Each amino acid in the putative transmembrane
helix III and its flanking regions (from Gly-62 to Tyr-98) of the
Tn10-encoded metal-tetracycline/H+ antiporter
(Tet(B)) was individually replaced with Cys. Out of these 37 cysteine-scanning mutants, the mutants from G62C to R70C and from S92C
to Y98C showed high or intermediate reactivity with [14C]N-ethylmaleimide (NEM) except for the
M64C mutant. On the other hand, the mutants from R71C to S91C showed
almost no reactivity with NEM except for the P72C mutant. These results
confirm that the transmembrane helix III is composed of 21 residues
from Arg-71 to Ser-91. The majority of Cys replacement mutants retained
high or moderate tetracycline transport activity. Cys replacements for
Gly-62, Asp-66, Ser-77, Gly-80, and Asp-84 resulted in almost inactive
Tet(B) (less than 3% of the wild-type activity). The Arg-70
Cys
mutant retained very low activity due to a mercaptide between
Co2+ and a SH group (Someya, Y., and Yamaguchi, A. (1996)
Biochemistry 35, 9385-9391). Three of these six important
residues (Ser-77, Gly-80, and Asp-84) are located in the transmembrane
helix III and one (Arg-70) is located in the flanking region. These
four functionally important residues are located on one side of the helical wheel. Only two of the residual 31 Cys mutants were inactivated by NEM (S65C and L97C). Ser-65 and Leu-97 are located on the
cytoplasmic and periplasmic loops, respectively, in the topology of
Tet(B). The degree of inactivation of these Cys mutants with SH
reagents was dependent on the volume of substituents. In the presence
of tetracycline, the reactivity of the S65C mutant with NEM was
significantly increased, in contrast, the reactivity of L97C was
greatly reduced, indicating that the cytoplasmic and periplasmic loop
regions undergo substrate-induced conformational change in the mutually
opposite direction.
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INTRODUCTION |
The transposon Tn10-encoded Tet(B) protein is one of the family of
bacterial tetracycline exporters of Gram-negative bacteria that
includes Tet(A) to (H) (1). It is a polytopic cytoplasmic membrane
protein that catalyzes stoichiometric metal-tetracycline/H+
antiport (2-4). It is composed of 401 amino acid residues (5, 6).
Tet(B) belongs to a major facilitator superfamily (7) that includes
uniporters and symporters in addition to bacterial multidrug exporters
such as NorA (8) and Bmr (9). Most of the major facilitator family
transporters are predicted to have a 12-membrane-spanning structure
(7). With respect to tetracycline/H+ antiporters, Eckert
and Beck (10) and Henderson and Maiden (11) proposed similar
12-membrane-spanning topologies of Tet(B) and pBR322-Tet(C) based on
the results of hydropathy analysis. Allard and Bertrand (12)
experimentally confirmed the topology of pBR322-Tet(C) by means of
alkaline-phosphatase (PhoA) gene fusions. Recently, we experimentally
established the 12-membrane-spannning structure of Tet(B) by using the
intact protein on the basis of the results of the competitive binding
of membrane permeable and impermeable SH reagents to the cysteine
residues introduced by site-directed mutagenesis (13).
The cysteine-scanning mutagenesis of lactose permease (14-22) and
other membrane transporters (23) has been extensively studied and
revealed to be useful for determination of the distribution of
functionally essential residues and the arrangement of transmembrane helices (24, 25). We showed that the reactivity of the
cysteine-scanning mutants of membrane proteins with
NEM1 is useful to determine
the range of the transmembrane segments and the exact boundaries
between membrane-embedded regions and the loop regions exposed to the
aqueous phase (26). We reported the exact range of the transmembrane
helix IX and experimentally showed the presence of a short loop
composed of three amino acid residues between helices IX and X exposed
to the aqueous phase (26).
