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Originally published In Press as doi:10.1074/jbc.M000555200 on April 14, 2000

J. Biol. Chem., Vol. 275, Issue 27, 20399-20405, July 7, 2000
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The Effect of Lipid Environment and Retinoids on the ATPase Activity of ABCR, the Photoreceptor ABC Transporter Responsible for Stargardt Macular Dystrophy*

Jinhi Ahn, Jason T. Wong, and Robert S. MoldayDagger

From the Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

Received for publication, January 27, 2000, and in revised form, March 17, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ABCR is a photoreceptor-specific ATP-binding cassette transporter that has been linked to various retinal diseases, including Stargardt macular dystrophy, and implicated in retinal transport across rod outer segment (ROS) membranes. We have examined the ATPase and GTPase activity of detergent-solubilized and reconstituted ABCR. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonic acid-solubilized ABCR had ATPase and GTPase activity (Km ~75 µM; Vmax ~ 200 nmol/min/mg) that was stimulated 1.5-2-fold by all-trans-retinal and dependent on phospholipid and dithiothreitol. The Km for ATP decreased to ~25 µM after reconstitution, whereas the Vmax was strongly dependent on the lipid used for reconstitution. ABCR reconstituted in ROS phospholipid had a Vmax for basal and retinal activated ATPase activity that was 4-6 times higher than for ABCR in soybean or brain phospholipid. This enhanced activity was mainly due to the high phosphatidylethanolamine (PE) content of ROS membranes. PE was also required for retinoid-stimulated ATPase activity. ATPase activity of ABCR was stimulated by the addition of N-retinylidene-PE but not the reduced derivative, retinyl-PE. ABCR expressed in COS-1 cells also exhibited retinal-stimulated ATPase activity similar to that of the native protein. These results support the view that ABCR is an active retinoid transporter, the nucleotidase activity of which is strongly influenced by its lipid environment.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ABCR, also known as the rim protein, is an abundant high molecular weight membrane glycoprotein found in photoreceptor outer segment disc membranes (1-3). Primary structural analysis indicates that ABCR is a member of the superfamily of ATP-binding cassette proteins that typically function in the active transport of various substances across cell membranes (2, 4, 5). Like other eukaryotic ABC transporters, such as P-glycoprotein and cystic fibrosis transmembrane conductance regulator, ABCR is organized in two homologous, tandem-arranged halves, each containing a cytoplasmic nucleotide binding domain preceded by a hydrophobic domain consisting of multiple membrane spanning segments.

The gene encoding ABCR has been implicated in a variety of retinal degenerative diseases associated with a loss in vision. Over 80 different mutations in ABCR have been found in patients with Stargardt macular dystrophy, a juvenile onset, autosomal recessive disease characterized by decreased visual acuity, bilateral atrophy of the central (macula) retina, accumulation of fluorescent yellow deposits in the retinal pigment epithelium, and delayed dark adaptation (4, 6-8). Mutations in ABCR have also been linked to individuals with fundus flavimaculatus, a late-onset variant of Stargardt macular dystrophy (9), autosomal recessive retinitis pigmentosa (10), cone-rod dystrophy (11), and age-related macular dystrophy (12, 13).

The substrate(s) transported by ABCR is not yet known. However, localization of ABCR to photoreceptor outer segment disc membranes led to the initial suggestion that ABCR may function to transport retinoids across the disc membrane (2-4). The putative role of ABCR as a retinal transporter is supported by two recent studies. In one study, purified ABCR reconstituted into brain lipid vesicles displayed ATPase activity that was stimulated up to 5-fold by retinal (14). Substrates that are actively transported across cell membranes by P-glycoprotein, histidine permease, multidrug resistance-associated protein, and the canalicular multispecific organic anion transporter (cMOAT/multidrug resistance-associated protein 2) also activate the ATPase activity of these proteins (15-22). In a second study, an abcr knockout mouse has been produced that displays delayed dark adaptation, a light-dependent increase in all-trans-retinal and protonated N-retinylidene-PE1 in ROS, and an accumulation of the pyridinium bis-retinoid compound, A2E, in photoreceptors and retinal pigment epithelial cells (23). Many of these characteristics are observed in individuals with Stargardt disease and are consistent with an accumulation of retinal-PE derivatives in photoreceptor membranes presumably due to defective transport of retinoid compounds across disc membranes.

As part of an ongoing study to characterize the structural and functional properties of ABCR, we have investigated the effect of various phospholipids and retinoid compounds on the nucleotidase activity of ABCR from ROS membranes. Here, we report that detergent-solubilized and reconstituted ABCR displays both ATPase and GTPase activity that is strongly influenced by the lipid environment and the presence of retinoid compounds. We also show that ABCR expressed in monkey kidney COS-1 cells exhibits retinal-stimulated ATPase activity comparable to that of the native protein.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- All-trans-retinal, all-trans-retinol, reduced glutathione, soybean phospholipid, SDPC, CHAPS, and N-ethylmaleimide were purchased from Sigma. Citral, 2,4-all-trans-nonadienal, 2-trans-6-cis-nonadienal, and nonylaldehyde were from Aldrich. 11-cis-Retinal was a generous gift of Dr. Rosalie Crouch. The following phospholipids were obtained from Avanti Polar Lipids: SDPE, DOPE, DOPC, and brain polar lipid extract.

