title: Substrate selectivity in glutamate-dependent acid resistance in enteric bacteria.
abstract: The bacterial antiporter GadC plays a central role in the glutamate (Glu)-dependent acid resistance system, which protects enteric bacteria against the extreme acidity of the human stomach. Upon acid shock, GadC imports Glu into the cytoplasm, where Glu decarboxylases consume a cytoplasmic proton, which ends up as a "virtual" proton in the decarboxylated product γ-aminobutyric acid (GABA) and is then exported via GadC. It was therefore proposed that GadC counters intracellular acidification by continually pumping out virtual protons. This scenario, however, is oversimplified. In gastric environments, GadC encounters substrates in multiple carboxyl protonation forms (outside: Glu(-), Glu(0), Glu(+); inside: GABA(0), GABA(+)). Of the six possible combinations of antiport partners, Glu(+)/GABA(0) results in proton influx, Glu(0)/GABA(0) and Glu(+)/GABA(+) are proton neutral, and Glu(-)/GABA(0), Glu(-)/GABA(+), or Glu(0)/GABA(+) lead to proton extrusion. Which of these exchanges does GadC catalyze? To attack this problem, we developed an oriented GadC liposome system holding a three-unit inward pH gradient to mimic the conditions facing bacteria in the stomach. By assessing the electrogenicity of substrate transport, we demonstrate that GadC selectively exchanges Glu(-) or Glu(0) with GABA(+), resulting in effective proton extrusion of >0.9 H(+) per turnover to counter proton invasion into acid-challenged bacteria. We further show that GadC selects among protonated substrates using a charge-based mechanism, rather than directly recognizing the protonation status of the carboxyl groups. This result paves the way for future work to identify the molecular basis of GadC's substrate selectivity.
authors.affiliation: Department of Biochemistry, Brandeis University, Waltham, MA 02454, USA.
authors.forename: Ming-Feng
authors.initials: MF
authors.lastname: Tsai
authors.forename: Patrick
authors.initials: P
authors.lastname: McCarthy
authors.forename: Christopher
authors.initials: C
authors.lastname: Miller
mesh_terms: D001692:Biological Transport; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D018698:Glutamic Acid / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry*; D008954:Models, Biological; D017494:Proton Pumps; D011522:Protons; D013379:Substrate Specificity; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; C491226:GadC protein, E coli; D008081:Liposomes; D008565:Membrane Proteins; D017494:Proton Pumps; D011522:Protons; D018698:Glutamic Acid; D005680:gamma-Aminobutyric Acid