Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

The retromer complex mediates retrograde transport of transmembrane cargo from endosomes to the trans-Golgi network (TGN). Mammalian retromer is composed of a sorting nexin (SNX) dimer that binds to phosphatidylinositol 3-phosphate–enriched endosomal membranes and a vacuolar protein sorting (Vps) 26/29/35 trimer that participates in cargo recognition. The mammalian SNX dimer is necessary but not sufficient for recruitment of the Vps26/29/35 trimer to membranes. In this study, we demonstrate that the guanosine triphosphatase Rab7 contributes to this recruitment. The Vps26/29/35 trimer specifically binds to Rab7–guanosine triphosphate (GTP) and localizes to Rab7-containing endosomal domains. Interference with Rab7 function causes dissociation of the Vps26/29/35 trimer but not the SNX dimer from membranes. This blocks retrieval of mannose 6-phosphate receptors to the TGN and impairs cathepsin D sorting. Rab5-GTP does not bind to the Vps26/29/35 trimer, but perturbation of Rab5 function causes dissociation of both the SNX and Vps26/29/35 components from membranes through inhibition of a pathway involving phosphatidylinositol 3-kinase. These findings demonstrate that Rab5 and Rab7 act in concert to regulate retromer recruitment to endosomes.     

SNX17 protects integrins from degradation by sorting between lysosomal and recycling pathways

The FERM-like domain-containing sorting nexins of the SNX17/SNX27/SNX31 family have been proposed to mediate retrieval of transmembrane proteins from the lysosomal pathway. In this paper, we describe a stable isotope labeling with amino acids in culture-based quantitative proteomic approach that allows an unbiased, global identification of transmembrane cargoes that are rescued from lysosomal degradation by SNX17. This screen revealed that several integrins required SNX17 for their stability, as depletion of SNX17 led to a loss of β1 and β5 integrins and associated a subunits from HeLa cells as a result of increased lysosomal degradation. SNX17 bound to the membrane distal NPXY motif in β integrin cytoplasmic tails, thereby preventing lysosomal degradation of β integrins and their associated a subunits. Furthermore, SNX17-dependent retrieval of integrins did not depend on the retromer complex. Consistent with an effect on integrin recycling, depletion of SNX17 also caused alterations in cell migration. Our data provide mechanistic insight into the retrieval of internalized integrins from the lysosomal degradation pathway, a prerequisite for subsequent recycling of these matrix receptors.     

SNX-BAR-mediated endosome tubulation is co-ordinated with endosome maturation

Endosomal sorting is essential for cell homeostasis. Proteins targeted for degradation are retained in the maturing endosome vacuole while others are recycled to the cell surface or sorted to the biosynthetic pathway via tubular transport carriers. Sorting nexin (SNX) proteins containing a BAR (for Bin-Amphiphysin-Rvs) domain are key regulators of phosphoinositide-mediated, tubular-based endosomal sorting, but how such sorting is co-ordinated with endosomal maturation is not known. Here, using well-defined Rab GTPases as endosomal compartment markers, we have analyzed the localization of SNX1 [endosome-to-trans-Golgi network (TGN) transport as part of the SNX-BAR-retromer complex], SNX4 (cargo-recycling from endosomes to the plasma membrane) and SNX8 (endosomes-to-TGN trafficking in a retromer-independent manner). We show that these SNX-BARs are primarily localized to early endosomes, but display the highest frequency of tubule formation at the moment of early-to-late endosome transition: the Rab5-to-Rab7 switch. Perturbing this switch shifts SNX-BAR tubulation to early endosomes, resulting in SNX1-decorated tubules that lack retromer components VPS26 and VPS35, suggesting that both early and late endosomal characteristics of the endosome are important for SNX-BAR-retromer-tubule formation. We also establish that SNX4, but not SNX1 and SNX8, is associated with the Rab11-recycling endosomes and that a high frequency of SNX4-mediated tubule formation is observed as endosomes undergo Rab4-to-Rab11 transition. Our study therefore provides evidence for fine-tuning between the processes of endosomal maturation and the formation of endosomal tubules. As tubulation is required for SNX1-, SNX4- and SNX8-mediated sorting, these data reveal a previously unrecognized co-ordination between maturation and tubular-based sorting.     

