Identification and characterization of SNX15, a novel sorting nexin involved in protein trafficking

Sorting nexins are a family of phox homology domain containing proteins that are homologous to yeast proteins involved in protein trafficking. We have identified a novel 342-amino acid residue sorting nexin, SNX15, and a 252-amino acid splice variant, SNX15A. Unlike many sorting nexins, a SNX15 ortholog has not been identified in yeast or Caenorhabditis elegans. By Northern blot analysis, SNX15 mRNA is widely expressed. Although predicted to be a soluble protein, both endogenous and overexpressed SNX15 are found on membranes and in the cytosol. The phox homology domain of SNX15 is required for its membrane association and for association with the platelet-derived growth factor receptor. We did not detect association of SNX15 with receptors for epidermal growth factor or insulin. However, overexpression of SNX15 led to a decrease in the processing of insulin and hepatocyte growth factor receptors to their mature subunits. Immunofluorescence studies showed that SNX15 overexpression resulted in mislocalization of furin, the endoprotease responsible for cleavage of insulin and hepatocyte growth factor receptors. Based on our data and the existing findings with yeast orthologs of other sorting nexins, we propose that overexpression of SNX15 disrupts the normal trafficking of proteins from the plasma membrane to recycling endosomes or the trans-Golgi network.     

Cargo selectivity of yeast sorting nexins

Sorting nexins are PX domain‐containing proteins that bind phospholipids and often act in membrane trafficking where they help to select cargo. However, the functions and cargo specificities of many sorting nexins are unknown. Here, a high‐throughput imaging screen was used to identify new sorting nexin cargo in the yeast Saccharomyces cerevisiae. Deletions of 9 different sorting nexins were screened for mislocalization of a set of green fluorescent protein (GFP)‐tagged membrane proteins found at the plasma membrane, Golgi or endosomes. This identified 27 proteins that require 1 or more sorting nexins for their correct localization, 23 of which represent novel sorting nexin cargo. Nine hits whose sorting was dependent on Snx4, the sorting nexin‐containing retromer complex, or both retromer and Snx3, were examined in detail to search for potential sorting motifs. We identified cytosolic domains of Ear1, Ymd8 and Ymr010w that conferred retromer‐dependent sorting on a chimeric reporter and identified conserved residues required for this sorting in a functional assay. This work defined a consensus sequence for retromer and Snx3‐dependent sorting.      

Trying to make sense of retromer

Retromer is a cytosolic protein complex which binds to post-Golgi organelles involved in the trafficking of proteins to the lytic compartment of the cell. In non-plant organisms, retromer mediates the recycling of acid hydrolase receptors from early endosomal (EE) compartments. In plants, retromer components are required for the targeting of vacuolar storage proteins, and for the recycling of endocytosed PIN proteins. However, there are contradictory reports as to the localization of the sorting nexins and the core subunit of retromer. There is also uncertainty as to the identity of the organelles from which vacuolar sorting receptors (VSRs) and endocytosed plasma membrane (PM) proteins are recycled. In this review we try to resolve some of these conflicting observations.     

Sorting nexins--unifying trends and new perspectives

The sorting nexins (SNXs) are a family of PX domain-containing proteins found in yeast and mammalian cells that have been proposed to regulate intracellular trafficking. Mammalian SNXs have been suggested to function variously in pro-degradative sorting, internalization, endosomal recycling, or simply in endosomal sorting. In yeast, the defining function for these proteins is a regulation of cargo retrieval. Here we examine recent data on the SNX family of proteins and attempt to draw out unifying themes between the work performed in yeast and mammalian systems.     

