A conserved motif, GXXX(D/E)(R/K)XG(R/K)(R/K), has been identified among a large group of evolutionarily related membrane proteins involved in the transport of small molecules across the membrane. To determine the importance of this motif within the lactose permease of Escherichia coli, a total of 28 site-directed mutations at the conserved first, fifth, sixth, eighth, ninth, and tenth positions were analyzed. A dramatic inhibition of activity was observed with all bulky mutations at the first-position glycine. Based on these results, together with sequence comparisons within the superfamily, it seems likely that small side chain volume (and possibly high beta-turn propensity) may be structurally important at this position. The acidic residue at the fifth position was also found to be very important for transport activity and even a conservative glutamate at this location exhibited marginal transport activity. In contrast, many substitutions at the eighth-position glycine, even those with a high side chain volume and/or low beta-turn propensity, still retained high levels of transport activity. Similarly, none of the basic residues within the motif were essential for transport activity when replaced individually by nonbasic residues. However, certain substitutions at the basic residue sites as well as the eighth-position glycine were observed to have moderately reduced levels of active transport of lactose. Taken together, the results of this study confirm the importance of the conserved loop 2/3 motif in transport function. It is suggested that this motif may be important in promoting global conformational changes within the permease.

A conserved motif, GXXX(D/E)(R/K)XG(R/K)(R/K), isfound in a large group of evolutionarily related membrane proteins involved in the transport of small molecules across the membrane. This motif is located within the cytoplasmic side of transmembrane domain 2 (TM-2) and extends through the hydrophilic loop that connects transmembrane domains 2 and 3. The motif is repeated again in the second half of the protein. In a previous study concerning the loop 2/3 motif (Jessen-Marshall, A. E., Paul, N. J., and Brooker, R. J. (1995) J. Biol. Chem. 270, 16251-16257), it was shown that the conserved aspartate at the fifth position in the motif is critical for transport activity since a variety of site-directed mutations were found to greatly diminish the rate of transport. In the current study, two of these mutations, in which the conserved aspartate was changed to threonine or serine, were used as parental strains to isolate second site suppressor mutations that restore transport function. A total of 10 different second site mutations were identified among a screen of 19 independent mutants. One of the suppressors was found within loop 1/2 in which Thr-45 was changed to arginine. Since the conserved aspartate and position 45 are at opposite ends of TM-2, these results suggest that the role of the conserved aspartate residue in loop 2/3 is to influence the topology of TM-2. Surprisingly, the majority of suppressor mutations were found in the second half of the permease. All of these are expected to alter helix topology in either of two ways. Some of the mutations involved residues within transmembrane segments 7 and 11 that produced substantial changes in side chain volume: TM-7 (Cys-234 3 Trp or Phe, Gln-241 3 Leu, and Phe-247 3 Val) and TM-11 (Ser-366 3 Phe). Alternatively, other mutations were highly disruptive substitutions at the ends of transmembrane segments or within hydrophilic loops (Gly-257 3 Asp, Val-367 3 Glu, Ala-369 3 Pro, and a 5-codon insertion into loop 11/12). It is hypothesized that the effects of these suppressor mutations are to alter the helical topologies in the second half of the protein to facilitate a better interaction with the first half. Overall, these results are consistent with a transport model in which TM-2 acts as an important interface between the two halves of the lactose permease. According to our tertiary model, this interaction occurs between TM-2 and TM-11.

) has shown that the conserved 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 transport activity. In this study, three mutants in which glycine-64 was changed to cysteine, serine, and valine were used as parental strains to isolate 64 independent suppressor mutations that restored transport function. Of these 64 isolates, 39 were first-site revertants to glycine or alanine, while 25 were second-site mutations that restored transport activity yet retained a cysteine, serine, or valine at position 64. The second-site mutations were found to be located at several sites within the lactose permease (Pro-28 3 Ser, Leu, or Thr; Phe-29 3 Ser; Ala-50 3 Thr, Cys-154 3 Gly; Cys-234 3 Phe; Gln-241 3 Leu; Phe-261 3 Val; Thr-266 3 Iso; Val-367 3 Glu; and Ala-369 3 Pro). A kinetic analysis was conducted which compared lactose uptake in the three parental strains and several suppressor strains. The apparent K m values of the Cys-64, Ser-64, and Val-64 parental strains were 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 max values of the Cys-64, Ser-64, and Val-64 strains were sharply reduced (3.9, 10.1, and 13.2 nmol of lactose/min ⅐ mg of protein, respectively) compared with the wild-type strain (676 nmol of lactose/min ⅐ mg of protein). 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 support the notion that Gly-64 in the wild-type permease is at a site in the protein which is important in facilitating conformational changes that are necessary for lactose translocation across the membrane. According to our tertiary model, this site is at an interface between the two halves of the protein.

A peptide motif, GXXX(D/E)(R/K)XG(R/K)(R/K), has been conserved in a large group of evolutionarily related membrane proteins that transport small molecules across the membrane. Within the superfamily, this motif is located in two cytoplasmic loops that connect transmembrane segments 2 and 3 and transmembrane segments 8 and 9. In a previous study concerning the loop 2-3 motif of the lactose permease (A. E. JessenMarshall, N. J. Paul, and R. J. Brooker, J. Biol. Chem. 270:16251-16257, 1995), it was shown that the firstposition glycine and the fifth-position aspartate are critical for transport activity since a variety of site-directed mutations greatly diminished the rate of transport. In the current study, a similar approach was used to investigate the functional significance of the conserved residues in the loop 8-9 motif. In the wild-type lactose permease, however, this motif has been evolutionarily modified so that the first-position glycine (an ␣-helix breaker) has been changed to proline (also a helix breaker); the fifth position 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 functional importance of this loop. With regard to transport activity, amino acid substitutions within the loop 8-9 motif tend to be fairly well tolerated. Most substitutions produced permeases with normal or mildly defective transport activities. However, three substitutions at the first position (i.e., position 280) resulted in defective lactose transport. Kinetic analysis of position 280 mutants indicated that the defect decreased the V max for lactose uptake. Besides substitutions at position 280, a Gly-288-to-Thr mutant had the interesting property that the kinetic parameters for lactose uptake were normal yet the rates of lactose efflux and exchange were approximately 10-fold faster than wildtype rates. The results of this study suggest that loop 8-9 may facilitate conformational changes that translocate lactose.