CYSTEINE SCANNING MUTAGENESIS OF PUTATIVE HELIX-XI IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI

Using a functional lactose permease mutant devoid of Cys residues (C-l ess permease), each amino acid in putative transmembrane helix XI was individually replaced with Cys (from Ala347 to Ser366). Fifteen of the 20 mutants are highly functional and accumulate lactose to >60% of th e level achieved by C-less permease, and an additional three mutants, all located at the cytoplasmic end of the helix, exhibit lower but sig nificant lactose accumulation. Cys replacements for Thr348 or Lys358 r esult in virtually inactive permease. Lys358, however, is not essentia l for active lactose transport but plays a role in permease folding or membrane insertion by interacting with Asp237. Immunoblots reveal tha t all mutant proteins are present in the membrane in amounts comparabl e to C-less with the exception of Lys358-->Cys which is hardly detecta ble, as expected. The results highlight Thr348 as a potentially import ant residue for further analysis. Finally, all active mutants were ass ayed after treatment with the sulfhydryl reagent N-ethylmaleimide, and results range from nearly complete inhibition to almost 2-fold stimul ation. Remarkably, all of the strongly inhibited positions lie on one face of helix XI. The implications of the findings for packing of tran smembrane helices in the C-terminal half of the permease are discussed .