Solutions-- The composition of buffers was as follows: homogenization buffer, 20 mM Tris acetate, pH 7.4, 10 mM taurine, 10 mM glucose, 0.25 mM MgCl2, 20% (w/v) sucrose; column buffer (Buffer C), 50 mM HEPES, pH 7.5, 0.1 M NaCl, 10 mM CHAPS, 1 mg/ml sonicated soybean phospholipid, 1 mM DTT, 3 mM MgCl2, 10% (v/v) glycerol; Buffer B, 50 mM HEPES, pH 7.5, 0.1 M NaCl, 0.5 mM EDTA, 10 mM CHAPS, 1 mM DTT, 10% glycerol; dialysis buffer (Buffer D1), 10 mM HEPES, pH 7.5, 0.1 M NaCl, 1 mM DTT; Buffer D2, 50 mM HEPES, pH 7.5, 0.1 M NaCl, 1 mM DTT, 3 mM MgCl2, 10% glycerol; and reconstitution buffer (Buffer E), 25 mM HEPES, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol. Retinoid compounds were dissolved in ethanol and diluted at least 200-fold in Buffer C or Buffer E (final ethanol concentration, <0.5% in reaction). All-trans-retinal, 11-cis-retinal, and all-trans-retinol concentrations were determined spectrophotometrically in ethanol using molar extinction coefficients of 42,880 (lambda max = 383 nm), 24,935 (lambda max = 380 nm), and 52,770 (lambda max = 325 nm), respectively (24).

Preparation and Purification of All-trans-retinal-PE Conjugates-- N-Retinylidene-PE, the Schiff base conjugate of retinal and PE, was prepared by the method of Anderson and Maude (25). All-trans-retinal (2.0 µmol) was mixed with 2.0 µmol of DOPE in 1.0 ml of a solvent consisting of chloroform, methanol, and triethylamine (12:6:1, by volume). The reaction was shielded from light and allowed to proceed for at least 30 min at room temperature. For the preparation of the reduction product of N-retinylidene-PE, N-retinyl-PE, a 1000-fold molar excess of NaBH4 was added following the initial incubation period. The retinal-PE conjugates were purified by HPLC on a Phenomenex Primesphere 5 C18 HC column (150 × 3.2 mm) by a procedure adapted from Parish et al. (26). The samples were eluted using a continuous gradient of 85% methanol in water to 100% methanol over a period of 30 min, followed by isocratic elution with 100% methanol (all solvents also contained 0.1% trifluoroacetic acid), at a flow rate of 0.5 ml/min (Fig. 1). Retinal eluted at approximately 12 min, N-retinylidene-PE at 48 min, and N-retinyl-PE at 60 min. N-Retinylidene-PE eluted from the column as the protonated Schiff base with an absorption maximum of 450 nm. Upon deprotonation of the Schiff base by addition of 5 N NaOH, the absorption maximum shifted to 370 nm, consistent with earlier observations (25). The absorption maximum of the N-retinyl-PE peak was 329 nm. The mass of the N-retinyl-DOPE compound was verified by electrospray mass spectrometry.


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  		Fig. 1.   HPLC analysis of reaction products of all-trans-retinal and phosphatidylethanolamine. Samples of retinal-PE reaction mixtures were injected onto a reversed phase C18 column and eluted with a gradient of 85-100% methanol. A, analysis of reaction of all-trans-retinal and PE. Absorbance was monitored at 450 nm. Peak 1, retinal peak (residual absorbance from maximum at 365 nm); peak 2, protonated N-retinylidene-PE; inset, absorbance spectrum of peak 2. B, analysis of N-retinyl-PE. Absorbance was monitored at 330 nm. Peak 3, N-retinyl-PE; inset, absorbance spectrum of peak 3.

Isolation of Rod Outer Segments-- ROS membranes were isolated from previously frozen bovine retinas on a continuous sucrose density gradient as described previously (27) and stored in homogenization buffer (4-8 mg protein/ml) at -80 °C.

Extraction of Phospholipids from ROS Membranes-- Phospholipids were extracted from ROS membranes by the method of Folch et al. (28), with precautions to prevent the reaction of endogenous retinal with amine-containing phospholipids and to limit the oxidation of the polyunsaturated acyl chains that are abundant in ROS phospholipids (29, 30). ROS membranes (16 mg) were washed three times in 10 mM potassium phosphate, pH 7.0, and suspended in 0.8 ml of the same buffer. A solution of 0.8 ml of 1 M NH2OH (pH adjusted to 7.0 using NaHCO3) and 4.2 ml of methanol was added to the membranes, and the mixture was incubated on ice for 10 min. NH2OH derivatized retinal to the corresponding oxime. The mixture was extracted with 4 ml of water and 7.5 ml of chloroform containing 50 µg/ml butylated hydroxytoluene as an antioxidant. The organic (lower) phase was separated from the aqueous phase and then washed with 5.6 ml of 0.3 M NaCl and 4.2 ml of methanol. The organic solvent was evaporated under nitrogen, and the lipids were resuspended in 0.2 ml of chloroform/methanol (1:1, by volume) and applied to a thin-layer chromatography plate (0.5-mm Silicagel G) under a nitrogen atmosphere. The plate was developed in a tank filled with nitrogen, using solvent system consisting of hexane/ether (1:1, by volume). The retinal oxime migrated near the solvent front, whereas the phospholipids remained at the origin. The phospholipids were scraped with a spatula and eluted from the Silicagel using chloroform/methanol (1:1, by volume) containing butylated hydroxytoluene. The yield of phospholipid (3.6 mg) was determined by analyzing lipid phosphorus using the method of Zhou and Arthur (31).