Endosomal receptor trafficking: Retromer and beyond

The tubular endolysosomal network is a quality control system that ensures the proper delivery of internalized receptors to specific subcellular destinations in order to maintain cellular homeostasis. Although retromer was originally described in yeast as a regulator of endosome‐to‐Golgi receptor recycling, mammalian retromer has emerged as a central player in endosome‐to‐plasma membrane recycling of a variety of receptors. Over the past decade, information regarding the mechanism by which retromer facilitates receptor trafficking has emerged, as has the identification of numerous retromer‐associated molecules including the WASH complex, sorting nexins (SNXs) and TBC1d5. Moreover, the recent demonstration that several SNXs can directly interact with retromer cargo to facilitate endosome‐to‐Golgi retrieval has provided new insight into how these receptors are trafficked in cells. The mechanism by which SNX17 cargoes are recycled out of the endosomal system was demonstrated to involve a retromer‐like complex termed the retriever, which is recruited to WASH positive endosomes through an interaction with the COMMD/CCDC22/CCDC93 (CCC) complex. Lastly, the mechanisms by which bacterial and viral pathogens highjack this complex sorting machinery in order to escape the endolysosomal system or remain hidden within the cells are beginning to emerge. In this review, we will highlight recent studies that have begun to unravel the intricacies by which the retromer and associated molecules contribute to receptor trafficking and how deregulation at this sorting domain can contribute to disease or facilitate pathogen infection.      

Regulation of P2Y1 Receptor Traffic by Sorting Nexin 1 is Retromer Independent

The activity and traffic of G‐protein coupled receptors (GPCRs) is tightly controlled. Recent work from our laboratory has shown that P2Y₁ and P2Y₁₂ responsiveness is rapidly and reversibly modulated in human platelets and that the underlying mechanism requires receptor trafficking as an essential part of this process. However, little is known about the molecular mechanisms underlying P2Y receptor traffic. Sorting nexin 1 (SNX1) has been shown to regulate the endosomal sorting of cell surface receptors either to lysosomes where they are downregulated or back to the cell surface. These functions may in part be due to interactions of SNX1 with the mammalian retromer complex. In this study, we investigated the role of SNX1 in P2Y receptor trafficking. We show that P2Y₁ receptors recycle via a slow recycling pathway that is regulated by SNX1, whereas P2Y₁₂ receptors return to the cell surface via a rapid route that is SNX1 independent. SNX1 inhibition caused a dramatic increase in the rate of P2Y₁ receptor recycling, whereas inhibition of Vps26 and Vps35 known to be present in retromer had no effect, indicating that SNX1 regulation of P2Y₁ receptor recycling is retromer independent. In addition, inhibition of SNX4, 6 and 17 proteins did not affect P2Y₁ receptor recycling. SNX1 has also been implicated in GPCR degradation; however, we provide evidence that P2Y receptor degradation is SNX1 independent. These data describe a novel function of SNX1 in the regulation of P2Y₁ receptor recycling and suggest that SNX1 plays multiple roles in endocytic trafficking of GPCRs.     

A Novel,Retromer‐Independent Role for Sorting Nexins 1 and 2 in RhoG‐Dependent Membrane Remodeling

The sorting nexins SNX1 and SNX2 are members of the retromer complex involved in protein sorting within the endocytic pathway. While retromer‐dependent functions of SNX1 and SNX2 have been well documented, potential retromer‐independent roles remain unclear. Here, we show that SNX1 and SNX2 interact with the Rac1 and RhoG guanine nucleotide exchange factor Kalirin‐7. Simultaneous overexpression of SNX1 or SNX2 and Kalirin‐7 in epithelial cells causes partial redistribution of both SNX isoforms to the plasma membrane, and results in RhoG‐dependent lamellipodia formation that requires functional Phox homology (PX) and Bin/Amphiphysin/Rvs (BAR) domains of SNX, but is Rac1‐ and retromer‐independent. Conversely, depletion of endogenous SNX1 or SNX2 inhibits Kalirin‐7‐mediated lamellipodia formation. Finally, we demonstrate that SNX1 and SNX2 interact directly with inactive RhoG, suggesting a novel role for these SNX proteins in recruiting an inactive Rho GTPase to its exchange factor.     

