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.