Functional expression of multidrug resistance protein 1 in Pichia pastoris.

Overexpression of the multidrug resistance-associated protein (MRP1) causes multidrug resistance in cultured cells. MRP1 transports a large number of glutathione, glucuronide, and sulfate-conjugated organic anions by an ATP-dependent efflux mechanism. Six other MRP proteins exist (MRP2-7), and mutations in some of these genes cause major pathological conditions in humans. A detailed characterization of the structure and mechanism of action of these proteins requires an efficient expression system from which large amounts of active protein can be obtained. We report the expression of a recombinant MRP1 in the methylotrophic yeast Pichia pastoris. The protein is expressed in the membrane fraction of these cells, as a stable and underglycosylated 165 kDa peptide. Expression levels are very high, and 30 times superior to those seen in multidrug-resistant HeLa/MRP1 transfectants. MRP1 expressed in P. pastoris binds 8-azido[alpha-(32)P]ATP in a Mg(2+)-dependent and EDTA-sensitive fashion, which can be competed by a molar excess of ADP and ATP. Under hydrolysis conditions (at 37 degrees C), orthovanadate induces trapping of the 8-azido[alpha-(32)P]nucleotide in MRP1, which can be further modulated by known MRP1 ligands. MRP1 is also labeled by a photoactive analogue of rhodamine 123 (IAARh123) in P. pastoris/MRP1 membranes, and this can be competed by known MRP1 ligands. Finally, MRP1-positive membrane vesicles show ATP-dependent uptake of LTC(4). Thus, MRP1 expressed in P. pastoris is active and shows characteristics of MRP1 expressed in mammalian cells, including drug binding, ligand-modulated formation of the MRP1-MgADP-P(i) intermediate (ATPase activity), and ATP-dependent substrate transport. The successful expression of catalytically active and transport-competent MRP1 in P. pastoris should greatly facilitate the efficient production and isolation of the wild type or inactive mutants of MRP1, or of other MRP proteins for structural and functional characterization.     

Immobilized palladium(II) ion affinity chromatography for recovery of recombinant proteins with peptide tags containing histidine and cysteine

Fusion of peptide‐based tags to recombinant proteins is currently one of the most used tools for protein production. Also, immobilized metal ion affinity chromatography (IMAC) has a huge application in protein purification, especially in research labs. The combination of expression systems of recombinant tagged proteins with this robust chromatographic system has become an efficient and rapid tool to produce milligram‐range amounts of proteins. IMAC‐Ni(II) columns have become the natural partners of 6xHis‐tagged proteins. The Ni(II) ion is considered as the best compromise of selectivity and affinity for purification of a recombinant His‐tagged protein. The palladium(II) ion is also able to bind to side chains of amino acids and form ternary complexes with iminodiacetic acid and free amino acids and other sulfur‐containing molecules. In this work, we evaluated two different cysteine‐ and histidine‐containing six amino acid tags linked to the N‐terminal group of green fluorescent protein (GFP) and studied the adsorption and elution conditions using novel eluents. Both cysteine‐containing tagged GFPs were able to bind to IMAC‐Pd(II) matrices and eluted successfully using a low concentration of thiourea solution. The IMAC‐Ni(II) system reaches less than 20% recovery of the cysteine‐containing tagged GFP from a crude homogenate of recombinant Escherichia coli, meanwhile the IMAC‐Pd(II) yields a recovery of 45% with a purification factor of 13. Copyright © 2014 John Wiley & Sons, Ltd.     

多核酶表达系统在HEK293细胞中对多药耐药相关蛋白（MRP1）的表达抑制作用

为了研究多核酶表达系统在HEK293细胞中对多药耐药相关蛋白表达抑制的作用．我们构建了含有20个可以自身切割的顺式作用核酶和10个靶向MRP1基因特定位点的反式作用核酶的多核酶表达系统。利用RT—PCR、Westem blot和MTT分析了多核酶系统分别与MRP1靶基因质粒和MRP1 全长基因质粒共转染的HEK293细胞。结果显示．多核酶表达系统能够明显降低荧光融合蛋白在HEK293细胞中的表达。RT—PCR分析表明．懈用靶mRNA降低程度与多核酶表达系统所含的反式作用核酶数目有关。Westernblot分析显示了与RT—PCR相似的结果。Mrrr分析表明,多核酶表达系统能够逆转由MRP1基因转染HEK293细胞产生的多药耐药性。结果提示．含有多个核酶的表达系统对MRP1基因的抑制效应优于单核酶的表达系统。因此．该策略可能用于基因治疗肿瘤或其他疾病．     

