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J. Biol. Chem., Vol. 279, Issue 51, 53571-53583, December 17, 2004

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Identification and Characterization of Functionally Important Elements in the Multidrug Resistance Protein 1 COOH-terminal Region^*

Christopher J. Westlake¶, Lea Payen, Mian Gao, Susan P. C. Cole||**, and Roger G. Deeley, The Stauffer Professor of Basic Oncology at Queen's University||

From the Department of Biochemistry, the ^||Department of Pathology and Molecular Medicine, and the Cancer Research Institute, Queen's University, Kingston, Ontario K7L 3N6, Canada

Received for publication, March 5, 2004 , and in revised form, August 31, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The ATP binding cassette (ABC) transporter, multidrug resistance protein 1 (MRP1/ABCC1), transports a broad spectrum of conjugated and unconjugated compounds, including natural product chemotherapeutic agents. In this study, we have investigated the importance of the COOH-terminal region of MRP1 for transport activity and basolateral plasma membrane trafficking. The COOH-terminal regions of some ABCC proteins have been implicated in protein trafficking, but the function of this region of MRP1 has not been defined. In contrast to results obtained with other ABCC proteins, we found that the COOH-proximal 30 amino acids of MRP1 can be removed without affecting trafficking to basolateral membranes. However, the truncated protein is inactive. Furthermore, removal of as few as 4 COOH-terminal amino acids profoundly decreases transport activity. Although amino acid sequence conservation of the COOH-terminal regions of ABC proteins is low, secondary structure predictions indicate that they consist of a broadly conserved helix-sheet-sheet-helix-helix structure. Consistent with a conservation of secondary and tertiary structure, MRP1 hybrids containing the COOH-terminal regions of either the homologous MRP2 or the distantly related P-glycoprotein were fully active and trafficked normally. Using mutated proteins, we have identified structural elements containing five conserved hydrophobic amino acids that are required for activity. We show that these are important for binding and hydrolysis of ATP by nucleotide binding domain 2. Based on crystal structures of several ABC proteins, we suggest that the conserved amino acids may stabilize a helical bundle formed by the COOH-terminal three helices and may contribute to interactions between the COOH-terminal region and the protein's two nucleotide binding domains.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Multidrug resistance protein 1 (MRP1/ABCC1)¹ is a member of the C branch of the ATP binding cassette (ABC) transporter family that was discovered by virtue of its overexpression in a multidrug-resistant, doxorubicin-selected human small cell lung cancer cell line, H69AR (1). The protein has been shown to confer multidrug resistance to structurally diverse, natural product cytotoxic drugs and to mediate their efflux in an ATP-dependent manner (1, 2). MRP1 has also been detected in tumors with many different cellular origins (3). Although the spectrum of drugs to which MRP1 confers resistance is similar to that of P-glycoprotein (P-gp) (4), the two transporters differ functionally in some important respects. For example, MRP1 but not P-gp transports a wide range of conjugated hydrophilic molecules such as the GSH conjugate leukotriene C₄ (LTC₄), 17-estradiol-17--D-glucuronide (E₂17G), and estrone 3-sulfate, as well as many other substrates, the transport of which is either stimulated by GSH or GSH-dependent (5–13).

Other members of the ABCC branch of the superfamily include the more recently identified MRP2 to -9 as well as the cystic fibrosis transmembrane conductance regulator (CFTR), the sulfonylurea receptors (SUR1 and SUR2), and ABCC13 (2, 14). Structurally, the "core" region of the ABCC proteins is similar to that of many eukaryotic ABC proteins, with the exception of ABCC13, and consists of two membrane-spanning domains (MSD), each containing six transmembrane helices followed by a nucleotide binding domain (NBD). MRPs 1, 2, 3, 6, and 7 and SUR1 and SUR2 are atypical among ABC proteins in that they have a third NH₂-terminal MSD (MSD1) that, based on topology predictions and, in some cases, strong experimental data, contains five transmembrane helices (15–19). This domain is required for the trafficking or activity of MRP2 and the SURs but in MRP1 appears to be dispensable for the binding and transport of at least some substrates, and its function is presently not well defined (20–26). However, a region of the cytoplasmic loop (cytoplasmic loop 3) of MRP1 that connects MSD1 to the remainder of the protein is essential for both substrate binding and appropriate protein trafficking (22, 23, 25, 26). Based on alignment with ABCC proteins lacking the third MSD, the region in cytoplasmic loop 3 begins at a location that aligns closely with the predicted NH₂ terminus of the core structure of MRP1 (27).

In this study, we have examined the importance of the COOH-terminal region of MRP1 for the trafficking and activity of the protein. In some ABCC proteins, such as MRP2, CFTR, and SUR1, mutations in, or deletions of, their COOH-terminal regions have been shown to affect targeting to the plasma membrane (28–30). Mutations of ABC proteins that result in aberrant trafficking in mammalian cells frequently result in retention and rapid degradation in the endoplasmic reticulum (22, 25, 28, 31). This precludes analysis of their effect on the function of the protein. However, when expressed in insect cells, some of these mutant proteins are stable and active, indicating that the mutations have not caused gross perturbations in the structure of the protein (25, 31). These observations are presumed to be attributable to the lower temperatures at which the insect cells are cultured and a less stringent proofreading machinery for monitoring protein folding in the endoplasmic reticulum (31). Consequently, we have carried out parallel studies of the COOH-terminal region of MRP1 using polarized Madin-Darby canine kidney (MDCK)-I cells to screen for mutations and deletions that affect protein trafficking and insect Sf21 cells to examine the effects of these structural alterations on protein function.

