Insights into cargo sorting by SNX32 in neuronal and non-neuronal cells: physiological implications in neurite outgrowth

Abstract

Sorting nexins (SNX) are a family of proteins containing the Phox homology domain, which shows a preferential endo-membrane association and regulates cargo sorting processes. Even with the vast amount of information unveiled systematically, the underlying mechanism of sorting remains elusive. Here, we established that SNX32, a SNX-BAR (Bin/Amphiphysin/Rvs) sub-family member, is associated with SNX4 via its BAR domain. We identified A226, Q259, E256, R366 of SNX32, and Y258, S448 of SNX4 at the interface of these two SNX proteins that are important for maintaining the association. Via its PX domain, SNX32 interacts with the Transferrin receptor (TfR) and Cation Independent Mannose-6-Phosphate Receptor (CIMPR). We showed that the conserved F131 in its PX domain is important in stabilising the above interactions. Silencing of SNX32 led to a defect in intracellular trafficking of TfR and CIMPR, which could be rescued by overexpressing shRNA-resistant snx32. We also showed that both individual domains play an essential role in trafficking. Our results indicate that SNX4, SNX32 and Rab11 may participate in a common pathway regulating transferrin trafficking; however, the existence of an independent pathway for Rab11 and SNX32 could not be completely ruled out. Further, we established that the PX domain of SNX32 could bind to PI(3)P and PI(4)P, suggesting a possible explanation for its sub-cellular localization. Taken together, our study showed that SNX32 mediate the trafficking of specific cargo molecules along distinct pathway via its PX domain-directed binding to phosphoinositides and its BAR domain-mediated association with other SNX family members. Further, using SILAC-based differential proteomics of the wild type and the mutant SNX32, impaired in cargo binding, we identified Basigin (BSG), an immunoglobulin super family member, as a potential interactor of SNX32 in SH-SY-5Y cells. We then demonstrated that SNX32 binds to BSG through its PX domain and facilitates its trafficking to the cell surface. In Neuro-Glial cell lines, the silencing of SNX32 led to defects in neuronal differentiation. Moreover, abrogation in lactate transport in the SNX32 depleted cells led us to propose that the SNX may contribute to maintaining the neuro-glial coordination via its role in BSG trafficking and the associated Monocarboxylate transporter activity.

SNX32 Regulates Sorting and Trafficking of Activated EGFR to the Lysosomal Degradation Pathway

“…Lipid blot overlay assay showed that unlike SNX6, which preferentially binds PI4P [17], binding of His-tagged SNX32 PX domain to PIPs is rather promiscuous (Figure 2E). This observation is in line with a previous study [16] and suggests that SNX32 does not recognize organellar or vesicular membrane via interaction with organellespecific PIP. Next, we determined whether its membrane association requires other SNXs.…”

“…Compared with wildtype (WT) cells, colocalization analysis did not detect change in the distribution of CI-MPR to the TGN in two independent SNX32 KO cell lines (Figure S3A S3E,F). In good agreement with most recent studies investigating roles of SNX32 in vesicle trafficking by gene knockdown [16], these data indicate that SNX32 functions redundantly in SNX-BAR-mediated transport of CI-MPR from endosomes to the TGN.…”

“…Consistently, coIP from HEK293T cells overexpressing epitope-tagged SNX-BAR protein and EGFR fused to EGFP detected interaction of EGFR with SNX32 but not other SNX-BARs (Figure 4F). Unexpectedly, although previous studies reported that SNX5/6/32 recognize cargoes via their PX domains [14][15][16], binding of SNX32 to EGFR requires either the PX or BAR domain (Figure 4G,H). Notably, although previous studies reported that SNX1 binds to EGFR through its C-terminus and promotes sorting of EGFR to lysosomes for degradation [20], we did not detect interaction between SNX1 and EGFR (Figure 4F).…”

“…The SNX-BAR proteins interact with each other via their BAR domains [2,16]. Indeed, coIP analysis verified that both SNX6 and SNX32 bind to SNX5 via the BAR domain (Figure 6A).…”

“…Among the SNX-BAR proteins, SNX32 shares the highest sequence homology with SNX6 (Table 1). Although it was established by gene knockdown studies that SNX32 functions in endosome-to-TGN transport of CI-MPR [13,16], and cooperates with SNX4 to regulate recycling of internalized transferrin receptors (TfR) to the plasma membrane [16], whether it is also a part of the SNX-BAR complex and plays roles similar to or distinct from that of SNX5/6 in endosomal trafficking of various cargoes other than CI-MPR and TfR remains largely unexplored. In this study, we demonstrate that SNX32 directly interacts with EGFR and coordinates with SNX5 and SNX6 to mediate EGF-stimulated sorting and trafficking of EGFR into the lysosomal degradation pathway.…”