Kinetic mechanism of Na+-coupled aspartate transport catalyzed by GltTk

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.

Structural and mechanistic analysis of a tripartite ATP-independent periplasmic TRAP transporter

“…Depending on their binding epitope, VHHs can influence the function of their target proteins, and examples of transportinhibiting VHHs are known, also for elevator-type transporters [50][51][52][53] . To test this possibility for our HiSiaQM VHHs, we used a modified in vivo transport assay for TRAP transporters based on E. coli strain SEVY3 8,54 .…”

High-speed atomic force microscopy reveals a three-state elevator mechanism in the citrate transporter CitS

“…In Fig. 1 B , the slope at low-Na + concentrations approaches −1, and therefore, we conclude that citrate-binding is preceded by an ordered step involving one Na + ( 20 , 22 ).…”

“…1 B , a double logarithmic plot is presented of these apparent affinity constants as function of the concentration of Na + . In such plots, linear relations are approached asymptotically in both the high– and low–Na + concentration extremes, with slopes of either zero or negative integers ( 20 – 22 ). The negative value of the slopes in the low- and high-concentration regime indicate the number of Na + ions that must bind before and after the citrate-binding event, respectively.…”

“…This is a well-known phenomenon in other sodium-coupled transporters such as Glt Ph ( 10 ), in which sodium ions bind weakly and cooperative binding of aspartate increases the apparent affinity for Na + . It is noteworthy that the equilibrium binding constant of Na + in the absence of citrate (defined by the K D ) is difficult to determine but likely is of much-lower affinity than the apparent K M values for Na + observed under transport (nonequilibrium) conditions ( 11 , 22 , 33 ). Thus, in the presence of only Na + , a fraction of the transporters is likely unoccupied and adopts an apo conformation, which has not been resolved in the available crystal structures.…”

Picornavirus infection enhances aspartate by the SLC38A8 transporter to promote viral replication

“…Dysregulation of AATs leads to metabolic reprogramming and changes in intracellular amino acid levels [ 32 ]. Aspartate transporters contain a large number of proteins, including SLC1A1, SLC1A2, SLC1A3, SLC1A6, SLC1A7, SLC25A12, SLC25A13,SLC38A7, and SLC38A8 [ 33 , 34 ]. To explore the mechanism by which FMDV infection promotes the increase of aspartate, the mRNA expression of aspartate transporters was detected to screen amino acid transporters involved in aspartate metabolism during viral infection.…”