J. Mol. Biol.

(2010) 402, 127138

doi:10.1016/j.jmb.2010.07.019

Available online at www.sciencedirect.com

Orientation of Small Multidrug Resistance Transporter

Subunits in the Membrane: Correlation with the
Positive-Inside Rule
Magdalena A. Kolbusz 1 , Ramon ter Horst 1 , Dirk-Jan Slotboom 2
and Juke S. Lolkema 1
1

Department of Molecular
Microbiology, Groningen
Biomolecular Sciences and
Biotechnology Institute,
University of Groningen,
Kerklaan 30, 9751 NN Haren,
The Netherlands
2

Department of Membrane
Enzymology, Groningen
Biomolecular Sciences and
Biotechnology Institute,
University of Groningen,
Nijenborgh 4, 9747 AG
Groningen, The Netherlands
Received 17 May 2010;
received in revised form
7 July 2010;
accepted 8 July 2010
Available online
17 July 2010

Small multidrug resistance (SMR) transport proteins provide a model for

the evolution of larger two-domain transport proteins. The orientation in
the membrane of 27 proteins from the SMR family was determined using
the reporter fusion technique. Nine members were encoded monocistronically (singles) and shown to insert in both orientations (dual topology).
Eighteen members were encoded in pairs on the chromosome and shown to
insert in fixed orientations; the two proteins in each pair invariably had
opposite orientations in the membrane. Interaction between the two
proteins in pairs was demonstrated by copurification. The orientation in
the membrane of either protein in the pair was affected only marginally by
the presence of the other protein.
For the proteins in pairs, the orientation in the membrane correlated well
with the distribution of positively charges residues (R + K) over the
cytoplasmic and extracellular loops (positive-inside rule). In contrast,
dual-topology insertion of the singles was predicted less well by the
positive-inside rule. Three singles were predicted to insert in a single
orientation with the N-terminus and the C-terminus at the extracellular side
of the membrane. Analysis of charge distributions suggests the requirement
of a threshold number of charges in the cytoplasmic loops for the positiveinside rule to be of predictive value. It is concluded that a combined analysis
of gene organization on the chromosome and phylogeny is sufficient to
distinguish between fixed or dual topology of SMR members and, probably,
similar types of membrane proteins. The positive-inside rule can be used to
predict the orientation of members in pairs, but is not suitable as a sole
predictor of dual topology.
2010 Elsevier Ltd. All rights reserved.

Edited by I. B. Holland

Keywords: EmrE; SMR family; dual topology; positive-inside rule; insertion

Introduction
Transporters of the small multidrug resistance
(SMR) family are widespread among prokaryotes.13
They are responsible for extruding various cationic
*Corresponding author. E-mail address:
j.s.lolkema@rug.nl.
Abbreviations used: SMR, small multidrug resistance;
TMS, transmembrane segments; GFP, green fluorescent
protein; PhoA, alkaline phosphatase.

toxicants from cells such as ethidium and tetraphenyl phosphonium, and also render the cells resistant
to antibiotics such as -lactams, cephalosporins, and
aminoglycosides. Extrusion is driven by the electrochemical proton gradient across the membrane. The
transporters differ from most other secondary
transporters in that the proteins are small, built
from 100 to 140 amino acid residues ( 1115 kDa)
that fold into four transmembrane segments (TMS)
connected by short hydrophilic loop regions. The
most extensively characterized member of the SMR
family is EmrE of Escherichia coli.

0022-2836/$ - see front matter 2010 Elsevier Ltd. All rights reserved.

Orientation of SMR Transporters

128
SMR transporters are dimeric proteins that come in
two types: homodimers encoded by a single gene (e.g.,
EmrE of E. coli) or heterodimers encoded by a
cistronically transcribed pair of genes. In all cases,
the two protomers of the heterodimeric proteins are
homologous proteins. Both subunits are required to
form an active transporter, which was demonstrated
for three different pairs of Bacillus subtilis (YkkC/
YkkD, YvdS/YvdR, and EbrA/EbrB).47 No increased
resistance was observed when the proteins were
expressed separately. Remarkably, mutant versions
of EbrA and EbrB of B. subtilis,6 and EbrB (but not
EbrA) expressed in an EmrE-deficient E. coli strain8
were reported to confer resistance when expressed
alone. The pairs are believed to have originated
during evolution from a single-gene type by duplication, followed by diversification of the two genes.912
Structures of EmrE of E. coli obtained by cryoelectron microscopy13,14 and X-ray diffraction15
suggested that the two subunits in the homodimeric
complex would have opposite orientations in the
membrane (antiparallel), which would be in line
with the predicted orientation of the two subunits of
the heterodimeric complexes encoded by the pairs of
genes. In case of the single-gene type, or singles in
short, the two chemically identical protomers are
thought to insert into the membrane in both orientations; this has led to the concept of dual-topology
proteins.9,10 The active dimer would be formed by
the association of two oppositely oriented copies of
the protein. Biochemical experimental evidence has
been put forward in favor of, and against, an
antiparallel orientation of the subunits of the EmrE
dimer, but the matter is still under debate.8,1618
The dual-topology concept is thought provoking
and controversial because membrane proteins usually insert into the membrane in one single orientation. Asymmetry with respect to the plane of the
membrane makes sense, since it allows for functional
asymmetry with respect to the two compartments
separated by the membrane. Already in the mid1980s, von Heijne discovered a correlation between
the amino acid sequence of membrane proteins and
the orientation in the membrane.19 The so-called
positive-inside rule states that loops connecting
TMSs facing the cytoplasm contain more positively
charged arginine and lysine residues than loops
facing the external medium. Although a comprehensive mechanistic explanation has never been given for
the correlation, the predictive power of the positiveinside rule is evident. In fact, compelling evidence for
an antiparallel orientation of the heterodimers of the
SMR transporters is based on the positive-inside rule:
The subunits encoded by the gene pairs (pairs) are
predicted to have opposite orientations in the
membrane. Moreover, the proteins encoded by the
singles genes (singles) were shown to largely lack a
positive charge bias over the membrane, in line with
the proposed insertion in both orientations.9,12

