Eukaryotic ATP-binding Cassette Transporter Research Articles

Expression Analysis of VvPDR Genes in Grape Berries

ATP bağlayıcı kaset (ABC) taşıyıcı membran proteinleri, tüm organizmalarda büyük oranda korunmuş en büyük membran protein ailelerindendir. Ökaryotik ABC taşıyıcılarının yapısı, bir transmembran alanı (TMD) ve bir nükleotit bağlama alanı (NBD) olarak adlandırılan iki bölgeden oluşmaktadır. Bu proteinler, ökaryot canlılarda ABCA’dan ABCG’ye kadar devam eden toplam yedi ana aileye ayrılmaktadırlar ve bu ana aileler de kendi içlerinde alt ailelere ayrılmaktadır. Plazma zarında ve tonoplast, kloroplast, mitokondri ve peroksizomlar gibi organların zarlarında lokalizedirler ve çok sayıda işlevi yerine getirmektedirler. Başlangıçta detoksifikasyon işlemlerine katılan taşıyıcılar olarak tanımlanmış olmalarına rağmen daha sonraki yapılan çalışmalarda bu proteinlerin bitki büyümesi ve gelişim dönemlerinde ve çevresel streslere karşı tepkilerde de etkili oldukları gösterilmiştir. ABCG'nin tam molekül üyelerinden PDR alt grubu sadece mantar ve bitkilerde tanımlanmıştır. Plant pleiotropic drug resistance (PDR) alt ailesine ait proteinlerin çeşitli lipidlerin ve hormonların taşınmasında görev aldıkları ve aynı zamanda bu proteinlerin abiyotik ve biyotik streslere karşı tepkiler sırasında da görevlerinin olduğu yapılan farklı çalışmalarla bildirilmiştir. Yapılan bu çalışmada ise Vitis vinifera sp. genomunda tanımlanan ve PDR alt ailesine ait VvABCG35, VvABCG36, VvABCG37 genlerinin asmada meyve gelişim dönemleri boyunca gösterdiği ifadeler incelenmiş ve bu genlerin fonksiyonları hakkında fikir sahibi olmak için STRING veritabanı kullanılarak etkileşime girdiği diğer proteinler belirlenmiştir.

Structures of multidrug resistance protein MRP4 reveal basis of substrate specificity

ATP-binding cassette (ABC) transporters are a ubiquitous family of transmembrane proteins which hydrolyze ATP to facilitate the movement of substrates across cell membranes. Despite common architectural features, individual eukaryotic ABC transporters have evolved to transport a broad spectrum of substrates. The multidrug resistance protein MRP4, a member of the MRP sub-family of transporters, has been linked to the efflux of diverse endogenous and exogenous substrates, including prostaglandins, cyclic nucleotides, conjugated steroids, and other chemical scaffolds.

MsbA: an ABC transporter paradigm.

ATP-binding cassette (ABC) transporters play an important role in various cellular processes. They display a similar architecture and share a mechanism which couples ATP hydrolysis to substrate transport. However, in the light of current data and recent experimental progress, this protein superfamily appears as multifaceted as their broad substrate range. Among the prokaryotic ABC transporters, MsbA can serve as a paradigm for research in this field. It is located in the inner membrane of Gram-negative bacteria and functions as a floppase for the lipopolysaccharide (LPS) precursor core-LPS, which is involved in the biogenesis of the bacterial outer membrane. While MsbA shows high similarity to eukaryotic ABC transporters, its expression in Gram-negative bacteria makes it conveniently accessible for many experimental approaches from spectroscopy to 3D structure determination. As an essential protein for bacterial membrane integrity, MsbA has also become an attractive target for the development of novel antibiotics. Furthermore, it serves as a model for multidrug efflux pumps. Here we provide an overview of recent findings and their relevance to the field, highlight the potential of methods such as solid-state NMR and EPR spectroscopy and provide a perspective for future work.

Crystal structure of the Escherichia coli transcription termination factor Rho.

