An analysis of suppressor mutations suggests that the two halves of the lactose 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 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 Vmax for lactose transport was over 30-fold lower. The primary effect of the second-site mutations was to increase the Vmax 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.
Pazdernik
N J
NJ
Department of Genetics and Cell Biology and the Bioprocess Technology Institute, University of Minnesota, St. Paul, Minnesota 55108, USA.
Cain
S M
SM
Brooker
R J
RJ
D001692:Biological Transport; D029968:Escherichia coli Proteins; D016153:Genes, Suppressor; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D007785:Lactose