(Received for publication, August 26, 1996, and in revised form, October 12, 1996)
From the Department of Cell Membrane Biology,
Institute of Scientific and Industrial Research, Osaka University,
Mihogaoka, Ibaraki, Osaka 567, the
Faculty of Pharmaceutical
Sciences, Osaka University, Yamadaoka, Suita, Osaka 565, and
§ Division of Microbial Chemistry, Faculty of
Pharmaceutical Sciences, Chiba University, Chiba 263, Japan
The transposon (Tn) 10-encoded
metal-tetracycline/H+ antiporter (Tn10-TetA) is predicted
to have a membrane topology involving 12 transmembrane domains on the
basis of the hydropathy profile of its sequence and the results of
limited proteolysis; however, the experimental results of limited
proteolysis are not enough to confirm the topology because proteases
cannot gain access from the periplasmic side (Eckert, B., and Beck, C. F. (1989) J. Biol. Chem. 264, 11663-11670). One or
two cysteine residues were introduced into each predicted hydrophilic
loop or the N-terminal segment of Tn10-TetA by site-directed
mutagenesis, and then the topology of the protein was determined by
examining whether labeling of the introduced Cys residue by
membrane-permeant [14C]N-ethylmaleimide
([14C]NEM) was prevented by preincubation of intact cells
with the membrane-impermeant maleimide,
4-acetamido-4-maleimidylstilbene-2,2
-disulfonic acid (AMS). The
binding of [14C]NEM to the S36C (loop 1-2), L97C (loop
3-4), S156C (loop 5-6), R238C (loop 7-8), S296C (loop 9-10), Y357C,
and D365C (loop 10-11) mutants was completely blocked by pretreatment
with AMS, indicating that these residues are located on the periplasmic
surface. In contrast, [14C]NEM binding to the S4C
(N-terminal segment), S65C (loop 2-3), D120C (loop 4-5), S199C and
S201C (loop 6-7), T270C (loop 8-9), and S328C (loop 10-11) mutants
was not affected by pretreatment with AMS, indicating that these
residues are on the cytoplasmic surface. These results for the first
time thoroughly confirm the 12-transmembrane topology of the
metal-tetracycline/H+ antiporter.
The transposon (Tn)1 10-encoded
metal-tetracycline/H+ antiporter (Tn10-TetA) is a typical
bacterial drug-extruding antiporter coupled with proton influx (2, 3, 4).
It is composed of 401 amino acid residues (5, 6). Tn10-TetA is
structurally similar to the bacterial multidrug efflux transporters,
NorA (7) and Bmr (8), which exhibit 24-25% sequence identity with
Tn10-TetA. Tn10-TetA is regarded as a member of the major facilitator
family including uniporters and symporters because of the structural similarity and the conserved sequence motif, DRXGRR (9).
Tn10-TetA is important for elucidating the common structural and
functional features of the major facilitator family because it is the
only antiporter in this family of which the molecular mechanism of the
transport has been extensively studied in detail. Using inside-out membrane vesicles, it has been shown that Tn10-TetA mediates the 1:1
antiport of a divalent cation-tetracycline complex with a proton (4,
10). Site-directed mutagenesis studies on Tn10-TetA revealed the
presence of some functionally essential aspartic acid residues (11,
12). One of these aspartic acid residues is located in the sequence
motif common to the major facilitator family transporters (13). The
functional and/or structural importance of the aspartic acid conserved
in this motif was also confirmed in -ketoglutarate permease (14) and
lactose permease (15), suggesting a common molecular mechanism for
symporters and antiporters.
Most major facilitator family transporters are predicted to contain 12 membrane-spanning domains (9). As to tetracycline/H+ antiporters, Eckert and Beck (1), and Henderson and Maiden (16) proposed similar 12-transmembrane segment models for the membrane topologies of Tn10-TetA and pBR322-TetA, respectively, based on the results of hydropathy analysis. The model of Tn10-TetA was partially confirmed by biochemical and immunological studies. (i) 4 of the 5 predicted cytoplasmic loops were cleaved by the proteases used from the inner side of the membrane (1). (ii) The N-terminal methionine is accessible for cyanate modification in inside-out vesicles but not in spheroplasts (1). (iii) The anti-C-terminal peptide antibody could bind to Tn10-TetA only from the inner side of the membrane (17). However, there is no biochemical evidence for the periplasmic location of any of the predicted periplasmic segments of Tn10-TetA, because it is resistant to protease digestion from the periplasmic side of the membrane (1). Allard and Bertrand (18) studied the membrane topology of pBR322-TetA, which is a tetracycline-resistant protein encoded by pBR322, by means of alkaline phosphatase (PhoA) gene fusions. With this technique, they succeeded in determining the periplasmic location of three of the six predicted periplasmic loops, whereas the fusion proteins connected at the predicted periplasmic loop 1-2, loop 3-4, and loop 9-10 showed unexpectedly low enzyme activity (18). Since the hydropathy plot of Tn10-TetA (1) showed no distinct hydrophilic segment between TM3 and TM4, or TM9 and TM10, the periplasmic location of loop 3-4 and loop 9-10 was not supported by either the biochemical evidence or the predictive algorithm.