Evidence for the role of glycosylation in accessibility of the extracellular domains of human MRP1 (ABCC1)

To enable cell surface localization of the human multidrug resistance protein (MRP1, ABCC1) and to assess the role of the extracellular domains of this transporter, the FLAG epitope tag was introduced into different extracellular loops of the three membrane-spanning domains (MSDs) of the transporter. We constructed and expressed various partially and fully glycosylation-deficient, FLAG-tagged MRP1 proteins in a Vaccinia virus-based transient expression system, and the cell surface expression level of MRP1 on intact cells was followed by flow cytometry, using the FLAG tag specific monoclonal antibody M2. We also expressed the wild-type MRP1 protein and some of the FLAG-tagged mutants in stably transfected HEK293 cells, and followed the cell surface expression and the transport function of MRP1 both by monitoring the efflux of fluorescent substrate and by their ability to confer resistance to HEK293 transfectants to anticancer agents such as daunorubicin and etoposide. When we inserted the FLAG epitope in extracellular loops of the MSD1 or MSD3, the tag was accessible upon removal of N-glycosylation sites (N --> Q at positions 17, 23, and 1006, respectively), whereas the FLAG epitope placed in the MSD2 was not accessible even after removal of all three N-glycosylation sites, indicating that MSD2 region is deeply buried in the plasma membrane. However, all FLAG tagged MRP1 mutants were expressed at the cell surface to the same extent as the wild-type protein and also exhibited normal transport function. Our results demonstrate that the accessibility of the external FLAG epitope is strongly dependent on the position of the tag and the glycosylation state of the different FLAG-tagged MRP1s, and the conformation of extracellular loops in MSD1 and MDS3 does not appear to contribute to the functional status of MRP1.     

Mutagenesis of SNM1, Which Encodes a Protein Component of the Yeast RNase MRP, Reveals a Role for This Ribonucleoprotein Endoribonuclease in Plasmid Segregation

RNase MRP is a ribonucleoprotein endoribonuclease that has been shown to have roles in both mitochondrial DNA replication and nuclear 5.8S rRNA processing. SNM1 encodes an essential 22.5-kDa protein that is a component of yeast RNase MRP. It is an RNA binding protein that binds the MRP RNA specifically. This 198-amino-acid protein can be divided into three structural regions: a potential leucine zipper near the amino terminus, a binuclear zinc cluster in the middle region, and a serine- and lysine-rich region near the carboxy terminus. We have performed PCR mutagenesis of the SNM1 gene to produce 17 mutants that have a conditional phenotype for growth at different temperatures. Yeast strains carrying any of these mutations as the only copy of snm1 display an rRNA processing defect identical to that in MRP RNA mutants. We have characterized these mutant proteins for RNase MRP function by examining 5.8S rRNA processing, MRP RNA binding in vivo, and the stability of the RNase MRP RNA. The results indicate two separate functional domains of the protein, one responsible for binding the MRP RNA and a second that promotes substrate cleavage. The Snm1 protein appears not to be required for the stability of the MRP RNA, but very low levels of the protein are required for processing of the 5.8S rRNA. Surprisingly, a large number of conditional mutations that resulted from nonsense and frameshift mutations throughout the coding regions were identified. The most severe of these was a frameshift at amino acid 7. These mutations were found to be undergoing translational suppression, resulting in a small amount of full-length Snm1 protein. This small amount of Snm1 protein was sufficient to maintain enough RNase MRP activity to support viability. Translational suppression was accomplished in two ways. First, CEN plasmid missegregation leads to plasmid amplification, which in turn leads to SNM1 mRNA overexpression. Translational suppression of a small amount of the superabundant SNM1 mRNA results in sufficient Snm1 protein to support viability. CEN plasmid missegregation is believed to be the result of a prolonged telophase arrest that has been recently identified in RNase MRP mutants. Either the SNM1 gene is inherently susceptible to translational suppression or extremely small amounts of Snm1 protein are sufficient to maintain essential levels of MRP activity.     