Regulation of endosomal clathrin and retromer‐mediated endosome to Golgi retrograde transport by the J‐domain protein RME‐8

After endocytosis, most cargo enters the pleiomorphic early endosomes in which sorting occurs. As endosomes mature, transmembrane cargo can be sequestered into inwardly budding vesicles for degradation, or can exit the endosome in membrane tubules for recycling to the plasma membrane, the recycling endosome, or the Golgi apparatus. Endosome to Golgi transport requires the retromer complex. Without retromer, recycling cargo such as the MIG‐14/Wntless protein aberrantly enters the degradative pathway and is depleted from the Golgi. Endosome‐associated clathrin also affects the recycling of retrograde cargo and has been shown to function in the formation of endosomal subdomains. Here, we find that the Caemorhabditis elegans endosomal J‐domain protein RME‐8 associates with the retromer component SNX‐1. Loss of SNX‐1, RME‐8, or the clathrin chaperone Hsc70/HSP‐1 leads to over‐accumulation of endosomal clathrin, reduced clathrin dynamics, and missorting of MIG‐14 to the lysosome. Our results indicate a mechanism, whereby retromer can regulate endosomal clathrin dynamics through RME‐8 and Hsc70, promoting the sorting of recycling cargo into the retrograde pathway.     

Retromer recycles vacuolar sorting receptors from the trans-Golgi network

Receptor-mediated sorting processes in the secretory pathway of eukaryotic cells rely on mechanisms to recycle the receptors after completion of transport. Based on this principle, plant vacuolar sorting receptors (VSRs) are thought to recycle after dissociating of receptor–ligand complexes in a pre-vacuolar compartment. This recycling is mediated by retromer, a cytosolic coat complex that comprises sorting nexins and a large heterotrimeric subunit. To analyse retromer-mediated VSR recycling, we have used a combination of immunoelectron and fluorescence microscopy to localize the retromer components sorting nexin 1 (SNX1) and sorting nexin 2a (SNX2a) and the vacuolar sorting protein VPS29p. All retromer components localize to the trans -Golgi network (TGN), which is considered to represent the early endosome of plants. In addition, we show that inhibition of retromer function in vivo by expression of SNX1 or SNX2a mutants as well as transient RNAi knockdown of all sorting nexins led to accumulation of the VSR BP80 at the TGN. Quantitative protein transport studies and live-cell imaging using fluorescent vacuolar cargo molecules revealed that arrival of these VSR ligands at the vacuole is not affected under these conditions. Based on these findings, we propose that the TGN is the location of retromer-mediated recycling of VSRs, and that transport towards the lytic vacuole downstream of the TGN is receptor-independent and occurs via maturation, similar to transition of the early endosome into the late endosome in mammalian cells.     

Reggies/flotillins interact with Rab11a and SNX4 at the tubulovesicular recycling compartment and function in transferrin receptor and E-cadherin trafficking

