Mutations of the Walker B Motif in the First Nucleotide Binding Domain of Multidrug Resistance Protein MRP1 Prevent Conformational Maturation

Archives of Biochemistry and Biophysics Vol. 392, No. 1, August 1, pp. 153–161, 2001 doi:10.1006/abbi.2001.2441, available online at http://www.idealibrary.com on

Mutations of the Walker B Motif in the First Nucleotide Binding Domain of Multidrug Resistance Protein MRP1 Prevent Conformational Maturation Liying Cui, Yue-Xian Hou, John R. Riordan, and Xiu-bao Chang 1 Mayo Clinic Scottsdale, S. C. Johnson Medical Research Center, 13400 East Shea Boulevard, Scottsdale, Arizona 85259

Received March 14, 2001, and in revised form April 30, 2001; published online July 5, 2001

ATP-binding cassette (ABC) transporters couple the binding and hydrolysis of ATP to the translocation of solutes across biological membranes. The so-called “Walker motifs” in each of the nucleotide binding domains (NBDs) of these proteins contribute directly to the binding and the catalytic site for the MgATP substrate. Hence mutagenesis of residues in these motifs may interfere with function. This is the case with the MRP1 multidrug transporter. However, interpretation of the effect of mutation in the Walker B motif of NBD1 (D792L/D793L) was confused by the fact that it prevented biosynthetic maturation of the protein. We have determined now that this latter effect is entirely due to the D792L substitution. This variant is unable to mature conformationally as evidenced by its remaining more sensitive to trypsin digestion in vitro than the mature wild-type protein. In vivo, the coreglycosylated form of that mutant is retained in the endoplasmic reticulum and degraded by the proteasome. A different substitution of the same residue (D792A) had a less severe effect enabling accumulation of approximately equal amounts of mature and immature MRP1 proteins in the membrane vesicles but still resulted in defective nucleotide interaction and organic anion transport, indicating that nucleotide hydrolysis at NBD1 is essential to MRP1 function. © 2001 Academic Press

Key Words: MRP1; nucleotide binding domain (NBD); conformational maturation; proteasome; trypsin digestion; photoaffinity labeling; ATP-dependent LTC 4 uptake.

1 To whom correspondence and reprint requests should be addressed. Fax: (480) 301-7017. E-mail: [email protected].

0003-9861/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

The nucleotide binding domains (NBDs) 2 of ATP binding cassette (ABC) transporters are characterized by highly conserved sequences including the Walker A and B motifs (1) involved in the hydrolysis of ATP which drives the active transport of solute (2–5). This is the case with the MRP1 organic anion transporter where mutagenesis of either of the Walker A lysine residues involved in binding ATP phosphates or the Walker B aspartates involved in coordination of Mg 2⫹ ions of the MgATP substrate inhibits ATP binding and hydrolysis and organic anion transport (6 – 8). However, in NBD1 of MRP1 two aspartate residues (D792 and D793) follow the four canonical hydrophobic residues that precede the aspartic acid coordinating Mg 2⫹ in other proteins of this type (5). Therefore to determine the contribution of NBD1 Walker B aspartate in MRP1 function, we had previously made substitutions at both positions simultaneously, i.e., D792L/D793L (8). This almost completely ablated organic anion transport by MRP1 but also prevented maturation and transport of the protein to the cell surface. Therefore it was important to determine if the loss of function is due solely to the processing defect or if it also reflects a loss of the inherent ability of the polypeptide to accomplish ATP-dependent active transport. This was the goal of this study. The results showed that the D792L mutation was responsible for the defective maturation and function while D793L had minimal effect on either. A different substitution of the important residue (D792A) only partially impaired maturation yet still 2 MRP1, multidrug resistance protein; NBD, nucleotide binding domain; ABC, ATP binding cassette; BHK, baby hamster kidney; LTC 4, leukotriene C 4; ALLN, N-acetyl-L-leucinyl-L-leucinal-L-norleucinal; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; EDTA, ethylenediaminetetraacetic acid; EGTA, Ethylene glycolbis(␤-aminoethyl ether)N,N,N,N-tetraacetic acid; SDS, sodium dodecyl sulfate.

