Electrophysiological Characterization of Uncoupled Mutants of LacY Olga Gaiko, † Andre Bazzone, ∥ Klaus Fendler, ∥ and H. Ronald Kaback* ,†,‡,§ † Departments of Physiology and ‡ Microbiology, Immunology & Molecular Genetics, § Molecular Biology Institute, University of California−Los Angeles, Los Angeles, California 90095, United States ∥ Department of Biophysical Chemistry, Max Planck Institute of Biophysics, D-60438 Frankfurt am Main, Germany ABSTRACT: In this study of the lactose permease of Escherichia coli (LacY), five functionally irreplaceable residues involved specifically in H + translocation (Arg302 and Glu325) or in the coupling between protonation and sugar binding (Tyr236, Glu269, and His322) were mutated individually or together with mutant Glu325 → Ala. The wild type and each mutant were purified and reconstituted into proteoliposomes, which were then examined using solid-supported-membrane-based electrophysiology. Mutants Glu325 → Ala or Arg302 → Ala, in which H + symport is abolished, exhibit a weakly electrogenic rapid reaction triggered by sugar binding. The reaction is essentially absent in mutant Tyr236 → Phe, Glu269 → Ala, and His322 → Ala, and each of these mutations blocks the electrogenic reaction observed in the Glu325 → Ala mutant. The findings are consistent with the interpretation that the electrogenic reaction induced by sugar binding is due to rearrangement of charged residues in LacY and that this reaction is blocked by mutation of each member of the Tyr236/Glu269/His322 triad. In addition, further support is provided for the conclusion that deprotonation is rate limiting for downhill lactose/H + symport. T he major facilitator superfamily (MFS) is arguably the largest family of membrane transport proteins known at present. 1,2 The members are single-polypeptides with mostly 12 transmembrane helices that catalyze transport of small solutes into (uniport, symport) or out of (antiport) the cell. The lactose permease of Escherichia coli (LacY), a paradigm for the MFS, transduces free energy stored in an H + electrochemical gradient (Δμ̃ H + ; interior negative and/or alkaline) into a galactoside concentration gradient. However, because transport is obligatorily coupled (symport), LacY will also transduce free energy stored in an imposed sugar concentration gradient into a Δμ̃ H + , the polarity of which depends upon the direction of the sugar gradient. 3,4 LacY has been solubilized from the membrane and purified to homogeneity in a completely functional state, 5 and it is structurally, 6,7 as well as functionally, 8 a monomer. X-ray crystal structures of LacY 9−12 and various independent biochemical and spectroscopic findings 13−20 provide converg- ing evidence for an alternating access mechanism. By this means, H + and sugar binding induce coordinated opening and closing of periplasmic and cytoplasmic cavities, respectively, thereby allowing alternating accessibility of sugar- and H + - binding sites to either side of the membrane (the alternating access model) (reviewed in refs 21 and 22). It is also likely that the alternating access model for LacY involves formation of an occluded intermediate(s), 23−25 which is consistent with the highly dynamic nature of the protein. 18,26−30 Cys-scanning and site-directed mutagenesis of each residues in LacY demonstrate that only a few side chains that are located in a deep cavity in the middle of the molecule are irreplaceable for lactose/H + symport (Figure 1) (reviewed in refs 4 and 31). Thus, a carboxyl group at position 126 (helix IV), a guanidino group at position 144 (helix V), and an aromatic side chain at position 151 (helix V) are critical for sugar binding, 32−34 Tyr236 (helix VII), Glu269 (helix VIII), and His322 (helix X) are essential with respect to protonation of LacY and galactoside-binding affinity, 35−37 and Arg302 (helix IX) and Glu325 (helix X) are required for deprotonation. 38−40 Using site-directed alkylation, it was shown recently 41 that replace- ment of Tyr236 (helix VII), Glu269 (helix VIII), or His322 (helix X) causes spontaneous opening of the periplasmic cavity in the absence of sugar and decreased closing of the cytoplasmic cavity in the presence of a galactoside. In contrast, mutation of Arg302 (helix IX) or Glu325 (helix X) has no such Received: September 25, 2013 Revised: October 22, 2013 Published: October 23, 2013 Article pubs.acs.org/biochemistry © 2013 American Chemical Society 8261 dx.doi.org/10.1021/bi4013269 | Biochemistry 2013, 52, 8261−8266