The lipid raft proteins reggie-1 and -2 (flotillins) are implicated in membrane protein trafficking but exactly how has been elusive. We find that reggie-1 and -2 associate with the Rab11a, SNX4, and EHD1–decorated tubulovesicular recycling compartment in HeLa cells and that reggie-1 directly interacts with Rab11a and SNX4. Short hairpin RNA–mediated down-regulation of reggie-1 (and -2) in HeLa cells reduces association of Rab11a with tubular structures and impairs recycling of the transferrin–transferrin receptor (TfR) complex to the plasma membrane. Overexpression of constitutively active Rab11a rescues TfR recycling in reggie-deficient HeLa cells. Similarly, in a Ca²⁺ switch assay in reggie-depleted A431 cells, internalized E-cadherin is not efficiently recycled to the plasma membrane upon Ca²⁺ repletion. E-cadherin recycling is rescued, however, by overexpression of constitutively active Rab11a or SNX4 in reggie-deficient A431 cells. This suggests that the function of reggie-1 in sorting and recycling occurs in association with Rab11a and SNX4. Of interest, impaired recycling in reggie-deficient cells leads to de novo E-cadherin biosynthesis and cell contact reformation, showing that cells have ways to compensate the loss of reggies. Together our results identify reggie-1 as a regulator of the Rab11a/SNX4-controlled sorting and recycling pathway, which is, like reggies, evolutionarily conserved.     

EHD1 interacts with retromer to stabilize SNX1 tubules and facilitate endosome-to-Golgi retrieval

Endosome-to-Golgi retrieval of the cation-independent mannose 6-phosphate receptor (CIMPR) requires the function of the retromer complex. Retromer is localized to endosomes and comprises two distinct sub complexes: the vacuolar protein sorting 35/29/26 sub complex that binds cargo and the sorting nexin (SNX)1/2 sub complex that tubulates endosomal membranes. To identify up- or down-stream regulatory factors of retromer, a comparative proteomic strategy was employed. Protein profiles of endosomally enriched membranes, from either wild-type or retromer-deficient mouse cells, were compared to identify proteins with either elevated or reduced expression levels. Eps15 homology domain-containing protein-1 (EHD1) was identified in endosomally enriched membrane fractions from retromer-deficient cells and was found to be approximately threefold upregulated in the absence of retromer. EHD1 is localized to tubular and vesicular endosomes, partially colocalizes with retromer and is associated with retromer in vivo. Mutation of the nucleotide-binding P-loop of EHD1 results in a dominant-negative effect upon retromer localization and endosome-to-Golgi retrieval, while loss of EHD1 expression by RNA interference destabilizes SNX1-positive tubules and inhibits endosome-to-Golgi retrieval. The interaction between EHD1 and retromer and the requirement for EHD1 to stabilize SNX1-tubules establish EHD1 as a novel facilitating component of endosome-to-Golgi retrieval.     

An essential role for SNX1 in lysosomal sorting of protease-activated receptor-1: evidence for retromer-, Hrs-, and Tsg101-independent functions of sorting nexins

Sorting nexin 1 (SNX1) and SNX2 are the mammalian homologues of the yeast Vps5p retromer component that functions in endosome-to-Golgi trafficking. SNX1 is also implicated in endosome-to-lysosome sorting of cell surface receptors, although its requirement in this process remains to be determined. To assess SNX1 function in endocytic sorting of protease-activated receptor-1 (PAR1), we used siRNA to deplete HeLa cells of endogenous SNX1 protein. PAR1, a G-protein-coupled receptor, is proteolytically activated by thrombin, internalized, sorted predominantly to lysosomes, and efficiently degraded. Strikingly, depletion of endogenous SNX1 by siRNA markedly inhibited agonist-induced PAR1 degradation, whereas expression of a SNX1 siRNA-resistant mutant protein restored agonist-promoted PAR1 degradation in cells lacking endogenous SNX1, indicating that SNX1 is necessary for lysosomal degradation of PAR1. SNX1 is known to interact with components of the mammalian retromer complex and Hrs, an early endosomal membrane-associated protein. However, activated PAR1 degradation was not affected in cells depleted of retromer Vps26/Vps35 subunits, Hrs or Tsg101, an Hrs-interacting protein. We further show that SNX2, which dimerizes with SNX1, is not essential for lysosomal sorting of PAR1, but rather can regulate PAR1 degradation by disrupting endosomal localization of endogenous SNX1 when ectopically expressed. Together, our findings establish an essential role for endogenous SNX1 in sorting activated PAR1 to a distinct lysosomal degradative pathway that is independent of retromer, Hrs, and Tsg101.     

