinc. Isolated glyoxysomes were incubated with radiolabeled thiolase (THL) or mature thiolase (mTHL) at either 25°C (lanes 1 and 2) or 4°C (lanes 3 and 4). Zinc was added to samples in lanes 2 and 4. All subsequent treatments were as described in "Materials and Methods." None of the samples was treated with protease. The results shown are from representative experiments that were repeated three times with mature thiolase and four times with full-length thiolase.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
In this report, we used an established in vitro import assay (Brickner et al., 1997; Brickner and Olsen, 1998; Crookes and Olsen, 1998; Pool et al., 1998) to study the mechanisms of PTS2 protein import into peroxisomes. We successfully and consistently reconstituted PTS2 protein import into isolated pumpkin glyoxysomes. Its transport into isolated glyoxysomes was different from the transport of proteins via the PTS1 pathway in several ways.

In vitro peroxisomal protein import assays showed that the import of thiolase was temperature dependent, similar to that seen with the control PTS1 protein GLO (Fig. 2), whose import has been studied extensively (Brickner et al., 1997; Brickner and Olsen, 1998). Time courses (Fig. 3) revealed that the initial import of the PTS2 protein was much slower than PTS1 protein import. The maximum level of import of both proteins was about the same (10% of the protein presented to the glyoxysomes, "100% relative import"), but thiolase reached this level much later than GLO. It is possible that the levels of soluble PTS2 receptor are lower or that the affinity of the PTS2 receptor for docking sites on the membrane is less, such that PTS2 import is simply less efficient. It may be that another factor is limiting PTS2 protein import compared with PTS1 protein import. This observed difference in rate of import cannot be explained by the oligomerization state of the cargo proteins (see below). Further experiments are needed to understand this apparent difference in initial rates of PTS1 and PTS2 protein import.

We have previously shown that PTS1 protein import is saturable and that PTS1 proteins compete for components in the import pathway (Brickner et al., 1997). Thus, it was not unexpected that increasing levels of nonradioactive thiolase equivalents in the presence of a constant amount of radiolabeled thiolase decreased the amount of protease-resistant (imported) radiolabeled thiolase observed (Fig. 4A), suggesting that PTS2 proteins also compete for import. It was more surprising that not only did the PTS1 protein GLO not inhibit import of thiolase, but it actually stimulated import of the PTS2 protein (Fig. 4B). Two other PTS1 proteins, isocitrate lyase and Ala:glyoxylate aminotransferase, stimulated the import of the PTS2 protein thiolase similarly (data not shown). Legakis and Terlecky (2001) recently reported comparable results using semipermeabilized mammalian cells. They found that a PTS1-containing peptide did not block the import of a PTS2 reporter protein into peroxisomes, whereas introduction of a PTS2-containing peptide did block PTS2 import. Their immunofluorescence-based assay would not detect stimulation of import, however. Our results with a bona fide PTS2 protein are consistent with their observations and extend them to include the stimulation of PTS2 protein import by full-length PTS1 proteins. This suggests that PTS1 and PTS2 proteins do not compete with each other for import, but rather the two import pathways cooperate in some way.

