ATP-binding Cassette Multidrug Transporters Research Articles

Multidrug resistance ABC transporters

Clinical multidrug resistance is caused by a group of integral membrane proteins that transport hydrophobic drugs and lipids across the cell membrane. One class of these permeases, known as multidrug resistance ATP binding cassette (ABC) transporters, translocate these molecules by coupling drug/lipid efflux with energy derived from the hydrolysis of ATP. In this review, we examine both the structures and conformational changes of multidrug resistance ABC transporters. Together with the available biochemical and structural evidence, we propose a general mechanism for hydrophobic substrate transport coupled to ATP hydrolysis.

The ATP Binding Cassette Multidrug Transporter LmrA and Lipid Transporter MsbA Have Overlapping Substrate Specificities

LmrA is an ATP binding cassette (ABC) multidrug transporter in Lactococcus lactis that is a structural and functional homologue of the human multidrug resistance P-glycoprotein MDR1 (ABCB1). LmrA is also homologous to MsbA, an essential ABC transporter in Escherichia coli involved in the trafficking of lipids, including Lipid A. We have compared the substrate specificities of LmrA and MsbA in detail. Surprisingly, LmrA was able to functionally substitute for a temperature-sensitive mutant MsbA in E. coli WD2 at non-permissive temperatures, suggesting that LmrA could transport Lipid A. LmrA also exhibited a Lipid A-stimulated, vanadate-sensitive ATPase activity. Reciprocally, the expression of MsbA conferred multidrug resistance on E. coli. Similar to LmrA, MsbA interacted with photoactivatable substrate [3H]azidopine, displayed a daunomycin, vinblastine, and Hoechst 33342-stimulated vanadate-sensitive ATPase activity, and mediated the transport of ethidium from cells and Hoechst 33342 in proteoliposomes containing purified and functionally reconstituted protein. Taken together, these data demonstrate that MsbA and LmrA have overlapping substrate specificities. Our observations imply the presence of structural elements in the recently published crystal structures of MsbA in E. coli and Vibrio cholera (Chang, G., and Roth, C. B. (2001) Science 293, 1793-1800; Chang, G. (2003) J. Mol. Biol. 330, 419-430) that support drug-protein interactions and suggest a possible role for LmrA in lipid trafficking in L. lactis.

Population genomics of drug resistance in Candida albicans.

We followed adaptation in experimental microbial populations to inhibitory concentrations of an antimicrobial drug. The evolution of drug resistance was accompanied in all cases by changes in gene expression that persisted in the absence of the drug; the new patterns of gene expression were constitutive. The changes in gene expression occurred in four replicate populations of the pathogenic fungus Candida albicans during 330 generations of evolution in the presence of the antifungal drug fluconazole. Genome-wide expression profiling of over 5,000 ORFs identified 301 whose expression was significantly modulated. Cluster analysis identified three distinct patterns of gene expression underlying adaptation to the drug. One pattern was unique to one population and included up-regulation of the multidrug ATP-binding cassette transporter gene, CDR2. A second pattern occurred at a late stage of adaptation in three populations; for two of these populations profiled earlier in their evolution, a different pattern was observed at an early stage of adaptation. The succession of early- and late-stage patterns of gene expression, both of which include up-regulation of the multidrug major facilitator transporter gene, MDR1, must represent a common program of adaptation to this antifungal drug. The three patterns of gene expression were also identified in fluconazole-resistant clinical isolates, providing further evidence that these patterns represent common programs of adaptation to fluconazole.

Bacterial multidrug resistance mediated by a homologue of the human multidrug transporter P-glycoprotein.

Most ATP-binding cassette (ABC) multidrug transporters known to date are of eukaryotic origin, such as the P-glycoproteins (Pgps) and multidrug resistance-associated proteins (MRPs). Only one well-characterized ABC multidrug transporter, LmrA, is of bacterial origin. On the basis of its structural and functional characteristics, this bacterial protein is classified as a member of the P-glycoprotein cluster of the ABC transporter superfamily. LmrA can even substitute for P-glycoprotein in human lung fibroblast cells, suggesting that this type of transporter is conserved from bacteria to man. The functional similarity between bacterial LmrA and human P-glycoprotein is further exemplified by their currently known spectrum of substrates, consisting mainly of hydrophobic cationic compounds. In addition, LmrA was found to confer resistance to eight classes of broad-spectrum antibiotics, and homologs of LmrA have been found in pathogenic bacteria, supporting the clinical and academic value of studying this bacterial protein. Current studies are focused on unraveling the mechanism by which ABC multidrug transporters, such as LmrA, couple the hydrolysis of ATP to the translocation of drugs across the membrane. Recent evidence indicates that LmrA mediates drug transport by an alternating two-site transport mechanism.

