From the Department of Cell Membrane Biology,
Institute of Scientific and Industrial Research, Osaka University,
Ibaraki, Osaka 567-0047, § Faculty of Pharmaceutical
Science, Osaka University, Suita, Osaka 565-0871, and
CREST,
Japan Science and Technology Corporation, Osaka 567-0047, Japan
Received for publication, August 31, 2000, and in revised form, January 22, 2001
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ABSTRACT |
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Bacterial Tn10-encoded
metal-tetracycline/H+ antiporter was the first
found drug exporter and has been studied as a paradigm of
antiporter-type major facilitator superfamily transporters. Here the
400 amino acid residues of this protein were individually replaced by
cysteine except for the initial methionine. As a result, we could
obtain a complete map of the functionally or structurally important
residues. In addition, we could determine the precise boundaries of all
the transmembrane segments on the basis of the reactivity with
N-ethylmaleimide (NEM). The NEM binding results indicated
the presence of a transmembrane water-filled channel in the
transporter. The twelve transmembrane segments can be divided into
three groups; four are totally embedded in the hydrophobic interior,
four face a putative water-filled channel along their full length, and
the remaining four face the channel for half their length, the other
halves being embedded in the hydrophobic interior. These three types of
transmembrane segments are mutually arranged with a 4-fold symmetry.
The competitive binding of membrane-permeable and -impermeable
SH reagents in intact cells indicates that the transmembrane
water-filled channel has a thin barrier against hydrophilic molecules
in the middle of the transmembrane region. Inhibition and stimulation
of NEM binding in the presence of tetracycline reflects the
substrate-induced protection or conformational change of the
Tn10-encoded metal-tetracycline/H+ antiporter. The
mutations protected from NEM binding by tetracycline were mainly
located around the permeability barrier in the N-terminal half,
suggesting the location of the substrate binding site.
The transposon Tn10-encoded metal-tetracycline/H+
antiporter (TetA(B))1 (1) was the first found bacterial
drug exporter (2) and has been studied as
a paradigm of bacterial drug efflux proteins. It belongs to a major
facilitator superfamily (3), and its 12-membrane-spanning structure
(Fig. 1) was established by site-directed competitive chemical
modification of cysteine mutants of a cysteine-free TetA(B) (5).
We previously reported that putative transmembrane helices (TM) 3 (6),
6 (7), and 9 (8) are totally embedded in a highly hydrophobic
environment, because none of the cysteine-scanning mutants as to
these transmembrane helices reacted with a maleimide derivative,
N-ethylmaleimide (NEM), whereas cysteine mutants as to
putative loop regions are generally highly reactive with NEM, except
for those as to a small number of non-reactive positions (9). Similar
transmembrane segments totally embedded in a hydrophobic interior are
known for the erythrocyte anion exchanger (10) and for the bacterial
small multidrug efflux protein EmrE (11). On the other hand, some
transmembrane cysteine mutants of major facilitator superfamily
transporters such as lactose permease (12-18) and UhpT (19) are
certainly inactivated by SH reagents, indicating that in these
transporters SH reagents can possibly gain access to some of the
residues located in the interior of the transmembrane region.
Afterward, we found that some cysteine-scanning mutants as to TM2 of
TetA(B) are reactive with NEM (20). The NEM-reactive positions
periodically appeared along the full-length of TM2, indicating
that one side of this transmembrane helix faces a water-filled transmembrane channel. It is impossible that such a water-filled channel is composed of only one amphiphilic helix. Some counterparts should be present in the transmembrane region of TetA(B). Very recently, we analyzed cysteine-scanning mutants of TM1 and TM11 (21)
and TM4 and TM5 (22) and found that TM5 and TM11 also face the channel
along their full length, whereas only the periplasmic (C-terminal) and
cytoplasmic (C-terminal) halves of TM1 and TM4 face the channel,
respectively. Considering the amphiphilic nature of the
metal-tetracycline chelation complex (1), the water-filled channel may
be at least a part of a substrate translocation pathway.
