An analysis of suppressor mutations suggests that the two halves of the lactose permease function in a symmetrical manner

Nanette J. Pazdernik, Shane M. Cain, Robert J. Brooker

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21 Scopus citations


A conserved motif, GXXX(D/E)(R/K)XG[X](R/K)(R/K), is located in loop 2/3 and loop 8/9 in the lactose permease, and also in hundreds of evolutionarily related transporters. The importance of conserved residues in loop 8/9 was previously investigated (Pazdernik, N.J., Jessen-Marshall, A. E., and Brooker, R. J. (1997) J. Bacteriol. 179, 735-741). Although this loop was tolerant of many substitutions, a few mutations in the first position of the motif were shown to dramatically decrease lactose transport. In the current study, a mutant at the first position in the motif having very low lactose transport, Leu280, was used as a parental strain to isolate second-site revertants that restore function. A total of 23 independent mutants were sequenced and found to have a second amino acid substitution at several locations (G46C, G46S, F49L, A50T, L212Q, L216Q, S233P, C333G, F354C, G370C, G370S, and G370V). A kinetic analysis revealed that the first-site mutation, Leu280, had a slightly better affinity for lactose compared with the wild- type strain, but its V(max) for lactose transport was over 30-fold lower. The primary effect of the second-site mutations was to increase the V(max) for lactose transport, in some cases, to levels that were near the wild-type value. When comparing this study to second-site mutations obtained from loop 2/3 defective strains, a striking observation was made. Mutations in three regions of the protein, codons 45-50, 234-241, and 366-370, were able to restore functionality to both loop 2/3 and loop 8/9 defects. These results are discussed within the context of a C1/C2 alternating conformation model in which lactose translocation occurs by a conformational change at the interface between the two halves of the protein.

Original languageEnglish (US)
Pages (from-to)26110-26116
Number of pages7
JournalJournal of Biological Chemistry
Issue number42
StatePublished - Oct 17 1997


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