Effects of pressure-induced membrane phase transitions on inactivation of HorA, an ATP-dependent multidrug resistance transporter, in Lactobacillus plantarum.

The effects of pressure on cultures of Lactobacillus plantarum were characterized by determination of the viability and activity of HorA, an ATP-binding cassette multidrug resistance transporter. Changes in the membrane composition of L. plantarum induced by different growth temperatures were determined. Furthermore, the effect of the growth temperature of a culture on pressure inactivation at 200 MPa was determined. Cells were characterized by plate counts on selective and nonselective agar after pressure treatment, and HorA activity was measured by ethidium bromide efflux. Fourier transform-infrared spectroscopy and Laurdan fluorescence spectroscopy provided information about the thermodynamic phase state of the cytoplasmic membrane during pressure treatment. A pressure-temperature diagram for cell membranes was established. Cells grown at 37 degrees C and pressure treated at 15 degrees C lost >99% of HorA activity and viable cell counts within 36 and 120 min, respectively. The membranes of these cells were in the gel phase region at ambient pressure. In contrast, cells grown at 15 degrees C and pressure treated at 37 degrees C lost >99% of HorA activity and viable cell counts within 4 and 8 min, respectively. The membranes of these cells were in the liquid crystalline phase region at ambient pressure. The kinetic analysis of inactivation of L. plantarum provided further evidence that inactivation of HorA is a crucial step during pressure-induced cell death. Comparison of the biological findings and the membrane state during pressure treatment led to the conclusion that the inactivation of cells and membrane enzymes strongly depends on the thermodynamic properties of the membrane. Pressure treatment of cells with a liquid crystalline membrane at 0.1 MPa resulted in HorA inactivation and cell death more rapid than those of cells with a gel phase membrane at 0.1 MPa.

Modulation by flavonoids of cell multidrug resistance mediated by P-glycoprotein and related ABC transporters.

Cancer cell resistance to chemotherapy is often mediated by overexpression of P-glycoprotein, a plasma membrane ABC (ATP-binding cassette) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis. P-glycoprotein (ABCB1, according to the human gene nomenclature committee) consists of two homologous halves each containing a transmembrane domain (TMD) involved in drug binding and efflux, and a cytosolic nucleotide-binding domain (NBD) involved in ATP binding and hydrolysis, with an overall (TMD-NBD)2 domain topology. Homologous ABC multidrug transporters, from the same ABCB family, are found in many species such as Plasmodiumfalciparum and Leishmania spp. protozoa, where they induce resistance to antiparasitic drugs. In yeasts, some ABC transporters involved in resistance to fungicides, such as Saccharomyces cerevisiae Pdr5p and Snq2p, display a different (NBD-TMD)2 domain topology and are classified in another family, ABCG. Much effort has been spent to modulate multidrug resistance in the different species by using specific inhibitors, but generally with little success due to additional cellular targets and/or extrusion of the potential inhibitors. This review shows that due to similarities in function and maybe in three-dimensional organization of the different transporters, common potential modulators have been found. An in vitro 'rational screening' was performed among the large flavonoid family using a four-step procedure: (i) direct binding to purified recombinant cytosolic NBD and/or full-length transporter, (ii) inhibition of ATP hydrolysis and energy-dependent drug interaction with transporter-enriched membranes, (iii) inhibition of cell transporter activity monitored by flow cytometry and (iv) chemosensitization of cell growth. The results indicate that prenylated flavonoids bind with high affinity, and strongly inhibit drug interaction and nucleotide hydrolysis. As such, they constitute promising potential modulators of multidrug resistance.

Hop resistance in the beer spoilage bacterium Lactobacillus brevis is mediated by the ATP-binding cassette multidrug transporter HorA.

Lactobacillus brevis is a major contaminant of spoiled beer. The organism can grow in beer in spite of the presence of antibacterial hop compounds that give the beer a bitter taste. The hop resistance in L. brevis is, at least in part, dependent on the expression of the horA gene. The deduced amino acid sequence of HorA is 53% identical to that of LmrA, an ATP-binding cassette multidrug transporter in Lactococcus lactis. To study the role of HorA in hop resistance, HorA was functionally expressed in L. lactis as a hexa-histidine-tagged protein using the nisin-controlled gene expression system. HorA expression increased the resistance of L. lactis to hop compounds and cytotoxic drugs. Drug transport studies with L. lactis cells and membrane vesicles and with proteoliposomes containing purified HorA protein identified HorA as a new member of the ABC family of multidrug transporters.