During the crystal structure analysis of an ATP-binding cassette (ABC) transporter overexpressed in Escherichia coli, a contaminant protein was crystallized. The identity of the contaminant was revealed by mass spectrometry to be the Escherichia coli transcription terminator factor Rho, structures of which had been previously determined in different conformational states. Although Rho was present at only ∼1% of the target protein (a bacterial homolog of the eukaryotic ABC transporter of mitochondria from Novosphingobium aromaticivorans; NaAtm1), it preferentially crystallized in space group C2 as thin plates that diffracted to 3.30 Å resolution. The structure of Rho in this crystal form exhibits a hexameric open-ring staircase conformation with bound ATP; this characteristic structure was also observed on electron-microscopy grids of the NaAtm1 preparation.

Mechanics and pharmacology of substrate selection and transport by eukaryotic ABC exporters

Much structural information has been amassed on ATP-binding cassette (ABC) transporters, including hundreds of structures of isolated domains and an increasing array of full-length transporters. The structures capture different steps in the transport cycle and have aided in the design and interpretation of computational simulations and biophysics experiments. These data provide a maturing, although still incomplete, elucidation of the protein dynamics and mechanisms of substrate selection and transit through the transporters. We present an updated view of the classical alternating-access mechanism as it applies to eukaryotic ABC transporters, focusing on type I exporters. Our model helps frame the progress in, and remaining questions about, transporter energetics, how substrates are selected and how ATP is consumed to perform work at the molecular scale. Many human ABC transporters are associated with disease; we highlight progress in understanding their pharmacology through the lens of structural biology and describe how this knowledge suggests approaches to pharmacologically targeting these transporters.

Learning the ABCs one at a time: structure and mechanism of ABC transporters.

ATP-binding cassette (ABC) transporters are essential proteins that are found across all kingdoms of life. ABC transporters harness the energy of ATP hydrolysis to drive the import of nutrients inside bacterial cells or the export of toxic compounds or essential lipids across bacteria and eukaryotic membranes. Typically, ABC transporters consist of transmembrane domains (TMDs) and nucleotide-binding domains (NBDs) to bind their substrate and ATP, respectively. The TMDs dictate what ligands can be recognised, whereas the NBDs are the power engine of the ABC transporter, carrying out ATP binding and hydrolysis. It has been proposed that they utilise the alternating access mechanism, inward- to outward-facing conformation, to transport their substrates. Here, we will review the recent progress on the structure determination of eukaryotic and bacterial ABC transporters as well as the novel mechanisms that have also been proposed, that fall out of the alternating access mechanism model.

The Role of Eukaryotic and Prokaryotic ABC Transporter Family in Failure of Chemotherapy.

Over the years chemotherapy failure has been a vital research topic as researchers have been striving to discover reasons behind it. The extensive studies carried out on chemotherapeutic agents confirm that resistance to chemotherapy is a major reason for treatment failure. “Resistance to chemotherapy,” however, is a comprehensive phrase that refers to a variety of different mechanisms in which ATP-binding cassette (ABC) mediated efflux dominates. The ABC is one of the largest gene superfamily of transporters among both eukaryotes and prokaryotes; it represents a variety of genes that code for proteins, which perform countless functions, including drug efflux – a natural process that protects cells from foreign chemicals. Up to date, chemotherapy failure due to ABC drug efflux is an active research topic that continuously provides further evidence on multiple drug resistance (MDR), aiding scientists in tackling and overcoming this issue. This review focuses on drug resistance by ABC efflux transporters in human, viral, parasitic, fungal and bacterial cells and highlights the importance of the MDR permeability glycoprotein being the mutual ABC transporter among all studied organisms. Current developments and future directions to overcome this problem are also discussed.

Tracing the structural evolution of eukaryotic ATP binding cassette transporter superfamily.