In this study, we constructed cysteine-scanning mutants as to the
remaining four putative transmembrane segments, TM7, TM8, TM10, and
TM12, and the central large loop region, loop6-7. Now, we have
obtained a complete set of the 400 cysteine-scanning mutants of
TetA(B), except for the mutant as to the initial methionine. In this
manuscript, we report the results of NEM binding and competitive binding of NEM and a membrane-impermeable sulfhydryl reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS), in
comparison with the results for the other eight transmembrane helices.
We first drew a complete map of the wall of the transmembrane water-filled channel and demonstrated the presence of a permeability barrier for hydrophilic molecules in the middle of the channel.
Materials--
N-[Ethyl-1-14C]maleimide
(1.5 giga becquerel/mmol) was purchased from PerkinElmer
Life Sciences. All other materials were of reagent grade and obtained
from commercial sources.
Site-directed Mutagenesis--
Cysteine-scanning mutants were
constructed by oligonucleotide-directed site-specific mutagenesis
according to the method of Kunkel (23) using synthetic
oligonucleotides. For the mutagenesis, plasmid pCTC377A (8), which
carries the 2.45-kilobase Tn10 tetA and tetR gene
fragments, was used as a template. All of the mutations were detected
as the appearance of a newly introduced restriction site and then
verified by DNA sequencing.
Low copy number mutant plasmids were constructed through exchange of
EcoRV-BamHI fragments (R186C to S201C) or
EcoRI-BamHI fragments (V202C to A401C) of
cysteine-scanning mutant tetA(B) genes with the
corresponding fragments of low copy number plasmid pLGC377A, which
encodes a cysteine-free mutant TetA(B) (8), and were then used for
tetA(B) gene expression.
Determination of Tetracycline Resistance--
Tetracycline
resistance was determined by means of the 2-fold agar dilution method
as described previously (24) and expressed as the minimum inhibitory concentration.
Assaying of the Reaction of [14C]N-Ethylmaleimide
with TetA(B) Proteins--
The [14C]NEM binding
experiment was performed as described previously (9). In brief, a
membrane suspension prepared by brief sonication of
Escherichia coli W3104 cells carrying pLGC377A or one
of its derivatives was incubated with 0.5 mM
[14C]NEM in 50 mM MOPS-KOH buffer (pH 7.0)
containing 0.1 M KCl for 5 min at 30 °C. The membrane
protein was solubilized with 1% Triton X-100 and 0.1% SDS in
phosphate-buffered saline containing 5 mM unlabeled NEM and
then TetA(B) proteins were immunoprecipitated with anti-TetA(B)
C-terminal peptide antiserum (25) and Pansorbin Staphylococcus
aureus cells (26). The TetA(B) protein was separated by SDS
polyacrylamide gel electrophoresis followed by Coomassie Brilliant Blue
staining. The dried gel was exposed to an imaging plate for
visualization with a BAS-1000 bioimaging analyzer (Fuji Film Co.,
Tokyo, Japan).
Prevention of NEM Binding with AMS--
5 µl of 100 mM AMS (final concentration, 5 mM) or the same
volume of distilled water was added to 100 µl of a suspension of E. coli W3104 cells expressing a mutant TetA(B) or a
sonicated membrane suspension containing a mutant TetA(B), followed by
incubation for 30 min at 30 °C. Subsequently, 2 µl of 25 mM [14C]NEM (final concentration, 0.5 mM) was added to the reaction mixture, followed by
incubation for 5 min and then the mixture was diluted with 900 µl of
the same buffer containing 5 mM unlabeled NEM. Immediately
after the dilution, the cells were collected and washed once with the
same buffer. Then the cells were disrupted by brief sonication, and
membranes were collected. The membranes were solubilized and TetA(B)
proteins were immunoprecipitated as described above. The radioactive
band densities on pretreatment with AMS were compared with those
without AMS treatment.