Evidence for the Vectorial Nature of Drug (Substrate)-stimulated ATP Hydrolysis by Human P-glycoprotein

P-glycoprotein (Pgp), the ATP-binding cassette multidrug transporter, exhibits a drug (substrate)-stimulatable ATPase activity, and vanadate (Vi) inhibits this activity by stably trapping the nucleoside diphosphate in the Pgp.ADP.Vi conformation. We recently demonstrated that Vi-induced 8-azido-[alpha-(32)P]ADP trapping into Pgp in the absence of substrate occurs both in the presence of 8-azido-[alpha-(32)P]ATP (following 8-azido-ATP hydrolysis) or 8-azido-[alpha-(32)P]ADP (without hydrolysis) and, the transition state intermediates generated under either condition are functionally indistinguishable. In this study, we compare the effect of substrates on Vi-induced 8-azido-[alpha-(32)P]ADP trapping into Pgp under both non-hydrolysis and hydrolysis conditions. We demonstrate that whereas substrates stimulate the Vi-induced trapping of 8-azido-[alpha-(32)P]ADP under hydrolysis conditions, they strongly inhibit Vi-induced trapping under non-hydrolysis conditions. This inhibition is concentration-dependent, follows first order kinetics, and is effected by drastically decreasing the affinity of nucleoside diphosphate for Pgp during trapping. However, substrates do not affect the binding of nucleoside diphosphate in the absence of Vi, indicating that the substrate-induced conformation exerts its effect at a step distinct from nucleoside diphosphate-binding. Our results demonstrate that during the catalytic cycle of Pgp, although the transition state, Pgp x ADP x P(i) (Vi), can be generated both via the hydrolysis of ATP or by directly providing ADP to the system, in the presence of substrate the reaction is driven in the forward direction, i.e. hydrolysis of ATP. These data suggest that substrate-stimulated ATP hydrolysis by Pgp is a vectorial process.

Multidrug transport by ATP binding cassette transporters: a proposed two-cylinder engine mechanism

The elevated expression of ATP binding cassette (ABC) multidrug transporters in multidrug-resistant cells interferes with the drug-based control of cancers and infectious pathogenic microorganisms. Multidrug transporters interact directly with the drug substrates. This review summarizes current insights into the mechanism(s) by which ATP hydrolysis is coupled to drug transport in bacterial LmrA and its human homolog P-glycoprotein. In addition, the relevance of these insights for other ABC transporters will be discussed.

Identification and expression of multidrug resistance-related ABC transporter genes in Candida krusei.

Infections with Candida krusei have increased in recent years as a consequence of its intrinsic resistance to fluconazole, an antifungal azole widely used in immunocompromised individuals to suppress infections due to azole-susceptible C. albicans. One established mechanism for azole resistance is drug efflux by ATP binding cassette (ABC) transporters. Since these transporters recognize structurally diverse drugs, their overexpression can lead to multidrug resistance (MDR). To identify C. krusei genes potentially involved in azole resistance, PCR was performed with primers corresponding to conserved sequences of MDR-related ABC transporters from other fungi. Two genes, ABC1 and ABC2, were identified; Southern blots suggested that both have one or two related gene copies in the C. krusei genome. ABC1 RNA was constitutively expressed at low levels in log phase cells while ABC2 RNA was undetectable. However, both genes were upregulated as cultures approached stationary phase, and this upregulation was correlated with decreased susceptibility to the lethal activity of the azole derivative miconazole. Furthermore, ABC1 was upregulated following brief treatment of C. krusei with miconazole and clotrimazole (but not other azoles), and the unrelated compounds albendazole and cycloheximide. The latter two compounds antagonized fluconazole activity versus C. krusei, supporting a role for the ABC1 transporter in azole efflux. Finally, miconazole-resistant mutants selected in vitro demonstrated increased constitutive expression of ABC1. Based on these expression data, genetic and functional characterization of the ABC1 transporter to directly test its role in C. krusei azole resistance would appear to be warranted.

