Plant Physiol. (1998) 116: 309-317

Nucleotide Triphosphates Are Required for the Transport of Glycolate Oxidase into Peroxisomes¹

Donna G. Brickner and Laura J. Olsen^*

Department of Biology, University of Michigan, Ann Arbor, Michigan 48109-1048

	ABSTRACT

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All peroxisomal proteins are nuclear encoded, synthesized on free cytosolic ribosomes, and posttranslationally targeted to the organelle. We have used an in vitro assay to reconstitute protein import into pumpkin (Cucurbita pepo) glyoxysomes, a class of peroxisome found in the cotyledons of oilseed plants, to study the mechanisms involved in protein transport across peroxisome membranes. Results indicate that ATP hydrolysis is required for protein import into peroxisomes; nonhydrolyzable analogs of ATP could not substitute for this requirement. Nucleotide competition studies suggest that there may be a nucleotide binding site on a component of the translocation machinery. Peroxisomal protein import also was supported by GTP hydrolysis. Nonhydrolyzable analogs of GTP did not substitute in this process. Experiments to determine the cation specificity of the nucleotide requirement show that the Mg²⁺ salt was preferred over other divalent and monovalent cations. The role of a putative protonmotive force across the peroxisomal membrane was also examined. Although low concentrations of ionophores had no effect on protein import, relatively high concentrations of all ionophores tested consistently reduced the level of protein import by approximately 50%. This result suggests that a protonmotive force is not absolutely required for peroxisomal protein import.

	INTRODUCTION

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Peroxisomes are ubiquitous organelles involved in a variety of important cellular processes, including the degradation of hydrogen peroxide and the -oxidation of fatty acids (for review, see Olsen and Harada, 1995). The single peroxisomal membrane surrounds a dense matrix, sometimes containing paracrystalline inclusions (Frederick et al., 1975). New organelles are thought to arise by the fission of preexisting peroxisomes, with the subsequent incorporation of additional membrane lipids and peroxisomal proteins. There are several classes of peroxisomes found in higher plants (Kindl, 1992; Gietl, 1996). Glyoxysomes are abundant in the cotyledons of most plants and are involved in lipid mobilization to provide nutrients during germination and seedling growth (Trelease, 1984). In leaves, peroxisomes contain enzymes such as GLO that are necessary for photorespiration (Ogren, 1984).

All peroxisomal proteins must be nuclear encoded because peroxisomes do not contain DNA. Peroxisomal proteins are synthesized on free, cytosolic ribosomes and imported posttranslationally into the organelle. The majority of matrix proteins, including GLO, are targeted to peroxisomes via a carboxyl-terminal tripeptide comprising the amino acids Ser-Lys-Leu or conserved variants (Gould et al., 1987; McNew and Goodman, 1996). Other peroxisomal proteins, such as malate dehydrogenase and thiolase, use an amino-terminal targeting signal (Gietl, 1990; Swinkels et al., 1991; Preisig-Müller and Kindl, 1993). Considerable genetic and biochemical evidence indicates the involvement of a proteinaceous receptor in peroxisomal protein import (Wolins and Donaldson, 1994; Rachubinski and Subramani, 1995; Dodt and Gould, 1996; McNew and Goodman, 1996; Brickner et al., 1997). However, a detailed description of the targeting mechanism and an understanding of receptor localization remain elusive. Protein import into yeast peroxisomes may also use other proteins (Dodt and Gould, 1996), including a cytosolic ATPase (Yahraus et al., 1996), a membrane-bound docking factor (Elgersma et al., 1996; Erdmann and Blobel, 1996; Gould et al., 1996), an N-ethylmaleimide-sensitive factor (Wendland and Subramani, 1993), and a molecular chaperone such as hsp70 (Walton et al., 1994).

Protein trafficking requires the investment of energy. The hydrolysis of ATP and/or GTP is required for protein transport into mitochondria (Pfanner et al., 1990), chloroplasts (Keegstra et al., 1989; Theg et al., 1989; Kessler et al., 1994), nuclei (Powers and Forbes, 1994), and the ER (Walter and Johnson, 1994). ATP hydrolysis is known to be necessary for peroxisomal protein import (Imanaka et al., 1987; Wendland and Subramani, 1993; Horng et al., 1995; Brickner et al., 1997). A careful analysis of the energy requirements for protein binding to chloroplasts revealed that other NTPs can support this process, although less efficiently than ATP (Olsen et al., 1989). Exactly where and how the energy is being used during translocation is currently unknown. It is likely that NTP hydrolysis induces conformational changes in the translocation machinery or in the targeted protein (perhaps by interaction with an energy-dependent molecular chaperone), thereby facilitating the protein's entry into the organelle (Pfanner et al., 1990).

