ROLE OF CONSERVED RESIDUES IN HYDROPHILIC LOOP-8-9 OF THE LACTOSE PERMEASE

A peptide motif, GXXX(D/E)(R/K)XG(R/K)(R/K), has been conserved in a l arge group of evolutionarily related membrane proteins that transport small molecules across the membrane, Within the superfamily, this moti f is located in two cytoplasmic loops that connect transmembrane segme nts 2 and 3 and transmembrane segments 8 and 9, In a previous study co ncerning the loop 2 3 motif of the lactose permease (A. E. Jessen-Mars hall, N. J. Paul, and R. J. Brooker, J. Biol. Chem. 270:16251-16257, 1 995), it was shown that the first-position glycine and the fifth-posit ion aspartate are critical for transport activity since a variety of s ite-directed mutations greatly diminished the rate of transport. In th e current study, a similar approach was used to investigate the functi onal significance of the conserved residues in the loop 8-9 motif, In the wild-type lactose permease, however, this motif has been evolution arily modified so that the first-position glycine (an alpha-helix brea ker) has been changed to proline (also a helix breaker); the fifth pos ition has been changed to an asparagine; and one of the basic residues has been altered. In this investigation, we made a total of 28 single and 7 double mutants within the loop 8-9 motif to explore the functio nal importance of this loop, With regard to transport activity, amino acid substitutions within the loop 8-9 motif tend to be fairly well to lerated. Most substitutions produced permeases with normal or mildly d efective transport activities, However, three substitutions at the fir st position (i.e., position 280) resulted in defective lactose transpo rt, Kinetic analysis of position 280 mutants indicated that the defect decreased the V-max for lactose uptake, Besides substitutions at posi tion 280, a Gly-288-to-Thr mutant had the interesting property that th e kinetic parameters for lactose uptake were normal yet the rates of l actose efflux and exchange were approximately 10-fold faster than wild -type rates, The results of this study suggest that loop 8-9 may facil itate conformational changes that translocate lactose.