The ATP binding cassette (ABC) transporters superfamily is one of the largest classes of membrane proteins. The core of the ABC transporter protein is composed of transmembrane domains (TMDs) and nucleotide binding domains (NBD). Eukaryotes ABC transporters are classified into seven main families (ABCA to ABCG) based on sequence similarity and domain organizations. With different domain number and domain organizations, eukaryote ABC transporters show diverse structures: the single structure (NBD or TMD), the ABC2 structure (NBD-NBD), the half structure (TMD-NBD or NBD-TMD) and the full structure (TMD-NBD-TMD-NBD or NBD-TMD-NBD-TMD). However, studies on how various ABC transporter gene structures evolved is still absent. Therefore, in this study, we comprehensively investigated the structural evolution of eukaryotic ABC transporters. The seven eukaryote ABC transporter families (A to G) fell into three groups: A&G group, B,C&D group and E&F group. There were at least four times the number of NBD and TMD fusion events in the origin of the half structure transporter. Two fusion modes were found in the full and ABC2 structure origination. Based on these findings, we present a putative structural evolutionary path of eukaryote ABC transporters that will increase our understanding on their origin, divergence and function.

Detergent-free purification of ABC (ATP-binding-cassette) transporters.

ABC (ATP-binding-cassette) transporters carry out many vital functions and are involved in numerous diseases, but study of the structure and function of these proteins is often hampered by their large size and membrane location. Membrane protein purification usually utilizes detergents to solubilize the protein from the membrane, effectively removing it from its native lipid environment. Subsequently, lipids have to be added back and detergent removed to reconstitute the protein into a lipid bilayer. In the present study, we present the application of a new methodology for the extraction and purification of ABC transporters without the use of detergent, instead, using a copolymer, SMA (polystyrene-co-maleic acid). SMA inserts into a bilayer and assembles into discrete particles, essentially solubilizing the membrane into small discs of bilayer encircled by a polymer, termed SMALPs (SMA lipid particles). We show that this polymer can extract several eukaryotic ABC transporters, P-glycoprotein (ABCB1), MRP1 (multidrug-resistance protein 1; ABCC1), MRP4 (ABCC4), ABCG2 and CFTR (cystic fibrosis transmembrane conductance regulator; ABCC7), from a range of different expression systems. The SMALP-encapsulated ABC transporters can be purified by affinity chromatography, and are able to bind ligands comparably with those in native membranes or detergent micelles. A greater degree of purity and enhanced stability is seen compared with detergent solubilization. The present study demonstrates that eukaryotic ABC transporters can be extracted and purified without ever being removed from their lipid bilayer environment, opening up a wide range of possibilities for the future study of their structure and function.