A PMF is required, in addition to ATP, for the export of proteins from bacteria (Yamane et al., 1987; Wong and Buckley, 1989); a pH gradient is needed to transport some proteins across thylakoid membranes (Cline et al., 1992; Theg and Scott, 1993); and the electrical component of the PMF facilitates the import of some proteins into mitochondria (Pfanner and Neupert, 1986). Addition of inhibitors or ionophores that collapse the PMF abolishes protein transport across each of these membranes. A PMF may exist across the peroxisomal membrane. Some researchers have found that ionophores inhibit protein import into peroxisomes (Bellion and Goodman, 1987), whereas others observed no effect on peroxisomal protein import (Imanaka et al., 1987; Wendland and Subramani, 1993). An ATPase, analogous to the V-class H⁺-ATPase found on vacuolar membranes, may be present on the peroxisomal membrane (Douma et al., 1987; del Valle et al., 1988; Wolvetang et al., 1990; Whitney and Bellion, 1991). ATP hydrolysis on the cytosolic face would make the peroxisomal matrix acidic (Nicolay et al., 1987), thus establishing a PMF that could be used to drive protein translocation. Alternatively, nonspecific pores may allow small ions and metabolites to diffuse freely across the membrane, thereby dissipating an electrochemical or pH gradient (for review, see van den Bosch et al., 1992).

We have extensively characterized the energy requirements for protein import into glyoxysomes using an optimized in vitro assay to reconstitute the transport event. First, we examined the ability of various NTPs to support the import of GLO into isolated pumpkin (Cucurbita pepo) glyoxysomes. The competence of nonhydrolyzable analogs of ATP and GTP to substitute for the NTP requirement was also analyzed. Next, we briefly investigated the cation specificity of the nucleotide requirement for protein import. Finally, in an effort to characterize the role that a putative PMF might play, we explored the effect of ionophores on GLO import. Ionophores were chosen that collapsed the pH gradient, the electrical gradient, or both components of the PMF. A thorough study of the energetics of protein import into peroxisomes is necessary to facilitate the understanding of the molecular mechanisms involved in the transport process.

	MATERIALS AND METHODS

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All NTPs, NTP analogs, ionophores, and general chemicals were purchased from Sigma. Sephadex G-25-80 (fine) was purchased from Pharmacia. Pumpkin (Cucurbita pepo var Half Moon) seeds were purchased from Petoseed Co., Inc. (Saticoy, CA). Redi-vue [³⁵S]Met (specific activity, 43.5 TBq/mmol) was purchased from Amersham.

Preparation of Radiolabeled GLO

Isolation of Pumpkin Glyoxysomes

In Vitro Import Assays

The Mg²⁺ salt of ATP was used in most standard import reactions, except that Na₂ATP was used for the cation specificity experiments (see Fig. 2). Other import reactions were supplemented with additional cations as indicated in the legend to Figure 2. Import reactions containing other NTPs and/or NTP analogs were supplemented with equimolar MgCl₂ in addition to the 1 mm MgCl₂ present in the import buffer. The presence of Mg²⁺ by itself had no effect on import (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 2. Import of GLO into isolated glyoxysomes requires Mg2+. Standard import reactions were performed as described in Figure 1 and ``Materials and Methods'' except that 5 mm Na2ATP was present in each reaction instead of MgATP. All other import reactions were supplemented with additional cations (supplied by MgCl2, MnCl2, CaCl2, or KCl) at 5 mm final concentration. The level of import observed with 5 mm MgCl2 (and 5 mm Na2ATP) added to the import reaction was set at 100% for comparison with import levels in the presence of the other cations. The values presented are the average ± se of two experiments.

Glyoxysomes were preincubated with ionophores and 5 mm ATP for 20 min at room temperature before radiolabeled GLO was added to start the import reaction. The endogenous NTPs were not removed from the radiolabeled GLO used in the ionophore experiments. Modified import buffer (containing only 25 mm Mes-KOH, pH 6.0, and 0.5 m Suc; i.e. lacking the 10 mm KCl, 1 mm MgCl₂, and azide) was used in the cation specificity experiments.

