title: Electrophysiological characterization of uncoupled mutants of LacY.
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.
authors.affiliation: Departments of Physiology and ‡Microbiology, Immunology & Molecular Genetics, §Molecular Biology Institute, University of California-Los Angeles , Los Angeles, California 90095, United States.
authors.forename: Olga
authors.initials: O
authors.lastname: Gaiko
authors.forename: Andre
authors.initials: A
authors.lastname: Bazzone
authors.forename: Klaus
authors.initials: K
authors.lastname: Fendler
authors.forename: H Ronald
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D055724:Electrophysiological Phenomena; D029968:Escherichia coli Proteins / Q000235:genetics / Q000502:physiology; D007785:Lactose / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000502:physiology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011510:Proteolipids / Q000378:metabolism; D027981:Symporters / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D008081:Liposomes; D026901:Membrane Transport Proteins; D011510:Proteolipids; D027981:Symporters; C020485:proteoliposomes; C021499:lactose permease; D007785:Lactose