The 3-4 Loop of GltPh, an Archaeal Glutamate Transporter Homolog Mediates the Movement of the Transport Domain

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Excitatory amino acid transporters (EAATs) are a family of secondary transporters responsible for the cessation of glutamatergic synaptic transmission; these proteins catalyze the Na+ gradient-dependent transport of glutamate out of the synaptic cleft. Recent structural studies of the archaeal EAAT homolog, GltPh (which transports aspartate) suggest that transport is achieved by a rigid body, piston-like movement of the transport domain, which houses the substrate binding site in its entirety, across the membrane. The transport domain is connected to an immobile scaffold by 3 loops, one of which, the 3-4 loop, undergoes a substrate sensitive conformational change. Proteolytic cleavage of the 3-4 loop was found to abolish transport activity indicating an essential function for this loop in the transport mechanism. In this work, we demonstrate that despite cleavage of the 3-4 loop, GltPh is still able to sample transport relevant conformations. Optimization of the reconstitution conditions reveal that fully cleaved GltPh can catalyze some residual transport. Analysis of the kinetics and temperature dependence reveals that this decreased transport activity is not due to alteration of the substrate binding characteristics, but is caused by significantly reduced turnover rate. By measuring crosslink formation rates and solute counterflow activity, we demonstrate that cleaving the 3-4 loop severely compromises the transition of the transport domain between the inward- and outward-facing conformational states. These results reveal a hitherto unknown role for the 3-4 loop in a fundamental step in the transport mechanism.