Complete cysteine-scanning mutagenesis and site-directed chemical modification of the Tn10-encoded metal-tetracycline/H+ antiporter.
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.
Tamura
N
N
Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.
Konishi
S
S
Iwaki
S
S
Kimura-Someya
T
T
Nada
S
S
Yamaguchi
A
A
D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003545:Cysteine / Q000235:genetics*; D004251:DNA Transposable Elements; D005033:Ethylmaleimide / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
D017920:Antiporters; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; C079051:tetA protein, Bacteria; D003545:Cysteine; D005033:Ethylmaleimide