Prenyl-Flavonoids as Potent Inhibitors of the Pdr5p Multidrug ABC Transporter from Saccharomyces cerevisiae

The Pdr5p multidrug ABC ("ATP-binding cassette) transporter was highly overexpressed in plasma membranes from a yeast strain exhibiting both pdr1-3 gain-of-function mutation in the transcription factor-encoding gene PDR1 and disruption of genes encoding other plasma membrane ABC transporters. Solubilized and purified Pdr5p displayed a tryptophan-characteristic intrinsic fluorescence, whose quenching was used to monitor interactions with substrates and effectors. The transporter exhibited a magnesium-dependent binding affinity for ATP and its fluorescent analogue 2'(3')-N-methylanthraniloyl-ATP, producing a marked fluorescence resonance-energy transfer. It also bound a series of known drug substrates and modulators. Interestingly, yeast Pdr5p interacted with flavonoids recently found to bind to cancer cell P-glycoprotein and to the protozoan parasite multidrug transporter. The extent of high-affinity binding of prenyl-flavonoids to purified Pdr5p was correlated to their efficiency to inhibit energy-dependent quenching of rhodamine 6G fluorescence catalyzed by Pdr5p-enriched plasma membranes. The hydrophobic flavonoid derivative 6-(3, 3-dimethylallyl)galangin was the most efficient, with a K(i) of 0.18 microM for competitive inhibition of the MgATP-dependent quenching of rhodamine 6G fluorescence. In contrast, inhibition of either ATP or UTP hydrolysis occurred at much higher concentrations and appeared to be noncompetitive. Prenyl-flavonoids therefore behave as potent inhibitors of drug binding to the yeast Pdr5p ABC transporter.

Multidrug ABC transporters from bacteria to man: an emerging hypothesis for the universality of molecular mechanism and function

. he ATP-binding cassette (ABC) superfamily of membrane-associated transporters encompasses a diverse group of proteins in a wide variety of organisms and cell types, many of which have been implicated in human diseases. In addition to highly homologous ATP-binding domains, most of these proteins have multiple transmembrane (TM) spanning domains and function as ATP-dependent transportersF z In eukaryotic cells, members of this family include the multidrug transporter, P-glycoprotein (P-gp), the related multidrug resistance-associated protein (mrpl) encoded by the MRP1 gene, 3 the cystic fibrosis transmembrane regulator (CFTR), ~ the STE6 a-mating factor transporter in the yeast Saccbaromyces cerevisiae, 5,6 and the ABCR protein in retina implicated in macular degeneration. 7,8 Additional members of this family are the peroxisomal membrane pumps, PMPTO 9-11 and ALDP ~2 and the related ALDRP I3 whose mutated versions have been implicated in the cerebro-hepato-renal autosomal recessive disorder Zellweger syndrome and PERSPECTIVES

The ATP Binding Cassette Transporters Pdr5 and Snq2 of Saccharomyces cerevisiae Can Mediate Transport of Steroids in Vivo

Multiple or pleiotropic drug resistance in the yeast Saccharomyces cerevisiae can arise from overexpression of the Pdr5 and Snq2 ATP binding cassette multidrug transporters. Expression of Pdr5 and Snq2 is regulated by the two transcription factors Pdr1 and Pdr3, as multidrug-resistant pdr1 and pdr3 gain-of-function mutants overexpress both drug efflux pumps. One such pdr1 mutant allele was previously cloned in a genetic screen by its ability to suppress the squelching toxicity mediated by an estradiol-inducible chimeric VP16-human estrogen receptor (VEO) expressed in yeast (Gilbert, D. M. , Heery, D. M., Losson, R., Chambon, P., and Lemoine, Y. (1993) Mol. Cell. Biol. 13, 462-472). In this study, we demonstrate that relief of estradiol toxicity in yeast cells expressing VEO requires functional PDR5 and SNQ2 genes, since a Deltapdr5 Deltasnq2 double deletion leads to an increased estradiol toxicity. Furthermore, using URA3 as an estradiol-inducible reporter gene, we show that Pdr5 and Snq2, when overexpressed from high-copy plasmids, can reduce the intracellular concentration of estradiol. In contrast, a Deltapdr5 Deltasnq2 double deletion mutant accumulates almost 30-fold more intracellular estradiol than the isogenic wild type. Indirect immunofluorescence showed that a pdr1-3 mutant massively overexpresses Pdr5 at the plasma membrane, suggesting that estradiol efflux from the cells occurs across the plasma membrane. Our data demonstrate that Pdr5 and Snq2 can transport steroid substrates in vivo and suggest that steroids and/or related membrane lipids could represent physiological substrates for certain yeast ABC transporters, which are otherwise involved in the development of pleiotropic drug resistance.

