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J. Biol. Chem., Vol. 277, Issue 42, 40142-40147, October 18, 2002

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ATP Binding/Hydrolysis by and Phosphorylation of Peroxisomal ATP-binding Cassette Proteins PMP70 (ABCD3) and Adrenoleukodystrophy Protein (ABCD1)^*

Arowu R. Tanaka^§, Kouichi Tanabe^§, Masashi Morita^¶, Mikinori Kurisu^¶, Yoshinori Kasiwayama^¶, Michinori Matsuo, Noriyuki Kioka, Teruo Amachi, Tsuneo Imanaka^¶, and Kazumitsu Ueda

From the  Laboratory of Cellular Biochemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan and ^¶ Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan

Received for publication, May 23, 2002, and in revised form, August 5, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The 70-kDa peroxisomal membrane protein (PMP70) and adrenoleukodystrophy protein (ALDP), half-size ATP-binding cassette transporters, are involved in metabolic transport of long and very long chain fatty acids into peroxisomes. We examined the interaction of peroxisomal ATP-binding cassette transporters with ATP using rat liver peroxisomes. PMP70 was photoaffinity-labeled at similar efficiencies with 8-azido-[-³²P]ATP and 8-azido-[-³²P]ATP when peroxisomes were incubated with these nucleotides at 37 °C in the absence Mg²⁺ and exposed to UV light without removing unbound nucleotides. The photoaffinity-labeled PMP70 and ALDP were co-immunoprecipitated together with other peroxisomal proteins, which also showed tight ATP binding properties. Addition of Mg²⁺ reduced the photoaffinity labeling of PMP70 with 8-azido-[-³²P]ATP by 70%, whereas it reduced photoaffinity labeling with 8-azido-[-³²P]ATP by only 20%. However, two-thirds of nucleotide (probably ADP) was dissociated during removal of unbound nucleotides. These results suggest that ATP binds to PMP70 tightly in the absence of Mg²⁺, the bound ATP is hydrolyzed to ADP in the presence of Mg²⁺, and the produced ADP is dissociated from PMP70, which allows ATP hydrolysis turnover. Properties of photoaffinity labeling of ALDP were essentially similar to those of PMP70. Vanadate-induced nucleotide trapping in PMP70 and ALDP was not observed. PMP70 and ALDP were also phosphorylated at a tyrosine residue(s). ATP binding/hydrolysis by and phosphorylation of PMP70 and ALDP are involved in the regulation of fatty acid transport into peroxisomes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

ATP-binding cassette (ABC)¹ superfamily proteins are composed of two homologous halves, each of which typically contains six transmembrane  helices and a nucleotide binding fold (NBF). Prominent members of eukaryotic ABC superfamily proteins, such as the multidrug efflux pump MDR1 (ABCB1) and the cystic fibrosis transmembrane conductance regulator CFTR (ABCC7), are full size and contain 12 transmembrane  helices and 2 NBFs. On the other hand, most of the organelle ABC superfamily proteins, such as antigen transporters TAP1 (ABCB1) and TAP2 (ABCB2) on endoplasmic reticulum membranes and peroxisomal ABC proteins, are half size and contain six transmembrane  helices and one NBF.

To date, four peroxisomal ABC proteins have been identified in mammalian peroxisomes: the 70-kDa peroxisomal membrane protein (PMP70, ABCD3), adrenoleukodystrophy protein (ALDP, ABCD1), ALDP-related protein (ALDRP, ABCD2), and PMP70-related protein (P70R, ABCD4) (1-7). These half-size ABC proteins are supposed to work after dimerization. Disruption of the half-size ABC protein gene of Saccharomyces cerevisiae PAT1 (Pxa1) or PAT2 (Pxa2), whose products were identified on peroxisomes, resulted in impaired growth in oleic acid medium, suggesting that Pat1p and Pat2p function as heterodimers (8-11). Indeed, Liu et al. (12) have shown that homo- as well as heterodimerization occurred among the ALDP, ALDRP, and PMP70 by using the yeast two-hybrid system and co-immunoprecipitation.

