From the Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel
Received for publication, December 23, 2002, and in revised form, January 27, 2003
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ABSTRACT |
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The Smr family of multidrug transporters consists
of small membrane proteins that extrude various drugs in exchange with
protons rendering cells resistant to these drugs. Smr proteins
identified to date have been found only in Eubacteria. In this work we
present the cloning and characterization of an Smr protein from the
archaeon Halobacterium salinarum, the first Smr in the
archaeal kingdom. The protein, named Hsmr, was identified through
sequence similarity to the Smr family, and the DNA sequence was cloned
into an Escherichia coli expression system. Hsmr is
heterologously expressed in a functional form despite the difference in
lipid composition of the membrane and the lower salt in the cell and
its environment. Cells harboring the Hsmr plasmid transport ethidium
bromide in an uncoupler-sensitive process and gain resistance to
ethidium bromide and acriflavine. Hsmr binds tetraphenylphosphonium
(TPP+) with a relatively low affinity
(KD ~200 nM) at low salt
concentration that increases (KD ~40
nM) upon the addition of 2 M of either NaCl or
KCl. The Hsmr protein contains many of the signature sequence elements
of the Smr family and also a high content of negative residues in the
loops, characteristic of extreme halophiles. Strikingly, Hsmr is
composed of over 40% valine and alanine residues. These residues are
clustered at certain regions of the protein in domains that are not
important for activity, as judged from lack of conservation and from
previous studies with other Smr proteins. We suggest that this
high content of alanine and valine residues is a reflection of a
"natural" alanine and valine scanning necessitated by
the high GC content of the gene. This phenomenon reveals
significant sequence elements in small multidrug transporters.
Multidrug transporters actively remove toxic compounds from the
cytoplasm of cells. They are widespread from bacteria to man and have
been associated with multidrug resistance (1, 2). Many multidrug
transporters have been identified to date and classified into several
protein families (3, 4). Proteins in the Smr family of small multidrug
resistance proteins are ~110-amino acids long and extrude various
drugs in exchange with protons, thereby rendering bacteria resistant to
these compounds (5, 6). Several Smr proteins have been characterized,
purified, and reconstituted in a functional form (7, 8). Most posses a
single membrane-embedded charged residue, Glu-14, conserved in more
than sixty homologous proteins (8), which was shown to be part of a
binding site common to protons and substrates (9, 10). The basic
oligomeric structure detected in two-dimensional crystals of
EmrE, an E. coli Smr, is a dimer (11). Radiolabeled
substrate binding to purified EmrE and negative dominance experiments
support the contention that the functional unit of the protein is an
oligomer formed by two dimers (12-14).
In the absence of high-resolution structure, it would be useful to
study the effect of multiple changes on the function of the protein.
This is a complicated task to perform using site-directed mutagenesis,
because designing an effective combination of mutations is not
straightforward. One way to tackle this problem is by analyzing a set
of homologues that provide natural, more complex, and less biased
mutants (8). However, also with these mutants it is not always possible
to identify suppressor or complimentary substitutions throughout the
protein. Here we describe a special case that demonstrates an approach
to overcome this problem. We report the first identification and
characterization of an Smr protein from Archaea, the third domain of the universal phylogenetic tree (15). This protein, named
Hsmr, was cloned from the archaeon H. salinarum
based on sequence similarity to the Smr family. Hsmr was heterologously expressed in E. coli cells in a functional form despite the
difference in lipid composition of the membrane and the lower salt in
the cell and its environment. Hsmr renders cells resistant to the effect of multiple drugs and catalyzes the active efflux of ethidium from the cells. Detergent-solubilized Hsmr binds the high affinity substrate tetraphenylphosphonium
(TPP+)1 in a
salt-dependent manner: at low salt with a
KD of 200 nM, and 40 nM at
high salt. The Hsmr protein contains many of the
signature sequence elements defining the Smr family. However, it also
has a highly unusual sequence displaying some unique characteristics. It is composed of over 40% valine and alanine residues, which are
distributed throughout the protein, often concentrated at areas where
there is no sequence conservation. We suggest that this phenomenon is
the outcome of a natural process of alanine and valine "scanning"
that is necessitated by the high GC content of the gene.