To determine the effects of the PTS2 signal on the saturation of PTS2 import and the stimulation of PTS1 import, a mature form of the protein was synthesized. When the analogous truncation was constructed from the plant canola thiolase (Fig. 1) and overexpressed in Escherichia coli, it exhibited the same enzymatic activity as the full-length protein (Olesen et al., 1997). Mature Arabidopsis thiolase, lacking the PTS2 signal peptide, decreased the import of full-length thiolase into isolated glyoxysomes (Fig. 5A). The level of inhibition observed, however, was never more than a 50% reduction in the import of full-length thiolase. This result suggests that when the full-length protein was present in the import reaction, the mature form of thiolase was able to cross the peroxisome membrane. When non-radiolabeled full-length thiolase was added to imports containing radiolabeled mature thiolase, mature thiolase was observed inside the organelle (data not shown). Mature thiolase by itself was not imported into the organelle (Fig. 2). An explanation for this observation might be found in the dimeric nature of the thiolase protein (Glover et al., 1994). In contrast to other organelle translocation systems, such as the mitochondrion, which only transport unfolded polypeptides, peroxisomes are capable of importing remarkably large structures. There is substantial evidence that proteins folded outside the organelle can be imported across the membrane (McNew and Goodman, 1994; Faber et al., 2002), although import of oligomeric matrix proteins is not a universal pathway (Faber et al., 2002). Peroxisomal matrix proteins with their PTS1 signal removed have been shown to import into the organelle through piggybacking with proteins that do contain a PTS1 (Lee et al., 1997). The fusion of a PTS2 to non-peroxisomal passenger proteins leads to the in vivo import of "mixed" oligmers containing some subunits lacking the signal (Flynn et al., 1998; Kato et al., 1999). Our data is consistent with a model in which a thiolase dimer, consisting of one full-length protein and one mature protein, was capable of import directed by the presence of the PTS2 signal on the full-length thiolase. The dimeric state of thiolase, however, does not seem to explain our observation that PTS2 proteins import at a slower rate than PTS1 proteins (Fig. 3). Although thiolase has been shown to import as a dimer (Glover et al., 1994), the PTS1 protein GLO exists in solution as an octamer (Stenberg and Lindquist, 1997).

The mature form of thiolase, however, did not compete with or stimulate the import of the PTS1 protein GLO (Fig. 5B). This contrasts with the results observed with full-length thiolase and GLO, where full-length thiolase stimulated the import of the PTS1 protein (Fig. 4). Because mature thiolase was not imported without the presence of full-length thiolase (Fig. 2), it is likely that mature thiolase does not bind to the PTS2 receptor, Pex7p. If mature thiolase were not bound to the PTS2 receptor, it would be unlikely to interact with the PTS1 import pathway to stimulate (or otherwise affect) the import of GLO.

Evidence from Terlecky and colleagues (2001) indicates that PTS1 import is stimulated by zinc. In this report, we quantitatively demonstrated that the addition of zinc to import reactions increased the level of thiolase imported into the peroxisome by severalfold. This effect was specific to zinc, because the respective potassium salts (potassium chloride, potassium acetate, and potassium sulfate) had no effect on import (Fig. 7). Addition of the ion chelator 1-10-phenanthroline completely inhibited the import stimulation (Fig. 6B, last bar). Thus, zinc is affecting the import of both PTS1 and PTS2 proteins, suggesting that it acts at a step that is common to the two pathways.

The import of matrix proteins can be considered in three steps: receptor/cargo complex assembly in the cytosol, docking of the receptor/cargo complex at the peroxisome membrane, and translocation of the complex into the peroxisomal matrix. Given the dramatic effect of zinc on the level of thiolase imported into glyoxysomes, we next examined where in the import pathway zinc may be acting. By assaying the level of thiolase bound to the peroxisomal membrane with and without the addition of zinc, we showed that addition of zinc increased the amount of full-length thiolase docked at the peroxisomal membrane (Fig. 8). The increased amount of thiolase binding at the membrane may not completely account for the severalfold increase thiolase import, however. It is therefore reasonable to conclude that zinc may be acting at more than one step of the import pathway.

Pex2p, Pex10p, and Pex12p are all members of the RING finger superfamily of zinc-binding proteins located on the peroxisomal membrane. Both Pex10p and Pex12p from mammals have been shown to interact directly with Pex5p, but not with Pex7p. Although several authors have proposed that after docking on the membrane protein Pex14p, the Pex5p/cargo complex is transferred to the RING finger proteins; it has recently been shown by Reguenga et al. (2001) that Pex5p, Pex14p, Pex12p, and Pex2p are all subunits of the same protein assembly. It is therefore possible that zinc could play a role in the regulation of receptor docking and matrix protein import by binding to the RING finger proteins. Our zinc binding and import results (Fig. 8) support this hypothesis. Although a direct interaction between Pex7p and the RING finger proteins has not been shown, it is possible that an effect on PTS2 import could be mediated through Pex5p, especially if the two pathways are interacting. Additional experiments should be performed to understand further the role of zinc in matrix protein import, but it is reasonable to propose a role for zinc binding to the RING finger proteins as a regulatory mechanism during Pex5p/Pex7p binding and subsequent matrix protein import.