Evidence for a role of SNX16 in regulating traffic between the early and later endosomal compartments

Sorting nexins (SNXs) are a growing family of proteins characterized by the presence of a PX domain. The PX domain mediates membrane association by interaction with phosphoinositides. The SNXs are generally believed to participate in membrane trafficking, but information regarding the function of individual proteins is limited. In this report, we describe the major characteristics of one member, SNX16. SNX16 is a novel 343-amino acid protein consisting of a central PX domain followed by a potential coiled-coil domain and a C-terminal region. Like other sorting nexins, SNX16 associates with the membrane via the PX domain which interacts with the phospholipid phosphatidylinositol 3-phosphate. We show via biochemical and cellular studies that SNX16 is distributed in both early and late endosome/lysosome structures. The coiled-coil domain is necessary for localization to the later endosomal structures, as mutant SNX16 lacking this domain was found only in early endosomes. Trafficking of internalized epidermal growth factor was also delayed by this SNX16 mutant, as these cells showed a delay in the segregation of epidermal growth factor in the early endosome for its delivery to later compartments. In addition, the coiled-coil domain is shown here to be important for homo-oligomerization of SNX16. Taken together, these results suggest that SNX16 is a sorting nexin that may function in the trafficking of proteins between the early and late endosomal compartments.     

The SNXy flavours of endosomal sorting

The retromer complex coordinates retrograde transport of cargo proteins between endosomes and the trans-Golgi network. The sorting nexin SNX3 is required for the retrograde trafficking of Wntless, but not of other retrograde cargo proteins, revealing that the cargo specificity of retromer is provided by the sorting nexins.     

Identification of a Family of Sorting Nexin Molecules and Characterization of Their Association with Receptors

Sorting nexin 1 (SNX1) is a protein that binds to the epidermal growth factor (EGF) receptor and is proposed to play a role in directing EGF receptors to lysosomes for degradation (R. C. Kurten, D. L. Cadena, and G. N. Gill, Science 272:1008–1010, 1996). We have obtained full-length cDNAs and deduced the amino acid sequences of three novel homologous proteins, which were denoted human sorting nexins (SNX2, SNX3, and SNX4). In addition, we identified a presumed splice variant isoform of SNX1 (SNX1A). These molecules contain a conserved domain of ~100 amino acids, which was termed the phox homology (PX) domain. Human SNX1 (522 amino acids), SNX1A (457 amino acids), SNX2 (519 amino acids), SNX3 (162 amino acids), and SNX4 (450 amino acids) are part of a larger family of hydrophilic molecules including proteins identified in Caenorhabditis elegans and Saccharomyces cerevisiae. Despite their hydrophilic nature, the sorting nexins are found partially associated with cellular membranes. They are widely expressed, although the tissue distribution of each sorting nexin mRNA varies. When expressed in COS7 cells, epitope-tagged sorting nexins SNX1, SNX1A, SNX2, and SNX4 coimmunoprecipitated with receptor tyrosine kinases for EGF, platelet-derived growth factor, and insulin. These sorting nexins also associated with the long isoform of the leptin receptor but not with the short and medium isoforms. Interestingly, endogenous COS7 transferrin receptors associated exclusively with SNX1 and SNX1A, while SNX3 was not found to associate with any of the receptors studied. Our demonstration of a large conserved family of sorting nexins that interact with a variety of receptor types suggests that these proteins may be involved in several stages of intracellular trafficking in mammalian cells.     

Deficiency of sorting nexin 27 (SNX27) leads to growth retardation and elevated levels of N-methyl-D-aspartate receptor 2C (NR2C)

Phox (PX) domain-containing sorting nexins (SNXs) are emerging as important regulators of endocytic trafficking. Sorting nexin 27 (SNX27) is unique, as it contains a PDZ (Psd-95/Dlg/ZO1) domain. We show here that SNX27 is primarily targeted to the early endosome by interaction of its PX domain with PtdIns(3)P. Although targeted ablation of the SNX27 gene in mice did not significantly affect growth and survival during embryonic development, SNX27 plays an essential role in postnatal growth and survival. N-Methyl-d-aspartate (NMDA) receptor 2C (NR2C) was identified as a novel SNX27-interacting protein, and this interaction is mediated by the PDZ domain of SNX27 and the C-terminal PDZ-binding motif of NR2C. Increased NR2C expression levels, together with impaired NR2C endocytosis in SNX27(-/-) neurons, indicate that SNX27 may function to regulate endocytosis and/or endosomal sorting of NR2C. This is consistent with a role of SNX27 as a general regulator for sorting of membrane proteins containing a PDZ-binding motif, and its absence may alter the trafficking of these proteins, leading to growth and survival defects.     