Experimental approaches used in studies of the

orientation of SMR transporters in the membrane
and the orientation of the subunits in the dimeric
complexes they form include reporter fusion
studies, 9,10 mutational studies to redirect the
orientation,6,10 fusion of subunits, 20 single Cys
labelling,7,8,21 cross-linking,8,22 and electron paramagnetic resonance spectroscopy.23 Almost all of
these studies were performed with a single protein:
EmrE of E. coli. In the current study, an approach
that is less dependent on the properties of a
particular protein is taken. For a set of nine singles
and nine pairs (27 proteins in total) randomly
selected from the SMR family, the correlation
between the experimentally determined orientation
of the proteins in the membrane and the positiveinside rule was studied. For the pairs, the orientation
has been determined in the presence and in the
absence of the partner subunit.

Results
SMR family: Singles and pairs
Members of the SMR protein family are widely
distributed over bacteria and archaea. Two groups
are discriminated: those that are encoded by a
single gene on the chromosome (singles) and those
that are encoded in pairs of genes that form a
transcriptional unit (pairs). In this study, nine
singles and nine pairs (18 proteins) were selected
from various bacteria in the phyla Firmicutes,
Proteobacteria, Actinobacteria, and Deinococcus/
Thermus (Table 1). The pairwise sequence identity
of the selected proteins was below 60% in all cases. In
the group of 10 singles, most pairwise sequence
identities were between 30% and 45%, while in the
group of pairs, the identities were between 25% and
35% (data not shown). The sequence identity of the
two proteins in the same pair (Table 1) ranged
between 26% for pair ab-DGEO1956dgeo (see
Materials and Methods for naming conventions) of
Deinococcus geothermalis in the phylum Deinococcus/
Thermus and 51% for pair ab-SMEG3670msme of
Mycobacterium smegmatis in the phylum Actinobacteria. The large number of proteins included in the
study, the phylogenetic distance of the organisms,
and the sequence diversity of the proteins allow for
the extraction of general information on the protein
family independent of a particular sequence.
K + R charge distribution
An averaged membrane topology model of SMR
family proteins was constructed based on
TMHMM24 predictions of individual members (see
Materials and Methods).12,25 The model shows four
TMSs connected by short hydrophilic loops. The

Orientation of SMR Transporters

129

Table 1. Pairs and singles from the SMR family used in this study
Name

Lineagea

Organism

Strain

GI number

SIb (%)

Pairs
a-YKKCbsub/b-YKKCbsub
a-YVDSbsub/b-YVDSbsub
a-SUGE1efae/b-SUGE1efae
a-DGEO1956dgeo/b-DGEO1956dgeo
a-YDGFstyp/b-YDGFstyp
a-SMEG3670msme/b-SMEG3670msme
a-EBRAbsub/b-EBRAbsub
a-ECP4595ecol/b-ECP4595ecol
a-QACHlpla/b-QACHlpla

B-F-b
B-F-b
B-F-l
B-D
B-P-c
B-A
B-F-b
B-P-c
B-F-l

B. subtilis
B. subtilis
En. faecalis
D. geothermalis
Salmonella typhimurium
M. smegmatis
B. subtilis
E. coli
Lactobacillus plantarum

168
168
V583
DSM 11300
LT2
MC2 155
168
536
WCFS1

16078374/16078375
16080502/16080503
29374998/29374999
94986055/94986056
16764827/16764828
118472281/118469651
16078793/16078792
110644702/110644701
28379650/28379649

29
31
28
26
33
51
45
43
33

Singles
SCO1918scoe
RRUA3353rrub
CAC3666cace
STM1653styp
DGEO2170dgeo
RRUA0272rrub
SUGEllac
AAVE4701aave
EMREecol

B-A
B-P-a
B-F-c
B-P-c
B-D
B-P-a
B-F-l
B-P-b
B-P-c

S. coelicolor
Rhodospirillum rubrum
Clostridium acetobutylicum
Sa. typhimurium
D. geothermalis
R. rubrum
L. lactis
A. avenae
E. coli

A(3)2
ATCC 11170
ATCC 824
LT2
DSM 11300
ATCC 11170
IL1403
AAC00-1
K12

21220405
83594682
15896899
16764997
94986269
83591612
15672104
120613332
16128526

For naming conventions, see Materials and Methods.

a
First character: kingdom B, Bacteria. Second character: F, Firmicutes; P, Proteobacteria; D, Deinococcus; A, Actinobacteria. Third
character: class F-b, Bacillales; F-l, Lactobacillales; P-a, -subdivision; P-b, -subdivision; P-c, -subdivision.
b
Sequence identity between a-protein and b-protein.

distribution of positive charges in the loops of

selected singles and pairs was analyzed by plotting
the densities of positively charged residues (R + K)
in the odd-numbered and even-numbered loops
against one another, as described before.12 The 18
proteins in the group of pairs distribute over two
clusters: one above the diagonal and one below the
diagonal (Fig. 1). The numbers of proteins in both
clusters are the same, with one member of each
pair found in the cluster below the diagonal and
with the other found in the cluster above the
diagonal. Proteins in the cluster above the diagonal
have a surplus of positively charged residues in the
even loops. According to the positive-inside rule,
these proteins would have the N-terminus and the
C-terminus located at the external face of the membrane (NoutCout orientation). The group below the
diagonal has a surplus of positive charges in the
odd-numbered loops and, consequently, both the
N-terminus and the C-terminus are predicted to be at
the cytoplasmic side (NinCin orientation). It follows
that the two proteins in each pair are predicted to
have opposite orientations in the membrane. Seven
of the proteins with the NinCin orientation are
encoded by the first gene on the chromosome (open
symbols, named NinCin/NoutCout pairs), whereas
the other two are encoded by the second gene (closed
symbols, named NoutCout/NinCin pairs). The latter
two groups represent different duplication events in
evolution.12
The proteins in the group of singles are located
between the two clusters formed by the proteins in
pairs, suggesting a more even distribution of

positive charges over the odd and even loops

(Fig. 1). However, AAVE4701aave of Acidovorax
avenae, SCO1918scoe of Streptomyces coelicolor, and
EMREecol of E. coli have a clear charge bias

Fig. 1. Distribution of positive charges (K + R) in the

odd and even loops of SMR proteins. The density of
positive charges (K + R) in the even loops of singles
(stars) and pairs (open squares, a-protein; closed squares,
b-protein) in the SMR family was plotted against the
density in the odd loops. The family members are listed
in Table 1. Insets: Topology models for family members
in NinCin and NoutCout orientations. The cytoplasm is
below.

