THE JOURNAL OF BIOLOGICAL CHEMISTRY
12000 –12005, 2003
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of an Archaeal Multidrug Transporter with a
Unique Amino Acid Composition*
Received for publication, December 23, 2002, and in revised form, January 27, 2003
Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M213119200
Shira Ninio‡ and Shimon Schuldiner§
From the Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel
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 ar-
chaeon Halobacterium salinarum, the first Smr in the
archaeal kingdom. The protein, named Hsmr, was iden-
tified through sequence similarity to the Smr family,
and the DNA sequence was cloned into an Escherichia
coli expression system. Hsmr is heterologously ex-
pressed 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 bro-
mide and acriflavine. Hsmr binds tetraphenylphospho-
nium (TPPⴙ) 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 ele-
ments of the Smr family and also a high content of neg-
ative residues in the loops, characteristic of extreme
halophiles. Strikingly, Hsmr is composed of over 40%
valine and alanine residues. These residues are clus-
tered 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 con-
tent 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 character-
* This work was supported by grants from the Deutsche-Israeli Pro-
gram (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. This
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.
12000
Vol. 278, No. 14, Issue of April 4, pp.
Printed in U.S.A.
ized, 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 use-
ful to study the effect of multiple changes on the function of the
protein. This is a complicated task to perform using site-di-
rected mutagenesis, because designing an effective combina-
tion of mutations is not straightforward. One way to tackle this
problem is by analyzing a set of homologues that provide nat-
ural, more complex, and less biased mutants (8). However, also
with these mutants it is not always possible to identify sup-
pressor or complimentary substitutions throughout the pro-
tein. Here we describe a special case that demonstrates an
approach to overcome this problem. We report the first identi-
fication 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 func-
tional 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. Deter-
gent-solubilized Hsmr binds the high affinity substrate tetra-
phenylphosphonium (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 defin-
ing the Smr family. However, it also has a highly unusual se-
quence 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.
EXPERIMENTAL PROCEDURES
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
1
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.
This paper is available on line at http://www.jbc.org
 An Archaeal Multidrug Transporter 12001
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-␤-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 ␮g/ml and 40 ␮M, 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.
Overexpression and Purification of Hsmr—E. coli BL21(DE3) cells
bearing plasmid pT7–7 Hsmr-His were grown at 37 °C in 2xYT me-
dium. 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 ␮g/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 sepa-
rated by centrifugation, and the membranes were collected by ultracen-
trifugation at 300,000 ⫻ g for 60 min. The membrane pellet was resus-
pended in the same buffer, frozen in liquid air, and stored at ⫺70 °C.
Hsmr-His was purified by solubilizing membranes (0.25 mg of total FIG. 1. Secondary structure and amino acid composition of
protein) in SDS-Urea buffer (2% SDS, 6 M urea, 15 mM Tris, pH 7.5) for Hsmr. A, model of the secondary structure of Hsmr based on hydrop-
15 min at 25 °C. Solubilized material was added to equilibrated Ni-NTA athy calculations (29) and on analogy to EmrE. Putative transmem-
beads (Qiagene, GmbH, Hilden, Germany), and incubated for one hour brane helices are shown as gray cylinders. Positively charged residues
at room temperature in the presence of 25 mM imidazole. Unbound are highlighted, and negatively charged residues are marked in black
material was discarded, the beads were washed once in the same buffer, circles. B, the number of selected residues is given for EmrE (of a total
and the protein was eluted in 200 mM imidazole. A sample was analyzed of 110) and Hsmr (112). In the cloned Hsmr the initiation codon was
on SDS-PAGE. modified so that it codes for methionine.
TPP⫹ Binding Assay—TPP⫹ binding was assayed essentially as de-
scribed previously (12). Membranes were solubilized with 1% n-dodecyl-
␤-maltoside (DDM), (Anatrace, Inc., Maumee, Ohio) in Na-buffer (150
of the protein, four of them in pairs, a pattern found in other
mM NaCl, 15 mM Tris-Cl, pH 7.5) and immobilized on Ni-NTA beads. proteins of halophilic organisms (Fig. 1, A and B). The overall
After two washes with Na buffer supplemented with 0.08% DDM, 200 number of charged residues in Hsmr is eleven, eight of which
␮l of buffer containing 10 nM [3H]TPP⫹ (27 Ci/mmol, Amersham Bio- are negative, two histidines and one highly conserved lysine at
sciences) were added and the samples were incubated for 15 min at position 22. A high content of negatively charged amino acids
4 °C. In each experiment, the values obtained in a control reaction, with has been reported before in other halophilic proteins, and is
100 ␮M unlabeled TPP⫹, were subtracted. All binding reactions were
performed in duplicate.
suggested to have a stabilizing effect at high salt concentra-
tions (19).