Because the defect of the ALDP gene resulted in impaired peroxisomal -oxidation and accumulation of very long chain fatty acids (VLCFAs) (13-17), and because overexpression of PMP70 in Chinese hamster ovary cells increased -oxidation of palmitic acid (18), ALDP and PMP70 are suggested to be involved in ATP-dependent import of VLCFAs and long chain fatty acids (LCFAs) into peroxisomes. Contreras et al. (19) reported that the NBF of ALDP was located on the cytoplasmic surface of peroxisomal membranes, and that the NBF released from peroxisomes by protease treatment bound to ATP (19). The NBFs of ALDP and PMP70, fused with the maltose-binding protein, were reported to be photoaffinity-labeled with 8-azido-[-³²P]ATP and showed ATPase activities (20). However, the ATP binding and hydrolysis properties of isolated NBF do not necessarily represent those of complete native proteins. In this study, we analyzed the interaction of peroxisomal ABC transporters with ATP using rat liver peroxisomes. We found that ATP bound to PMP70 and ALDP tightly and that PMP70 and ALDP formed a stable complex with other peroxisomal membranes proteins, which also showed tight ATP binding properties. Furthermore, ATP occluded to homo- and heterodimer of PMP70 and ALDP in the absence of Mg²⁺ was shown to be hydrolyzed in the presence of Mg²⁺. PMP70 and ALDP were also phosphorylated at a tyrosine residue(s).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- 8-Azido-[-³²P]ATP and 8-azido-[-³²P]ATP (18-22 Ci/mmol) were purchased from Affinity Labeling Technologies (Lexington, KY). [-³²P]ATP (800 Ci/mmol) was obtained from ICN Biomedicals (Costa Mesa, CA). H-89 and CKI-7 were from Seikagaku Co. (Tokyo, Japan). Protein G-agarose, H-7, and lavendustin A were from Sigma. Rabbit anti-PMP70 antibody was raised against the COOH-terminal 15 amino acids of rat PMP70 (21). Rabbit anti-ALDP antibody raised against the COOH-terminal 24 amino acids of human ALDP (22) was kindly provided by Dr. T. Yamada (Kyushu University).

Preparation of Rat Liver Peroxisomes-- Peroxisomes were purified from rat liver by differential centrifugation in a buffer containing sucrose followed by isopycnic centrifugation in Nycodentz (23) with some modifications. About 1.0 ml of a light mitochondrial fraction was layered onto a 10 ml linear Nycodentz gradient (density span, 1.15-1.25 g/ml) in a Beckman NVT65T rotor (Beckman, Fullerton, CA). The gradient rested on a 0.5-ml cushion of 1.3 g/ml Nycodentz. All solutions contained 0.25 M sucrose, 1 mM EDTA, 0.1% (v/v) ethanol, and 5 mM Hepes-KOH, pH 7.4. The centrifugation was carried out at 51,200 rpm (193,000 × g) for 120 min at 4 °C. Fractions of ~1.0 ml were collected in preweighed microtubes, and the density of each fraction was determined by refractometry. The peroxisomal fraction was identified by the density and the distribution of catalase (18).

Photoaffinity Labeling of Peroxisome Proteins with 8-Azido-[-- ³²P]ATP and 8-Azido-[-³²P]ATPRat liver peroxisomes (30 µg) were incubated with 50 µM 8-azido-[-³²P]ATP or 8-azido-[-³²P]ATP, 2 mM ouabain, 0.1 mM EGTA, and 40 mM Tris-Cl, pH 7.5, in a total volume of 6 µl for 10 min at 37 °C in the presence of 3 mM MgSO₄ or EDTA. After unbound nucleotides were removed before UV irradiation, 500 µl of ice-cold TEM buffer (40 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, and 1 mM MgSO₄) or TEE buffer (40 mM Tris-HCl, pH 7.5, 0.1 mM EGTA, and 1.5 mM EDTA) was added to the samples, and the supernatants were removed from peroxisome pellets after centrifugation (15,000 × g, 10 min, 4 °C). This wash step was done again, and then the peroxisomes were resuspended in 8 µl of TEM buffer and exposed to UV light for 3 min (at 254 nm, 5.5 mW/cm²) on ice. The peroxisomes were solubilized, and PMP70 and ALDP were immunoprecipitated as described in the following section. Samples were electrophoresed on a 7% SDS-polyacrylamide gel and autoradiographed. 8-Azido-[³²P]ATP bound to PMP70 and ALDP was measured by scanning with a radioimaging analyzer (BAS2000; Fuji Photo Film Co.). Experiments were carried out at least four times.

