The Atg6/Vps30/Beclin 1 ortholog BEC-1 mediates endocytic retrograde transport in addition to autophagy in C. elegans

Autophagy and endocytosis are dynamic and tightly regulated processes that contribute to many fundamental aspects of biology including survival, longevity, and development. However, the molecular links between autophagy and endocytosis are not well understood. Here, we report that BEC-1, the C. elegans ortholog of Atg6/Vps30/Beclin1, a key regulator of the autophagic machinery, also contributes to endosome function. In particular we identify a defect in retrograde transport from endosomes to the Golgi in bec-1 mutants. MIG-14/Wntless is normally recycled from endosomes to the Golgi through the action of the retromer complex and its associated factor RME-8. Lack of retromer or RME-8 activity results in the aberrant transport of MIG-14/Wntless to the lysosome where it is degraded. Similarly, we find that lack of bec-1 also results in mislocalization and degradation of MIG-14::GFP, reduced levels of RME-8 on endosomal membranes, and the accumulation of morphologically abnormal endosomes. A similar phenotype was observed in animals treated with dsRNA against vps-34. We further identify a requirement for BEC-1 in the clearance of apoptotic corpses in the hermaphrodite gonad, suggesting a role for BEC-1 in phagosome maturation, a process that appears to depend upon retrograde transport. In addition, autophagy genes may also be required for cell corpse clearance, as we find that RNAi against atg-18 or unc-51 also results in a lack of cell corpse clearance.     

C. elegans AP-2 and retromer control Wnt signaling by regulating mig-14/Wntless

While endocytosis can regulate morphogen distribution, its precise role in shaping these gradients is unclear. Even more enigmatic is the role of retromer, a complex that shuttles proteins between endosomes and the Golgi apparatus, in Wnt gradient formation. Here we report that DPY-23, the C. elegans mu subunit of the clathrin adaptor AP-2 that mediates the endocytosis of membrane proteins, regulates Wnt function. dpy-23 mutants display Wnt phenotypes, including defects in neuronal migration, neuronal polarity, and asymmetric cell division. DPY-23 acts in Wnt-expressing cells to promote these processes. MIG-14, the C. elegans homolog of the Wnt-secretion factor Wntless, also acts in these cells to control Wnt function. In dpy-23 mutants, MIG-14 accumulates at or near the plasma membrane. By contrast, MIG-14 accumulates in intracellular compartments in retromer mutants. Based on our observations, we propose that intracellular trafficking of MIG-14 by AP-2 and retromer plays an important role in Wnt secretion.     

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.     

Serum- and Glucocorticoid-Inducible Kinase-1 (SGK-1) Plays a Role in Membrane Trafficking in Caenorhabditis elegans

The mammalian serum- and glucocorticoid-inducible kinase SGK1 regulates the endocytosis of ion channels. Here we report that in C. elegans sgk-1 null mutants, GFP-tagged MIG-14/Wntless, the sorting receptor of Wnt, failed to localize to the basolateral membrane of intestinal cells; instead, it was mis-sorted to lysosomes. This effect can be explained in part by altered sphingolipid levels, because reducing glucosylceramide biosynthesis restored the localization of MIG-14::GFP. Membrane traffic was not perturbed in general, as no obvious morphological defects were detected for early endosomes, the Golgi apparatus, and the endoplasmic reticulum (ER) in sgk-1 null animals. The recycling of MIG-14/Wntless through the Golgi might be partially responsible for the observed phenotype because the subcellular distribution of two plasma membrane cargoes that do not recycle through the trans-Golgi network (TGN) was affected to a lesser degree. Consistently, knockdown of the ArfGEF gbf-1 altered the distribution of SGK-1 at the basolateral membrane of intestinal cells. In addition, we found that sgk-1(RNAi) induced unfolded protein response in the ER, suggesting at least an indirect role of SGK-1 early in the secretory pathway. We propose that SGK-1 function is required for lipid homeostasis and that it acts at different intracellular trafficking steps.     

Receptor-mediated Endocytosis 8 Utilizes an N-terminal Phosphoinositide-binding Motif to Regulate Endosomal Clathrin Dynamics

Receptor-mediated endocytosis 8 (RME-8) is a DnaJ domain containing protein implicated in translocation of Hsc70 to early endosomes for clathrin removal during retrograde transport. Previously, we have demonstrated that RME-8 associates with early endosomes in a phosphatidylinositol 3-phosphate (PI(3)P)-dependent fashion. In this study, we have now identified amino acid determinants required for PI(3)P binding within a region predicted to adopt a pleckstrin homology-like fold in the N terminus of RME-8. The ability of RME-8 to associate with PI(3)P and early endosomes is largely abolished when residues Lys¹⁷, Trp²⁰, Tyr²⁴, or Arg²⁶ are mutated resulting in diffuse cytoplasmic localization of RME-8 while maintaining the ability to interact with Hsc70. We also provide evidence that RME-8 PI(3)P binding regulates early endosomal clathrin dynamics and alters the steady state localization of the cation-independent mannose 6-phosphate receptor. Interestingly, RME-8 endosomal association is also regulated by the PI(3)P-binding protein SNX1, a member of the retromer complex. Wild type SNX1 restores endosomal localization of RME-8 W20A, whereas a SNX1 variant deficient in PI(3)P binding disrupts endosomal localization of wild type RME-8. These results further highlight the critical role for PI(3)P in the RME-8-mediated organizational control of various endosomal activities, including retrograde transport.     

