Phosphoinositide binding by the SNX27 FERM domain regulates localisation at the immune synapse of activated T-cells

Abstract

Sorting nexin 27 (SNX27) controls the endosomal-to-cell-surface recycling of diverse transmembrane protein cargos. Crucial to this function is the recruitment of SNX27 to endosomes which is mediated by the binding of phosphatidylinositol-3-phosphate (PtdIns3P) by its phox homology (PX) domain. In T-cells, SNX27 localizes to the immunological synapse in an activation-dependent manner, but the molecular mechanisms underlying SNX27 translocation remain to be clarified. Here, we examined the phosphoinositide-lipid-binding capabilities of full-length SNX27, and discovered a new PtdInsPbinding site within the C-terminal 4.1, ezrin, radixin, moesin (FERM) domain. This binding site showed a clear preference for bi-and tri-phosphorylated phophoinositides, and the interaction was confirmed through biophysical, mutagenesis and modeling approaches. At the immunological synapse of activated T-cells, cell signaling regulates phosphoinositide dynamics, and we find that perturbing phosphoinositide binding by the SNX27 FERM domain alters the SNX27 distribution in both endosomal recycling compartments and PtdIns(3,4,5)P 3 -enriched domains of the plasma membrane during synapse formation. Our results suggest that SNX27 undergoes dynamic partitioning between different membrane domains during immunological synapse assembly, and underscore the contribution of unique lipid interactions for SNX27 orchestration of cargo trafficking.

A role for novel lipid interactions in the dynamic recruitment of SNX27 to the T-cell immune synapse

“…Although initial recruitment of the mutant to the IS was similar compared to that of wild type SNX27, deficiency in lipid binding by the FERM domain impaired the subsequent retrieval of the mutant into endosomal compartments following IS maturation. These results correlate with previous studies showing that PM localization of SNX27 during IS formation is absolutely PDZ domaindependent, 4 and suggest that this additional lipid binding site with a preference for the bi-and triphosphorylated PtdIns derivatives known to be involved in endocytosis, 13 favors SNX27 recycling.…”

“…Based on our results in HeLa cells, the FERM-located PtdInsbinding site is not necessary for SNX27 accumulation at the endoplasmic recycling compartment (ERC), a property dependent exclusively on the PX domain. 13 This coincides with results from Tseng et al, who compared SNX27 vesicle-binding abilities to those of other SNX-FERM proteins, and suggested an additional lipid-binding site in SNX27 that was absent in both SNX17 and SNX31. 8 The relevance of this alternative lipid binding site for SNX27 endosomal localization was only apparent in HeLa cells treated with the PI3K inhibitor wortmannin, or when the PX domain was mutated.…”

“…The interaction was confirmed through biophysical, mutagenesis and modeling approaches. 13 The SNX27 FERM domain PI binding site is entirely distinct from those found in other FERM-domains that recognize PtdIns phosphorylated forms, such as those present in talin or radixin. The PI binding pocket in SNX27 is close to the FxNPxY cargo recognition site, suggesting that cargo and lipid binding may be allosterically linked or favor coincidence detection of specific membrane sites.…”

Structural Mechanism for Cargo Recognition by the Retromer Complex

“…This conservation is not so evident in the VPS27 PX domain; yet, the binding sites for the PDZ and PX domains on VPS26 are ~80 Å apart, a distance compatible with the 33-residue linker that connects both domains (Figure 7B). This arrangement would place the FERM domain of VPS27 at the C-terminal end of the PX domain, parallel to the membrane (Ghai et al, 2015), and on the concave side of VPS35. In the case of SNX-BAR proteins, the only PX-BAR tandem structure solved to date corresponds to SNX9 (Pylypenko et al, 2007).…”

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

“…SNX27, a member of sorting nexin (SNX) family, is known to regulate the trafficking of endosomal cargo proteins from early endosomes to the cell surface (14-16). SNX27's FERM domain interacts with NPXY motif containing cargoes, whereas its PDZ domain can interact with PDZbinding motifs (PDZbm) at the C-terminus of cargo proteins (15,17). Proteomic studies revealed that the cell surface levels of numerous SLC family proteins including ASCT2 were modulated by SNX27 (15), while structural bioinformatics found that SLC family proteins were one of the most abundant classes of PDZbm-containing SNX27 cargos (18).…”