Azide (10 mm NaN₃) was included in the organelle isolation buffer and import buffer in all experiments except the cation specificity (see Fig. 2) and the ionophore experiments (see Fig. 7). The presence of azide during organelle isolation and in import reactions did not affect import levels (data not shown). The purpose of the azide was to inhibit any ATP synthesis by the small amounts of mitochondria that may have been present in the glyoxysome preparation (Brickner et al., 1997), and thus remove any endogenous ATP supplied by the mitochondria. Azide was not included in the cation-specificity and ionophore experiments because the exact level of ATP present was not critical. In addition, we wanted to control precisely the amount of Na present in the import reactions when testing for cation effects on import.

View larger version (15K): [in this window] [in a new window]   Figure 7. Ionophores reduce the level of glyoxysomal protein import. To determine whether a PMF plays a role in peroxisomal protein import, isolated glyoxysomes were preincubated for 20 min at room temperature with various ionophores (1-20 µm final concentration). Import reactions were performed and analyzed as described in Figure 1. The level of protease-resistant GLO present in samples that had no ionophore added was set as 100% relative import for comparison with the ionophore-treated samples. A representative experiment is presented. A, GLO import into glyoxysomes in the presence of nigericin or valinomycin, ionophores that collapse a single component of the PMF. B, GLO import into glyoxysomes in the presence of ionophores that collapse the total PMF.

Each of the ionophore stock solutions was prepared in ethanol; dilutions of the stocks were used in the import reactions presented in Figure 7. Ethanol by itself, equivalent to the highest concentration present in the ionophore treatments (approximately 0.4%), had no effect on the level of GLO import (data not shown). Import reactions testing the effect of the Ca²⁺ ionophore A23187 also contained 10 mm CaCl₂; 10 mm CaCl₂ was also present in the controls and all import samples testing the Ca²⁺ ionophore A23187; 10 mm KCl was included in the controls and import samples testing valinomycin.

Quantitation

View larger version (19K): [in this window] [in a new window]   Figure 1. Import of GLO into glyoxysomes is energy dependent. To characterize the NTP dependence of protein transport, increasing concentrations of ATP () or GTP () were added to standard import reactions (see ``Materials and Methods''). Before addition to the import reactions, GLO translation products were desalted on a Sephadex G-25 column to remove endogenous nucleotides and other small molecules. The amount of radiolabeled GLO that remained protease protected after import in the presence of 5 mm ATP was set as 100% relative import for comparison with the other samples. The average ± se of three independent experiments is shown.

View larger version (18K): [in this window] [in a new window]   Figure 3. Nonhydrolyzable ATP analogs cannot support GLO import. To determine whether ATP hydrolysis is required for peroxisomal protein import, increasing amounts of ATP, AMP-PCP, or AMP-PNP (nonhydrolyzable analogs of ATP) were added to standard import reactions. Before addition to the import reactions, GLO translation products were desalted on a Sephadex G-25 column to remove endogenous nucleotides and other small molecules (see ``Materials and Methods''). The amount of GLO imported into glyoxysomes in the presence of 5 mm ATP was set at 100% for comparison with the other samples in the same experiment. The average ± se of three independent experiments is shown.

View larger version (15K): [in this window] [in a new window]   Figure 4. GTP hydrolysis is required for peroxisomal protein import. To determine whether the hydrolysis of GTP was required for peroxisomal protein import, increasing amounts of either GTP or GTP--S were added to NTP-depleted import reactions, as described in Figure 1. The average ± se of two independent experiments is shown.

View larger version (18K): [in this window] [in a new window]   Figure 5. AMP-PCP competes with ATP during protein import. Isolated glyoxysomes were incubated with increasing amounts of ATP and challenged with the nonhydrolyzable ATP analog, AMP-PCP, at the concentrations indicated. Subsequent import reactions were performed as described in Figure 1. The results shown are the average ± se of three separate experiments.

View larger version (15K): [in this window] [in a new window]   Figure 6. GTP--S inhibits ATP-dependent protein import. To examine the effects of GTP--S on ATP-dependent import, ATP and/or excess GTP--S were preincubated with isolated glyoxysomes for 5 min. Protein import was initiated by the addition of radiolabeled GLO proteins. The amount of radiolabeled GLO imported in the presence of 5 mm ATP was set as 100% relative import. Because of minor differences in the ways in which each replicate experiment was performed, a representative experiment is presented.

