Evidence for structural symmetry and functional asymmetry in the lactose permease of Escherichia coli.
Previous work on the lactose permease of Escherichia coli has shown that mutations along a face of predicted transmembrane segment 8 (TMS-8) play a critical role in conformational changes associated with lactose transport (Green, A. L., and Brooker, R. J. [2001] Biochemistry 40, 12220-12229). Substitutions at positions 261, 265, 268, 272, and 276, which form a continuous stripe along TMS-8, were markedly defective for lactose transport velocity. In the current study, three single mutants (F261D, N272Y, N272L) and a double mutant (T265Y/M276Y) were chosen as parental strains for the isolation of mutants that restored transport function. A total of 68 independent mutants were isolated and sequenced. Forty-four were first-site revertants in which the original mutation was changed back to the wild-type residue or to a residue with a similar side-chain volume. The other 24 mutations were second-site suppressors in TMS-2 (Q60L, Q60P), loop 2/3 (L70H), TMS-7 (V229G/A), TMS-8 (F261L), and TMS-11 (F354V, C355G). On the basis of their locations, the majority of the second-site suppressors can be interpreted as improving the putative TMS-2/TMS-7/TMS-11 interface to compensate for conformational defects imposed by mutations in TMS-8 that disrupt the putative TMS-1/TMS-5/TMS-8 interface. Overall, this paper suggests that the TMS-2/TMS-7/TMS-11 interface is more important from a functional point of view, even though there is compelling evidence for structural symmetry between the two halves of the permease.
D019943:Amino Acid Substitution; D001692:Biological Transport / Q000235:genetics; D004926:Escherichia coli / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005838:Genotype; D007785:Lactose / Q000378:metabolism; D008553:Melibiose / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D013489:Suppression, Genetic; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D008553:Melibiose; D007785:Lactose