Construction of Cysteine-scanning Mutants and Their Drug
Resistance--
We have hitherto constructed 260 cysteine-scanning
mutants as to TM1 to TM6, TM9 and TM11, and the connecting loop regions of TetA(B) and reported their reactivity with sulfhydryl reagents (6,
7, 8, 20, 21, 22). In this study, we constructed 140 cysteine-scanning
mutants as to TM7, TM8, TM10, and TM12 and their connecting loop
regions. As a result, we have now obtained a complete set of
cysteine-scanning mutants of TetA(B) comprising 400 cysteine mutants
except for one as to the initial methionine. Three of these 140 cysteine mutants, S199C, S201C, and R238C, had been previously
constructed (5); therefore, the number of newly constructed mutants was
137. With respect to the 11 residues, Leu213,
Ile216 to Gly224, and Pro227,
we had constructed glutamic acid and/or aspartic acid mutants (27), and
alanine mutants of the other two residues, His257 and
Gln261, were also reported previously (28).
The tetracycline resistance levels of these 140 cysteine-scanning mutants are shown in Table
I. Surprisingly, the essential residues
of which cysteine mutants showed host-level resistance (0.8 µg/ml)
numbered only one (G247C). The cysteine mutants that showed drastically
reduced drug resistance of no more than 50 µg/ml were D190C, E192C,
S201C, and M210C for the putative central loop region, F234C and R238C
for putative loop7-8, G254C, H257C, and S258C for putative TM8, L305C
and A317C for putative TM10, Q319C for putative loop10-11, and Y376C,
L382C, and T385C for putative TM12. In summary, the number of mutants
that showed reduced or no resistance was 16 (11%). The other 124 mutants (89%) retained almost full resistance comparable with that of
the wild type.
[14C]NEM Binding to Cysteine-scanning Mutants--
A
maleimide derivative reacts with a deprotonated form of a sulfhydryl
group, and this reaction requires a water molecule as a proton acceptor
(20); thus the degree of NEM binding will depend on the presence of a
water molecule. Therefore, a region embedded in a hydrophobic
environment can be distinguished on the basis of the NEM reactivity of
a cysteine mutant (6, 7, 8, 20).
Fig. 2 shows the [14C]NEM binding to the
cysteine-scanning mutants. There were no significant differences in the
expression levels of these 138 cysteine-scanning mutants (data not
shown). The NEM binding results were confirmed to be reproducible. On the basis of the hydropathy profiles, the boundary between the central
loop region and TM7 was predicted to be Pro211 (Fig.
1); however, the I212C and L213C mutants
showed significantly high reactivity with NEM (Fig.
2). The reactivity
of mutants R186C to L213C with NEM was
generally high except in the case of the four scattered mutants, S201C, Y203C, I204C, and P211C. In comparison with the results of NEM binding to the mutants as to around TM6 (7), it
can be concluded that the central hydrophilic loop region comprises the
35 residues between Phe179 and Leu213.
Similarly, although the periplasmic boundary of TM7 was predicted to be
Phe234 (Fig. 1), it should be shifted to
Val232, because the NEM reactivity of L233C and F234C was
very high (Fig. 2), and that of the following mutants was also high. As a result, TM7 comprises 19 residues, this length being four residues shorter than the original prediction (Fig. 1), whereas the central position of TM7 was not shifted. TM7 included two highly reactive positions as to NEM, G224C and A228C. These positions were clustered in
the periplasmic (C-terminal) half of TM7 and located on one side of the
helical wheel (Fig. 3). This pattern is
similar to that of TM1 (21). Although the number of NEM-reactive
positions in TM1 was greater than that in TM7, the reactive positions
were also concentrated on one side of the periplasmic half of TM1. These observations indicated that TM7 is tilted away from a
water-filled channel, and only its periplasmic half faces the channel,
similar to the case of TM1.
The mutants as to the putative loop7-8 region, T235C to M245C, were
all highly or moderately reactive with NEM (Fig. 2). Thus, there is no
doubt that the N-terminal boundary of TM8 is Val246 (Fig.
