Insights into cargo sorting by SNX32 in neuronal and non-neuronal cells: physiological implications in neurite outgrowth
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This manuscript presents a series of important findings about the roles of the BAR-domain containing protein SNX32 in endosomal cargo sorting and in neurite outgrowth. The authors provide convincing evidence for their claims, which will be of interest for those working not only in membrane trafficking but also for cell biologists in general with interest in neurobiology.
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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 SHSY5Y 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.
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eLife assessment
This manuscript presents a series of important findings about the roles of the BAR-domain containing protein SNX32 in endosomal cargo sorting and in neurite outgrowth. The authors provide convincing evidence for their claims, which will be of interest for those working not only in membrane trafficking but also for cell biologists in general with interest in neurobiology.
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Reviewer #1 (Public Review):
In this manuscript Sugatha et al. present a comprehensive study on sorting nexin 32 (SNX32) with a wide-spectrum of methodologies and model systems. Authors investigate binding to other sorting nexins involved in the same pathways (SNX1 and SNX4) as well as to its cargo in biochemical and cell-based experiments. They show the importance and explore mechanisms of SNX32 in Transferrin Receptor and Cation Independent Mannose-6-Phosphate Receptor trafficking. Moreover, this work also demonstrates the role of SNX32 in concert with Basigin in neuron differentiation.
Authors with the help of structure modelling and subsequent biochemical experiments find specific residues within the BAR domain of SNX32 that are crucial for heterodimer formation with its interaction partners on endosomal membranes: SNX1 and SNX4. …
Reviewer #1 (Public Review):
In this manuscript Sugatha et al. present a comprehensive study on sorting nexin 32 (SNX32) with a wide-spectrum of methodologies and model systems. Authors investigate binding to other sorting nexins involved in the same pathways (SNX1 and SNX4) as well as to its cargo in biochemical and cell-based experiments. They show the importance and explore mechanisms of SNX32 in Transferrin Receptor and Cation Independent Mannose-6-Phosphate Receptor trafficking. Moreover, this work also demonstrates the role of SNX32 in concert with Basigin in neuron differentiation.
Authors with the help of structure modelling and subsequent biochemical experiments find specific residues within the BAR domain of SNX32 that are crucial for heterodimer formation with its interaction partners on endosomal membranes: SNX1 and SNX4. Moreover, this study, by using various microscopy techniques, also demonstrates localization of SNX32 to early endosomes as well as its co-trafficking with Rab11 and Golgi marker. Furthermore, authors with knock-down and rescue experiments investigate the role of SNX32 in Transferrin Receptor and Cation Independent Mannose-6-Phosphate Receptor trafficking. With co-immunoprecipitation they show that the cargo interaction occurs via the conserved stretch in the PX domain and that single amino acid substitution can disrupt this binding. This feature is utilized in a subsequent neuroblastoma cell-based SILAC screen for SNX32 interactome that identifies Basigin (a transmembrane receptor belonging to the superfamily of immunoglobulins) as one of the most prominent interactors in these cells. Finally, authors identify SNX32 and Basigin as crucial factors involved in neurite outgrowth and network formation. Experiments demonstrate that SNX32, but not its homolog SNX6, assists in the surface localization of Basigin where this protein could potentially interact with monocarboxylate transporters crucial for neuro-glial coordination.
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Reviewer #2 (Public Review):
This manuscript presents a thorough set of investigations on the roles of a previously poorly-studied protein, SNX32. SNX32 is a sorting nexin involved in cargo sorting along the endosomal system. SNX32 contains a BAR domain and a PX domain, and the authors have convincingly shown that, by interacting with SNX4 and with phosphoinositides (PI(3)P or PI(4)P), SNX32 localizes to early endosomes and regulates the trafficking of different cargo receptors (transferrin receptor and cation independent mannose-6 phosphate receptor). In a second part, the authors moved to a more physiological context, in which they studied the functions of SNX32 in neuronal differentiation, which they suggest that is linked to the role of SNX32 in mediating the trafficking of Basigin (BSG).
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Reviewer #3 (Public Review):
The paper by Sugatha et al. examines the role of SNX32 in membrane trafficking. They found SNX32 interacts with SNX4, SNX32 binds the TfR and CIMPR and is required for their intracellular trafficking, and the intracellular location of SNX32 to endosomes was through lipid binding to PI(3)P or PI(4)P. This study further demonstrated that SNX32 plays a role in BSG trafficking to the cell surface. And lastly, they demonstrated SNX32 plays a role in neuronal differentiation likely through its regulation of BSG trafficking.
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