From the Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel
Received for publication, December 6, 2000, and in revised form, January 17, 2001
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ABSTRACT |
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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.
Multidrug transporters recognize a broad range of substrates with
a relatively high affinity and actively remove them from the cytoplasm
(1, 2). In many cases, the substrates are toxic to the cells, and their
removal confers resistance. For example, multidrug transporters are
responsible for resistance of cancer cells and bacteria to
antineoplastic agents and antibiotics, respectively (1, 3,
4).
The SMR are the smallest multidrug transporters known (5, 6).
They are about 100 amino acids long, widespread in the eubacterial
kingdom. EmrE, a member of this family, is a 12-kDa transporter from
Escherichia coli (7). This transporter is unique in terms of
its size and properties and can be easily expressed and purified.
Therefore, the protein can serve as a model system to study ion-coupled
transporters (8).
Hydrophobicity analysis of EmrE predicts four EmrE was shown to be a homo-oligomer by mixing of wild type and
inactive mutants, both in vivo and in vitro, in
which negative dominance has been observed (11). The projection
structure of two-dimension crystals of EmrE revealed a nonsymmetric
dimeric structure as the basic oligomeric unit (12).
The protein is tightly packed, without any continuous aqueous domain
(9, 13). This suggests the existence of a hydrophobic pathway in the
membrane region through which the substrates are translocated.
EmrE contains eight charged residues, seven of them in the hydrophilic
loops, and only one, Glu-14, is embedded in the putative membrane
domain (Fig. 1). Substitution of this
highly conserved residue totally abolishes resistance to EmrE
substrates and dramatically affects transport activity (5, 14-16). The
results support the contention that this residue is an essential part
of the binding domain shared by substrates and protons. The occupancy
of this site is mutually exclusive and provides the basis of the
simplest coupling for two fluxes (17).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical transmembrane
segments. Results from transmission Fourier transform infrared
measurements and high resolution NMR studies agree remarkably well with
this prediction (9, 10).
View larger version (43K):
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Fig. 1.
Model of the secondary structure of
EmrE. The model is based on the hydropathic profile as calculated
according to Engelman et al. (28) and experimental data (9,
10, 13). Putative transmembrane segments are shown in boxes,
connected by hydrophilic segments. Charged residues are shown in
diamonds, the carboxyls in the hydrophilic segments are
highlighted in gray, and the Glu-14
membrane-embedded carboxyl is highlighted in
black.
Carboxylic residues embedded in the membrane were shown to be important
for activity in various ion-coupled transporters (17, 18). In some
cases, these carboxyls are involved in substrate recognition and
binding, and in others, they are part of the coupled ion binding site.
EmrE is unique in that a single carboxyl is involved in recognition of
both substrate and the coupling ion. In this work we provide strong
support for the role of Glu-14; a mutant bearing a single carboxylic
residue (Glu-14) is shown to be active.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains and Plasmids-- E. coli TA15 cells (19) with pT7-7 plasmid (20) containing wild type EmrE-Myc/His and pGP1-2 plasmid are used throughout all the experiments (15). The E25C/D84C mutant, in which Glu-25 and Asp-84 were replaced by Cys, was also constructed into the pT7-7 plasmid with the c-Myc and His tags by using polymerase chain reaction and standard methods of molecular cloning.