In a continuing effort to understand the cellular and biochemical mechanisms of peroxisomal matrix protein import, we have characterized the in vitro processing and import of the PTS2 protein thiolase into glyoxysomes. The ability to study PTS2 protein import reliably in vitro will allow us to dissect more fully the molecular mechanisms of the peroxisomal matrix protein import process, especially with respect to the interactions between the PTS1 and PTS2 protein import pathways.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Preparation of Radiolabeled Peroxisomal Proteins

The plasmid (pMV2) containing the full-length cDNA insert for spinach (Spinacia oleracea) GLO was provided by Dr. Chris Somerville (Carnegie Institution, Stanford, CA). The plasmid (pTHL) containing the full-length cDNA insert coding for thiolase was obtained from the Arabidopsis Biological Resource Center at Ohio State University (Columbus; expressed sequence tag stock 91A18T7; GenBank accession no. T20943; Arabidopsis Genome Initiative locus At2g33150) and fully sequenced on both strands to confirm its identity. To engineer a mature form of the thiolase protein, beginning with a Met in place of the Leu at the putative cleavage site (see Fig. 1) and thus lacking the first 35 amino acids, the full-length thiolase cDNA was used as template for PCR with the forward primer 5'-ACCATGGGCTGGGGACAGTGCTGC-3' and the reverse primer SP6. The resulting PCR product was ligated into the pCRII-TOPO vector (Invitrogen, Carlsbad, CA) to create pMTL and fully sequenced. pMV2 was linearized with HindIII. pTHL was linearized with NotI. pMTL was linearized with BamHI. Transcription of the linearized DNA with T7 RNA polymerase RNA was performed by modification of the manufacturer's instructions (Promega, Madison, WI). Each reaction mixture contained transcription buffer, 2.5 µg of bovine serum albumin, 0.5 mM GpppG cap analog, 10 mM dithiothreitol, 0.5 mM rNTPs, 0.15 mM GTP, 20 units of RNase inhibitor, 2.5 µg of linearized DNA, and 30 units of RNA polymerase. Transcription reactions were incubated at 37°C for 90 min. Radiolabeled GLO and both forms of thiolase were synthesized in a cell-free wheat germ lysate or rabbit reticulocyte lysate system in the presence of [³⁵S]Met. Redi-vue [³⁵S]Met (specific activity 43.5 TBq mmol^-1) was purchased from Amersham Biosciences (Uppsala). The efficiency of translation was assessed by trichloroacetic-acid precipitation onto glass fiber filters, followed by ethanol washes and then quantitation in a liquid scintillation counter (model LS 6800, Beckman Coulter, Fullerton, CA).

Isolation of Pumpkin Glyoxysomes

Pumpkin seeds (Cucurbita pepo var Connecticut Fields) were purchased from Siegers Seed Co. (Zeeland, MI). Pumpkin seedlings were grown in moist vermiculite for 5 to 7 d in the dark at 25°C to 28°C. Glyoxysomes were isolated from pumpkin cotyledons as described previously (Brickner et al., 1997). The glyoxysomal pellet was resuspended in isolation buffer (10 mM HEPES-KOH, pH 6, and 0.3 M mannitol) to a final concentration of 30 to 50 mg mL^-1 total protein.

