From the Departamento de Analises Clinicas e
Toxicologicas, Faculdade de Farmacia and Departemento de
Microbiologia/Instituto de Microbiologia Profesor Paulo de Góes,
Universidade Federal do Rio de Janeiro, CEP 21949-900, Rio
de Janeiro, Brazil, the § Institut Curie, Section Recherche,
Unité Mixte de Recherche-CNRS 168 et Laboratoire de Recherche
Correspondant-Commissariat à l'Energie 34V, 11 Rue Pierre
et Marie Curie, 75231 Paris Cedex 05, France, and the
Unité de Biochimie Physiologique, Université
Catholique de Louvain, Place Croix du Sud 2-20,
B-1348 Louvain-la-Neuve, Belgium
Received for publication, December 2, 2002, and in revised form, January 27, 2003
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ABSTRACT |
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Pdr5p, the major multidrug exporter in
Saccharomyces cerevisiae, is a member of the ATP-binding
cassette (ABC) superfamily. Pdr5p shares similar mechanisms of
substrate recognition and transport with the human MDR1-Pgp, despite an
inverted topology of transmembrane and ATP-binding domains. The
hexahistidine-tagged Pdr5p multidrug transporter was highly
overexpressed in yeast strains where other ABC genes have been deleted.
After solubilization and purification, the 160-kDa recombinant Pdr5p
has been reconstituted into a lipid bilayer. Controlled detergent
removal from Pdr5p-lipid-detergent micelles allowed the production of
peculiar square-shaped particles coexisting with liposomes and
proteoliposomes. These particles having 11 nm in side were well suited
for single particle analysis by electron microscopy. From such
analysis, a computed volume has been determined at 25-Å resolution,
giving insight into the structural organization of Pdr5p. Comparison
with the reported structures of different bacterial ABC transporters
was consistent with a dimeric organization of Pdr5p in the square
particles. Each monomer was composed of three subregions corresponding
to a membrane region of about 50 Å in height that joins two well separated protruding stalks of about 40 Å in height, ending each one
with a cytoplasmic nucleotide-binding domain (NBD) lobe of about 50-60
Å in diameter. The three-dimensional reconstruction of Pdr5p revealed
a close arrangement and a structural asymmetric organization of the two
NBDs that appeared oriented perpendicularly within a monomer. The
existence of different angular positions of the NBDs, with respect to
the stalks, suggest rotational movements during the catalytic cycle.
Drug resistance is a crucial clinical problem in the treatment of
human cancers and infection of bacterial or fungal origin (1-5). The
most important resistance mechanism, ubiquitous from bacteria to man,
which leads to multidrug resistance
(MDR)1 is the overexpression
of membrane-associated transporters that extrude drugs out of the cell.
The best characterized and the clinically most important MDR
transporters are members of the ATP-binding cassette (ABC) superfamily
such as the human P-glycoprotein (Pgp) and the MDR-associated proteins
(6-9).
The identification of yeast genes sharing homology with the
mammalian drug resistance genes provided interesting possibilities for
genetic and molecular manipulations (10, 11). The yeast Saccharomyces cerevisiae genome project revealed the
existence of 31 distinct genes encoding ABC proteins, several of which
are implicated in multidrug resistance (5, 12, 13). In the yeast
S. cerevisiae, a phenotype resembling the mammalian
multidrug resistance phenotype and known as pleiotropic drug resistance (PDR), confers resistance to most currently available classes of
clinically and agriculturally important fungicides and also to many
antibiotics and herbicides (14-19). The first and, by now, the best
characterized yeast PDR transporter is the PDR 5 gene product. The protein Pdr5p has been shown to share nucleotide triphosphatases activities, as well as substrates and modulators, with
the human MDR1-Pgp (13, 17, 18, 20-23). The predicted topography of
Pdr5p comprises two hydrophobic domains, each composed of six
trans-membrane segments (TMS6), and two cytoplasmic
nucleotide-binding domains (NBD), corresponding to a named
(TMS6-NBD)2 "full transporter" (24, 25).