Purification of ABCR from ROS Membranes-- The Rim 3F4 antibody was used to isolate the ABCR protein from CHAPS-solubilized ROS membranes as described previously (2, 14). All steps were carried out in the dark or under dim red light at 4 °C. ROS membranes (2 mg of protein) were diluted in 10 mM HEPES, pH 7.4, and centrifuged at 86,000 × g for 10 min in a TLA100.4 rotor (Optima TL Ultracentrifuge, Beckman, Palo Alto, CA). After two more washes in 10 mM HEPES, pH 7.4, the membrane suspension was added to 1 ml of Buffer C containing 18 mM CHAPS and stirred for 20 min. The mixture was centrifuged described as above, and the soluble fraction (supernatant) was mixed for 1 h with 100 µl of Rim3F4-Sepharose 2B preequilibrated in Buffer C. The Sepharose beads were transferred to a 0.45 µm filter Ultrafree-MC spin column (Millipore) and washed with Buffer C (six washes of 0.4 ml each). ABCR was eluted by shaking the beads vigorously for 15 min in 60 µl of Buffer C containing 0.2 mg/ml 3F4 peptide (YDLPLHPRTG). The elution step was repeated, and the beads were washed with an additional 60 µl of Buffer C without peptide to yield a final volume of 180 µl (20-40 ng of ABCR/µl).

Reconstitution of ABCR in Lipid Vesicles-- ABCR was reconstituted into soybean phospholipid vesicles at 4 °C by mixing equal volumes of purified ABCR (in Buffer C) and 20 mg/ml sonicated soybean lipid (~40% PC) in Buffer B. After stirring for 15 min, the mixture was dialyzed against Buffer D1 for 24 h (3 × 500 ml) and subsequently against 500 ml of Buffer D2. ABCR was reconstituted into brain and ROS phospholipids using the procedure of Sun et al. (14). Briefly, 18 µl of 20 mg/ml sonicated brain polar lipid extract (33% PE, 18% phosphatidylserine, 13% PC, 36% other phospholipids) or ROS phospholipid extract (~ 40% PE, ~ 40% PC, ~10% phosphatidylserine, 10% other lipids) were mixed with 6 µl of 15% n-octylglucoside (w/v) in 25 mM HEPES, pH 7.4, 140 mM NaCl, 10% glycerol. Purified ABCR (48 µl) was added, and the mixture was incubated on ice for 30 min. Buffer E (400 µl) was added rapidly, and the vesicles were passed through 400 µl of Extracti-gel resin in a 10 µm pore size filter Mobicol mini-column (MoBiTec, Gottingen, Germany) that had been equilibrated in Buffer E. The flow-through containing reconstituted ABCR was collected at 0.8 ml/min by applying gentle pressure with a syringe, and 5 mM MgCl2 was added prior to measuring ATPase activity.

ATPase Assay-- The hydrolysis of [alpha -32P]ATP (NEN Life Science Products) in a 10-µl reaction volume was detected by thin layer chromatography as described before (32). Eight µl (20-40 ng) of CHAPS-solubilized ABCR (diluted in Buffer C) or reconstituted sample (undiluted) were pipetted into 0.5-ml microcentrifuge tubes. Retinal and other compounds were added from 10× solutions. The reaction was initiated with the addition of 1 µl of a 10× ATP solution (0.2 µCi). Unless otherwise noted, the final ATP concentration was 1 mM for solubilized ABCR and 50 µM for reconstituted ABCR. After 30 min at 37 °C, 4 µl of 10% SDS were added, and the tube was centrifuged briefly. One µl of the reaction mixture was spotted onto a polyethyleneimine cellulose plate (Aldrich) and chromatographed in 0.5 M LiCl/1 M formic acid. The plate was exposed to a storage phosphor screen for 3 h and scanned in a PhosphorImager SI (Molecular Dynamics, Sunnyvale, CA). Spots corresponding to ATP and ADP were quantified using IPLab Gel Analysis software (Signal Analytics Corp., Vienna, VA). The ratio of the amount of ADP produced to the initial amount of ATP present in the reaction mixture was calculated. Each sample was assayed in triplicate. Buffer blanks were included to determine nonenzymatic ATP hydrolysis, which was subtracted from the total. GTPase activity was measured in an identical manner using [alpha -32P]GTP.