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strongly inhibited transport indicating that it is essential to function. EXPERIMENTAL PROCEDURES Materials. Anti-mouse Ig conjugated with horseradish peroxidase was purchased from Amersham. Chemiluminescent substrate for Western Blotting was from Pierce. Sodium orthovanadate, ALLN, TPCK-treated trypsin and MgCl 2 were purchased from Sigma. 8-Azido-[␣- 32P]ATP was purchased from ICN. The Stratalinker UV Crosslinker 2400 model (wavelength 254-nm) and QuikChange Site Directed Mutagenesis kit were from Stratagene. [14,15,19,20- 3H(N)]leukotriene C 4 and [ 35S]methionine were from NEN Life Science Products. Lactacystin was from the E.J. Corey laboratory, Department of Biochemistry, Harvard University. In vitro mutagenesis of MRP1 cDNA. The coding sequence of human MRP1 cDNA in the pNUT expression vector was employed (9). The aspartic acid residues at positions of 792 and 793 were mutated either to alanine (Fig. 1B, D792A) or leucine residues (Fig. 1B, D792L and D793L) using the QuikChange Site Directed Mutagenesis kit. Due to the fact that a conservative D to N substitution in Walker motif B of the cystic fibrosis transmembrane conductance regulator (CFTR) was still active (our unpublished results), we decided to make less conservative changes in MRP1, i.e., D to L or A. In order to ensure that no other mutations were introduced into the cDNA during mutagenesis, fragments covering the mutations were sequenced completely and used to replace their counterparts in the wild-type MRP1 cDNA in pNUT. The mutations were verified after insertion into the pNUT expression vector. Cell culture and stable transfection of MRP1 in BHK cells. Baby hamster kidney (BHK-21) cells were cultured at 37°C in 5% CO 2. Stable cell lines expressing wild-type and mutant MRP1s, K684L, D792L/D793L, K1333L, and D1454L/E1455L were established previously (8). The cell lines expressing D792A, D792L, and D793L were generated using the same procedures (9). [ 35S]Methionine labeling and immunoprecipitation. Monolayers of cells were starved for 30 min in methionine-free media and then pulse labeled for 20 min with 100 ␮Ci/ml L-[ 35S]methionine. After removing the L-[ 35S]methionine media the cells were incubated for another 20 min in the methionine-free media. The labeled proteins were chased with regular media containing 1 mM methionine. The starving, labeling, and chasing were performed in a 37°C incubator with 5% CO 2. Cells were lysed in RIPA buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), and 1⫻ protease inhibitors (2 ␮g/ml aprotinin, 121 ␮g/ml benzamidine, 3.5 ␮g/ml E64, 1 ␮g/ml leupeptin and 50 ␮g/ml Pefabloc). The soluble supernatant obtained after centrifugation at 16,000g for 15 min at 4°C was incubated with 2 ␮l of 897.2 ascites (8) for overnight in cold room. Then protein G-agarose beads were added to the mixture and incubated for 60 min in the cold room. The complex formed with the protein G-agarose beads were washed four times with RIPA buffer and then eluted with 1⫻ electrophoresis sample buffer for 60 min at room temperature. SDS–PAGE and immunoblotting. SDS–PAGE and immunoblotting were performed as described earlier (9). The primary antibody used was mouse anti-human MRP1 monoclonal antibody 897.2 and the secondary antibody used was anti-mouse Ig conjugated with horseradish peroxidase. Chemiluminescent film detection was performed according to the manufacturer’s recommendations. TPCK-treated trypsin digestion of wild-type and mutant MRP1s. Membrane vesicle proteins (2.2 ␮g) were digested with TPCKtreated trypsin at 37°C for 15 min in a 60-␮l solution containing 40 mM Tris–HCl, pH 7.4, and 1 mM EDTA. TPCK-treated trypsin was prepared and diluted in 10 mM HCl. The ratio of trypsin to total