Phosphorylation of Peroxisomal Membrane Proteins-- All procedures were carried out at 4 °C unless otherwise stated. Peroxisomes (200 µg) were suspended in 200 µl of a phosphorylation buffer (25 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 2 mM MnCl₂, 0.4 mM EDTA, 1 mM dithiothreitol, 2 mM orthovanadate, 10 mM NaF, 5 mM -glycerophosphate, and 10 µM ATP) containing a protease inhibitor mixture (1 mM phenylmethylsulfonyl fluoride and 10 µg/ml antipain, chymostatin, leupeptin, and pepstatin A). 50 µCi of [-³²P]ATP was then added to the mixture and incubated for 20 min at 30 °C. In some experiments, protein kinase inhibitors at a concentration of 50 µM were preincubated for 10 min before the addition of [-³²P]ATP. After centrifugation at 15,000 × g for 5 min, the pellet was washed with the same buffer and suspended with 600 µl of RIPA buffer. Immunoprecipitates, prepared as described below, were analyzed on a 7-12% SDS-polyacrylamide gradient gel and autoradiographed.

Immunoprecipitation of PMP70 and ALDP-- The peroxisome pellets were solubilized with 100 µl of RIPA buffer (20 mM Tris-HCl, pH 7.5, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 0.15 M NaCl, 10 µg/ml leupeptin, and 100 µg/ml (p-amidinophenyl)methanesufonyl fluoride). The lysates were kept on ice or rotated for 1 h, and the debris was removed by centrifugation at 15,000 × g for 5 min. The supernatants were precleared with an appropriate amount of protein G-agarose that was prewashed in RIPA buffer. After centrifugation at 10,000 × g for 10 s, the supernatant was incubated with 5 µl of anti-PMP70 or anti-ALDP antibodies for 1 h and then incubated with 20 µl of a 50% (v/v) suspension of protein G-agarose for 1 h.

Other Methods-- Protein and catalase were assayed as described previously (18, 21). Western blot analysis was done using ECL+Plus, a Western blotting detection system.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Specific Interaction of Peroxisomal Proteins with ATP-- To examine the interaction of peroxisomal membrane proteins with ATP, rat liver peroxisomes were incubated at 26 °C with 8-azido-[-³²P]ATP. Then, the peroxisomes were exposed to UV light after removing unbound 8-azido-[-³²P]ATP by washing with excess buffer. Several peroxisomal proteins between 60 and 100 kDa were found to be specifically photoaffinity-labeled (Fig. 1A, lane 1). The intensity of the bands decreased in the presence of an excess amount of ATP in a concentration-dependent manner (Fig. 1A, lanes 4-7), suggesting that ATP bound to these peroxisomal proteins specifically. Under these conditions, peroxisomal matrix proteins such as hydratase-dehydrogenase, acyl-CoA oxidase, catalase, and urate oxidase were not released from peroxisomal particles (data not shown). Indeed, all the photoaffinity-labeled proteins were recovered in the membrane pellet when the peroxisomes were separated into soluble and membrane fractions by the sodium carbonate procedure (24) (data not shown), suggesting that photoaffinity-labeled proteins were located on the peroxisomal membranes. Western hybridization of the blot, run in parallel, with anti-PMP70 or ALDP antibody showed a single band with a molecular mass of 65 or 75 kDa (Fig. 1A, lanes 2 and 3), which could correspond to one of the radiolabeled proteins.

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Specific interaction of peroxisomal proteins with ATP. A, rat liver peroxisomes were incubated with 50 µM 8-azido-[-32P]ATP in the absence (lanes 1 and 4) or presence of 1 (lane 5), 5 (lane 6), or 10 mM ATP (lane 7) at 26 °C for 10 min and exposed to UV light after removing unbound nucleotide by washing with excess buffer. Western hybridization of the blot was run in parallel with anti-PMP70 (lane 2) and anti-ALDP (lane 3) antibodies. B, rat liver peroxisomes were incubated with 50 µM 8-azido-[-32P]ATP at 37 °C for 10 min. UV irradiation was done after removing unbound nucleotides. Peroxisomes were solubilized with RIPA buffer, and immunoprecipitation was done with the preimmune antibody (lane 1), the anti-PMP70 antibody (lane 2), and anti-ALDP antibody (lane 3), respectively. Western hybridization of the blot was run in parallel with anti-PMP70 (lane 4) and anti-ALDP (lane 5) antibodies. The photoaffinity-labeled proteins were separated by electrophoresis on a 7% SDS-polyacrylamide gel and autoradiographed. Free 8-azido-[32P]ATP was seen below the molecular mass marker of 50 kDa in A. The band of ~45 kDa in B seems to be nonspecific because it was also detected with preimmune serum.

