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J. Biol. Chem., Vol. 276, Issue 22, 18905-18907, June 1, 2001
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From the Department of Biochemistry and Biophysics, Stockholm
University, S-106 91 Stockholm, Sweden
Received for publication, February 23, 2001
We have determined the topology of the
Escherichia coli inner membrane protein YrbG, a putative
Na+/Ca2+ exchanger with homology to a family of
eukaryotic ion exchangers. Our results show that the two homologous
halves of YrbG both have five transmembrane segments but opposite
membrane orientations. This has implications for our understanding of
the function of Na+/Ca2+ exchangers and
provides an example of "divergent" evolution of membrane protein topology.
The Escherichia coli YrbG protein belongs to a
superfamily of membrane transporters that includes both prokaryotic and
eukaryotic Na+/Ca2+ exchangers (1). All
proteins in this family have an internal repeat in their membrane
domain that presumably has arisen from a primordial gene duplication
event. A strongly conserved and functionally important so-called
In general, both prediction and experimental determination of membrane
protein topology is easier for bacterial than for mammalian proteins,
and we have thus undertaken a study of the E. coli family member YrbG. The sequence alignment in Fig. 1A shows clear
evidence of the internal repeat, including the two Interestingly, topology predictions using TOPPRED II (7), TMHMM (8),
and HMMTOP (9) suggest a topology with 10 transmembrane segments
for YrbG, with the first and second internal repeat having opposite
orientations in the inner membrane; see Fig. 1B. This is
reminiscent of a recently described case of "divergent" topology evolution where two homologous E. coli inner membrane
proteins, YdgQ and ORF193, each with 6 transmembrane segments,
were shown to have opposite membrane orientations (10). The predicted
topology for YrbG likewise suggests that a primordial protein with 5 transmembrane segments has evolved, after an internal gene duplication,
into a protein with 10 transmembrane segments where the first and
second halves insert into the membrane with opposite orientations. From this perspective, it is particularly noteworthy that the distribution of positively charged amino acids is different between the two halves
of YrbG; in the N-terminal half, Arg and Lys residues are predominantly
found in the loops between transmembrane segments 1/2 and 3/4, whereas
in the C-terminal half they are more prevalent in the loop preceding
the first transmembrane segment and between transmembrane segments 2/3
and 4/5; see Fig. 1B. The YrbG protein may thus be a second
example of divergent topology evolution, this time following an
internal gene duplication event.
Given the biological importance of the Na+/Ca2+
exchanger family and the topology evolution issue, we decided to
experimentally map the topology of YrbG using the PhoA fusion
approach (11). Our results provide strong support for the predicted
topology with 5+5 transmembrane segments, placing the two Enzymes and Chemicals--
Unless otherwise stated, all enzymes
were from Promega (Madison, WI). T7 DNA polymerase, Taq
polymerase, and [35S]Met were from Amersham
Pharmacia Biotech. T4 ligase was from Life Technologies, Inc.
Oligonucleotides were from CyberGene (Stockholm, Sweden). PhoA
antiserum was from 5 Prime Strains and Plasmids--
Experiments were performed in E. coli strains MC1061 ( DNA Techniques--
All plasmid constructs were confirmed by DNA
sequencing using T7 DNA polymerase. The yrbG gene was
amplified by PCR1 from
E. coli JM109 chromosomal DNA using Taq
polymerase and the Expand Long Template PCR system (Roche
Molecular Biochemicals). By use of appropriate PCR primers, a 5'
XhoI and a 3' KpnI site were introduced in the
regions flanking the amplified gene. The PCR products were cleaved with
XhoI and KpnI and cloned behind the
ara promoter in a XhoI-KpnI restricted
plasmid derived from pING1 containing a lep gene with a 5'
XhoI site just upstream of the initiator ATG and a
KpnI site in codon 78. Relevant parts of the yrbG
gene were amplified by PCR from the pING1 plasmid with a 5'
SalI and a 3' KpnI site encoded in the primers.
Finally, the amplified SalI-KpnI fragment
carrying the lep upstream region and the relevant
yrbG segment was cloned into a previously constructed plasmid (15) carrying a phoA gene lacking the 5' segment
coding for the signal sequence and the first 5 residues of the mature protein and immediately preceded by a KpnI site. In all
constructs, an 18-amino acid linker (VPDSYTQVASWTEPFPFC) was present
between the YrbG part and the PhoA moiety.