Multidrug ATP‐binding cassette transporters are essential for hepatic development of Plasmodium sporozoites

Multidrug resistance‐associated proteins (MRPs) belong to the C‐family of ATP‐binding cassette (ABC) transport proteins and are known to transport a variety of physiologically important compounds and to be involved in the extrusion of pharmaceuticals. Rodent malaria parasites encode a single ABC transporter subfamily C protein, whereas human parasites encode two: MRP1 and MRP2. Although associated with drug resistance, their biological function and substrates remain unknown. To elucidate the role of MRP throughout the parasite life cycle, Plasmodium berghei and Plasmodium falciparum mutants lacking MRP expression were generated. P. berghei mutants lacking expression of the single MRP as well as P. falciparum mutants lacking MRP1, MRP2 or both proteins have similar blood stage growth kinetics and drug‐sensitivity profiles as wild type parasites. We show that MRP1‐deficient parasites readily invade primary human hepatocytes and develop into mature liver stages. In contrast, both P. falciparum MRP2‐deficient parasites and P. berghei mutants lacking MRP protein expression abort in mid to late liver stage development, failing to produce mature liver stages. The combined P. berghei and P. falciparum data are the first demonstration of a critical role of an ABC transporter during Plasmodium liver stage development.     

Expression and purification of isotopically labeled peptide inhibitors and substrates of cAMP-dependant protein kinase A for NMR analysis

Extensive X-ray crystallographic studies carried out on the catalytic-subunit of protein kinase A (PKA-C) enabled the atomic characterization of inhibitor and/or substrate peptide analogues trapped at its active site. Yet, the structural and dynamic transitions of these peptides from the free to the bound state are missing. These conformational transitions are central to understanding molecular recognition and the enzymatic cycle. NMR spectroscopy allows one to study these phenomena under functionally relevant conditions. However, the amounts of isotopically labeled peptides required for this technique present prohibitive costs for solid-phase peptide synthesis. To enable NMR studies, we have optimized both expression and purification of isotopically enriched substrate/inhibitor peptides using a recombinant fusion protein system. Three of these peptides correspond to the cytoplasmic regions of the wild-type and lethal mutants of the membrane protein phospholamban, while the fourth peptide correspond to the binding epitope of the heat-stable protein kinase inhibitor (PKI_5–24). The target peptides were fused to the maltose binding protein (MBP), which is further purified using a His₆ tag approach. This convenient protocol allows for the purification of milligram amounts of peptides necessary for NMR analysis.     

Mutation of proline residues in the NH(2)-terminal region of the multidrug resistance protein,MRP1 (ABCC1): effects on protein expression,membrane localization,and transport function

The Multidrug Resistance Protein, MRP1 (ABCC1) confers drug resistance and transports organic anions such as leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-D-glucuronide) (E(2)17betaG). Previous studies showed that portions of the first membrane spanning domain (MSD1) and the cytoplasmic loop (CL3) connecting it to MSD2 are important for MRP1 transport function. We have replaced 12 prolines in MSD1 and CL3 with alanine and determined the effects of these substitutions on MRP1 expression and transport activity. All singly substituted MRP1-Pro mutants could be expressed in HeLa cells, except MRP1-P104A. The expressed mutants also transported LTC(4) and E(2)17betaG, and their K(m) (LTC(4)) values were similar to wild-type MRP1. Expression of the double mutant MRP1-P42/51A was reduced by >80% although it localized to the plasma membrane and transported organic anions. MRP1 expression was also reduced when the first transmembrane helix (amino acids 37-54) was deleted. In contrast, the phenotypes of the multiply substituted CL3 mutants MRP1-P196/205/207/209A and MRP1-P235/255A were comparable to wild-type MRP1. However, Pro(255)-substituted MRP1 mutants showed reduced immunoreactivity with a monoclonal antibody (MAb) whose epitope is located in CL3. We conclude that certain prolines in MSD1 and CL3 play a role in the expression and structure of MRP1.     

Mutational analysis of ionizable residues proximal to the cytoplasmic interface of membrane spanning domain 3 of the multidrug resistance protein, MRP1 (ABCC1): glutamate 1204 is important for both the expression and catalytic activity of the transporter

