Two-component bacterial multidrug transporter, EbrAB: Mutations making each component solely functional

EbrAB in Bacillus subtilis belongs to a novel small multidrug resistance (SMR) family of multidrug efflux pumps. EmrE in Escherichia coli, a representative of SMR, functions as a homo-oligomer in the membrane. On the other hand, EbrAB requires a hetero-oligomeric configuration consisting of two polypeptides, EbrA and EbrB. Although both polypeptides have a high sequence similarity, expression of either single polypeptide does not confer the multidrug-resistance. We performed mutation studies on EbrA and B to determine why EbrAB requires the hetero-oligomerization. Mutants of EbrA and B lacking both the hydrophilic loops and the C-terminus regions conferred the multidrug-resistance solely by each protein. This suggests that the hydrophilic loops and the C-terminus regions constrain them to their respective conformations upon the formation of the functional hetero-oligomer.     

Anti-parallel membrane topology of two components of EbrAB, a multidrug transporter

EbrAB is a multidrug-resistance transporter in Bacillus subtilis that belongs to the small multidrug resistance, and requires two polypeptides of both EbrA and EbrB, implying that it functions in the hetero-dimeric state. In this study, we investigated the transmembrane topologies of EbrA and EbrB. Various single-cysteine mutants were expressed in Escherichia coli cells, and the efflux activity was measured. Only mutants having a high activity were used for the topology experiments. The reactivity of a membrane impermeable NEM-fluorescein against the single cysteine of these fully functional mutants was examined when this reactive fluorophore was applied either from the outside or both sides of the cell membrane or in the denatured state. The results clearly showed that EbrA and EbrB have the opposite orientation within the membrane or an anti-parallel configuration.     

Functional characterization of the heterooligomeric EbrAB multidrug efflux transporter of Bacillus subtilis

The Bacillus subtilis genome contains two tandem genes, ebrA and ebrB, which encode two homologues of the SMR family of multidrug efflux transporters. The sequences of EbrA and EbrB are highly similar to each other and to that of EmrE, the prototypical SMR transporter of Escherichia coli. Drug resistance profiling and drug binding experiments showed that the presence of both EbrA and EbrB is required for proper transport function. EbrA and EbrB directly interact and combine to form a functional transporter. They likely form a heterodimer in analogy to the EmrE homodimer. Mutagenesis experiments indicate that the conserved membrane-embedded glutamates in the first transmembrane helices of both EbrA and EbrB are required for multidrug efflux activity. However, the two glutamates are nonequivalent since EbrA E15 is required for substrate binding while EbrB E14 is not. Our studies support a model in which functional residues in EbrAB are relegated to at least two sets that participate in distinct steps of the active drug transport process.     

A two-component multidrug efflux pump, EbrAB, in Bacillus subtilis

Genes (ebrAB) responsible for ethidium resistance were cloned from chromosomal DNA of Bacillus subtilis ATCC 9372. The recombinant plasmid produced elevated resistance against ethidium bromide, acriflavine, pyronine Y, and safranin O not only in Escherichia coli but also in B. subtilis. It also caused an elevated energy-dependent efflux of ethidium in E. coli. EbrA and EbrB showed high sequence similarity with members of the small multidrug resistance (SMR) family of multidrug efflux pumps. Neither ebrA nor ebrB was sufficient for resistance, but introduction of the two genes carried on different plasmids conferred drug resistance. Thus, both EbrA and EbrB appear to be necessary for activity of the multidrug efflux pump. In known members of the SMR family, only one gene produces drug efflux. Thus, EbrAB is a novel SMR family multidrug efflux pump with two components.     

Characterization and functional reconstitution of the multidrug transporter

P-Glycoprotein, the multidrug transporter, is isolated from the plasma membrane of CH^RC5 cells using a selective two-step detergent extraction procedure. The partially purified protein displays a high level of ATPase activity, which has a highK_M for ATP, is stimulated by drugs, and can be distinguished from that of other membrane ATPases by its unique inhibition profile. Delipidation completely inactivates ATPase activity, which is restored by the addition of fluid lipid mixtures. P-Glycoprotein was reconstituted into lipid bilayers with retention of both drug transport and ATPase activity. Proteoliposomes containing P-glycoprotein display osmotically sensitive ATP-dependent accumulation of³H-colchicine in the vesicle lumen. Drug transport is active, generating a stable 5.6-fold concentration gradient, and can be blocked by compounds in the multidrug resistance spectrum. Reconstituted P-glycoprotein also exhibits a high level of ATPase activity which is further stimulated by various drugs. P-Glycoprotein therefore functions as an active drug transporter with constitutive ATPase activity.     

