A triple mutant, K319N/H322Q/E325Q, of the lactose permease cotransports H+ with thiodigalactoside.
In a previous study, we characterized a lactose permease mutant (K319N/E325Q) that can transport H+ ions with sugar. This result was surprising because other studies had suggested that Glu-325 plays an essential role in H+ binding. To determine if the lactose permease contains one or more auxiliary H+ binding sites, we began with the K319N/E325Q strain, which catalyzes a sugar-dependent H+ leak, and isolated third site suppressor mutations that blocked the H+ leak. Three types of suppressors were obtained: H322Y, H322R, and M299I. These mutations blocked the H+ leak and elevated the apparent Km value for lactose. The M299I and H322Y suppressors could still transport H+ with beta-d-thiodigalactoside (TDG), but the H322R strain appeared uncoupled for H+/sugar cotransport. Four mutant strains containing a nonionizable substitution at codon 322 (H322Q) were analyzed. None of these were able to catalyze uphill accumulation of lactose, however, all showed some level of substrate-induced proton accumulation. The level seemed to vary based on the substrate being analyzed (lactose or TDG). Most interestingly, a triple mutant, K319N/H322Q/E325Q, catalyzed robust H+ transport with TDG. These novel results suggest an alternative mechanism of lactose permease cation binding and transport, possibly involving hydronium ion (H3O+).
Johnson
J L
JL
Department of Genetics, Cell Biology and Development, and the Bioprocess Technology Institute, University of Minnesota, St. Paul, MN 55108, USA.
Lockheart
M S
MS
Brooker
R J
RJ
D019943:Amino Acid Substitution / Q000235:genetics; D002414:Cations, Monovalent / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005973:Glutamine / Q000235:genetics / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010641:Phenotype; D017354:Point Mutation / Q000235:genetics*; D011522:Protons; D013489:Suppression, Genetic / Q000235:genetics; D027981:Symporters; D013862:Thiogalactosides / Q000378:metabolism*
D002414:Cations, Monovalent; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011522:Protons; D027981:Symporters; D013862:Thiogalactosides; D005973:Glutamine; D006859:Hydrogen; C031803:thiodigalactoside; C021499:lactose permease; D007785:Lactose