Site-directed chemical labeling of cysteine residues with a sulfhydryl reagent is useful for analyzing the topology of a polytopic membrane protein (19). Tn10-TetA contains only one cysteine residue at position 377; however, SH reagents did not bind to Cys-377 due to it being buried in the hydrophobic interior of the membrane (20). There was no difference in [14C]NEM binding between the wild-type and the C377A (Cys-less) mutant (30). Therefore, the reactivity of an SH reagent to a mutant TetA protein represents the reactivity of the reagent to an introduced cysteine residue (20). In this study, we constructed 15 site-directed mutants of Tn10-TetA in which cysteine residues were introduced into putative periplasmic or cytoplasmic loop regions, followed by chemical labeling with the membrane-permeant SH reagent, [14C]N-ethylmaleimide, after preincubation with or without a membrane-impermeant SH reagent to determine their sidedness.
N-[Ethyl-1-14C]maleimide
(1.5 GBq/mmol) was purchased from DuPont NEN.
4-Acetamido-4-maleimidylstilbene-2,2
-disulfonic acid (AMS) was from
Molecular Probes Inc. All other materials were of reagent grade and
obtained from commercial sources.
Escherichia coli CJ236 (21), TG1 (22), and W3104 (23) were used for the preparation of single-stranded DNA, transformation after mutagenesis, and inverted vesicle preparation, respectively. E. coli JM109 (21) and BMH71-18 mutS (21) were used for oligonucleotide-directed mutagenesis by the Kunkel method (21). pCT1183 (24) and pLGT2 (11) are plasmids carrying 2.45-kilobase Tn10-tetA and tetR gene fragments cloned into pUC118 (purchased from Takara, Kyoto, Japan) and pLG339 (25), respectively. A multicopy plasmid, pCT1183, was used for mutagenesis, and a low copy number plasmid, pLGT2, was used for expression of the mutant tet gene.
Site-directed MutagenesisCysteine mutants were constructed by oligonucleotide-directed site-specific mutagenesis according to the method of Kunkel (21) using pCT1183 as a template, and a synthetic oligonucleotide containing mismatches generating a codon change and silent mismatches to generate a new restriction site in order to check the mutation. Mutations were at first detected as the appearance of a newly introduced restriction site and then verified by DNA sequencing using a Shimadzu DNA sequencer DSQ-1000. Then the mutant tetA gene was transferred to low copy number plasmid pLGT2 by corresponding fragment exchange.
Determination of Tetracycline ResistanceThe tetracycline resistance of E. coli W3104 cells harboring pLGT2 or its mutant plasmid was determined by the agar dilution method and expressed as the minimum inhibitory concentration.
Labeling of Mutant TetA Proteins with [14C]N-Ethylmaleimide (NEM) and Its Prevention by 4-Acetamido-4E. coli W3104 cells harboring mutant plasmids were grown in the minimal medium supplemented with 0.1% casamino acids and 0.2% glucose. At the middle of the logarithmic phase, tetA gene expression was induced with 0.25 µg/ml heat-inactivated chlortetracycline (26) for 2 h. The cells were then harvested and washed with 50 mM MOPS-KOH buffer (pH 7.0) containing 0.1 M KCl, followed by suspension in the same buffer and adjustment of the absorbance at 530 nm to 85. The absorbance was determined by dilution of an aliquot. Five µl of 100 mM AMS (final concentration 5 mM) or the same volume of distilled water was added to 100 µl of the cell suspension, 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. After removal of unbroken cells, the membrane fraction was collected by ultracentrifugation with a Beckman Ultracentrifuge Optima TL. The resultant precipitate was solubilized with 1% Triton X-100 and 0.1% SDS in phosphate-buffered saline containing unlabeled NEM, and then immunoprecipitated with anti-TetA C-terminal peptide antiserum (17) and Pansorbin Staphylococcus aureus cells (27), as described previously (28). Then the precipitate was subjected to SDS-polyacrylamide gel electrophoresis, followed by Coomassie Brilliant Blue staining. The resulting gel was soaked in Amplify (Amersham Corp.) prior to being dried. The dried gel was exposed to an imaging plate for visualization with a BAS-1000 Bio-Imaging Analyzer (Fuji Film Co., Tokyo, Japan).
