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

• DOI: 10.1038/s42003-021-02267-y
• Publication Date: 2021-06-17T10:03:09Z
• Authors: Gianluca Trinco; Valentina Arkhipova; Alisa A. Garaeva; Cedric A. J. Hutter; Markus A. Seeger; Albert Guskov; Dirk J. Slotboom
• Keywords: Aspartic Acid; 0303 health sciences; 10179 Institute of Medical Microbiology; QH301-705.5; Proteolipids; Sodium; 610 Medicine & health; 2701 Medicine (miscellaneous); Biological Transport; 1100 General Agricultural and Biological Sciences; Article; Thermococcus; 03 medical and health sciences; Excitatory Amino Acid Transporter 3; 1300 General Biochemistry, Genetics and Molecular Biology; 570 Life sciences; biology; Biology (General); Pyrococcus horikoshii

Abstract It is well-established that the secondary active transporters Glt Tk and Ph catalyze coupled uptake of aspartate three sodium ions, but insight in kinetic mechanism transport fragmentary. Here, we systematically measured rates proteoliposomes containing purified , derived rate equation for a which two ions bind before another after aspartate. Re-analysis existing data on using this allowed determination turnover number (0.14 s −1 ), without need error-prone protein quantification. To overcome complication may adopt right-side-out or inside-out membrane orientations upon reconstitution, thereby confounding analysis, employed rapid method synthetic nanobodies to inactivate one population. Oppositely oriented proteins showed same kinetics, consistent with use an identical gating element both sides membrane. Our work underlines value bona fide experiments reveal mechanistic features Na + -aspartate symport cannot be observed detergent solution. Combined previous pre-equilibrium binding studies, full structurally characterized SLC1A family now emerging.