Identification of the functional domains of yeast sorting nexins Vps5p and Vps17p

Sorting nexins (Snxs) are a recently discovered family of conserved hydrophilic cytoplasmic proteins that have been found associated with membranes of the endocytic system and that are implicated in the trafficking of many endosomal membrane proteins, including the epidermal growth factor receptor and transferrin receptor. Snx proteins are partly defined by the presence of a p40 phox homology domain that has recently been shown to bind phosphatidylinositol 3-phosphate. Most Snx proteins also contain a predicted coiled-coils domain in the carboxyl-terminal half of the protein and have been shown to form dimers with other members of the Snx family. The yeast sorting nexins Vps5p and Vps17p form a dimer and are also components of the retromer complex that mediates endosome-to-Golgi transport of the carboxypeptidase Y receptor Vps10p. To functionally define the different domains of the yeast sorting nexins Vps5p and Vps17p, we have generated various truncations to examine the role that the different domains of Vps5p/Vps17p play in their respective functions. Herein, we show that the C-terminal halves of Vps5p and Vps17p, which contain the coiled-coils domains, are necessary and sufficient for their interaction. We have also mapped the retromer assembly domain to the N-terminal half of Vps5p and found that binding of Vps5p by Vps17p synergizes the interaction between Vps5p and other retromer components. Additionally, we have examined which domain(s) of Vps5p is necessary for membrane association.     

Endosomal sorting and signalling: an emerging role for sorting nexins

The endocytic network comprises a series of interconnected tubulo-vesicular membranous compartments that together regulate various sorting and signalling events. Although it is clear that defects in endocytic function underlie a variety of human diseases, our understanding of the molecular entities that regulate these sorting and signalling events remains limited. Here we discuss the sorting nexins family of proteins and propose that they have a fundamental role in orchestrating the formation of protein complexes that are involved in endosomal sorting and signalling.     

分选连接蛋白及其相关细胞信号通路

分选连接蛋白（sorting nexins,SNXs）是一类包含PX（phox homology）结构域的高度保守真核生物蛋白,其功能主要是参与负载蛋白的内吞、分选和降解过程,以维持细胞信号的稳态和平衡。SNXs参与调控与肿瘤等疾病相关的重要信号通路,如SNX3介导分泌型糖蛋白Wnt受体Wntless的胞内循环|SNX1、SNX5等众多SNXs介导表皮生长因子受体（epidermal growth factor receptor, EGFR）和转化因子β受体（TGF β）等的内吞、分选和降解等过程。其中,对EGFR降解的调控研究最多,尤其是在肿瘤方面的进展令人鼓舞,可也较为复杂,仍有许多未解之谜。随着SNXs的深入研究,将对疾病的发生机制产生新的认识。     

Analyses of sorting nexins reveal distinct retromer-subcomplex functions in development and protein sorting in Arabidopsis thaliana

Sorting nexins (SNXs) are conserved eukaryotic proteins that associate with three types of vacuolar protein sorting (VPS) proteins to form the retromer complex. How SNXs act in this complex and whether they might work independently of the retromer remains elusive. Here, we show by genetic and cell imaging approaches that the Arabidopsis thaliana SNX1 protein recruits SNX2 at the endosomal membrane, a process required for SNX1-SNX2 dimer activity. We report that, in contrast with the mammalian retromer, SNXs are dispensable for membrane binding and function of the retromer complex. We also show that VPS retromer components can work with or independently of SNXs in the trafficking of seed storage proteins, which reveals distinct functions for subcomplexes of the plant retromer. Finally, we provide compelling evidence that the combined loss of function of SNXs and VPS29 leads to embryo or seedling lethality, underlining the essential role of these proteins in development.     

