An analysis of the side chain requirement at position 177 within the lactose permease which confers the ability to recognize maltose.
In previous work (Brooker, R. J., and Wilson, T. H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 3959-3963), lactose permease mutants were isolated which possessed an enhanced recognition for maltose. In some of these mutants, the wild-type alanine residue at position 177 was changed to valine or threonine. To gain further insight into the side chain requirement at position 177 that confers maltose recognition, further substitutions of isoleucine, leucine, phenylalanine, proline, and serine have been made via site-directed mutagenesis. Permeases containing alanine or serine exhibited poor maltose recognition whereas those containing isoleucine, leucine, phenylalanine, proline, or valine showed moderate or good recognition. As far as galactosides are concerned, the Val-177, Pro-177, and Ser-177 mutants were able to transport lactose as well as, or slightly better than, the wild-type strain. The other mutants displayed moderately reduced levels of lactose transport. For example, the Phe-177 mutant, which was the most defective, showed a level of downhill transport which was approximately 20% that of the wild-type strain. In uphill transport assays, all of the position 177 mutants were markedly defective in their ability to accumulate beta-D-thiomethylgalactopyranoside against a concentration gradient. Finally, the position 177 mutants were analyzed for their ability to catalyze an H+ leak. Interestingly, even though the wild-type permease does not leak H+ across the bacterial membrane, all of the position 177 mutants were shown to transport H+ in the absence of sugars. For most of the mutants, this H+ leak was blocked by the addition of beta-D-thiodigalactoside. Overall, these results are discussed with regard to the effects of position 177 substitutions on the sugar recognition site and H+ transport.
Gram
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CD
Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108.
Brooker
R J
RJ
D001483:Base Sequence; D001565:Benzoates / Q000378:metabolism; D019817:Benzoic Acid; D001692:Biological Transport; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D029968:Escherichia coli Proteins; D005960:Glucosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008320:Maltose / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D027981:Symporters
D001565:Benzoates; D029968:Escherichia coli Proteins; D005960:Glucosides; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D008320:Maltose; D019817:Benzoic Acid; C021499:lactose permease