In this study, we established cysteine-scanning mutants around the
putative helix III and its flanking regions. Tet(B) contains a
conserved sequence motif, GXXXDRXGRR, in the
putative cytoplasmic loop2-3, which is common to the major facilitator
superfamily transporters (7). Site-directed mutagenesis studies of
Tet(B) suggest that the region containing this motif plays a role as an
entrance gate for tetracycline translocation (27). The importance of
this motif was also confirmed in
-ketoglutarate permease (28) and
lactose permease (29). In addition, there is an Asp-84 in the middle of
helix III, which is one of three essential transmembrane aspartic acid
residues (30). The conserved quartet of residues is located around this
region and in helix II, which may be related to the substrate binding
(31). Therefore, loop2-3 and the helix III region possibly form a
substrate translocation pathway of Tet(B). The results in this study
confirm that the reactivity of the cysteine-scanning mutants with SH
reagents is very useful for determining the boundary between the
transmembrane region and the region exposed to the aqueous phase.
 |
EXPERIMENTAL PROCEDURES |
Materials--
N-[1-14C]ethylmaleimide
([14C]NEM) (1.5 GBq/mmol) and
[7-3H]tetracycline were purchased from NEN Life Science
Products. [
-32P]dCTP was purchased from Amersham Corp.
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 (32). For the mutagenesis, plasmid
pCT1183 (33) was used as a template that carries the 2.45-kilobase Tn10
tetA(B) and tetR gene fragments.
Mutations were detected based on the appearance of a newly introduced
restriction site and then verified by DNA sequencing.
Low copy number mutant plasmids were constructed through exchange of
the EcoRV-EcoRI fragment of the cysteine-scanning
mutant tetA(B) genes with the corresponding
fragment of the low copy number plasmid, pLGT2 (30), and used for
determination of the reactivity with [14C]NEM.
Assay of the Reaction of [14C]NEM with Tet(B)
Proteins--
Sonicated membranes were prepared from Escherichia
coli W3104 carrying pLGT2 or its derivatives as described
previously (34) except that the cells were disrupted by brief
sonication. The reaction of [14C]NEM with Tet(B) proteins
in the sonicated membranes were performed as described previously (34).
After solubilization, the [14C]NEM-bound Tet(B) proteins
were immunoprecipitated with anti-Tet(B) C-terminal peptide antiserum
(35) and Pansorbin Staphylococcus aureus cells (36). The
resultant pellet was subjected to SDS-polyacrylamide gel
electrophoresis, followed by Coomassie Brilliant Blue staining. The
resultant gels were soaked in Amplify prior to drying. The dried gels
were exposed to an imaging plate for visualization using a BAS-1000
Bio-Imaging Analyzer (Fuji Film Co., Tokyo).
Assay of Tetracycline Transport by Everted Membrane
Vesicles--
Everted membrane vesicles were prepared from E. coli TG1 cells carrying a high copy number plasmid pCT1183 or its
derivatives as described previously (37).
[3H]Tetracycline uptake by everted membrane vesicles was
assayed in the presence of 10 µM
[3H]tetracycline and 50 µM
CoCl2 in 50 mM MOPS-KOH buffer (pH 7.0) containing 0.1 M KCl as described in our previous paper
(30).
 |
RESULTS |
Construction of Cysteine-scanning Mutants--
Each amino acid in
the putative transmembrane helix III and its flanking regions (from
Gly-62 to Tyr-98) (Fig. 1) was
individually replaced with Cys by site-directed mutagenesis as
described under "Experimental Procedures" using the mutagenic
primers. Mutants from P72C to Y98C were constructed in this study,
whereas those from G62C to R71C were constructed previously (27, 34,
38, 39). Membranes were prepared by brief sonication from E. coli W3104/pLGT2 after induction of tetA(B) gene
expression. Resulting membranes were solubilized in 1% Triton X-100
and 0.1% SDS, and then Tet(B) proteins were precipitated with C
terminus-specific antiserum (35). After SDS-gel electrophoresis, Tet(B)
proteins were detected by Coomassie Brilliant Blue staining
(Fig. 2A). There was no
significant alteration observed in the level of expression of the
Cys-scanning mutants.