Bacterial Strains and Plasmids--
E. coli JM109
(16) and E. coli BL21(DE3) (Novagen, Madison, WI) were used
throughout this work. The plasmids used are pT7-7 (17), pT7-7
EmrE-His (12), and pT7-7 Hsmr-His. The latter was constructed as
follows: the hsmr gene (OE3652F in the H. salinarum data base www.halolex.mpg.de) was cloned by polymerase
chain reaction using as template genomic DNA from H. salinarum (provided by Prof. M. Mevarech, Faculty of Life
Sciences, Tel Aviv University, Israel). Primers were designed to
overlap with each end of the gene and included sites for restriction
enzymes NdeI and EcoRI. The gene was cloned into
the pT7-7-His vector, which was obtained by removing the
emrE gene from vector pT7-7-EmrE-His with restriction
enzymes NdeI and EcoRI. The start codon was
modified from GTG to ATG.
Resistance to Toxic Compounds--
Resistance to toxic compounds
was tested after overnight growth in 2xYT liquid media consisting of
1.6% (w/v) tryptone, 1% (w/v) yeast extract, and 0.5% (w/v) NaCl.
Efflux of Ethidium in Whole Cells--
Efflux of ethidium in
whole cells was assayed essentially as described (7), except that
E. coli BL21(DE3) cells were used. The cells were grown at
37 °C in LB medium, when the culture reached A600 ~0.4,
isopropyl-1-thio- Overexpression and Purification of Hsmr--
E. coli
BL21(DE3) cells bearing plasmid pT7-7 Hsmr-His were grown at 37 °C
in 2xYT medium. When the culture reached A600
~0.8, IPTG was added to 0.5 mM. Two hours later the cells
were harvested by centrifugation, washed once with buffer containing
150 mM NaCl, 10 mM Tris, pH 7.5, 250 mM sucrose, 2.5 mM MgSO4, and 0.5 mM dithiothreitol, and resuspended in the same
buffer containing 15 µg/ml DNaseI (5 ml buffer/g cells).
Membranes were prepared by disrupting the cells using a Microfluidics
microfluidizer processor (M-110EHi). Non-disrupted cells were separated
by centrifugation, and the membranes were collected by
ultracentrifugation at 300,000 × g for 60 min. The membrane
pellet was resuspended in the same buffer, frozen in liquid air, and
stored at TPP+ Binding Assay--
TPP+ binding was
assayed essentially as described previously (12). Membranes were
solubilized with 1% n-dodecyl- Hsmr Has a Remarkable Amino Acid Composition--
The Hsmr protein
(Fig. 1A) was identified based
on sequence similarity to EmrE, an E. coli protein belonging
to the Smr family of multidrug resistance proteins. The gene encoding
for Hsmr is very rich in GC (68% GC), whereas the percentage of
coding GC in the H. salinarum genome is slightly lower
(65%). At the amino acid level Hsmr shares 54% similarity and 43%
identity with the EmrE protein. Hsmr is of particular interest because
it is the first Smr protein found in the Archaea kingdom. A simple
comparison of the amino acid composition of Hsmr and EmrE reveals some
striking differences, which are summarized in Fig. 1B. The
number of charged residues greatly differs between the two proteins.
Both proteins have a glutamate at position 14, the only charged residue
in the trans-membrane domain. This glutamate residue is conserved
throughout the Smr family (3, 8) and was shown to have a central role in the transport mechanism of EmrE (12, 18). However, although EmrE has
only two additional negatively charged residues, Hsmr has seven. They
are located in putative hydrophilic loops of the protein, four of them
in pairs, a pattern found in other proteins of halophilic organisms
(Fig. 1, A and B). The overall number of charged
residues in Hsmr is eleven, eight of which are negative, two histidines
and one highly conserved lysine at position 22. A high content of
negatively charged amino acids has been reported before in other
halophilic proteins, and is suggested to have a stabilizing effect at
high salt concentrations (19).
