Sorting nexin 8 regulates endosome-to-Golgi transport

Sorting nexin 8 (SNX8) belongs to the sorting nexin protein family, whose members are involved in endocytosis and endosomal sorting and signaling. The function of SNX8 has so far been unknown. Here, we have investigated the role of SNX8 in intracellular transport of the bacterial toxin Shiga toxin (Stx) and the plant toxin ricin. After being endocytosed, these toxins are transported retrogradely from endosomes, via the Golgi apparatus and the endoplasmic reticulum (ER), into the cytosol, where they exert their toxic effect. Interestingly, our experiments show that SNX8 regulates the transport of Stx and ricin differently; siRNA-mediated knockdown of SNX8 significantly increased the Stx transport to the trans-Golgi network (TGN), whereas ricin transport was slightly inhibited. We also found that SNX8 colocalizes with early endosome antigen 1 (EEA1) and with retromer components, suggesting an endosomal localization of SNX8 and supporting our finding that SNX8 is involved in endosomal sorting.     

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.     

SNX12 role in endosome membrane transport

In this paper, we investigated the role of sorting nexin 12 (SNX12) in the endocytic pathway. SNX12 is a member of the PX domain-containing sorting nexin family and shares high homology with SNX3, which plays a central role in the formation of intralumenal vesicles within multivesicular endosomes. We found that SNX12 is expressed at very low levels compared to SNX3. SNX12 is primarily associated with early endosomes and this endosomal localization depends on the binding to 3-phosphoinositides. We find that overexpression of SNX12 prevents the detachment (or maturation) of multivesicular endosomes from early endosomes. This in turn inhibits the degradative pathway from early to late endosomes/lysosomes, much like SNX3 overexpression, without affecting endocytosis, recycling and retrograde transport. In addition, while previous studies showed that Hrs knockdown prevents EGF receptor sorting into multivesicular endosomes, we find that overexpression of SNX12 restores the sorting process in an Hrs knockdown background. Altogether, our data show that despite lower expression level, SNX12 shares redundant functions with SNX3 in the biogenesis of multivesicular endosomes.     

Receptor and membrane recycling can occur with unaltered efficiency despite dramatic Rab5(q79l)-induced changes in endosome geometry

Current models for sorting in the endosomal compartment suggest that endosomal geometry plays a significant role as membrane-bound proteins accumulate in tubular regions for recycling, and lumenal markers accumulate in large vacuolar portions for delivery to lysosomes. Rab5, a small molecular weight GTPase, functions in the formation and maintenance of the early/sorting endosome. Overexpression of the constitutively active form, Rab5(Q79L), leads to enhanced endosome fusion resulting in the enlargement of early endosomes. Using an adenoviral expression system to regulate the time and level of Rab5(Q79L) overexpression in HeLa cells, we find that although endosomes are dramatically enlarged, the rates of transferrin receptor-mediated endocytosis and recycling are unaffected. Moreover, despite the enlarged endosome phenotype, neither the rate of internalization of a fluid phase marker nor the rate of recycling of a bulk lipid marker were affected. These results suggest that GTP hydrolysis by Rab5 is rate-limiting for endosome fusion but not for endocytic trafficking and that early endosome geometry may be a less critical determinant of sorting efficiencies than previously thought.     

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.     

The big brain aquaporin is required for endosome maturation and notch receptor trafficking

Activity of the big brain (bib) gene influences Notch signaling during Drosophila nervous system development. We demonstrate that Bib, which belongs to the aquaporin family of channel proteins, is required for endosome maturation in Drosophila epithelial cells. In the absence of Bib, early endosomes arrest and form abnormal clusters, and cells exhibit reduced acidification of endocytic trafficking organelles. Bib acts downstream of Hrs in early endosome morphogenesis and regulates biogenesis of endocytic compartments prior to the formation of Rab7-containing late endosomes. Abnormal endosome morphology caused by loss of Bib is accompanied by overaccumulation of Notch, Delta, and other signaling molecules as well as reduced intracellular trafficking of Notch to nuclei. Analysis of several endosomal trafficking mutants reveals a correlation between endosomal acidification and levels of Notch signaling. Our findings reveal an unprecedented role for an aquaporin in endosome maturation, trafficking, and acidification.     

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.      

