Abstract
It is well-established that the secondary active transporters GltTk and GltPh catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic mechanism of transport is fragmentary. Here, we systematically measured aspartate uptake rates in proteoliposomes containing purified GltTk, and derived the rate equation for a mechanism in which two sodium ions bind before and another after aspartate. Re-analysis of existing data on GltPh using this equation allowed for determination of the turnover number (0.14 s−1), without the need for error-prone protein quantification. To overcome the complication that purified transporters may adopt right-side-out or inside-out membrane orientations upon reconstitution, thereby confounding the kinetic analysis, we employed a rapid method using synthetic nanobodies to inactivate one population. Oppositely oriented GltTk proteins showed the same transport kinetics, consistent with the use of an identical gating element on both sides of the membrane. Our work underlines the value of bona fide transport experiments to reveal mechanistic features of Na+-aspartate symport that cannot be observed in detergent solution. Combined with previous pre-equilibrium binding studies, a full kinetic mechanism of structurally characterized aspartate transporters of the SLC1A family is now emerging.
Highlights
It is well-established that the secondary active transporters GltTk and GltPh catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic mechanism of transport is fragmentary
To study the kinetic mechanism of Na+-coupled aspartate transport by GltTk, we used a classical enzymology method, in which we measured the initial uptake rates of radiolabelled L-aspartate into proteoliposomes reconstituted with purified GltTk, as a function of the external concentrations of L-aspartate and Na+
For secondary active transporters, which can readily operate in both directions, the coexistence of right-side-out and inside-out oriented proteins is problematic for kinetic analysis
Summary
It is well-established that the secondary active transporters GltTk and GltPh catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic mechanism of transport is fragmentary. The closely related archaeal transporters GltPh from Pyrococcus horikoshii and GltTk from Thermococcus kodakarensis of the SLC1A family (78% sequence identity to each other, ~36% sequence identity to EAATs) take up aspartate rather than glutamate in symport with three sodium ions and are not coupled to potassium or proton transport[7,8,9,10,11]. We measured initial rates of transport at a wide range of substrate and co-ion concentrations, a method that has been used extensively for the mechanistic characterization of enzymes, leading to insight into the order of binding of substrates[38] This method has not been used much on purified membrane transporters, in part because it is often impossible to control the orientation of the reconstituted transporters in proteoliposomes, leading to mixed populations, complicating kinetic analysis
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