Identification of Photoaffinity-labeled ABC Proteins-- PMP70 is a major constituent of peroxisomal membranes of normal rat liver. As compared with the amounts of PMP70, ALDP, ALDRP, and P70R on the membranes, ALDP was about one-seventh the amount of PMP70, and ALDRP and P70R were roughly less than one-tenth the amount of ALDP.² To examine whether the photoaffinity-labeled 65- and 75-kDa proteins were PMP70 and ALDP, the proteins labeled with 8-azido-[-³²P] were immunoprecipitated with antibodies against PMP70 and ALDP (Fig. 1B). Both antibodies precipitated mainly four photoaffinity-labeled proteins from 65 to 90 kDa. The anti-PMP70 antibody mainly precipitated a photoaffinity-labeled 65-kDa protein. The photoaffinity-labeled 65-kDa protein was also precipitated by the anti-ALDP antibody, but less efficiently than by the anti-PMP70 antibody. The other three labeled proteins were precipitated at similar efficiencies by these antibodies. Western hybridization of the blot, run in parallel, with anti-PMP70 and anti-ALDP antibodies showed that photoaffinity-labeled 65- and 75-kDa proteins were PMP70 and ALDP, respectively (Fig. 1B, lanes 4 and 5). Photoaffinity labeling of these proteins was scarcely observed when peroxisomes were incubated with 8-azido-[-³²P]ATP at 0 °C and irradiated by UV light after removing unbound nucleotide (data not shown). These results suggest that PMP70 and ALDP bind to ATP tightly in a temperature-dependent manner and form a stable complex together with other peroxisomal membrane proteins, which also bind ATP.

ATP Binding Properties of PMP70 and ALDP-- To examine the ATP binding properties of PMP70 and ALDP, peroxisomes were incubated with 8-azido-[-³²P]ATP or 8-azido-[-³²P]ATP of the same specific radioactivities at 37 °C for 10 min in the presence or absence of Mg²⁺ and exposed to UV light before or after unbound nucleotides were removed by washing with excess buffer. Proteins were then immunoprecipitated with anti-PMP70 antibody (Fig. 2), and intensities of labeled PMP70 and ALDP were quantitated (Fig. 3). The 65-kDa PMP70 was photoaffinity-labeled at similar efficiencies with 8-azido-[-³²P]ATP and 8-azido-[-³²P]ATP (Fig. 2, lanes 1 and 5) when peroxisomes were incubated with nucleotides in the absence Mg²⁺ and exposed to UV light without removing unbound nucleotides. The addition of Mg²⁺ to the reaction mixture reduced photoaffinity labeling with 8-azido-[-³²P]ATP by 70% (lane 6 versus lane 5 in Fig. 2 and the left panel of Fig. 3), whereas it reduced photoaffinity labeling with 8-azido-[-³²P]ATP by only 20% (lane 2 versus lane 1 in Fig. 2 and the left panel of Fig. 3). These results suggest that ATP binds to PMP70 in the absence of Mg²⁺ and that -phosphate is released from ATP bound to PMP70 in the presence of Mg²⁺.

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Photoaffinity labeling of PMP70 and ALDP with 8-azido-[-32P]ATP and 8-azido-[- 32P]ATP. Rat liver peroxisomes were incubated with 8-azido-[-32P]ATP (lanes 1-4) or 8-azido-[-32P]ATP (lanes 5-8) of the same specific radioactivities at 37 °C for 10 min in the presence (lanes 2, 4, 6, and 8) or absence (lanes 1, 3, 5, and 7) of Mg2+ and exposed to UV light before (lanes 1, 2, 5, and 6) or after (lanes 3, 4, 7, and 8) removing unbound nucleotides by washing with excess buffer. Proteins were then immunoprecipitated with anti-PMP70 antibody. Free 8-azido-[32P]ATP was seen below the 50 kDa molecular mass marker.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   ATP occlusion and hydrolysis in PMP70 (left panel) and ALDP (right panel). 8-Azido-[32P]ATP bound to PMP70 and ALDP (a representative result was shown as Fig. 2) was measured by scanning with a radioimaging analyzer (BAS2000; Fuji Photo Film Co.). Relative photoaffinity labeling of PMP70 and ALDP is expressed as a percentage of that with 8-azido-[-32P]ATP in the absence of Mg2+ plus UV irradiation without removal of unbound nucleotides (shown in lane 1). The intensity of photoaffinity labeling of ALDP with 8-azido-[-32P]ATP in the absence of Mg2+ and UV irradiation without removal of unbound nucleotides (shown in lane 1) was about 20% of that of PMP70. Experiments were carried out at least four times, and the average values are represented with S.E.