130

Orientation of SMR Transporters

suggesting an NoutCout orientation. The other

singles are located more closely to the diagonal,
indicating no strong preference for one orientation in
the membrane, in agreement with a dual orientation
in the membrane.
Orientation in the membrane
The orientation of the 9 singles and 18 proteins
from the pairs in the cytoplasmic membrane was
deduced from the cellular disposition of the Cterminus that was experimentally determined by
reporter fusions. The 28 genes were cloned in
appropriate vectors, resulting in translational
fusions with the genes coding for green fluorescent
protein (GFP) and mature alkaline phosphatase
(PhoA),26 and the fusion proteins were expressed
in E. coli SF100. GFP matures and folds properly
only when located in the cytoplasm, and it is not
fluorescent when translocated to the periplasm,2729
while PhoA is enzymatically active only when
exported to the periplasmic space and not in the
cytoplasm.30 A protein with a periplasmic localization of the C-terminus (e.g., the NoutCout orientation
in case of SMR family proteins) thus has high PhoA
activity when fused to PhoA and low fluorescence
when fused to GFP, while conversely, a protein with
a cytoplasmic localization of the C-terminus (NinCin
orientation) has low PhoA activity and high GFP
fluorescence.
The normalized PhoA activities and GFP fluorescence (see Materials and Methods) of the individual
proteins of the pairs and the singles fused to the
reporter proteins were plotted against one another
(Figs. 2a and 3a, respectively).9,10,26 Similarly as
observed for the K + R density distribution over the
even-numbered and odd-numbered loops, the proteins from the pairs were distributed over two
clusters of nine proteins each, one below the
diagonal and one above the diagonal, corresponding to high GFP/low PhoA (NinCin) and
high PhoA/low GFP (NoutCout), respectively. The
separation between the two clusters is more
pronounced than in the case of the K + R density
distribution (Fig. 1). Many of the proteins from the
pairs that resulted in a positive GFP signal have
negligible PhoA activity, while most of the proteins
from the pairs with high PhoA activity have low but
significant GFP fluorescence. The results for the two
proteins from each of the nine pairs are visualized in
Fig. 2b, which shows the logarithm of the ratio of the
normalized PhoA activity and GFP fluorescence.
The log(nAP/GFP) plot eliminates the effect of
differences in the expression levels of the proteins.26
In each pair, the log(nAP/GFP) value is positive for
one of the proteins (NoutCout) and negative for the
other (NinCin). It follows that the two proteins in
each pair have opposite orientations in the membrane, and there is a good correlation with the K + R

Fig. 2. Orientation in the membrane of pairs (a)

Normalized PhoA activities of C-terminal PhoA fusions
were plotted against the GFP fluorescence of C-terminal
GFP fusions for all proteins in pairs listed in Table 1.
(b) Log(nAP/GFP) plot for each individual protein. The
a-proteins (first genes in operons) are indicated with open
squares (a) and bars (b); the b-proteins are indicated with
closed squares (a) and bars (b).

charge distribution over the odd and even loops. In

addition, it follows that there are seven NinCin/
NoutCout pairs and two NoutCout/NinCin pairs.
Eight singles showed significant PhoA activity
and GFP fluorescence when fused to the respective
fusion partners (Fig. 3a), resulting in log(nAP/GFP)
values close to zero (Fig. 3b). One single, SUGEllac
of Lactococcus lactis, showed low activities with both
reporters. High PhoA activity and high GFP
fluorescence for the same protein indicate that part
of the population is inserted into the membrane in
one orientation and part of the population is
inserted into the membrane in the opposite orientation (i.e., dual topology).

Orientation of SMR Transporters

Fig. 3. Orientation in the membrane of singles (a)

Normalized PhoA activities of C-terminal PhoA fusions
were plotted against the GFP fluorescence of C-terminal
GFP fusions for the singles listed in Table 1. (b) Log(nAP/
GFP) plot for each individual protein.

Coexpression of pairs
In the experiments described so far, the orientation in the membrane of the proteins in pairs (Fig. 2)
was determined in the absence of the other protein
from the same pair. The possibility that the coexpression of the two proteins of a pair would affect
the orientation assay was investigated by cloning
the operons encoding four NinCin/NoutCout pairs
and two NoutCout/NinCin pairs into the reporter
fusion vectors. In the resulting expression vectors,
the first encoded protein in the operon (a-protein)
contains a His-tag at the N-terminus but no reporter
fused to the C-terminus, while the second protein
(b-protein) is not His-tagged but contains the
reporter protein at the C-terminus (see Fig. 4a and
c for the NinCin/NoutCout and NoutCout/NinCin