Overexpression of EmrE-His--
E. coli TA15 cells
with pT7-7 EmrE-His were grown at 30 °C in minimal medium
supplemented with 0.01% MgSO4, 2.5 µg/ml thiamine, 0.5%
glucose, 50 µg/ml ampicillin, and 50 µg/ml kanamycin, to an
A600 nm of about 0.8. The temperature was then
increased to 42 °C to induce the T7 polymerase; 15 min later the
culture was shifted back to 30 °C for 2 h. Cells were collected
by centrifugation and washed in TSCD buffer (250 mM
sucrose, 150 mM choline chloride, 10 mM
Tris-Cl, pH 7.5, 0.5 mM dithiothreitol, and 2.5 mM MgSO4). At this stage the cell pellets can
be kept at 70 °C until further processing. For preparation of
membrane vesicles, the cells were thawed and resuspended in TSCD buffer
containing DNaseI. The cells were broken up by two passages through a
French Press at 20,000 p.s.i. Undisrupted cells were removed by
centrifugation at 12,000 × g for 5 min at 4 °C. The
supernatant was then pelleted by centrifugation at 311,000 × g for 60 min at 4 °C. The pellet was resuspended in TSCD
buffer and stored at
70 °C.
Purification and Reconstitution of EmrE-His--
EmrE-His
protein was purified using ion chelate chromatography essentially as
described in Muth and Schuldiner (15), except that size exclusion
chromatography was omitted as the last purification step. Membranes
were solubilized in buffer containing 150 mM NaCl, 15 mM Tris-Cl, pH 7.5,1.5% n-dodecyl--maltoside
(DM,1 Anatrace, Inc. Maumee,
OH), 0.5 mM phenylmethylsulfonyl fluoride, and 15 mM
-mercaptoethanol. After a 20-min incubation at
25 °C the extract was centrifuged at 435,000 × g
for 20 min at 4 °C. The supernatant was incubated with the Ni-NTA
beads (Qiagene, GmbH, Hilden, Germany) in the presence of 20 mM imidazole for 1 h at 4 °C and then washed with
buffer containing 150 mM NaCl, 15 mM Tris-Cl,
pH 7.5, 0.08% DM, 15 mM
-mercaptoethanol, and 30 mM imidazole. For reconstitution the beads bound to
EmrE-His (EmrE beads) were washed first in the same buffer, with 0.08% DM and then in buffer containing 1%
n-octyl-
-D-glucopyranoside (Calbiochem-Novabiochem). EmrE-His was eluted from the beads with the
same buffer containing 200 mM imidazole and DM for
purification or n-octyl-
-D-glucopyranoside
for reconstitution.
Reconstitution was performed essentially as previously described for
NhaA (21). Purified EmrE-His was mixed with a solution containing 25 mg/ml E. coli phospholipids (Avanti Inc. Alabaster, AL), 150 mM NaCl, 15 mM Tris-Cl, pH 7.5, 1%
n-octyl--D-glucopyranoside. After sonication
the mixture was diluted into NH4 buffer containing 190 mM NH4Cl, 15 mM Tris-Cl, pH 7, and
1 mM dithiothreitol. For proteoliposome formation the
diluted mixture was incubated for 20 min in 25 °C and then
centrifuged at 257,000 × g for 1 h.
Proteoliposomes were resuspended in NH4 buffer, frozen, and
kept at
70 °C. Before the assay, the proteoliposome suspension was
thawed and sonicated in a bath-type sonicator for a few seconds until clear.
Uptake Assay-- Uptake of [14C]methyl viologen into proteoliposomes was assayed essentially as described in Yerushalmi et al. (7). 3 µl of the ammonium chloride-containing proteoliposomes (about 600 ng of EmrE) were diluted into 200 µl of an ammonium-free solution. The latter contained 37.2 µM [14C]methyl viologen (64-74 nCi/assay), 140 mM KCl, 10 mM Tricine, 5 mM MgCl2, and 10 mM Tris-Cl, pH 8.5. At given times the reaction was stopped by dilution with 2 ml of the same ice-cold solution. The samples were filtered through Millipore filters (0.22 µm) and washed with an additional 2 ml of solution. The radioactivity on the filters was measured by liquid scintillation. In each experiment the values obtained in a control reaction, with 15 µM nigericin, were subtracted from all experimental points. This background was no more than 10% of most experimental values. The kinetics of uptake was measured in duplicate at 25 °C.
