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.     

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.      

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.     

Sorting nexin-1 mediates tubular endosome-to-TGN transport through coincidence sensing of high- curvature membranes and 3-phosphoinositides

BACKGROUND: Sorting nexins (SNXs) are phox homology (PX) domain-containing proteins thought to regulate endosomal sorting of internalized receptors. The prototypical SNX is sorting nexin-1 (SNX1), a protein that through its PX domain binds phosphatidylinositol 3-monophosphate [PtdIns(3)P] and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P(2)]. SNX1 is associated with early endosomes, from where it has been proposed to regulate the degradation of internalized epidermal growth factor (EGF) receptors through modulating endosomal-to-lysosomal sorting. RESULTS: We show here that SNX1 contains a BAR (Bin/Amphiphysin/Rvs) domain, a membrane binding domain that endows SNX1 with the ability to form dimers and to sense membrane curvature. We present evidence that through coincidence detection, the BAR and PX domains efficiently target SNX1 to a microdomain of the early endosome defined by high curvature and the presence of 3-phosphoinositides. In addition, we show that the BAR domain endows SNX1 with an ability to tubulate membranes in-vitro and drive the tubulation of the endosomal compartment in-vivo. Using RNA interference (RNAi), we establish that SNX1 does not play a role in EGF or transferrin receptor sorting; rather it specifically perturbs endosome-to-trans Golgi network (TGN) transport of the cation-independent mannose-6-phosphate receptor (CI-MPR). Our data support an evolutionarily conserved function for SNX1 from yeast to mammals and provide functional insight into the molecular mechanisms underlying lipid-mediated protein targeting and tubular-based protein sorting. CONCLUSIONS: We conclude that through coincidence detection SNX1 associates with a microdomain of the early endosome-characterized by high membrane curvature and the presence of 3-phosphoinositides-from where it regulates tubular-based endosome-to-TGN retrieval of the CI-MPR.     

Specific amino acids in the BAR domain allow homodimerization and prevent heterodimerization of sorting nexin 33

SNX33 (sorting nexin 33) is a homologue of the endocytic protein SNX9 and has been implicated in actin polymerization and the endocytosis of the amyloid precursor protein. SNX33 belongs to the large family of BAR (Bin/amphiphysin/Rvs) domain-containing proteins, which alter cellular protein trafficking by modulating cellular membranes and the cytoskeleton. Some BAR domains engage in homodimerization, whereas other BAR domains also mediate heterodimerization between different BAR domain-containing proteins. The molecular basis for this difference is not yet understood. Using co-immunoprecipitations we report that SNX33 forms homodimers, but not heterodimers, with other BAR domain-containing proteins, such as SNX9. Domain deletion analysis revealed that the BAR domain, but not the SH3 (Src homology 3) domain, was required for homodimerization of SNX33. Additionally, the BAR domain prevented the heterodimerization between SNX9 and SNX33, as determined by domain swap experiments. Molecular modelling of the SNX33 BAR domain structure revealed that key amino acids located at the BAR domain dimer interface of the SNX9 homodimer are not conserved in SNX33. Replacing these amino acids in SNX9 with the corresponding amino acids of SNX33 allowed the mutant SNX9 to heterodimerize with SNX33. Taken together, the present study identifies critical amino acids within the BAR domains of SNX9 and SNX33 as determinants for the specificity of BAR domain-mediated interactions and suggests that SNX9 and SNX33 have distinct molecular functions.     

Inhibitory regulation of EGF receptor degradation by sorting nexin 5

Endosomal trafficking of EGF receptor (EGFR) upon stimulation is a highly regulated process during receptor-mediated signaling. Recently, the sorting nexin (SNX) family has emerged as an important regulator in the membrane trafficking of EGFR. Here, we report the identification of a novel interaction between two members of the family, SNX1 and SNX5, which is mediated by the newly defined BAR domain of both SNXs. We have also shown that the PX domain of SNX5 binds specifically to PtdIns other than to PtdIns(3)P. Furthermore, the BAR domain but not the PX domain of SNX5 is sufficient for its subcellular membrane association. Functionally, overexpression of SNX5 inhibits the degradation of EGFR. This process appears to be independent of its interaction with SNX1. However, overexpression of SNX1 is able to attenuate the effect of SNX5 on EGFR degradation, suggesting the two proteins may play antagonistic roles in regulating endosomal trafficking of the receptor.     