In contrast to results obtained with other ABCC proteins (28, 29), we found that removal of up to 30 amino acids from the COOH terminus had no effect on protein trafficking or binding of a substrate such as LTC₄ but essentially eliminated transport activity. Furthermore, deletion of as few as 4 amino acids from the COOH terminus was sufficient to decrease activity by 75%. We show that this reduction in transport function is accompanied by a decrease in ATP binding and hydrolysis by NBD2 and an impaired ability to shift from a high to low affinity substrate binding state. Despite little amino acid sequence identity, transport activity can be restored by substitution of the COOH-terminal region of MRP1, with the comparable regions of MRP2 and the distantly related P-gp, indicating that the secondary and tertiary structures of these regions must be very similar. Alanine scanning mutagenesis revealed that five hydrophobic residues within the COOH-terminal region that are broadly conserved among ABC proteins are critical for activity of MRP1. Comparison of the predicted tertiary structure of MRP1 in this region with the crystal structures of distantly related ABC proteins suggests that these residues contribute to the stability of a highly conserved -helical bundle, elements of which are in close proximity to functionally important residues in both NBDs.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—[14,15,19,20-³H₄]LTC₄ (182 Ci mmol^-) and [6,7-³H₂]E₂17G (45 Ci mmol^-1) were purchased from PerkinElmer Life Sciences. 8-azido-[-³²P]N₃ATP, 8-azido-[-³²P]N₃ATP, and 8-azido-[-³²P]N₃ADP were from Affinity Labeling Technologies Inc. (Lexington, KY). LTC₄ was purchased from Calbiochem. Orthovanadate, AMP, ATP, ATPS, and anti-calnexin mAb were from Sigma. mAb MRPr1 and MRPm5 were obtained from Alexis (San Diego, CA), and 7-aminoactinomycin D and fluorescent labeled secondary antibodies Alexa 488 and Alexa 546 were purchased from Molecular Probes, Inc. (Eugene, OR). Canine Na⁺/K⁺-ATPase polyclonal antibody was purchased from ABCAM. Fugene6 and Complete EDTA-free protease inhibitors were purchased from Roche Applied Science. Geneticin was obtained from Invitrogen. N-(4-Azido-3-[¹²⁵I]phenyl)-2-[3-(9-chloro-3-methyl-4-oxo-4H-isoxazolo[4,3-c]quinolin-5-yl)-cyclohexyl]-acetamide ([¹²⁵I]LY475776) (295.2 µCi/ml) was synthesized and purified by Lilly and generously provided as a gift.

Generation of MRP1 COOH-terminal Truncation and Chimeric Constructs and Site-directed Mutagenesis—Full-length MRP1 in the pFastBac (pFB) vector (pFB-MRP1) and pCDNA3.1 (pCDNA-MRP1) have been previously described (25, 32). MRP1 NH₂-(aa 1–932) and COOH-proximal (aa 932–1531) halves cloned into the pFastBac DUAL expression vector (pFBdh-MRP1) have been previously described (33). PCR was used to generate constructs lacking the COOH-terminal region of MRP1 (Fig. 1A) with pFB-MRP1 as a template. The forward primer D1241 was designed to amplify MRP1 nucleic acids upstream of the unique ClaI site at bp 4058. The reverse primers added a stop codon and a KpnI site after MRP1 codons for aa 1493, 1498, 1501, 1509, 1522, and 1527. A reverse primer was also designed to delete codons for Ser¹⁵²³–Asp¹⁵²⁷ (MRP1_{1–1522+AGLV}). The DNA products were digested with ClaI and KpnI and ligated into ClaI/KpnI pFB-MRP1 and pFBdh-MRP1, which had the COOH-terminal regions excised. The fidelity of the COOH-terminal truncated MRP1 constructs was confirmed by sequencing. Mutant MRP1 constructs were subsequently moved into NotI-KpnI-digested pCDNA3.1(-) via an EagI-KpnI digest of the pFB DNA.

View larger version (66K): [in this window] [in a new window]   FIG. 1. Trafficking of MRP1 COOH-terminal deletion constructs in MDCK-I cells. A, MRP1 COOH-terminal truncation constructs engineered by PCR. B and C, MRP11–1498 (left) and MRP11–1501 (right) were expressed in polarized MDCK-I cells grown at either 37 °C (B) or 27 °C (C) and immunodetected with mAb MRPr1 and Alexa 488 goat anti-rat (green signal) using confocal microscopy as described under "ExperimentalProcedures." Cells were stained with either the endoplasmic reticulum marker mAb anti-calnexin (B), detected with Alexa 546 goat anti-rabbit (red signal), or the polyclonal antibody Na+/K+-ATPase basolateral membrane marker (C), immunodetected with Alexa 546 goat anti-chicken (red signal). Nuclei were counterstained with 7-aminoactinomycin D (blue signal). The arrows mark the position of the vertical x/z sections made through the cells shown in the lower panels. White signals shown in the middle and bottom panels indicate areas of colocalization of the truncated MRP1 proteins and the cellular marker.

All COOH-terminal mutations were generated using the QuikChange^TM site-directed mutagenesis kit (Stratagene, La Jolla, CA). MRP1 template DNA was prepared by digesting pFB-MRP1 with EcoRI and KpnI and cloning the COOH-terminal 713 base pairs into pBS. Oligonucleotides bearing mismatched bases at the COOH-terminal residues to be mutated were generated, and mutagenesis was performed according to the instructions from the manufacturer. Mutations were confirmed by sequencing. DNA fragments containing the desired mutations were cloned into the pFB-MRP1 as described above for the MRP1 COOH-terminal truncation constructs, and the fidelity of constructs was confirmed by sequencing.

Expression and Localization of MRP1 in MDCK-I Cells—Generation of MDCK-I cells stably expressing COOH-terminal truncation constructs cloned into the pCDNA3.1(-) vector has been previously described (25). For localization studies, stable expressing MDCK-I cells were seeded on glass coverslips and polycarbonate filters and grown for 10–14 days at 37 °C to allow polarization. In some cases, after cells had polarized on glass coverslips and polycarbonate filters they were incubated at 27 °C for 48 h. Ethanol fixation of MDCK-I cells and immunofluorescent detection with the mAb MRPr1 and either the mAb anti-calnexin or a polyclonal antibody Na⁺/K⁺-ATPase have been previously described (25). mAb MRPr1 was immunodetected with Alexa 488 goat anti-rat, whereas Alexa 546 goat anti-rabbit and Alexa 546 goat anti-chicken were used to detect antibody interactions with the cellular markers calnexin and Na⁺/K⁺ ATPase, respectively. The fluorescent secondary antibodies and the 7-aminoactinomycin D nuclear stain were excited with a Leica TCS SP2 dual photon confocal microscope.