protein and the amount of the total protein loaded in each lane is indicated in the figure legend. Endoglycosidase H treatment of membrane proteins. Membrane proteins (10 ␮g) were incubated in the presence or absence of 10 mU endoglycosidase H at 37°C for 14 h in 200 ␮l of solution containing 50 mM sodium acetate (pH 5.3), 0.5% NP-40, 1% ␤-mercaptoethanol, 0.2% SDS, and 1⫻ protease inhibitors (2 ␮g/ml aprotinin, 121 ␮g/ml benzamidine, 3.5 ␮g/ml E64, 1 ␮g/ml leupeptin, and 50 ␮g/ml Pefabloc). At the end of incubation 800 ␮l of cold ethanol was added to precipitate the proteins. The pellet was collected by centrifugation at 4°C for 15 min. Membrane vesicle preparations. MRP1-containing membrane vesicles were prepared according to the procedure described previously (8). Briefly, the cells collected by centrifugation were resuspended in membrane vesicle preparation buffer (10 mM Tris–HCl, pH 7.5, 250 mM sucrose, 0.2 mM MgCl 2, and 1⫻ protease inhibitors) and equilibrated on ice for 20 min at 800 p.s.i. in a Parr N 2 cavitation bomb. After release of the pressure, the cell homogenate was adjusted to 1 mM EDTA. The homogenate was diluted fivefold with 10 mM Tris–HCl and 25 mM sucrose, pH 7.5, and centrifuged at 1000g to remove nuclei and unbroken cells. The supernatant was overlaid on a 35% sucrose solution containing 10 mM Tris–HCl, pH 7.5, and 1 mM EDTA and centrifuged at 16,000g for 30 min. The interface membrane was collected, diluted fivefold with a solution containing 10 mM Tris–HCl, pH 7.5, and 250 mM sucrose and then centrifuged at 100,000g for 45 min. The pellet was resuspended in a solution containing 40 mM Tris–HCl, pH 7.5, 0.1 mM EGTA, and 1⫻ protease inhibitors. After passage through a Liposofast vesicle extruder (200 nm filter, Avestin, Ottawa, Canada) they were aliquoted and stored in ⫺80°C. Membrane Vesicle Transport. ATP-dependent transport of [ 3H]Leukotriene C 4 into the membrane vesicles was assayed by a rapid filtration technique (10, 11). The assays were performed with 3 ␮g of membrane protein in a 30 ␮l reaction volume containing 50 mM Tris–HCl (pH 7.5), 250 mM sucrose, 10 mM MgCl 2, 200 nM LTC 4 (17.54 nCi of 3H-LTC 4) and 4 mM ATP (or 4 mM AMP). After incubation at 37°C for 6 min, the samples were diluted with 1 ml of ice cold 1X transport buffer (50 mM Tris–HCl, pH 7.5, 250 mM sucrose, and 10 mM MgCl 2) and filtered through a nitrocellulose membrane (0.2 ␮m), which had been equilibrated with 1⫻ transport buffer. The filter was then washed with 10 ml cold 1⫻ transport buffer, air-dried, and placed in 10 ml of biodegradable counting scintillant (Amersham). The radioactivity bound to the nitrocellulose membrane was determined by liquid scintillation counting (Beckman LS 6000SC). Photoaffinity labeling of MRP1 protein. Vanadate preparation and photoaffinity labeling of MRP1 protein were performed according the procedures described previously (8). Briefly, the photolabeling experiments were carried out in a 10 ␮l of solution containing 40 mM Tris–HCl (pH 7.5), 2 mM ouabain, 0.1 mM EGTA, 10 mM MgCl 2, 5 ␮M of 8-azido[␣- 32P]-ATP (1 ␮Ci), and 800 ␮M vanadate for 10 min at 37°C. The samples were then transferred to ice and diluted with 400 ␮l of ice-cold Tris-EGTA buffer (0.1 mM EGTA and 40 mM Tris–HCl, pH 7.5). The membranes were pelleted in a microfuge in the cold room (4°C), washed again with 400 ␮l of ice-cold Tris-EGTA buffer, resuspended in 10 ␮l of Tris-EGTA buffer, placed on ice and irradiated for 2 min in a Stratalinker UV Crosslinker (␭ ⫽ 254 nm). The labeled proteins were separated on a polyacrylamide gel (7%).