SNX4 coordinates endosomal sorting of TfnR with dynein-mediated transport into the endocytic recycling compartment

SNX-BAR proteins are a sub-family of sorting nexins implicated in endosomal sorting. Here, we establish that through its phox homology (PX) and Bin-Amphiphysin-Rvs (BAR) domains, sorting nexin-4 (SNX4) is associated with tubular and vesicular elements of a compartment that overlaps with peripheral early endosomes and the juxtanuclear endocytic recycling compartment (ERC). Suppression of SNX4 perturbs transport between these compartments and causes lysosomal degradation of the transferrin receptor (TfnR). Through an interaction with KIBRA, a protein previously shown to bind dynein light chain 1, we establish that SNX4 associates with the minus end-directed microtubule motor dynein. Although suppression of KIBRA and dynein perturbs early endosome-to-ERC transport, TfnR sorting is maintained. We propose that by driving membrane tubulation, SNX4 coordinates iterative, geometric-based sorting of the TfnR with the long-range transport of carriers from early endosomes to the ERC. Finally, these data suggest that by associating with molecular motors, SNX-BAR proteins may coordinate sorting with carrier transport between donor and recipient membranes.     

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.     

Sorting nexin 3 (SNX3) is a component of a tubular endosomal network induced by Salmonella and involved in maturation of the Salmonella‐containing vacuole

Salmonella enterica serovar Typhimurium is an intracellular pathogen that grows within a modified endomembrane compartment, the Salmonella‐containing vacuole (SCV). Maturation of nascent SCVs involves the recruitment of early endosome markers and the remodelling of phosphoinositides at the membrane of the vacuole, in particular the production of phosphatidylinositol 3‐phosphate [PI(3)P]. Sorting nexins (SNXs) are a family of proteins characterized by the presence of a phox homology (PX) domain that binds to phosphoinositides and are involved in intracellular trafficking in eukaryotic cells. We therefore studied whether sorting nexins, particularly sorting nexin 3 (SNX3), play a role in Salmonella infection. We found that SNX3 transiently localized to SCVs at early times post invasion (10 min) and presented a striking tubulation phenotype in the vicinity of SCVs at later times (30–60 min). The bacterial effector SopB, which is known to promote PI(3)P production on SCVs, was required for the formation of SNX3 tubules. In addition, RAB5 was also required for the formation of SNX3 tubules. Depletion of SNX3 by siRNA impaired RAB7 and LAMP1 recruitment to the SCV. Moreover, the formation of Salmonella‐induced filaments (Sifs) was altered by SNX3 knock‐down. Therefore, SNX3 plays a significant role in regulating the maturation of SCVs.     

Fission of SNX-BAR–coated endosomal retrograde transport carriers is promoted by the dynamin-related protein Vps1

Retromer is an endosomal sorting device that orchestrates capture and packaging of cargo into transport carriers coated with sorting nexin BAR domain proteins (SNX-BARs). We report that fission of retromer SNX-BAR–coated tubules from yeast endosomes is promoted by Vps1, a dynamin-related protein that localizes to endosomes decorated by retromer SNX-BARs and Mvp1, a SNX-BAR that is homologous to human SNX8. Mvp1 exhibits potent membrane remodeling activity in vitro, and it promotes association of Vps1 with the endosome in vivo. Retrograde transport carriers bud from the endosome coated by retromer and Mvp1, and cargo export is deficient in mvp1- and vps1-null cells, but with distinct endpoints; cargo export is delayed in mvp1-null cells, but cargo export completely fails in vps1-null cells. The results indicate that Mvp1 promotes Vps1-mediated fission of retromer- and Mvp1-coated tubules that bud from the endosome, revealing a functional link between the endosomal sorting and fission machineries to produce retrograde transport carriers.     

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 .      

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.     

Towards a molecular understanding of endosomal trafficking by Retromer and Retriever

Endosomes are dynamic intracellular compartments that control the sorting of a constant stream of different transmembrane cargos either for ESCRT‐mediated degradation or for egress and recycling to compartments such as the Golgi and the plasma membrane. The recycling of cargos occurs within tubulovesicular membrane domains and is facilitated by peripheral membrane protein machineries that control both membrane remodelling and selection of specific transmembrane cargos. One of the primary sorting machineries is the Retromer complex, which controls the recycling of a large array of different cargo molecules in cooperation with various sorting nexin (SNX) adaptor proteins. Recently a Retromer‐like complex was also identified that controls plasma membrane recycling of cargos including integrins and lipoprotein receptors. Termed “Retriever,” this complex uses a different SNX family member SNX17 for cargo recognition, and cooperates with the COMMD/CCDC93/CCDC22 (CCC) complex to form a larger assembly called “Commander” to mediate endosomal trafficking. In this review we focus on recent advances that have begun to provide a molecular understanding of these two distantly related transport machineries.     

