A triple mutant, K319N/H322Q/E325Q, of the lactose permease cotransports H+ with thiodigalactoside

J. L. Johnson, M. S.K. Lockheart, R. J. Brooker

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13 Scopus citations


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 β-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+).

Original languageEnglish (US)
Pages (from-to)215-224
Number of pages10
JournalJournal of Membrane Biology
Issue number3
StatePublished - Jun 1 2001


  • Cotransporter
  • Lactose permease
  • Sugar transporter
  • Symporter


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