When peroxisomes were incubated with the nucleotides in the absence of Mg²⁺ and exposed to UV light even after removal of unbound nucleotides, PMP70 was strongly photoaffinity-labeled with 8-azido-[-³²P]ATP (Fig. 2, lane 3). More than 60% of 8-azido-[-³²P]ATP remained bound to PMP70 during the washing step (lane 3 versus lane 1 in Fig. 2 and the left panel of Fig. 3). However, more than two-thirds of bound nucleotide was dissociated from PMP70 during the washing step when peroxisomes were reacted with the nucleotide in the presence of Mg²⁺ (lane 4 versus lane 2 in Fig. 2 and the left panel of Fig. 3). Experiments with 8-azido-[-³²P]ATP showed similar results in the absence of Mg²⁺ (lane 7 versus lane 5 in Fig. 2 and the left panel of Fig. 3), although they fluctuated in different experiments. These results suggest that (i) ATP binds to PMP70 tightly in the absence of Mg²⁺, (ii) the bound ATP is hydrolyzed to ADP in the presence of Mg²⁺, (iii) the hydrolysis product, 8-azido-ADP, is retained before washing with excess buffer, and (iv) 8-azido-ADP is dissociated from PMP70 during the washing step. Dissociation of 8-azido-ADP from PMP70 suggests a low binding affinity of ADP to PMP70.

Properties of photoaffinity labeling of ALDP were similar to those of PMP70, although the intensities of labeled bands of ALDP were about one-fifth of those of PMP70 (Fig. 2 and the right panel of Fig. 3). ATP bound to ALDP tightly in the absence of Mg²⁺, and that bound ATP was hydrolyzed in the presence of Mg²⁺, and 8-azido-ADP was dissociated from ALDP.

No Vanadate-induced Nucleotide Trapping in PMP70 or ALDP-- It is well known that MDR1 and MRP1 (ABCC1), the multidrug transporters, trap ADP in the presence of orthovanadate, an analog of phosphate, and Mg²⁺. As a result, the labeling intensity of these proteins by photoaffinity labeling with 8-azido-[-³²P]ATP was increased when membranes containing these proteins were reacted with the nucleotide in the presence of orthovanadate and Mg²⁺ (25-27). Based on this observation, we tried to examine vanadate-induced nucleotide trapping of PMP70 and ALDP. However, no increase of photoaffinity labeling of PMP70 or ALDP was observed when peroxisomes were incubated with 8-azido-[-³²P]ATP in the presence of orthovanadate and Mg²⁺ and exposed to UV light before (data not shown) or after unbound nucleotide was removed by washing with excess buffer (Fig. 4).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   No vanadate-induced nucleotide trapping in PMP70 or ALDP. Rat liver peroxisomes were incubated with 8-azido-[-32P]ATP at 37 °C for 10 min in the presence of 3 mM Mg2+ and 1 mM sodium vanadate and then UV-irradiated after unbound nucleotides were removed by washing with excess buffer. Proteins were then immunoprecipitated with anti-PMP70 antibody.

Phosphorylation of PMP70 and ALDP-- Because PMP70 was labeled strongly with 8-azido-[-³²P]ATP (Fig. 2) and because some members of the ABC superfamily have been reported to be phosphorylated, we examined whether PMP70 and ALDP were phosphorylated. Peroxisomes were incubated with [-³²P]ATP for 20 min at 30 °C in the presence of Mg²⁺, and immunoprecipitation was performed with either anti-PMP70 antibody or anti-ALDP antibody. As shown in Fig. 5A, both antibodies precipitated two phospho-proteins corresponding to PMP70 and ALDP (lanes 2 and 7). Co-precipitation of PMP70 and ALDP was revealed by immunoprecipitation followed by Western blot analysis. As shown in Fig. 5B, after immunoprecipitation with anti-PMP70 antibody, PMP70 and ALDP were detected in the immunoprecipitate by Western blotting of either anti-PMP70 antibody or anti-ALDP antibody (lane 2 of the left and right panels). Both proteins were also detected in the immunoprecipitate with anti-ALDP antibody (lane 3 of the left and right panels). In addition, the phosphorylation of PMP70 and ALDP was markedly inhibited in the presence of lavendustin A, a tyrosine kinase inhibitor, but not in the presence of H-7 (a protein kinase C inhibitor), H-89 (a protein kinase A inhibitor), and CKI-7 (a casein kinase I inhibitor) (Fig. 5A, lanes 3-6). These results suggest that PMP70 and ALDP were tyrosine-phosphorylated and existed as a complex.