131
pairs, respectively). The effect of the presence of the
a-protein on the orientation of the b-protein was
determined by comparing the activities of the
reporter proteins fused to the b-protein in the
presence and in the absence of the a-protein.
When expressed alone, the PhoA activity of the
PhoA fusion of the b-protein from the ab-YVDSbsub
pair of B. subtilis was high, but the GFP fusion also
showed significant fluorescence (Fig. 4b, closed
square). In contrast, the a-protein showed strong
GFP fluorescence and no PhoA activity (open
square). Coexpression of the a-protein and the bprotein resulted in a slightly higher PhoA activity of
the b-protein PhoA fusion. In addition, GFP fluorescence of the b-protein GFP fusion was lower than
when it was expressed alone. Consequently, the data
point for the b-protein moves to the upper left corner,
making a stronger case for the NoutCout orientation
of the b-protein. Similar results were obtained for the
NinCin/NoutCout pairs ab-YKKCbsub of B. subtilis
and ab-ECP4595ecol of E. coli. In the latter case, the
increase in PhoA activity of the b-protein was
stronger with little effect on the GFP fluorescence.
The coexpression of the a-protein had no effect on the
orientation of the b-protein for pair ab-SUGE1efae of
Enterococcus faecalis.
The His-tag at the N-terminus of the solitary bproteins of NinCin/NoutCout pairs may hamper the
insertion of the proteins into the membrane, since
the (hydrophilic) tag has to be exported across the
membrane. To exclude the possibility that the
improved orientation signal for the b-protein
(when coexpressed with the a-protein) might be
caused by the absence of the His-tag, we also
assayed the orientation of the b-YVDSbsub and bECP4595ecol proteins from which the His-tags had
been removed in the absence of the corresponding aproteins. The PhoA activities and GFP fluorescence
of these fusion proteins were not significantly
different from the His-tagged versions, suggesting
that the His-tag does not affect the insertion of the
proteins into the membrane (data not shown).
Coexpression of NoutCout/NinCin pairs showed
similar results as observed for the NinCin/Nout
Cout pairs (Fig. 4d). The GFP fluorescence of the bprotein in the ab-EBRAbsub pair of B. subtilis
increased slightly, while the PhoA activity slightly
decreased when coexpressed with the a-protein. In
the case of ab-QACHlpla, the effects were even
smaller. It follows for both NinCin/NoutCout and
NoutCout/NinCin pairs that the orientation of the bprotein is consistently better determined in the
presence of the a-protein, but the effects are small.
Subunit interactions of pairs
Some SMR family members encoded in a pair of
genes have been shown to form heterodimers. Here,
interaction between the two proteins of the four

132

Orientation of SMR Transporters

Fig. 4. Orientation of one protein in a pair in the presence and in

the absence of the other protein. (a
and c) Models for the proteins in
NinCin/NoutCout pairs (a) and
NoutCout/NinCin pairs (c) when
expressed alone or together. Encircled H symbolizes the His-tag,
and the C-terminal star symbolizes
the reporter protein. The cytoplasm
is below (b and d) Normalized
PhoA activity and GFP fluorescence
of the solitary proteins and the
coexpressed proteins for the four
NinCin/NoutCout pairs (b), as indicated, and the two NoutCout/
NinCin pairs (d), as indicated.

NinCin/NoutCout pairs and the two NoutCout/

NinCin pairs from the previous paragraph was
demonstrated by copurification. The separately
expressed a-protein and b-protein have His-tags at
the N-terminus and bind to Ni2+ -NTA affinity resin.
When coexpressed as pairs, the b-protein lacks a

His-tag and will only be bound to the resin when

bound to the His-tagged a-protein (see Fig. 5).
Solubilized membranes prepared from standardized amounts of cells expressing the different
constructs were run through a small-scale batchwise
purification procedure (see Materials and Methods).

Orientation of SMR Transporters

133

Fig. 5. Subunit interactions of the proteins in pairs. (a) PhoA activity of the purified PhoA fusion proteins of the
indicated NinCin/NoutCout pairs when expressed and purified alone (both proteins with His-tag) and when coexpressed
and copurified (His-tag only on a-protein). Values were corrected for the background activity observed with elution
buffer (0.01 a.u.). (b) GFP fluorescence of purified GFP fusion proteins of the indicated NoutCout/NinCin pairs when
expressed and purified alone (both proteins with His-tag) and when coexpressed and copurified (His-tag only on aprotein). Values were corrected for the background fluorescence observed with elution buffer (0.1 a.u.). (a and b) Models
show the orientation of the a-protein and the b-protein. Encircled H symbolizes the His-tag, and the C-terminal star
symbolizes PhoA (a) or GFP (b). The cytoplasm is below.

The combination of low expression levels and smallscale purification resulted in amounts of purified
proteins that were too low to make them visible by
Coomassie-brilliant-blue-stained SDS-PAGE. Instead, the PhoA activity and GFP fluorescence of
the eluted fractions were measured. As expected,
PhoA activity was high in the purified fraction of the
solitary b-protein samples of the four NinCin/Nout
Cout pairs, while no activity was observed for the aproteins (Fig. 5). Significant activity was observed in
the samples of the coexpressed a-protein and bprotein, indicating copurification of the b-proteins
with the a-proteins. Similarly, GFP fluorescence was
measured in both of the purified samples when the
NoutCout/NinCin pairs where coexpressed. It
follows for both types of pairs that the two proteins
interact to form a multimeric complex.

Discussion
In this study, the orientation in the membrane of
27 proteins from the SMR family which is believed
to contain accessible information on early events in
the evolution of large two-domain transport proteins, was determined using the reporter fusion

technique to correlate the results with the positiveinside rule for these small membrane proteins. The
orientation in the membrane was determined from
the ratio of the normalized activities of PhoA and
GFP fused at the C-terminus of the proteins. In a
similar way, the preference for one particular
orientation based on the positive-inside rule was
deduced from the ratio of the positive charge
density in the loop regions at both sides of the
membrane. By analysis of a large number of proteins
with significant sequence divergence from the same
family, artifacts due to a particular sequence were
minimized, and the insertion behavior of the whole
family was investigated. Eighteen of the studied
proteins were encoded in pairs of genes and
believed to insert into the membrane in opposite
orientations, and the remaining nine proteins were
encoded monocistronically (singles) and believed
to insert in both orientations.
The orientation of the proteins in pairs was clearly
determined by fusions with the reporters GFP and
PhoA; high PhoA activity correlated with low GFP
fluorescence and vice versa, indicating that the
proteins were inserted with a strong preference for
one orientation (Fig. 2a). In each pair, when one
protein was NinCin, the other was NoutCout, in

