Mammalian retromer is composed of cargo‐recognition trimer comprised of Vps26, Vps29 and Vps35 and membrane recruiting subcomplex formed by sorting nexin (SNXs), which has been shown to play a critical role in sorting various receptors (cargoes) from the endosome to the TGN. Recently, several studies have been focused on retromer in complex with SNX27 and its function in retrieval of receptors from the endosome to the plasma membrane. Here we investigate functional consequence of SNX27‐retromer‐mediated trafficking of one such cargo, ASCT2.

ASCT2 is a major glutamine transporter, allowing the uptake of glutamine into the cells as one of the major fuel sources and facilitating amino acid stimulated mTOR activation, and ASCT2 overexpression has been observed in several human cancers. We here identify ASCT2 as an SNX27‐retromer cargo mediated by SNX27 PDZ domain, as shown by co‐immunoprecipitation that a SNX27 truncated construct, lacking of PDZ domain, abolished the binding, despite their co‐localization was unperturbed. Using both biochemical (cell surface biotinylation) and subcellular localisation methodologies, we further demonstrate that ASCT2 mainly resides at the cell surface, and this plasma membrane localisation is significantly lost upon CRISPER‐mediated SNX27 knockout (KO). SNX27 KO leads to the missorting of ASCT2 to the degradation pathways, which can be partially reversed by the presence of chloroquine, an inhibitor for vacuolar type H⁺‐ATPase, or MG132, a proteasome inhibitor. ASCT2 reduction in SNX27 KO cells leads to the decreased glutamine uptake as measured by L‐H³‐glutamine incorporation, associated with the decreased cell proliferation rate and the increased cell cycle arrest. Furthermore, SNX27 KO cells showed the reduced activation of amino acid‐stimulated mTOR pathway, yet the enhanced autophagy activation as measured by LC3 levels. Taken together, this data indicates a role of SNX27‐retromer in glutamine homeostasis via the modulation of ASCT2 sorting.

Australian Research Council Grant DP160101573