Abstract
The lactose permease is an integral membrane protein that cotransports H(+) and lactose into the bacterial cytoplasm. Previous work has shown that bulky substitutions at glycine 64, which is found on the cytoplasmic edge of transmembrane segment 2 (TMS-2), cause a substantial decrease in the maximal velocity of lactose uptake without significantly affecting the K(m) values (Jessen-Marshall, A. E., Parker, N. J., and Brooker, R. J. (1997) J. Bacteriol. 179, 2616-2622). In the current study, mutagenesis was conducted along the face of TMS-2 that contains glycine-64. Single amino acid substitutions that substantially changed side-chain volume at codons 52, 57, 59, 63, and 66 had little or no effect on transport activity, whereas substitutions at codons 49, 53, 56, and 60 were markedly defective and/or had lower levels of expression. According to helical wheel plots, Phe-49, Ser-53, Ser-56, Gln-60, and Gly-64 form a continuous stripe along one face of TMS-2. Several of the TMS-2 mutants (S56Y, S56L, S56Q, Q60A, and Q60V) were used as parental strains to isolate mutants that restore transport activity. These mutations were either first-site mutations or second-site suppressors in TMS-1, TMS-2, TMS-7 or TMS-11. A kinetic analysis showed that the suppressors had a higher rate of lactose transport compared with the corresponding parental strains. Overall, the results of this study are consistent with the notion that a face on TMS-2, containing Phe-49, Ser-53, Ser-56, Gln-60, and Gly-64, plays a critical role in conformational changes associated with lactose transport. We hypothesize that TMS-2 slides across TMS-7 and TMS-11 when the lactose permease interconverts between the C1 and C2 conformations. This idea is discussed within the context of a revised model for the structure of the lactose permease.
Highlights
The lactose permease is an integral membrane protein that cotransports H؉ and lactose into the bacterial cytoplasm
The results of this study are consistent with the notion that a face on transmembrane segment 2 (TMS-2), containing Phe-49, Ser-53, Ser-56, Gln-60, and Gly-64, plays a critical role in conformational changes associated with lactose transport
The NEM-inhibited strains were not subjected to a kinetic analysis, so it is not known if NEM modification blocks sugar binding and/or prevents conformational changes associated with lactose transport
Summary
The lactose permease is an integral membrane protein that cotransports H؉ and lactose into the bacterial cytoplasm. The results of this study are consistent with the notion that a face on TMS-2, containing Phe-49, Ser-53, Ser-56, Gln-60, and Gly-64, plays a critical role in conformational changes associated with lactose transport. Several topological studies are consistent with a secondary structural model in which the lactose permease contains 12 transmembrane segments in an ␣-helical conformation [7,8,9]. Analysis of the MFS for hydrophobicity, amphipathicity, loop length, and potential salt bridges between helices of the lactose permease provided enough information for us to propose a tertiary structure model [14]. In secondary models of the lactose permease, Gly-64 is predicted to lie along the cytoplasmic edge of TMS-2 This observation is consistent with the idea that a face on TMS-2 is important for conformational changes, which alternate the Hϩ. Mutagenesis was conducted along the face of TMS-2 that contains glycine 64 to see if such mutations have an effect on lactose transport
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