Mutations affecting substrate specificity of the Bacillus subtilis multidrug transporter Bmr.

The Bacillus subtilis multidrug transporter Bmr, a member of the major facilitator superfamily of transporters, causes the efflux of a number of structurally unrelated toxic compounds from cells. We have shown previously that the activity of Bmr can be inhibited by the plant alkaloid reserpine. Here we demonstrate that various substitutions of residues Phe143 and Phe306 of Bmr not only reduce its sensitivity to reserpine inhibition but also significantly change its substrate specificity. Cross-resistance profiles of bacteria expressing mutant forms of the transporter differ from each other and from the cross-resistance profile of cells expressing wild-type Bmr. This result strongly suggests that Bmr interacts with its transported drugs directly, with residues Phe143 and Phe306 likely to be involved in substrate recognition.     

Conformational and functional characterization of trapped complexes of the P-glycoprotein multidrug transporter

The Pgp (P-glycoprotein) multidrug transporter couples ATP hydrolysis at two cytoplasmic NBDs (nucleotide-binding domains) to the transport of hydrophobic compounds. Orthovanadate (V(i)) and fluoroaluminate (AlF(x)) trap nucleotide in one NBD by forming stable catalytically inactive complexes (Pgp-M2+-ADP-X), which are proposed to resemble the catalytic transition state, whereas the complex formed by beryllium fluoride (BeF(x)) is proposed to resemble the ground state. We studied the trapped complexes formed via incubation of Pgp with ATP (catalytically forward) or ADP (reverse) and V(i), BeF(x) or AlF(x) using Mg2+ or Co2+ as the bivalent cation. Quenching of intrinsic Pgp tryptophan fluorescence by acrylamide, iodide and caesium indicated that conformational changes took place upon formation of the trapped complexes. Trapping with V(i) and ATP led to a 6-fold increase in the acrylamide quenching constant, K(SV), suggesting that large conformational changes take place in the Pgp transmembrane regions on trapping in the forward direction. Trapping with V(i) and ADP gave only a small change in quenching, indicating that the forward- and reverse-trapped complexes are different. TNP (trinitrophenyl)-ATP/TNP-ADP interacted with all of the trapped complexes, however, the fluorescence enhancement differed for the trapped states, suggesting a change in polarity in the nucleotide-binding sites. The nucleotide-binding site of the BeF(x)-trapped complex was much more polar than that of the V(i) and AlF(x) complexes. Functionally, all the trapped complexes were able to bind drugs and TNP-nucleotides with unchanged affinity compared with native Pgp.     

Photosensitized labeling of a functional multidrug transporter in living drug-resistant tumor cells

A 170,000-Da glycoprotein (P170 multidrug transporter) becomes specifically labeled in multidrug-resistant human KB carcinoma cells by the photolabile lipophilic membrane probe 5-[125I]iodonaphthalene-1-azide ([125I]INA) when photoactivation of the probe is triggered by energy transfer from intracellular doxorubicin or rhodamine 123. In contrast, in drug-sensitive cells, drug-induced specific labeling of membrane proteins with [125I]INA was not observed. Instead, multiple membrane proteins became labeled in a nonspecific manner. This phenomenon of drug-induced specific labeling of P170 by [125I]INA is observed only in living cells, but not in purified membrane vesicles or lysed cells. It is generated by doxorubicin and rhodamine 123, drugs that are chromophores and to which the cells exhibit resistance; but it is not observed with other drugs or dyes. Verapamil, a calcium channel blocker which reverses resistance to doxorubicin, also abolishes doxorubicin-induced specific [125I]INA labeling of P170. These results reveal that a specific interaction between P170 and doxorubicin takes place in living cells and demonstrate that P170 is directly involved in the mechanism of drug resistance in vivo. They also provide a possible means to label functional domains in the multidrug transporter. The results demonstrate that photosensitized [125I]INA labeling is a technique which provides sufficient spatial and time resolution to detect specific intracellular interactions between chromophores and proteins in vivo.     