RESULTS

Substitution of D792 but not D793 in the NBD1 Walker B motif interferes with MRP1 maturation. The influence of the amino acid substitutions in the Walker B motif of the first nucleotide binding domain of MRP1 indicated in Fig. 1 on the steady-state

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FIG. 1. Schematic diagram of MRP1 protein and the Walker B aspartic acid mutations in NBD1. (A) The diagram of MRP1 protein indicates the 17 putative transmembrane segments, three potential glycosylation sites, and two nucleotide binding domains, NBD1 and NBD2. The diagram also indicates the location of the monoclonal antibody, 897.2, binding site (mAb, from 1318 to 1388) and trypsin sensitive site (TS). If the protein is partially digested with trypsin and probed with the monoclonal antibody 897.2, two main bands are detected by the antibody, the intact 190-kDa protein and the Cterminal 65-kDa digestion product. (B) Mutations in Walker B motif (underlined) in NBD1 are shown. Alanine or leucine residues replacing the aspartates are indicated in black boxes.

amounts of the immature and mature forms of the protein is shown in Fig. 2A. The larger band of approximately 190 kDa represents the mature glycoprotein with complex oligosaccharide chains and the smaller band (⬃170 kDa) its immature core-glycosylated precursor that is sensitive to endoglycosidase H digestion. At this level of exposure the mature band is predominantly detected in the wild-type, while none of this band is present in the double mutant, D792L/D793L. D793L is indistinguishable from wild-type, whereas D792L is similar to the double mutant indicating that the substitution at this position is primarily responsible for the misprocessing. However, when this residue was replaced by alanine (D792A) instead of leucine a detectable mature band was also present. Faint smaller bands were also detectable in the lanes of the mutants which did not mature completely (Fig. 2A). These were more apparent in a longer exposure of the same blot (Fig. 2B), especially in D792L/D793L, where, in addition to the major 170-kDa species, bands of approximately 160, 130, 100, and 30 kDa can be seen. Of these four only the 130- and 30-kDa bands are seen