However, the disposition of the two hydrophobic domains and of the two
hydrophilic NBDs of Pdr5p mirrors that of the major eucaryotic ABC
proteins. Each half-Pdr5p starts with an NH2-terminal NBD
followed by the first TMS6 tract, whereas in Pgp, the
TMS6 tracts precede the nucleotide binding domains. Thus,
despite similar mechanisms of substrate recognition and transport, the
significance of such domain inversion in yeast ABC transporters is
unknown. This is mainly related to the lack of structural information
on yeast ABC transporters as compared with mammalian
full-transporters (26-30) or bacterial "half-transporters" consisting of one TMS6 domain connected to one NBD and
assembled as a (TMS6-NBD)2 homodimer
(31-33).
Here, we report the first three-dimensional reconstruction of an
ABC transporter from S. cerevisiae. The overexpression of Pdr5p in the yeast pdr1-3 mutant allowed the production of high amounts of the transporter (12, 16). The overexpressed His-tagged Pdr5p
plasma membrane protein of 160 kDa was solubilized and purified through
Ni-NTA chromatography. Controlled detergent removal from a
lipid-detergent-Pdr5p micellar solution led to the production of
square-shaped particles of about 11 nm in side that have been analyzed
by electron microscopy and single particle techniques. A
three-dimensional structure of Pdr5p has been computed at 25-Å resolution from negatively stained lipid-reconstituted samples. This
volume showed a dimeric organization of Pdr5p, in which each monomer
protruded out of the membrane through two stalks ending each one by a
cytoplasmic NBD lobe. Comparison with the reported structures of
bacterial ABC transporters (31, 32) showed a common cone-shaped
organization that leaves a large chamber between the two stalks bearing
the NBDs. The present work also revealed that the two NBDs are
spatially close and asymmetrically organized, appearing oriented
perpendicularly in the Pdr5p monomer. Such an observation supports the
hypothesis of a functional asymmetry of ABC transporters (34, 35) and
suggests catalytic mechanisms in which the NBDs can move around the
major axe of the stalk.
Materials--
Phospholipids of the highest purity were
purchased from Avantis Polar Lipids,
n-dodecyl- Protein Overexpression--
Overexpression of PDR5-6HISp has
been achieved by integration of the PDR5-6HIS allele at the PDR5 locus
in the AD12345678 strain (pdr1-3, yor1D::hisG,
snq2D::hisG, pdr5D::hisG, pdr10D::hisG, pdr11D::hisG, ycf1D::hisG, pdr15D::hisG,
pdr3D::hisG, ura3
The PDR5-6HIS allele was constructed by PCR retrieval on the
PDR3.3deltaK plasmid of 615 bp of the PDR5 gene spanning from the
internal MluI site (+3939/ATG) to the last codon before the STOP, using the primer sequences 5'-GAACGCGTCTGCAGCTGGCCAGT and 5'-CGCCAGGTTAACTTAGTGATGGTGATGGTGATGTTTCTTGGAGAGTTTACCGTTCTTTTT, to which the restriction site AvrII, the STOP codon,
and the His6-tag had been included. The
MluI/AvrII PCR fragment was used for replacement of the original 807-bp MluI/AvrII fragment of
PDR5. This replacement resulted in the loss of PDR5 terminator from
position +4531(STOP) to 4745 and yielded pAD-YE/PDR5-6HIS in which the
His6-tag substituted the last 3B4-terminal PDR5 codon. The
6.8-kb KpnI/BamHI fragment containing the
PDR5-6HIS allele was cloned into pSK to give pAD-SK/PDR5-6HIS. The
AD12345678 strain was transformed with the
KpnI/SpeI-cleaved pAD-SK/PDR5-6HIS plasmid.
Yeast transformants were selected onto 0.05 µg/ml cycloheximide, and
recombination in the yeast genome of the PDR5-6HIS allele was checked
by PCR.
Protein Purification--
The recombinant strains were grown
overnight at 30 °C in 5.8% glucose, 2% yeast extract, pH 5.2, until late exponential phase (300 million cells/ml). Enriched plasma
membrane fraction (including the acidic precipitation of contaminating
mitochondrial membranes) were prepared as described in Goffeau and
Dufour (37) and stored before use at Protein Reconstitution--
Purified His-Pdr5p was mixed with
phospholipids in the same buffer used for protein purification.