View larger version (41K): [in this window] [in a new window]   Fig. 5.   Phosphorylation of PMP70 and ALDP. A, rat liver peroxisomes were incubated with [-32P]ATP at 30 °C for 20 min in the presence of MgCl2. After solubilization with RIPA buffer, proteins were immunoprecipitated with preimmune antibody (lane 1), anti-PMP70 antibody (lanes 2-6), and anti-ALDP antibody (lane 7), and labeled proteins were analyzed by SDS-PAGE followed by autoradiography. Protein kinase inhibitors (50 µM) H-7 (lane 3), H-89 (lane 4), lavendustin A (lane 5), and CKI-7 (lane 6) were preincubated for 10 min and included in the reaction mixture for the labeling period. B, co-immunoprecipitation of PMP70 and ALDP. Immunoprecipitates with preimmune antibody (lane 1), anti-PMP70 antibody (lane 2), and anti-ALDP antibody (lane 3) as described in A were electrophoresed and subjected to immunoblotting using anti-PMP70 antibody (left panel) or anti-ALDP antibody (right panel).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Peroxisomes are involved in oxidative degradation of LCFAs and VLCFAs. LCFAs are known to be activated into CoA derivatives outside of peroxisomes by a LCFA-CoA synthetase before peroxisomal -oxidation (28). Recently, we demonstrated that overexpression of PMP70 in Chinese hamster ovary cells increased the rate of palmitic acid -oxidation into peroxisomes by 2- to 3-fold and that the peroxisomes prepared from the cells stimulated palmitoyl-CoA -oxidation. These results suggest that PMP70 is involved in the transport of LCFA-CoA across peroxisomal membranes (18). On the other hand, VLCFAs are thought to be activated into CoA derivatives inside of peroxisomes because VLCFA-CoA synthetase locates in peroxisomes facing its active site to the lumimal side (16). From several laboratories, it has been reported that oxidation of VLCFAs was reduced by 60-80% in ALD fibroblasts and that oxidation was restored by transfection of ALDP cDNA into the cells, suggesting that ALDP is involved in the metabolism of VLCFA. ALDP could transport VLCFAs across peroxisomal membranes (13-18).

ATP was shown to bind to the NBF of ALDP released from peroxisomes by trypsin digestion (19). It was also reported that ATP bound to and was hydrolyzed by the recombinant NBFs of PMP70 and ALDP (20). However, the properties of these proteins against ATP in native states have not yet been characterized. In the present study, we have used photoaffinity labeling of peroxisomes with 8-azido-[-³²P]ATP and 8-azido-[-³²P]ATP under different conditions, and we have found the following characteristics of native PMP70 and ALDP. (i) 8-azido-ATP is occluded in PMP70 and ALDP in the absence of Mg²⁺ and is hydrolyzed in the presence of Mg²⁺. (ii) The hydrolysis product, 8-azido-ADP, is retained in PMP70 and ALDP before removing the excess nucleotides. (iii) PMP70 or ALDP does not form a stable inhibitory complex E-MgADP-Vi after hydrolysis in the presence of orthovanadate to trap 8-azido-ADP. (iv) PMP70 and ALDP exist as homo- or heterodimer and form a stable complex together with other peroxisomal membrane proteins, which also showed tight ATP binding properties. (v) PMP70 and ALDP are tyrosine-phosphorylated by some protein kinase(s) on peroxisomal membranes.

ABC proteins have divergent functions and can be classified as transporters, channels, and regulators, although their predicted domain structures and amino acid sequences of their NBFs in a region of about 200 amino acids are well conserved (29, 30). ABC proteins show various properties in ATP binding and hydrolysis, which might explain the divergent functions of ABC proteins. We compared the nucleotide binding/hydrolysis properties of PMP70 and ALDP with those of other eukaryotic ABC proteins.