Posttranslational regulation of the bile salt export pump

Bile plays an essential role in the nutrient uptake of the human body. It consists mostly of amphipathic bile salts, phospholipids and cholesterol in mixed micelles. The bile solubilizes lipophilic nutrients, such as lipids and fat-soluble vitamins, and facilitates their uptake. The formation of bile depends on the activity of various ATP-binding cassette (ABC) transporters localized in the apical membrane of hepatocytes. The main components of bile, phosphatidylcholine, cholesterol and bile salts, are transported by multidrug resistance protein 3 (MDR3, ABCB4), ABCG5/G8 and the bile salt export pump (BSEP, ABCB11), respectively. BSEP, as the transporter of the main solute in bile, is considered to be the primary driving force of bile formation. BSEP belongs to the family of ABC transporters, which constitute one of the largest protein families. They are primary active transporters with a common functional unit: two nucleotide–binding domains and two transmembrane domains. The nucleotide–binding domains fuel the transport by hydrolyzing ATP, while the transmembrane domains provide the translocation pathway over the membrane and are thought to be responsible for substrate recognition. Eukaryotic ABC transporters are usually encoded by one gene as in the case of BSEP (full-size transporters) or by two genes coding for one nucleotide-binding domain and one transmembrane domain each. In the latter case the transporter is called a half-size transporter and the two polypeptides homo- or heterodimerize to form a functional unit. An example for such a half-size transporter is ABCG5/G8. Mutations in ABC transporters can lead to severe diseases. For example an impairment of targeting or activity in BSEP leads to the accumulation of bile salts in the hepatocytes. Bile salts, in addition to their detergent function, can furthermore act as signaling molecules and thereby cause different forms of cholestasis, such as progressive familial intrahepatic cholestasis type 2 (PFIC2) or benign recurrent intrahepatic cholestasis type 2 (BRIC2). An additional influence that can affect the development of diseases is the post-translational regulation of ABC transporter functionality. The regulation can occur directly in the form of chemical modifications or can be mediated for example by protein-protein interactions. For several ABC transporters of the apical hepatocyte membrane an influence of protein-protein interactions on targeting, turnover or activity could be shown. MRP2 for example interacts with NHERF-1, a scaffold protein that is responsible for linking its interacting partners to the cytoskeleton. It has furthermore been reported that the interaction of MRP2 with NHERF-1 influences the apical membrane expression of MRP2. For example, NHERF-1 knock–out mice show reduced abundance of MRP2 in the membrane [1]. For BSEP two interaction partners are known to date. One of them is the AP2 adaptor related protein complex, which is involved in internalization of BSEP [2]. To study ABC transporters in their isolated form a heterologous overexpression system is required to obtain sufficient amounts of protein. Initially, toxicity of the BSEP cDNA for Escherichia coli was an obstacle. Therefore a Saccharomyces cerevisiae based cloning technique has been developed, which is independent of E. coli[3]. Subsequently, with the optimized cloning procedure an overexpression of BSEP could be established in the methylotrophic yeast Pichia pastoris. With the help of a GFP-fusion protein BSEP could be traced to the P. pastoris plasma membrane. Additionally, sucrose density centrifugation experiments confirmed the correct localization of BSEP. A subsequent detergent screen led to the identification of detergents suitable for BSEP solubilization. The transporter appears to be in a detergent-resistant environment as only the lipid–like, zwitter–ionic detergents of the Fos–choline and Cyclofos series, comparatively “harsh” detergents, could efficiently extract BSEP. Finally, a purification protocol was established to obtain BSEP in a detergent-solubilized form. With a dual–affinity tag tandem affinity purification could be employed to obtain the ABC transporter with approximately 75% purity [4]. In summary, the optimized cloning strategy for BSEP cDNA in S. cerevisiae as well as the efficient expression and purification protocols enable us to investigate the interaction of BSEP with other proteins in vitro.

Retinoid Binding Properties of Nucleotide Binding Domain 1 of the Stargardt Disease-associated ATP Binding Cassette (ABC) Transporter, ABCA4

The retina-specific ATP binding cassette transporter, ABCA4 protein, is associated with a broad range of inherited macular degenerations, including Stargardt disease, autosomal recessive cone rod dystrophy, and fundus flavimaculatus. In order to understand its role in retinal transport in rod out segment discs, we have investigated the interactions of the soluble domains of ABCA4 with both 11-cis- and all-trans-retinal. Using fluorescence anisotropy-based binding analysis and recombinant polypeptides derived from the amino acid sequences of the four soluble domains of ABCA4, we demonstrated that the nucleotide binding domain 1 (NBD1) specifically bound 11-cis-retinal. Its affinity for all-trans-retinal was markedly reduced. Stargardt disease-associated mutations in this domain resulted in attenuation of 11-cis-retinal binding. Significant differences in 11-cis-retinal binding affinities were observed between NBD1 and other cytoplasmic and lumenal domains of ABCA4. The results suggest a possible role of ABCA4 and, in particular, the NBD1 domain in 11-cis-retinal binding. These results also correlate well with a recent report on the in vivo role of ABCA4 in 11-cis-retinal transport.