Different lipidic compositions, including
L- Electron Microscopy--
Structures produced during protein
reconstitution experiments were analyzed by negative staining using 1%
uranyl acetate, after sample adsorption onto glow-discharged 300-mesh
carbon-coated grids. For three-dimensional reconstruction, untilted
electron micrographs and tilted pairs (0 and 45°) were recorded onto
a Philips CM120 electron microscope at an accelerating voltage of 120 kV and nominal magnification of ×35,000, using a low dose system and
Image Analysis--
A total number of 2 × 910 projections
from tilted pairs and 767 from untilted images were windowed and
centered by using X-MIPP software (40) and PSPC algorithm (41) before
classification. Self-organizing mapping based on the Kohonen neural
network (42) was performed over untilted images of tilted pairs.
Multivariate statistical analysis (43, 44) of dictionary vectors was
used to perform the map segmentation and to identify the homogeneous groups of projections. The average image of each group was computed and
a rotational analysis was performed before three-dimensional reconstruction of each homogeneous class. Volume computing, merging, and refinement (45, 46) were performed using SPIDER software (47).
Volume symmetry was analyzed by searching the maximum correlation
coefficient value between the refined volume and itself when rotated at
different angles. Final volume was computed after symmetry applying,
refinement, and filtering at the resolution estimated by the
Fourier ring correlation method (48, 49). Volume rendering was
performed using ETDIPS v2 software (50).
The plasma membrane preparation, from recombinant yeast cells,
contains high amounts of Pdr5p similar to those of Pma1p, the major
constitutive component in wild type yeast plasma membranes in which
Pdr5p is undetectable. It was verified that the Pdr5-H6p-enriched plasma membranes contained a novel UTPase activity (about 0.8 µmol·min
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-D-maltopyranoside (DDM) was from Sigma and Bio-Beads SM2 (20-50 mesh) were from Bio-Rad. Ni-NTA columns
were from Qiagen. All other reagents were of analytical grade.
) described in Decottignies
et al. (36). In this strain, the pdr1-3 gain-of-function mutation activates the PDR5-6HISgene transcription, which is
controlled by its own promotor.
70 °C. Plasma membranes were
solubilized by DDM (protein/detergent ratio = 1.2 w/w) in a 10 mM Tris buffer, pH 7.5, during 15 min at room temperature
under stirring. After centrifugation the solubilized membrane proteins
were loaded onto a Ni-NTA resin column pre-equilibrated in 0.1% DDM,
10 mM Tris, pH 7.5. After 2-h incubation at 4 °C for
His-tagged protein binding, the column was washed extensively with 10 mM imidazole buffer, pH 7.5, 0.1% DDM. Purified protein
was eluted with 250 mM imidazole buffer, pH 7.5, 0.1% DDM.
The purified protein eluted at a concentration of about 0.5 mg of
protein/ml, and samples were stored at
70 °C. The protein content
was assayed according to Bradford (38) using bovine serum albumin as a
standard. Proteins were analyzed on 10% SDS-PAGE gels, according to
the method of Laemmli (39), and the gels were stained with Coomassie
Blue R-250 or silver nitrate. Proteins were transferred onto
nitrocellulose membrane, and Pdr5-6HISp was detected by
chemiluminescence using the INDIA HisProbe-HRP (Pierce). UTPase
activity was measured at pH 6.3 as described in Decottignies et
al. (36).
-dimyristoylphosphatidylcholine,
dioleoylphophatidylcholine, Escherichia coli lipids, and
egg phosphatidylcholine with or without 10% negatively charged
phospholipids were analyzed. The best results were obtained using
L-
-dimyristoylphosphatidylcholine at lipid to
protein ratios ranging from 0.5 to 1 w/w. After 1-h incubation at
25 °C, allowing micellar equilibration, detergent was removed by addition of 5 mg Bio-Beads per 50 µl and aliquots taken as a
function of time for electron microscopy analysis (32).