Binding Assay-- Tetraphenylphosphonium (TPP+) binding was assayed essentially as described in Muth and Schuldiner (15). EmrE-His membranes were solubilized with 0.8% DM in NH4 buffer (190 mM NH4Cl, 15 mM Tris-Cl, pH 7) at 25 °C for 15 min. Ni-NTA beads were washed twice in distilled H2O and once in NH4 buffer with 0.08% DM (NH4-DM buffer). The beads (10 µl per assay) were bound to EmrE-His protein from solubilized membranes or to purified protein by incubation at 4 °C for 45 min. The unbound material was discarded, and the EmrE-His bound to beads (EmrE beads) was washed with NH4-DM buffer. After two washes with NH4-DM buffer, 200 µl of buffer containing 12.5 nM [3H]TPP+ (5 Ci/mmol, Amersham Pharmacia Biotech) were added, and the samples were incubated for 15 min at 4 °C. In each experiment the values obtained in a control reaction, with 25 µM unlabeled TPP+, were subtracted. The binding reaction was stopped by separating the beads from the supernatant by pulse centrifugation and then removing the supernatant. The bead fraction was then incubated for 10 min at room temperature with 450 µl of NH4-DM buffer containing 150 mM imidazole to release the EmrE-His and [3H]TPP+ from the beads. After spinning down the beads, the [3H]TPP+-associated radioactivity was measured by liquid scintillation. All binding reactions were performed in triplicate.
In experiments that tested the effect of EmrE substrates on [3H]TPP+ binding, the substrates were added with the [3H]TPP+ to the EmrE beads.
For examining the effect of dicyclohexylcarbodiimide (DCCD) on [3H]TPP+ binding, EmrE beads (10 µl) were incubated at 4 °C in a 100-µl suspension of NH4-DM buffer containing different concentrations of DCCD. In some of the experiments EmrE substrates (ethidium bromide, acriflavine, methyl viologen, 1-methyl-4-phenylpyridinium (MPP+), or benzalkonium) were added to the buffer as well as DCCD. [3H]TPP+ binding was assayed on the EmrE beads after the reaction was stopped by the addition of 1.4 ml of NH4-DM buffer and immediate spinning down.
When the effect of pH on TPP+ binding was tested, the solutions contained 140 mM KCl, 10 mM Tricine, 5 mM MgCl2, and 0.08% DM. The different solutions were buffered with 30 mM MES (pH range 6.5-7.5) and Tris-Cl (pH range 7.5-9.5) and titrated with either KOH or HCl. In these experiments the binding buffer contained 6 nM [3H]TPP+ (30 Ci/mmol, Amersham Pharmacia Biotech).
For measurement of the Kd calculations of E25C/D84C
EmrE,1 µg of purified protein was used. TPP+ binding was
measured in a range of concentrations (3-300 nM) essentially as described in Muth and Schuldiner (15).
Kd and Bmax were calculated
from the Scatchard plot.
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RESULTS |
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EmrE with a Single Carboxyl (Glu-14) Displays Properties Similar to
Those of the Wild Type--
In previous studies Glu-14 was shown to be
central for activity. This residue is involved in binding both
substrate and proton but separately in time (15, 16). To further
characterize the importance of Glu-14 for EmrE activity, a single
carboxyl mutant (E25C/D84C) was constructed. This mutant confers
resistance to EmrE substrates, although it is less resistant compared
with the wild type, especially in the presence of methyl viologen.
(16). Because of the uniqueness of such a protein with a single
carboxyl, we tagged it with a His6 tag, purified it, and
characterized its activity. After purification on a Ni-NTA column the
protein was reconstituted with E. coli lipids by detergent
dilution. The proteoliposomes loaded with NH4Cl were
diluted into an ammonium-free buffer to generate a pH gradient (7). The
E25C/D84C mutant shows significant uptake activity (50% of the wild
type activity, Fig. 2A), and transport is inhibited by the ionophore nigericin that prevents generation of a pH gradient (not shown). The pH dependence of the
uptake reaction is practically identical to that of the wild type (not
shown). This demonstrates again that Glu-14 is indeed central for
transport activity whereas the other carboxylic residues are less
important.
