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.