1 µm as the defocus value. All micrographs were recorded on a
1024 × 1024 pixel Gatan ssCCD camera resulting in a pixel size of 5.78 Å/pixel.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1·mg
1), which was previously
shown to be a specific marker for Pdr5p (12). The solubilization of the
membranes by DDM and the protein purification through a Ni-NTA column
allowed us to obtain samples containing a major polypeptide of
160 kDa (Fig. 1A), which
corresponds to the expected molecular weight of Pdr5-H6p, cleared out
from the major Pma1p contaminant.
View larger version (122K):
[in a new window]
Fig. 1.
Reconstitution of Pdr5p by detergent removal.
A, SDS-PAGE analysis of Pdr5p purification. Lane
a, solubilized membrane fraction ater centrifugation (Coomassie
Blue); lanes b (Comassie Blue) and c (silver
staining), purified Pdr5p after Ni-NTA chromatography. The major band
corresponds to a polypeptide with an apparent molecular mass of
160 kDa. B, electron micrography of a negatively stained
sample taken after detergent removal from lipid-detergent-protein
micellar solutions. Circumferences surround characteristic
square-shaped particles coexisting with liposomes and proteoliposomes.
Scale bar = 50 nm. C, gallery showing some
of the 910 windowed images used for classification. Scale
bar = 20 nm.
When analyzed by electron microscopy, the purified preparation only shows small and large (about 11 × 11 nm) micellar particles with no aggregates observable. In addition, due to the presence of high detergent concentration, negative staining of these preparations produces noisy images, precluding detailed single particle analysis. Addition of phospholipids to the micellar purified protein, followed by the addition of Bio-Beads for detergent removal, led to a partial reconstitution of Pdr5p into proteoliposomes. Interestingly, after 1-h treatment with Bio-Beads, which decreases the detergent concentration to below its critical micellar concentration (data not shown; see Ref. 32), the proteins non-incorporated into proteoliposomes appear as clearly visible square particles having 11 nm in side and suitable for single particle analysis (Fig. 1B). The partial reconstitution of Pdr5p into proteoliposomes is dependent upon the lipid composition and the best results, in terms of the number of individual particles non-incorporated into proteoliposomes, have been obtained with dimyristoylphosphatidylcholine. Longer incubation with Bio-Beads to remove residual detergent leads to the stacking of the square-shaped particles into larger globular lipid-protein aggregates.
A gallery of representative images windowed from negatives is shown
in Fig. 1C. Self-organizing mapping and multivariable statistical analysis on these images result in the identification of
three major classes representing 91% of the total population. The
first class (56% of images) is composed by 4-fold symmetrical projections presenting four circular stain excluding regions, of about
5 nm in diameter, arranged around a central stain-penetrating area
(Fig. 2A). The second class
(20% of images) corresponds to 2-fold rectangular shape views of Pdr5p
having about 11 nm × 13 nm (Fig. 2B). Finally, the
third class (15% of images) is a 4-fold cross pattern presenting four
pear-shaped stain-excluding regions, about 5 × 4 nm, that join at
the center of the particle (Fig. 2C). Comparison of the
non-symmetrical volumes, computed from the corresponding tilted
images belonging to each class,
demonstrates that the three classes
corresponded to different projections of the same object (Table
I). Thus, projections belonging to the second class (Fig.
2B) correspond to a volume perpendicularly oriented
(
90°,
0°) to that computed from the
projections of the first class (Fig. 2A). The volume from
the third class correlates well with an orientation intermediate
between those of the two other classes.
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The existence of two perpendicular volumes allowed us to compute
a merged reconstruction for refinement and symmetry analysis. The
reconstruction obtained after two cycles of refinement from the merged
volume presents a maximum correlation value (0.75), with itself when
rotated
180°,
=
0°,
demonstrating the existence of a 2-fold symmetry. Combination of the
whole image set, including images belonging to the third class, allows
us to compute a final 25-Å resolution three-dimensional
reconstruction after 2-fold symmetry imposition (Fig.
