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doi:10.1016/j.jmb.2010.07.019
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
Edited by I. B. Holland
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.
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
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
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
130
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
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
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
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
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.
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.