The proportion of hydrophobic residues is similar in the two proteins;
however, the composition is very different. Hsmr contains more alanine
and valine residues, 19 and 26 compared with 9 and 5 in EmrE,
respectively. Thus, Hsmr displays a remarkable amino acid composition
of over 40% valine and alanine residues. In parallel, the amount of
isoleucine residues also changes from 15 in EmrE to only 3 in Hsmr. A
small difference is observed in the number of glycines (11 in Hsmr, 12 in EmrE), leucines (12 and 16, respectively) and phenylalanines (4 and
5, respectively) (not shown). Hsmr contains no cysteine residues and a
single tryptophan at position 63, a fully conserved residue. Stevens
and Arkin (20) found a strong correlation between the GC content of a
genome, and the ratio of (Val+Ala)/(Ile+Phe) residues of its membrane
proteins. The higher the GC content of a genome, the higher the amount
of valine and alanine residues. This correlation is explained by a
codon bias toward GC-rich codons, together with a need to retain a
certain level of hydrophobicity in a membrane-spanning region. In
hsmr the codons used for alanine and valine are,
exclusively, the ones with the highest GC content: GTG and GTC for
valine, and GCG and GCC for alanine. The distribution of valine and
alanine residues within the trans-membrane helices of Hsmr is not
random. Many of these abundant residues appear to be clustered in
structural domains, as can be seen in the helical-wheel projection of
the trans-membrane helices of Hsmr (Fig.
2). Particularly stunning is the
situation in helices 1 and 4. Residues that are highly conserved
throughout the Smr family (8) are located on the opposite face of the
helix from where the valine and alanine residues are clustered (Fig. 2,
arrows). A similar distribution is observed in additional
Smr proteins from other GC-rich organisms (not shown). This dramatic
phenomenon may reflect the outcome of an evolutionary process leading
to the replacement of all residues not essential for function with
either valine or alanine. In a sense this resembles the result of an
alanine scan mutagenesis experiment, pointing out instantaneously the
residues that are important for the function of the protein, and
therefore cannot be replaced with valine or alanine.
Hsmr Confers Resistance Against Cytotoxic Compounds--
Such a
high alanine and valine content could be very useful in identifying
domains important for function in Smr proteins. Therefore, we tested
whether Hsmr can be functionally expressed. E. coli JM109
cells carrying the plasmids pT7-7 Hsmr, pT7-7 EmrE-His, or pT7-7
(vector alone) were tested for resistance to ethidium bromide,
acriflavine, and methyl viologen. Cells expressing Hsmr grow in the
presence of ethidium bromide up to 500 µM, to the same
extent as cells expressing EmrE, whereas cells with vector alone
display an IC50 of ~130 µM (Fig.
3A). In acriflavine, however, Hsmr cells display an intermediate resistance, with an IC50
of ~70 µM, whereas vector alone cells have an
IC50 of around 20 µM, and EmrE-His cells are
only inhibited by 25% at 120 µM acriflavine (Fig.
3B). Hsmr cells are not resistant to methyl viologen and display an IC50 of 75 µM, similar to that of
vector alone cells, whereas EmrE-His cells can grow in the presence of
up to 200 µM methyl viologen (Fig. 3C). Hsmr,
therefore, functions as a multidrug resistance protein but displays a
different substrate-specificity relative to that of EmrE.
Hsmr Catalyzes Ethidium Efflux in Whole Cells--
We further
explored Hsmr function by directly testing its ability to catalyze the
transport of ethidium bromide in whole cells. Entry of ethidium into
cells is manifested by an increase in the fluorescence due to binding
to nucleic acids (7, 21). Cells overexpressing either Hsmr or mock
vector were treated with the uncoupler CCCP to facilitate the entry of
ethidium into the cells, as previously described (7). After 60 min of
incubation with ethidium (1 µg/ml) and CCCP (40 µM) the
cells were collected by centrifugation and rapidly resuspended in
medium containing the same concentration of ethidium, but without
uncoupler. Upon removal of the uncoupler from Hsmr overexpressing
cells, a rapid decrease in fluorescence is observed, even before the
tracing begins, and is completed in less than 7 min (Fig.