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Fig. 1.
The topology of the putative transmembrane
helix III and its flanking loop regions of Tn10-Tet(B). A,
predicted by hydropathy analysis (10). B, revised topology
based on the results of this study. The positions at which the cysteine
mutants showed high reactivity with NEM are indicated by closed
circles or squares. Moderately reactive positions are
indicated by gray circles or squares. The
positions at which the cysteine mutants showed loss of or greatly
reduced tetracycline transport activity are enclosed by
squares. Arrowheads indicate the positions of the
cysteine mutants sensitive to NEM inactivation.
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Fig. 2.
The binding of [14C]NEM to the
cysteine-scanning mutants of Tn10-Tet(B). The sonicated membranes
(0.5 mg of protein) of cysteine-scanning mutants were incubated with
0.5 mM [14C]NEM for 5 min at 30 °C,
followed by solubilization and immunoprecipitation of Tet(B)
proteins as described under "Experimental Procedures." A, after SDS-PAGE, the protein bands and the radioactive
bands were visualized by Coomassie Brilliant Blue staining
(upper panels) and by use of a Bio-Imaging Analyzer BAS-1000
(lower panels), respectively. B, relative amount
of bound [14C]NEM per Tet(B) protein calculated from band
densities in panel A.
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Reactivity of the Cys-Scanning Mutants with
[14C]NEM--
The [14C]NEM binding to the
Cys-scanning mutants is shown in Fig. 2. The result is a typical one
out of at least three independent experiments. Since the density of the
radioactive bands of the mutant proteins was increased in proportion to
the incubation time with [14C]NEM, at least up to 10 min
(data not shown), and wild-type Tet(B) did not show any radioactive
band, the density reflects the reactivity of each Cys residue
introduced in Tet(B). As shown in Fig. 2, Cys residues introduced into
positions 62, 63, from 65 to 69, from 92 to 94, and 97 were highly
reactive with NEM, and those introduced into positions 70, 72, 95, 96, and 98 showed moderate reactivity, indicating that the former residues
are exposed to the aqueous phase and the latter ones are partially
exposed. In contrast, Cys residues introduced into positions 64, 71, and from 73 to 91 showed almost no significant reactivity with NEM,
indicating that these residues are embedded in the hydrophobic
interior. As judged by these observations, the transmembrane helix III
is likely to range from Arg-71 to Ser-91. Out of nine residues of the
putative cytoplasmic loop2-3, only Met-64 is very slightly reactive
with NEM as reported in our previous paper (27, 40), probably due to
the fact that the side chain of this position is oriented to the
protein interior. On the other hand, there was no moderately reactive
position in the mid-transmembrane helix region.
Tetracycline Transport Activity of the Cys-scanning
Mutants--
The initial rate (for a period of 30 s) of the
tetracycline uptake by everted membrane vesicles containing each
Cys-scanning mutant was measured (Fig.
3). The majority of the Cys-scanning mutants exhibited high or moderate
tetracycline transport activity (>25% of the wild-type). Only three
mutants (D66C, G80C, and D84C) exhibited complete loss of the activity.
Two mutants (G62C and S77C) exhibited greatly reduced activity (less
than 3% of the wild-type). Another one mutant (R70C) showed
significant activity (about 8% of the wild-type); however, since other
Arg-70 mutants, except for R70K and R70C, showed no activity (39),
Arg-70 is also a functionally important residue. As reported
previously, the residual activity of the R70C mutant is due to the
mercaptide formed between Co2+ and the SH group acting as a
functional positive charge (39). Thus, out of 37 residues of helix III
and its flanking regions, only six residues are important for
tetracycline transport. The importance and the possible roles of these
six residues have been investigated in detail in our previous studies
(27, 30, 31, 41). The new finding in this work is that there is no
additional essential residue in this region. As shown in
Fig. 4, the functionally important
residues cluster on one side of helix III in a helical wheel plot,
probably supporting the presence of the substrate translocation pathway
along this face of helix III.