In the case of labeling using everted membrane vesicles, the vesicles were prepared from E. coli W3104 cells harboring mutant plasmids after tetA gene induction as described previously (28). Then the vesicles (0.5 mg of protein) were used in place of intact cells as described above.
Transport AssayTetracycline uptake by everted membrane vesicles was assayed as described in our previous paper (4) in the presence of 10 µM [3H]tetracycline and 50 µM CoCl2.
First of
all, we examined the binding of [14C]NEM to the S65C and
L97C mutants of Tn10-TetA in either intact cells or everted membrane
vesicles after preincubation in the presence or absence of AMS. Ser-65
and Leu-97 are located in predicted cytoplasmic loop 2-3 and
periplasmic loop 3-4, respectively. Ser-65 is not important for the
transport function, but position 65 is a unique hot spot in loop 2-3
for inactivation by an SH reagent (28). Leu-97 is a hot spot in loop
3-4 similar to position 65.2 It is certain
that the S65C and L97C mutants retain the wild-type conformation of
TetA, because they showed wild-type drug resistance. A binding
experiment was performed as described under "Experimental Procedures." Since NEM is a membrane-permeant reagent, both the S65C
and L97C mutants were equally labeled by [14C]NEM in
either intact cells or everted membrane vesicles in the absence of AMS
(Fig. 1), indicating that these positions are exposed to
the aqueous phase. Under these conditions containing 0.5 mM NEM, about 90% of the tetracycline transport activity was abolished (data not shown), indicating that the labeling reaction was almost saturated. With 1.0 mM NEM, the degree of inactivation no
longer increased, whereas 0.2 mM NEM abolished only 65%
activity. The degree of binding of NEM to the S65C mutant was not
affected by preincubation with a membrane-impermeant reagent, AMS, in
intact cells, whereas the binding of NEM in everted membrane vesicles was prevented by pretreatment with 5 mM AMS (Fig. 1),
indicating that position 65 is located inside of intact cells and
exposed to the outside medium in everted membrane vesicles. This
confirms the cytoplasmic location of position 65. In contrast, the
binding of [14C]NEM to the L97C mutant was completely
prevented by preincubation with 5 mM AMS for 30 min in
intact cells (Fig. 1), indicating that position 97 is located on the
outside surface of the cytoplasmic membrane. With 2 mM AMS,
the degree of protection decreased to about half, while various
preincubation times, from 5 to 30 min, did not have a significant
effect on the degree of protection. The binding of NEM to the L97C
mutant was partially blocked in everted membrane vesicles (Fig. 1),
probably due to contamination by right-side-out or unsealed membrane
vesicles. These results clearly showed that an experiment on the
binding of [14C]NEM to a cysteine mutant and the
protection by AMS is useful for determining whether a position is
located on the cytoplasmic or periplasmic surface.
Construction and Activity of Cysteine Mutants
We constructed 15 different cysteine mutants to determine the sidedness of the hydrophilic loop regions of Tn10-TetA. In addition to positions 65 and 97, cysteine residues were introduced into positions 4, 36, 120, 156, 199, 201, 238, 270, 296, 328, 357, 360, and 365 using the mutagenic oligonucleotide primers listed in Table I. The locations of the mutations are depicted in Fig. 2. These positions covered all of the 11 putative hydrophilic loop regions and the N-terminal segment. The cytoplasmic location of the C-terminal is evident because the anti-C-terminal peptide antibody is only accessible from the inner side of the membrane (17). Mutagenesis was performed using pCT1183 as a template, and the sequences were determined by DNA sequencing, followed by transfer of the mutant tetA gene to a low copy number plasmid, pLGT2, by fragment exchange. No additional unexpected mutations occurred within the fragment transferred to pLGT2. The expression of the mutant TetA proteins was detected by 1) immunoblotting using anti-C-terminal peptide antiserum, and 2) Coomassie Brilliant Blue staining of gels after SDS electrophoresis of the immunoprecipitated TetA proteins. The degrees of expression of the TetA proteins were not significantly affected by these mutations (data not shown).