Protein Determination-- The protein concentration of ROS membrane preparations was determined by the BCA method (Pierce). The amount of protein in detergent extracts was determined by comparing the intensity of Coomassie Brilliant Blue staining with that of bovine serum albumin standards after SDS-polyacrylamide gel electrophoresis. Protein content of reconstituted ABCR was estimated by Western blot analysis using known amounts of purified ABCR protein. Gels and film were scanned with an Ultroscan XL laser densitometer (LKB, Bromma, Sweden), and relative peak areas were used to determine protein concentration.

Western Blot Analysis-- Proteins were separated by SDS gel electrophoresis on 6% polyacrylamide gels and transferred to Immobilon P membranes (Millipore) at 300 mA for 40 min in a semidry transfer apparatus (Bio-Rad) using a buffer consisting of 25 mM Tris, 192 mM glycine, 10% methanol, pH 8.3. The membrane was incubated in 1% skim milk, PBS (140 mM NaCl, 3 mM KCl, 10 mM phosphate, pH 7.4) containing 0.05% Tween 20 (PBS-T) for 30 min and incubated first with Rim 3F4 monoclonal antibody diluted in 0.1% milk, PBS-T for 1 h and then with peroxidase-conjugated sheep anti-mouse IgG (diluted 1:5000 in 0.1% milk, PBS-T) for detection by enhanced chemiluminescence (Amersham Pharmacia Biotech).

COS-1 Cell Expression-- For expression studies, the human ABCR cDNA (4, 6) was subcloned into the NotI and blunted XbaI restriction sites of pcDNA3 (Invitrogen). COS-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 units/ml penicillin, and 50 µg/ml streptomycin and passaged twice a week. Cells (one 10-cm dish) were transfected at 80% confluency with 30 µg of pcDNA3-ABCR and 60 µl of SuperFect transfection reagent (Qiagen) for 3 h. After 48 h, cells were washed twice in PBS and harvested by scraping in PBS. The cells were centrifuged at 2800 × g for 10 min, and the pellet was solubilized in 0.5 ml of Buffer C containing 18 mM CHAPS and 0.2 mM phenylmethylsulfonyl fluoride. ABCR was purified on a Rim3F4-Sepharose 2B column as described above for ABCR from ROS membranes, except that the procedure was carried out under normal laboratory light. Typically, four 10-cm dishes of transfected cells were used for 100 µl of packed Rim3F4 beads.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Nucleotidase Activity of CHAPS-solubilized ABCR-- The ATPase activity of CHAPS-solubilized, immunoaffinity-purified ABCR from ROS membranes was measured under various conditions. When the purification and activity measurements were carried out in the presence of soybean phospholipids, DTT, and glycerol, ABCR exhibited a basal ATPase activity that was stimulated up to 2-fold by 11-cis- and all-trans-retinal (Figs. 2 and 3A). All-trans-retinol, reduced glutathione, and four structurally related unsaturated aldehydes (citral, 2,4-nonadienal, 2,6-nonadienal, and nonyl aldehyde) had no significant effect on the basal ATPase activity of ABCR. The activity of CHAPS-solubilized ABCR was dependent on the presence of DTT and lipid. Over 70% of the basal ATPase activity was lost when DTT or soybean phospholipid was omitted during purification and activity determination. N-Ethylmaleimide also inhibited the ATPase activity of ABCR.


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  		Fig. 2.   ATPase activity of CHAPS-solubilized ABCR is stimulated by retinal. ABCR was solubilized and purified from bovine ROS membranes by immunoaffinity chromatography in the presence of CHAPS. The ATPase activity of the eluate was assayed in the presence of 100 µM 11-cis-retinal, 100 µM all-trans-retinal, 100 µM all-trans-retinol, 100 µM citral, 100 µM 2,4-all-trans-nonadienal, 100 µM 2-trans-6-cis-nonadienal, 100 µM nonylaldehyde, 1 mM reduced glutathione (GSH), or 1 mM N-ethylmaleimide (NEM). The right two columns represent ATPase activity associated with ABCR purified and assayed in the absence of DTT (w/o DTT) and in the absence of soybean phospholipids (w/o lipid). Each value is the mean of three determinations ± S.D.


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  		Fig. 3.   Retinal stimulates the nucleotide triphosphatase activity of CHAPS-solubilized ABCR. A, the ATPase activity of purified ABCR was measured as a function of the all-trans-retinal () or all-trans-retinol (black-square) concentration. Results shown represent the means ± S.D. of three experiments. B, the stimulation of GTPase activity by all-trans-retinal in a typical experiment (100% activity = 150 nmol/min/mg). In both cases, half-maximal stimulation of ATPase activity was obtained with 10 µM all-trans-retinal.

The ATPase activity of solubilized ABCR was highly labile. ATPase stimulation by all-trans-retinal was lost when CHAPS-solubilized ABCR was stored overnight at 4 °C. Preincubation of ABCR at 37 °C for 30 min resulted in the loss of over 90% of the ATPase activity of ABCR. Addition of ATP during preincubation diminished this inactivation. Approximately 40% of the basal ATPase activity remained if the preincubation step was carried out in the presence of 1 mM ATP.

