Evidence that the asparagine 322 mutant of the lactose permease transports protons and lactose with a normal stoichiometry and accumulates lactose against a concentration gradient.
The single asparagine 322 mutant of the lactose permease was made by constructing a hybrid plasmid which contained the amino-terminal coding sequence from the wild-type permease gene and the carboxyl-terminal coding sequence from a previously characterized double mutant permease which contained an asparagine residue at position 322. Since histidine at position 322 has been postulated to be critically involved with H+ transport and the active accumulation of sugars, the ability of the Asn-322 mutant to couple H+ and sugar transport was carefully examined. Measurements of proton/lactose stoichiometries gave very similar values for the wild-type (0.78) and the Asn-322 strain (0.82). Moreover, the Asn-322 mutant was able to effectively accumulate lactose against a concentration gradient although the levels of accumulation in the Asn-322 mutant (approximately 5-7-fold) were significantly less than that of the wild-type strain (approximately 30-40-fold). Overall, these results are inconsistent with the notion that an ionizable histidine residue at position 322 is obligatorily required for H+ transport or the active accumulation of galactosides against a concentration gradient. The ability of the Asn-322 mutant to recognize a variety of sugars was compared with wild-type, Val-177, and Val-177/Asn-322 strains. The Asn-322 mutant exhibited an ability to recognize and transport maltose (an alpha-glucoside) which was significantly better than the wild-type strain but not as good as either the single Val-177 mutant or the double Val-177/Asn-322 mutant. Both the Asn-322 and the Val-177/Asn-322 strain showed a relatively poor recognition for alpha-galactosides (i.e. melibiose), beta-galactosides (lactose and thiodigalactoside), and beta-glucosides (cellobiose). In contrast, the single Val-177 strain exhibited a normal recognition for these sugars.
Franco
P J
PJ
Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108.
Brooker
R J
RJ
D001216:Asparagine; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D005838:Genotype; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007785:Lactose / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D010957:Plasmids; D011522:Protons; D015183:Restriction Mapping; D013379:Substrate Specificity; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011522:Protons; D027981:Symporters; D001216:Asparagine; C021499:lactose permease; D007785:Lactose