Cysteine scanning mutagenesis of putative transmembrane helices IX and X in the lactose permease of Escherichia coli

Abstract

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino-acid residue in putative transmembrane helices IX and X and the short intervening loop was systematically replaced with Cys (from Asn-290 to Lys-335). Thirty-four of 46 mutants accumulate lactose to high levels (70-100% or more of C-less), and an additional 7 mutants exhibit lower but highly significant lactose accumulation. As expected (see Kaback, H.R., 1992, Int. Rev. Cytol. 137A, 97-125), Cys substitution for Arg-302, His-322, or Glu-325 results in inactive permease molecules. Although Cys replacement for Lys-319 or Phe-334 also inactivates lactose accumulation, Lys-319 is not essential for active lactose transport (Sahin-Tóth, M., Dunten, R.L., Gonzalez, A., & Kaback, H.R., 1992, Proc. Natl. Acad. Sci. USA 89, 10547-10551), and replacement of Phe-334 with leucine yields permease with considerable activity. All single-Cys mutants except Gly-296 → Cys are present in the membrane in amounts comparable to C-less permease, as judged by immunological techniques. In contrast, mutant Gly-296 → Cys is hardly detectable when expressed at a relatively low rate from the lac promoter/operator but present in the membrane in stable form when expressed at a high rate from the T7 promoter. Finally, studies with N-ethylmaleimide (NEM) show that only a few mutants are inactivated significantly. Remarkably, the rate of inactivation of Val-315 → Cys permease is enhanced at least 10-fold in the presence of β-galactopyranosyl 1-thio-β,d-galactopyranoside (TDG) or an H⁺ electrochemical gradient (Δ). The results demonstrate that only three residues in this region of the permease–Arg-302, His-322, and Glu-325–are essential for active lactose transport. Furthermore, the enhanced reactivity of the Val-315 → Cys mutant toward NEM in the presence of TDG or Δ probably reflects a conformational alteration induced by either substrate binding or Δ.