CHAPS-solubilized ABCR exhibited both ATPase and GTPase activities. As shown in Fig. 3, the basal ATPase and GTPase activities were activated by all-trans-retinal with half-maximal stimulation at 10 µM all-trans-retinal. The specific ATPase and GTPase activities were similar.

ATPase Activity of ABCR Reconstituted into Phospholipid Vesicles-- Previously, Sun et al. (14) reported that ABCR reconstituted into brain polar lipid and brain PE possesses basal and retinal-stimulated ATPase activity. We have now reconstituted purified ABCR into various phospholipid mixtures in order to examine further the effect of the lipid environment on the basal and retinal-stimulated ATPase activity of ABCR. Fig. 4 shows the dependence of ATP hydrolysis on ATP concentration for ABCR reconstituted into ROS phospholipid vesicles. The Km and Vmax values for ATP hydrolysis by detergent-solubilized and reconstituted ABCR are compared in Table I. The Km values for basal ATPase activity of ABCR reconstituted in soybean, brain, and ROS phospholipid are similar (Km ~ 25 µM ATP) but generally lower than for CHAPS-solubilized ABCR (Km ~ 75 µM ATP). The lipid composition had a significant influence on the Vmax values of the reconstituted enzyme. The Vmax of ABCR reconstituted into ROS phospholipid (Vmax = 202 ± 45 nmol/min/mg) is four times higher than ABCR reconstituted into soybean phospholipid and over six times higher than ABCR in brain polar lipid but comparable to that observed for the solubilized protein (Table I). Retinal increased both the Vmax and Km for ATP hydrolysis by ABCR reconstituted into each type of lipid. Although retinal stimulation was somewhat variable between preparations, in general, retinal increased the Vmax by 2.5-5-fold for ABCR in ROS and brain lipids but only 1.5-2-fold for ABCR in soybean phospholipids.


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  		Fig. 4.   Kinetics of ATP hydrolysis for ABCR reconstituted in ROS lipid vesicles. A, basal () and 60 µM all-trans-retinal-stimulated (black-square) ATPase activities were measured as a function of ATP concentration. B, the double-reciprocal plot was used to determine Km and Vmax. Each data point is the mean ± S.D. of triplicate values from a single experiment. Basal Km = 19 µM, Vmax = 230 nmol/min/mg; retinal-stimulated Km = 71 µM, Vmax = 750 nmol/min/mg. Similar graphs were obtained after reconstitution in soybean and brain polar lipid in at least three experiments for each lipid.

                              
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Table I
Comparison of ATPase activity of purified ABCR reconstituted in different lipid preparations
ATPase activity was measured immediately following purification or after reconstitution in soybean, brain polar, or rod outer segment lipids. Km and Vmax values were determined from the double reciprocal plots of ATP hydrolysis versus ATP concentration in the absence (basal) or presence of 60 µM all-trans-retinal. Shown are the means of three experiments with S.D., except where noted with a 2 in parentheses (two experiments).

In addition to affecting the kinetics of ATP hydrolysis, the lipid environment also influenced the stability of ABCR. Unlike CHAPS-solubilized ABCR, the basal and retinal-stimulated activity of reconstituted ABCR was unaffected by storage at 4 °C for at least 4 days.

The effect of all-trans-retinal and all-trans-retinol concentration on the ATPase activity of ABCR in ROS and brain phospholipids was also measured. Fig. 5 shows that ATPase activation reached a maximum at about 50 µM all-trans-retinal. At high retinal concentration (200 µM), an inhibition of ATPase activity was typically observed for ABCR reconstituted in ROS lipids. All-trans-retinol also stimulated the ATPase activity of ABCR reconstituted in brain and ROS lipids, but this activation was less pronounced and occurred only at higher retinol concentration.


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  		Fig. 5.   Effect of all-trans-retinal () and all-trans-retinol (black-square) concentration on ATPase activity of ABCR after reconstitution in lipid vesicles. Half-maximal stimulation of ATPase activity was obtained at 10 µM retinal. Shown is a representative experiment repeated three times.

Effect of Different Lipids on ATPase Activity-- ROS membranes are known to contain a relatively high content of PE and docosahexaenoic acid (C22:6) containing phospholipids (25, 30). To determine whether these lipids are responsible for the increased ATPase activity of ABCR in ROS lipids, we measured the basal and retinal-stimulated ATPase activity of ABCR reconstituted into brain lipid vesicles containing added SDPE, SDPC, DOPE, or DOPC. As shown in Fig. 6, replacing 50% of the brain lipid extract with pure SDPE or DOPE resulted in a 2-3-fold increase in basal and retinal-stimulated ATPase activity, with SDPE showing the largest increase. In contrast, the addition of SDPC or DOPC to brain lipid resulted in a decrease in both basal and retinal-stimulated ATPase activity.