3D, left). This decrease in fluorescence, which
represents removal of ethidium from the cells against a concentration
gradient, is completely reversed upon addition of CCCP (Fig.
3D, left). As expected, in cells harboring a
control vector no decrease in fluorescence was observed (Fig. 3D, right). These results demonstrate that Hsmr
catalyzes the active removal of ethidium in whole cells.
The Hsmr Dimer Is Resistant to Denaturant
Treatment--
Expression levels were tested in membranes prepared
from IPTG induced E. coli BL21 (DE3) cells harboring the
plasmid pT7-7 Hsmr-His. The His-tagged Hsmr protein was purified from
the membranes using a Ni-NTA column. The purified protein was analyzed
on SDS-PAGE, alongside purified samples of EmrE (Fig.
4A). Hsmr reaches high levels
of expression in E. coli membranes, up to 2 mg per 1 liter culture. As seen in Fig. 4A, the EmrE monomer
displays an apparent Mr of 14,000. This
value is close to the theoretical value of molecular mass calculated
for a protein the size of EmrE or Hsmr (not shown). Surprisingly, Hsmr
displays an apparent Mr of 30,000, suggesting
that the dimeric complex of Hsmr does not dissociate, even in the
presence of SDS. Furthermore, a small amount of protein corresponding
to the Hsmr tetramer can also be observed (Fig. 4A). The
protein was challenged with an anti-His antibody and also specifically
radiolabeled, and in both cases it co-migrated with the
Coomassie-stained protein (not shown). Attempts to dissociate the dimer
were made by modifying the treatment of the sample before loading on
the gel. These treatments included solubilization in various detergents
and boiling of the purified protein; however, they yielded no visible
monomers on the gel. Monomers were observed only after extraction of
diluted protein in a mixture of chloroform and methanol (not
shown).
Solubilized Hsmr Binds
[3H]TPP+ and Interacts with Several Other
Drugs--
A very convenient functional assay of detergent-solubilized
EmrE has been previously developed (12). Here we show that
DDM-solubilized Hsmr also specifically binds
[3H]TPP+ in a dose-dependent
manner (Fig. 4B) in the range up to 50 µg of protein,
leveling off at the high protein amounts, because of substrate
depletion. To test interaction with other known substrates of EmrE, the
compounds were tested for their ability to inhibit the binding
reaction. The results are shown in Fig.
5. Methyl viologen had no effect on
[3H]TPP+ binding to Hsmr, as expected from
the inability of Hsmr to confer resistance against this compound in
living cells (see Fig. 3C). Benzalkonium, ethidium, and
acriflavine inhibit [3H]TPP+ binding to Hsmr
in the µM range (Fig. 5), suggesting that they specifically interact with Hsmr. These results are in accordance with
the phenotype presented in Fig. 3, and together they demonstrate the
function of Hsmr as a multidrug protein, which can interact in a
specific manner with different drugs.
Salt Dependence of Hsmr--
H. salinarum,
from which the Hsmr protein originates, normally resides in extreme
environments where the salt concentration reaches very high values of
4-5 M (22). It was therefore interesting to examine the
effect of salt on the activity of Hsmr. For that purpose solubilized
membranes from cells expressing either Hsmr or EmrE were assayed for
[3H]TPP+ binding at increasing concentrations
of NaCl, KCl, or LiCl. Hsmr-binding activity is markedly stimulated in
the presence of KCl, NaCl, and LiCl (Fig.
6A). As the concentration of
salt rises from zero to 3.2 M an increase in the binding
activity is observed, saturating around the high salt concentration.
Both KCl and NaCl brought about a 6-7-fold increase in the binding
upon addition of about 3.0 M salt. A smaller effect is seen
in LiCl that induces only a 4-fold increase at similar concentrations.
The binding activity of EmrE is not affected by the salt concentration
and remains constant up to 3 M KCl, NaCl, or LiCl (Fig.
