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 Nethylmaleimide (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.

Cysteine-scanning mutants, E32C to G62C, of the metal-tetracycline/H؉ antiporter were constructed in order to precisely determine the membrane topology around putative transmembrane segment II. None of the mutants lost the ability to confer tetracycline resistance, indicating that the cysteine mutation in each mutant did not alter the protein conformation. [14 C]N-Ethylmaleimide (NEM) binding to these cysteine mutants in isolated membranes was then investigated. The peptide chain of this region passes through the membrane at least once because residues 36 and 65 are exposed on the outside and inside surfaces of the membrane, respectively (Kimura, T., Ohnuma, M., Sawai, T., and Yamaguchi, A. (1997) J. Biol. Chem. 272, 580 -585). However, there was no continuous segment in which all of the introduced cysteine residues showed almost no reactivity with [14 C]NEM. The proportion of the unbound positions in the second half downstream from position 45 was 55% (10/18), which was clearly higher than that in the first half (21%; 3/14), suggesting that the second half is a transmembrane segment. Positions reactive to NEM appear periodically in the second half. They are located on one side of the helical wheel, suggesting that this side of the transmembrane helix faces a water-filled channel. The cysteine mutants as to the reactive positions in the second half were severely inactivated by NEM except for the P59C mutant, whereas the A40C mutant was the only one inactivated by NEM in the first half. These results suggest that the water-filled channel along this helical region may be a substrate translocation pathway.The tetracycline/H ϩ antiporter (Tet(B)) is a tetracycline exporter existing in the cytoplasmic membrane of Gram-negative bacteria (1-3). This transporter is a member of the major facilitator superfamily (4, 5) including transporters for sugars, amino acids, antibiotics, and neurotransmitters. These transporters were predicted to have 12 transmembrane segments in common (4). The secondary structures of most intrinsic membrane proteins have been determined by the gene fusion method (6, 7). We established a new method for determining the membrane topology on the basis of the reactivity of SH reagents to cysteine-scanning mutants of the tetracycline/H ϩ antiporter ( Fig. 1) (8). Cysteine-scanning mutagenesis of intrinsic membrane proteins, especially lactose permease, has been widely used for site-directed chemical modification analysis (9 -21). We found that Tet(B) has some segments composed of about 20 consecutive residues at which cysteine-scanning mutants were hardly reactive with NEM 1 (22, 23). These segments are embedded in the hydrophobic interior of the membrane. On the basis of this finding, we succeeded in determining the precise boundaries of transmembrane segment (TM) III (23) and TM IX (22). Similar results were obtained for the second transmembrane segment of EmrE.2 However, these observations were inconsistent with the results for lactose permease (10 -16) and UhpT (9), of which some cysteine mutants ...

Cysteine-scanning mutants as to putative transmembrane segments 4 and 5 and the flanking regions of Tn10-encoded metal-tetracycline/H ؉ antiporter (TetA (B)) were constructed. All mutants were normally expressed. Among the 57 mutants (L99C to I155C), nine conserved arginine-, aspartate-, and glycine-replaced ones exhibited greatly reduced tetracycline resistance and almost no transport activity, and five conserved glycine-and proline-replaced mutants exhibited greatly reduced tetracycline transport activity in inverted membrane vesicles despite their high or moderate drug resistance. All other cysteine-scanning mutants retained normal drug resistance and normal tetracycline transport activity except for the L142C and I143C mutants. The transmembrane (TM) regions TM4 and TM5 were determined to comprise 20 amino acid residues, Leu-99 to Ile-118, and 17 amino acid residues, Ala-136 to Ala-152, respectively, on the basis of N-[ 14 C]ethylmaleimide ([ 14 C]NEM) reactivity. The NEM reactivity patterns of the TM4 and TM5 mutants were quite different from each other. TM4 could be divided into two halves, that is, a NEM nonreactive periplasmic half and a periodically reactive cytoplasmic half, indicating that TM4 is tilted toward a water-filled transmembrane channel and that only its cytoplasmic half faces the channel. On the other hand, NEM-reactive mutations were observed periodically (every two residues) along the whole length of TM5. A permeability barrier for a membrane-impermeable sulfhydryl reagent, 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid, was present in the middle of TM5 between Leu-142 and Gly-145, whereas all the NEM-reactive mutants as to TM4 were not accessible to 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid, indicating that the channel-facing side of TM4 is located inside the permeability barrier. Tetracycline protected the G141C mutant from the NEM binding, whereas the other mutants in TM4 and TM5 were not protected by tetracycline.