Import Reactions

Standard in vitro protein imports reactions were initiated by the addition of radiolabeled GLO or thiolase (5 x 10⁵ cpm trichloroacetic acid-precipitable protein) to 300 to 500 µg of glyoxysomes in the presence of import buffer (25 mM MES-KOH, pH 6, 0.5 M Suc, 10 mM KCl, 1 mM MgCl₂, and 5 mM MgATP) in a final volume of 200 µL. All import reactions were performed at 25°C unless otherwise noted. GLO import reactions were incubated for 30 min, and then the glyoxysomes were treated with thermolysin (10 µg mL^-1 in 0.5 mM CaCl₂) to remove proteins that were not imported. Thiolase import reactions were incubated for 60 min and subsequently treated with thermolysin (50 µg mL^-1 in 0.5 mM CaCl₂). Protease treatments were performed for 30 min on ice; reactions were stopped by the addition of EDTA (25 mM final concentration) to inhibit the thermolysin. After protease treatment, the glyoxysomes were repurified on a 0.7 M Suc cushion, solubilized in SDS-PAGE sample buffer, and subjected to SDS-PAGE as described previously (Brickner et al., 1997). Note that only proteins protected by an intact glyoxysomal membrane would be protease resistant. Lysed or compromised organelles would not be recovered from the Suc cushion. Some samples (indicated in the figures) were treated with Triton X-100 (1% [v/v] final concentration) following import and protease treatment as described (Brickner et al., 1997) to lyse the organelles. Binding assays were performed at 4°C as described above but without any subsequent protease treatments.

The level of import was determined by phosphor imaging analysis (Bio-Rad Laboratories, Hercules, CA) as described previously (Crookes and Olsen, 1998). In brief, the radioactivity present in each lane of an SDS-PAGE gel was quantitated by exposing the gel in a cassette compatible with the instrument (Bio-Rad Laboratories). The associated software allowed the user to identify the bands whose density should be determined, including background signal to be removed from the calculations. One hundred percent relative import was set as the maximum level of radiolabeled protein detected, as indicated in the figure legends. In most cases, this represents approximately 10% of the protein originally presented to the organelle. Because each of the proteins used in these experiments contain roughly the same number of Met residues (i.e. GLO and mature thiolase have 13 Met residues, full-length thiolase has 12 Met residues), equal counts of radioactivity represent equal amounts of protein presented for import or binding.

For the competition experiments, import was initiated by the simultaneous addition of radiolabeled protein (thiolase) and non-radiolabeled protein (GLO or thiolase). Non-radiolabeled proteins were synthesized in a cell-free wheat germ lysate system or rabbit reticulocyte lysate, as before, except 0.8 mM Met (final concentration) was present instead of [³⁵S]Met. Equivalent amounts of proteins to be used were determined by performing translation reactions (with nonradioactive amino acids) simultaneously with standard translation reactions (containing [³⁵S]Met). The amount of trichloroacetic acid-precipitable protein obtained for the standard translation reaction was used to calculate the equivalent amount of unlabeled translation product to be added to import reactions. After import, samples were treated with the appropriate amount of protease (the higher concentration of protease if two different proteins were used) to degrade any proteins that were not imported. All subsequent treatments were described as above.

Distribution of Materials

Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permission will be the responsibility of the requestor.

	ACKNOWLEDGMENTS

 
We thank Drs. Wendy Crookes and Aaron Liepman for many helpful discussions. We are especially grateful to Dr. Stanley Terlecky (Wayne State University, Detroit) for his encouragement and critical reading of this manuscript.

Received June 6, 2003; returned for revision July 9, 2003; accepted September 4, 2003.

	FOOTNOTES

 
¹ This work was supported in part by the U.S. Department of Agriculture (grant to L.J.O.). T.L.J. was supported by a Regents fellowship from The University of Michigan and the University of Michigan Human Genetics Training Program (National Institutes of Health National Research Service Award no. 5-T32-GM07544).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.028217.

^* Corresponding author; email ljo{at}umich.edu; fax 734-647-0884.

	LITERATURE CITED

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Behari R, Baker A (1993) The carboxyl terminus of isocitrate lyase is not essential for import into glyoxysomes in an in vitro system. J Biol Chem 268: 7315-7322[Abstract/Free Full Text]

Brickner DG, Harada JJ, Olsen LJ (1997) Protein transport into higher plant peroxisomes: in vitro import assay provides evidence for receptor involvement. Plant Physiol 113: 1213-1221[Abstract]

Brickner DG, Olsen LJ (1998) Nucleotide triphosphates are required for the transport of glycolate oxidase into peroxisomes. Plant Physiol 116: 309-317[Abstract/Free Full Text]