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The single carboxyl mutant binds the high affinity substrate TPP+ with properties comparable with those of the wild type protein (Kd = 28 nM, Bmax = 0.18 mol/mol). The affinity of the mutant protein is 3 times lower than that of the wild type (Kd = 10 nM (15)) suggesting that the two hydrophilic carboxyls have only a minor effect on substrate binding. The total number of binding sites determined from the experiment is 0.15-0.2 mol/mol. This value is slightly lower than that of the wild type (0.25-0.3 mol/mol (15)). The difference can result from the fact that the mutant protein was only partially purified, as compared with the wild type EmrE that was purified to homogeneity.
The pH dependence of binding activity of this mutant is practically identical to that of the wild type as well (Fig. 2B). TPP+ binding increases dramatically between pH 6.7 and 7.8 and is maximal above pH 8.3 (50% at pH 7.3). This similarity supports our previous suggestion that Glu-14, and not the other charged residues, is responsible for the pH dependence. In addition, the findings indicate the presence of a residue with an apparent pK of 7.3-7.5 in the binding site of the wild type as well as the mutant.
EmrE substrates inhibit TPP+ binding of the E25C/D84C mutant at the same concentration range as for the wild type (Fig. 2C (15)). Benzalkonium, ethidium, and acriflavine inhibit binding with a relatively high affinity; 10 µM benzalkonium or 100 µM ethidium and acriflavine fully inhibit TPP+ binding. As previously shown for the detergent-solubilized wild type EmrE, the affinity for MPP+ is lower, and even at 10 mM it does not inhibit binding totally, whereas methyl viologen does not inhibit at all, at the concentrations tested.
Single carboxyl EmrE shows uptake and binding properties similar to those of the wild type. This similarity between the E25C/D84C mutant and the wild type protein supports the contention that Glu-14 is the only carboxyl essential for transport activity and for proton and substrate binding.
DCCD Inhibits TPP+ Binding of Wild Type EmrE in a Substrate-protectable Manner-- Characterization of the single carboxyl mutant emphasizes the importance of this residue. The role of Glu-14 in substrate recognition and binding was further examined by chemical modification by carbodiimides. In previous studies we showed that DCCD, a carbodiimide that is known to react with carboxyls in hydrophobic environments, inhibits uptake by wild type EmrE in a dose-dependent manner (16). In addition, it was shown that substitution of each of the two other carboxylic residues, Glu-25 and Asp-84, does not modify the profile of inhibition. Glu-14 is the only carboxylic residue common to the wild type and the two other mutants; therefore it was suggested that this residue is the site of action of DCCD.
In this work the DCCD effect on binding of the high affinity substrate,
TPP+, was tested directly. The binding to wild type EmrE is
inhibited in a time-dependent manner (Fig.
3A). The reaction with the
detergent-solubilized protein can be controlled and manipulated very
easily. To slow down the reaction it was carried out at 4 °C. Under
these conditions 50% of inhibition can be seen after 25 min of
incubation in 4 °C, whereas 2 h are needed for full inhibition
(Fig. 3A). Addition of 150 µM ethidium, one of
the EmrE substrates, during the incubation with DCCD prevents this
inactivation almost completely. Lower ethidium bromide concentration
has only a partial protective effect. Other substrates of EmrE prevent
inhibition by DCCD as well (Fig. 3B). Very low
concentrations of benzalkonium (1 µM) prevent most of the
DCCD effect on binding, whereas ethidium and acriflavine protect
against DCCD inhibition at higher concentrations. MPP+
shows only 50% protection, even at high concentration (20 mM), whereas methyl viologen does not prevent DCCD
inhibition even at concentrations as high as 100 mM. The
effect of various substrates on DCCD prevention of binding is in the
same range and order of potency as the inhibition of TPP+
binding.