Expression of Fusion Proteins--
E. coli strain
CC118 transformed with the pING1 vector carrying the relevant construct
under control of the arabinose promoter was grown at 37 °C in M9
minimal medium supplemented with 100 µg/ml ampicillin, 0.5%
fructose, 100 µg/ml thiamin, and all amino acids (50 µg/ml each)
except methionine. An overnight culture was diluted 1:25 in fresh
medium, shaken for 3.5 h at 37 °C, induced with arabinose
(0.2%) for 5 min, and labeled with [35S]methionine (75 µCi/ml). After 2 min, samples were acid-precipitated with
trichloroacetic acid (10% final concentration), resuspended in
10 mM Tris/2% SDS, immunoprecipitated with antisera to
PhoA, washed, and analyzed by SDS polyacrylamide gel electrophoresis. Gels were scanned in a FUJIX Bas 1000 phosphorimager and analyzed using
the MacBAS software (version 2.31).
PhoA Activity Assay--
Alkaline phosphatase activity was
measured by growing strain CC118 transformed with the appropriate
pING1-derived plasmid in liquid culture for 2 h in the absence of
arabinose and then for 1 h in the presence of 0.2% arabinose
(16). Mean activity values were obtained from three independent
measurements and were normalized by the rate of synthesis (mean of
three experiments) of the fusion protein determined by pulse labeling
of arabinose-induced CC118 cells as described above. Normalized
activities were calculated as in Equation 1,
Topology Prediction and Sequence Alignment--
Topology
predictions were done using TOPPRED II (7), TMHMM (8), and
HMMTOP (9). Sequence alignments were done using BLASTP and PSI-BLAST
(6) at the NCBI website and LFASTA (17) at the Biology Workbench
3.0 website on the Internet. Default parameter settings were used in
all cases.
Determination of the Topology of YrbG by PhoA Fusions--
To
determine the membrane topology of YrbG, a series of PhoA fusions were
made. As recommended (18), the fusion joints were generally placed near
the C-terminal end of the putative periplasmic and cytoplasmic loops;
see Fig. 1B. All fusions could
be expressed in the phoA
The alkaline phosphatase activities are in general in very good
agreement with the predicted topology. The only apparent exception is
the somewhat high activity of fusion 1 (placed in the loop between
transmembrane segments 1 and 2). PhoA fusions directly after the first
transmembrane segment in polytopic proteins with an
Nout orientation (i.e., with a periplasmic N
terminus) often give high activities, and there are now a number of
examples where it has been found that more than one transmembrane
segment needs to be present to ensure translocation of an N-terminal
tail across the membrane (19). Because the hydrophobicity profile is
very distinct in this area of the protein, and because all the other fusions are consistent with the predicted overall
Nout-Cout topology, it seems likely that the
high "uncorrected" activity of the most N-terminal PhoA fusion does
not properly reflect the orientation of the first transmembrane segment
in the full-length protein.
We conclude that YrbG has a two-domain structure where each domain has
five transmembrane segments but where the two domains have opposite
orientations in the inner membrane. This also places the two conserved
In this study, we have mapped the topology of the E. coli inner membrane protein YrbG, which belongs to a large family
of Na+/Ca2+ exchangers that includes both
prokaryotic and eukaryotic proteins. Sequence alignment, topology
prediction, and experimental topology mapping using PhoA fusions
strongly suggest a two-domain structure, each with five transmembrane
segments and with opposite orientations in the membrane. As shown in
Fig. 1B, this places the two copies of the conserved
Equally interesting, YrbG provides a clear example of divergent
topology evolution where the two homologous halves of the protein have
evolved opposite orientations in the membrane. Presumably, the
redistribution of positively charged Arg and Lys residues between the
different loops in the protein underlies this evolutionary process, as
has been suggested earlier for the two homologous E. coli
inner membrane proteins YdgQ and ORF193 (10). In fact, the bacterial
homologues of YrbG found by a PSI-BLAST search of the Entrez protein
sequence data base all have the same predicted Nout-Cout topology with 10 transmembrane
segments, except for the Synechocystis homologue (slr0681),
which has an extra predicted C-terminal transmembrane segment not
present in the other proteins, and all have the same enrichment of
positively charged residues in their predicted cytoplasmic loops as
seen for YrbG (data not shown).
Whether the postulated primordial YrbG half-protein with five
transmembrane segments had a single orientation in the membrane or
could insert both ways (Nin-Cout and
Nout-Cin) as is the case for,
e.g. ductin (21), is unknown. If it had only a
single orientation, it is unlikely that a topology with 10 transmembrane segments would be adopted by anything but a very small
fraction of the molecules immediately after the gene duplication.
Instead, the majority would most likely adopt a so-called "leave one
out" topology where one of the hydrophobic segments would not insert
across the membrane, and all highly charged loops would remain on the cytoplasmic side (22). A considerable number of mutational events would
then be required before most molecules would insert into the membrane
with the Nout-Cout topology seen today. In any
case, our results underscore the importance of the "positive
inside" rule (23, 24) for determining membrane protein topology and suggest that divergent topology evolution or even topology inversion may be a more prevalent phenomenon than hitherto thought.