ABCA4 is an N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine importer

ATP-binding cassette (ABC) transporters comprise a superfamily of proteins, which actively transport a variety of compounds across cell membranes. Mammalian and most eukaryotic ABC transporters function as exporters, flipping or extruding substrates from the cytoplasmic to the extracellular or lumen side of cell membranes. Prokaryotic ABC transporters function either as exporters or importers. Here we show that ABCA4, an ABC transporter found in retinal photoreceptor cells and associated with Stargardt macular degeneration, is a novel importer that actively flips N-retinylidene-phosphatidylethanolamine from the lumen to the cytoplasmic leaflet of disc membranes, thereby facilitating the removal of potentially toxic retinoid compounds from photoreceptors. ABCA4 also actively transports phosphatidylethanolamine in the same direction. Mutations known to cause Stargardt disease decrease N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine transport activity of ABCA4. These studies provide the first direct evidence for a mammalian ABC transporter that functions as an importer and provide insight into mechanisms underlying substrate transport and the molecular basis of Stargardt disease.

Functional Rescue of a Misfolded Eukaryotic ATP-binding Cassette Transporter by Domain Replacement

ATP-binding cassette (ABC) transporters are integral membrane proteins that couple ATP binding/hydrolysis with the transport of hydrophilic substrates across lipid barriers. Deletion of Phe-670 in the first nucleotide-binding domain (NBD1) of the yeast ABC transporter, Yor1p, perturbs interdomain associations, reduces functionality, and hinders proper transport to the plasma membrane. Functionality of Yor1p-ΔF was restored upon co-expression of a peptide containing wild-type NBD1. To gain insight into the biogenesis of this important class of proteins, we defined the requirements for this rescue. We show that a misfolding lesion in NBD1 of the full-length protein is a prerequisite for functional rescue by exogenous NBD1, which is mediated by physical replacement of the dysfunctional domain by the soluble NBD1. This association does not restore trafficking of Yor1p-ΔF but instead confers catalytic activity to the small population of Yor1p-ΔF that escapes to the plasma membrane. An important coupling between the exogenous NBD1 and ICL4 within full-length aberrant Yor1p-ΔF is required for functional rescue but not for the physical interaction between the two polypeptides. Together, our genetic and biochemical data reveal that it is possible to modulate activity of ABC transporters by physically replacing dysfunctional domains.

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Mapping of Interdomain Interfaces Required for the Functional Architecture of Yor1p, a Eukaryotic ATP-binding Cassette (ABC) Transporter

ATP-binding cassette (ABC) transporters are a large superfamily of proteins that mediate substrate translocation across biological membranes. Our goal was to define the intramolecular interactions that contribute to quaternary assembly of a eukaryotic ABC transporter and determine how the architecture of this protein influences its biogenesis within the secretory pathway. We used chemical cross-linking approaches to map interdomain interactions in the yeast ABC transporter, Yor1p, which functions as a pleiotropic drug pump at the plasma membrane. We have defined interactions between the two nucleotide-binding domains (NBDs) and between the NBDs and specific intracellular loops (ICLs) that are consistent with current structural models of bacterial ABC exporters. Furthermore, we detected relatively weak NBD-NBD and ICL-ICL interactions that may correspond to transient sites of cross-talk between domains required for coupling of ATP hydrolysis with substrate translocation. Mutation of a key residue in ICL2 caused misassembly of the altered protein, leading to increased sensitivity to the mitochondrial poison, oligomycin. We identified intragenic suppressing mutations that rescued the oligomycin resistance associated with this aberrant protein and demonstrated that the suppressing mutations restored multiple interdomain interfaces. Together, our biochemical and genetic approaches contribute to a greater understanding of the architecture of this important class of proteins and provide insight into the quality control surveillance that regulates their biogenesis and deployment within the eukaryotic cell.