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  		Fig. 6.   Basal and retinal-stimulated ATPase activity of ABCR in different lipid mixtures. Purified ABCR was reconstituted in brain polar lipid extract containing SDPE, DOPE, SDPC, or DOPC (% of brain lipid extract replaced by pure PE or PC) and assayed for ATPase activity in the absence or presence of 50 µM all-trans retinal. All lipids were dissolved in chloroform:methanol (1:1), mixed together, and dried under nitrogen before dissolving in buffer and bath sonication. The final lipid concentration was kept constant at 0.7 mg/ml. The means of three experiments ± S.D. are shown.

Effect of N-Retinylidene-PE on ATPase Activity of ABCR-- It has long been known that retinal reacts with PE to form the Schiff base conjugate, N-retinylidene-PE (25, 33). To determine the extent to which retinal reacts with PE in lipid vesicle preparations used for reconstitution, 50 µM all-trans-retinal was added to brain polar lipid vesicles (lipid concentration, 0.8 mg/ml) at 37 °C. After a 30-min incubation period, the reaction was stopped by the addition of NaBH4, and the retinoids and N-retinyl-PE products were separated by HPLC (Fig. 7) and quantified from the peak absorbances and extinction coefficients of these compounds (25, 33). Approximately 55% (mole ratio) of the all-trans-retinal added to the vesicles reacted with PE to form N-retinylidene-PE derivatives.


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  		Fig. 7.   HPLC analysis of all-trans-retinal in brain polar lipid vesicles. All-trans-retinal (50 µM) was added to brain polar lipid vesicles (lipid concentration, 0.8 mg/ml), and the mixture was incubated at 37 °C for 30 min. The reaction was stopped by the addition of NaBH4, and chloroform and methanol were added to cause phase separation. The products in the organic phase were analyzed by HPLC. Absorbance was monitored at 330 nm. Peak 1, retinol; peak 2, N-retinyl-PE.

Given that retinal can form a conjugate with PE in vesicles, it was of interest to determine whether exogenously added N-retinylidene-PE would have any effect on the ATPase activity of ABCR. As shown in Fig. 8, the addition of N-retinylidene-PE to ABCR reconstituted in brain polar lipid resulted in stimulation of the ATPase activity by approximately 3-fold. The addition of 1.0-25.0 µM N-retinyl-PE (NaBH4-reduced compound), instead of N-retinylidene-PE, resulted in a small inhibition in ATPase activity of about 25%.2 There was no significant stimulation of ATPase activity by 25 µM N-retinylidene-PE, all-trans retinal, or PE when ABCR was reconstituted in pure SDPC or DOPC vesicles (Fig. 9).


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  		Fig. 8.   Effect of N-retinylidene-PE on the ATPase activity of ABCR. ABCR was reconstituted into brain polar lipid vesicles. N-Retinylidene-PE was added at the final concentrations as indicated, and ATPase hydrolysis was determined. These data represent the means ± S.D. from a typical experiment performed in triplicate. Basal ATPase activity was 4.2 ± 0.1 nmol/min/mg. This experiment was repeated four times, with similar results.


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  		Fig. 9.   The effect of retinoids on the ATPase activity of ABCR reconstituted in PC vesicles. ABCR reconstituted in brain polar lipid, SDPC, or DOPC was assayed for ATPase activity in the presence of 25 µM all-trans-retinal, 25 µM N-retinylidene-PE (N-RPE), or 25 µM PE.

The Schiff base formed between retinal and PE is known to be labile. Therefore, we investigated whether N-retinylidene-PE was stable when added to brain polar lipid vesicles or whether it dissociated into its parent compounds. To this end, N-retinylidene-PE at an initial concentration of 10 µM was incubated with vesicles for up to 30 min. Subsequent HPLC analysis revealed that approximately 40% of the N-retinylidene-PE was recovered as the retinal-PE conjugate, whereas the rest was recovered as the free retinoid.

Expression of the Human ABCR in COS-1 Cells-- To determine whether the retinal-stimulated ATPase activity observed in purified ABCR preparations is a property of ABCR itself, rather than a contaminating ATPase of ROS membranes, ABCR was expressed in monkey kidney COS-1 cells, and the ATPase activity of the immunoaffinity-purified protein was measured in the presence and absence of retinal. Fig. 10 shows a Coomassie Blue-stained gel and a Western blot of ABCR purified from transfected COS-1 cells. As in the case of ABCR from ROS membranes, the expressed and purified ABCR migrated on SDS gels as a single protein with an apparent molecular mass of 220 kDa. Over 50% of ABCR expressed in COS-1 cells bound to the Rim 3F4 immunoaffinity matrix. After reconstitution into brain phospholipid vesicles, ABCR showed basal and retinal-stimulated ATPase activity similar to that of the native protein (Fig. 11).


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  		Fig. 10.   Expression of the human ABCR in COS-1 cells. Cells transfected with pcDNA3 (lanes 1-3 in both panels) or pcDNA3-ABCR (lanes 4-6) were solubilized in 18 mM CHAPS and purified by immunoaffinity chromatography. The CHAPS detergent extract (lanes 1 and 4; <FR><NU>1</NU><DE>50</DE></FR> of total), flow-through fraction (lanes 2 and 5; <FR><NU>1</NU><DE>50</DE></FR> of total), and eluate (lanes 3 and 6; <FR><NU>1</NU><DE>3</DE></FR> of total) were loaded on 6% polyacrylamide gels and stained with Coomassie Brilliant Blue or transferred to Immobilon membrane for Western blot analysis with 3F4 antibody (for detection of ABCR). Only cells transfected with ABCR cDNA expressed the 220-kDa protein.