5). On the other hand, it is difficult to determine the precise
boundary between TM8 and loop8-9, because the N-terminal half of TM8
includes many NEM-reactive positions. Two alternative boundaries are
possible, that is, Leu256 and Phe260. On the
basis of only the NEM binding results, Leu256 seems to be
more natural than Phe260 as a boundary, because the
following three positions (H257C, S258C, and V259C) are highly reactive
with NEM. However, if Val246 and Leu256 are at
the two ends of TM8, it comprises only 11 residues, which is far less
than the length required for a polypeptide chain to cross the membrane
once in an
We previously reported that the periplasmic boundary of TM10 is
undoubtedly Trp299 (8). In this study, we constructed
cysteine mutants as to TM10 downstream from P303C. As shown in Fig. 2
and by our previous results (8), TM10 can be clearly divided into two
parts. The periplasmic (N-terminal) half is highly hydrophobic and
includes no NEM-reactive position. In contrast, the cytoplasmic half
includes some NEM-reactive positions (Fig. 2). The NEM-reactive
positions are also distributed periodically, and as a result, they are
located on one side of the wheel (Fig. 3). Therefore, the cytoplasmic half of TM10 seems to face the water-filled channel. This situation is
similar to that of TM4 (22). These two helices seem to be tilted away
from the channel in opposite directions to TM1 and TM7. On the basis of
the NEM binding results, Leu318 seems to be the most
natural boundary between TM10 and loop10-11. If so, TM10 comprises
just 20 amino acid residues, whereas the possibility cannot be excluded
that the C-terminal boundary is Met322 or
Thr326. However, if the former or latter is the case, TM10
comprises 24 or 28 amino acid residues, respectively.
Previously, we reported that the mutants as to L361C to G366C were all
reactive with NEM (22). In this study, we observed that the 19 mutants
as to W367C to T385C were all not or only slightly reactive with NEM,
and the following mutants, as to F386C to the C-terminal end of
TetA(B), were highly reactive with NEM, except for that as to T389C.
Therefore, it is clear that the boundaries of TM12 are
Trp367 and Thr385 (Fig. 5). This
conclusion is almost the same as the original prediction (Fig. 1)
except that one end was shortened by one residue (Phe386).
TM12 was revealed to be a highly hydrophobic transmembrane segment
similar to TM3, TM6, and TM9.
Prevention of [14C ]NEM Binding by
Membrane-impermeable Maleimide Derivative AMS in Intact Cells--
The
results of [14C]NEM binding indicated that three of the
four transmembrane segments examined in this study face the
water-filled channel. One of them (TM8) faces the channel along its
full length, indicating that the channel completely crosses the
membrane. If such a water-filled channel crosses the membrane, TetA(B)
must cause uncoupling. We previously showed that a permeability barrier for hydrophilic molecules exists in the middle of the channel (21,
22). Thus, we examined whether the permeability barrier is in TM7, TM8,
or TM10. Fig. 4 shows the effect of
pretreatment of intact cells and sonicated membranes with AMS on the
[14C]NEM binding to the NEM-reactive cysteine mutants.
Because AMS molecules cannot pass through the cytoplasmic membrane,
they should not have access to cysteine residues located inside the
permeability barrier in intact cells, and therefore they should not
prevent [14C]NEM binding in intact cells. In contrast,
the NEM binding to cysteine residues located outside the permeability
barrier should be prevented by AMS in intact cells. In the case of
unsealed sonicated membranes, AMS molecules prevent NEM binding to
cysteine residues regardless of their location inside or outside the
permeability barrier, because AMS molecules can gain access from both
sides to TetA(B). If a cysteine residue is embedded in the narrow gap to which NEM molecules can gain access but AMS molecules cannot, AMS
molecules should not prevent NEM binding to cysteine residues in either
intact cells or sonicated membranes.
With respect to TM7, NEM binding to G224C was not affected by AMS in
intact cells (Fig. 4A, upper panel). The binding
to A228C was significantly prevented, and the binding to V232C and
L233C was completely blocked. On the other hand, NEM binding to
all of these mutants was completely prevented by AMS in sonicated membranes (Fig. 4A, lower panel). Thus, the
permeability barrier seems to exist between Gly224 and
Ala228.
In TM8, NEM binding to the NEM-reactive mutants as to upstream from
L250C was completely prevented by AMS, whereas that to the mutants as
to downstream from L253C was not affected in intact cells (Fig.
4B, upper panel). Therefore, it can be concluded
that the permeability barrier is located between Leu250 and
Leu253. Interestingly, the NEM binding to the G254C mutant
was not affected by AMS even in sonicated membranes, whereas that to
the other mutants was completely prevented in sonicated membranes (Fig. 4B, lower panel), indicating that
Gly254 may be embedded in the narrow gap in the channel
wall. For TM10, NEM binding to all the NEM-reactive mutants was not
affected by AMS in intact cells and completely prevented in sonicated
membranes (Fig. 4C). These results indicated that TM10 faces
the channel only inside the permeability barrier.
