(Received for publication, December 9, 1994)
From the
The smallest membrane protein shown to catalyze ion-coupled
transport is documented in this report. A gene coding for a small
110-amino acid membrane protein (emrE or mvrC) has
been previously identified and cloned and shown to render Escherichia coli cells resistant to methyl viologen and to
ethidium. In this report, it is shown that the resistance is due to
extrusion of the toxic compounds in a process that requires a proton
electrochemical gradient rather than ATP. For this purpose, cells in
which the unc gene was inactivated were used so that the
interconversion between the proton gradient and ATP is not possible,
and the effect of agents, which specifically affect either of them, was
tested on transport of ethidium in the intact cell. In addition, EmrE
has been overexpressed and metabolically labeled with
[S]methionine. Strikingly, the protein can be
quantitatively extracted with a mixture of organic solvents such as
chloroform:methanol and is practically pure after this extraction.
Moreover, after addition of E. coli lipids to the
chloroform:methanol extract, EmrE has been reconstituted in
proteoliposomes loaded with ammonium chloride. Upon dilution of the
proteoliposomes in ammonium-free medium, a pH gradient was formed that
drove transport of ethidium and methyl viologen into the
proteoliposome. Both substrates compete with each other and exchange
with previously transported solute. EmrE is a multidrug transporter of
a novel type, and, because of its size and its solubility properties,
it provides a unique model to study structure-function aspects of
transport reactions in ion-coupled processes.
Toxic compounds are part of the natural environment in which living cells dwell, and development of strategies for life in this environment is a necessary trait for survival. Resistance to a wide range of cytotoxic compounds is a common phenomenon observed in many organisms throughout the evolutionary scale. Removal of toxic substances by transport is one of the strategies that have evolved. Multidrug transporters can actively remove a wide variety of substrates in an energy-dependent process and decrease the concentration of the offending compounds near their target. The proteins responsible for performing this task have been found in many organisms from bacteria to man. From analysis of their structure and properties, three families can be distinguished. One of them (ABC type) is best known for the P-glycoprotein in its various forms, which confers multidrug resistance to cancerous cells (Doige and Ames, 1993; Endicott and Ling, 1989; Gottesman and Pastan, 1993; Gros et al., 1986; Higgins, 1992). This family includes also many bacterial transport proteins. The ABC transporters utilize ATP to actively transport a wide variety of compounds. In some of the bacterial transporters, several subunits are required for activity, but in the P-glycoprotein and many others, one large polypeptide seems to be sufficient to perform all of the functions. The other family (TEXANs) includes proteins that render fungi and bacteria resistant to antibiotic treatment (Lewis, 1994; Neyfakh et al., 1991; Nikaido, 1994; Paulsen and Skurray, 1993). The TEXANs are antiporters, 400-500 amino acids long, which exchange various drugs with one or more protons and utilize in this way the proton electrochemical gradient to actively transport a variety of substrates. Proteins in mammals, located in intracellular organelles, which usually transport neurotransmitters such as dopamine, adrenaline, serotonin, and acetylcholine, also behave as multidrug transporters and belong to the TEXAN family (Schuldiner, 1994; Schuldiner et al., 1995).
Very little attention has been paid to bacterial proteins of the third family (MiniTEXANs) (Grinius et al., 1992). They are very small, about 100 amino acids long, and, by analogy to the other known systems, it has been hypothesized that they confer resistance to several antibiotics presumably also by active extrusion. Being the smallest putative ion-coupled transporters known, they roused our attention since they could provide a simple model to study.
A variety of genes coding for MiniTEXANs has been identified in several bacteria based on their ability to confer resistance to several drugs (Grinius et al., 1992). One of these genes, emrE or mvrC, is an Escherichia coli gene, which has been identified and cloned on the basis of its ability to confer resistance to ethidium (Purewal, 1991) and to methyl viologen (paraquat) (Morimyo et al., 1992). Previous studies have suggested the existence of an efflux system for toxic cationic compounds in E. coli (Midgley, 1987), and, because of the hydrophobic nature of the predicted polypeptide, it was suggested that EmrE is indeed an efflux system (Purewal, 1991). However, no direct evidence for this suggestion has yet been presented, and the nature of the energy required for extrusion has not been studied.