A single carboxyl mutant of the multidrug transporter EmrE is fully functional

EmrE, a multidrug transporter from Escherichia coli removes toxic compounds from the cell in exchange with protons. Glu-14 is the only charged residue in the putative membrane domains and is fully conserved in more than 50 homologues of the protein. This residue was shown to be an essential part of the binding site, common to protons and substrate. EmrE bearing a single carboxylic residue, Glu-14, shows uptake and binding properties similar to those of the wild type. This suggests that a small protein bearing only 110 amino acids with a single carboxyl in position 14 is the most basic structure that shows ion-coupled transport activity. The role of Glu-14 in substrate binding was examined by using dicyclohexylcarbodiimide, a hydrophobic carbodiimide that is known to react with carboxyls. Tetraphenylphosphonium binding to both wild type and the single carboxyl mutant is inhibited by dicyclohexylcarbodiimide in a dose-dependent manner. Ethidium and other substrates of EmrE prevent this inhibition with an order of potency in accord with their apparent affinities. This suggests that dicyclohexylcarbodiimide binding is sterically prevented by the substrate, supporting the contention that Glu-14, the reactive residue, is part of the substrate-binding site.     

Molecular analysis of the multidrug transporter, P-glycoprotein

Inherent or acquired resistance of tumor cells to cytotoxic drugs represents a major limitation to the successful chemotherapeutic treatment of cancer. During the past three decades dramatic progress has been made in the understanding of the molecular basis of this phenomenon. Analyses of drug-selected tumor cells which exhibit simultaneous resistance to structurally unrelated anti-cancer drugs have led to the discovery of the human MDR1 gene product, P-glycoprotein, as one of the mechanisms responsible for multidrug resistance. Overexpression of this 170 kDa N-glycosylated plasma membrane protein in mammalian cells has been associated with ATP-dependent reduced drug accumulation, suggesting that P-glycoprotein may act as an energy-dependent drug efflux pump. P-glycoprotein consists of two highly homologous halves each of which contains a transmembrane domain and an ATP binding fold. This overall architecture is characteristic for members of the ATP-binding cassette or ABC superfamily of transporters. Cell biological, molecular genetic and biochemical approaches have been used for structure-function studies of P-glycoprotein and analysis of its mechanism of action. This review summarizes the current status of knowledge on the domain organization, topology and higher order structure of P-glycoprotein, the location of drug- and ATP binding sites within P-glycoprotein, its ATPase and drug transport activities, its possible functions as an ion channel, ATP channel and lipid transporter, its potential role in cholesterol biosynthesis, and the effects of phosphorylation on P-glycoprotein activity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Combined localization and real-time functional studies using a GFP-tagged ABCG2 multidrug transporter

ABCG2 is a half-transporter which causes multidrug resistance when overexpressed in tumor cells. Availability of combined localization and functional assays would greatly improve cell biology and drug modulation studies for this transporter. Here we demonstrate that an N-terminally GFP-tagged version of the protein (GFP-G2) can be used to directly monitor ABCG2 expression, dimerization, localization and function in living cells. GFP-G2 is fully functional when tested for drug-stimulated ATPase activity, vesicular transport assay, subcellular localization or cell surface epitope conformational changes. By measuring both GFP and Hoechst 33342 dye fluorescence in HEK-293 cells, we provide evidence that a real-time transport assay can be reliably applied to identify ABCG2 substrates, transport modulators, as well as to monitor the cellular functions of this multidrug transporter protein. This approach also avoids the need of cloning, drug selection or other further separation or characterization of the transgene-expressing cells.     

Three-dimensional structure of the bacterial multidrug transporter EmrE shows it is an asymmetric homodimer

The small multidrug resistance family of transporters is widespread in bacteria and is responsible for bacterial resistance to toxic aromatic cations by proton-linked efflux. We have determined the three-dimensional (3D) structure of the Escherichia coli multidrug transporter EmrE by electron cryomicroscopy of 2D crystals, including data to 7.0 A resolution. The structure of EmrE consists of a bundle of eight transmembrane alpha-helices with one substrate molecule bound near the centre. The substrate binding chamber is formed from six helices and is accessible both from the aqueous phase and laterally from the lipid bilayer. The most remarkable feature of the structure of EmrE is that it is an asymmetric homodimer. The possible arrangement of the two polypeptides in the EmrE dimer is discussed based on the 3D density map.     