6B). To understand the mechanism underlying this salt effect
we measured the binding affinity at the different salt concentrations.
We found that the binding affinity increases 5-fold, from 200 to 40 nM, upon the addition of 2 M NaCl.
The first Smr protein from the archaeon H. salinarum
was identified based on sequence similarity. Its unusual alanine and valine content, and the clustering of these residues in certain domains, suggested possible functional implications. Therefore, we set
out to probe the activity of the protein. Hsmr was functionally expressed in E. coli membranes, to high levels. Proteins
from extremely halophilic bacteria are adapted to a very high salt environment (above 3 M), and usually denature in the
absence of high salt, resulting in a non-functional protein (22).
Nonetheless, expression of the hsmr gene in E. coli results in the production of a protein that is functional
in vivo as judged from drug resistance phenotype and from a
whole cell ethidium efflux assay. Hsmr is active in a wide range of
conditions because it does not strictly require salt for its activity.
It is also able to function in the E. coli membrane, which
has a lipid composition very different from the archaeal membrane it
originated from (23, 24). The stability of the protein is also apparent
when analyzed on SDS-PAGE. The Hsmr dimer stays intact even at high
concentrations of SDS. This unusual phenomenon of an SDS-stable
oligomer has been reported before with the dimer of glycophorine A (25)
as well as for the phospholamban pentamer (26). Attempts to dissociate
the Hsmr dimer have been made by using different solubilization
conditions and by heating the protein sample before loading on the gel.
The protein retained its "dimeric" mobility on the gel unless
diluted and extracted in an organic solvent. Hsmr is also functional
in vitro, in its DDM-solubilized form, where it specifically
binds the substrate TPP+ in a dose-dependent
manner. Although the protein retains its binding activity even in the
absence of salt, this activity is greatly enhanced at high salt
concentrations as a result of an increase in the affinity of the
protein to TPP+. This salt effect appears to be a general
one because it is observed in both NaCl and KCl as well as in LiCl and
MgCl2 (not shown) to a lesser extent.
The Smr proteins are not as ubiquitous in the Archaea as in the
Eubacteria. Only three other homologues can be identified until now,
all of them in various Methanosarcinoma species (not shown).
Hsmr was identified based on sequence similarity to the Smr family, yet
was found to have a unique sequence composition. Compared with other
Smr proteins, Hsmr contains many negative charges, located mostly at
the hydrophilic loops of the protein. This phenomenon is observed in
many halophilic proteins and is said to contribute much to their
stability in high salt (27). Hsmr has only one positive charge, Lys-22,
which is fully conserved in the Smr family, and two histidine residues
facing the opposite side of the membrane. Because there are so few
positive charges, which are distributed quite symmetrically on both
sides of the membrane, it is difficult to apply the von Heijne rule for
topology prediction (28) to this protein. The inapplicability of this rule with Smr proteins has been demonstrated before and was suggested to result from their small size (18).
The most striking feature of Hsmr is the fact that it is built of over
40% valine and alanine residues, clustered at certain regions of the
protein. This clustering is in domains that do not seem important for
activity. According to Stevens and Arkin (20), and discussed earlier,
there exists a strong positive correlation between the GC content of a
genome and the amount of valine and alanine residues found in membrane
proteins it codes for. This correlation is explained by a codon bias
toward amino acids coded predominately by GC nucleotides, together with
a need to retain certain physical properties within a trans-membrane domain. The rise in the abundance of valine residues is also thought to
be compensating for the lack of AT-coded amino acids, particularly isoleucine, with which it shares similar physical properties. The
alanine and valine codons are GCN and GTN, respectively, of which only
GCG and GCC (for alanine) and GTG and GTC (for valine) are present in
the hsmr gene. Although codon bias could be fulfilled also
by glycine and leucine, the frequency of these amino acids does not
increase in proteins from organisms with high GC content (20). We
speculate that glycine would not be hydrophobic enough and could allow
too much flexibility of the helix; leucine may be bulkier than necessary.