134
agreement with the quaternary structure of an
antiparallel dimer. Seven pairs were NinCin/Nout
C out , and two pairs were N out C out /N in C in ,
depending on the orientation of the protein encoded
by the first gene in the operon (Fig. 2b). Most of the
proteins from the pairs with high GFP intensity
showed little or no PhoA activity; in contrast, most
of the proteins with high PhoA activity showed low
but significant GFP activity. Most likely, with a
cytoplasmic C-terminus, the insertion machinery
will have no tendency to export the reporter moiety.
On the other hand, with a periplasmic C-terminus,
the reporter has to be actively translocated over the
membrane, which may not be successful for a
fraction of the GFP reporters that subsequently
mature in the cytoplasm.31
In consideration of background activities in the
two reporters estimated from the proteins in pairs,
eight of the nine singles showed significant activity
with both reporters, while one protein (SUGEllac)
showed low activity with both reporters, indicating
low levels of expression. The expressed singles
showed significant activity, with both reporters
showing that the singles were present in significant
proportions in both orientations in the membrane,
consistent with a dual-topology mode of insertion.
EmrE of E. coli in the singles group was previously
reported to adopt an NoutCout orientation based on
GFP and PhoA reporter studies9 and an NinCin
orientation when a Myc-tag was used,32 which was
explained by a reporter-making-the-news argument. In this study, EmrE is reported to insert in
both orientation, which is more in line with the
insertion behavior of the other singles in the set.
Possibly, the low levels of expression used in this
study contribute to the result. The proteins in the
pair ab-EBRAbsub (EbrAB) of B. subtilis were shown
to have opposite orientations by site-directed
cysteine labeling,7 in agreement with the present
study. This shows that, at least for this pair, the
reporters do not influence the orientation of the
proteins in the membrane. The consistent outcome
for all pairs suggests that this may be generally true
for the assay system used here.
The experimental data show that in the collection
of 27 SMR proteins, the proteins encoded in pairs all
insert into the membrane in opposite fixed orientations, while proteins encoded as single genes insert
in both orientations, strongly suggesting that this is
general for all proteins in the family. Analysis of the
positive charge distribution over the loops located
on either side of the membrane demonstrated to
what extent the orientation of the individual
proteins is correctly predicted by the positive-inside
rule. In general and at first sight, there seems to be a
good correlation between the experiment and the
positive-inside rule (Fig. S1). A plot of the density of
positively charged residues in the odd loops against
the even loops of the proteins in pairs revealed two

Orientation of SMR Transporters

clusters: one below the diagonal and one above the

diagonal (Fig. 1). Within each pair, one protein was
located in the cluster above the diagonal, and the
other was located in the cluster below the diagonal,
predicting opposite orientations for the two proteins, in agreement with the experimental data. In
addition, the orientation of the proteins is predicted
correctly. On average, the prediction of the Nout
Cout orientation is somewhat stronger than that of
the NinCin orientation. In the same analysis, six of
the singles cluster close to the diagonal, indicating
no strong preference for one particular orientation,
again in agreement with the experimental data.
Remarkably, in the plots of both the charge
distribution and the experimental data, the cluster
is slightly above the diagonal, indicating a bias
towards the NoutCout orientation. The significance
of this offset, if any, is not clear. Surprisingly, three
singles (AAVE4701aave of A. avenae at the y-axis,
EmrE of E. coli, and SCO1918scoe of S. coelicolor)
showed a clear bias in the positive charge distribution, which was not in line with the experimental
data. These three proteins are predicted to be in the
NoutCout orientation, but are found in both orientations in the membrane in the reporter fusion study.
Analysis of the positive charge distribution in
AAVE4701aave revealed the presence of three
positive charges in the even loops and none in the
odd loops, making a strong but erroneous prediction for the NoutCout orientation (see Table S1).
EMREecol has four positively charged residues in
the loops, three in the even loops, and one in the odd
loops. Since the total numbers of residues in the odd
and even loops are almost the same, the charge
density distribution predicts the NoutCout orientation. Finally, SCO1918scoe has four positive charges:
three in the even loops and one in the odd loops.
The discrepancies discussed above demonstrate
that the positive-inside rule is not a good predictor
for all proteins. In addition, the three clusters in the
positive charge density distribution plots formed by
singles, NinCin proteins, and NoutCout proteins are
not well separated, making it difficult for each
individual protein to predict the orientation in the
membrane by the positive-inside rule alone. Dual
topology of the singles appears to be more poorly
predicted than the fixed topology of the pairs, which
may be related to the low numbers of positive
charges in the loops of the former. The nine singles,
on average, contain 2.1 and 3.4 R + K residues in the
odd and even loops, respectively, while the NinCin
and NoutCout proteins in pairs contain 6.1 and 1.8,
and 0.9 and 6.4, respectively (Table S1 in Supplementary Data). It follows that in an evolutionary
path, as indicated in the Introduction, a fixed
orientation of the protein products of the duplicated
genes is largely obtained by inserting positive
charges into the loops at one side of the membrane,
increasing the total number of charges in the loops.