	RESULTS

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NTP Requirement for Peroxisomal Protein Import

As shown in Figure 1, the amount of GLO imported increased with higher concentrations of ATP or GTP. Maximal levels of import were observed at 5 mm ATP; no additional GLO was imported in the presence of 10 mm ATP (Brickner et al., 1997). Low levels of protein import (approximately 18-22%, relative to import at 5 mm ATP) were observed even when no exogenous NTP was added to the import reaction. This background level of import has been observed by others (Imanaka et al., 1987; Horng et al., 1995) and may be mediated by NTP bound to a component of the isolated organelles, to translation products, or to a cytosolic factor present in the wheat germ lysate; free NTPs were removed by the desalting procedure (Olsen et al., 1989). GTP supported GLO import nearly as well as ATP. Maximal GTP-dependent import was achieved at a lower NTP concentration; only 1 mm GTP was needed, compared with 5 mm ATP. This result suggests that GTP itself can be used as an energy source for protein import. It does not distinguish between a single energy-dependent process, which can use either ATP or GTP to drive import, and two separate energy-requiring steps, one dependent on ATP and the other dependent on GTP. Finally, the addition of both ATP and GTP to the same import reaction stimulated import only slightly more than the level in the control samples (data not shown).

In addition to ATP and GTP, other NTPs were assessed for their ability to support peroxisomal protein import (Table I). Standard import reactions were performed in the presence of 5 mm NTP; the radiolabeled GLO translation products were desalted to remove ATP added to the translation reaction or present in the wheat germ lysate (see ``Materials and Methods''). With the exception of ITP, all NTPs tested in our in vitro protein import system could serve as an alternative energy source for peroxisomal protein import, although much less efficiently than ATP.

Cation Specificity for Peroxisomal Protein Import

NTP Hydrolysis Is Required for Peroxisomal Protein Import

Because GTP was also capable of supporting peroxisomal protein import (Fig. 1), the necessity of GTP hydrolysis was examined. GTP--S, a nonhydrolyzable analog of GTP, was added to standard import reactions in the absence of other NTPs, but was unable to increase the import of GLO to greater than background levels (Fig. 4). Therefore, it appears that the hydrolysis of GTP may also provide energy to drive peroxisomal proteins across the membrane.

Two components of the chloroplast protein import machinery have been shown to bind GTP (Kessler et al., 1994). There are at least two proteins involved in peroxisome biogenesis or function that have ATP-binding domains (Swartzman et al., 1996; Yahraus et al., 1996). One approach to the determination of whether the NTP hydrolysis requirement for peroxisomal protein import is caused by direct binding of the NTP by a protein factor (rather than an indirect metabolic role for NTP) is the use of nucleotide competition experiments. For these experiments an increasing concentration of the nonhydrolyzable ATP analog AMP-PCP was preincubated with ATP and isolated glyoxysomes. The import reaction was then initiated by the addition of radiolabeled GLO protein. As shown in Figure 5, the amount of protease-protected GLO decreased with increasing amounts of nonhydrolyzable ATP analog even in the presence of ATP. This competitive interaction between ATP and AMP-PCP indicates that there may be a discrete nucleotide binding site on some component of the translocation apparatus, or on another factor that is required for import of proteins into peroxisomes.

To assess the effect of a nonhydrolyzable GTP analog on ATP-dependent protein import levels, NTP competition experiments were performed using GTP--S as the competitor. Competition was established by challenging an import reaction containing ATP with an excess of GTP--S (Fig. 6). The addition of 10 mm GTP--S significantly reduced the level of GLO imported into glyoxysomes in the presence of 5 mm ATP. This suggests that ATP and GTP are either competing for a common nucleotide binding site on some component required for protein translocation, or that both NTPs are required at different steps of protein import, one of which is inhibited by the binding of GTP--S.

Effects of Ionophores on Peroxisomal Protein Import

	DISCUSSION

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Energy is required to transport polypeptides through phospholipid membranes. At least three possible sources for that energy have been proposed: (a) energy released by induced conformational changes caused by the initial interaction between the protein and the membrane; (b) energy released by the hydrolysis of high-energy bonds found in ATP and other NTPs; and (c) a PMF consisting of a transmembrane electric potential and a pH gradient (Pugsley, 1989). Different protein import systems use each of these sources to varying degrees. We have addressed only the latter two in this report. Our in vitro assay for protein import into peroxisomes allows us to biochemically manipulate the exact reaction conditions. For most experiments, we first removed any endogenous NTPs from the GLO translation products and then added back different forms of energy to examine their effects on protein translocation.