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Fig. 3.
Tetracycline transport activity of
cysteine-scanning mutants of Tet(B). Initial rate of tetracycline
uptake by everted membrane vesicles containing cysteine-scanning
mutants was measured for 30 s in the presence of 10 µM [3H]tetracycline and 50 µM
CoCl2 as described under "Experimental Procedures."
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Fig. 4.
Helical wheel projection of residues in the
N-terminal half of the putative helix III. Residues of Cys mutants
that showed greatly reduced activity are highlighted by a dark
background.
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Effect of NEM on Tetracycline Transport Activity--
The initial
rate of tetracycline uptake by each vesicle after 5 min of treatment
with 2 mM NEM is indicated as a percentage of that in the
absence of NEM (Fig. 5). Fig. 5 shows the
results for 31 Cys-scanning mutants that showed significant
tetracycline transport activity in the absence of NEM. Out of these
mutants, the transport activities of only two (S65C and L97C) were
greatly inactivated by NEM. On the other hand, the activities of the
other 29 Cys mutants were hardly affected by NEM. Although the K63C, R67C to G69C, and S92C to L94C mutants were highly reactive with NEM
and the P72C, W95C, M96C, and Y98C mutants were moderately reactive as
shown in Fig. 2, the tetracycline transport activities of all these
mutants were not inhibited by NEM. This result indicates that positions
65 and 97 are unique hot spots for chemical modification.

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Fig. 5.
Effect of NEM on tetracycline transport by
everted membrane vesicles containing cysteine-scanning mutants of
Tet(B). Everted membrane vesicles were treated with 2 mM NEM for 5 min at 30 °C prior to the tetracycline
transport assay. The initial rate of [3H]tetracycline
transport was measured as described under "Experimental Procedures." Residual activities are presented as a percentage of the
initial rate measured in the absence of NEM.
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Fig. 6 shows the dependence of
inactivation of the transport activity of the S65C and L97C mutants on
the concentration of SH reagents. As reported in our previous paper
(37, 40, 42), the transport activity of the S65C mutant was completely
inactivated by NEM at less than 1 mM, whereas the
inactivation of the activity of the S65C mutant with methyl
methanethiosulfonate (MMTS), which substitutes a small thiomethyl
group, was saturated at the level of 40%. Since the activity of the
MMTS-pretreated S65C mutant was no longer inactivated by NEM, it is
clear that each molecule of the thiomethylated S65C mutant retains 60%
activity (Fig. 6A). In the case of the L97C mutant, the
maximum inactivation level by NEM was about 90%, which was reached at
2 mM NEM (Fig. 6B). MMTS did not affect the
tetracycline transport activity of the L97C mutant. The activity of the
MMTS-treated L97C mutant was also not inactivated by NEM, indicating
that the thiomethylated L97C mutant molecule retains full activity.
Such substituent-volume dependence of the inactivation level of the
activity of the S65C and L97C mutants with SH reagents indicates that
the inactivation is based on the steric hindrance caused by a bulky
substituent.

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Fig. 6.
Dose dependence of inactivation of the S65C
and L97C mutants with NEM and MMTS. Everted membrane vesicles were
preincubated with the indicated concentrations of NEM (closed
circles) or MMTS (open circles) for 5 min at 30 °C
prior to the tetracycline transport assay. In the case of sequential
treatment (open squares), everted membrane vesicles were
first pretreated with 2 mM MMTS for 5 min, and then the
incubation was continued in the presence of indicated concentrations of
NEM for 5 min. The initial rate of tetracycline transport was measured
for 30 s as described under "Experimental Procedures."
Residual activity is presented as a percentage of the initial rate in
the absence of SH reagents. A, S65C mutant cited from our
previous paper (43). B, L97C mutant.