Retromer and the sorting nexins Snx4/41/42 mediate distinct retrieval pathways from yeast endosomes

The endocytic pathway in yeast leads to the vacuole, but resident proteins of the late Golgi, and some endocytosed proteins such as the exocytic SNARE Snc1p, are retrieved specifically to the Golgi. Retrieval can occur from both a late pre-vacuolar compartment and early or 'post-Golgi' endosomes. We show that the endosomal SNARE Pep12p, and a mutant version that reaches the cell surface and is endocytosed, are retrieved from pre-vacuolar endosomes. As with Golgi proteins, this requires the sorting nexin Grd19p and components of the retromer coat, supporting the view that endosomal and Golgi residents both cycle continuously between the exocytic and endocytic pathways. In contrast, retrieval of Snc1p from post-Golgi endosomes requires the sorting nexin Snx4p, to which Snc1p can be cross-linked. Snx4p binds to Snx41p/ydr425w and to Snx42p/ydl113c, both of which are also required for efficient Snc1p sorting. Our findings suggest a general role for yeast sorting nexins in protein retrieval, rather than degradation, and indicate that different sorting nexins operate in different classes of endosomes.     

Sorting nexins provide diversity for retromer-dependent trafficking events

Sorting nexins are a large family of evolutionarily conserved phosphoinositide-binding proteins that have fundamental roles in orchestrating cargo sorting through the membranous maze that is the endosomal network. One ancient group of complexes that contain sorting nexins is the retromer. Here we discuss how retromer complexes regulate endosomal sorting, and describe how this is generating exciting new insight into the central role played by endosomal sorting in development and homeostasis of normal tissues.     

Structural Studies Reveal Unique Non-canonical Regulators of G Protein Signaling Homology (RH) Domains in Sorting Nexins

As a subgroup of sorting nexins (SNXs) that contain regulator of G protein signaling homology (RH) domain, SNX-RH proteins, including SNX13, SNX14 and SNX25, were proposed to play bifunctional roles in protein sorting and GPCR signaling regulation. However, mechanistic details of SNX-RH proteins functioning via RH domain remain to be illustrated. Here, we delineate crystal structures of the RH domains of SNX13 and SNX25, revealing a homodimer of SNX13 RH domain mediated by unique extended α4 and α5 helices, and a thiol modulated homodimer of SNX25-RH triggered by a unique cysteine on α6 helix. Further studies showed that RH domains of SNX-RH do not possess binding capacity toward Gα subunits, owing to the lack of critical residues for interaction. Thus, this study identifies a group of novel non-canonical RH domains that can act as a dimerization module in sorting nexins, which provides structural basis for mechanism studies on SNX-RH protein functions.     

The PX-domain protein SNX17 interacts with members of the LDL receptor family and modulates endocytosis of the LDL receptor

Sorting nexins (SNXs) comprise a family of proteins characterized by the presence of a phox-homology domain, which mediates the association of these proteins with phosphoinositides and recruits them to specific membranes or vesicular structures within cells. Although only limited information about SNXs and their functions is available, they seem to be involved in membrane trafficking and sorting processes by directly binding to target proteins such as certain growth factor receptors. We show that SNX17 binds to the intracellular domain of some members of the low-density lipoprotein receptor (LDLR) family such as LDLR, VLDLR, ApoER2 and LDLR-related protein. SNX17 resides on distinct vesicular structures partially overlapping with endosomal compartments characterized by the presence of EEA1 and rab4. Using rhodamine-labeled LDL, it was possible to demonstrate that during endocytosis, LDL passes through SNX17-positive compartments. Functional studies on the LDLR pathway showed that SNX17 enhances the endocytosis rate of this receptor. Our results identify SNX17 as a novel adaptor protein for LDLR family members and define a novel mechanism for modulation of their endocytic activity.     

SNX–BAR proteins in phosphoinositide-mediated,tubular-based endosomal sorting

The endocytic network is morphologically characterized by a wide variety of membrane bound compartments that are able to undergo dynamic re-modeling through tubular and vesicular structures. The precise molecular mechanisms governing such re-modeling, and the events that co-ordinated this with the major role of endosomes, cargo sorting, remain unclear. That said, recent work on a protein family of sorting nexins (SNX) - especially a subfamily of SNX that contain a BAR domain (SNX-BARs) – has begun to shed some much needed light on these issues and in particular the process of tubular-based endosomal sorting. SNX-BARs are evolutionary conserved in endosomal protein complexes such as retromer, where they co-ordinate membrane deformation with cargo selection. Furthermore a central theme emerges of SNX-BARs linking the forming membrane carrier to cytoskeletal elements for transport through motor proteins such as dynein. By studying these SNX-BARs, we are gaining an increasingly detailed appreciation of the mechanistic basis of endosomal sorting and how this highly dynamic process functions in health and disease.     

