Abstract
GadC, a central component of the Escherichia coli acid resistance system, is a Glu/GABA antiporter. A previous structural study and biochemical characterization showed that GadC exhibits a stringent pH dependence for substrate transport, with no detectable activity at pH values above 6.5. However, the substrate selectivity and the mechanism of pH-dependent transport activity of GadC remain enigmatic. In this study, we demonstrate that GadC selectively transports Glu with no net charge and GABA with a positive charge. A C-plug-truncated variant of GadC (residues 1-470) transported Gln (a mimic of Glu with no net charge), but not Glu, even at pH 8.0. The pH-dependent transport of Gln by this GadC variant was shifted ~1 unit toward a higher pH compared with Glu transport. Taken together, the results identify the substrate selectivity for GadC and show that the protonation states of substrates are crucial for transport.
Highlights
GadC is a glutamate/GABA antiporter in the Escherichia coli acid resistance system
We demonstrate that GadC selectively transports Glu with no net charge and GABA with a positive charge
On the basis of these results, we propose a working model to illustrate the mechanism of the pH-dependent transport by GadC in E. coli (Fig. 5)
Summary
GadC is a glutamate/GABA antiporter in the Escherichia coli acid resistance system. Results: GadC selectively transports glutamate with no net charge and GABA with a positive charge. Gln exists in two potential charged states: with no net charge (Gln0) and with one positive charge (Glnϩ) (Fig. 1C) Among these various states, Glnϩ and Gluϩ require protonation of their ␣-carboxyl groups (pKa ϳ 2.17–2.19) and are unlikely to populate within living cells, where the pH value cannot drop below 3.5 [20]. Previous studies show that the exchange of Glu and GABA is strongly influenced by membrane potential in the proteoliposome-based assay; GABA efflux and Glu influx are markedly increased at a positive potential inside the proteoliposomes and decreased at negative potentials inside the proteoliposomes [18]. We demonstrate that GadC transports Glu0, Gln0, and GABAϩ in a proteoliposome-based counterflow assay These results indicate that the protonation states of a given substrate are crucial for transport and that side chain deprotonation of Glu and GABA at neutral pH impedes Glu/ GABA exchange
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