The multidrug resistance protein MRP1 is an ATP-dependent transporter of organic anions and chemotherapeutic agents. A significant number of ionizable amino acids are found in or proximal to the 17 transmembrane (TM) helices of MRP1, and we have investigated 6 of these at the cytoplasmic interface of TM13-17 for their role in MRP1 expression and transport activity. Opposite charge substitutions of TM13 Arg(1046) and TM15 Arg(1131) did not alter MRP1 expression nor did they substantially affect activity. In contrast, opposite charge substitutions of TM16 Arg(1202) and Glu(1204) reduced protein expression by >80%; however, MRP1 expression was not affected when Arg(1202) and Glu(1204) were replaced with neutral or same-charge residues. In addition, organic anion transport levels of the R1202L, R1202G, and R1202K mutants were comparable with wild-type MRP1. In contrast, organic anion transport by E1204L was substantially reduced, whereas transport by E1204D was comparable with wild-type MRP1, with the notable exception of GSH. Opposite charge substitutions of TM16 Arg(1197) and TM17 Arg(1249) did not affect MRP1 expression but substantially reduced transport. Mutants containing like-charge substitutions of Arg(1197) or Arg(1249) were also transport-inactive and no longer bound leukotriene C(4). In contrast, substrate binding by the transport-compromised E1204L mutant remained intact. Furthermore, vanadate-induced trapping of azido-ADP by E1204L was dramatically increased, indicating that this mutation may cause a partial uncoupling of the catalytic and transport activities of MRP1. Thus, Glu(1204) serves a dual role in membrane expression of MRP1 and a step in its catalytic cycle subsequent to initial substrate binding.     

Functional and structural consequences of cysteine substitutions in the NH2 proximal region of the human multidrug resistance protein 1 (MRP1/ABCC1)

The 190 kDa multidrug resistance protein 1 (MRP1; ABCC1) is comprised of three membrane spanning domains (MSDs) and two nucleotide binding domains (NBDs) configured MSD1-MSD2-NBD1-MSD3-NBD2. MRP1 overexpression in tumor cells results in an ATP-dependent efflux of many oncolytic agents and arsenic and antimony oxyanions. MRP1 also transports GSSG and GSH as well as conjugated organic anions, including leukotriene C(4) and 17beta-estradiol 17-(beta-D-glucuronide) and certain xenobiotics in association with GSH. Previous studies have shown that portions of MSD1 and the cytoplasmic loop (CL3) connecting it to MSD2 are important for MRP1 transport function. In the present study, Cys residues at positions 43, 49, 85, 148, and 190 in MSD1 and positions 208 and 265 in CL3 were mutated to Ala and Ser, and the effects on protein expression, plasma membrane localization, trypsin sensitivity, organic anion transport, and drug resistance properties were investigated. Confocal microscopy showed that 11 of 14 mutants displayed significant levels of nonplasma membrane-associated MRP1. Most mutant proteins were also more resistant to trypsin proteolysis than wild-type MRP1. All Cys mutants transported organic anions (0.5-1.5-fold wild-type MRP1 activity), and cells expressing Ser-substituted but not Ala-substituted Cys43 and Cys265 MRP1 mutants exhibited a 2.5-fold decrease and a 3-fold increase in arsenite resistance, respectively; Cys43Ser MRP1 also conferred lower levels of vincristine resistance. These results indicate that certain Cys residues in the NH(2) proximal region of MRP1 can be important for its structure and selected transport activities.     

Discoordinate expression of the yeast mitochondrial ribosomal protein MRP1

We have examined expression of the protein coded within the MRP 1 locus of Saccharomyces cerevisiae. Direct evidence is provided for the assignment of the MRP1 gene product as a protein component of the small subunit of mitochondrial ribosomes. Further studies examined the extent to which the expression of the MRP1 protein is coordinated with the expression of other mitochondrial ribosomal components coded in the nuclear and mitochondrial genomes. Extra copies of the MRP1 gene were introduced into yeast cells to perturb expression from MRP1 relative to other mitochondrial ribosomal components to determine whether forms of regulation function to limit the accumulation of either MRP1 mRNA or protein under these conditions. Increases in MRP1 gene dosage were accompanied by substantial increases in both MRP1 mRNA and protein, indicating that their accumulation was not linked to the level of expression of other mitochondrial ribosomal components. This conclusion was confirmed by additional studies that showed that the accumulation of the MRP1 protein was unaffected in cells that did not express mitochondrially-encoded rRNAs. These results contrast with previous studies on the expression of two other mitochondrial ribosomal proteins indicating that regulatory properties of mitochondrial ribosomal proteins are quite diverse.     