We and others have shown that peroxisomal protein import is an ATP-dependent process (Imanaka et al., 1987; Soto et al., 1993; Horng et al., 1995; Brickner et al., 1997), but it is not yet known where in the targeting and translocation pathway the energy is required or how the energy is used. One possibility is that ATP is required by cytosolic chaperones or other cytosolic factors to ensure proper targeting of the protein to peroxisomes. In fact, the cytoplasmic ATPase PXAAA1 from humans apparently stabilizes a soluble receptor required for peroxisomal protein import (Yahraus et al., 1996). The Mg²⁺ salt of ATP has been shown to be specifically required by some chaperones for optimal function (Miernyk, 1997). The cation specificity experiments also showed that Mg²⁺ is the preferred salt for protein import into peroxisomes (Fig. 2). However, Mg²⁺ likely has multiple functions in this pathway. For instance, a yeast peroxisomal, membrane-associated, proton-translocating ATPase is also Mg²⁺ dependent (Douma et al., 1987).

A second possible role for NTPs during peroxisomal protein import is suggested by the nucleotide binding sites identified on several peroxisome-associated proteins (Verheyden et al., 1992; Swartzman et al., 1996) as well as on two components of the chloroplast translocation machinery (Kessler et al., 1994). When isolated glyoxysomes were preincubated with ATP and an excess of a nonhydrolyzable NTP analog, subsequent GLO import was significantly decreased (Figs. 5 and 6), suggesting that nucleotide binding to an unidentified factor may be important for peroxisomal protein transport.

Both ATP and GTP were able to support GLO import in our in vitro assay system (Figs. 1, 4-6). GTP-binding proteins are known to be involved in several other transport pathways (Balch, 1990; Pfeffer, 1992; Kessler et al., 1994) and three small GTP-binding proteins have been identified in rat peroxisomal membranes (Verheyden et al., 1992). Thus, it is possible that GTP has a direct role in peroxisomal protein import. Although Wendland and Subramani (1993) did not observe a similar involvement of GTP-hydrolyzing proteins when they examined peroxisomal protein import in permeabilized mammalian cells, they used only 100 µm GTP--S in the presence of 1 mm ATP and an ATP-regenerating system. The concentration of the GTP analog may have been too low to observe a clear effect on the localization of the microinjected peroxisomal protein.

It is possible that ATP and GTP are both necessary for protein import, but at distinct energy-requiring steps. When a nonhydrolyzable analog of GTP was added to import reactions in the presence of 5 mm ATP, the level of GLO import was lower than in reactions containing ATP only, but higher than the import seen with GTP--S alone (Fig. 6). This reduction in the amount of GLO imported in an ATP-dependent manner indicates that there is either a common NTP-binding site for which ATP and GTP compete, or that the addition of GTP--S inhibits a separate, GTP-requiring import step. At this time we are unable to distinguish between these two options. It is likely that ATP (and possibly GTP as well) has multiple roles and acts at several different steps along the import pathway. This will make it more complicated to determine exactly how each nucleotide is involved in peroxisomal protein import.

Some protein transport systems also require a PMF for maximal efficiency of translocation (Pfanner and Neupert, 1986; Yamane et al., 1987; Wong and Buckley, 1989; Cline et al., 1992; Theg and Scott, 1993), but it is not clear whether a PMF is present across the peroxisomal membrane. Our results, using a broad range of ionophores, suggest that a PMF is involved in peroxisomal protein transport, although it is not absolutely required (Fig. 7). Each of the ionophores tested consistently reduced the amount of GLO imported to approximately 50 to 60% of control import (in the absence of ionophores), indicating that these ionophores inhibited protein import but did not abolish the process. It appears that this inhibition is not caused by decreased efficiency of import; GLO import in the presence of ionophores does not recover even after longer reaction times (data not shown). It may be that a PMF-requiring factor is depleted in the presence of ionophores such that translocation into the matrix is compromised. Alternatively, the pH of the peroxisome matrix may be important for optimal protein import; addition of ionophores (except valinomycin) would make it difficult to maintain a constant pH in the matrix, resulting in lower levels of peroxisomal protein import. A proton-ATPase on the peroxisome membrane may be responsible for generating and maintaining the acidic matrix of yeast peroxisomes (Douma et al., 1987; Nicolay et al., 1987; del Valle et al., 1988; Waterham et al., 1990); however, neither an ATPase nor an acidic matrix has yet been described for plant peroxisomes.

