Abstract
Secondary transporters in the excitatory amino acid transporter family terminate glutamatergic synaptic transmission by catalyzing Na(+)-dependent removal of glutamate from the synaptic cleft. Recent structural studies of the aspartate-specific archaeal homolog, Glt(Ph), suggest that transport is achieved by a rigid body, piston-like movement of the transport domain, which houses the substrate-binding site, between the extracellular and cytoplasmic sides of the membrane. This transport domain is connected to an immobile scaffold by three loops, one of which, the 3-4 loop (3L4), undergoes substrate-sensitive conformational change. Proteolytic cleavage of the 3L4 was found to abolish transport activity indicating an essential function for this loop in the transport mechanism. Here, we demonstrate that despite the presence of fully cleaved 3L4, Glt(Ph) is still able to sample conformations relevant for transport. Optimized reconstitution conditions reveal that fully cleaved Glt(Ph) retains some transport activity. Analysis of the kinetics and temperature dependence of transport accompanied by direct measurements of substrate binding reveal that this decreased transport activity is not due to alteration of the substrate binding characteristics but is caused by the significantly reduced turnover rate. By measuring solute counterflow activity and cross-link formation rates, we demonstrate that cleaving 3L4 severely and specifically compromises one or more steps contributing to the movement of the substrate-loaded transport domain between the outward- and inward-facing conformational states, sparing the equivalent step(s) during the movement of the empty transport domain. These results reveal a hitherto unknown role for the 3L4 in modulating an essential step in the transport process.
Highlights
The extracellular loop, 3L4, plays an important, unknown role in the transport cycle of the glutamate transporter homolog, GltPh
Cleaving 3L4 Does Not Prevent GltPh from Sampling the Inward-facing Conformation—The observed reduction in transport activity by GltPh cleaved at residue 125 in the 3L4 may be due to a decrease in the protein’s ability to sample particular states in the transport cycle, to changes in its ability to bind substrate or Naϩ, to changes in transition rates between two or more cycle states, or a combination thereof
In the context of a simplified kinetic scheme, which captures the essential features of transport, we find strong evidence that the basic binding/unbinding reactions with substrate and Naϩ are minimally affected by cutting 3L4 and, surprisingly, that the transfer of the empty transport domain is preserved
Summary
The extracellular loop, 3L4, plays an important, unknown role in the transport cycle of the glutamate transporter homolog, GltPh. By measuring solute counterflow activity and cross-link formation rates, we demonstrate that cleaving 3L4 severely and compromises one or more steps contributing to the movement of the substrate-loaded transport domain between the outwardand inward-facing conformational states, sparing the equivalent step(s) during the movement of the empty transport domain These results reveal a hitherto unknown role for the 3L4 in modulating an essential step in the transport process. Loop Cleavage Inhibits Conformational Change in GltPh ing in a piston-like movement of the substrate-binding site across the membrane (Fig. 1B, step 2) In our scheme, this step includes the formation of an occluded outward-facing state (“closing,” perhaps by HP2 closure), the large scale translocation of the domain, and the transition from “inward-occluded” to “inward-open” substrate-bound states (“opening”). We demonstrate that cleaving 3L4 inhibits one of the components of the translocation step of the substrate-loaded transport domain of GltPh while sparing the equivalent component of the translocation of the substrate-free apo-translocation domain, a conclusion with important implications for the overall mechanism of transport
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