In Fig. 5, we depict the permeability
barrier (green line) in TM7, TM8, or TM10. They are located
at almost the same depth in the membrane. We also revealed the presence
of a permeability barrier in TM2 (data not shown). Therefore, in
comparison with the previously reported results (21, 22), all of the
channel-facing transmembrane segments revealed the presence of a
permeability barrier. These observations strongly indicated that these
helices face the same unique water-filled channel with a single
permeability barrier.
The Effect of Tetracycline on NEM Binding--
First of all, we
investigated the effect of tetracycline on the [14C]NEM
binding to 76 NEM-reactive cysteine-scanning mutants as to loop6-7,
TM7, loop7-8, TM10, TM12, the C terminus, and their flanking regions.
To our surprise, there was no significant effect on the NEM binding to
the Cys mutants except for the mutants as to TM8 and its flanking
regions. Fig. 6 shows only the results as
to TM8 and the flanking regions. Of 76 Cys mutants, the NEM binding
only to the S258C and V259C mutants was inhibited, and that to the
S243C mutant was stimulated (Fig. 6A). The degree of NEM
binding to the S258C and V259C mutants in the presence of 1 mM tetracycline was decreased to 40 and 50%, respectively, compared with those in the absence of tetracycline. In contrast, the
binding to the S243C mutant was increased to 230% in the presence of
tetracycline. The former two mutations are located in the cytoplasmic half of TM8, and the latter mutant is in periplasmic loop7-8 (Fig. 7).
Then, we reinvestigated the effect of tetracycline on the NEM binding
to all of the Cys mutants of TetA(B). As a result, the binding to the
eight Cys mutants including S258C and V259C was prevented by
tetracycline, and that to five Cys mutants was stimulated including
S243C (Fig. 7). Among them, we previously reported the prevention of
the NEM binding to the L97C (periplasmic loop3-4) (6), A109C (TM4),
and G141C (TM5) (22) mutants and the stimulation of that to the S65C
mutant (cytoplasmic loop2-3) (29). In the current study, we
found that the NEM binding to the G20C (TM1), M23C (TM1), and A51C
(TM2) was inhibited to 60, 60, and 50%, respectively, in the presence
of 1 mM tetracycline, whereas that to the L47C (TM2), L351C
(TM11), and A354C (TM11) was stimulated to 180, 230, and 220%,
respectively (Fig. 6B). Because all these Cys mutants retained their drug resistance activity except the G141C mutant, the
alterations in their NEM accessibility must reflect the
tetracycline-induced conformational change or the protection by
tetracycline binding during the substrate transport process. Five
prevented mutations were concentrated in the vicinity of the
permeability barrier in the N-terminal half, probably suggesting that
the region involves the substrate binding site.
In this study, we accomplished the construction of a complete set
of cysteine-scanning mutants of the tetracycline/H+
antiporter. To our surprise, only a small number of the mutants (around
14%) showed reduced drug resistance. The majority of the mutants
retained full level resistance. This is very advantageous for topology
determination by means of the site-directed chemical labeling method.
Fig. 7 shows a summary of the detailed topology of TetA(B) determined
on the basis of the results of this study and our previous studies (6,
7, 8, 21, 22). Each transmembrane segment comprises a number of amino
acid residues between 24 (TM11) and 15 (TM8); however, it should be
noted that this method tends to underestimate the numbers of amino acid
residues in "hydrophilic" or "amphiphilic" transmembrane
segments. Highly hydrophilic segments such as TM5 and TM8 are
followed by entirely hydrophobic segments, TM6 and TM9, respectively,
indicating that the membrane insertion of these hydrophilic segments
may be promoted by insertion of the following hydrophobic segments
during topogenesis. The topology shown in Fig. 7 clearly reveals the
presence of a striking 4-fold symmetry in TetA(B). That is, the totally
embedded segments, TM3, TM6, TM9, and TM12, are separated by two other segments, respectively. The "totally amphiphilic" segments, TM2, TM5, TM8, and TM11, and the "partly amphiphilic" segments, TM1, TM4, TM7, and TM10, are also separated by two other segments, respectively. All of the partly amphiphilic segments exhibit an amphiphilic nature in their C-terminal halves, whereas their N-terminal halves are entirely hydrophobic. The amphiphilic parts of the odd and
even numbered partly amphiphilic segments are at the periplasmic and
cytoplasmic ends, respectively. We previously reported that the
periplasmic ends of TM1 and TM2 are close to that of TM11 (30, 31).
