| Abstract
| - Previous work on the lactose permease of Escherichia coli has shown that mutations along aface of predicted transmembrane segment 2 (TMS-2) play a critical role in conformational changesassociated with lactose transport [Green, A. L., Anderson, E. J., and Brooker, R. J. (2000) J. Biol. Chem.275, 23240−23246]. In the current study, mutagenesis was conducted along the side of predicted TMS-8that contains the first amino acid in the conserved loop 8/9 motif. Several substitutions at positions 261,265, 272, and 276 were markedly defective for downhill lactose transport although these mutants werewell expressed. Substitutions along the entire side of TMS-8 containing the first amino acid in the loop8/9 motif displayed defects in uphill lactose transport. Again, substitutions at positions 261, 265, 268,272, and 276 were the most defective, with several of these mutants showing no lactose accumulationagainst a gradient. According to helical wheel plots, Phe-261, Thr-265, Gly-268, Asn-272, and Met-276form a continuous stripe along one face of TMS-8. These results are discussed according to our hypotheticalmodel, in which the two halves of the protein form a rotationally symmetrical dimer. In support of thismodel, alignment of predicted TMS-2 and TMS-8 shows an agreement between the amino acid residuesin these transmembrane segments that are critical for lactose transport activities.
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