RESULTS The proportion of hydrophobic residues is similar in the two
Hsmr Has a Remarkable Amino Acid Composition—The proteins; however, the composition is very different. Hsmr con-
Hsmr protein (Fig. 1A) was identified based on sequence sim- tains more alanine and valine residues, 19 and 26 compared
ilarity to EmrE, an E. coli protein belonging to the Smr family with 9 and 5 in EmrE, respectively. Thus, Hsmr displays a
of multidrug resistance proteins. The gene encoding for Hsmr remarkable amino acid composition of over 40% valine and
is very rich in GC (68% GC), whereas the percentage of coding alanine residues. In parallel, the amount of isoleucine residues
GC in the H. salinarum genome is slightly lower (65%). At the also changes from 15 in EmrE to only 3 in Hsmr. A small
amino acid level Hsmr shares 54% similarity and 43% identity difference is observed in the number of glycines (11 in Hsmr, 12
with the EmrE protein. Hsmr is of particular interest because in EmrE), leucines (12 and 16, respectively) and phenylala-
it is the first Smr protein found in the Archaea kingdom. A nines (4 and 5, respectively) (not shown). Hsmr contains no
simple comparison of the amino acid composition of Hsmr and cysteine residues and a single tryptophan at position 63, a fully
EmrE reveals some striking differences, which are summarized conserved residue. Stevens and Arkin (20) found a strong cor-
in Fig. 1B. The number of charged residues greatly differs relation between the GC content of a genome, and the ratio of
between the two proteins. Both proteins have a glutamate at (Val⫹Ala)/(Ile⫹Phe) residues of its membrane proteins. The
position 14, the only charged residue in the trans-membrane higher the GC content of a genome, the higher the amount of
domain. This glutamate residue is conserved throughout the valine and alanine residues. This correlation is explained by a
Smr family (3, 8) and was shown to have a central role in the codon bias toward GC-rich codons, together with a need to
transport mechanism of EmrE (12, 18). However, although retain a certain level of hydrophobicity in a membrane-span-
EmrE has only two additional negatively charged residues, ning region. In hsmr the codons used for alanine and valine are,
Hsmr has seven. They are located in putative hydrophilic loops exclusively, the ones with the highest GC content: GTG and
12002 An Archaeal Multidrug Transporter
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.
GTC for valine, and GCG and GCC for alanine. The distribu-
tion of valine and alanine residues within the trans-membrane
helices of Hsmr is not random. Many of these abundant resi-
dues 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 through-
out 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 evolution-
ary process leading to the replacement of all residues not es-
sential for function with either valine or alanine. In a sense this
resembles the result of an alanine scan mutagenesis experi-
ment, pointing out instantaneously the residues that are im-
portant 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 ex-
pressed. E. coli JM109 cells carrying the plasmids pT7–7 Hsmr,
pT7–7 EmrE-His, or pT7–7 (vector alone) were tested for re-
sistance to ethidium bromide, acriflavine, and methyl viologen.
Cells expressing Hsmr grow in the presence of ethidium bro-
mide up to 500 ␮M, to the same extent as cells expressing
EmrE, whereas cells with vector alone display an IC50 of ⬃130
␮M (Fig. 3A). In acriflavine, however, Hsmr cells display an
intermediate resistance, with an IC50 of ⬃70 ␮M, whereas
vector alone cells have an IC50 of around 20 ␮M, and EmrE-His
cells are only inhibited by 25% at 120 ␮M acriflavine (Fig. 3B).
Hsmr cells are not resistant to methyl viologen and display an
IC50 of 75 ␮M, similar to that of vector alone cells, whereas
EmrE-His cells can grow in the presence of up to 200 ␮M methyl
viologen (Fig. 3C). Hsmr, therefore, functions as a multidrug
resistance protein but displays a different substrate-specificity
relative to that of EmrE.
FIG. 3. Resistance to toxic compounds and drug efflux activity.
E. coli JM109 cells carrying plasmids pT7–7 Hsmr-His (Œ), pT7–7
EmrE-His (f), 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 Proce-
dures.” 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 ␮M CCCP was
added where indicated.
Hsmr Catalyzes Ethidium Efflux in Whole Cells—We further
explored Hsmr function by directly testing its ability to cata-
lyze the transport of ethidium bromide in whole cells. Entry of
ethidium into cells is manifested by an increase in the fluores-
cence due to binding to nucleic acids (7, 21). Cells overexpress-
ing either Hsmr or mock vector were treated with the uncou-
pler CCCP to facilitate the entry of ethidium into the cells, as
previously described (7). After 60 min of incubation with
ethidium (1 ␮g/ml) and CCCP (40 ␮M) the cells were collected
by centrifugation and rapidly resuspended in medium contain-
ing 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 dem-
onstrate 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 sam-
ples 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
 An Archaeal Multidrug Transporter 12003

FIG. 5. Effect of different drugs on the binding of [3H]TPPⴙ to

FIG. 4. Expression, purification and activity of Hsmr. E. coli Hsmr. Membranes from cells expressing His-tagged Hsmr (50 ␮g of
BL21 (DE3) cells carrying the plasmid pT7–7 Hsmr-His were grown in total membrane protein) were solubilized with 1% DDM and assayed for
rich media, and protein expression was induced using 0.5 mM IPTG for [3H]TPP⫹ binding as described under “Experimental Procedures.” To
two hours. The cells were harvested, and membranes were obtained by determine the effect of various drugs on the binding they were added to
a single pass through a microfluidizer device, followed by ultracentrif- the binding solution in the indicated concentrations.