Endosomal maturation by Rab conversion in Aspergillus nidulans is coupled to dynein-mediated basipetal movement

We exploit the ease with which highly motile early endosomes are distinguished from static late endosomes in order to study Aspergillus nidulans endosomal traffic. RabS(Rab7) mediates homotypic fusion of late endosomes/vacuoles in a homotypic fusion- and vacuole protein sorting/Vps41-dependent manner. Progression across the endocytic pathway involves endosomal maturation because the end products of the pathway in the absence of RabS(Rab7) are minivacuoles that are competent in multivesicular body sorting and cargo degradation but retain early endosomal features, such as the ability to undergo long-distance movement and propensity to accumulate in the tip region if dynein function is impaired. Without RabS(Rab7), early endosomal Rab5s-RabA and RabB-reach minivacuoles, in agreement with the view that Rab7 homologues facilitate the release of Rab5 homologues from endosomes. RabS(Rab7) is recruited to membranes already at the stage of late endosomes still lacking vacuolar morphology, but the transition between early and late endosomes is sharp, as only in a minor proportion of examples are RabA/RabB and RabS(Rab7) detectable in the same-frequently the less motile-structures. This early-to-late endosome/vacuole transition is coupled to dynein-dependent movement away from the tip, resembling the periphery-to-center traffic of endosomes accompanying mammalian cell endosomal maturation. Genetic studies establish that endosomal maturation is essential, whereas homotypic vacuolar fusion is not.     

Role of SNX16 in the dynamics of tubulo-cisternal membrane domains of late endosomes

In this paper, we report that the PX domain-containing protein SNX16, a member of the sorting nexin family, is associated with late endosome membranes. We find that SNX16 is selectively enriched on tubulo-cisternal elements of this membrane system, whose highly dynamic properties and formation depend on intact microtubules. By contrast, SNX16 was not found on vacuolar elements that typically contain LBPA, and thus presumably correspond to multivesicular endosomes. We conclude that SNX16, together with its partner phosphoinositide, define a highly dynamic subset of late endosomal membranes, supporting the notion that late endosomes are organized in distinct morphological and functional regions. Our data also indicate that SNX16 is involved in tubule formation and cholesterol transport as well as trafficking of the tetraspanin CD81, suggesting that the protein plays a role in the regulation of late endosome membrane dynamics.     

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.     

An ESCRT–spastin interaction promotes fission of recycling tubules from the endosome

Mechanisms coordinating endosomal degradation and recycling are poorly understood, as are the cellular roles of microtubule (MT) severing. We show that cells lacking the MT-severing protein spastin had increased tubulation of and defective receptor sorting through endosomal tubular recycling compartments. Spastin required the ability to sever MTs and to interact with ESCRT-III (a complex controlling cargo degradation) proteins to regulate endosomal tubulation. Cells lacking IST1 (increased sodium tolerance 1), an endosomal sorting complex required for transport (ESCRT) component to which spastin binds, also had increased endosomal tubulation. Our results suggest that inclusion of IST1 into the ESCRT complex allows recruitment of spastin to promote fission of recycling tubules from the endosome. Thus, we reveal a novel cellular role for MT severing and identify a mechanism by which endosomal recycling can be coordinated with the degradative machinery. Spastin is mutated in the axonopathy hereditary spastic paraplegia. Zebrafish spinal motor axons depleted of spastin or IST1 also had abnormal endosomal tubulation, so we propose this phenotype is important for axonal degeneration.     

Rab conversion as a mechanism of progression from early to late endosomes

The mechanisms of endosome biogenesis and maintenance are largely unknown. The small GTPases Rab 5 and Rab 7 are key determinants of early and late endosomes, organizing effector proteins into specific membrane subdomains. Whether such Rab machineries are indefinitely maintained on membranes or can disassemble in the course of cargo transport is an open question. Here, we combined novel image-analysis algorithms with fast live-cell imaging. We found that the level of Rab 5 dynamically fluctuates on individual early endosomes, linked by fusion and fission events into a network in time. Within it, degradative cargo concentrates in progressively fewer and larger endosomes that migrate from the cell periphery to the center where Rab 5 is rapidly replaced with Rab 7. The class C VPS/HOPS complex, an established GEF for Rab 7, interacts with Rab 5 and is required for Rab 5-to-Rab 7 conversion. Our results reveal unexpected dynamics of Rab domains and suggest Rab conversion as the mechanism of cargo progression between early and late endosomes.     