Viral Infection, Membrane Vesicle Preparation, and Immunoblotting—Baculoviruses generated from recombinant bacmids were used to infect Sf21 insect cells as described previously (32). To generate membrane vesicles, insect cells were disrupted by nitrogen cavitation, and the vesicles were isolated by sucrose density centrifugation (7). Full-length wild-type and COOH-terminal truncated, mutated, and hybrid membrane proteins were subjected to SDS-PAGE (7.5%), whereas dual half-expressed proteins were resolved using SDS-PAGE gradient gels (7.5–15%). Immunoblots of intact proteins were probed with mAb MRPr1 (35, 36), whereas the dual expressed NH₂-half was immunodetected using mAb MRPr1, and the COOH-half was immunodetected with mAb MRPm5 (37). Antibody-protein interactions were detected using horseradish peroxidase secondary antibodies utilizing an enhanced chemiluminescence, and the relative expression of MRP1 fragments was determined by densitometry.

LTC₄ and E₂17G Transport into Sf21 Membrane Vesicles—Uptake of [³H]LTC₄ (50 nM) and [³H]E₂17G (400 nM) was measured at 23 and 37 °C, respectively, as described previously (6, 32). Briefly, 4 µg (LTC₄ uptake) or 8 µg (E₂17G uptake) of Sf21 membrane vesicles were incubated with tritiated substrates in the presence of 4 mM ATP or AMP and 10 mM MgCl₂ in TB (50 mM Tris, pH 7.4, 250 mM sucrose). ATP-dependent transport was determined by subtracting uptake in the presence of ATP from uptake in the presence of AMP. For ATP-dependent kinetic determinations, [³H]LTC₄ (150 nM, 182 Ci mmol^-1) uptake after 1 min was measured at different ATP or AMP concentrations (25–2000 µM) and plotted by the Hanes-Woolf method.

Photoaffinity Labeling of MRP1 Constructs with [³H]LTC₄ and [¹²⁵I]LY475776—Labeling of MRP fragments by [³H]LTC₄ and [¹²⁵I]LY4775776 was carried out as described previously (24, 38). Briefly, Sf21 membrane vesicles (75 µg of protein) were incubated with 200 nM [³H]LTC₄ (0.25 µCi) for 30 min at room temperature. Samples were frozen in liquid nitrogen and immediately UV-irradiated at 302 nm for 30 s and repeated 10 times. For [¹²⁵I]LY475776 labeling, membrane vesicles (75 µg of protein) were incubated with the MRP1 inhibitor (0.5 nM) at 37 °C for 10 min before UV irradiation for 5 min at 302 nm on ice. Vesicle proteins labeled with radioactive LTC₄ and LY475776 were then resolved on SDS-PAGE (7.5%). Gels were dried for 2 h at 80 °C and exposed to film.

Photolabeling of MRP1 NBDs Using 8-Azido-[-³²P]ATP—8-Azido-[-³²P]ATP and 8-azido-[-³²P]ATP photolabeling was performed as described previously (33). Briefly, Sf21 membrane vesicles (20 or 30 µg of protein) were incubated with 5 µM 8-azido-[-³²P]ATP in TB containing 5 mM MgCl₂ for 5 min on ice, followed by UV irradiation at 302 nm for 8 min on ice. The reaction was stopped, and free radioactive nucleotide was removed by the addition of 500 µl of cold wash buffer (50 mM Tris-HCl, pH 7.4, 0.1 mM EGTA, 5 mM MgCl₂) before centrifugation at 14,000 rpm. The wash was repeated, and the pelleted vesicles were resuspended in Laemmli buffer (4x) containing dithiothreitol (100 mM). Under conditions where MRP1 wild-type and COOH-terminal truncated proteins were proteolytically digested, UV-irradiated radioactive nucleotide-labeled vesicle proteins were resuspended in TB containing 20 ng of trypsin (1:1000) and incubated for 30 min at 37 °C. Proteins were resolved on gradient SDS-PAGE (7.5–15%), and the gels were dried for 2 h at 80 °C before exposure to film. For K_d determinations, vesicle proteins were labeled with various concentrations of 8-azido-[³²P]ATP (1–128 µM). Densitometry was performed to determine the radioactivity incorporated into the NBDs. Because of the higher concentrations of 8-azido-[-³²P]ATP that were required to saturate the COOH-terminal NBD of the truncated proteins in K_d determinations, binding was corrected for the background observed in a parallel experiment using membrane vesicles prepared from Sf21 cells infected with a -gus expression vector.

Photolabeling of MRP1 NBDs Using 8-Azido-[-³²P]ADP—8-Azido[-³²P]ADP labeling was performed as described previously (39). Sf21 membrane vesicles (20 µg of protein) were incubated with 5 µM 8-azido[-³²P]ADP in TB containing 1 mM orthovanadate and 5 mM MgCl₂ for 15 min on ice, followed by UV irradiation at 302 nm for 8 min on ice. Unbound radionucleotides were removed, and proteins were resolved on gradient SDS-PAGE (7.5–15%). Gels were subsequently dried at 80 °C prior to exposure to film.

Vanadate and Beryllium Fluoride Trapping of 8-Azido-[-³²P]ATP at 37 °C—ADP orthovanadate and beryllium fluoride trapping was performed as described previously (33, 39). Briefly, Sf21 membrane vesicles (20 µg of protein) were incubated with of 8-azido-[-³²P]ATP (5–45 µM) for 15 min in TB containing 5 mM MgCl₂ for 15 min at 37 °C in the presence or absence of 1 mM orthovanadate or 200 µM beryllium fluoride. Wash buffer was added to the samples, followed by centrifugation, and the wash was repeated. The pelleted vesicles were resuspended in 15 µl of wash buffer and transferred to a 96-well plate, after which the samples were UV-irradiated at 302 nm for 8 min on ice. Vesicle proteins were then diluted in Laemmli buffer (4x) containing dithiothreitol (100 mM) and resolved on gradient SDS-PAGE (7.5–15%). The gels were dried for 2 h at 80 °C before audioradiography.

