In the present study, lactose permease mutants were isolated which have an enhanced recognition toward maltose (an alpha-glucoside) and diminished recognition for cellobiose (a beta-glucoside). Nine mutants were isolated from a strain encoding a wild-type permease (pTE18) and nine from a strain encoding a mutant permease which recognizes maltose (pB15). All 18 mutants were subjected to DNA sequencing, and it was found that all mutations are single base substitutions within the lac Y gene effecting single amino acid substitutions within the protein. From the pTE18 parent, substitutions involved Tyr-236 to Phe or His; Ser-306 to Thr; and six independent mutants in which Ala-389 was changed to Pro. From pB15, Tyr-236 was changed to Phe or Asn, Ser-306 to Thr or Leu, Lys-319 to Asn, and His-322 to Tyr, Asn, or Gln. All 18 mutants exhibited enhanced recognition for maltose (compared with the pTE18 strain) and a diminished recognition for cellobiose. In addition, all mutants showed a diminished recognition toward beta-galactosides as well. The Phe-236, His-236, Leu-306, Asn-319, Tyr-322, Asn-322, and Gln-322 mutants were completely defective in the uphill accumulation of methyl-beta-D-thiogalactopyranoside whereas the Asn-236, Thr-306, and Pro-389 mutants could effectively accumulate methyl-beta-D-thiogalactopyranoside against a concentration gradient. The mutants obtained in this study, together with previous lactose permease mutants, tend to be found on transmembrane segments, and those which are on the same transmembrane segment are often found three or four amino acids away from each other. This pattern is consistent with a protein structure in which important amino acid side chains project from several transmembrane segments in such a way as to form a hydrophilic channel for the recognition and transport of H+ and galactosides. It is proposed that the mechanism for H+/lactose cotransport is consistent with a "flanking gate" model in which the protein contains a single recognition site for galactosides within the channel which is flanked on either side by gates.
|Original language||English (US)|
|Number of pages||6|
|Journal||The Journal of biological chemistry|
|State||Published - Sep 5 1989|