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At first, the tetracycline resistance levels of E. coli W3104 cells harboring these mutant plasmids were measured on agar plates. As shown in Table II, 10 of the 15 mutants showed tetracycline resistance comparable with that of the wild type. S156C, S201C, and R238C showed moderate resistance, indicating that these mutations did not cause any significant alteration in the protein conformation. In contrast, the D120C and Y357C mutants had almost completely lost the drug resistance (Table II). As to the D120C mutant, it retained comparable tetracycline transport activity to that of the wild type when the activity was measured in everted membrane vesicles (Fig. 3). A similar discrepancy between transport activity and the resistance level was also observed for the D120N mutant (12), of which minimum inhibitory concentration was 19 µg/ml but the transport activity was comparable with that in the wild type. This discrepancy may be based on alteration of the kinetic properties of the transporter (12). Thus, some of the conformational change must be caused by the Asp-120 mutation. However, as judged from the high level transport activity of the D120C mutant, the conformational change should not be large enough to destroy the structure of the transporter. The Y357C mutant also showed a similar discrepancy. The mutant retained about 10% of the transport activity (Fig. 3), indicating that it also retains the structure of the transporter, whereas the conformational damage in Y357C may be larger than that in the D120C mutant. So, an additional mutant, D365C, was constructed as to the same predicted loop.
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Reactivity of Cysteine Residues Introduced into the N-terminal Half of Tn10-TetA with [14C]NEM and Its Prevention by AMS
The binding of [14C]NEM to the cysteine
residues introduced into the N-terminal half of Tn10-TetA was examined.
As shown in Fig. 4, the reactivity of the S4C, S36C,
S65C, L97C, and D120C mutants to [14C]NEM was high in the
absence of AMS, indicating that these positions are located in regions
exposed to the aqueous phase. On the other hand, the reactivity of the
S156C mutant was somewhat lower than those of the other five mutants;
however, binding was still detectable in the absence of AMS. After
preincubation with excess AMS, the [14C]NEM binding to
the S36C, L97C, and S156C mutants was completely blocked, whereas the
binding to the S4C, S65C, and D120C mutants was not affected at all by
AMS preincubation (Fig. 4). These results were highly reproducible, and
it should be noted that the protective effect of AMS was almost all or
none under these conditions, as shown on quantitative measurement
(Table III). These results clearly indicated that
positions 36, 97, and 156 are located on the periplasmic side of the
membrane, whereas positions 4, 65, and 120 are located on the
cytoplasmic surface of the membrane. Positions 36 and 97 are located in
predicted periplasmic loop 1-2 and loop 3-4, respectively. These
results are the first direct biochemical evidence for the periplasmic
location of these two loops. In particular, it is interesting that loop
3-4 is highly exposed to the aqueous medium, as judged from the
reactivity of the L97C mutant to [14C]NEM, although the
hydrophilicity of this loop is not distinct and neither protease
cleavage nor PhoA fusion data provide direct support for a periplasmic
location (1, 18).
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At first, cysteines were introduced into positions 201, 238, 270, 296, 328, and 360. Positions 201, 270, and 328 are located in
predicted cytoplasmic loops, whereas positions 238, 296, and 360 are
located in predicted periplasmic loops (Fig. 2). The reactivity of
[14C]NEM to four of these six mutants was high (Fig.
5), confirming that these positions are located in loops
exposed to the aqueous phase. However, the reactivity of
[14C]NEM to the S201C mutant was very low, and
[14C]NEM did not bind to the S360C mutant, indicating
that these positions are hardly exposed to the aqueous phase. There are
two alternative possibilities: 1) predicted loop 6-7 and loop 11-12 containing positions 201 and 360, respectively, might not be exposed to
the aqueous phase. If this is the case, the topology of Tn10-TetA should be greatly changed. 2) Positions 201 and 360 may be unique points in the water-exposed loops at which the side chains are oriented
into the protein interior. Our previous work on cysteine-scanning mutants as to loop 2-3 (29) indicated the presence of such a unique
cryptic point in this water-exposed loop. In order to determine whether
the segments containing these positions are exposed or not, we
introduced cysteine residues into several positions close to positions
201 and 360. The S199C and D365C mutants showed high reactivity to
[14C]NEM in the absence of AMS (Fig. 6 and
Table III). The Y357C mutant showed low but detectable reactivity to
NEM (Fig. 6 and Table III). When the residues are located in the
transmembrane region in TetA proteins, Cys mutants of these residues
show no or very low binding (less than 0.1 mol of bound
[14C]NEM per mol of TetA) (30). Thus, it was confirmed
that loop 6-7 and loop 11-12 are exposed to the aqueous phase.