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The Effect of Tetracycline on Reactivity of the Hot Spot Mutants
with NEM--
The substituent-volume dependence of inactivation of the
activity of the S65C and L97C mutants with NEM suggests that these residues are located in the substrate translocation pathway. Thus, the
effect of tetracycline on the reactivity of these mutants with
[14C]NEM was investigated. Tetracycline significantly
stimulates the reactivity of the S65C mutant with NEM as reported
previously (43). In contrast, the reactivity of the L97C mutant with
NEM was greatly inhibited by tetracycline
(Fig. 7), indicating that the degree of
exposure of the side chain at position 97 is reduced by the
substrate-induced conformational change. When a D66A mutation was added
to the L97C mutant, the substrate-induced change in the reactivity with
NEM disappeared. On the other hand, the L97C/R70A double mutants
retained the substrate-induced conformational change (Fig. 7). This
results indicate that Asp-66 confers to the substrate-induced conformational change while Arg-70 may contribute to the step after the
conformational change.

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Fig. 7.
Effect of tetracycline on the
[14C]NEM binding to the L97C mutant. Everted
membrane vesicles of the L97C single mutant or the L97C/D66A or
L97C/R70A double mutants were incubated with 0.5 mM
[14C]NEM in the absence or presence of 1 mM
tetracycline and 5 mM MgCl2 at 30 °C for 5 min. The radioactive bands were detected as described under
"Experimental Procedures." and + indicate the absence and
presence of tetracycline, respectively.
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 |
DISCUSSION |
The results presented here confirm that the membrane-embedded
regions and the aqueous phase-exposed regions can be distinguished on
the basis of the reactivity of the cysteine-scanning mutants with NEM
as reported previously (26). Out of 37 cysteine-scanning mutants
established in this study, the 21 mutants barely reactive with NEM were
clustered from Arg-71 to Ser-91, a region which corresponds to the
putative transmembrane helix III. Several isolated barely reactive
positions were presented in the aqueous phase-exposed regions, whereas
there was no reactive positions in the transmembrane regions. The
majority of the mutants retained transport activity. Only six mutants
showed loss of or greatly reduced activity. Two NEM-sensitive hot spots
were found in the water-exposed regions.
Cysteine-scanning mutants of lac permease were extensively studied by
Kaback and his co-workers (14-22). The important points revealed from
their studies are: 1) there are very few essential residues present in
the permease, 2) NEM inactivates the transport activity of a few of
these cysteine-scanning mutants, and 3) NEM-sensitive positions lie on
one face of a putative transmembrane helix. Yan and Maloney (23) also
reported that some cysteine mutants in the putative transmembrane
segments of G6P/Pi antiporter (UhpT) are sensitive to SH reagents.
Their results suggest that water-filled channels, which may contribute
to a substrate translocation pathway, are present in the permeases.
However, it should be noted that the cysteine mutants insensitive to SH
reagents may reflect two completely different situations; 1) the
residue is not reactive with SH reagents, or 2) the residue is reactive
but the modification does not affect the protein function. In fact,
Sahin-Toth and Kaback (14) observed that the rate of inactivation of
the activity of the V315C mutant of lac permease by NEM was greatly
stimulated in the presence of TDG due to the substrate-induced
conformational change, reflecting the change in the reactivity.
Therefore, it is required to directly determine the reactivity of each
cysteine mutant with NEM to determine the hydrophobicity of the regions flanking each position. In a previous study, we established the cysteine-scanning mutants of the putative helix IX of Tet(B) and its
flanking regions, and the NEM binding was directly investigated (26).
All of the mutants of the putative transmembrane helix IX were barely
reactive with NEM while the mutants of the flanking hydrophilic loop
regions were highly reactive (26). The results in this study support
the hypothesis that the situations are the same around helix III. It is
not known why the periodical appearance of moderately reactive
positions corresponding to the water-filled channel face of the
transmembrane helix was not observed in Tet(B). The substrate
translocation channel of Tet(B), if it exists, may be more hydrophobic
than that of lac permease and UhpT.