siRNA介导多药耐药相关蛋白(MRP1)的表达沉默

利用pSIREN-RetroQ载体构建了3个沉默多药耐药相关蛋白（MRP1）基因表达质粒pSI REN-siRNAs.并通过限制性内切酶酶切鉴定和DNA测序鉴定,将截断MRP和全长MRP1 cDNA分别克隆到真核表达载体pEGFP-N2和pcDNA3.1中,产生了pEGFP-MRP1T和pcDNA-MRP1表达质粒．质粒pEGFP-MRP1T分别与3个pSIREN-siRNAs共转染HEK293细胞沉默MRP1T-GFP靶基因,pSIREN-siRNA1作为阴性对照.荧光显微镜下显示结果表明,与pSIREN-siRNA1相比,pSIREN-siRNA2和pSIREN-siRNA3产生的siRNA能够有效沉默MRP1T-GFP融合蛋白的表达．为了沉默全长MRP1基因的表达,pcDNA-MRP1分别与3个pSIREN-siRNAs共转染HEK293细胞．Western印迹和MTT分析表明,pSIREN-siRNA2和pSIREN- siRNA3能有效抑制190 kD MRP1在HEK293细胞中的表达,而pSIREN-siRNA1则不能．pSIREN-siRNA2和pSIREN-siRNA3能逆转MRP1转染HEK293细胞产生的多药耐药性．RNA二级结构预测结果分析表明,siRNA1靶序列mRNA局部自由能热动力参数ΔG低于siRNA2和siRNA3靶序列mRNA局部自由能热动力参数,siRNA1的GC含量和Tm值高于siRNA2和siRNA3．这些数据提示,siRNA和局部靶结构可能影响siRNA对MRP1 mRNA表达的沉默作用．     

Characterization of the 5'-flanking region of the human multidrug resistance protein 2 (MRP2) gene and its regulation in comparison withthe multidrug resistance protein 3 (MRP3) gene.

The multidrug resistance proteins MRP2 (symbol ABCC2) and MRP3 (symbol ABCC3) are conjugate export pumps expressed in hepatocytes. MRP2 is localized exclusively to the apical membrane and MRP3 to the basolateral membrane. MRP2 mRNA is expressed at a high level under normal conditions, whereas MRP3 mRNA expression is low and increases only when secretion across the apical membrane by MRP2 is impaired. We studied some of the regulatory properties of the two human genes using transient transfection assays with promoter-luciferase constructs in HepG2 cells and cloned fragments of 1229 nucleotides and 1287 nucleotides of the MRP2 and MRP3 5'-flanking regions, respectively. The sequence between nucleotides -517 and -197 was decisive for basal MRP2 expression. Basal promoter activity of MRP3 was only 4% of that measured for MRP2. At submicromolar concentrations, the histone deacetylase inhibitor trichostatin A reduced the MRP2 reporter gene activity and expression of the protein. Disruption of microtubules with nocodazole decreased gene and protein expression of MRP2 and increased MRP3 reporter gene activity. The genotoxic 2-acetylaminofluorene decreased the activity of the human MRP2 reporter gene construct, but increased MRP3 gene activity and enhanced the amounts of mRNA and protein of MRP2 and MRP3. Thus, regulation of the expression of these ATP-dependent conjugate export pumps is not co-ordinate, but in part inverse. The inverse regulation of the two MRP isoforms is consistent with their distinct localization, their different mRNA expression under normal and pathophysiological conditions, and their different directions of substrate transport in polarized cells.     

Cloning and characterization of genes specifically expressed during infection stages in the rice blast fungus

We isolated promoters of 12 genes from the rice blast fungus based on the sequences of randomly selected expressed sequence tags (ESTs) (appressorium formation stage cDNA library of Magnaporthe available from GenBank). These promoters (and the 5' coding regions if any) were fused in frame with egfp, and their expression patterns were examined under the epifluorescence microscope. Among them, two turned out to be specifically active in structures necessary for infection, viz. a promoter of adenylate cyclase interacting protein 1-like gene expressed in conidia, germ tubes, and appressoria, and a promoter of putative membrane-associated or secreted protein gene specifically expressed in appressoria. Although targeted knockout mutants of either gene failed to show detectable phenotypic alterations under laboratory conditions, these ESTs should be useful for identification of genes expressed during infection stages.     

