Galactoside-dependent proton transport by mutants of the Escherichia coli lactose carrier: substitution of tyrosine for histidine-322 and of leucine for serine-306.
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.
King
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
Wilson
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D001665:Binding Sites; D001692:Biological Transport; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005690:Galactose / Q000494:pharmacology*; D026901:Membrane Transport Proteins / Q000235:genetics*; D008756:Methylgalactosides / Q000493:pharmacokinetics; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D011522:Protons; D027981:Symporters; D013862:Thiogalactosides / Q000493:pharmacokinetics
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D008756:Methylgalactosides; D009004:Monosaccharide Transport Proteins; D011522:Protons; D027981:Symporters; D013862:Thiogalactosides; C009468:thiomethylgalactoside; C021499:lactose permease; D005690:Galactose