The distribution of valine and alanine residues in the first
trans-membrane domain of Hsmr correlates well with information deduced
from extensive site-directed mutagenesis of the same trans-membrane domain in the homologous protein EmrE. EmrE mutations in residues in
the same face of the helix as Glu-14 have effects on the catalytic activity of the protein, whereas mutations on the other face of the
helix have no effect (31). Strikingly, the face tolerant to mutations
is the one predominated by alanine residues in Hsmr. Without a
high-resolution structure of an Smr we can only speculate about the
location of an alanine-rich face in a membrane protein. Alanine is a
small, relatively hydrophobic amino acid. Because of its size it could
provide an interface for tight packing of two helices (30). On the
other hand, its hydrophobicity is high enough for interaction with
lipids in the membrane.
Hsmr is therefore a case of natural alanine and valine
mutagenesis. Multiple sites in the protein, located in regions not important for activity, have become through evolution occupied by
valine or alanine residues. The advantage of analyzing sequences such
as that of Hsmr is that the outcome of replacements in more than one
position can be seen within the same protein. Multiple replacements are
more informative than single ones. The latter depend at times on the
properties of amino acids in positions other than the ones modified.
This could be because of size, charge, or other properties. Multiple
replacements are difficult to design, therefore the analysis of valine-
and alanine-rich sequences could greatly facilitate their planning
or even serve as ready made "mutants," a quick alternative for experimentation.
We found that the valine and alanine clustering is observed in
additional membrane proteins from H. salinarum, and
therefore may be part of a general phenomenon. A full genomic and
statistical analysis is required to conclude whether this phenomenon
can be used to reveal significant sequence elements in membrane
proteins in general. If this is indeed a general phenomenon it could be used to devise an algorithm predicting important regions within a
Val+Ala-rich membrane protein sequence, without the need for additional
information. This prediction, in turn, could be projected on to
non-Val+Ala-rich homologues, providing a general tool for a quick
preliminary prediction of significant regions within a given membrane protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside (IPTG) was added to 0.5 mM. Two hours later the cells were centrifuged and
resuspended in minimal medium A supplemented with 0.05% NaCl, 1 mM MgSO4, 0.1 mM CaCl2,
and 0.36% glucose. At this point, ethidium and carbonyl cyanide
m-chlorophenylhydrazone (CCCP) were added to a final
concentration of 1 µg/ml and 40 µM, respectively, and
the cells were incubated for 60 min at 37 °C. The cells were
collected by centrifugation and quickly resuspended in medium
containing the same ethidium concentration without CCCP, and
fluorescence was measured in a PerkinElmer fluorimeter (Luminescence
Spectrometer LS-5) with exciting light at 545 nm and emission at 610 nm.
70 °C. Hsmr-His was purified by solubilizing membranes
(0.25 mg of total protein) in SDS-Urea buffer (2% SDS, 6 M
urea, 15 mM Tris, pH 7.5) for 15 min at 25 °C.
Solubilized material was added to equilibrated Ni-NTA beads (Qiagene,
GmbH, Hilden, Germany), and incubated for one hour at room temperature
in the presence of 25 mM imidazole. Unbound material was
discarded, the beads were washed once in the same buffer, and the
protein was eluted in 200 mM imidazole. A sample was
analyzed on SDS-PAGE.
-maltoside (DDM),
(Anatrace, Inc., Maumee, Ohio) in Na-buffer (150 mM NaCl, 15 mM Tris-Cl, pH 7.5) and immobilized on Ni-NTA beads.
After two washes with Na buffer supplemented with 0.08% DDM, 200 µl of buffer containing 10 nM
[3H]TPP+ (27 Ci/mmol, Amersham Biosciences)
were added and the samples were incubated for 15 min at 4 °C. In
each experiment, the values obtained in a control reaction, with 100 µM unlabeled TPP+, were subtracted. All
binding reactions were performed in duplicate.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Secondary structure and amino acid
composition of Hsmr. A, model of the secondary
structure of Hsmr based on hydropathy calculations (29) and on analogy
to EmrE. Putative transmembrane helices are shown as gray
cylinders. Positively charged residues are highlighted,
and negatively charged residues are marked in black circles.