The predicted sequence of EmrE suggests that
it is a highly hydrophobic 12-kDa protein (Fig. 1). A
hydropathic analysis of the sequence reveals the presence of four
putative transmembrane segments with only one charged residue (Glu) in
the putative transmembrane domain and a total of eight charged amino
acids throughout the protein. A protein similar in size and number of
charges, which has been studied in detail by many groups, is subunit c,
a small membrane-bound subunit of the H-ATPase
(Fillingame, 1992). Subunit c is also known as the proteolipid because
of its solubility in organic solvents. Since subunit c is purified by
extraction in a mixture of chloroform and methanol, it was tempting to
test a similar extraction procedure for EmrE.
Figure 1: Model of the secondary structure of EmrE. The model shown is based on the predictions of the hydropathic profile as calculated according to Engelman et al.(1986). Putative transmembrane segments are shown in boxes connected by hydrophilic segments. The charged residues are shaded.
In this report, we
describe studies in whole cells that provide evidence that EmrE is an
antiporter that exchanges ethidium, methyl viologen, and a series of
other quaternary compounds with one or more protons. In addition, EmrE
displays unique properties that make it soluble in a mixture of organic
solvents such as chloroform:methanol. EmrE was overproduced so that it
was practically the only protein extractable in the above solvent
mixture. The protein thus purified was reconstituted in
proteoliposomes, and it catalyzed pH-driven uptake of ethidium and
[
C]methyl viologen. Uptake of both substrates
was inhibited by a series of toxic cations, and preaccumulated solute
exchanged with unlabeled excess substrate.
emrE was cloned by polymerase chain reaction using primers based on the published sequence of the gene (Morimyo et al., 1992; Purewal, 1991). The sense primer (CGGAATTCATATGAACCCTTATATTTATCTTG) overlapped with the first ATG in the coding area of the gene and included two restriction sites for cloning (NdeI and EcoRI). The reverse primer (CCGAATTCAAGCTTAATGTGGTGTGCTTCGTGAC) overlapped with the first termination codon after the open reading frame and included sites for the restriction enzymes HindIII and EcoRI. The polymerase chain reaction was performed using genomic DNA from E. coli EP432 (Pinner et al., 1992), a K12 derivative, as a template. The reaction yielded a 333-base pair segment that was purified, digested with the proper enzymes, and cloned into both pT7-7 (Tabor and Richardson, 1985) predigested with NdeI and HindIII and into pKK223-3 (Pharmacia Biotech Inc.) predigested with EcoRI and HindIII. The plasmids obtained were named pT7-32 and pKK56, respectively.
For overexpression, E. coli JM109/pKK56 was grown in minimal medium A with 0.5% glycerol,
thiamine, and ampicillin as above. When the culture reached an A = 0.5,
isopropyl-1-thio-
-D-galactopyranoside was added to 0.5
mM; 2 h later, the cells were chilled on ice and harvested by
centrifugation. Membranes were prepared by disrupting the cells using a
French pressure procedure (Rosen, 1986), except that the buffer used
was 10 mM Tris-Cl, pH 7.5, 250 mM sucrose, 150 mM choline chloride, 0.5 mM dithiothreitol, 2.5 mM MgSO
, and 15 µg/ml DNaseI. After
ultracentrifugation, membranes were resuspended at 10 mg of protein/ml,
frozen in liquid air, and stored at -70 °C.
For reconstitution, the volumes were increased (600 µl of
membranes and 5 mg of protein/ml were extracted with 4.5 ml of organic
solvent), and E. coli phospholipids (160 µl of 50 mg/ml)
were added prior to extraction. The dried extract was resuspended in a
solution containing 0.15 M NHCl, 0.015 M Tris-Cl, pH 7.5, 1 mM dithiothreitol, and 1 µg of
plasmid DNA. The suspension was frozen in liquid air and kept at
-70 °C. Before the assay, the proteoliposome suspension was
thawed at 37 °C and sonicated in a bath-type sonicator for a few
seconds until clear. In proteoliposomes prepared for assay of
[
C]methyl viologen, no DNA was added. Control
experiments showed no effect of DNA on transport of the radioactive
label.