Inhibition of transferrin recycling and endosome tubulation by phospholipase A2 antagonists

We report here that a broad spectrum of phospholipase A(2) (PLA(2)) antagonists produce a concentration-dependent, differential block in the endocytic recycling pathway of transferrin (Tf) and Tf receptors (TfRs) but have no acute affect on Tf uptake from the cell surface. At low concentrations of antagonists (approximately 1 microm), Tf and TfR accumulated in centrally located recycling endosomes, whereas at higher concentrations (approximately 10 microm), Tf-TfR accumulated in peripheral sorting endosomes. Several independent lines of evidence suggest that this inhibition of recycling may result from the inhibition of tubule formation. First, BFA-stimulated endosome tubule formation was similarly inhibited by PLA(2) antagonists. Second, endocytosed tracers were found in larger spherical endosomes in the presence of PLA(2) antagonists. And third, endosome tubule formation in a cell-free, cytosol-dependent reconstitution system was equally sensitive PLA(2) antagonists. These results are consistent with the conclusion that endosome membrane tubules are formed by the action of a cytoplasmic PLA(2) and that PLA(2)-dependent tubules are involved in intracellular recycling of Tf and TfR. When taken together with previous studies on the Golgi complex, these results also indicate that an intracellular PLA(2) activity provides a novel molecular mechanism for inducing tubule formation from multiple organelles.     

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.     

Fission of tubular endosomes triggers endosomal acidification and movement

The early endosome acts as a sorting station for internalized molecules destined for recycling or degradation. While recycled molecules are sorted and delivered to tubular endosomes, residual compartments containing molecules to be degraded undergo "maturation" before final degradation in the lysosome. This maturation involves acidification, microtubule-dependent motility, and perinuclear localization. It is currently unknown how sorting and the processes of maturation cooperate with each other. Here, we show that fission of a tubular endosome triggers the maturation of the residual endosome, leading to degradation. Use of the dynamin inhibitor dynasore to block tubular endosome fission inhibited acidification, endosomal motility along microtubules, perinuclear localization, and degradation. However, tubular endosome fission was not affected by inhibiting endosomal acidification or by depolymerizing the microtubules. These results demonstrate that the fission of recycling tubules is the first important step in endosomal maturation and degradation in the lysosome. We believe this to be the first evidence of a cascade from sorting to degradation.     

Arf6-independent GPI-anchored protein-enriched early endosomal compartments fuse with sorting endosomes via a Rab5/phosphatidylinositol-3'-kinase-dependent machinery

In the process of internalization of molecules from the extracellular milieu, a cell uses multiple endocytic pathways, consequently generating different endocytic vesicles. These primary endocytic vesicles are targeted to specific destinations inside the cell. Here, we show that GPI-anchored proteins are internalized by an Arf6-independent mechanism into GPI-anchored protein-enriched early endosomal compartments (GEECs). Internalized GPI-anchored proteins and the fluid phase are first visualized in GEECs that are acidic, primary endocytic structures, negative for early endosomal markers, Rab4, Rab5, and early endosome antigen (EEA)1. They subsequently acquire Rab5 and EEA1 before homotypic fusion with other GEECs, and heterotypic fusion with endosomes containing cargo from the clathrin-dependent endocytic pathway. Although, the formation of GEECs is unaffected by inhibition of Rab5 GTPase and phosphatidylinositol-3'-kinase (PI3K) activity, their fusion with sorting endosomes is dependent on both activities. Overexpression of Rab5 reverts PI3K inhibition of fusion, providing evidence that Rab5 effectors play important roles in heterotypic fusion between the dynamin-independent GEECs and clathrin- and dynamin-dependent sorting endosomes.     

Rab4 affects both recycling and degradative endosomal trafficking

The small GTPases Rab4, Rab5 and Rab7 are endosomal proteins which play important roles in the regulation of various stages of endosomal trafficking. Rab4 and Rab5 have both been localized to early endosomes and have been shown to control recycling and endosomal fusion, respectively. Rab7, a marker of the late endosomal compartment, is involved in the regulation of the late endocytic pathway. Here, we compare the role of Rab4, Rab5 and Rab7 in early and late endosomal trafficking in HeLa cells monitoring ligand uptake, recycling and degradation. Expression of the Rab4 dominant negative mutant (Rab4AS22N) leads to a significant reduction in both recycling and degradation while, as expected, Rab7 mutants exclusively affect epidermal growth factor (EGF) and low density lipoprotein degradation. As also expected, expression of the dominant negative Rab5 mutant perturbs internalization kinetics and affects both recycling and degradation. Expression of Rab4WT and dominant positive mutant (Rab4AQ67L) changes dramatically the morphology of the transferrin compartment leading to the formation of membrane tubules. These transferrin positive tubules display swellings (varicosities) some of which are positive for early endosomal antigen-1 and contain EGF. We propose that the Rab4GTPase is important for the function of the early sorting endosomal compartment, affecting trafficking along both recycling and degradative pathways.     

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 .      

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.     

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.