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We suggest that substrate protection against the DCCD reaction is most likely due to steric hindrance, and therefore, its target residue is in or near the binding site.
Substrates Prevent Inhibition by DCCD Also in the Single Carboxyl Mutant-- As was shown here the activity of the single carboxyl mutant E25C/D84C is comparable with that of the wild type EmrE. In addition, the only site of action for DCCD in this mutant is the single carboxylic residue, Glu-14. Therefore, this mutant can be used for further examining the role of Glu-14 in substrate recognition.
DCCD inhibits also TPP+ binding to E25C/D84C EmrE in a time-dependent manner (Fig. 3C). As previously shown for the single replacement D84C (16), this mutant is also more susceptible to DCCD than the wild type. After less than 5 min of incubation with 500 µM DCCD, 50% of the binding is inhibited, whereas 25 min are needed for the same inhibition of the wild type. Substitution of the carboxylic residues in the hydrophilic loops (Glu-25 and Asp-84) seems to increase DCCD accessibility to the protein and therefore increases the sensitivity. Ethidium bromide (150 µM) prevents most of the inhibition by DCCD. Other substrates of EmrE protect against the DCCD effect in a dose-dependent manner (Fig. 3D). As described above for the wild type, the substrates with the higher affinity are benzalkonium, ethidium bromide, and acriflavine that react in the micromolar range; MPP+ affinity is lower (milimolar range), and methyl viologen does not show any protection at all. Most of the substrates more efficiently prevent the inhibition of binding by DCCD in the wild type compared with the single carboxyl mutant. For example 100 µM acriflavine prevents the DCCD effect totally in the wild type, whereas in the mutant it prevents only 60% of the DCCD effect. This is in line with the observed higher rate of inhibition by DCCD in the mutant that decreases the efficiency of the protection.
The protection against the DCCD effect by substrate in the single
carboxyl mutant strengthens again the suggestion that Glu-14 is an
essential part of the substrate-binding site. Binding of substrate to
this residue decreases the accessibility of DCCD to this site and
therefore prevents its action. The substrate specificity and the
concentration range for inhibition of TPP+ binding and for
protection against the DCCD effect is similar for the single carboxyl
mutant. This confirms the above contention that the inhibition of the
DCCD reaction with Glu-14 by substrates is most likely due to a steric effect.
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DISCUSSION |
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EmrE has eight charged residues, three of which are carboxyls (Glu-14, Glu-25, and Asp-84, Fig. 1). Glu-14 is strictly conserved in more than 50 homologues of EmrE and is the only charged residue essential for transport (16). Replacement with an Asp has a profound effect on the behavior of the protein, because in the mutated protein the Asp residue has a much lower pK than the corresponding Glu (15). Our results support the notion that Glu-14 is an essential part of the binding domain shared by substrates and protons but mutually exclusive in time (17).
DCCD, a carbodiimide that was shown to react specifically with Glu-14, was previously shown to inhibit uptake of methyl viologen by proteoliposomes (16). In this work we tested the effect of DCCD directly on binding of the high affinity substrate, TPP+, to the detergent-solubilized transporter. The detergent-solubilized protein maintains its ability to recognize most substrates at high affinity; the pK of Glu-14 is not modified, and the sensitivity to DCCD is conserved. The inhibition of transport by DCCD is observed at lower concentrations, but this may reflect the partition of this hydrophobic compound in the membrane, reaching an effective concentration much higher than in the bulk. The binding assay allows for a very simple termination of the reaction by dilution and washing as opposed to the proteoliposomes system where the DCCD partitions into the lipid phase and is not removed. DCCD inhibits binding of TPP+ to EmrE in a time-dependent manner, and this is prevented by the presence of EmrE substrates (benzalkonium, ethidium, acriflavine, and partially by MPP+). The different substrates inhibit TPP+ binding and the DCCD effect in the same range of concentrations, indicating that the binding affinity of each substrate is comparable with its potency to prevent the DCCD effect on binding. Our explanation for this effect is that binding of substrate limits the accessibility of DCCD to its site of action, Glu-14, suggesting that this residue is part of the substrate binding site.