Photoaffinity Labeling with [³H]LTC₄ in the presence of ATP, ATP and Orthovanadate, or ATPS—Photoaffinity labeling of MRP fragments by LTC₄ in the presence of nucleotides has been described previously (24, 39). Briefly, membrane vesicles (50 µg) were incubated with either ATP (4 mM), ATP (4 mM), and orthovanadate (1 mM) or with ATPS (4 mM) with 5 mM MgCl₂ in TB for 30 min at room temperature, followed by incubation with 200 nM [³H]LTC₄ for 30 min at room temperature. Subsequently, proteins were UV-irradiated and resolved on SDS-PAGE (7.5%), and gels were dried before exposure to film.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Plasma Membrane Trafficking by COOH-terminal MRP1 Truncated Proteins in MDCK-I Cells—To determine whether or not the MRP1 COOH-terminal region is required for plasma membrane localization, we prepared stable MDCK-I transfectants expressing various MRP1 COOH-terminal truncated proteins (Fig. 1A). Deletion of the COOH-terminal 4, 9, 22 (not shown), or 30 amino acids of the protein did not prevent trafficking to the basolateral membranes in MDCK-I cells (Fig. 1B). However, further truncation to Ile¹⁴⁹⁸ (Fig. 1B) or Asp¹⁴⁹³ (not shown) resulted in accumulation of the mutant proteins in intracellular membranes that stained positively for calnexin, a well established marker for the endoplasmic reticulum (40). Previously, it has been demonstrated that short term growth at 27 °C can promote surface expression of CFTR mutant proteins that display trafficking defects (31). Growth under such conditions did not correct the trafficking defect of MRP1_1–1498, as assessed by its lack of colocalization with the Na⁺/K⁺-ATPase (Fig. 1C), a known marker for the basolateral membrane in this cell line (25). It has recently been reported that Ala substitution of Val¹⁴⁹⁷-Ile¹⁴⁹⁸-Val¹⁴⁹⁹-Leu¹⁵⁰⁰ in the COOH-terminal region of MRP1 impaired protein maturation in BHK-21 cells (41). The analogous tetramer of hydrophobic residues within the COOH-terminal region of CFTR, Phe¹⁴¹³-Leu¹⁴¹⁴-Val¹⁴¹⁵Ile¹⁴¹⁶, has also been shown to be important for protein processing (41). Our findings support the importance of this motif and demonstrate that elimination of the COOH-terminal region to include Val¹⁴⁹⁹ and Leu¹⁵⁰⁰ is sufficient to disrupt normal processing in MDCK-I cells.

LTC₄ and E₂17G Transport Activity by COOH-terminal Truncated MRP1—To assess the effect of COOH-terminal truncations on the activity of MRP1, the mutant proteins were expressed in Sf21 cells using a baculovirus expression system. Densitometry performed on immunoblots of membrane proteins from the infected Sf21 cells indicated that the MRP1 truncations were expressed at levels similar to the full-length protein, with the exception of MRP1_1–1493, the levels of which were 60% of wild-type (Fig. 2A). In all data shown, ATP-dependent LTC₄ uptake was corrected by subtracting the low levels of endogenous activity present in membranes from control cells infected with a -gus vector and then normalized for differences in expression levels (Fig. 2B). The transport activity of MRP1_1–1527 and MRP1_1–1522 was 30% of that observed for wild-type MRP1. Further truncation to Gly¹⁵⁰⁹ reduced transport to 15%, whereas MRP1_1–1501, MRP1_1–1498 (not shown), and MRP1_1–1493 had less than 10% of the transport activity of the full-length protein (Fig. 2B). The relative rates of E₂17G transport by the various truncated proteins were similar to those observed for LTC₄, with the exception of MRP1_1–1522, which, for reasons that are presently not known, consistently retained 50% activity (Fig. 2C). Thus, although the COOHterminal 30 amino acids of MRP1 are dispensable as far as trafficking of the protein is concerned, elimination of only the COOH-terminal 4 amino acids has a major effect on transport activity. However, the addition of Ala¹⁵²⁸–Val¹⁵³¹ to the COOH terminus of partially active MRP1_1–1522 had no effect on transport of either LTC₄ or E₂17G (not shown), indicating that the COOH-terminal 4 residues are not sufficient to restore transport activity to the more extensively deleted protein.

View larger version (20K): [in this window] [in a new window]   FIG. 2. Effect of COOH-terminal truncation on MRP1 LTC4 and E217G transport activity. A, immunoblot of wild-type (residues 1–1531) and COOH-terminal truncated MRP1 proteins expressed in Sf21 insect cells detected with mAb MRPr1. B and C, membrane vesicles were incubated with 50 nM [3H]LTC4 (B) or 400 nM [3H]E217G (C) and 4 mM ATP or AMP as described under "Experimental Procedures." ATP-dependent transport was normalized for differences observed in protein expression (A) and plotted as a percentage of the wild-type uptake after 1 min.

View larger version (17K): [in this window] [in a new window]   FIG. 3. Transport activity of MRP1/MRP2 and MRP1/P-gp COOH-terminal hybrid proteins. A, immunoblot of wild type, MRP11–1465/MRP21473–1545, and MRP11–1509/P-gp1255–1279 expressed in Sf21 insect cells detected with mAb MRPr1. B, normalized ATP-dependent uptake of membrane vesicles incubated with 50 nM [3H]LTC4 was performed as described under "Experimental Procedures."

Substrate and Inhibitor Binding by COOH-terminal Truncated MRP1 Proteins—To determine whether the effect of the COOH-terminal deletions on transport activity was a consequence of altered substrate binding, we carried out photoaffinity labeling experiments using LTC₄ and the recently developed, high affinity MRP1 inhibitor, LY475776 (38, 42, 43). We have shown previously that LTC₄ preferentially labels a site in MSD2 and to a lesser extent a site in MSD3 (24). In contrast, LY475776 displays GSH-dependent binding and almost exclusively labels a site in MSD3 (38, 43). The results of these studies indicated that binding of substrate and inhibitor was not affected by any of the COOH-terminal truncations examined that extended to Asp¹⁵⁰¹ (not shown).