Crookes WJ, Olsen LJ (1998) The effects of chaperones and the influence of protein assembly on peroxisomal protein import. J Biol Chem 273: 17236-17242[Abstract/Free Full Text]

Dammai V, Subramani S (2001) The human peroxisomal targeting signal receptor, Pex5p, is translocated into the peroxisomal matrix and recycled to the cytosol. Cell 105: 187-196[CrossRef][Web of Science][Medline]

Dodt G, Gould SJ (1996) Multiple PEX genes are required for proper subcellular distribution and stability of Pex5p, the PTS1 receptor: evidence that PTS1 protein is mediated by a cycling receptor. J Cell Biol 135: 1763-1774[Abstract/Free Full Text]

Erdmann R, Veenhuis M, Kunau W-H (1997) Peroxisomes: organelles at the crossroads. Trends Cell Biol 7: 400-407[CrossRef][Medline]

Faber KN, Keizer-Gunnink I, Pluim D, Harder W, Geert AB, Veenhuis M (1995) The N-terminus of amine oxidase of Hansenula polymorpha contains a peroxisomal targeting signal. FEBS Lett 357: 115-120[CrossRef][Medline]

Faber KN, van Dijk R, Keizer-Gunnink I, Koek A, van der Klei IJ, Veenhuis M (2002) Import of assembled PTS1 proteins into peroxisomes of the yeast Hansenula polymorpha: Yes and No! Biochim Biophys Acta 1591: 157-162[Medline]

Flynn CR, Mullen RT, Trelease RN (1998) Mutational analyses of a type 2 peroxisomal targeting signal that is capable of directing oligomeric protein import into tobacco BY-2 glyoxysomes. Plant J 16: 709-720[CrossRef][Web of Science][Medline]

Fransen M, Terlecky SR, Subramani S (1998) Identification of a human PTS1 receptor docking protein directly required for peroxisomal protein import. Proc Natl Acad Sci USA 95: 8087-8092[Abstract/Free Full Text]

Gebhardt JS, Wadsworth GJ, Matthews BF (1998) Characterization of a single soybean cDNA encoding cytosolic and glyoxysomal isozymes of aspartate aminotransferase. Plant Mol Biol 37: 99-108[CrossRef][Web of Science][Medline]

Germain V, Rylott EL, Larson TR, Sherson SM, Bechtold N, Carde J-P, Bryce JH, Graham IA, Smith SM (2001) Requirement for 3-ketoacyl-CoA thiolase-2 in peroxisome development, fatty acid -oxidation and breakdown of triacylglycerol in lipid bodies of Arabidopsis seedlings. Plant J 28: 1-12[CrossRef][Web of Science][Medline]

Gietl C (1990) Glyoxysomal malate dehydrogenase from watermelon is synthesized with an amino-terminal transit peptide. Proc Natl Acad Sci USA 87: 5773-5777[Abstract/Free Full Text]

Gietl C, Faber KN, van der Klei IJ, Veenhuis M (1994) Mutational analysis of the N-terminal topogenic signal of watermelon glyoxysomal malate dehydrogenase using the heterologous host Hansenula polymorpha. Proc Natl Acad Sci USA 91: 3151-3155[Abstract/Free Full Text]

Gietl C, Wimmer B, Adamec J, Kalousek F (1997) A cysteine endopeptidase isolated from castor bean endosperm microbodies processes the glyoxysomal malate dehydrogenase precursor protein. Plant Physiol 113: 863-871[Abstract]

Glover JR, Andrews DW, Rachubinski RA (1994) Saccharomyces cerevisiae peroxisomal thiolase is imported as a dimer. Proc Natl Acad Sci USA 91: 10541-10545[Abstract/Free Full Text]

Gould SJ, Keller G-A, Hosken N, Wilkinson J, Subramani S (1989) A conserved tripeptide sorts proteins to peroxisomes. J Cell Biol 108: 1657-1664[Abstract/Free Full Text]

Horng JT, Behari R, Burke ECA, Baker A (1995) Investigation of the energy requirement and targeting signal for the import of glycolate oxidase into glyoxysomes. Eur J Biochem 230: 157-163[Web of Science][Medline]