It has been reported that 8-azido-ATP is occluded in the absence Mg²⁺ in NBF1 of CFTR (ABCC7) (31-33). This binding is followed by minimal Mg²⁺-dependent hydrolysis and retention of the hydrolysis product, 8-azido-ADP, but orthovanadate does not stabilize 8-azido-ADP binding. 8-Azido-ATP occlusion in the absence of Mg²⁺ was also found in NBF1 of SUR1 and SUR2 (ABCC8, 9) (34-36), channel regulatory subunits of the ATP-sensitive K⁺ channel. However, it has not been clear whether NBF1 hydrolyzes ATP because NBF1 was photoaffinity-labeled with both 8-azido-[-³²P]ATP and 8-azido-[-³²P]ATP in the presence of Mg²⁺ under the conditions examined (35, 37). These observations suggest that the nucleotide binding/hydrolysis properties of PMP70 and ALDP resemble those of NBF1 of CFTR and partially resemble those of NBF1 of SUR.

Although the nucleotide binding/hydrolysis properties of PMP70 and ALDP resemble those of the NBF1s of CFTR and SUR1, PMP70 and ALDP are different in the combination of two NBFs from CFTR and SUR. In CFTR, in contrast to NBF1, 8-azido-ATP is not occluded at NBF2. 8-Azido-ATP, bound to NBF2, is hydrolyzed in the presence of Mg²⁺, and 8-azido-ADP dissociates immediately (31-33). NBF2 of SUR was photoaffinity-labeled with 8-azido-[-³²P]ATP but not with 8-azido-[-³²P]ATP, suggesting that NBF2 also hydrolyzes ATP immediately (35, 37). Thus, CFTR and SUR have two NBFs with different nucleotide binding/hydrolysis properties in a molecule. However, PMP70 works as a homodimer, and PMP70/ALDP works as a heterodimer that has two NBFs with similar nucleotide binding/hydrolysis properties.

The prominent eukaryotic ABC protein, which has two NBFs with similar nucleotide binding/hydrolysis properties, is MDR1, a primary active xenobiotics efflux pump. Both NBFs of MDR1 hydrolyze ATP and trap ADP in the presence of orthovanadate equivalently. One ADP-Vi has been reported to be trapped in a molecule (38), therefore the NBF at which the initial hydrolysis occurs is supposed to be chosen randomly. Two NBFs of PMP70 homodimer and PMP70/ALDP heterodimer would hydrolyze ATP equivalently. This combination of two NBFs may cause PMP70/PMP70 and PMP70/ALDP to be primary active transporters like MDR1, but not ion channels like CFTR. Recently, Kashiwayama et al. (39) reported that the cycle of ATP binding and hydrolysis induced conformational changes in PMP70.

The properties of ATP binding and the kinetics of its hydrolysis might be important to the transport of substrates by PMP70 and ALDP. Recently, we succeeded in purifying ABCA1, which is suggested to be involved in cholesterol homeostasis, and found that purified ABCA1 showed ATPase activity.³ ABCA1 shows 8-azido-ATP occlusion, but no vanadate-induced nucleotide trapping was observed (40).⁴ The kinetics of ATP hydrolysis of ABC proteins involved in lipid homeostasis, such as PMP70, ALDP, and ABCA1, could be different from those of MDR1, a xenobiotic transporter.

With regard to phosphorylation of ABC proteins, CFTR, channel gating itself, is known to be regulated by phosphorylation. Both TAP1 and TAP2, which are involved in major histocompatibility complex class I antigen processing and presentation, are phosphorylated under physiological conditions, and their function is modulated by reversible TAP phosphorylation (41). ATPase activities of CFTR have also been reported to be regulated by protein phosphorylation (42). Interestingly, PMP70 and ALDP were found to be phosphorylated by incubating peroxisomes with [-³²P]ATP (Fig. 5). The phosphorylation of PMP70 and ALDP was markedly inhibited in the presence of lavendustin A, a tyrosine kinase inhibitor. PMP70 and ALDP contain a few tyrosine residues that are potential phosphorylation sites in their amino acid sequence, and one of them is positioned in the cytosolic loop between transmembrane domains 4 and 5. Because purified peroxisomes were used for the experiment, a protein kinase(s) on the peroxisomal membranes is suggested to be involved in the tyrosine phosphorylation of PMP70 and ALDP. The phosphorylation mechanism of PMP70 and ALDP was distinct from those of CFTR and MDR1, which depend on protein kinase A. The tyrosine phosphorylation of PMP70 and ALDP could be involved in the modulation of their functions.