Kinetic Analysis of ATP-dependent LTC₄ Uptake and 8-Azido-[-³²P]ATP Photolabeling—Given that no major changes in substrate binding were caused by the COOH-terminal truncations, we examined the possibility that loss of transport activity was attributable to decreased nucleotide binding and/or hydrolysis. Kinetic analysis of LTC₄ uptake by MRP1_1–1527 revealed that the K_m for ATP (495 µM) was 3-fold higher than observed for the full-length protein (K_m(ATP) = 161 µM) (Fig. 4A). Furthermore, after normalizing for differences in protein expression levels, the V_max for LTC₄ transport by the truncated protein was 2.5-fold lower (75 pmol mg^-1 min^-1) than that of wild-type MRP1 (190 pmol mg^-1 min^-1). These results strongly suggest that the 70% reduction in LTC₄ and E₂17G transport observed after removal of the COOH-terminal 4 amino acids is attributable to impaired ATP binding and/or hydrolysis. Similar analyses of the more extensively deleted proteins was precluded by the very low levels of residual transport activity.

View larger version (23K): [in this window] [in a new window]   FIG. 4. Effect of COOH-terminal truncation on the Km and Vmax of ATP-dependent LTC4 transport and 8-azido-[-32P]ATP photolabeling. A, Michaelis-Menten plot showing AMP-subtracted ATP-dependent LTC4 uptake (150 nM) into MRP11–1531-expressing (•) and MRP11–1527-expressing () membrane vesicles (8 µg) at different concentrations of ATP/AMP (25–2000 µM) as described under "Experimental Procedures." Hanes-Woolf plots (inset) were used to determine Km and Vmax values. Shown are immunoblot (B) and audioradiograph (C) of wild-type and COOH-terminal truncated MRP1 vesicle proteins digested with trypsin for 30 min at 37 °C after photocross-linking with 8-azido-[-32P]ATP as described under "Experimental Procedures." The positions of 8-azido-[-32P]ATP-labeled NH2- and COOH-proximal half MRP1 fragments and endogenous proteins are indicated (E).

Photolabeling of Dual Expressed MRP1_1–932/MRP1_932–1527 with 8-Azido-[-³²P]ATP and 8-Azido-[-³²P]ADP under Nonhydrolytic Conditions—To confirm that nucleotide interaction with NBD2 was altered by COOH-terminal truncation, we co-expressed the NH₂- and COOH-proximal halves of both the wild-type protein and MRP1_1–1527. We have shown that it is possible to use a baculovirus dual expression system to produce stoichiometrically equivalent amounts of NH₂- and COOH-proximal fragments of MRP1 that reassociate to form a functional LTC₄ transporter with greater than 90% efficiency (24, 32, 33, 39). Densitometry of immunoblots (Fig. 5A) indicated that both fragments of MRP1_1–1527 (MRP1_1–932 and MRP1_932–1527) were consistently expressed at 2-fold lower levels than those obtained with the vector encoding the two wild-type halves, MRP1_1–932 and MRP1_932–1531. To compensate for this difference in expression levels, vesicles containing the wild-type half molecules were diluted with an equivalent amount, based on total protein, of vesicles from control cells infected with a -gus+CAT vector prior to analysis. LTC₄ transport assays were used to confirm that the addition of the control vesicles resulted in the predicted decrease in specific uptake activity (Fig. 5B).

View larger version (27K): [in this window] [in a new window]   FIG. 5. Transport activity of dual expressed MRP1 fragments, Kd determinations for 8-azido-[-32P]ATP binding, and azido-[-32P]ADP binding. A, immunoblot of pFastBac DUAL expressed wild-type (dh 1–1531) and MRP11–1527 (dh 1–1527) NH2- and COOH-proximal fragments immunodetected with mAb MRPr1 (top; 1 µg/lane) and mAb MRPm5 (bottom; 2 µg/lane), respectively. dh1–1531 and 1/2dh1–1531 are NH2- and COOH-proximal fragments that have approximately equivalent and half the expression levels of dh1–1527, respectively. Total protein content was maintained by mixing dh wild-type and dh-gus+CAT control vesicles. B, ATP-dependent LTC4 transport by dual expressed MRP1 fragments. LTC4 transport activity was determined for vesicles containing dh-gus+CAT with 25, 33.3, 50, and 100% total wild-type protein (inset). C, vesicle proteins (20 µg) containing dh1–1531 and dh1–1527 were labeled with various concentrations of 8-azido-[-32P]ATP (1–128 µM) labeling as described under "Experimental Procedures." Radioactive nucleotide incorporation in NBD1 and NBD2 was determined electronically following exposure of gels to phosphoscreens using a PhosphorImager (Amersham Biosciences). Kd values were determined by plotting the radioactive nucleotide signal for MRP11–1531 NBD1 () and MRP11–1531 NBD2 () and the -gus-subtracted NBD1 MRP11–1527 (•) and MRP11–1527 () against the concentration of 8-azido-[-32P]ATP. D, audioradiograph of dual expressed vesicle proteins (20 µg) after photolabeling with 8-azido-[-32P]ADP (5 µM) in the presence of 1 mM orthovanadate as described under "Experimental Procedures." The positions of labeled MRP1 NH2-half and COOH-half are indicated along with radioactive nucleotide cross-linked endogenous proteins (E).

To determine whether the reduced photolabeling of NBD2 of MRP1_1–1527 was the result of a decreased ability to stabilize binding via interactions with the -phosphate of ATP, we carried out similar experiments with 8-azido-[-³²P]ADP. Previously, we have shown that 8-azido-[-³²P]ADP strongly photolabels NBD2 of dhMRP1_1–1531 in the presence of vanadate at 4 °C (39). ADP binding at NBD2 of MRP1_1–1527 was weak in comparison with the strong binding of the wild-type NBD2 (Fig. 5D). Thus, removal of the COOH-terminal 4 amino acids decreases binding of both ATP and ADP to NBD2.

Nucleotide Trapping by Wild-type dhMRP1 and dhMRP1_1–1527 at 37 °C—Since ATP and ADP binding by NBD2 was impaired at 4 °C following removal of the COOH-terminal 4 amino acids, we next determined whether ATP hydrolysis was also affected. Comparison of the dhMRP1_1–1531 and dhMRP1_1–1527 proteins following vanadate trapping studies at 37 °C showed that ADP trapping was almost completely abolished at NBD2 of the COOH-terminal truncated protein (Fig. 6A). Similar experiments were also performed with beryllium fluoride, since ADP trapped in the presence of beryllium is believed to mimic an earlier step in catalysis and to correspond to an ATP binding ground state, as opposed to the posthydrolytic transition state formed in the presence of vanadate (47). Despite the fact that trapping was carried out at concentrations of 8-azido-[-³²P]ATP as high as 45 µM, no trapping at NBD2 of dhMRP1_1–1527 was detected (Fig. 6B). Comparison of the approximate 2-fold reduction in ATP binding at NBD2 of MRP1_1–1527 with the almost complete abolition of ADP trapping in the presence of either beryllium and vanadate suggests that hydrolysis is also compromised. This observation is consistent with the 2.5-fold reduction in V_max for LTC₄ transport by the truncated protein (Fig. 4A).