A SNX3-dependent retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is required for Wnt secretion

Wnt proteins are lipid-modified glycoproteins that play a central role in development, adult tissue homeostasis and disease. Secretion of Wnt proteins is mediated by the Wnt-binding protein Wntless (Wls), which transports Wnt from the Golgi network to the cell surface for release. It has recently been shown that recycling of Wls through a retromer-dependent endosome-to-Golgi trafficking pathway is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is poorly understood. Here, we report that Wls recycling is mediated through a retromer pathway that is independent of the retromer sorting nexins SNX1-SNX2 and SNX5-SNX6. We have found that the unrelated sorting nexin, SNX3, has an evolutionarily conserved function in Wls recycling and Wnt secretion and show that SNX3 interacts directly with the cargo-selective subcomplex of the retromer to sort Wls into a morphologically distinct retrieval pathway. These results demonstrate that SNX3 is part of an alternative retromer pathway that functionally separates the retrograde transport of Wls from other retromer cargo.     

Insights into the PX (phox-homology) domain and SNX (sorting nexin) protein families: structures, functions and roles in disease

The mammalian genome encodes 49 proteins that possess a PX (phox-homology) domain, responsible for membrane attachment to organelles of the secretory and endocytic system via binding of phosphoinositide lipids. The PX domain proteins, most of which are classified as SNXs (sorting nexins), constitute an extremely diverse family of molecules that play varied roles in membrane trafficking, cell signalling, membrane remodelling and organelle motility. In the present review, we present an overview of the family, incorporating recent functional and structural insights, and propose an updated classification of the proteins into distinct subfamilies on the basis of these insights. Almost all PX domain proteins bind PtdIns3P and are recruited to early endosomal membranes. Although other specificities and localizations have been reported for a select few family members, the molecular basis for binding to other lipids is still not clear. The PX domain is also emerging as an important protein-protein interaction domain, binding endocytic and exocytic machinery, transmembrane proteins and many other molecules. A comprehensive survey of the molecular interactions governed by PX proteins highlights the functional diversity of the family as trafficking cargo adaptors and membrane-associated scaffolds regulating cell signalling. Finally, we examine the mounting evidence linking PX proteins to different disorders, in particular focusing on their emerging importance in both pathogen invasion and amyloid production in Alzheimer's disease.     

The COG Complex, Rab6 and COPI Define a Novel Golgi Retrograde Trafficking Pathway that is Exploited by SubAB Toxin

Toxin trafficking studies provide valuable information about endogenous pathways of intracellular transport. Subtilase cytotoxin (SubAB) is transported in a retrograde manner through the endosome to the Golgi and then to the endoplasmic reticulum (ER), where it specifically cleaves the ER chaperone BiP/GRP78 (Binding immunoglobin protein/Glucose-Regulated Protein of 78 kDa). To identify the SubAB Golgi trafficking route, we have used siRNA-mediated silencing and immunofluorescence microscopy in HeLa and Vero cells. Knockdown (KD) of subunits of the conserved oligomeric Golgi (COG) complex significantly delays SubAB cytotoxicity and blocks SubAB trafficking to the cis Golgi. Depletion of Rab6 and β-COP proteins causes a similar delay in SubAB-mediated GRP78 cleavage and did not augment the trafficking block observed in COG KD cells, indicating that all three Golgi factors operate on the same 'fast' retrograde trafficking pathway. SubAB trafficking is completely blocked in cells deficient in the Golgi SNARE Syntaxin 5 and does not require the activity of endosomal sorting nexins SNX1 and SNX2. Surprisingly, depletion of Golgi tethers p115 and golgin-84 that regulates two previously described coat protein I (COPI) vesicle-mediated pathways did not interfere with SubAB trafficking, indicating that SubAB is exploiting a novel COG/Rab6/COPI-dependent retrograde trafficking pathway.