B, the number of selected residues is given for EmrE (of a
total of 110) and Hsmr (112). In the cloned Hsmr the initiation codon
was modified so that it codes for methionine.
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Fig. 2.
Alanine and valine clustering in Hsmr. A
helical-wheel projection of the four trans-membrane helices of Hsmr.
Alanine and valine residues are highlighted. Residues fully
conserved, or partially conserved, within the Smr-family are marked by
thick and thin arrows,
respectively.
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Fig. 3.
Resistance to toxic compounds and drug efflux
activity. E. coli JM109 cells carrying plasmids pT7-7
Hsmr-His ( ), pT7-7 EmrE-His (
), or pT7-7 mock vector (
),
were grown in liquid media containing the indicated amounts of ethidium
bromide (A), acriflavine (B), or Methyl viologen
(C), as described under "Experimental
Procedures." D, E. coli BL21 (DE3) cells
harboring the plasmid pT7-7 Hsmr-His (left), or pT7-7
(right) were grown in LB to mid-logarithmic phase and then
induced with 0.5 mM IPTG for 2 h. The cells were then
centrifuged and resuspended in minimal medium containing ethidium
bromide and CCCP, as described under "Experimental Procedures."
After 60 min of incubation at 37 °C, the cells were washed quickly
in CCCP-free medium and monitored for fluorescence. 40 µM
CCCP was added where indicated.
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Fig. 4.
Expression, purification and activity of
Hsmr. E. coli BL21 (DE3) cells carrying the plasmid
pT7-7 Hsmr-His were grown in rich media, and protein expression was
induced using 0.5 mM IPTG for two hours. The cells were
harvested, and membranes were obtained by a single pass through a
microfluidizer device, followed by ultracentrifugation. A,
His-tagged Hsmr was purified from the E. coli membranes by
solubilization in SDS-urea buffer and binding to Ni-NTA beads. A sample
(equivalent to 25 µg total membrane protein) was analyzed on SDS-PAGE
alongside a sample of purified EmrE (0.5 µg). B,
increasing amounts or the E. coli membranes expressing
Hsmr were solubilized in 1% DDM and assayed for
[3H]TPP+ binding as described under
"Experimental Procedures."
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Fig. 5.
Effect of different drugs on the binding of
[3H]TPP+ to Hsmr. Membranes from cells
expressing His-tagged Hsmr (50 µg of total membrane protein) were
solubilized with 1% DDM and assayed for
[3H]TPP+ binding as described under
"Experimental Procedures." To determine the effect of various drugs
on the binding they were added to the binding solution in the indicated
concentrations.
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Fig. 6.
Salt dependence of Hsmr. Solubilized
Hsmr-His (50 µg of total membrane protein) (A) or EmrE-His
(1 µg of total membrane protein) (B) were assayed for
[3H]TPP+ binding as described under
"Experimental Procedures." To test the salt dependence of the
binding, immobilized Hsmr was washed once in buffer containing 0.08%
DDM, 15 mM Tris, pH 8, and either NaCl ( ), KCl (
), or
LiCl (
) at the indicated concentrations, and the binding reaction
was carried on in the same buffer. Bound TPP+ is plotted as
the percent of TPP+ bound when no salt is added to the
binding solution.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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 Aerospace 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.
Recipient of a Yeshaya Horowitz Foundation fellowship.
§ To whom correspondence should be addressed. Tel.: 972-2-6585992; Fax: 972-2-5634625; E-mail: Shimon.Schuldiner@huji.ac.il.
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M213119200
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ABBREVIATIONS |
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The abbreviations used are:
TPP+, tetraphenylphosphonium;
IPTG, isopropyl-1-thio--D-galactopyranoside;
CCCP, carbonyl
cyanide m-chlorophenylhydrazone;
DDM, n-dodecyl-
-maltoside;
Ni-NTA, nickel-nitrilotriacetic
acid.
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