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Volume 272, Number 1, Issue of January 3, 1997 pp. 580-585
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Membrane Topology of the Transposon 10-encoded Metal-Tetracycline/H⁺ Antiporter as Studied by Site-directed Chemical Labeling

(Received for publication, August 26, 1996, and in revised form, October 12, 1996)

Tomomi Kimura ^§ , Masae Ohnuma ^§ , Tetsuo Sawai ^§ and Akihito Yamaguchi

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.

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