Fig. 8 shows a hypothetical model of the
helix arrangement of TetA(B) based on our previous and current results.
There is a water-filled channel at the center of the molecule. The four amphiphilic helices, TM2, TM5, TM8 and TM11, comprise the wall of the
channel. The partly amphiphilic helices, TM1, TM4, TM7, and TM10, are
tilted away from the channel, with only their cytoplasmic or
periplasmic halves facing it. The four "hydrophobic" helices, TM3,
TM6, TM9, and TM12, are probably located at the periphery of the helix
bundle. This model is fundamentally similar to the model for
lactose permease presented by Goswitz and Brooker (32). Recently,
Yin et al. (33) reported that the low resolution
two-dimensional image of TetA(B) was reported by electron microscopic
analysis, which also could be superimposed with the ring-like structure such as that of lactose permease.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
The tetracycline resistance levels of E. coli W3104 cells harboring
plasmids encoding cysteine-scanning mutant TetA(B)s
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Fig. 1.
Putative membrane topology of TetA(B).
This structure was constructed on the basis of the results of
hydropathy analysis (4) and our previous results as to site-directed
chemical modification of Cys mutants of TetA(B) (5-8, 21, 22).
Putative transmembrane segments are enclosed in squares.
Gray squares indicate the transmembrane segments of which
the precise boundaries were determined by site-directed chemical
modification of cysteine-scanning mutants (6-8, 21, 22). The residues
of the cysteine-scanning mutants constructed in this study are depicted
as encircled letters. The closed circle with a
white letter indicates the residue mutated in Cys-free
TetA(B).
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Fig. 2.
Reactivity of cysteine-scanning mutants with
[14C]N-ethylmaleimide. Sonicated
membranes (0.5 mg of protein) including cysteine-scanning mutants were
incubated with 0.5 mM [14C]NEM for 5 min at
30 °C, followed by solubilization and immunoprecipitation of the
TetA(B) proteins with anti-TetA(B) C-terminal peptide antiserum, and
then the protein bands were visualized after SDS polyacrylamide gel
electrophoresis as described under "Experimental Procedures."
A, the radioactive bands visualized with a BAS-1000
bioimaging analyzer. As to the C-terminal region, the NEM binding of
Q397C and E398C could not be measured, because these mutant proteins
were not immunoprecipitated with anti-C-terminal peptide antibody
probably because of these residues being the common part of the
epitopes of this polyclonal antibody. B, the amounts of
[14C]NEM binding per TetA(B) protein (mol/mol) were
calculated from the radioactive band densities in panel A
and the Coomassie Brilliant Blue-stained protein bands (data not shown)
as described previously (6).
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Fig. 3.
Helical wheel projection of the residues in
TM7, TM8, and TM10 viewed from the periplasmic side. The residues
of which cysteine mutants gave NEM reactivity higher than 0.1 mol of
NEM per mol of TetA(B) in Fig. 2B are depicted as
outlined letters on a black background. As to TM7
and TM10, only the N-terminal amphiphilic halves were shown.
-helical form. Such a situation is similar to the case of
TM5 (22), which also includes many NEM-reactive positions, and
determination of the boundary is difficult. In the three-dimensional
structure of TetA(B), the cytoplasmic entrance of the water-filled
channel may resemble a funnel, and the cytoplasmic halves of TM8 and
TM5 may be exposed to the aqueous phase on the funnel wall. Therefore,
the cytoplasmic end of TM8 may be extended to Phe260 or
even to Val264. In the former and latter cases, TM8
comprises 15 and 19 amino acid residues, respectively. In this study,
we tentatively adopted Phe260 as the boundary (Fig. 5).