Orientation of SMR Transporters

Initially, the (random) orientation would be determined by a combination of the intrinsic properties of
the amino acid sequence other than the positive
charges and the positive charge distribution over the
membrane where both would contribute equally.
Insertion of positively charged residues at one side
would pull the protein in one orientation; as their
number increases, they completely overrule the
other contributions to the orientation (i.e., the
orientation is determined by the positive-inside
rule). A threshold of about six residues in the
cytoplasmic loops appears to be essential for a fixed
orientation. A model like this would be supported
by a very recent study in which a single positively
charged residue added to any of the loops of
EMREecol shifted the protein to the orientation
with the added positive charge in the cytoplasm,
irrespective of the charge distribution over the
internal and external loops in the resulting mutant
protein.33
In conclusion, the insertion behavior of the
members of the SMR family may be predicted by
the embedding of the coding genes in the chromosome: singles insert in both orientations, and the two
proteins in pairs insert in opposite orientations.
Subsequently, the orientation of the two proteins in
pairs may be determined by an analysis of the
positive-inside rule when the whole family is
included. Examples such as AAVE4701aave of A.
avenae show that the positive-inside rule does not
mechanistically determine the orientation of this
type of proteins, and possibly of any membrane
protein, during insertion. This may not be surprising
as membrane proteins are inserted cotranslationally
and the orientation of the proteins will be determined following the insertion of the first TMS in one
orientation.
The proteins in the SMR family form homodimers
or heterodimers in the membrane, and the interaction between the two proteins in four NinCin/
NoutCout pairs and two NoutCout/NinCin pairs
was demonstrated (Fig. 5). Remarkably, there was
very little effect on the activities of the reporter
proteins fused to one protein by the presence or the
absence of the other protein (Fig. 4). For most of the
pairs, the ratio of the activities improved somewhat
to indicate one orientation. Possibly, the formation
of the dimer results in slight stabilization of the
intended orientation. In general, it follows that the
subunits by themselves are stably inserted into the
membrane, and no evidence for interaction between
the subunits during the early stages of insertion/
assembly was obtained. For two NoutCout proteins
in NinCin/NoutCout pairs, the effect of the His-tag
at the N-terminus on the orientation was investigated. There were no significant differences in the
GFP and PhoA activities of the b-YVDSbsub and bECP4595ecol proteins with and without the His-tag
at the N-terminus.

135

Materials and Methods

Strains and growth conditions
E. coli strain SF100 [recA lac ompT]34 harboring pLIC
vectors (see the text below) was grown in LuriaBerthani
broth medium supplemented with 50 g/ml ampicillin at
37 C with continuous shaking. Overnight cultures were
diluted 1:30 in 5 ml of fresh medium. Protein expression
from the pLIC vectors was induced by adding 0.004%
arabinose (wt/vol) when the optical density of the
cultures measured at 660 nm had reached a value of 0.6,
followed by additional growth for 1.5 h.
DNA techniques
The genes encoding SMR transporter subunits were
cloned by ligation-independent cloning35 in vectors pLIC1
and pLIC2 described before,26 resulting in PhoA and GFP
fusion proteins, respectively. Plasmid pLIC3, which does
not carry a reporter protein, was used as negative control.
The genes were amplified from the host organisms (Table 1)
by PCR using pfu polymerase (Fermentas). Forward
primers contained the overhang sequence 5-TCATCATCACCATCACCATTG-3 and the reverse primer 5-TACCACACCACTATTTTG-3 compatible with the ligationindependent cloning cassette. PCR products were treated
with T4 DNA polymerase (Fermentas) in the presence of
dCTP for 30 min at room temperature to create singlestranded tails. Similarly, plasmids pLIC1 and pLIC2, after
linearization with SwaI (Fermentas), were treated with T4
DNA polymerase, but in the presence of dGTP. T4
polymerase was inactivated by incubation at 75 C for
20 min. The T4 polymerase-treated PCR products and
vectors were combined and, after 5 min of incubation at
room temperature, transformed into E. coli SF100. The
pLIC1 and pLIC2 vectors encode the SMR transport protein
with an N-terminal His-tag linked with a leucine residue to
the protein, and at the C-terminus with the linker sequence
QNSGVVP followed by the reporter protein. Operons
containing pairs were cloned in the same way using the
forward primer for the a-protein and the backward primer
for the b-protein.
The His-tag encoding sequence in constructs pLIC1/
b-YVDSbsub and pLIC1/b-ECP4595ecol was removed by
PCR gene amplification, followed by restriction and
ligation. The sequence of the forward primers was 5GCGAAACCATGGCGGCATGGTTTTTATTAGTGATTGCCGGC-3 (p1/b-YVDSbsub-F) and 5-GCGAAACCATGGCGTTAAATATTGGATTTTTATGGCTG-3
(p1-ECP4595ecol-F) for b-YVDSbsub and b-ECP4595ecol,
respectively. The primers were complementary to the
sequence immediately following the His-tag sequence and
introduced a NcoI cleavage site at the 5 end. The reverse
primer was the same in both cases (pLIC1-R; 5TTGCGGCCGCTGCCAGCCATTTGCC-3). The primer
was targeted at a sequence downstream of the gene
encoding PhoA that contained a unique NotI cleavage site.
The PCR products and the pLIC1 vector were restricted by
the NcoI and NotI enzymes, followed by ligation using T4
DNA ligase (Fermentas). Treatment of the pLIC1 vector
with these enzymes removes the His-tag encoding
sequence.