Interchangeable but essential functions of SNX1 and SNX2 in the association of retromer with endosomes and the trafficking of mannose 6-phosphate receptors

The retromer is a cytosolic/peripheral membrane protein complex that mediates the retrieval of the cation-independent mannose 6-phosphate receptor from endosomes to the trans-Golgi network (TGN) in mammalian cells. Previous studies showed that the mammalian retromer comprises three proteins, named Vps26, Vps29, and Vps35, plus the sorting nexin, SNX1. There is conflicting evidence, however, as to whether a homologous sorting nexin, SNX2, is truly a component of the retromer. In addition, the nature of the subunit interactions and assembly of the mammalian retromer complex are poorly understood. We have addressed these issues by performing biochemical and functional analyses of endogenous retromers in the human cell line HeLa. We found that the mammalian retromer complex consists of two autonomously assembling subcomplexes, namely, a Vps26-Vps29-Vps35 obligate heterotrimer and a SNX1/2 alternative heterodimer or homodimer. The association of Vps26-Vps29-Vps35 with endosomes requires the presence of either SNX1 or SNX2, whereas SNX1/2 can be recruited to endosomes independently of Vps26-Vps29-Vps35. We also found that the presence of either SNX1 or SNX2 is essential for the retrieval of the cation-independent mannose 6-phosphate receptor to the TGN. These observations indicate that the mammalian retromer complex assembles by sequential association of SNX1/2 and Vps26-Vps29-Vps35 subcomplexes on endosomal membranes and that SNX1 and SNX2 play interchangeable but essential roles in retromer structure and function.     

Molecular Insights into Rab7‐Mediated Endosomal Recruitment of Core Retromer: Deciphering the Role of Vps26 and Vps35

Retromer, a peripheral membrane protein complex, plays an instrumental role in host of cellular processes by its ability to recycle receptors from endosomes to the trans‐Golgi network. It consists of two distinct sub‐complexes, a membrane recognizing, sorting nexins (SNX) complex and a cargo recognition, vacuolar protein sorting (Vps) complex. Small GTPase, Rab7 is known to recruit retromer on endosomal membrane via interactions with the Vps sub‐complex. The molecular mechanism underlying the recruitment process including the role of individual Vps proteins is yet to be deciphered. In this study, we developed a FRET‐based assay in HeLa cells that demonstrated the interaction of Rab7 with Vps35 and Vps26 in vivo. Furthermore, we showed that Rab7 recruits retromer to late endosomes via direct interactions with N‐terminal conserved regions in Vps35. However, the single point mutation, which disrupts the interaction between Vps35 and Vps26, perturbed the Rab7‐mediated recruitment of retromer in HeLa cells. Using biophysical measurements, we demonstrate that the association of Vps26 with Vps35 resulted in high affinity binding between the Vps sub‐complex and the activated Rab7 suggesting for a possible allosteric role of Vps26. Thus, this study provides molecular insights into the essential role of Vps26 and Vps35 in Rab7‐mediated recruitment of the core retromer complex.      

Distinct complexes of yeast Snx4 family SNX‐BARs mediate retrograde trafficking of Snc1 and Atg27