TSGIT: An N‐ and C‐terminal tandem tag system for purification of native and intein‐mediated ligation‐ready proteins

A large variety of fusion tags have been developed to improve protein expression, solubilization, and purification. Nevertheless, these tags have been combined in a rather limited number of composite tags and usually these composite tags have been dictated by traditional commercially‐available expression vectors. Moreover, most commercially‐available expression vectors include either N‐ or C‐terminal fusion tags but not both. Here, we introduce TSGIT, a fusion‐tag system composed of both N‐ and a C‐terminal composite fusion tags. The system includes two affinity tags, two solubilization tags and two cleavable tags distributed at both termini of the protein of interest. Therefore, the N‐ and the C‐terminal composite fusion tags in TSGIT are fully orthogonal in terms of both affinity selection and cleavage. For using TSGIT, we streamlined the cloning, expression, and purification procedures. Each component tag is selected to maximize its benefits toward the final construct. By expressing and partially purifying the protein of interest between the components of the TSGIT fusion, the full‐length protein is selected over truncated forms, which has been a long‐standing problem in protein purification. Moreover, due to the nature of the cleavable tags in TSGIT, the protein of interest is obtained in its native form without any additional undesired N‐ or C‐terminal amino acids. Finally, the resulting purified protein is ready for efficient ligation with other proteins or peptides for downstream applications. We demonstrate the use of this system by purifying a large amount of native fluorescent mRuby3 protein and bacteriophage T7 gp2.5 ssDNA‐binding protein.     

Functional importance of three basic residues clustered at the cytosolic interface of transmembrane helix 15 in the multidrug and organic anion transporter MRP1 (ABCC1)

The multidrug resistance protein 1 (MRP1) mediates drug and organic anion efflux across the plasma membrane. The 17 transmembrane (TM) helices of MRP1 are linked by extracellular and cytoplasmic (CL) loops of various lengths and two cytoplasmic nucleotide binding domains. In this study, three basic residues clustered at the predicted TM15/CL7 interface were investigated for their role in MRP1 expression and activity. Thus, Arg1138, Lys1141, and Arg1142 were replaced with residues of the same or opposite charge, expressed in human embryonic kidney cells, and the properties of the mutant proteins were assessed. Neither Glu nor Lys substitutions of Arg1138 and Arg1142 affected MRP1 expression; however, all four mutants showed a decrease in organic anion transport with a relatively greater decrease in leukotriene C4 and glutathione transport. These mutations also modulated MRP1 ATPase activity as reflected by a decreased vanadate-induced trapping of 8-azido-[32P]ADP. Mutation of Lys1141 to either Glu or Arg reduced MRP1 expression, and routing to the plasma membrane was impaired. However, only the Glu-substituted Lys1141 mutant showed a decrease in organic anion transport, and this was associated with decreased substrate binding and vanadate-induced trapping of 8-azido-ADP. These studies identified a cluster of basic amino acids likely at the TM15/CL7 interface as a region important for both MRP1 expression and activity and demonstrated that each of the three residues plays a distinct role in the substrate specificity and catalytic activity of the transporter.     

Mutagenesis of the putative nucleotide-binding domains of the multidrug resistance associated protein (MRP). Analysis of the effect of these mutations on MRP mediated drug resistance and transport

Studies were conducted to examine the functional role of the nucleotide-binding domains of MRP in drug resistance and drug transport in isolated membrane vesicles. In vivo studies were conducted by preparing stable transfectants of HeLa cells with wild-type MRP cDNA or MRP cDNAs which had been mutated at certain nucleotide binding domains (NBD). Stable transfectants producing equivalent amounts of the MRP encoded protein P190 were used in this study. The results demonstrated that deletions in the C-motif of NBD1 or the A-motif of NBD2 have a pronounced effect in reducing resistance levels to chemotherapeutic agents. Certain single-site mutations in lysines in these same motifs also reduce IC₅₀ values. It has also been observed that mutation of the MRP NBDs results in an increase in drug accumulation and a reduction in drug efflux. Additional studies have been carried out in which recombinant baculovirus containing either wild-type MRP or MRP containing mutated NBDs was prepared and used to infect SF21 insect cells. Using this system we have analyzed the effects of these mutations on in vitro transport of leukotriene C₄ (LTC₄) 17 β-estradiol 17 (β-D-glucuronide)(E₂17βG) and daunomycin in membrane vesicles prepared from baculovirus infected cells. The results demonstrate that deletions and site-specific mutations in MRP NBDs greatly reduce the ATP dependent transport of all three substrates. The results of these studies conducted both in vivo and in vitro demonstrate that the NBDs of MRP function in a cooperative manner and are critical for the transport activity of the MRP encoded protein P190. These studies also identify specific lysines in NBD1 and NBD2 which are important for optimal MRP activity. This revised version was published online in August 2006 with corrections to the Cover Date.