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  		Fig. 11.   ATPase activity of human ABCR purified from transfected COS-1 cells. ABCR was solubilized and purified from transiently transfected COS-1 cells (ABCR) and reconstituted in brain polar lipid vesicles. A mock purification using untransfected COS-1 cells (UNTR) was also performed to measure background ATPase activity. ATP hydrolysis was measured in the absence (open columns) or presence of 50 µM all-trans-retinal (solid columns). The amount of ATP hydrolyzed over a 30-min period by immunopurified and reconstituted protein from approximately 2 × 105 cells is shown ± S.D. (three experiments).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we have examined the effect of lipids and other compounds on the nucleotidase activity of ABCR purified from ROS disc membranes. Phospholipid and a reducing environment are required because ATPase activity is lost when detergent-solubilized ABCR is purified in the absence of either phospholipid or DTT. N-Ethylmaleimide, a sulfhydryl reactive agent, also abolished activity, indicating that the ATP hydrolysis is sensitive to covalent modification, as well as oxidation, of one or more cysteine residues of ABCR. These properties are shared with P-glycoprotein, an ABC transporter that shows only limited (~25% identity) sequence identity with ABCR, even within the nucleotide binding domains (17-19, 34).

In the presence of soybean phospholipid, CHAPS-solubilized ABCR exhibits basal ATPase activity that is stimulated up to 2-fold by all-trans- and 11-cis-retinal. This stimulatory effect on CHAPS-solubilized ABCR is specific for retinal because retinol and a series of unsaturated aldehydes lacking the retinoid ring structure had no effect on the basal activity of solubilized ABCR. These results, together with those of earlier studies (14), indicate that both the aldehyde group (possibly linked to PE via a Schiff base) and the retinoid ring structure are required for ATPase activation. Previously, Sun et al. (14) reported that the ATPase activity of CHAPS-solubilized ABCR in a mixture of brain PE and egg PC was not stimulated by retinal prior to reconstitution. The specific activity of ATP hydrolysis reported in the earlier paper was 7-fold lower. The difference between the present study and the earlier one (14) may be due to the different lipid mixtures used during purification of ABCR, the method of protein determination, or the labile nature of retinal-stimulated ATPase activity of solubilized ABCR.

The lipid environment influences the kinetics of ATP hydrolysis by ABCR. A 3-fold decrease in the Km for basal ATPase occurs upon reconstitution of ABCR into lipid vesicles. Removal of detergent and/or the presence of a lipid bilayer may favor the formation of the enzyme-substrate complex between ATP and ABCR. The lipid environment also affects the Vmax for ATP hydrolysis. The Vmax values for ABCR reconstituted into soybean or brain phospholipid vesicles are considerably lower than for detergent-solubilized ABCR (Table I). The soybean and brain phospholipid bilayer may constrain conformational changes in ABCR coupled to ATP hydrolysis, thereby resulting in a decrease in reaction rate. Interestingly, the Vmax of ABCR reconstituted into ROS disc lipids is considerably higher than the Vmax of ABCR reconstituted into brain or soybean lipids and comparable to the basal activity of purified P-glycoprotein, cystic fibrosis transmembrane conductance regulator, and multidrug resistance-associated protein (17, 21, 32, 35). We considered the possibility that the relatively high levels of PE (40% of total disc lipid is PE) and/or docosahexaenoic acid (37% of ROS fatty acids) may provide a more favorable environment for ABCR. This was investigated by determining the effect of added DOPE, SDPE, DOPC, and SDPC on the ATPase activity of ABCR reconstituted in brain lipid. Both DOPE and SDPE increased the ATPase activity of ABCR in brain lipid, whereas DOPC and SDPC decreased the activity, indicating that a high PE content enhances the basal and retinal-stimulated ATPase activity of ABCR. The decrease in activity observed with added PC lipids can be explained in part by the effective dilution of endogenous brain lipid PE by added PC lipids. The importance of PE in retinal activated ATPase activity of ABCR is underscored by the finding that ABCR reconstituted in pure PC lipid vesicles is largely devoid of retinoid activation. This indicates that the coupling of retinoid binding within the membrane domain of ABCR to the ATPase activity of the nucleotide binding domains of ABCR requires a lipid environment rich in PE. The lipid environment is known to influence the ATPase and drug binding activities of P-glycoprotein (18, 19, 36). In this case, the phospholipid polar head group is less important because both egg PC and dipalmitoyl-PE stimulate P-glycoprotein ATPase activity, whereas egg PE does not. Loo and Clarke (37) have also reported that the nucleotide binding domains of P-glycoprotein interact with the transmembrane domains. High PE content is known to destabilize the lipid bilayer through hexagonal II phase formation (17). The increased accessibility of ABCR for ATP in such membrane sheets may also contribute to the increased basal ATPase activity observed in lipids containing a high PE content.

