Galactoside-dependent proton transport by mutants of the Escherichia coli lactose carrier: substitution of tyrosine for histidine-322 and of leucine for serine-306

It is concluded that the presence of a histidine residue at position 322 of the lactose carrier is not obligatory for H+ transport per se, and the mutants cloned by a novel 'sucrose marker exchange' method retain the capacity to carry out facilitated diffusion with lactose or melibiose.

Abstract

The lac Y genes from two Escherichia coli mutants, MAB20 and AA22, have been cloned in a multicopy plasmid by a novel 'sucrose marker exchange' method. Characterization showed that the plasmids express a lactose carrier with poor affinity for lactose. Neither mutant carried out concentrative uptake with methyl beta-D-galactopyranoside, lactose, or melibiose as the substrate. Nor did the mutants catalyze counterflow or exchange with methyl beta-D-galactopyranoside. Both mutants did, however, retain the capacity to carry out facilitated diffusion with lactose or melibiose. DNA sequencing revealed that MAB20 (histidine-322 to tyrosine) and AA22 (serine-306 to leucine) have amino acid substitutions within the putative 'charge-relay' domain thought to be responsible for proton transport. Galactoside-dependent H+ transport was readily measured in both mutants. We conclude, therefore, that the presence of a histidine residue at position 322 of the lactose carrier is not obligatory for H+ transport per se.