View larger version (48K): [in this window] [in a new window]   FIG. 6. Nucleotide trapping by dual expressed MRP11–1527 using 8-azido-[-32P]ATP. A, under hydrolytic conditions (37 °C), ADP trapping by dh1–1531 (50% dh wild-type + 50% dh-gus+CAT), 1/2dh1–1531 (25% dh wild type + 75% dh-gus+CAT), and dh1–1527 were determined. Membrane vesicles (30 µg) were incubated with 8-azido-[-32P]ATP (15 µM) in the absence (-) and presence (+) of 1 mM orthovanadate for 15 min in TB containing 5 mM MgCl2. Unincorporated nucleotide was removed by centrifugation before UV-cross-linking and SDS-PAGE and audioradiography as described under "Experimental Procedures." B, under the conditions described in A, membrane vesicles (30 µg) were labeled with 8-azido-[-32P]ATP (5, 15, and 45 µM) in the absence (-) and presence (+) of 200 µM beryllium fluoride for 15 min. The positions of labeled MRP1 NH2- and COOH-proximal half are indicated along with radioactive nucleotide cross-linked endogenous proteins (E).

View larger version (31K): [in this window] [in a new window]   FIG. 7. Effects of the COOH-terminal region on the nucleotide-dependent LTC4 binding switch. A and B, vesicle proteins (50 µg) were preincubated for 30 min at room temperature in the absence (-) and presence (+) of 4 mM ATP, 4 mM ATP, and 1 mM orthovanadate or 4 mM ATPS, followed by the addition of 200 mM [3H]LTC4 and photoaffinity cross-linking as described under "Experimental Procedures."

View larger version (27K): [in this window] [in a new window]   FIG. 8. ABC protein alignment and structural model of the COOH-terminal region. Shown is alignment of predicted secondary structures (human MRP1, MRP2, MRP3, CFTR, and P-gp; hamster SUR1; and mouse SUR2A) and known secondary structures (hemolysin B, LmrA, and Tap1) of the COOH-terminal regions of ABC proteins, with -helical (boldface) and -sheet (underlined) elements indicated. Secondary structure predictions were performed using PSI-pred (60).

In addition to single mutations, Ala substitutions were made of the conserved hydrophobic tetramer Leu¹⁴⁹⁷-Ile¹⁴⁹⁸-Val¹⁴⁹⁹-Leu¹⁵⁰⁰ and the dileucine motif in predicted helix C2. The L1497A/I1498A/V1499A/L1500A mutation essentially inactivated the protein, and the double L1514A/L1515A mutation reduced transport by 80%. Similar to the MRP1_1–1527, the L1514A/L1515A mutant had a 4-fold increase in the K_m for ATP and an approximate 2.5-fold decrease in the V_max for LTC₄ transport. In contrast, the D1527A/G1529A double mutation increased LTC₄ uptake by 25% (Table I).

Comparison with more distantly related ABC proteins indicated that Val and Ile are also found at one or the other of the positions of the highly conserved helix C2 residues, Leu¹⁵¹⁴ and Leu¹⁵¹⁵ (Fig. 8). Consequently, we created two additional double mutations, L1514V/L1515V and L1514I/L1515I. The former decreased LTC₄ transport activity by 25% and the latter by only 5% (Table I). These results confirm the requirement for hydrophobic amino acids with side chains larger than that of Ala at this location. Similarly, the residue present at the analogous position of MRP1 Phe¹⁵²¹ in other ABC proteins is typically aromatic. Consistent with a requirement for an aromatic side chain, mutation of Phe¹⁵²¹ to Tyr had no effect on LTC₄ transport, whereas substitution with Gly, Gln, or Glu reduced LTC₄ transport activity by 60, 70, and 75%, respectively (Table I).

Since mutation of Met¹⁵²⁴ in helix C1 to Ala increased LTC₄ transport activity, we also mutated this residue to Gly, which, unlike Ala, would be expected to decrease the stability of the helix. In contrast to Ala substitution, the M1524G mutation decreased LTC₄ transport by 35%. Furthermore, although a single mutation of the adjacent Ala¹⁵²⁴ to Gly did not affect activity, the double mutation M1524G/A1525G reduced LTC₄ transport by 75% (Table I). Kinetic analysis of this mutant showed that the K_m for ATP was increased by 4.5-fold, whereas the V_max for LTC₄ transport was 3-fold lower (Table I). Photoaffinity binding studies indicate that this double glycine mutation impaired nucleotide binding and trapping at NBD2 to an extent comparable with that resulting from removal of the COOH-terminal 4 amino acids (not shown). These observations strongly suggest that the integrity of the -helix at the COOH terminus of MRP1 is important for efficient nucleotide binding and possibly hydrolysis by NBD2.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Previous studies have shown that the COOH-terminal regions of the ABCC proteins MRP2 and SUR1 are important for protein trafficking and/or maturation (28, 29). In contrast, up to 60 amino acids can be eliminated from the COOH terminus of CFTR without affecting protein maturation (48). However, it has also been reported that the terminal PDZ motif, which is in this region, is important for apical membrane localization (30). We found that the COOH-terminal region of MRP1 was not required for trafficking, but unlike CFTR, elimination of as few as 4 amino acids markedly reduced activity (41). Despite this observation, exchange of the COOH-terminal region of MRP1 for comparable regions of MRP2 and P-gp did not affect either activity or basolateral trafficking of the hybrid proteins. This result, particularly in the case of the hybrid involving the distantly related P-gp, strongly suggests that the higher order structure of the COOH-terminal regions of these proteins are sufficiently conserved to permit retention of function, despite their relatively low amino acid sequence conservation.

