Abstract
Acidic residues which are found on transmembrane segments within the lactose permease may play an important role in H+ and/or sugar recognition. To examine the functional roles of Glu-269 and Glu-325, we have constructed a variety of amino acid substitutions (e.g. aspartate, glycine, alanine, serine, or glutamine) via site-directed mutagenesis. At position 269, all mutations appear to have a detrimental effect on sugar affinity, downhill transport, and counterflow. The Asp-269 mutant was able to accumulate lactose against a concentration gradient, whereas all of the nonionizable substitutions at position 269 were completely defective. Nevertheless, in spite of their inability to actively accumulate sugars, Gly-269, Ala-269, and Gln-269 mutants were observed to transport H+ upon the addition of galactosides. Mutations at position 325 had a markedly different phenotype. For example, the Asp-325, Gly-325, and Gln-325 mutants exhibited an apparent Km for lactose transport (e.g. 0.21, 0.47, and 0.50 mM, respectively), which was actually lower than that of the wild-type strain (1.44 mM). In counterflow assays, all position 325 mutants also appear to catalyze lactose exchange. Similar to the results obtained at position 269, the Asp-325 mutant exhibited moderate levels of accumulation, whereas none of the nonionizable mutations at position 325 were able to accumulate galactosides against a concentration gradient. However, unlike the position 269 mutants, no H+ transport was observed in the Gly-325, Ala-325, Ser-325, or Gln-325 strains upon the addition of lactose, S-beta-D-galactopyranosyl-(1,1)-beta-thiogalactopyranoside, 1-O-methyl-beta-D-galactopyranoside, or melibiose. Furthermore, in these mutants, the efflux of lactose during counterflow assays became insensitive to delta pH. Overall, these results are consistent with the notion that an acidic residue at position 325 is required for H+ transport via the lactose permease. Alternative hypotheses are also discussed.
Highlights
Acidic residues which are found on transmembrane permease, l a c y, has been cloned on multicopy vectors and sesegments within the lactose permease may play an im- quenced [9, 10]
At position 269, all mutations In a variety of different types of transport proteins, acidic appear to have a detrimental effect on sugar affinity, amino acids have been implicated to be involved with cation downhill transport, and counterflow
F-type ion motive ATPases, Asp-61 within subunit c of the Fo. In spiteof their inability to actively accumulate sugars, Gly-269,Ala-269, and Gln-269 mutants were observed to transportH+upon the addition of galactosides
Summary
GAA to GGA nose), melibiose (O-a-D-galaCtOpyranOSyl-(1,6)-D-glUCOpyranose)T,DGI pE269A. HgCl, was included in the wash buffer to rapidly inhibit the lactose RF DNA was isolated and digested with EcoRI t o produce the permease and thereby minimize sugaerfflux during the removaolf the 2,300-base pair fragment containing thleacy gene. This fragment was extracellular medium.As a control,the lacy- strainH, S4006/F'IQZ+Y-/ ligated into tEhecoRI site of pACYC184(Ref. 31).Following transpACYC184 (no lacy insert), was assayed for radiolabeled sugar formation of E. coli strain T184, cells harboring hybrid plasmids with a uptake in order to obtain an accurate valuefor nonspecific sugar up- lacy insert weriedentified by their loss of chloramphenicol resistance. To initiate sugar-induced H' transport, an anaerobic solution containing
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