Putative transmembrane helices (TM) 1 and 11 in the metal-tetracycline/H؉ antiporter are predicted to be close to each other on the basis of disulfide cross-linking experiments of the double-cysteine mutants in the periplasmic loop regions (Kubo, Y., Konishi, S., Kawabe, T., Nada, S., and Yamaguchi, A. (2000) J. Biol. Chem. 275, 5270 -5274). In this study, each amino acid from Asn-2 to Gly-44 in the putative TM1 and loop1-2 regions or that from Ser-328 to Gly-366 in TM11 and its flanking regions was individually replaced with cysteine. With respect to the TM1 region, 10 mutants, from T5C to L14C, were all not reactive with N-ethylmaleimide (NEM), and from D15C to I22C, NEM-reactive and non-reactive mutations periodically appeared every two residues. Three mutants, M23C to V25C, were all NEM-reactive, but the degree of the latter two mutants was very low. Seven mutants, from L26C to E32C, were all highly reactive with NEM. Therefore, the region of TM1 is composed of the 21 amino acid residues from Thr-5 to Val-25. It is a partially amphiphilic helix, that is, the N-terminal (cytoplasmic) half is embedded in the hydrophobic interior, and the C-terminal (periplasmic) half faces a waterfilled channel. With respect to TM11, nine mutants, from S328C to G336C, and six mutants, from L361C to G366C, were all reactive with NEM. On the other hand, out of the 24 mutants, from L337C to S360C, 17 were not reactive with NEM, and the 7 NEM-reactive mutants were scattered, indicating that this region is a transmembrane segment. The 7 residues from Val-347 to Phe-353 including Pro-350 formed a central hydrophobic core, and the 7 NEM-reactive mutations were periodically distributed in its flanking regions, indicating that both ends of TM11 face a water-filled channel. Ala-354 is located at about 1/3 of the length from the periplasmic end of TM11. Disulfide cross-linking experiments on doublecysteine mutants having the combination of A354C and a cysteine-scanning mutation in the loop1-2 region indicated that loop1-2 is very flexible and close to the periplasmic end of TM11. Tetracycline prevented the cross-linking formation between the periplasmic ends of TM1 and TM11; however, it did not affect the crosslinking between loop1-2 and TM11, indicating that the substrate-induced conformational change involves a shift in the relative locations of TM1 and TM11.The transposon Tn10-encoded metal-tetracycline/H ϩ antiporter TetA(B) (2, 3) has been studied as a paradigm of antiporters belonging to a major facilitator superfamily (MFS). Its 12-membrane-spanning structure was at first determined on the basis of the results of hydropathy analysis and limited protease digestion (4). Afterward, the 12-membrane-spanning structure of TetA(C), which is a homolog of TetA(B) encoded by pBR322, was confirmed by a PhoA fusion experiment (5). We finally established the topology of TetA(B) by means of sitedirected competitive chemical modification of cysteine-introduced mutants, in which a cysteine residue was introduced into each putative loop region of ...

Each amino acid in putative transmembrane helix VI and its flanking regions, from Ser-156 to Thr-185, of a Cys-free mutant of the Tn10-encoded metal-tetracycline/H + antiporter (TetA(B)) was individually replaced by Cys. All of the cysteinescanning mutants showed a normal level of tetracycline resistance except for the S156C mutant, which showed moderate resistance, indicating that there is no essential residue located in this region. All 20 mutants from S159C to W178C showed no reactivity with N-ethylmaleimide (NEM), whereas the mutants of the flanking regions from S156C to H158C and F179C to T185C were highly or moderately reactive with NEM. These results indicate that like transmembrane helices III and IX, the transmembrane helix VI comprising residues Ser-159^Trp-178 is totally embedded in the hydrophobic environment.z 1999 Federation of European Biochemical Societies.