Orientation of SMR Transporters

136
GFP and PhoA activity of whole cells
GFP fluorescence
Cells from 2 ml of culture were washed once and
resuspended in 50 mM TrisHCl (pH 8.0), 200 mM NaCl,
and 15 mM ethylenediaminetetraacetic acid to an OD660 of
0.2. N-Dodecyl--D-maltoside was added to the suspension to a final concentration of 0.5% (wt/vol). Fluorescence was measured with an Aminco-Bowman Series 2
Spectrometer using an excitation wavelength of 468 nm
and an emission wavelength of 507 nm. For each sample,
the background fluorescence of cells harboring the
plasmid pLIC3 carrying no insert was subtracted. Experiments were performed in triplicate.
PhoA activity
Cells from 2 ml of culture were washed once and
resuspended in 1 ml of 1 M TrisHCl (pH 8.0), and OD660
was measured. Following equilibration of 500 l of the
suspension at 37 C, p-nitrophenyl phosphate (SigmaAldrich) was added to a final concentration of 1.4 mg/ml.
The reaction was stopped by addition of 1 M K2HPO4
when a yellow color appeared. PhoA activity was
expressed in Miller units.36 Measurements were performed by three independent measurements.
GFP fluorescence and PhoA activities were related to
each other by a normalization factor n, which represents
the ratio of the averages of the positive PhoA and GFP
activities of the proteins in the data set.26 Log(nAP/GFP)
values were truncated at 2.5 and 2.5.
GFP and PhoA activities of purified proteins
His-tagged proteins were purified by Ni2+-NTA affinity
chromatography following a small-scale purification
protocol described before.25 Briefly, E. coli SF100 cells
were washed twice and then resuspended in 2 ml of
50 mM KPi (pH 7.0). Cells were broken by sonication
(Soniprep 150) using nine cycles of 15 s on and 45 s off
while keeping the suspension on ice. Unbroken cells and
debris were removed by centrifugation at 9000 rpm for
10 min. Membranes were collected by ultracentrifugation
using a Beckman TLA 100.4 rotor ( 80,000 rpm for 25 min
at 4 C) and washed once with 50 mM KPi (pH 7.0).
Subsequently, the membranes were solubilized for 30 min
on ice in 50 mM KPi (pH 8), 400 mM NaCl, and 10%
glycerol containing 1% Triton X-100. Undissolved material
was removed by ultracentrifugation at 80,000 rpm for
25 min. The supernatant was mixed with 100 l of Ni2+NTA resin. After overnight incubation, the column
material was pelleted by pulse centrifugation using a
tabletop centrifuge, and the supernatant was removed.
The resin was washed once with 500 l of 50 mM KPi
(pH 8.0), 300 mM NaCl, and 10% glycerol containing
40 mM imidazole and 0.1% Triton X-100. Bound proteins
were eluted with 50 l of 50 mM KPi (pH 8.0), 300 mM
NaCl, and 10% glycerol containing 150 mM imidazole and
0.1% Triton X-100.
PhoA activity in the eluted fraction was measured by
50-fold dilution in 50 mM KPi (pH 7), 300 mM NaCl,
150 mM imidazole, 10% glycerol, and 0.1% Triton X-100.
Subsequently, pNPP was addend to a final concentration

1.4 mg/ml. After incubation for 1 h at 37 C, the reaction

was stopped by adding 100 l of 1 M K2HPO4, and OD420
was measured. GFP fluorescence in the eluted fraction
was measured as described above using proper dilution of
the sample.
Computational methods
Proteins in pairs were named after the name of the
protein encoded by the first gene as annotated in the
database and preceded by a. The second protein was
given the same name, but was preceded by b. A
consensus topology model was constructed by combining
secondary structure prediction by TMHMM2.024 and
multiple-sequence alignment, as described before.12,25
Multiple-sequence alignments were computed using the
command line version of CLUSTAL W37 for the Windows
XP platform. The positions of the TMS were manually
adjusted to make sure that R and K residues at the loop/
TMS interfaces were part of the loops rather than the TMS.
The positive charge density in the loops at the two sides of
the membrane was determined by counting the positive
charges (R + K) in the loops defined in the consensus
topology model by the total number of residues in the
loops. The N-terminus and the C-terminus were treated
similarly but, obviously, extended at one side only.

Acknowledgements
We would like to thank Nicolas Pinel for
providing the genomic DNA of A. avenae subsp.
AAC00-1; Elena Gaidamakova for providing the
genomic DNA of D. geothermalis DSM 11300; Ulrich
Dobrindt for providing the genomic DNA of E. coli
536; Gary Roberts for providing the genomic DNA
of R. rubrum ATCC 11170; and Anne Wiersma for
providing the strain Lactob. plantarum WCFS1.38
This work was supported by a grant from the Dutch
Organization for Scientific Research (NWO-CW).

Supplementary Data
Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.jmb.2010.07.019

References
1. Bay, D. C., Rommens, K. L. & Turner, R. J. (2007).
Small multidrug resistance proteins: a multidrug
transporter family that continues to grow. Biochim.
Biophys. Acta, 1778, 18141838.

Downloaded from ftp://ftp.ebi.ac.uk/pub/software/

dos/clustalw/

Orientation of SMR Transporters

2. Bay, D. C. & Turner, R. J. (2009). Diversity and
evolution of the small multidrug resistance protein
family. BMC Evol. Biol. 9, 127.
3. Schuldiner, S. (2009). EmrE, a model for studying
evolution and mechanism of ion-coupled transporters.
Biochim. Biophys. Acta, 1794, 748762.
4. Jack, D. L., Storms, M. L., Tchieu, J. H., Paulsen, I. T. &
Saier, M. H., Jr. (2000). A broad-specificity multidrug
efflux pump requiring a pair of homologous SMRtype proteins. J. Bacteriol. 182, 23112313.
5. Zhang, Z., Ma, C., Pornillos, O., Xiu, X., Chang, G. &
Saier, M. H., Jr. (2007). Functional characterization of
the heterooligomeric EbrAB multidrug efflux transporter of Bacillus subtilis. Biochemistry, 46, 52185225.
6. Kikukawa, T., Nara, T., Araiso, T., Miyauchi, S. &
Kamo, N. (2006). Two-component bacterial multidrug
transporter, EbrAB: mutations making each component
solely functional. Biochim. Biophys. Acta, 1758, 673679.
7. Kikukawa, T., Miyauchi, S., Araiso, T., Kamo, N. &
Nara, T. (2007). Anti-parallel membrane topology of
two components of EbrAB, a multidrug transporter.
Biochem. Biophys. Res. Commun. 358, 10711075.
8. Nasie, I., Steiner-Mordoch, S., Gold, A. & Schuldiner, S.
(2010). Topologically random insertion of EmrE supports a pathway for evolution of inverted repeats in ioncoupled transporters. J. Biol. Chem. 285, 1523415244.
9. Rapp, M., Granseth, E., Seppl, S. & von Heijne, G.
(2006). Identification and evolution of dual-topology
membrane proteins. Nat. Struct. Mol. Biol. 13, 112116.
10. Rapp, M., Seppl, S., Granseth, E. & von Heijne, G.
(2007). Emulating membrane protein evolution by
rational design. Science, 315, 12821284.
11. Poolman, B., Geertsma, E. & Slotboom, D. J. (2007). A
missing link in membrane protein evolution. Science,
315, 12291231.
12. Lolkema, J. S., Dobrowolski, A. & Slotboom, D. J.
(2008). Evolution of antiparallel two-domain membrane proteins: tracing multiple gene duplication
events in the DUF606 family. J. Mol. Biol. 378, 596606.
13. Ubarretxena-Belandia, I., Baldwin, J. M., Schuldiner,
S. & Tate, C. G. (2003). Three-dimensional structure of
the bacterial multidrug transporter EmrE shows it is
an asymmetric homodimer. EMBO J. 22, 61756181.
14. Ubarretxena-Belandia, I. & Tate, C. G. (2004). New
insights into the structure and oligomeric state of the
bacterial multidrug transporter EmrE: an unusual
asymmetric homo-dimer. J. Mol. Biol. 340, 797808.
15. Chen, Y. J., Pornillos, O., Lieu, S., Ma, C., Chen, A. P. &
Chang, G. (2007). X-ray structure of EmrE supports
dual topology model. Proc. Natl Acad. Sci. USA, 104,
1899919004.
16. Schuldiner, S. (2007). When biochemistry meets
structural biology: the cautionary tale of EmrE.Trends
Biochem. Sci. 32, 252258; Erratum in Trends Biochem
Sci., 32, 300.
17. Korkhov, V. M. & Tate, G. M. (2009). An emerging
consensus for the structure of EmrE. Acta Crystallogr.
Sect. D, 65, 186192.
18. Schuldiner, S. (2007). Controversy over EmrE structure. Science, 317, 748751.
19. von Heijne, G. (1986). The distribution of positively
charged residues in bacterial inner membrane proteins correlates with the trans-membrane topology.
EMBO J. 5, 30213027.