In the single carboxyl mutant (E25C/D84C) the substrate effect on inhibition by DCCD is comparable with what was shown for the wild type. In this mutant, the carboxylic residue Glu-14 is the only site of action for DCCD. Therefore, preventing this reaction between DCCD and Glu-14 by substrates strongly strengthens the role of Glu-14 in the substrate-binding site.
Carboxylic residues embedded in the transmembrane region were shown to be part of the substrate- or coupling ion-binding site in a range of larger transporters that are more complex than EmrE (reviewed in Refs. 17 and 18)). In many ion-coupled transporters this carboxyl is found in the first transmembrane segment. This is the case for MdfA, another E. coli multidrug transporter (22), the Tet transporter Tet(B) (23, 24), and eukaryotic systems as well. In the vesicular and plasma membrane monoamine transporters, an Asp in transmembrane segment 1 is important for substrate recognition (25-27).
EmrE and other transporters in the SMR family are the smallest
ion-coupled transporters known (5, 6). This means that a simple
structure of 110 amino acids, organized as an oligomer, is capable of
transporting a wide variety of substrates. In this work, we show that
even a simpler structure, which includes only one carboxylic residue,
is active. E. coli cells expressing the single carboxyl
mutant, E25C/D84C, show significant resistance to EmrE substrates (16).
The purified protein reconstituted in proteoliposomes catalyzes
pH-driven transport of [14C]methyl viologen with
properties similar to those of the wild type protein. In addition, the
affinity of binding as well as the number of binding sites is
comparable with that of the wild type protein.
Both wild type and mutant show similar pH dependence of TPP+ binding. We have previously shown that this pH dependence reflects the fact that binding and release of the substrate occur only upon the corresponding release and binding of protons (15).
It is quite surprising that in such a small protein, with only eight charged residues, removal of two negative charges has relatively little effect on activity, implying that the overall structure must be maintained. In addition, the negative charges in the loop seem to play a minor role in "guiding" the positively charged substrates, because the affinity of TPP+ is not dramatically altered in the single carboxyl mutant. The results imply also very little, if any, effect of these two residues on insertion into the membrane and on protein stability. The single carboxyl mutant has four net positive charges. We therefore speculate that to prevent electrostatic repulsion with the cationic substrates most of the positive charges in the protein may be neutralized by ion pairing to species such as phospholipids for example.
The simplicity and size of EmrE allow for a detailed dissection of the
role of Glu-14. The construction of an active transporter that has a
single carboxyl residue at position 14 provides even stronger support
for the previous conclusions. This mutant also gives us the opportunity
to try to find the most basic structure that shows transport activity.
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ACKNOWLEDGEMENT |
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We thank Y. Mordoch for performing the binding experiments.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche-Israeli Program (Federal Ministry of Education, Science and Research-BMBF-International Bureau at the German Aerospcace Center Technology), Grant NS16708 from the National Institutes of Health, and Grant 463/00 from the Israel Science Foundation.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. Tel.: 972-2-6585992;
Fax: 972-2-5634625; E-mail: shimons@leonardo.ls.huji.ac.il.
Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M010979200
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ABBREVIATIONS |
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The abbreviations used are:
DM, n-dodecyl--maltoside;
TPP+, tetraphenylphosphonium;
MPP+, 1-methyl-4-phenylpyridinium;
DCCD, N,N'-dicyclohexylcarbodiimide;
EmrE-His, EmrE fused to Myc/His epitope;
Ni-NTA, nickel-nitrilotriacetic acid;
MES, 4-morpholineethanesulfonic acid.
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