HELIX PACKING IN THE SUCROSE PERMEASE OF ESCHERICHIA-COLI - PROPERTIES OF ENGINEERED CHARGE PAIRS BETWEEN HELIX-VII AND HELIX-XI

Of four putative intramembrane charge pairs in lactose permease, only three are conserved in the homologous sucrose permease of Escherichia coli [Bockmann, J., Heuel, H., and Lengeler, J. W. (1992) Mol. Gen. Ge net. 235, 22-32]. The missing charge pair was introduced into wild-typ e sucrose permease by site-directed mutagenesis of Asn234 (helix VII) and Ser356 (helix XI). Individual replacement of either residue with a charged amino acid abolishes active sucrose transport with the except ion of the Asn234-->Asp mutant. However, simultaneous replacement of A sn234 with Asp or Glu and Ser356 with Arg or Lys results in high activ ity. Thus, an acidic residue at position 234 rescues the activity of t he Ser356-->Arg or Ser356-->Lys mutant, and a basic residue at positio n 356 rescues the activity of the Asn234-->Glu mutant. Furthermore, wh en expressed at a relatively low rate, the double mutant Asn234-->Asp/ Ser356-->Arg is present in the membrane in a significantly greater amo unt than wild-type, suggesting that the charge pair improves insertion of sucrose permease into the membrane. The results indicate that heli ces VII and XI of sucrose permease are in close proximity and that a c harge pair interaction can be established between residues 234 (helix VII) and 356 (helix XI). However, interchange of the acidic residue at position 234 with the basic residue at position 356 abolishes sucrose transport. Clearly, therefore, the interaction between the engineered residues in sucrose permease is more complex than the corresponding A sp237-Lys358 interaction in lactose permease where reversal of the cha rge pair has little or no effect on activity [Sahin-Toth, M., Dunten, R. L., Gonzalez, A., and Kaback, H. R. (1992) Proc. Natl. Acad. Sci. U .S.A. 89, 10547-10551].