Transport of [C]methyl viologen into
proteoliposomes was assayed by dilution of 3 µl of the ammonium
chloride containing proteoliposomes into 200 µl of an ammonium-free
medium containing 42 µM [
C]methyl
viologen (77 nCi), 140 mM KCl, 10 mM tricine, and 5
mM MgCl
(pH 8). At given times, the reaction was
stopped by dilution with 2 ml of the same ice-cold solution, filtered
through Schleicher and Schull filters (0.2 µm), and washed with an
additional 2-ml solution. The radioactivity on the filters was
estimated by liquid scintillation.
Transport of ethidium into proteoliposomes was assayed by dilution of 10-15 µl of proteoliposomes to 2.5 ml of a medium as above except that ethidium (1 µg/ml) replaced the methyl viologen. Fluorescence was measured in a Perkin-Elmer fluorimeter (Luminescence Spectrometer LS-5) with exciting light at 545 nm and emission at 610 nm.
Protein determination was according to Bradford(1976).
Phospholipids were prepared from E. coli as described (Viitanen, et al., 1986).
Figure 2:
Energy-dependent transport of ethidium in
whole cells. E. coli JM109/pT7-7 and E. coli JM109/pT7-32 grown in LB medium to A = 0.4 were collected by centrifugation and resuspended in
minimal salt medium as described under ``Experimental
Procedures.'' A, cells treated with CCCP (40
µM) prior to the addition of ethidium (1 µg/ml). If
CCCP was omitted, no time-dependent increase in fluorescence was
observed. B, cells incubated for 60 min at 37 °C with CCCP
and ethidium as in A were centrifuged and quickly resuspended
in medium containing the same ethidium concentration with (+CCCP) or without CCCP. C and D, E. coli TA15
unc/pT7-32 were treated as in A except that the following additions were made: in C, no additions or 10 mM KCN (+KCN); in D, 40 µM CCCP (+CCCP) or 12.5
mM arsenate (+ARS) and then 40 µM CCCP.
It is difficult to identify the driving force for active
transport in a strain possessing the H-ATPase since
the main forms of cell energy (oxidation energy,
, and ATP) can be interconverted by
this enzyme. To demonstrate the nature of the energy source required
for ethidium extrusion, we used a strain carrying a deletion in the unc operon so that the F
-F
H
-ATPase was inactive, and it was then possible
to manipulate independently both
and
ATP. In these experiments, the effect of various agents on ethidium
entry into the cell was tested. As in Fig. 2A, ethidium
entry is due to failure of the extrusion machinery to remove it from
the cell. The results of such an experiment are summarized in Fig. 2, C and D. Addition of CCCP and cyanide,
two agents that inhibit
but did not
affect ATP levels in this strain under these conditions (data not
shown), induced ethidium entry into the cell as judged from the
increase in its fluorescence. On the other hand, arsenate, an agent
that decreased dramatically the ATP level (data not shown) without any
effect on
, had no effect on ethidium
uptake either. The data presented strongly supported the contention
that EmrE is an H
-ethidium antiporter, which utilizes
the
generated by the bacterial
primary pumps to extrude toxic cations and reduce in this way their
concentration in the cytoplasm.
Figure 3:
Labeling of EmrE with
[S]methionine and solubilization with
chloroform-methanol. Membranes (15-20 µg of protein) from E. coli JM109 cells carrying pT7-32 (A), pKK56 (B), pT7-7 (C), or pKK223-3 (D)
were solubilized with sample buffer and analyzed by SDS-PAGE. In
parallel, chloroform:methanol extracts from pT7-32 (E)
and pKK56 (F) were analyzed. In lanesG and H, the material shown in lanesA and E was also analyzed for radioactivity by phosphor autoradiography
(Fuji Film Co. Ltd).