Earlier studies have shown that all-trans-retinal derived from photobleached rhodopsin reacts with PE in disc membranes to produce the Schiff base conjugate, N-retinylidene-PE (25, 38). This raises the possibility that either free retinal or N-retinylidene-PE is responsible for stimulation of ATPase activity of ABCR. In an effort to resolve this issue, we measured the effect of added N-retinylidene-PE on the ATPase activity of reconstituted ABCR. N-Retinylidene-PE, like retinal, resulted in over 3-fold stimulation of ATP hydrolysis. However, HPLC analysis following the addition of either free retinal or N-retinylidene-PE to brain lipid membranes suggests that an equilibrium is established between free retinal and N-retinylidene-PE, thereby complicating the identification of the substrate responsible for ATPase activation of ABCR. We have attempted to resolve this issue by determining whether N-retinyl-PE, the stable reduced form of N-retinylidene-PE, can stimulate the ATPase activity of ABCR. This compound, however, failed to stimulate the ATPase activity of ABCR. The identity of the retinoid substrate responsible for ATPase activation of ABCR was also investigated by measuring the effect of free retinal and retinylidene-PE on the ATPase activity of ABCR reconstituted into pure PC vesicles. However, neither retinoid stimulated ATP hydrolysis by ABCR in this lipid environment. Although this experiment failed to identify the retinal derivative responsible for ATPase activation of ABCR, it did reveal that such activation requires membranes containing substantial amounts of PE. This strongly suggests that PE is required to couple the binding of specific retinoids within the transmembrane domain of ABCR to the ATPase activity of the nucleotide binding domains.

Several indirect observations now support the view that ABCR may function to make retinal more accessible to all-trans-retinol dehydrogenase by either flipping N-retinylidene-PE from the lumenal to the cytoplasmic side of the disc membrane or extruding retinal from the disc membrane. Substances that activate the ATPase activity of P-glycoprotein and other ABC transporters are also substrates for transport by these proteins. By analogy, activation of the ATPase activity of ABCR by retinal compounds suggests that these physiological compounds may also be actively transported across disc membranes by ABCR. Patients with Stargardt disease-linked mutations in ABCR are known to exhibit delayed dark adaptation and accumulate diretinal-pyridinium (A2E) compounds in the form of lipofuscin (26, 39-42). These characteristics can be explained on the basis of a diminished rate of 11-cis-retinal regeneration and accumulation of retinal-PE derivatives in disc membranes. Finally, recent studies indicate that abcr knockout mice also show reduced dark adaptation kinetics, elevated levels of protonated N-retinylidene-PE, and accumulation of A2E compounds upon prolonged exposure to light (23). Efforts are now under way to directly identify the substrate for ABCR and the mechanism of substrate transport across disc membranes.

Unlike most cells, rod photoreceptors contain similar levels of ATP and GTP. Therefore, it was of interest to determine whether ABCR can catalyze GTP as well as ATP hydrolysis. Studies carried out here revealed that the kinetics of ATP and GTP hydrolysis by purified ABCR are similar. Moreover, basal GTPase activity is stimulated by similar concentrations of retinal. These results are consistent with earlier photoaffinity labeling studies showing that ABCR binds both ATP and GTP (2). GTP is a poor substrate for P-glycoprotein (18, 34) and does not support taurocholate transport by the bile salt transporter, SPGP (43). It remains to be determined whether GTP hydrolysis is able to support the active transport function of ABCR.

Finally, we have demonstrated that ABCR can be expressed in COS-1 cells. The purified and reconstituted protein exhibits basal and retinal-stimulated ATPase activity similar to ABCR from native ROS membranes. This provides strong evidence that the ATPase activity observed in immunoaffinity-purified ABCR from ROS is due to ABCR and not a retinal-sensitive ATPase contaminant. This cell expression system should be useful in the structure-function analysis of ABCR and understanding how selective mutations in ABCR cause Stargardt disease and related retinopathies.

    ACKNOWLEDGEMENTS

We thank Hui Sun, Philip Smallwood, and Jeremy Nathans for helpful discussions and the complete human ABCR cDNA used in transfection studies and Phil Owen for expert assistance with mass spectrometry.

    FOOTNOTES

* This work was supported by a grant from the Ruth and Milton Steinbach Foundation, NEI Grant EY 02422, and a grant from the Medical Research Council.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.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, 2146 Health Sciences Mall, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada. Tel.: 604-822-6173; Fax: 604-822-5227; E-mail: molday@interchange.ubc.ca.

Published, JBC Papers in Press, April 14, 2000, DOI 10.1074/jbc.M000555200

2 J. Ahn, J. T. Wong, and R. S. Molday, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: PE, phosphatidylethanolamine; ROS, rod outer segment; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DOPE, dioleoylphosphatidylethanolamine; DOPC, dioleoylphosphatidylcholine; SDPC, 1-stearoyl-2-docosahexaenoylphosphatidylcholine; SDPE, 1-stearoyl-2-docosahexaenoylphosphatidylethanolamine; DTT, dithiothreitol; PC, phosphatidylcholine; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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