Cardiac sulfonylurea receptor short form-based channels confer a glibenclamide-insensitive K ATP activity

The cardiac sarcolemmal ATP-sensitive potassium channel (K ATP) consists of a Kir6.2 pore and an SUR2 regulatory subunit, which is an ATP-binding cassette (ABC) transporter. K ATP channels have been proposed to play protective roles during ischemic preconditioning. An SUR2 mutant mouse was previously generated by disrupting the first nucleotide-binding domain (NBD1), where a glibenclamide action site was located. In the mutant ventricular myocytes, a non-conventional glibenclamide-insensitive (10 μM), ATP-sensitive current ( I KATPn) was detected in 33% of single-channel recordings with an average amplitude of 12.3 ± 5.4 pA per patch, an IC 50 to ATP inhibition at 10 μM and a mean burst duration at 20.6 ± 1.8 ms. Newly designed SUR2 isoform- or variant-specific antibodies identified novel SUR2 short forms in the sizes of 28 and 68 kDa in addition to a 150-kDa long form in the sarcolemmal membrane of wild-type (WT) heart. We hypothesized that channels constituted by these short forms that lack NBD1 confer I KATPn. The absence of the long form in the mutant corresponded to loss of the conventional glibenclamide-sensitive K ATP currents ( I KATP) in isolated cardiomyocytes and vascular smooth muscle cells but the SUR2 short forms remained intact. Nested exonic RT-PCR in the mutant indicated that the short forms lacked NBD1 but contained NBD2. The SUR2 short forms co-immunoprecipitated with Kir6.1 or Kir6.2 suggesting that the short forms may function as hemi-transporters reported in other eukaryotic ABC transporter subgroups. Our results indicate that different K ATP compositions may co-exist in cardiac sarcolemmal membrane.

Insight in eukaryotic ABC transporter function by mutation analysis

With regard to structure–function relations of ATP-binding cassette (ABC) transporters several intriguing questions are in the spotlight of active research: Why do functional ABC transporters possess two ATP binding and hydrolysis domains together with two ABC signatures and to what extent are the individual nucleotide-binding domains independent or interacting? Where is the substrate-binding site and how is ATP hydrolysis functionally coupled to the transport process itself? Although much progress has been made in the elucidation of the three-dimensional structures of ABC transporters in the last years by several crystallographic studies including novel models for the nucleotide hydrolysis and translocation catalysis, site-directed mutagenesis as well as the identification of natural mutations is still a major tool to evaluate effects of individual amino acids on the overall function of ABC transporters. Apart from alterations in characteristic sequence such as Walker A, Walker B and the ABC signature other parts of ABC proteins were subject to detailed mutagenesis studies including the substrate-binding site or the regulatory domain of CFTR. In this review, we will give a detailed overview of the mutation analysis reported for selected ABC transporters of the ABCB and ABCC subfamilies, namely HsCFTR/ABCC7, HsSUR/ABCC8,9, HsMRP1/ABCC1, HsMRP2/ABCC2, ScYCF1 and P-glycoprotein (Pgp)/MDR1/ABCB1 and their effects on the function of each protein.

Three-dimensional Structure of P-glycoprotein: THE TRANSMEMBRANE REGIONS ADOPT AN ASYMMETRIC CONFIGURATION IN THE NUCLEOTIDE-BOUND STATE

Multidrug resistance of cancer cells and pathogens is a serious clinical problem. A major factor contributing to drug resistance in cancer is the over-expression of P-glycoprotein, a plasma membrane ATP-binding cassette (ABC) drug efflux pump. Three-dimensional structural data with a resolution limit of approximately 8 A have been obtained from two-dimensional crystals of P-glycoprotein trapped in the nucleotide-bound state. Each of the two transmembrane domains of P-glycoprotein consists of six long alpha-helical segments. Five of the alpha-helices from each transmembrane domain are related by a pseudo-2-fold symmetry, whereas the sixth breaks the symmetry. The two alpha-helices positioned closest to the (pseudo-) symmetry axis at the center of the molecule appear to be kinked. A large loop of density at the extracellular surface of the transporter is likely to correspond to the glycosylated first extracellular loop, whereas two globular densities at the cytoplasmic side correspond to the hydrophilic, nucleotide-binding domains. This is the first three-dimensional structure for an intact eukaryotic ABC transporter. Comparison with the structures of two prokaryotic ABC transporters suggests significant differences in the packing of the transmembrane alpha-helices within this protein family.