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in the D792A and D792L lanes. Since none of these bands appear in lanes when the protein is fully mature (Fig. 2B, wild-type and D793L), we assume that they are degradation products of the immature forms of the misprocessed mutants. When membranes were isolated and analyzed instead of whole lysates from these cells, the smallest 30 kDa degradation band was no longer detected (Fig. 2C and lane 1 of Figs. 5C, 5D, and 5F). This is not surprising as it must be a C-terminal hydrophilic part of the protein reactive with an antibody recognizing an epitope in NBD2 (Fig. 1A). However, the presence of the 160-, 130-, and 100-kDa fragments in membrane vesicles (lane 1 of Figs. 5C, 5D, and 5F) indicates that these degradation products are still membrane-bound and therefore the cleavages that created them must have occurred before retrograde translocation out of the membrane. It is also apparent that the mature form of MRP1 is enriched in the membrane fraction compared with the whole cell lysate; this is especially obvious with D792A, where there appears to be about equal amounts of the immature and mature bands in the membrane fraction (Fig. 2C). Evidence that the upper band represents mature band that has moved to the Golgi and acquired complex oligosaccharide chains is provided by its insensitivity to endoglycosidase H (Fig. 2D). It is also notable that the 160-kDa band present only in the double mutant is as apparent in the membrane (Fig. 2C) as in the lysate (Fig. 2A). To determine if it might be an unglycosylated form of the intact polypeptide endoglycosidase H digestion was performed (Fig. 2D). As a result both this band and the larger major core glycosylated band (170 kDa) were decreased in size indicating that they both contained high mannose oligosaccharide chains. Since the enzyme removes these chains completely, the smaller size of the 160-kDa band cannot be due to the presence of less carbohydrate and therefore must reflect absence of a portion of the polypeptide. To confirm kinetically that D792L and D792L/D793L were unable to mature, pulse chase experiments were performed (Fig. 3). The core-glycosylated forms of these mutants and the wild-type formed during the 20-min pulse all disappeared with T 1/ 2 values of less than 1 h. By 3 h of chase the wild-type was completely converted to the larger mature form, whereas very little mature D792L and no mature D792L/D793L appeared. Although faint immature mutant bands were still detectable at 3 h, their rate of disappearance was not much less than that of the wild-type. Thus the mutant coreglycosylated polypeptides appear to be degraded at a rate only slightly slower than the rate of maturation of the wild-type. Proteasomal inhibition causes Walker B aspartate mutants to form insoluble aggregates. Many secretory and membrane proteins unable to assume correct na-

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FIG. 2. Expression of the wild-type and mutant MRP1s detected in Western blots. (A) Relative amounts of MRP1 protein in whole cell lysates. Whole cell lysates of stable BHK cell lines expressing each variant were subjected to Western blotting with monoclonal antibody, 897.2, against NBD2 (Fig. 1A) as probe. The following amounts of protein were loaded in each lane: 0.5 ␮g of wild-type MRP1; 8 ␮g of D792A; 18 ␮g of D792L; 0.5 ␮g of D793L; 20 ␮g of D792L/D793L. (B) The degradation products of MRP1 proteins in whole cell lysates. The same gel in part A was exposed longer against X-ray film. 190 indicates the complex-glycosylated mature 190-kDa MRP1 protein. 170 indicates the core-glycosylated immature 170-kDa MRP1 protein. 160, 130, 100, and 30 indicate the degradation products in the whole cell lysates. (C) Enrichment of the mature protein in membrane vesicles. Membrane vesicles were prepared according the procedure described under Experimental Procedures. The following amounts were loaded in each lane: 0.5 ␮g of wild-type MRP1; 2 ␮g of D792A; 4 ␮g of D792L; 0.35 ␮g of D793L; 8 ␮g of D792L/D793L. The ratio of mature to immature protein in membrane vesicles of D792A and D792L are much higher than in whole cell lysates (Fig. 2A). (D) The smaller fragment of D792L/D793L is also core-glycosylated. The samples were incubated in the absence (⫺) or in the presence of endoglycosidase H (⫹) according to the method described under Experimental Procedures. Lanes 1 and 2, 1 ␮g of D793L cell lysates in each lane; Lanes 3 and 4, 4 ␮g of D792A cell lysates in each lane; Lanes 5 and 6, 10 ␮g of D792L/D793L cell lysates in each lane. Both the 170-kDa core-glycosylated MRP1 protein from either D793L, D792A, or D792L/D793L and 160-kDa degradation product from D792L/D793L were decreased in size by treatment with endoglycosidase H.

tive structures in the endoplasmic reticulum are ubiquitinated, retrotranslocated from the ER membrane and degraded by the 26S proteasome (12–25). To de-

termine whether the MRP1 Walker B mutants are degraded by the proteasome, the influence of inhibitors was tested (Fig. 4). Strikingly, treatment of cells ex-

