CYSTEINE SCANNING MUTAGENESIS OF THE N-TERMINAL 32 AMINO-ACID-RESIDUES IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI

Using a functional lactose permease mutant devoid of Cys residues (C-l ess permease), each amino acid residue in the hydrophilic N-terminus a nd the first putative transmembrane helix was systematically replaced with Cys (from Tyr-2 to Trp-33). Twenty-three of 32 mutants exhibit hi gh lactose accumulation (70-100% or more of C-less), and an additional 8 mutants accumulate to lower but highly significant levels. Surprisi ngly, Cys replacement for Gly-24 or Tyr-26 yields fully active permeas e molecules, and permease with Cys in place of Pro-28 also exhibits si gnificant transport activity, although previous mutagenesis studies on these residues suggested that they may be required for lactose transp ort. As expected, Cys replacement for Pro-31 completely inactivates, i n agreement with previous findings indicating that ''helix-breaking'' propensity at this position is necessary for full activity (Consler TG , Tsolas O, Kaback HR, 1991, Biochemistry 30:1291-1297). Twenty-nine m utants are present in the membrane in amounts comparable to C-less per mease, whereas membrane levels of mutants Tyr-3 --> Cys and Phe-12 --> Cys are slightly reduced, as judged by immunological techniques. Dram atically, mutant Phe-9 --> Cys is hardly detectable when expressed fro m the lac promoter/operator at a relatively low rate, but is present i n the membrane in a stable form when expressed at a high rate from the T7 promoter. Finally, studies with N-ethylmaleimide show that 6 Cys-r eplacement mutants that cluster at the C-terminal end of putative heli x I are inactivated significantly. The results demonstrate that althou gh no residue per se in this region is essential for activity, the str uctural integrity of the periplasmic half of the first transmembrane h elix is important for active lactose transport.