Alterations in the primary structure of the active transport carrier and changes in the function

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Several transport systems for nutrients in cell membranes are Na^+-coupled cotransport systems. One of the best characterized Na^+-coupled cotransport systems is the melibiose transport system in Esherichia coli.We isolated many mutants which showed altered cation specificity in the melibiose carrier. There were two types of mutant. The melibiose carrier of one type of the mutants lost the ability to couple to H^+ and acquired the ability to couple to Li^<> when melibiose was the substrate. The coupling to Na^+ in those mutants was normal. The melibiose carrier of another type of the mutants lost the ability to couple to H^<> and could utilize only Na^+ as the coupling csation. We analysed the second type mutants in this project.Mutated gene, melB, of the mutants was cloned, and the nucleotide sequence was determined. The nucleotide replacements caused the following substitutions of amino acid residues in the melibiose carrier; Pro-142 with Ser, Leu-232 with Phe, or Ala-236 with Thr or Val. These amino acid residues are located in slightly hydrophobic regions of the melibiose carier. The results provide a strong support for the idea that such regions or their vicinities which contain those amino acid residues play an important role in H^+ recognition or H^+ transport by the melibiose carrier.