Johnson TL, Olsen LJ (2001) Building new models for peroxisome biogenesis. Plant Physiol 127: 731-739[Free Full Text]

Kato A, Hayashi M, Kondo M, Nishimura M (1996) Targeting and processing of a chimeric protein with the N-terminal presequence of the precursor to glyoxysomal citrate synthase. Plant Cell 8: 1601-1611[Abstract]

Kato A, Hayashi M, Nishimura M (1999) Oligomeric proteins containing N-terminal targeting signals are imported into peroxisomes in transgenic Arabidopsis. Plant Cell Physiol 40: 586-591[Abstract/Free Full Text]

Kindl H (1992) Plant peroxisomes: recent studies on function and biosynthesis. Cell Biochem Funct 10: 153-158[CrossRef][Medline]

Kunau W-H (2001) Peroxisomes: the extended shuttle to the peroxisome matrix. Curr Biol 11: R659-R662[CrossRef][Web of Science][Medline]

Lee MS, Mullen RT, Trelease RN (1997) Oilseed isocitrate lyases lacking their essential type 1 peroxisomal targeting signal are piggybacked to glyoxysomes. Plant Cell 9: 185-197[Abstract]

Legakis JE, Terlecky SR (2001) PTS2 protein import into mammalian peroxisomes. Traffic 2: 252-260[Web of Science][Medline]

McNew JA, Goodman JM (1994) An oligomeric protein is imported into peroxisomes in vivo. J Cell Biol 127: 1245-1257[Abstract/Free Full Text]

Miura S, Miyazawa S, Osumi T, Hashimoto T, Fujiki Y (1994) Posttranslational import of 3-ketoacyl-CoA thiolase into rat liver peroxisomes in vitro. J Biochem 115: 1064-1068[Abstract/Free Full Text]

Olesen C, Brandt A (1996) A full length cDNA encoding 3-ketoacyl-CoA thiolase from Brassica napus. Plant Physiol 110: 714

Olesen C, Thomsen K, Svendsen I, Brandt A (1997) The glyoxysomal 3-ketoacyl-CoA thiolase precursor from Brassica napus has enzymatic activity when synthesized in Escherichia coli. FEBS Lett 412: 138-140[CrossRef][Medline]

Olsen LJ (1998) The surprising complexity of peroxisome biogenesis. Plant Mol Biol 38: 163-189[CrossRef][Web of Science][Medline]

Osumi T, Tsukamoto T, Hata S, Yokota S, Miura S, Fujiki Y, Hijikata M, Miyazawa S, Hashimoto T (1991) Amino-terminal presequence of the precursor of peroxisomal 3-ketoacyl-CoA thiolase is a cleavable signal peptide for peroxisomal targeting. Biochem Biophys Res Commun 181: 947-954[CrossRef][Web of Science][Medline]

Pool MR, Lopez-Huertas E, Baker A (1998) Characterization of intermediates in the process of plant peroxisomal protein import. EMBO J 17: 6854-6862[CrossRef][Web of Science][Medline]

Reguenga C, Oliveria MEM, Gouveia AMM, Sa-Miranda C, Azevedo JE (2001) Characterization of the mammalian peroxisomal import machinery. J Biol Chem 276: 29935-29942[Abstract/Free Full Text]

Stenberg K, Lindquist Y (1997) Three dimensional structures of glycolate oxidase with bound active site inhibitors. Protein Sci 6: 1009-1015[Web of Science][Medline]

Swinkels BW, Gould SJ, Bodnar AG, Rachubinski RA, Subramani S (1991) A novel, cleavable peroxisomal targeting signal at the amino-terminus of the rat 3-ketoacyl-CoA thiolase. EMBO J 10: 3255-3262[Web of Science][Medline]

Terlecky SR, Legakis JE, Hueni SE, Subramani S (2001) Quantitative analysis of peroxisomal protein import in vitro. Exp Cell Res 263: 98-106[CrossRef][Medline]

Wiemer EA, Subramani S (1994) Protein import deficiencies in human peroxisomal disorders. Mol Gen Med 4: 119-153

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