Sequence alignments of the COOH-terminal regions of a number of prokaryotic and human ABC transporters are shown in Fig. 8. Secondary structure algorithms predict a conserved helix-sheet-sheet-helix-helix motif in the region COOH-proximal to the highly conserved "switch" histidine in NBD2 (Fig. 8). Comparison with analogous regions of the crystal structures of several distantly related ABC proteins reveals broad conservation of these secondary structure elements. Based on the model depicted in Fig. 8, the studies of MRP2 and SUR1 suggest that elimination of part of helix C1 together with the downstream COOH-terminal region of the protein is sufficient to impair localization to the plasma membrane (28, 29). In contrast, elimination of helix C1 of MRP1 by truncation to amino acid 1522 or even more extensive truncation to Asp¹⁵⁰¹ did not prevent targeting to the basolateral plasma membrane in polarized cells, indicating that necessary trafficking signals must reside elsewhere in the protein. Thus, despite the predicted conservation of secondary structure, the requirement for various conserved elements in the COOH-terminal region for protein processing and/or plasma membrane trafficking differs among the ABCC proteins. Further truncation of MRP1 to Ile¹⁴⁹⁸ did result in retention in the endoplasmic reticulum, and it has been shown recently that mutation of the hydrophobic tetramer Val¹⁴⁹⁷-Ile¹⁴⁹⁸-Val¹⁴⁹⁹-Leu¹⁵⁰⁰ in MRP1 as well as the corresponding Phe-Leu-Val-Ile sequence in CFTR impaired maturation of both proteins (41). Together, these results indicate that the integrity of sheet C2 is critical for trafficking of MRP1, whereas the COOH-terminal sheet C1 and -helices C2 and C1 are not essential (Fig. 8).

Studies with MRP1 and other ABC proteins, notably CFTR, have shown that mutant proteins that fail to traffic in mammalian cells may be functional when expressed in insect cells (25, 31). At present, it is not known whether COOH-terminal truncations of MRP2 and SUR1 that prevent trafficking also affect the activity of the protein, since comparable studies in insect cells have not been reported (28, 29). Nevertheless, the results described here represent the converse situation in which truncations of the COOH-terminal region of MRP1 that do not affect trafficking profoundly decrease activity. Notably, removal of only the 4 (MRP1_1–1527) or 9 (MRP_1–1522) COOH-terminal amino acids reduced transport activity by 70%. Since elimination of either 4 or 9 amino acids had a similar effect, we examined whether the COOH-terminal 4 amino acids might comprise a discrete element required for activity. However, their addition to MRP1_1–1522 failed to restore function (not shown). Consequently, it appears likely that the decrease in activity of the protein following elimination of the COOH-terminal 4 or 9 amino acids is a result of destabilizing helix C1. This suggestion is further supported by the effect of introducing destabilizing Gly mutations into this helix, as exemplified by the double M1524G/A1525G mutation, which had the same effect on activity as deletion of the terminal 4 or 9 amino acids.

Truncation of MRP1 to Asp¹⁵⁰¹, which removes sheet C1, helix C2, and helix C1 and eliminates transport activity, did not affect substrate binding, as evidenced by photolabeling with LTC₄ and the MRP1 reversing agent, LY475776 (not shown). Rather, the loss of activity following COOH-terminal truncation appears to be the consequence of a reduced ability of NBD2 to bind nucleotide and hydrolyze ATP. Removal of the terminal 4 amino acids increased the K_m(ATP) for LTC₄ uptake 3-fold and decreased the V_max 2.5-fold. Photolabeling studies of wild-type and truncated proteins with 8-azido-[-³²P]ATP, followed by limited tryptic digestion, suggested that nucleotide binding to NBD1 was unaffected, whereas binding to NBD2 of MRP1_1–1527 appeared to be impaired relative to the wild-type protein and to be even lower for the more severely compromised MRP1_1–1501. More quantitative analyses using a dual vector to express the NH₂- and COOH-halves of MRP1_1–1527 indicated that the K_d of NBD2 for ATP was increased by 2-fold, whereas the binding of nucleotide to NBD1 was unaffected (Fig. 5C). Furthermore, the trapping of ADP in the presence of vanadate or beryllium was barely detectable even at relatively high concentrations of ATP, suggesting that nucleotide hydrolysis as well as binding is impaired in the truncated protein (Fig. 6, A and B). Similarly, truncation of CFTR to Lys¹⁴²⁰ (which aligns with MRP1 Glu¹⁵⁰⁴) reduces channel activity 4–5-fold, and it has been suggested that this is attributable to reduced ATP binding and hydrolysis by NBD2 (41). This CFTR truncation would be predicted to eliminate the analogous sheet C1 and helices C2 and C1 (Fig. 8).

As confirmation that the decreased nucleotide binding by NBD2 of the COOH-terminal truncations was of functional significance, we examined the effect of ATP and ATPS on substrate binding by the truncated proteins. We have shown that binding of nucleotide to NBD2 of the wild-type protein results in transition from a high to low affinity substrate binding state (24, 39). The transition can be readily demonstrated as a marked nucleotide-dependent decrease in photolabeling with a substrate such as LTC₄ or the inhibitor LY475776 (24, 39, 43). In the absence of nucleotide, the binding of substrate by MRP_1–1501 was similar to that of wild-type protein. However, unlike full-length MRP1, preincubation of MRP1_1–1501 with ATP and vanadate completely failed to decrease LTC₄ binding. This is consistent with severely impaired ATP binding by NBD2 in the truncated protein. Comparison of LTC₄ photolabeling of wild-type MRP1 and the partially active MRP1_1–1527 demonstrated that ATP alone failed to shift the mutant protein from a high to a low affinity binding state as efficiently as the wild-type protein. Furthermore, no detectable decrease in LTC₄ labeling was observed for MRP1_1–1527 in the presence of the poorly hydrolyzable ATP analog, ATPS. These results support the conclusion that the integrity of the COOH-terminal region of MRP1 is important for ATP binding and hydrolysis at NBD2 and the consequential transition from a high to a low affinity LTC₄ binding state.