Anyway, the NEM-reactive positions were distributed along the
full-length of TM8, and they were located on one side of the helical
wheel (Fig. 3), indicating that TM8 faces the water-filled channel
along its full length.
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Fig. 4.
Effect of AMS on [14C]NEM
binding to the cysteine-scanning mutant TetA(B) proteins in intact
cells and sonicated membranes. Upper panels, intact
cells expressing the cysteine-scanning mutants were preincubated in the
presence (+) or absence ( ) of 5 mM AMS for 30 min at
30 °C, followed by incubation with 0.5 mM
[14C]NEM for 5 min. After stopping the labeling by
dilution with excess non-labeled NEM, the cells were disrupted by brief
sonication. The membrane fractions were solubilized, and then the
TetA(B) proteins were immunoprecipitated. The radioactive bands on SDS
polyacrylamide gel electrophoresis gels were visualized with a BAS-1000
bioimaging analyzer. Lower panels, sonicated membranes were
preincubated in the presence (+) or absence (
) of AMS, followed by
labeling with [14C]NEM, and then the radioactive bands
were visualized as described in Fig. 2. A, TM7;
B, TM8; and C, TM10.
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Fig. 5.
The detailed topology of the C-terminal half
of TetA(B) determined in this study. Transmembrane regions
are shown in boxes. The residues of which Cys mutants
exhibited the NEM binding more than 0.1 mol/mol of TetA(B) are
indicated by blue bold letters, and those of which Cys
mutants were almost not reactive with NEM (less than 0.1 mol/mol of
TetA(B)) are indicated by red bold letters. Pink and
light blue regions indicate the portions embedded in the
hydrophobic environment and those facing the water-filled channel,
respectively. Yellow circles indicate the residues to which
AMS can gain access in intact cells, and open circles
indicate those to which AMS cannot gain access in intact cells. The
open square indicates the residue to which AMS cannot gain
access in either intact cells or sonicated membranes. The green
line indicates the permeability barrier for AMS.
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Fig. 6.
Effect of tetracycline on
[14C]NEM binding to the cysteine-scanning mutants as to
TM8 and its flanking regions of TetA(B). Sonicated membranes
containing Cys-scanning mutant TetA(B) were labeled with
[14C]NEM in the presence (+) or absence ( ) of 1 mM tetracycline (TC) as described previously
(22). The radioactive bands were visualized as in Fig. 4.
A, the effect of tetracycline on NEM binding to the
NEM-reactive Cys mutants as to TM8 and its flanking regions.
B, the effect of tetracycline on NEM binding to the Cys
mutants that were newly found in the current study to have NEM binding
sensitivity to tetracycline.
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Fig. 7.
The entire topology of TetA(B) determined by
the site-directed chemical modification method.
Transmembrane regions are shown in boxes. The
residues of which Cys mutants exhibited high or moderate NEM reactivity
are indicated by blue bold letters, and those of which Cys
mutants were almost not reactive with NEM are indicated by red
bold letters. Pink and light blue regions indicate the
portions embedded in the hydrophobic environment and those facing the
water-filled channel, respectively. The green line indicates
the permeability barrier for AMS. Yellow circles and
white squares indicate the functionally or
structurally important residues of which cysteine mutants exhibited
drastically reduced tetracycline resistance (less than 10 µg/ml) and
moderately reduced resistance (less than 50 µg/ml), respectively. The
green upward and downward arrows indicate the
residues of which NEM binding to Cys mutants were stimulated and
inhibited by tetracycline, respectively. Outlined letters in
brown ovals indicate the numbers of residues in the
boundaries in each transmembrane segment.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 8.
Model of the helix arrangement of
TetA(B). All -helices project from the periplasmic side.
White letters in a blue background indicate
NEM-reactive positions. Helices in pink,
light blue, and light green backgrounds indicate
the entirely hydrophobic, amphiphilic, and partially amphiphilic
helices described in the text. A, helix arrangement on the
cytoplasmic side. B, helix arrangement on the periplasmic
side. In each wheel, residues in the cytoplasmic or periplasmic half
are depicted.