137
20. Steiner-Mordoch, S., Soskine, M., Solomon, D., Rotem,
D., Gold, A., Yechieli, M. et al. (2007). Parallel topology
of genetically fused EMRE. EMBO J. 27, 1726.
21. Nara, T., Kouyama, T., Kurata, Y., Kikukawa, T.,
Miyauchi, S. & Kamo, N. (2007). Anti-parallel membrane topology of a homo-dimeric multidrug transporter, EmrE. J. Biochem. 142, 621625.
22. Soskine, M., Steiner-Mordoch, S. & Schuldiner, S.
(2002). Crosslinking of membrane-embedded
cysteines reveals contact points in the EmrE oligomer.
Proc. Natl Acad. Sci. 99, 1204312048.
23. Mchaourab, H. S., Mishra, S., Koteiche, H. A. & Amadi,
S. H. (2008). Role of sequence bias in the topology of
the multidrug transporter EmrE. Biochemistry, 47,
79807982.
24. Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer,
E. L. (2001). Predicting transmembrane protein topology with a hidden Markov model: application to
complete genomes. J. Mol. Biol. 305, 567580.
25. Dobrowolski, A., Sobczak-Elbourne, I. & Lolkema,
J. S. (2007). Membrane topology prediction by
hydropathy profile alignment: membrane topology
of the Na+-glutamate transporter GltS. Biochemistry,
46, 23262332.
26. ter Horst, R. & Lolkema, J. S. (2010). Rapid screening
of membrane topology of secondary transport proteins. Biochim. Biophys. Acta, 1798, 672680.
27. Feilmeier, B. J., Iseminger, G., Schroeder, D., Webber,
H. & Phillops, G. J. (2000). Green fluorescent protein
functions as a reporter for protein localization in
Escherichia coli. J. Bacteriol. 182, 40684076.
28. Daley, D. O., Rapp, M., Granseth, E., Meln, K., Drew,
D. & von Heijne, G. (2005). Global topology analysis
of the Escherichia coli inner membrane proteome.
Science, 308, 13211323.
29. Cassel, M., Seppl, S. & von Heijne, G. (2008).
Confronting fusion protein-based membrane protein
topology mapping with reality: the Escherichia coli
ClcA H+/Cl- exchange transporter. J. Mol. Biol. 381,
860866.
30. Manoil, C. & Beckwith, J. (1986). A genetic approach
to analyzing membrane protein topology. Science, 233,
14031408.
31. Van Geest, M. & Lolkema, J. S. (2000). Membrane
topology and insertion of membrane proteins: search
for topogenic signals. Microbiol. Mol. Biol. Rev. 64,
1333.
32. Ninio, S., Elbaz, Y. & Schuldiner, S. (2004). The
membrane topology of EmrEa small multidrug
transporter from Escherichia coli. FEBS Lett. 562,
193196.
33. Seppl, S., Slusky, J. S., Lloris-Garcer, P., Rapp, M. &
von Heijne, G. (2010). Control of membrane protein
topology by a single C-terminal residue. Science, 328,
16981700.
34. Baneyx, F. & Georgiou, G. (1990). In vivo degradation
of secreted fusion proteins by the Escherichia coli outer
membrane protease OmpT. J. Bacteriol. 172, 491494.
35. Geertsma, E. R. & Poolman, B. (2007). High-throughput cloning and expression in recalcitrant bacteria.
Nat. Methods, 4, 705707.
36. Manoil, C. (1991). Analysis of membrane protein topology using alkaline phosphatase and beta-galactosidase
gene fusions. Methods Cell Biol. 34, 6175.

138
37. Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994).
CLUSTAL W: improving the sensitivity of progressive
multiple sequence alignment through sequence
weighting, position-specific gap penalties and weight
matrix choice. Nucleic Acids Res. 22, 46734680.

Orientation of SMR Transporters

38. Kleerebezem, M., Boekhorst, J., van Kranenbur,
R., Molenaar, D., Kuipers, O. P., Leer, R. et al.
(2003). Complete genome sequence of Lactobacillus
plantarum WCFS1. Proc. Natl Acad. Sci. USA, 18,
19901995.