To identify specific amino acids that might be critical for MRP1 activity, we carried out Ala scanning mutagenesis of the COOH-terminal region. None of the single Ala mutations were predicted to alter the secondary structure of the region, and, as might be expected from the low level of sequence conservation, most had no effect on activity (Table I). However, alignment of the COOH-terminal regions of a number of distantly related ABC proteins reveals several broadly conserved amino acids, notably the dileucine motif in helix C2 and a Phe or Tyr at the start of helix C1 (Fig. 8). In SUR1, both the highly conserved dileucine motif and the COOH-proximal Phe have been implicated as trafficking signals (29). In contrast, substitution of the comparable dileucine residues in MRP1 with Ala had no affect on plasma membrane localization (not shown). Instead, mutation of these helix C2 residues reduced LTC₄ transport activity (Table I) and nucleotide binding to NBD2 (not shown) to an extent similar to that caused by removal of the COOH-terminal 4 amino acids. Consistent with the presence of Leu-Val and Leu-Ile, rather than di-Leu, in some ABC proteins, substitution of Leu¹⁵¹⁴ and Leu¹⁵¹⁵ with Ile or Val rather than Ala had minimal effect on LTC₄ transport activity. Similarly, mutation of the conserved Phe¹⁵²¹ in helix C1 to Tyr had no effect on activity, whereas mutation to any of several nonaromatic amino acids diminished LTC₄ transport to an extent comparable with that resulting from Ala substitution of the dileucine motif. In addition, two residues in helix C3, Leu¹⁴⁸⁸ and Ile¹⁴⁹¹ are also relatively highly conserved, and mutation of either one to Ala decreased LTC₄ transport activity by 40% (Fig. 8).

The crystal structures of hemolysin B (49), LmrA (Protein Data Bank number 1MV5 [PDB] ), and Tap1 (50) indicate that the COOH-terminal regions of these proteins contain an -helical bundle composed of helices C3, C2, and C1 (following the nomenclature in Fig. 8) that appears to be stabilized by hydrophobic interactions between certain residues that project into the core of the bundle. These residues correspond to the conserved Leu¹⁵¹⁴-Leu¹⁵¹⁵, Phe¹⁵²¹, Leu¹⁴⁸⁸, and Ile¹⁴⁹¹ in MRP1, mutation of which decreased LTC₄ transport (Table I). Thus, the truncations and mutations we have described that decrease the activity of MRP1 may do so by destabilizing this COOH-terminal helical bundle. However, it should be noted that although the COOH-terminal helical bundle is widely conserved among ABC proteins, some prokaryotic NBDs, such as MJ0796, lack helices analogous to helices C3 and C2 but still bind ATP with relatively high affinity (K_m = 50 µM) (51, 52).

Based on the known structures of ABC proteins, there is no evidence to suggest that COOH-terminal helical elements interact directly with bound nucleotide (49, 50, 53–55). However, examination of the known ABC crystal structures supports the possibility of interactions between residues in sheet C2 and the Walker A motif in NBD2. Furthermore, a naturally occurring Val mutation of a Leu in SUR1 (Leu¹⁵⁵¹) that is analogous to Leu¹⁵⁰⁰ in MRP1, impairs MgADP activation of K_ATP channels and results in congenital hyperinsulinism (56, 57). The published model of SUR1 NBD suggests that mutation of Leu¹⁵⁵¹ may influence nucleotide binding to NBD2 because of its proximity (<3.5 Å) to Ser¹³⁸² and Lys¹³⁸⁴, which correspond to Ala¹³³¹ and Lys¹³³³ in the Walker A motif of MRP1 (58). In addition, nonconserved COOH-terminal residues have been identified recently in SUR2A and SUR2B splice variants that contribute to differences in ADP sensitivity (59).

Finally, the COOH-terminal region may be important for transduction of conformational changes from NBD1 to NBD2, since ATP binding to MRP1 NBD1 is essential for binding and hydrolysis of ATP at NBD2 (33, 44). The requirement for binding to NBD1 is believed to reflect a nucleotide-dependent alteration in the interaction between the two NBDs that enables nucleotide binding by NBD2. The crystal structure of the BtuD NBD dimer indicates that the COOH-terminal residues Tyr²³⁰ and Met²³² near the end of what we have termed helix C1 are within 4–5 Å of Val¹⁶⁶ from the adjoining NBD (54). Val¹⁶⁶ is adjacent to the highly conserved Asp residue of the D loop. Helix C1 in MRP1 contains Tyr (Tyr¹⁵²²) and Met (Met¹⁵²⁴) at corresponding positions, as do the analogous helices in MRP2 and P-gp (Fig. 8). We are currently investigating the possibility that interactions between the COOH-terminal helical structures in MRP1 and elements near the D loop of NBD1 may be involved in transmitting ATP binding-induced conformational changes from one NBD to the other.

	FOOTNOTES

 
^* This work was supported by a grant from the National Cancer Institute of Canada with funds from the Terry Fox Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Recipient of an Ontario Graduate Scholarship.

^** The Canada Research Chair in Cancer Biology.

To whom correspondence should be addressed: Cancer Research Institute, Botterell Hall, Queen's University, Kingston, Ontario K7L 3N6, Canada. Tel.: 613-533-2979; Fax: 613-533-6830; E-mail: deeleyr{at}post.queensu.ca.

¹ The abbreviations used are: MRP, multidrug resistance protein; ABC, ATP-binding cassette; P-gp, P-glycoprotein; CFTR, cystic fibrosis transmembrane conductance regulator; SUR, sulfonylurea receptor; MSD, membrane-spanning domain; NBD, nucleotide binding domain; mAb, monoclonal antibody; LTC₄, leukotriene C₄; E₂17G, 17-estradiol-17--(D-glucuronide); MDCK, Madin-Darby canine kidney; ATPS, adenosine 5'-O-(thiotriphosphate); pBS, pBluescript-KS+; pFB, pFast-Bac; aa, amino acids; [¹²⁵I]LY475776, N-(4-azido-3-[¹²⁵I]phenyl)-2-[3-(9-chloro-3-methyl-4-oxo-4H-isoxazolo[4,3-c]quinolin-5-yl)-cyclohexyl]-acetamide; dhMRP1, dual half-expressed MRP1; nt, nucleotides.

	ACKNOWLEDGMENTS

 
We thank Dr. Robert Campbell from the Protein Function Discovery Facility (Queen's University) for advice on protein modeling. Assistance from Dr. Yue-ming Qian, Monika Vasa, and Ashley Theis is also much appreciated.

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