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FIG. 3. Stability of [ 35S]methionine-labeled nascent MRP1s. The MRP1 cells expressing the variants were pulse labeled with [ 35S]methionine and chased with methionine as described under Experimental Procedures. MRP1 proteins were immunoprecipitated with monoclonal antibody 897.2 and separated on a 6% polyacrylamide gel. Lane 1, 20-min pulse labeling without chase. Lane 2, 0.5-h chase. Lane 3, 1-h chase. Lane 4, 3-h chase. Lane 5, 9-h chase. The molecular weight standards (kDa) are indicated on the left. 170 indicates core-glycosylated immature 170-kDa MRP1 protein. 190 indicates the complex-glycosylated mature 190-kDa MRP1 protein.

pressing either the D792L or D792L/D793L mutants with either lactacystin or ALLN resulted in the total disappearance of the immature forms from nonionic detergent soluble fractions and appearance in insoluble pellets. The banding patterns of the insoluble fractions indicate species both larger and smaller than immature MRP1. The larger species are probably polyubiquitinated but may also be aggregates not fully dissociated by SDS. The smaller forms must be degradation products produced by proteases other than the proteasome. This may occur as a consequence of the toxic effects of the long term proteasome inhibitor treatment of the cells. Nevertheless these results support the view that the proteasome is responsible for degradation of the immature mutant MRP1 polypeptides that are unable to mature. Maturation-incompetent MRP1 mutants exhibit increased susceptibility to trypsin digestion. To determine if the conformational maturation of MRP1 can be detected in vitro, sensitivity to trypsin digestion in its natural environment, in membrane vesicles, was tested. As detected by immunoblots probed with the 897.2 Ab recognizing an epitope in the C-terminal NBD, the disappearance of the mature wild-type band and concomitant appearance of a band of approximately 65 kDa was observed (Fig. 5A). This proteolysis product was first detected at a trypsin to membrane protein mass ratio of 1:64 (lane 7). This was also true in the case of D793L (Fig. 5E) and mutations of the Walker A lysine residues in both NBDs (Fig. 5B, K684L, and Fig. 5G, K1333L), where the protein matured normally as did a variant in which the Walker B aspartate in NBD2 was mutated (Fig. 5H, D1454L/ E1455L). Hence the wild-type protein and variants where mutations in either NBD were known to inhibit MRP1 function but not its maturation all exhibited a very similar pattern of digestion by tryprin. In contrast the pattern of disappearance and appearance of bands

was entirely different in the case of the D792L mutant (Fig. 5D), which matured poorly and the D792L/D793L mutant that did not mature at all (Fig. 5F). First the major degradation band was smaller (⬃58 kDa compared to 65 kDa). This is expected since it is a product of digestion of the dominant 170-kDa core glycosylated immature species rather than the major 190-kDa species digested by trypsin in the case of the wild-type and other fully processed variants. The extension of the single oligosaccharide chain in the sixth extracytoplasmic loop (Fig. 1A) would account for this apparent size difference of approximately 7 kDa between the 65- and 58-kDa products. This smaller product of digestion of the immature D792L polypeptide appeared at a lower trypsin to membrane protein ratio (Fig. 5D, lane 6, 1:128). Consistent with this the intact 170-kDa core glycosylated band disappeared at lower trypsin concentration (Fig. 5D) than did the mature forms of the wild-type (Fig. 5A) and variants that did mature (Figs. 5B, 5E, 5G, and 5H). This comparison is most obvious with the D792A mutant where the membranes subjected to trypsin digestion contain approximately equal amounts of immature and mature species (Fig. 5C). The principle immature band is entirely proteolyzed at a trypsin to membrane protein ratio of 1:16 (lane 9), whereas the mature 190-kDa band is still present at a ratio of 1:2 (lane 12). The smaller C-terminal coreglycosylated product of digestion of the immature band appears at a much lower ratio (58-kDa digestion product appeared at a ratio of 1:128) than does the larger complex-glycosylated product (65 kDa appeared at a ratio of 1:16) of digestion of the mature band. These results provide direct evidence of a major conformation change between the polypeptide bearing core and complex oligosaccharide chains. The D792L mutant is nearly and the D792L/D792L mutant is completely unable to undergo this conformation change.