As described above, there are some
similarities in the overall properties of EmrE and the subunit c of the
F-F
-H
-ATPase (Fillingame,
1992). Both are small polypeptides (11.9 and 8.2 kDa, respectively)
with a relatively high percentage of hydrophobic amino acids typical of
intrinsic membrane proteins and a small number of charges. Subunit c
was also called the proteolipid because it was extracted with solvents
used to extract membrane lipids and it was purified after extraction
with these solvents. When membranes in which EmrE was labeled with
[
S]methionine were similarly extracted with a
mixture of chloroform and methanol (1:1), more than 85% of the
radioactivity was detected in the organic phase. When the organic phase
was analyzed in SDS-PAGE and the radioactivity was assessed by imaging,
again a single polypeptide was observed, identical to the one detected
in the intact membrane (Fig. 3, laneH). As
expected, most other proteins are not extracted by organic solvents as
evident from the pattern of the polypeptide composition analyzed by
SDS-PAGE and visualized by staining with Coomassie Blue (Fig. 3, laneE). Practically no other proteins are observed,
while a faint band corresponding to EmrE can now be clearly detected.
These results are even more dramatic when EmrE was overexpressed using
another vector, pKK223-3, in which the gene was fused to the
strong and highly regulated tac promoter (plasmid pKK56). When
membranes prepared from cells induced by the addition of
isopropyl-1-thio-
-D-galactopyranoside were extracted as
described above, a strong band corresponding to EmrE was observed (Fig. 3, laneF). This band was absent in
extracts from membranes prepared from cells that were transformed with
plasmid with no insert (not shown).
Figure 4: Transport of ethidium into proteoliposomes reconstituted with EmrE. A, proteoliposomes (Lipo, 5 µg of protein) reconstituted with EmrE in ammonium chloride medium were diluted into an ammonium-free medium as described under ``Experimental Procedures.'' Ethidium (Eth) was added to 1 µg/ml. Nigericin (nig) was added to 4 µM to collapse the pH gradient. B, as in A except that the liposomes were added last. C, as in A except that methyl viologen (MV) was added to 1 mM prior to the liposomes. D, the liposomes used contained no EmrE.
A
more quantitative documentation of EmrE-catalyzed transport was
obtained by following accumulation of another substrate,
[C]methyl viologen. In these experiments, the pH
gradient is generated as above, and a time-dependent accumulation of
[
C]methyl viologen was observed in
proteoliposomes reconstituted with EmrE (Fig. 5A, opensquares). Accumulation was linear during the
first 5 min, reached a maximal level, and slowly leaked back to the
medium (Fig. 5B, opensquares) as
expected from the transient nature of the driving force. Transport was
almost completely abolished when nigericin was added to the suspension
so that no pH gradient was formed (Fig. 5A, filledsquares). No accumulation over the nigericin levels was
detectable when liposomes reconstituted without EmrE were diluted to
the reaction medium (Fig. 5A, filledcircles). The gradient of
[
C]methyl viologen that the proteoliposomes
maintain was about 100 (in > out). Thus, about 10% of the total
radioactivity accumulated in the proteoliposomes while, based on
measurements in similar systems (Taglicht et al., 1993), their
internal volume represented only 0.1% of the total. The accumulated
substrate was free in solution rather than irreversibly bound to
internal structures since it could be rapidly released after
equilibrium was reached; at the plateau level, addition of nigericin (Fig. 5B, filledsquares) caused a
rapid release of all of the preaccumulated substrate within less than a
minute. Also, addition of an excess (1 mM) of unlabeled methyl
viologen, which exchanged with the intraliposomal isotope (filledcircles), caused the release of all of the radioactivity
within 5-10 min.
Figure 5:
Transport of
[C]methyl viologen into proteoliposomes
reconstituted with EmrE. A, proteoliposomes (1 µg of
protein) reconstituted with EmrE in ammonium chloride medium were
diluted into an ammonium-free medium containing
[
C]methyl viologen as described under
``Experimental Procedures'' (
-
). In
parallel series, nigericin was added to 15 µM (
-
), or liposomes with no EmrE were used in the
assay (
-
). B, experiment was as in A except that after 7.5 min, 15 µM nigericin
(
-
) or 1 mM methyl viologen
(
-
) was added. To the control series, no additions
were made (
-
). C, initial rates of
uptake (1 min) were measured at the indicated concentrations of methyl
viologen. The straightline was obtained by linear
regression. Each experimental point is the average value of
duplicates.
The kinetics of uptake were further analyzed
by monitoring the initial rate of uptake at various concentrations of
methyl viologen. The initial rate of uptake increased with the
concentration of the substrate and then saturated (not shown). When the
results of such an experiment were plotted according to Lineweaver-Burk (Fig. 5C), an apparent K of 247
µM and a V
of 1572 nmol/min/mg
protein were calculated from the linear regression of the plot.
