Sorting nexin 27 (SNX27) regulates the trafficking and activity of the glutamine transporter ASCT2

Abstract

The Alanine, Serine, Cysteine-preferring Transporter 2 (ASCT2; SLC1A5) is responsible for the uptake of glutamine into cells, a major source of cellular energy and a key regulator of mammalian Target of Rapamycin (mTOR) activation. Furthermore, ASCT2 expression has reported in several human cancers making it a potential target for both diagnostic and therapeutic purposes. Here we identify ASCT2 as a membrane trafficked cargo molecule, sorted through a direct interaction with the PDZ domain of Sorting Nexin 27 (SNX27). Using both membrane fractionation and subcellular localisation approaches we demonstrate that the majority of ASCT2 resides at the plasma membrane. This is significantly reduced within CrispR-mediated SNX27 Knock-Out (KO) cell lines, as it is missorted into the lysosomal degradation pathway. The reduction of ASCT2 levels in SNX27 KO cells leads to a decreased glutamine uptake, which in turn inhibits cellular proliferation. SNX27 KO cells also present impaired activation of mTOR complex 1 (mTORC1) pathway and enhanced autophagy. Taken together, our data reveals a role for SNX27 in glutamine uptake and amino acidstimulated mTORC1 activation via the modulation of ASCT2 intracellular trafficking.

TFEB controls retromer expression in response to nutrient availability

“…2, A-C). Together, these data are consistent with evidence that ASCT2 requires binding to the retromer-associated cargo adaptor SNX27 for its retrieval and recycling (Kvainickas et al, 2017;Yang et al, 2018) and, moreover, establishes that SNAT2 may constitute a previously unrecognized cargo for retromer-mediated endosome to TGN retrograde transport.…”

“…Retromer-mediated sorting is required as part of an adaptive response to glutamine deprivation To date, mammalian studies of the role of retromer in the sorting of nutrient transporters have primarily focused on cells grown in nutrient-rich conditions (Steinberg et al, 2013;Kvainickas et al, 2017;Yang et al, 2018). For glutamine transporters, nutrient withdrawal induces an adaptive response in which transporters reconfigure their cell surface expression to scavenge extracellular amino acids in an attempt to rebalance nutrient supply with cellular demand (Hyde et al, 2001;Nardi et al, 2015;Bröer et al, 2018;Hoffmann et al, 2018).…”

Towards a molecular understanding of endosomal trafficking by Retromer and Retriever

“…The FERM domain may also possess cargo‐interacting capacity, although this remains to be functionally verified . The PDZ domain of SNX27, however, has a demonstrated cargo‐binding role, interacting with literally hundreds of different transmembrane proteins including ion channels, solute carriers and GPCRs via a highly specific PDZ‐binding motif (PDZbm) (Figure B). These PDZbm sequences are found at the extreme C‐terminus of the target proteins and the presence of acidic side‐chains that interact with a conserved arginine within SNX27 enhance their affinity.…”

A role of GCC88 in the retrograde transport of CI‐M6PR and the maintenance of lysosomal activity

“…The mTORC1 activation assay was performed as previously described (Yang et al, ). HeLa cells were subjected to amino acid‐free DMEM medium (D9800‐13; USBiological) for 2 h before stimulated by MEM essential AA solution (final concentration: 2×; 11130051; Thermo Fisher Scientific) for 30 min.…”