Coupling mechanism and ion selectivity in Na+/H+-driven membrane transport systems
The SLC1 family of glutamate transporters conserved across prokaryotes and eukaryotes, which mediate the uptake of negatively charged amino acids coupled to Na⁺ or H⁺ gradients. In humans, excitatory amino acid transporters (EAATs) use Na⁺ to remove glutamate from synapses, whereas Escherichia coli GltP, the focus of this study, operates strictly through H⁺ coupling.
This divergence highlights the evolutionary of ion selectivity within a conserved structural framework. Using purified GltP reconstituted into liposomes, strict proton coupling and electrogenic transport were demonstrated, with no sodium dependence. GltP transported glutamate and aspartate with comparable affinities, and transport was strongly stimulated by a proton gradient and negative-inside potential. Stoichiometric analysis revealed co-transport of three protons per substrate, paralleling the three Na⁺ ions coupled in sodium-dependent homologs. Cryo-EM analysis yielded the first structural insights into a proton-coupled glutamate transporter: while the scaffold domain resembled Na⁺-coupled counterparts, the transport domain remained unresolved, indicating high conformational flexibility.
Sybodies developed to stabilize GltP inhibited approximately 80% of activity but failed to resolve the transport domain, suggesting the need for additional stabilizing conditions. Beyond mechanistic characterization, the thesis explores synthetic biology applications by integrating transport with bioenergetics. A chemiosmotic module comprising MleP (L-malate/lactate antiporter) and MleS (L-malate decarboxylase) generated a self-sustaining proton motive force that powered GltP- and LacY-mediated nutrient uptake, establishing a synthetic platform coupling energy transduction, transport, and metabolism in minimal cells.