ugation. 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 ␮g total membrane protein) was analyzed on
SDS-PAGE alongside a sample of purified EmrE (0.5 ␮g). 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.”

calculated for a protein the size of EmrE or Hsmr (not shown).

Surprisingly, Hsmr displays an apparent Mr of 30,000, suggest-
ing 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 in-
cluded 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 di-
luted 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 FIG. 6. Salt dependence of Hsmr. Solubilized Hsmr-His (50 ␮g of
to 50 ␮g of protein, leveling off at the high protein amounts, total membrane protein) (A) or EmrE-His (1 ␮g of total membrane
because of substrate depletion. To test interaction with other protein) (B) were assayed for [3H]TPP⫹ binding as described under
“Experimental Procedures.” To test the salt dependence of the binding,
known substrates of EmrE, the compounds were tested for their immobilized Hsmr was washed once in buffer containing 0.08% DDM,
ability to inhibit the binding reaction. The results are shown in 15 mM Tris, pH 8, and either NaCl (●), KCl (Œ), or LiCl (f) at the
Fig. 5. Methyl viologen had no effect on [3H]TPP⫹ binding to indicated concentrations, and the binding reaction was carried on in the
Hsmr, as expected from the inability of Hsmr to confer resistance same buffer. Bound TPP⫹ is plotted as the percent of TPP⫹ bound when
no salt is added to the binding solution.
against this compound in living cells (see Fig. 3C). Benzalko-
nium, ethidium, and acriflavine inhibit [3H]TPP⫹ binding to
Hsmr in the ␮M range (Fig. 5), suggesting that they specifically observed, saturating around the high salt concentration. Both
interact with Hsmr. These results are in accordance with the KCl and NaCl brought about a 6 –7-fold increase in the binding
phenotype presented in Fig. 3, and together they demonstrate upon addition of about 3.0 M salt. A smaller effect is seen in LiCl
the function of Hsmr as a multidrug protein, which can interact that induces only a 4-fold increase at similar concentrations. The
in a specific manner with different drugs. binding activity of EmrE is not affected by the salt concentration
Salt Dependence of Hsmr—H. salinarum, from which the and remains constant up to 3 M KCl, NaCl, or LiCl (Fig. 6B). To
Hsmr protein originates, normally resides in extreme environ- understand the mechanism underlying this salt effect we meas-
ments where the salt concentration reaches very high values of ured the binding affinity at the different salt concentrations. We
4 –5 M (22). It was therefore interesting to examine the effect of found that the binding affinity increases 5-fold, from 200 to 40
salt on the activity of Hsmr. For that purpose solubilized mem- nM, upon the addition of 2 M NaCl.
branes from cells expressing either Hsmr or EmrE were assayed
for [3H]TPP⫹ binding at increasing concentrations of NaCl, KCl, DISCUSSION
or LiCl. Hsmr-binding activity is markedly stimulated in the The first Smr protein from the archaeon H. salinarum was
presence of KCl, NaCl, and LiCl (Fig. 6A). As the concentration of identified based on sequence similarity. Its unusual alanine
salt rises from zero to 3.2 M an increase in the binding activity is and valine content, and the clustering of these residues in
12004 An Archaeal Multidrug Transporter
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 lev-
els. 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 con-
ditions 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 phe-
nomenon of an SDS-stable oligomer has been reported before
with the dimer of glycophorine A (25) as well as for the phos-
pholamban pentamer (26). Attempts to dissociate the Hsmr
dimer have been made by using different solubilization condi-
tions and by heating the protein sample before loading on the
gel. The protein retained its “dimeric” mobility on the gel un-
less diluted and extracted in an organic solvent. Hsmr is also
functional in vitro, in its DDM-solubilized form, where it spe-
cifically 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 con-
tains 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 distrib-
uted 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 pro-
teins has been demonstrated before and was suggested to re-
sult 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 cor-
relation 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 informa-
tion 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 pro-
tein. Alanine is a small, relatively hydrophobic amino acid.
Because of its size it could provide an interface for tight pack-
ing 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 mu-
tagenesis. 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 se-
quences 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 dif-
ficult 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 ele-
ments 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 prelimi-
nary prediction of significant regions within a given membrane
protein.
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