3A). This final volume reveals
the presence of four 140-Å-long components, each one composed of three
domains corresponding, from top to bottom, to a near globular domain
(50-Å diameter and 60-Å height) compatible in dimensions with a NBD,
a 40-Å stalk and, finally, a 50-Å height domain compatible in
dimensions with a lipid bilayer. According to the predicted
(TMS6-NBD)2 structural organization of the
Pdr5p monomer, it is concluded that the final reconstruction represents a dimer of this full ABC transporter with the four globular domains corresponding to the four NBD domains. The membrane domains provide contacts between the four hydrophobic components. As these contacts are
not equivalent, the interpretation of a dimeric structure, in which the
two dimers are separated by the region of lowest stain penetration, is
favored (see Fig. 3C).
|
To assign in more detail each region of the Pdr5p molecule in the three-dimensional reconstruction obtained in negative staining, it has been compared with our recent cryo-electron microscopy three-dimensional reconstruction structure of the YvcC homodimer from Bacillus subtilis (32) and to the x-ray crystallographic data on the MsbA homodimer from E. coli (31). As depicted in Fig. 3, A-C, the dimensions and shape of the three-dimensional reconstruction of the Pdr5p dimer are similar to a dimer of YvcC homodimers, while the x-ray structure of MsbA matches well into the volume reconstruction of a Pdr5p monomer (see also Ref. 32 for a fit of MsbA into the volume reconstruction of YvcC). This allows us to conclude that these bacterial and Pdr5p have a common structure and that a monomer of the yeast ABC transporter protrudes out of the membrane through two well separated stalks to a height of about 4 nm, each one ending by a cytoplasmic lobe which correlates with a NBD domain. In addition, the three-dimensional reconstructions of Pdr5p and YvcC reveal a close arrangement of the NBDs, appearing slightly disconnected in Pdr5p due to the use of negative staining.
It is also interesting to compare our data with structural features reported from electron microscopy analysis of single molecules and different two-dimensional crystals of hamster and mouse Pgp (26, 27, 30). Single particle analysis, in negative staining, of these detergent-solubilized ABC transporters has been interpreted assuming a monomeric state of these ABC transporters. However, all reported structures approximate a cylinder of about 100 Å in diameter, which in fact corresponds to the size of a Pdr5p dimer or to the size of a dimer of an YvcC homodimer. On the other hand, the size and shape of Pgp monomers, deduced from projection maps of two-dimensional crystals at 25-Å resolution, varied with the origin of Pgp, the method used for the production of two-dimensional crystals as well as with the method of specimen embedding (28, 30). Interestingly, the projection structure of the mouse Pgp was compared with the projection structure deduced from the X-Ray model of MsbA in the "open" conformation, with well separated nucleotide-binding domains as they appear in the three-dimensional crystal (31), or in the "closed" conformation, with the nucleotide-binding domains brought into contact by modifying the MsbA co-ordinate file (30). As the dimensions of the projection of MsbA were roughly 115 × 45 Å in the open conformation and 70 × 50 Å in the manually closed conformation, Lee et al. (30) suggested that the mouse Pgp (68 × 45 Å) crystallized in the lipid bilayer in a closed conformation with the two NBDs and the two stalks in close contact. In contrast, our data on Pdr5p, as well as on YvcC, demonstrate that the NBDs can be in contact, or only slightly disconnected, while the stalks are spatially separated. This infers that the two NBDs can be in contact while preserving an open V-shaped structure of the transporter without the need for large movements of the trans-membrane segments to bring the two stalks in close contact. In this context, it should be stressed that the model proposed by Lee et al. (30) does not take into account that (i) in the reported MsbA open structure, the disordered NBDs are not resolved accurately and are expected to be much closer than 50 Å, and (ii) a projection structure in negative staining does not give any information about the organization of the trans-membrane domains. Accordingly, it was reported that, in frozen-hydrated two-dimensional crystals, the hamster Pgp monomer had an elliptical shape, 91 × 60 Å, larger than the dimensions, 70 × 70 Å, observed in negatively stained two-dimensional crystals (28) and more compatible with the dimensions of MsbA in the open conformation.
