ALTERATION OF NA-COUPLED TRANSPORT IN SITE-DIRECTED MUTANTS OF THE MELIBIOSE CARRIER OF ESCHERICHIA-COLI()

Asn-58 of the Escherichia coli melibiose carrier was replaced by Ala, Leu, Ser, and Gin. Trp-54 was replaced by Leu and a double mutant Leu- 54/Ala-58 was constructed using site-directed mutagenesis. Cation/suga r cotransport and sugar-induced cation uptake were studied for each mu tant. The change of Asn-58 to Ala results in a nearly complete loss of Na+-stimulated galactoside transport as well as sugar-stimulated Nauptake. Substitutions of Leu, Gin, and Ser for Asn-58 were also defect ive in Na+-stimulated sugar transport. The Trp-54 to Leu mutant shows moderate sugar accumulation with cation selectivity similar to wild-ty pe. The double mutant Leu-54/Ala-58 shows elevated H+-melibiose cotran sport as well as reduced Na+-stimulated melibiose cotransport. These r esults suggest that Asn-58 is important for Na+ recognition.