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P]ATP, which reflects both binding of the intact nucleoside triphosphate and trapping of its hydrolysis product, 8-azido-[␣- 32P] ADP. Figure 6 shows labeling of the wild-type and the innocuous D793L variant which matures normally. However, there was greatly reduced labeling of the variants in which D792 was substituted including D792A where there is considerable mature protein. Therefore the D792 residue is essential to nucleotide binding and trapping by MRP1. Since hydrolysis is believed to drive MRP1 transport it would be expected that the mature D792A protein would not be capable of active transport. The data in Fig. 7 confirm this expectation, i.e., there is not significantly more ATP-dependent LTC 4 uptake by vesicles containing D792A protein that does mature than by the other variants that do not mature (Fig. 7, D792L and D792L/D793L), nor by the NBD2 mutants (Fig. 7, K1333L and D1454L/E1455L) that do mature but have difficulties to hydrolyze ATP and to trap the hydrolysis product, ADP (8). DISCUSSION

FIG. 4. Influence of proteasome inhibitors on MRP1 variants. Subconfluent cells were treated with potent proteasome inhibitors overnight in a 37°C incubator. The cells were lysed with cold buffer containing 10 mM Tris–HCl (pH 7.5), 5 mM EDTA, 1% Triton X-100 and a protease inhibitor cocktail for 30 min at 4°C. The cell lysates were centrifuged at 4°C for 15 min to separate soluble (A) and insoluble proteins (B). Each were solubilized with 1⫻ electrophoresis sample buffer. 25-␮g proteins of each were electrophoresed, blotted and probed with the monoclonal antibody 897.2. The cells were treated without proteasome inhibitor (control), with 30 ␮M lactacystin, or 150 ␮M ALLN. The molecular weight standards (kDa) are indicated on the left. 170 indicates core-glycosylated immature 170kDa MRP1 protein. 160 indicates the major degradation product of D792L/D793L in the absence of proteasome inhibitor.

NBD1 Walker B aspartate is essential for nucleotide occlusion and LTC 4 transport by MRP1. It was impossible to tell if the dysfunction of the D792L mutation was secondary to its inability to mature conformationally. However, since approximately half of the D792A protein in the membrane was mature it was possible to assay its functional capability. To do this we first performed photoaffinity labeling with 8-azido-[␣-

It has become increasingly apparent that missense mutations in many different ABC proteins results in loss of their function due to misfolding, failure to mature and retention and degradation by the ER quality control system (26 –32). This has been most extensively studied in the case of CFTR, where the ⌬F508 mutation which occurs most frequently in patients has this effect (27–32). In the case of this and other diseases referenced above, understanding and manipulating the steps in the quality control is of potential therapeutic relevance. However, in addition the sensitivity of this family of proteins to misfolding on in vitro mutagenesis to explore structure–function relationship can complicate interpretations. This is exemplified by the influence of substitution of the Walker B aspartate in NBD1 of MRP1. The D792L mutant had minimal ability to trap nucleotide and transport an organic anion but it was also unable to mature during biosynthesis. The immature core-glycosylated form was apparently degraded at the ER by the proteasome because proteasome inhibitors caused its accumulation in insoluble aggregates as occurs with ⌬F508 CFTR (30). Hence on the basis of these findings alone it was not possible to conclude whether or not the Walker B aspartate of the first nucleotide binding domain of MRP1 is essential to its transport function. However, when a different substitution, D792A, was made considerable maturation occurred although still less than wild-type (Figs. 2B and 2C). Nevertheless, this mutant was not capable of greater transport activity than the completely misprocessed D792L (Fig. 7). On this basis it can be concluded that this aspartic acid is essential to the active transport function of the protein, perhaps by coordinat-