We thank Dr. Kyle Cunningham, Johns Hopkins
University, for drawing our attention to the
Na+/Ca2+ exchanger family.
*
This work was supported by grants from the Swedish Cancer
Foundation and the Swedish Natural and Technical Sciences Research Councils (to G.v.H.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, March 19, 2001, DOI 10.1074/jbc.M101716200
The abbreviation used is:
PCR, polymerase chain
reaction.
The Internal Repeats in the Na+/Ca2+
Exchanger-related Escherichia coli Protein
YrbG Have Opposite Membrane Topologies*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-motif that spans two of the predicted transmembrane helices is
present in each copy of the internal repeat (1, 2). Attempts to
determine the membrane topology of the mammalian
Na+/Ca2+ exchanger have yielded conflicting
results (3-5), and it is not clear whether the two conserved
-motifs are located on the same or opposite sides of the membrane.
Obviously, a resolution of this point will have important functional
implications for this class of proteins.
-motifs. Overall,
there is ~34% identity between the N- and C-terminal halves of YrbG, and a BLASTP (6) search of the Entrez protein sequence data bank using
the N-terminal half (residues 1-155) finds the C-terminal half
(residues 172-320) with an E-value of 2 × 10
16 (data not shown).
-domains
on opposite sides of the membrane and suggesting that divergent
topology evolution may be more prevalent than hitherto thought.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
3 Prime, Inc. (Boulder, CO). The
alkaline phosphatase chromogenic substrate PNPP (Sigma 104 phosphatase substrate) was from Sigma.
lacX74, araD139,
(ara, leu)7697, galU, galK, hsr, hsm, strA) (12) and
CC118 ((ara-leu)7697, lacX74,
phoA20, galE, galK, thi, rpsE, rpoB,
argE(am), recA1) (13). All constructs were expressed from the pING1 plasmid (14) by induction with arabinose.
where A0 is the measured activity,
OD600 is the cell density at the time of pulse labeling,
nMet is the number of Met residues in the fusion protein,
and CPM is the intensity of the relevant band measured on the phosphorimager.
(Eq. 1)
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
strain CC118 (13),
could be immunoprecipitated by a polyclonal PhoA antiserum, and were of
the expected sizes; see Fig.
2A. Alkaline phosphatase
activities were measured according to Ref. 16 and are shown in
Fig. 2B.
View larger version (35K):
[in a new window]
Fig. 1.
A, LFASTA alignment of the N- and
C-terminal halves of YrbG. The predicted transmembrane segments are
underlined, and the conserved -motifs are
boxed. Note that each -motif consists of two parts
separated by a short, less well conserved loop. B, proposed
topology for YrbG. The two homologous halves are indicated by
black and hatched transmembrane segments,
respectively, and the two -motifs are shown by thick
lines. The number of Lys + Arg residues is indicated in each
loop. Each fusion is indicated by its number; the
exact position of each fusion joint is given in the legend to Fig.
2.
View larger version (47K):
[in a new window]
Fig. 2.
A, expression of YrbG-PhoA fusion
proteins in E. coli CC118 cells. Cells were labeled for 2 min by [35S]Met, and fusion proteins were
immunoprecipitated by a PhoA antiserum and analyzed by SDS
polyacrylamide gel electrophoresis. Lane 1,
YrbG37-PhoA; lane 2, YrbG67-PhoA;
lane 3, YrbG104-PhoA; lane 4,
YrbG128-PhoA; lane 5, YrbG171-PhoA;
lane 6, YrbG209-PhoA; lane 7,
YrbG239-PhoA; lane 8, YrbG275-PhoA;
lane 9, YrbG300-PhoA; lane 10,
YrbG325-PhoA. B, alkaline phosphatase activities
for the different YrbG-PhoA fusions. Black bars indicate the
measured activities (in Miller units) before correcting for expression
levels, and white bars indicate activities after this
correction (with the expression level for fusion 1 set to 1; see
"Materials and Methods").
-motifs on opposite sides of the membrane. The 5+5 topology implies
that YrbG has undergone a process of "internal" divergent topology
evolution after the primordial gene duplication event.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-motif on opposite sides of the membrane, suggesting that this will
also be the case for the eukaryotic family members. Some experimental
support for this idea has been obtained previously (5). In terms of
three-dimensional structure, this means that proteins in the
Na+/Ca2+ exchanger family will have a
quasi-symmetric organization relative to the plane of the membrane,
somewhat reminiscent of the aquaporins where two copies of the
so-called NPA loop, one entering the membrane from the cytoplasmic side
and one from the extra-cytoplasmic side, are thought to interact in the
middle of the pore (20).
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed. Tel.: 46-8-16-25-90;
Fax: 46-8-15-36-79; E-mail: gunnar@dbb.su.se.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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