The yeast multidrug transporter Pdr5 of the plasma membrane is ubiquitinated prior to endocytosis and degradation in the vacuole

We have recently demonstrated that the Pdr5 ATP binding cassette multidrug transporter is a short-lived protein, whose biogenesis involves cell surface targeting followed by endocytosis and delivery to the vacuole for proteolytic turnover [Egner, R., Mahé, Y., Pandjaitan, R., and Kuchler, K. (1995) Mol. Cell. Biol. 15, 5879–5887]. Using c- myc epitope-tagged ubiquitin, we now have shown that Pdr5 is a ubiquitinated plasma membrane protein in vivo. Ubiquitination of Pdr5 was detected in both wild type and conditional end4 mutants defective in endocytic vesicle formation. Likewise, the Ste6 a-factor pheromone transporter, which represents another short-lived ABC transporter whose turnover requires vacuolar proteolysis, was also found to be ubiquitinated, and ubiquitin-modified Ste6 massively accumulated in end4 mutants at the restrictive temperature. By contrast, the plasma membrane ATPase Pma1, a long-lived and metabolically very stable protein, was found not to be ubiquitinated. Our results imply a novel function for ubiquitin in protein trafficking and suggest that ubiquitination of certain short-lived plasma membrane proteins may trigger their endocytic delivery to the vacuole for proteolytic turnover.

Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters.

Azole antifungal agents, and especially fluconazole, have been used widely to treat oropharyngeal candidiasis in patients with AIDS. An increasing number of cases of clinical resistance against fluconazole, often correlating with in vitro resistance, have been reported. To investigate the mechanisms of resistance toward azole antifungal agents at the molecular level in clinical C. albicans isolates, we focused on resistance mechanisms related to the cellular target of azoles, i.e., cytochrome P450(14DM) (14DM) and those regulating the transport or accumulation of fluconazole. The analysis of sequential isogenic C. albicans isolates with increasing levels of resistance to fluconazole from five AIDS patients showed that overexpression of the gene encoding 14DM either by gene amplification or by gene deregulation was not the major cause of resistance among these clinical isolates. We found, however, that fluconazole-resistant C. albicans isolates failed to accumulate 3H-labelled fluconazole. This phenomenon was reversed in resistant cells by inhibiting the cellular energy supply with azide, suggesting that resistance could be mediated by energy-requiring efflux pumps such as those described as ATP-binding cassette (ABC) multidrug transporters. In fact, some but not all fluconazole-resistant clinical C. albicans isolates exhibited up to a 10-fold relative increase in mRNA levels for a recently cloned ABC transporter gene called CDR1. In an azole-resistant C. albicans isolate not overexpressing CDR1, the gene for another efflux pump named BENr was massively overexpressed. This gene was cloned from C. albicans for conferring benomyl resistance in Saccharomyces cerevisiae. Therefore, at least the overexpression or the deregulation of these two genes potentially mediates resistance to azoles in C. albicans clinical isolates from AIDS patients with oropharyngeal candidiasis. Involvement of ABC transporters in azole resistance was further evidenced with S. cerevisiae mutants lacking specific multidrug transporters which were rendered hypersusceptible to azole derivatives including fluconazole, itraconazole, and ketoconazole.

Endocytosis and vacuolar degradation of the plasma membrane-localized Pdr5 ATP-binding cassette multidrug transporter in Saccharomyces cerevisiae.

Multidrug resistance (MDR) to different cytotoxic compounds in the yeast Saccharomyces cerevisiae can arise from overexpression of the Pdr5 (Sts1, Ydr1, or Lem1) ATP-binding cassette (ABC) multidrug transporter. We have raised polyclonal antibodies recognizing the yeast Pdr5 ABC transporter to study its biogenesis and to analyze the molecular mechanisms underlying MDR development. Subcellular fractionation and indirect immunofluorescence experiments showed that Pdr5 is localized in the plasma membrane. In addition, pulse-chase radiolabeling of cells and immunoprecipitation indicated that Pdr5 is a short-lived membrane protein with a half-life of about 60 to 90 min. A dramatic metabolic stabilization of Pdr5 was observed in delta pep4 mutant cells defective in vacuolar proteinases, and indirect immunofluorescence showed that Pdr5 accumulates in vacuoles of stationary-phase delta pep4 mutant cells, demonstrating that Pdr5 turnover requires vacuolar proteolysis. However, Pdr5 turnover does not require a functional proteasome, since the half-life of Pdr5 was unaffected in either pre1-1 or pre1-1 pre2-1 mutants defective in the multicatalytic cytoplasmic proteasome that is essential for cytoplasmic protein degradation. Immunofluorescence analysis revealed that vacuolar delivery of Pdr5 is blocked in conditional end4 endocytosis mutants at the restrictive temperature, showing that endocytosis delivers Pdr5 from the plasma membrane to the vacuole.