Another important feature of the Pdr5p structure is that the orientations of the four NBD lobes in the Pdr5p dimer reconstruction are not the same. As shown by the arrows in Fig. 3A, two contiguous NBD lobes appear to be oriented perpendicularly. This observation correlates with the clear asymmetry of the NBDs observed in the YvcC homodimer (Fig. 3B). Functional asymmetry has been proposed for the NBDs of other transporters including Pgp (51, 52), CFTR (34), and Ste6 (53). Noteworthy, a transport model of a two-cylinder engine has been proposed (35). In such mechanistic model, a (TMS6-NBD)2 structure, corresponding to a full transporter such as Pdr5 or to an homodimer of a half-transporter such as YvcC, would work co-operatively by using each (TMS6-NBD) alternatively to couple drug transport to ATP hydrolysis. The ATP-bound state is thought to be associated with a high affinity drug-binding site, while the ADP-bound state would be associated with a low affinity site and the ADP-Pi-bound state with an occluded drug-binding site. Moreover, differential interaction of nucleotides at the two NBD transporters has been recently described in the cystic fibrosis trans-membrane conductance regulator (54). According to the two-cylinder engine model, the two NBDs are never equivalent and appear asymmetric in the snapshot structure. In this framework, the clear asymmetry and the different orientations of the NBDs observed in the three-dimensional reconstruction of Pdr5 and YvcC suggest mechanisms in which the NBDs can move around the major axes of the stalks during the catalytic cycle. Such conformational changes could be coupled to a rotation of the trans-membrane segments as suggested from hydrogen/deuterium exchange kinetics and limited proteolysis on MDR-associated protein (MRP)-1 (55). Such a hypothetical mechanism would not necessarily imply large movements of the trans-membrane segments going from an open to a closed state bringing the two stalks in close contact.
A last point that deserves some comments is that Pdr5p tends to dimerize upon membrane reconstitution by detergent removal, as already observed for YvcC homodimers (Ref. 32; Fig. 3B). The appearance of dimers may simply reflect favorable protein-protein interactions during reconstitution rather than reflecting a native dimer. However, the possibility of an interaction between the first cytolosic loops (CL1) of two neighboring Pdr5p molecules has been reported recently and could possibly have implications for dimerization of this ABC transporter (56). In addition, a dimeric organization of different ABC transporters in native membranes has been proposed based on radiation inactivation and cross-linking experiments, as well as on the use of fusion proteins or monoclonal antibodies (57-61).
In summary, the present study reports the first three-dimensional
reconstruction, at 25-Å resolution, of Pdr5p, an ABC transporter from
S. cerevisiae. The dimensions and shape of the
full-transporter Pdr5p compare well with the three-dimensional
reconstruction of the B. subtilis half-transporter YvcC (32)
and with the x-ray data on the E. coli half-transporter MsbA
(31). This suggests a common architecture of these ABC transporters,
which could be extended to other MDRs such as the mammalian
P-glycoproteins. The most important features of our three-dimensional
reconstruction are 1) the close arrangement of the two NBDs while the
stalks are spatially separated and 2) the different orientations of the two NBDs. Our data suggest mechanisms involving significant rotational movements of the NBDs during the catalytic cycle. Three-dimensional reconstructions in the presence of different substrates and/or inhibitors may provide more detailed understanding of the
conformational changes associated with the pumping mechanisms of ABC transporters.
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FOOTNOTES |
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* This work was supported by grants from the CNRS (France) and Conselho Nacional de Desenvolvimiento Científico e Tecnológico (Brazil) (to A. P. and J.-L. R.) and by grants from the CNRS (ACI post-génomique), the European Community (HPRN-CT-2002-00269), and the Interuniversity Pole d'Attraction Programmes of the Belgium government office for scientific, technical, and cultural affairs.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. E-mail: sergio.marco@curie.fr.
** Supported by the Curie Institute (Bourse Mayenz Rotschild) and the Ecole Normale Supérieure (Chaire Internationale de Recherche Blaise Pascal).
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M212198200
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ABBREVIATIONS |
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The abbreviations used are:
MDR, multidrug
resistance;
ABC, ATP-binding cassette;
PDR, pleiotropic drug
resistance;
Pgp, P-glycoprotein;
DDM, n-dodecyl--D-maltoside;
NBD, nucleotide-binding domain;
TMS, trans-membrane segment;
Ni-NTA, nickel-nitrilotriacetic acid.
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