To
test the substrate specificity of EmrE, the ability of several drugs to
inhibit the initial rate of [C]methyl viologen
uptake was measured, and the results are shown in Fig. 6. Of the
compounds tested, the most potent was the lipophilic cation
TPP
with an IC
of 8 nM, while
erythromycin had no effect up to 100 µM. The other
inhibitors were, in order of potency, ethidium (2.5 µM)
> acriflavine (8 µM) > tetracycline=
reserpine (25 µM) = MPP
(30
µM).
Figure 6:
Transport of
[C]methyl viologen is inhibited by a variety of
drugs. Initial rates of uptake (1-3 min) were measured at the
indicated concentrations of the following compounds: TPP
(
-
), ethidium (
-
), acriflavine
(
-
), reserpine (
-
),
tetracycline (
-
), erythromycin
(
-
), and MPP
(
-
). Each experimental point is the average
value of duplicates.
The results described in this report demonstrate that EmrE, a
110-amino acid polypeptide, is an antiporter that exchanges
H with toxic cations such as ethidium and methyl
viologen. The studies with whole cells confirmed that expression of
EmrE confers resistance to a variety of toxic cations most likely due
to the increased ability of the cell to remove them from the cell and
therefore lower their concentration in the cytoplasm. This assumption
was supported by the demonstration that active removal of ethidium is
markedly faster in cells overexpressing EmrE. In addition, the use of a
unc strain, which cannot interconvert phosphate bond
energy with the electrochemical gradient of protons, allowed to
establish conditions in which the source of energy available for
transport could be controlled. The studies with this strain
demonstrated that EmrE uses the electrochemical gradient of protons to
extrude ethidium. We concluded at this stage that most likely the basis
of the resistance to the toxicity of the other compounds tested is also
due to their active extrusion by the same mechanism.
The studies in
the intact cells were extended and confirmed by purification and
reconstitution of EmrE. The reconstituted protein catalyzed the
accumulation of ethidium and methyl viologen in a process dependent on
pH generated by an ammonium diffusion gradient. These studies
directly demonstrated that EmrE functions as an antiporter that
exchanges one or more H
with toxic cations. Transport
of methyl viologen and ethidium was demonstrated directly, while
transport of the other cations was inferred from their ability to
inhibit the accumulation of [
C]methyl viologen.
As predicted from its ability to confer resistance to a variety of
drugs, EmrE also showed a wide range of specificity. Transport was
inhibited very potently by TPP
and also by ethidium,
acriflavine, reserpine, tetracycline, and MPP
. As for
the first three drugs, EmrE was already shown to confer resistance to
them (Midgley, 1987; Purewal et al., 1990). Tetracycline was
tested because of the partial but significant resistance observed in
this study (Table 1). The anti-hypertensive reserpine is a potent
inhibitor (K
in the subnanomolar range) of
vesicular monoamine transporters, a protein from the TEXAN family of
multidrug transporters (Schuldiner, 1994; Schuldiner et al.,
1995). It also inhibits BMR, the Bacillus subtilis multidrug
transporter, the closest bacterial homolog of the vesicular monoamine
transporters. BMR is able to efflux out of bacterial cells structurally
diverse compounds such as ethidium, TPP
, and acridine
and is inhibited by reserpine at the low micromolar range (Ahmed et
al., 1993; Neyfakh et al., 1991). Interestingly,
sensitivity to reserpine is characteristic also of the mammalian
multidrug efflux transporter, P-glycoprotein (Pearce et al.,
1989). EmrE displays the lowest sensitivity to reserpine, with an
IC
of 25 µM. The neurotoxin
MPP
, an agent that causes Parkinsonism in model
systems, is a phenyl-pyridinium with structural similarity to the
dipyridinium methyl viologen and is also a substrate of the vesicular
monoamine transporter (Liu et al., 1992). It inhibits EmrE
with an IC
of 30 µM. Although a more
comprehensive survey is needed, it seems that there is a significant
overlap in the specificity range of the various multidrug transporters
known.