The [14C]NEM binding to the R238C, S296C, Y357C, and D365C mutants was completely prevented by preincubation with excess AMS, whereas the binding to the S199C, T270C, and S328C mutants was not affected by AMS at all (Figs. 5 and 6 and Table III). Binding of [14C]NEM to the S201C mutant was also observed after preincubation with AMS (Fig. 5 and Table III). These results clearly indicated that large central loop 6-7, loop 8-9, and loop 10-11 are located on the cytoplasmic surface, whereas loop 7-8, loop 9-10, and loop 11-12 are located on the outside surface of the cytoplasmic membrane. It may be surprising that position 296 is highly exposed to the outside medium, as judged from the reactivity of the S296C mutant to [14C]NEM (Fig. 5B), in spite of the low hydrophilicity of the segment around this position and the lack of direct biochemical evidence of the periplasmic location of this loop (1, 18).
The membrane topologies of Tn10-TetA and its homolog, pBR322-TetA, have been predicted to include 12-transmembrane segments, and the N and C termini are located on the cytoplasmic side, as judged from the hydropathy profile (1, 16). However, the predicted 12-transmembrane structure is not obvious because there are no distinct hydrophilic segments between predicted transmembrane segment (TM) 3 and TM4, and TM9 and TM10 (1). Hydropathy analysis of Tn10-TetA indicated the presence of 10 hydrophobic clusters of different sizes (1). Neither the presence of water-extruding loops nor the periplasmic location of segments between TM3 and TM4 and TM9 and TM10 has been directly supported by any biochemical data (1, 18). The results presented in this article for the first time clearly confirmed the validity of the 12-transmembrane topology of the TetA protein.
We performed site-directed chemical labeling of cysteine mutants with sulfhydryl reagents to determine the membrane topology of Tn10-TetA. This method is applicable to Tn10-TetA because wild-type Tn10-TetA has only one cysteine residue at position 377, but maleimide derivatives are not reactive to this cysteine (20) due to it being buried in the hydrophobic interior of the membrane. The criteria used for determining the location of an introduced cysteine are as follows. 1) When cysteine residues are not labeled with [14C]NEM, they are embedded in the hydrophobic interior of the membrane or the folded loops, because the active species of a sulfhydryl group for reaction with maleimide is the deprotonated form. 2) When the labeling of cysteine residues with [14C]NEM is not blocked by membrane-impermeant AMS, the residues are located on the cytoplasmic surface. 3) When the labeling with [14C]NEM is blocked by AMS, the residues are located on the periplasmic surface.
We constructed 15 site-directed mutants in which cysteine residues were introduced into predicted periplasmic or cytoplasmic loops. Except for the S201C and S360C mutants, all mutants were highly labeled by [14C]NEM in the absence of AMS. Since cysteine residues introduced into positions nearby 201 and 360 also showed good reactivity to [14C]NEM, positions 201 and 360 are likely to be local cryptic points in the water-extruding loop regions. The binding of [14C]NEM to the cysteine residues introduced into the predicted periplasmic loops was completely blocked by preincubation of intact cells with a membrane-impermeant SH reagent, AMS, whereas the binding to the cysteine residues introduced into the N-terminal segment and the predicted cytoplasmic loops was not affected by AMS, clearly indicating that the former cysteine residues are located on the periplasmic surface and the latter ones on the cytoplasmic surface. These results obviously support the 12-transmembrane structure of Tn10-TetA. Since the intervals between introduced cysteine residues are enough for the protein to traverse the membrane once but shorter than required to traverse it more than twice, the model of Tn10-TetA composed of more than 13 transmembrane segments is impossible.
The predicted periplasmic loops tend to be shorter than the cytoplasmic loops (Fig. 2). It has been suspected that TetA proteins are hardly exposed to the periplasmic surface of the membrane because no proteases can gain access from the periplasmic side (1). However, as judged from our results as to [14C]NEM binding to the cysteines introduced into the periplasmic loops, they were certainly exposed to the aqueous phase, similar to the loops located on the cytoplasmic surface. The inaccessibility for proteases from the periplasmic side may be due to the protein conformation.