Another important point addressed in the present study is the complex of PMP70 and ALDP together with other peroxisomal proteins. A stable complex of PMP70 and ALDP was supported by the evidence that PMP70 and ALDP were co-immunoprecipitated even after solubilization with RIPA buffer. Western blot analysis after immunoprecipitation (Fig. 5) revealed that more than half of ALDP formed a complex with PMP70 and that at least one-tenth of PMP70 formed a complex with ALDP. The amount of ALDP is about one-seventh that of PMP70 in rat peroxisome,² and the intensity of photoaffinity labeling of ALDP with 8-azido-[-³²P]ATP (Fig. 3, lane 1) was about 20% of that of PMP70. Therefore, the labeling stoichiometry of incorporated azido-ATP per mole of PMP70 and ALDP would be roughly calculated to be 1:1. Interestingly, other peroxisomal proteins contained in PMP70·ALDP complexes were also photoaffinity-labeled with 8-azido-[-³²P]ATP and 8-azido-[-³²P]ATP (Figs. 1 and 2) but were not phosphorylated (Fig. 5). Among them, a 90-kDa protein was photoaffinity-labeled most strongly. LCFA-CoA and VLCFA-CoA synthetases were candidates for the proteins. However, Western blotting with antibodies against these enzymes did not recognize the photoaffinity-labeled proteins (data not shown). ALDRP and P70R are not likely to be the strongly photoaffinity-labeled proteins because their molecular masses are lower than that of ALDP and because their amounts in peroxisomal membranes are less than one-tenth that of ALDP.² Although we have not yet identified the proteins, one of the photoaffinity-labeled proteins could be a tyrosine kinase, which phosphorylates PMP70 and ALDP.

It has been reported that tapasin, class I heterodimers, and a protein kinase are contained in the phosphorylated TAP-containing complexes and that phosphorylation is involved in the formation of protein complexes (41). Recently, it has been shown that several proteins involved in the biogenesis of peroxisomes interacted with each other and formed stable protein complexes in the peroxisomal membranes (43). The complex solubilized by the detergents showed a molecular mass of ~400 kDa on sucrose gradient and ~700 kDa on blue-native PAGE. Previously, we found that PMP70 solubilized with peroxisomal membranes with 0.5% C₁₂E₉ showed two peaks with molecular mass of ~500 kDa and 160-200 kDa by gel filtration on superose 6 prep HR15/50 column (44). It is possible that the former is a complex of several proteins including PMP70, ALDP, and other proteins and that the latter is homo- or heterodimer of PMP70 and ALDP.

In this study, we found that PMP70 and ALDP formed a stable complex together with some peroxisomal proteins and bound to and hydrolyzed ATP with a unique manner. Furthermore, we found that PMP70 and ALDP were tyrosine-phosphorylated. Formation of the complex and phosphorylation of PMP70 and ALDP are expected to be involved in the regulation of fatty acid transport into peroxisomes.

	ACKNOWLEDGEMENT

We thank Dr. T. Yamada for anti-ALDP antibody.

	FOOTNOTES

^* This work was supported by Grant-in-aid for Scientific Research 10217205 on Priority Areas ABC Proteins from the Ministry of Education, Science, Sports, and Culture of Japan and by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists (to K. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Both authors contributed equally to this work.

To whom correspondence should be addressed. Tel.: 81-75-753-6105; Fax: 81-75-753-6104; E-mail: uedak@kais.kyoto-u.ac.jp.

Published, JBC Papers in Press, August 9, 2002, DOI 10.1074/jbc.M205079200

² D. Tomimoto, Y. Murasaki, M. Morita, and T. Imanaka, unpublished observations.

³ K. Takahashi, Y. Kimura, M. Matsuo, and K. Ueda, manuscript in preparation.

⁴ A. R. Tanaka and K. Ueda, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: ABC, ATP-binding cassette; NBF, nucleotide binding fold; PMP70, the 70-kDa peroxisomal membrane protein; ALDP, adrenoleukodystrophy protein; ALDRP, ALDP-related protein; P70R, PMP70-related protein; VLCFA, very long chain fatty acid; LCFA, long chain fatty acid; RIPA, radioimmune precipitation assay.

	REFERENCES

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