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FIG. 5. TPCK-treated trypsin digestion of MRP1 variants. The trypsin digestion was performed as described under Experimental Procedures. The ratio of trypsin to membrane protein are: lane 1, without trypsin; 2, 1:2048; 3, 1:1024; 4, 1:512; 5, 1:256; 6, 1:128; 7, 1:64; 8, 1:32; 9, 1:16; 10, 1:8; 11, 1:4; 12, 1:2; 13, 1:1. (A) Wild-type MRP1, 0.3 ␮g of digested protein was loaded in each lane. (B) K684L, 0.6 ␮g protein in each lane. (C) D792A, 0.73 ␮g protein in each lane. (D) D792L, 1.1 ␮g protein in each lane. (E) D793L, 0.3 ␮g protein in each lane. (F) D792L/D793L, 1.1 ␮g protein in each lane. (G) K1333L, 0.3 ␮g protein in each lane. (H) D1454L/E1455L, 0.3 ␮g protein in each lane. As described in the legend of Fig. 2, 190, 170, 160, 130, and 100 indicate the mature, core-glycosylated MRP1 protein and the degradation products of the protein in the original membrane vesicles. 65 indicates the 65-kDa fragment, a partial digestion product from the complex-glycosylated mature protein. 58 indicates the 58-kDa fragment, a partial digestion product from the core-glycosylated immature protein.

ing the divalent cation of the MgATP during hydrolysis. The lack of hydrolysis is presumably reflected in the inability of this variant to be photolabeled by

8-azido-[␣- 32P]ATP under conditions where trapping of the hydrolysis product, 8-azido-[␣- 32P]ADP occurs (Fig. 6).

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Impairment of the trafficking of mutant proteins beyond the ER in the secretory pathway is believed to be due to their inability to mature conformationally (32). The immature core-glycosylated species that have not yet achieved a compact native structure are ubiquitinated and degraded by the 26S proteasome (30). If this proteolysis is inhibited these immature polypeptides form insoluble cellular inclusions (33). This appears to occur with the D792L variant of MRP1 (Fig. 4). The fact that the immature and mature forms of MRP1 are conformationally distinct was confirmed by comparing their sensitivity to limited trypsin digestion in vitro. Much more of the protease was required to digest the mature forms of either the wild-type or the mutant

FIG. 7. ATP-dependent LTC 4 uptake by membrane vesicles prepared from wild-type and mutant MRP1s. The membrane vesicle transport experiments were performed according to Experimental Procedures. Each experiment was performed in triplicate in the presence of 4 mM AMP or 4 mM ATP. The amount of LTC 4 bound to the membrane vesicles in the presence of 4 mM AMP was considered as background and subtracted from the amount of LTC 4 in the presence of 4 mM ATP. The percentages of the LTC 4 transported in this figure are the average of the results of three experiments.

proteins (Fig. 5), consistent with the idea that maturation involves the assumption of a more compact structure in which protease sensitive sites are less exposed. ACKNOWLEDGMENTS We thank Susan Bond and Sharon Fleck for preparation of the manuscript and Marv Ruona for preparation of the graphics.

REFERENCES

FIG. 6. Photolabeling of membrane-associated MRP1 protein with 8-azido-[␣- 32P]ATP. Membrane vesicles from MRP1-expressing cells were incubated for 10 min at 37°C in a 10-␮l solution containing 40 mM Tris–HCl, pH 7.5, 2 mM ouabain, 0.1 mM EGTA, 10 mM MgCl 2, 800 ␮M vanadate, and 5 ␮M 8-azido-[␣- 32P]ATP. Membranes were pelleted, washed, and irradiated at 254 nm for 2 min in a Stratalinker prior to SDS–polyacrylamide electrophoresis. The samples are 10 ␮g of wild-type MRP1, 20 ␮g of D792A, 40 ␮g of D792L, 10 ␮g of D793L, and 40 ␮g of D792L/D793L. Molecular weights of standard proteins are indicated on the left. 190 indicates the complex-glycosylated 190-kDa MRP1 protein.

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