AN ANALYSIS OF SUPPRESSOR MUTATIONS SUGGESTS THAT THE 2 HALVES OF THELACTOSE PERMEASE FUNCTION IN A SYMMETRICAL MANNER

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 evolu tionarily related transporters, The importance of conserved residues i n loop 8/9 was previously investigated (Pazdernik, N. J., Jessen-Marsh all, A, E., and Brooker, R. J, (1997) J, Bacteriol. 179, 735-741), Alt hough this loop was tolerant of many substitutions, a few mutations in the first position of the motif were shown to dramatically decrease l actose transport, In the current study, a mutant at the first position in the motif having very low lactose transport, Leu(230), was used as it parental strain to isolate second-site revertants that restore fun ction, A total of 23 independent mutants were sequenced and found to h ave a second amino acid substitution at several locations (G46C, G46S, F49L, A50T, L212Q, L216Q, S233P, C333G, F354C, G370C, G370S, and G370 V). A kinetic analysis revealed that the first-site mutation, Leu(230) , had a slightly better affinity for lactose compared with the wild-ty pe 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 th e wild-type value. When comparing this study to second-site mutations obtained from loop 2/3 defective strains, a striking observation was m ade, 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 oc curs by a conformational change at the interface between the two halve s of the protein.