The Unique Mechanism of SNX9 BAR Domain for Inducing Membrane Tubulation

Sorting nexin 9 (SNX9) is a member of the sorting nexin family of proteins and plays a critical role in clathrin-mediated endocytosis. It has a Bin-Amphiphysin-Rvs (BAR) domain which can form a crescent-shaped homodimer structure that induces deformation of the plasma membrane. While other BAR-domain containing proteins such as amphiphysin and endophilin have an amphiphatic helix in front of the BAR domain which plays a critical role in membrane penetration, SNX9 does not. Thus, whether and how SNX9 BAR domain could induce the deformation of the plasma membrane is not clear. The present study identified the internal putative amphiphatic stretch in the 1^st α-helix of the SNX9 BAR domain and proved that together with the N-terminal helix (H₀) region, this internal putative amphiphatic stretch is critical for inducing membrane tubulation. Therefore, our study shows that SNX9 uses a unique mechanism to induce the tubulation of the plasma membrane which mediates proper membrane deformation during clathrin-mediated endocytosis.     

Sorting Nexin 31 Binds Multiple β Integrin Cytoplasmic Domains and Regulates β1 Integrin Surface Levels and Stability

Trafficking of α5β1 integrin to lysosomes and its subsequent degradation is influenced by ligand occupancy and the binding of SNX17 via its protein 4.1, ezrin, radixin, moesin (FERM) domain to the membrane-distal NPxY motif in the cytoplasmic domain of β1 integrin in early endosomes. Two other sorting nexin (SNX) family members, namely SNX27 and SNX31, share with SNX17 next to their obligate phox domain a FERM domain, which may enable them to bind β integrin tails. Here we report that, in addition to SNX17, SNX31 but not SNX27 binds several β integrin tails in early endosomes in a PI3 (phosphatidylinositide 3)-kinase-dependent manner. Similarly like SNX17, binding of SNX31 with β1 integrin tails in early endosomes occurs between the FERM domain and the membrane-distal NPxY motif in the β1 integrin cytoplasmic domain. Furthermore, expression of SNX31 rescues β1 integrin surface levels and stability in SNX17-depleted cells. In contrast to SNX17, expression of SNX31 is restricted and found highly expressed in bladder and melanoma tissue. Altogether, these results demonstrate that SNX31 is an endosomal regulator of β integrins with a restricted expression pattern.     

EFA6A,a guanine nucleotide exchange factor for Arf6, interacts with sorting nexin‐1 and regulates neurite outgrowth

The membrane trafficking and actin cytoskeleton remodeling mediated by ADP ribosylation factor 6 (Arf6) are functionally linked to various neuronal processes including neurite formation and maintenance, neurotransmitter release, and receptor internalization. EFA6A is an Arf6‐specific guanine nucleotide exchange factor that is abundantly expressed in the brain. In this study, we identified sorting nexin‐1 (SNX1), a retromer component that is implicated in endosomal sorting and trafficking, as a novel interacting partner for EFA6A by yeast two‐hybrid screening. The interaction was mediated by the C‐terminal region of EFA6A and a BAR domain of SNX1, and further confirmed by pull‐down assay and immunoprecipitation from mouse brain lysates. In situ hybridization analysis demonstrated the widespread expression of SNX1 in the mouse brain, which overlapped with the expression of EFA6A in the forebrain. Immunofluorescent analysis revealed the partial colocalization of EFA6A and SNX1 in the dendritic fields of the hippocampus. Immunoelectron microscopic analysis revealed the overlapping subcellular localization of EFA6A and SNX1 at the post‐synaptic density and endosomes in dendritic spines. In Neuro‐2a neuroblastoma cells, expression of either EFA6A or SNX1 induced neurite outgrowth, which was further enhanced by co‐expression of EFA6A and SNX1. The present findings suggest a novel mechanism by which EFA6A regulates Arf6‐mediated neurite formation through the interaction with SNX1.

     

Sorting nexin 27 interacts with the Cytohesin associated scaffolding protein (CASP) in lymphocytes

CASP is a small cytokine-inducible protein, primarily expressed in hematopoetic cells, which associates with members of the Cytohesin/ARNO family of guanine nucleotide-exchange factors. Cytohesins activate ARFs, a group of GTPases involved in vesicular initiation. Functionally, CASP is an adaptor protein containing a PDZ domain, a coiled-coil, and a potential carboxy terminal PDZ-binding motif that we sought to characterize here. Using GST pulldowns and mass spectrometry we identified the novel interaction of CASP and sorting nexin 27 (SNX27). In lymphocytes, CASP's PDZ-binding motif interacts with the PDZ domain of SNX27. This protein is a unique member of the sorting nexin family of proteins, a group generally involved in the endocytic and intracellular sorting machinery. Endogenous SNX27 and CASP co-localize at the early endosomal compartment in lymphocytes and also in transfection studies. These results suggest that endosomal SNX27 may recruit CASP to orchestrate intracellular trafficking and/or signaling complexes.     

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.     

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.