SUPPRESSOR ANALYSIS OF MUTATIONS IN THE LOOP-2-3 MOTIF OF LACTOSE PERMEASE - EVIDENCE THAT GLYCINE-64 IS AN IMPORTANT RESIDUE FOR CONFORMATIONAL-CHANGES

A superfamily of transport proteins, which includes the lactose permea se of Escherichia coli, contains a highly conserved motif, G-X-X-X-D/E -R/K-X-G-R/K-R/K, in the loops that connect transmembrane segments 2 a nd 3 and transmembrane segments 8 and 9. Previous analysis of this mot if in the lactose permease (A. E. Jessen-Marshall, N. J. Paul, and R. J. Brooker, J. Biol. Chem. 270:16251-16257, 1995) has shown that the c onserved glycine residue found at the first position in the motif (i.e ., Gly-64) is important for transport function. Every substitution at this site, with the exception of alanine, greatly diminished lactose t ransport activity. In this study, three mutants in which glycine-64 wa s changed to cysteine, serine, and valine were used as parental strain s to isolate 64 independent suppressor mutations that restored transpo rt function. Of these 63 isolates, 39 were first-site revertants to gl ycine or alanine, while 25 were second-site mutations that restored tr ansport activity yet retained a cysteine, serine, or valine at positio n 64. The second-site mutations were found to be located at several si tes within the lactose permease (Pro-28 --> Ser, Leu, or Thr; Phe-29 - -> Ser; Ala-50 --> Thr, Cys-154 --> Gly; Cys-234 --> Phe; Gln-241 --> Leu; Phe-261 --> Val; Thr-266 --> Iso; Val-367 --> Glu; and Ala-369 -- > Pro). A kinetic analysis was conducted which compared lactose uptake in the three parental strains and several suppressor strains. The app arent K-m values of the Cys-64, Ser-64, and Val-64 parental strains we re 0.8 mM, 0.7 mM, and 4.6 mM, respectively, which was similar to the apparent K-m of the wild-type permease (1.4 mM). In contrast, the V-ma x values of the Cys-64, Ser-64, and Val-64 strains were sharply reduce d (3.9, 10.1, and 13.2 nmol of lactose/min.mg of protein, respectively ) compared with the wild-type strain (676 nmol of lactose/min.mg of pr otein). The primary effect of the second-site suppressor mutations was to restore the maximal rate of lactose transport to levels that were similar to the wild-type strains. Taken together, these results suppor t the notion that Gly-64 in the wild-type permease is at a site in the protein which is important in facilitating conformational changes tha t are necessary for lactose translocation across the membrane. Accordi ng to our tertiary model, this site is at an interface between the two halves of the protein.