Abstract
Alanine-serine-cysteine transporter 2 (ASCT2, SLC1A5) is the primary transporter of glutamine in cancer cells and regulates the mTORC1 signaling pathway. The SLC1A5 function involves finely tuned orchestration of two domain movements that include the substrate-binding transport domain and the scaffold domain. Here, we present cryo-EM structures of human SLC1A5 and its complex with the substrate, L-glutamine in an outward-facing conformation. These structures reveal insights into the conformation of the critical ECL2a loop which connects the two domains, thus allowing rigid body movement of the transport domain throughout the transport cycle. Furthermore, the structures provide new insights into substrate recognition, which involves conformational changes in the HP2 loop. A putative cholesterol binding site was observed near the domain interface in the outward-facing state. Comparison with the previously determined inward-facing structure of SCL1A5 provides a basis for a more integrated understanding of substrate recognition and transport mechanism in the SLC1 family.
Highlights
SLC1A5 (ASCT2) catalyzes an obligatory Na+-dependent antiport in which Na+ together with an extracellular neutral amino acid are exchanged with an intracellular neutral amino acid (Kanai and Hediger, 2004; Utsunomiya-Tate et al, 1996)
The presence of affinity tags used for purification did not affect the sodium-dependent glutamine uptake when HAP1 SLC1A5 knock-out cells were transiently transfected with full-length SLC1A5 but the observed transport activity was low, compared to the background
Negative-stain electron microscopy revealed that one molecule of Fab was bound to each protomer of a SLC1A5 homotrimer (Figure 1—figure supplement 1)
Summary
SLC1A5 (ASCT2) catalyzes an obligatory Na+-dependent antiport in which Na+ together with an extracellular neutral amino acid are exchanged with an intracellular neutral amino acid (Kanai and Hediger, 2004; Utsunomiya-Tate et al, 1996). The structure and function of SLC1 transporters have been studied using bacterial aspartate transporter homologues GltPH from Pyrococcus horikoshii (Yernool et al, 2004; Boudker et al, 2007; Reyes et al, 2009; Georgieva et al, 2013; Akyuz et al, 2015; Ji et al, 2016), GltTK from Thermococcus kodakarensis (Jensen et al, 2013; Guskov et al, 2016), human glutamate transporter SLC1A3 (Canul-Tec et al, 2017) and glutamine transporter SLC1A5 (Garaeva et al, 2018; Garaeva et al, 2019) All these transporters form a trimer of independently functioning protomers that uses an elevator mechanism to carry amino acids across membranes To gain insights into structural features that permit substrate recognition in an outward-facing state, using an antibody fragment as a fiducial marker we have solved the cryo-EM structures of the unliganded transporter and its complex with glutamine at 3.5 and 3.8 Aresolution, respectively
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