A unique feature of EmrE is its size; a 110-amino acid
protein with four putative transmembrane segments catalyzes the same
activities that have been previously observed in proteins three to five
times larger. The fact that the small proteins of this family are
transporters has been hypothesized before but without experimental
support (Grinius et al., 1992). Also, several putative
transporters with only one transmembrane segment have been cloned in
recent years based on expression cloning in oocytes (Wells and Hediger,
1992). However, the overall size of these proteins is high, and the
possibility that they may influence transport indirectly has not been
ruled out. In this report we directly demonstrated that purified EmrE
catalyzes the H-cation exchange activity, and no other
polypeptides are required for this activity. The following question can
then be raised: why are the other transporters so large when the
minimal subunit necessary is at the most 110 amino acids? One
possibility is that EmrE functions as a homooligomer to form at least
the 12 transmembrane segments usually observed in other transporters.
On the other hand, it is also possible that only some domains of the
larger proteins are needed for transport, and the rest functions for
regulation of the activity and/or interaction with other proteins.
Another very unique feature of EmrE, is its solubility in
chloroform:methanol, which provides us with a powerful and unique tool
for purification and characterization. Since only a handful of minor
proteins is soluble in these organic solvents, after overproduction of
EmrE it was possible to extract it highly purified. Most importantly,
EmrE was active after this treatment, suggesting that the extraction
did not induce major irreversible changes in its structure. The
reconstituted protein has an apparent K for methyl
viologen of 247 µM and a V
of 1572
nmol/min/mg protein. The latter represents a turnover number of 14
min
, which is in the same order of magnitude of many
ion-coupled transporters (Taglicht et al., 1991). No
quantitative data is available on the native protein, but the
specificity range of the protein is practically identical to the range
of resistance reported. The solubility of EmrE and subunit c in organic
solvents does not reside in a specially high proportion of hydrophobic
amino acids in both proteins; the percentage in both proteins is 49 and
44%, respectively, which is not different from other classical
ion-coupled transporters, which is between 41 and 49.6% in seven
H
-coupled bacterial transporters analyzed based on the
sequences available from the data bank. (
)On the other hand,
the percent of charged amino acids is 7.2 and 9.6%, respectively, while
in the 12 transmembrane helix transporters scanned as above, it is
between 11 and 14.8%. Another important difference is the number of net
charges, which is only +1 in EmrE and -2 in subunit c from E. coli. The range in the other transporters is between
-7 and +7, with the lowest absolute number of net charges in
the group analyzed being 4. It is therefore suggested that the
existence of unpaired charges on the protein represent the main energy
barrier for solubilization in the highly hydrophobic milieu of the
organic solvents. This has been extensively discussed in the case of
charge insertion in membranes (Engelman et al., 1986) and has
also received some experimental support in experiments in which
neutralization of charges of membrane proteins with H
or Ca
rendered them soluble in organic solvents
(Gitler, 1977).
Multidrug resistance is a major concern in medical and agricultural diseases. In medicine, the emergence of resistance to multiple drugs is a major obstacle in the treatment of several tumors as well as many infectious diseases. In agriculture, the control of resistance of plant pathogens is of major economic importance. However, besides being an economical and health-threatening phenomenon, it seems that the great diversity of transporters that play a role in drug resistance points to evolution of these systems as a necessary trait for survival in a variety of adverse environments. Outside the protected atmosphere of the laboratory, all organisms have to cope with a large variety of toxic compounds. Some of these compounds are produced by living organisms as part of their fight for survival, while others are toxic wastes, either natural or man made. Obviously, only those organisms that have developed through evolution the ability to cope with a wide variety of compounds, have been able to survive. EmrE is a representative of one of the strategies that evolved, most likely to provide protection from the toxic compounds. Because of their small size and the mechanistic similarity with the TEXANs, we propose to call this family MiniTEXANs. As described here, the MiniTEXANSs seem to be a unique, effective, and very exciting family of proteins. Because of their size and properties, they may provide a very useful model to understand structure-function aspects of transport reactions in ion-coupled processes.
Note Added in Proof-After submission of this article, L. L. Grinius and E. B. Goldberg ((1994) J. Biol. Chem.269, 29998-30004) reported the purification of Smr, a protein from Staphylococcus aureus homologous to Emr.