MEMBRANE TOPOLOGY OF THE TRANSPOSON 10-ENCODED METAL-TETRACYCLINE H+ ANTIPORTER AS STUDIED BY SITE-DIRECTED CHEMICAL LABELING/

The transposon (Tn) 10-encoded metal-tetrarcycline/H+ antiporter (Tn10 -TetA) is predicted to have a membrane topology involving 12 transmemb rane domains on the basis of the hydropathy profile of its sequence an d the results of limited proteolysis; however, the experimental result s of limited proteolysis are not enough to confirm the topology becaus e proteases cannot gain access from the periplasmic side (Eckert, B., and Beck, C. F. (1989) J. Biol. Chem. 264, 11663-11670). One or two cy steine residues were introduced into each predicted hydrophilic loop o r the N-terminal segment of Tn10-TetA by site directed mutagenesis, an d then the topology of the protein was determined by examining whether labeling of the introduced Cys residue by membrane-permeant [C-14]N-e thylmaleimide ([C-14]NEM) was prevented by preincubation of intact cel ls with the membrane-impermeant maleimide, 4-acetamido-4'-maleimidylst ilbene-2,2'-disulfonic acid (AMS). The binding of [C-14]NEM to the S36 C (loop 1-2), L97C (loop 3-4), S156C (loop 5-6), R238C (loop 7-8), S29 6C (loop 9-10), Y357C, and D365C (loop 10-11) mutants was completely b locked by pretreatment with AMS, indicating that these residues are lo cated on the periplasmic surface, In contrast, [C-14]NEM binding to th e S4C (N-terminal segment), S65C (loop 2-3), D120C (loop 4-5), S199C a nd S201C (loop 6-7), T270C (loop 8-9), and S328C (loop 10-11) mutants was not affected by pretreatment with AMS, indicating that these resid ues are on the cytoplasmic surface, These results for the first time t horoughly confirm the 12-transmembrane topology of the metal-tetracycl ine/H+ antiporter.