There are two additional factors that must be considered. First, it is important to note that low concentrations of nigericin or valinomycin did not inhibit GLO import in vitro (Fig. 7A). There was slightly greater inhibition of import when nigericin and valinomycin were added together, but the results were not strictly additive (data not shown). Nicolay et al. (1987) found that 1 to 2 µm nigericin and valinomycin together was sufficient to dissipate the pH gradient across yeast peroxisomal membranes. GLO import into plant peroxisomes was clearly inhibited by 5 to 10 µm nigericin (Fig. 7A). When used at 10 µm, neither valinomycin nor nigericin inhibited in vitro protein transport into rat liver peroxisomes, although no quantitation of these results was presented (Imanaka et al., 1987). In most systems, nigericin and valinomycin are expected to be active at very low concentrations, i.e. 1 µm or less (Pfanner and Neupert, 1986; Cline et al., 1992; Theg and Scott, 1993). Concentrations of nigericin or valinomycin greater than 10 µm may have nonspecific or surfactant effects (Reed, 1979).

Second, there may be some differences in the ways in which peroxisomes from different organisms respond to ionophores. When the total PMF was collapsed by 10 µm CCCP, protein import into plant peroxisomes was inhibited (Fig. 7B). Wendland and Subramani (1993) found that 10 µm CCCP does not inhibit import into peroxisomes in semipermeabilized mammalian cells; Imanaka et al. (1987) observed no inhibition of in vitro protein transport into rat liver peroxisomes in the presence of 10 µm CCCP. Neither group presented any quantitation of these results. CCCP may have more effect on yeast peroxisomes, although it is difficult to know for sure because higher concentrations of CCCP have been used. The assembly of alcohol oxidase is prevented by 25 µm CCCP, leading the authors to conclude that a PMF is required for the import and maturation of this yeast protein (Bellion and Goodman, 1987). Using a ³¹P NMR assay, Nicolay et al. (1987) found that the pH gradient across yeast peroxisomal membranes is destroyed by 2 mm CCCP.

Thus, it may be that a single component of the PMF alone does not affect peroxisomal protein import in plants; neither nigericin nor valinomycin at low (active) concentrations inhibited GLO import (Fig. 7A). However, even 10 µm CCCP showed maximal inhibition of GLO import (Fig. 7B), suggesting that collapsing the total PMF decreases protein import into peroxisomes in vitro. It seems unlikely that the PMF is directly providing necessary energy for the translocation event. A secondary role for a PMF, such as in maintaining a pH gradient across the membrane, may be responsible for the consistent level of inhibition we observed in the presence of each ionophore. It is not possible to conclude anything definitive based solely on results provided by experiments with a single ionophore at a single concentration. Variables such as the size of the compartment and the magnitude of the pH gradient across the membrane may also influence the behavior of individual ionophores (Reed, 1979). We have tested the effects on peroxisomal protein import of a wide range of ionophores at many concentrations, and we conclude that a PMF appears to play a role in peroxisomal protein import, perhaps through an indirect effect, but that the energy of a PMF is not absolutely required for protein translocation.

In a continuing effort to understand the cellular requirements and mechanisms of higher plant peroxisomal protein import, we have extensively characterized the energy requirements for GLO import into glyoxysomes in vitro. We have firmly established that energy from NTPs is required during protein translocation in peroxisomes. However, future studies are needed to define exactly where and how this energy is being used. Detailed investigations may lead to mechanistic models that include energy-requiring cytosolic factors and/or peroxisome-specific factors that are necessary to translocate peroxisomal proteins through the lipid bilayer.

	FOOTNOTES

   Received June 18, 1997; accepted October 8, 1997.

	ABBREVIATIONS

Abbreviations: A23187, calcinomycin. AMP-PCP, methylene adenosine 5-triphosphate. AMP-PNP, 5-adenylylimidodiphosphate. CCCP, carbonyl cyanide m-chloro-phenylhydrazone. GLO, glycolate oxidase. NTP, nucleotide triphosphate. PMF, protonmotive force.

	ACKNOWLEDGMENTS

We thank Jason Brickner, Wendy Crookes, Yan Lin, and Aaron Liepman for many helpful discussions. Olivia Bottum and Jessica McHie provided excellent technical assistance. Drs. Charles Yocum and Eran Pichersky contributed useful advice and comments during the preparation of the manuscript.

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