Domain interactions in the yeast ATP binding cassette transporter Ycf1p: intragenic suppressor analysis of mutations in the nucleotide binding domains.

The yeast cadmium factor (Ycf1p) is a vacuolar ATP binding cassette (ABC) transporter required for heavy metal and drug detoxification. Cluster analysis shows that Ycf1p is strongly related to the human multidrug-associated protein (MRP1) and cystic fibrosis transmembrane conductance regulator and therefore may serve as an excellent model for the study of eukaryotic ABC transporter structure and function. Identifying intramolecular interactions in these transporters may help to elucidate energy transfer mechanisms during transport. To identify regions in Ycf1p that may interact to couple ATPase activity to substrate binding and/or movement across the membrane, we sought intragenic suppressors of ycf1 mutations that affect highly conserved residues presumably involved in ATP binding and/or hydrolysis. Thirteen intragenic second-site suppressors were identified for the D777N mutation which affects the invariant Asp residue in the Walker B motif of the first nucleotide binding domain (NBD1). Two of the suppressor mutations (V543I and F565L) are located in the first transmembrane domain (TMD1), nine (A1003V, A1021T, A1021V, N1027D, Q1107R, G1207D, G1207S, S1212L, and W1225C) are found within TMD2, one (S674L) is in NBD1, and another one (R1415G) is in NBD2, indicating either physical proximity or functional interactions between NBD1 and the other three domains. The original D777N mutant protein exhibits a strong defect in the apparent affinity for ATP and V(max) of transport. The phenotypic characterization of the suppressor mutants shows that suppression does not result from restoring these alterations but rather from a change in substrate specificity. We discuss the possible involvement of Asp777 in coupling ATPase activity to substrate binding and/or transport across the membrane.

Mutation of a Single Conserved Tryptophan in Multidrug Resistance Protein 1 (MRP1/ABCC1) Results in Loss of Drug Resistance and Selective Loss of Organic Anion Transport

Multidrug resistance protein 1 (MRP1/ABCC1) belongs to the ATP-binding cassette transporter superfamily and is capable of conferring resistance to a broad range of chemotherapeutic agents and transporting structurally diverse conjugated organic anions. In this study, we found that substitution of a highly conserved tryptophan at position 1246 with cysteine (W1246C-MRP1) in the putative last transmembrane segment (TM17) of MRP1 eliminated 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) transport by membrane vesicles prepared from transiently transfected human embryonic kidney cells while leaving the capacity for leukotriene C(4)- and verapamil-stimulated glutathione transport intact. In addition, in contrast to wild-type MRP1, leukotriene C(4) transport by the W1246C-MRP1 protein was no longer inhibitable by E(2)17betaG, indicating that the mutant protein had lost the ability to bind the glucuronide. A similar phenotype was observed when Trp(1246) was replaced with Ala, Phe, and Tyr. Confocal microscopy of cells expressing Trp(1246) mutant MRP1 molecules fused at the C terminus with green fluorescent protein showed that they were correctly routed to the plasma membrane. In addition to the loss of E(2)17betaG transport, HeLa cells stably transfected with W1246C-MRP1 cDNA were not resistant to the Vinca alkaloid vincristine and accumulated levels of [(3)H]vincristine comparable to those in vector control-transfected cells. Cells expressing W1246C-MRP1 were also not resistant to cationic anthracyclines (doxorubicin, daunorubicin) or the electroneutral epipodophyllotoxin VP-16. In contrast, resistance to sodium arsenite was only partially diminished, and resistance to potassium antimony tartrate remained comparable to that of cells expressing wild-type MRP1. This suggests that the structural determinants required for transport of heavy metal oxyanions differ from those for chemotherapeutic agents. Our results provide the first example of a tryptophan residue being so critically important for substrate specificity in a eukaryotic ATP-binding cassette transporter.