The yeast SNX4 sub‐family of sorting nexin containing a Bin‐Amphiphysin‐Rvs domain (SNX‐BAR) proteins, Snx4/Atg24, Snx41 and Atg20/Snx42, are required for endocytic recycling and selective autophagy. Here, we show that Snx4 forms 2 functionally distinct heterodimers: Snx4‐Atg20 and Snx4‐Snx41. Each heterodimer coats an endosome‐derived tubule that mediates retrograde sorting of distinct cargo; the v‐SNARE, Snc1, is a cargo of the Snx4‐Atg20 pathway, and Snx4‐Snx41 mediates retrograde sorting of Atg27, an integral membrane protein implicated in selective autophagy. Live cell imaging of individual endosomes shows that Snx4 and the Vps5‐Vps17 retromer SNX‐BAR heterodimer operate concurrently on a maturing endosome. Consistent with this, the yeast dynamin family protein, Vps1, which was previously shown to promote fission of retromer‐coated tubules, promotes fission of Snx4‐Atg20 coated tubules. The results indicate that the yeast SNX‐BAR proteins coat 3 distinct types of endosome‐derived carriers that mediate endosome‐to‐Golgi retrograde trafficking.      

Retromer

The retromer is a heteropentameric complex that associates with the cytosolic face of endosomes and mediates retrograde transport of transmembrane cargo from endosomes to the trans-Golgi network. The mammalian retromer complex comprises a sorting nexin dimer composed of a still undefined combination of SNX1, SNX2, SNX5 and SNX6, and a cargo-recognition trimer composed of Vps26, Vps29 and Vps35. The SNX subunits contain PX and BAR domains that allow binding to PI(3)P enriched, highly curved membranes of endosomal vesicles and tubules, while Vps26, Vps29 and Vps35 have arrestin, phosphoesterase and alpha-solenoid folds, respectively. Recent studies have implicated retromer in a broad range of physiological, developmental and pathological processes, underscoring the critical nature of retrograde transport mediated by this complex.     

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.     

SNX-3 mediates retromer-independent tubular endosomal recycling by opposing EEA-1-facilitated trafficking

Early endosomes are the sorting hub on the endocytic pathway, wherein sorting nexins (SNXs) play important roles for formation of the distinct membranous microdomains with different sorting functions. Tubular endosomes mediate the recycling of clathrin-independent endocytic (CIE) cargoes back toward the plasma membrane. However, the molecular mechanism underlying the tubule formation is still poorly understood. Here we screened the effect on the ARF-6-associated CIE recycling endosomal tubules for all the SNX members in Caenorhabditis elegans (C. elegans). We identified SNX-3 as an essential factor for generation of the recycling tubules. The loss of SNX-3 abolishes the interconnected tubules in the intestine of C. elegans. Consequently, the surface and total protein levels of the recycling CIE protein hTAC are strongly decreased. Unexpectedly, depletion of the retromer components VPS-26/-29/-35 has no similar effect, implying that the retromer trimer is dispensable in this process. We determined that hTAC is captured by the ESCRT complex and transported into the lysosome for rapid degradation in snx-3 mutants. Interestingly, EEA-1 is increasingly recruited on early endosomes and localized to the hTAC-containing structures in snx-3 mutant intestines. We also showed that SNX3 and EEA1 compete with each other for binding to phosphatidylinositol-3-phosphate enriching early endosomes in Hela cells. Our data demonstrate for the first time that PX domain-only C. elegans SNX-3 organizes the tubular endosomes for efficient recycling and retrieves the CIE cargo away from the maturing sorting endosomes by competing with EEA-1 for binding to the early endosomes. However, our results call into question how SNX-3 couples the cargo capture and membrane remodeling in the absence of the retromer trimer complex.     

Sorting of plant vacuolar proteins is initiated in the ER

Transport of soluble cargo molecules to the lytic vacuole of plants requires vacuolar sorting receptors (VSRs) to divert transport of vacuolar cargo from the default secretory route to the cell surface. Just as important is the trafficking of the VSRs themselves, a process that encompasses anterograde transport of receptor–ligand complexes from a donor compartment, dissociation of these complexes upon arrival at the target compartment, and recycling of the receptor back to the donor compartment for a further round of ligand transport. We have previously shown that retromer‐mediated recycling of the plant VSR BP80 starts at the trans‐Golgi network (TGN). Here we demonstrate that inhibition of retromer function by either RNAi knockdown of sorting nexins (SNXs) or co‐expression of mutants of SNX1/2a specifically inhibits the ER export of VSRs as well as soluble vacuolar cargo molecules, but does not influence cargo molecules destined for the COPII‐mediated transport route. Retention of soluble cargo despite ongoing COPII‐mediated bulk flow can only be explained by an interaction with membrane‐bound proteins. Therefore, we examined whether VSRs are capable of binding their ligands in the lumen of the ER by expressing ER‐anchored VSR derivatives. These experiments resulted in drastic accumulation of soluble vacuolar cargo molecules in the ER. This demonstrates that the ER, rather than the TGN, is the location of the initial VSR–ligand interaction. It also implies that the retromer‐mediated recycling route for the VSRs leads from the TGN back to the ER.     

