THE ROLE OF TRANSMEMBRANE DOMAIN-III IN THE LACTOSE PERMEASE OF ESCHERICHIA-COLI

Deletion of putative transmembrane helix III from the lactose permease of Escherichia coli results in complete loss of transport activity. S imilarly, replacement of this region en bloc with 23 contiguous Ala, L eu, or Phe residues abolishes active lactose transport. The observatio ns suggest that helix III may contain functionally important residues; therefore, this region was subjected to Cys-scanning mutagenesis. Usi ng a functional mutant devoid of Cys residues (C-less permease) each r esidue from Tyr 75 to Leu 99 was individually replaced with Cys. Twent y-one of the 25 mutants accumulate lactose to >70% of the steady-state exhibited by C-less permease, and an additional 3 mutants transport t o lower, but significant levels (40-60% of C-less). Cys replacement fo r Leu 76 results in low transport activity (18% of C-less). However, w hen placed in the wild-type background, mutant Leu 76 --> Cys exhibits highly significant rates of transport (55% of wild type) and steady-s tate levels of lactose accumulation (65% of wild type). Immunoblots re veal that the mutants are inserted into the membrane at concentrations comparable to wild type. Studies with N-ethylmaleimide show that muta nt Gly 96 --> Cys is rapidly inactivated, whereas the other single-Cys mutants are not altered significantly by the alkylating agent, Moreov er, the rate of inactivation of Gly 96 --> Cys permease is enhanced at least 2-fold in the presence of P-galactopyranosyl 1-thio-beta,D-gala ctopyranoside. The observations demonstrate that although no residue p er se appears to be essential, structural properties of helix III are important for active lactose transport.