Small multidrug resistance proteins: a multidrug transporter family that continues to grow

The small multidrug resistance (SMR) protein family is a bacterial multidrug transporter family. As suggested by their title, SMR proteins are composed of four transmembrane alpha-helices of approximately 100-140 amino acids in length. Since their designation as a family, many homologues have been identified and characterized both structurally and functionally. In this review the topology, structure, drug resistance, drug binding, and transport mechanisms of the entire SMR protein family are examined. Additionally, updated bioinformatic analysis of predicted and characterized SMR protein family members was also conducted. Based on SMR sequence alignments and phylogenetic analysis of current members, we propose that this small multidrug resistance transporter family should be expanded into three subclasses: (i) the small multidrug pumps (SMP), (ii) suppressor of groEL mutation proteins (SUG), and a third group (iii) paired small multidrug resistance proteins (PSMR). The roles of these three SMR subclasses are examined, and the well-characterized members, such as Escherichia coli EmrE and SugE, are described in terms of their function and structural organization.     

Small multidrug resistance proteins: A multidrug transporter family that continues to grow

The small multidrug resistance (SMR) protein family is a bacterial multidrug transporter family. As suggested by their title, SMR proteins are composed of four transmembrane α-helices of approximately 100-140 amino acids in length. Since their designation as a family, many homologues have been identified and characterized both structurally and functionally. In this review the topology, structure, drug resistance, drug binding, and transport mechanisms of the entire SMR protein family are examined. Additionally, updated bioinformatic analysis of predicted and characterized SMR protein family members was also conducted. Based on SMR sequence alignments and phylogenetic analysis of current members, we propose that this small multidrug resistance transporter family should be expanded into three subclasses: (i) the small multidrug pumps (SMP), (ii) suppressor of groEL mutation proteins (SUG), and a third group (iii) paired small multidrug resistance proteins (PSMR). The roles of these three SMR subclasses are examined, and the well-characterized members, such as Escherichia coli EmrE and SugE, are described in terms of their function and structural organization.     

Fungi short-chain carboxylate transporter: shift from microbe hereditary functional component to metabolic engineering target

Applied Microbiology and Biotechnology - Short-chain carboxylic acids and their derivatives are widely utilized in all aspects of our daily life. Given their specific functional groups, these...     

Anti-parallel membrane topology of a homo-dimeric multidrug transporter, EmrE

EmrE in Escherichia coli belongs to the small multidrug resistance (SMR) transporter family. It functions as a homo-dimer, but the orientation of the two monomers in the membrane (membrane topology) is under debate. We expressed various single-cysteine EmrE mutants in E. coli cells lacking a major efflux transporter. Efflux from cells expressing the P55C or T56C mutant was blocked by the external application of membrane-impermeable SH-reagents. This is difficult to explain by the parallel topology configuration, because Pro55 and Thr56 are considered to be located in the cytoplasm. From both the periplasm and the cytoplasm, biotin-PE-maleimide, a bulky membrane-impermeable SH-reagent, could access the cysteine residue at the 25th position in the presence of transport substrates and at the 108th position. These observations support the anti-parallel topology in the membrane.     

New insights into the structure and oligomeric state of the bacterial multidrug transporter EmrE: an unusual asymmetric homo-dimer

EmrE is a small multidrug transporter that contains 110 amino acid residues that form four transmembrane alpha-helices. The three-dimensional structure of EmrE has been determined from two-dimensional crystals by electron cryo-microscopy. EmrE is an asymmetric homo-dimer with one substrate molecule bound in a chamber accessible laterally from one leaflet of the lipid bilayer. Evidence from substrate binding analyses and analytical ultracentrifugation of detergent-solubilised EmrE shows that the minimum functional unit for substrate binding is a dimer. However, it is possible that EmrE exists as a tetramer in vivo and plausible models are suggested based upon analyses of two-dimensional crystals.