SNX3 controls Wingless/Wnt secretion through regulating retromer-dependent recycling of Wntless

Drosophila Wingless (Wg) acts as a morphogen during development. Wg secretion is controlled by a seven-pass transmembrane cargo Wntless (Wls). We have recently identified retromer as a key regulator involved in Wls trafficking. As sorting nexin (SNX) molecules are essential components of the retromer complex, we hypothesized that specific SNX(s) is required for retromer-mediated Wnt secretion. Here, we generated Drosophila mutants for all of the eight snx members, and identified Drosophila SNX3 (DSNX3) as an essential molecule required for Wg secretion. We show that Wg secretion and its signaling activity are defective in Dsnx3 mutant clones in wing discs. Wg levels in the culture medium of Dsnx3-depleted S2 cells are also markedly reduced. Importantly, Wls levels are strikingly reduced in Dsnx3 mutant cells, and overexpression of Wls can rescue the Wg secretion defect observed in Dsnx3 mutant cells. Moreover, DSNX3 can interact with the retromer component Vps35, and co-localize with Vps35 in early endosomes. These data indicate that DSNX3 regulates Wg secretion via retromer-dependent Wls recycling. In contrast, we found that Wg secretion is not defective in cells mutant for Drosophila snx1 and snx6, two components of the classical retromer complex. Ectopic expression of DSNX1 or DSNX6 fails to rescue the Wg secretion defect in Dsnx3 mutant wing discs and in Dsnx3 dsRNA-treated S2 cells. These data demonstrate the specificity of the DSNX3-retromer complex in Wls recycling. Together, our findings suggest that DSNX3 acts as a cargo-specific component of retromer, which is required for endocytic recycling of Wls and Wg/Wnt secretion.     

A Sorting Nexin 17‐Binding Domain Within the LRP1 Cytoplasmic Tail Mediates Receptor Recycling Through the Basolateral Sorting Endosome

Sorting nexin 17 (SNX17) is an adaptor protein present in early endosomal antigen 1 (EEA1)‐positive sorting endosomes that promotes the efficient recycling of low‐density lipoprotein receptor‐related protein 1 (LRP1) to the plasma membrane through recognition of the first NPxY motif in the cytoplasmic tail of this receptor. The interaction of LRP1 with SNX17 also regulates the basolateral recycling of the receptor from the basolateral sorting endosome (BSE). In contrast, megalin, which is apically distributed in polarized epithelial cells and localizes poorly to EEA1‐positive sorting endosomes, does not interact with SNX17, despite containing three NPxY motifs, indicating that this motif is not sufficient for receptor recognition by SNX17. Here, we identified a cluster of 32 amino acids within the cytoplasmic domain of LRP1 that is both necessary and sufficient for SNX17 binding. To delineate the function of this SNX17‐binding domain, we generated chimeric proteins in which the SNX17‐binding domain was inserted into the cytoplasmic tail of megalin. This insertion mediated the binding of megalin to SNX17 and modified the cell surface expression and recycling of megalin in non‐polarized cells. However, the polarized localization of chimeric megalin was not modified in polarized Madin‐Darby canine kidney cells. These results provide evidence regarding the molecular and cellular mechanisms underlying the specificity of SNX17‐binding receptors and the restricted function of SNX17 in the BSE .