The green lines in Fig. 7 indicate the location of the permeability barrier in the channel wall-forming helices. All wall-forming helices are involved in the barrier, which means that there is no uncoupling bypass in the water-filled channel. The barrier is located in the middle of the transmembrane segment a little to the periplasmic end, except for that of TM1. In TM1, the barrier is very close to the periplasmic end. There are two possibilities, i.e. 1) the TM1 helix may extend toward loop1-2 and form a funnel-like exit of the channel, or 2) the TetA(B) molecule is dented on the periplasmic surface at TM1.
The effects of tetracycline on the NEM binding are indicated by downward (inhibitory) and upward (stimulatory) arrows in Fig. 7. Previously (6), we supposed that tetracycline may cause the TetA(B) conformational change from the inside-closed/outside-open form to the inside-open/outside-closed form, because the NEM binding to the S65C (inside) and L97C (outside) mutants was inhibited and stimulated by tetracycline (6). However, on the basis of our current results, the substrate-induced conformational change may be complicated. To our surprise, there was no region in which NEM binding to Cys mutants was continuously inhibited or stimulated by tetracycline, indicating that the substrate-induced occlusion or exposure of any domain(s) of TetA(B) did not occur. It should be noted that the mutations exhibiting inhibitory effects of tetracycline are mainly located around the permeability barrier in the N-terminal half, suggesting the presence of the substrate binding site.
Fig. 7 also shows the distribution of the functionally and/or structurally important residues of which cysteine mutants exhibit reduced drug resistance. Among them, the number of essential residues, the MIC of cysteine mutants of which were less than 10 µg/ml, is only 17 (4%), and that of moderately important residues (MIC < 50 µg/ml) is 41 (10%). In total, 58 (14%) of the 401 amino acid residues of TetA(B) contribute to the structure and/or function. The remaining 343 residues (86%) are apparently related to neither the structure nor the function. Of the 17 essential residues, 13 are located in the transmembrane region. In addition, of these 13 essential residues, eight are glycines, and two are prolines, confirming the importance of glycines and prolines in the transmembrane regions of transporter molecules. The other essential residues in the transmembrane regions number only three, Arg101, Asp285, and Tyr357. Essential residues are concentrated in TM4, TM5, TM8, and TM11. Among the loop regions, only three loops, loop2-3, loop4-5, and loop10-11 include essential residues, all of which are cytoplasmic. However, it should not be forgotten that the residues of which cysteine mutants exhibit moderately reduced resistance also play significant roles in the transport process, such as Asp84 (34), Arg70 (35), and His257 (36).
Complete cysteine-scanning mutants of lactose permease in
E. coli has been constructed by H. R. Kaback's laboratory
(37) and vigorously analyzed. Although lactose permease is
substrate/H+ symporter, the analysis of the complete
cysteine-scanning mutants of tetracycline/H+ antiporter
revealed interesting similarity to lactose permease, i.e.
the small numbers of essential residues, periodical distribution of NEM-reactive positions in the transmembrane segments, and the circular arrangement of transmembrane helices whereas the order of each
helix seems different in each other, indicating that the molecular
constructions of symporters and antiporters belonging to the major
facilitator superfamily are fundamentally common. Anyway, the
comparative analysis of the complete sets of cysteine-scanning mutants
of tetracycline/H+ antiporter and lactose permease will be
very useful for clearing the molecular structures and the transport
mechanism of secondary transporters.
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FOOTNOTES |
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* This work was supported by grants-in-aid from the Ministry of Education, Science, and Technology of Japan.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.
¶ Postdoctoral research fellow of the Japan Society for the Promotion of Science.
** To whom correspondence should be addressed: Inst. of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki-shi, Osaka 567-0047, Japan. Tel.: 81-6-6879-8545; Fax: 81-6-6879-8549; E-mail: akihito@sanken.osaka-u.ac.jp.
Published, JBC Papers in Press, February 6, 2001, DOI 10.1074/jbc.M007993200
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ABBREVIATIONS |
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The abbreviations used are: TetA(B), Tn10-encoded metal-tetracycline/H+ antiporter; TM, transmembrane helices; NEM, N-ethylmaleimide; AMS, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; MIC, minimum inhibitory concentration.
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