Retromer retrieves wntless

Wntless is a sorting receptor required for Wnt secretion. Wntless is retrieved from endosomes to the Golgi by retromer, permitting Wntless reutilization in Wnt transport. In the absence of retromer, Wntless is degraded in lysosomes and Wnt secretion is impaired.     

Analysis of Articulation Between Clathrin and Retromer in Retrograde Sorting on Early Endosomes

Clathrin and retromer have key functions for retrograde trafficking between early endosomes and the trans -Golgi network (TGN). Previous studies on Shiga toxin suggested that these two coat complexes operate in a sequential manner. Here, we show that the curvature recognition subunit component sorting nexin 1 (SNX1) of retromer interacts with receptor-mediated endocytosis-8 (RME-8) protein, and that RME-8 and SNX1 colocalize on early endosomes together with a model cargo of the retrograde route, the receptor-binding B-subunit of Shiga toxin (STxB). RME-8 has previously been found to bind to the clathrin uncoating adenosine triphosphatase (ATPase) Hsc70, and we now report that depletion of RME-8 or Hsc70 affects retrograde trafficking at the early endosomes–TGN interface of STxB and the cation-independent mannose 6-phosphate receptor, an endogenous retrograde cargo protein. We also provide evidence that retromer interacts with the clathrin-binding protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) not only via SNX1, as previously published (Chin Raynor MC, Wei X, Chen HQ, Li L. Hrs interacts with sorting nexin 1 and regulates degradation of epidermal growth factor receptor. J Biol Chem 2001;276:7069–7078), but also via the core complex component Vps35. Hrs codistributes at the ultrastructural level with STxB on early endosomes, and interfering with Hrs function using antibodies or mild overexpression inhibits retrograde transport. Our combined data suggest a model according to which the functions in retrograde sorting on early endosomes of SNX1/retromer and clathrin are articulated by RME-8, and possibly also by Hrs.     

RAB-6.1 and RAB-6.2 Promote Retrograde Transport in C. elegans

Retrograde transport is a critical mechanism for recycling certain membrane cargo. Following endocytosis from the plasma membrane, retrograde cargo is moved from early endosomes to Golgi followed by transport (recycling) back to the plasma membrane. The complete molecular and cellular mechanisms of retrograde transport remain unclear. The small GTPase RAB-6.2 mediates the retrograde recycling of the AMPA-type glutamate receptor (AMPAR) subunit GLR-1 in C. elegans neurons. Here we show that RAB-6.2 and a close paralog, RAB-6.1, together regulate retrograde transport in both neurons and non-neuronal tissue. Mutants for rab-6.1 or rab-6.2 fail to recycle GLR-1 receptors, resulting in GLR-1 turnover and behavioral defects indicative of diminished GLR-1 function. Loss of both rab-6.1 and rab-6.2 results in an additive effect on GLR-1 retrograde recycling, indicating that these two C. elegans Rab6 isoforms have overlapping functions. MIG-14 (Wntless) protein, which undergoes retrograde recycling, undergoes a similar degradation in intestinal epithelia in both rab-6.1 and rab-6.2 mutants, suggesting a broader role for these proteins in retrograde transport. Surprisingly, MIG-14 is localized to separate, spatially segregated endosomal compartments in rab-6.1 mutants compared to rab-6.2 mutants. Our results indicate that RAB-6.1 and RAB-6.2 have partially redundant functions in overall retrograde transport, but also have their own unique cellular- and subcellular functions.     

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.     

Wnt signaling requires retromer-dependent recycling of MIG-14/Wntless in Wnt-producing cells

Wnt proteins are secreted signaling molecules that play a central role in development and adult tissue homeostasis. We have previously shown that Wnt signaling requires retromer function in Wnt-producing cells. The retromer is a multiprotein complex that mediates endosome-to-Golgi transport of specific sorting receptors. MIG-14/Wls is a conserved transmembrane protein that binds Wnt and is required in Wnt-producing cells for Wnt secretion. Here, we demonstrate that in the absence of retromer function, MIG-14/Wls is degraded in lysosomes and becomes limiting for Wnt signaling. We show that retromer-dependent recycling of MIG-14/Wls is part of a trafficking pathway that retrieves MIG-14/Wls from the plasma membrane. We propose that MIG-14/Wls cycles between the Golgi and the plasma membrane to mediate Wnt secretion. Regulation of this transport pathway may enable Wnt-producing cells to control the range of Wnt signaling in the tissue.     

Inactivation of the autophagy gene bec-1 triggers apoptotic cell death in C. elegans

Programmed cell death (PCD) is an essential and highly orchestrated process that plays a major role in morphogenesis and tissue homeostasis during development. In humans, defects in regulation or execution of cell death lead to diabetes, neurodegenerative disorders, and cancer. Two major types of PCD have been distinguished: the caspase-mediated process of apoptosis and the caspase-independent process involving autophagy. Although apoptosis and autophagy are often activated together in response to stress, the molecular mechanisms underlying their interplay remain unclear. Here we show that BEC-1, the C. elegans ortholog of the yeast and mammalian autophagy proteins Atg6/Vps30 and Beclin 1, is essential for development. We demonstrate that BEC-1 is necessary for the function of the class III PI3 kinase LET-512/Vps34, an essential protein required for autophagy, membrane trafficking, and endocytosis. Furthermore, BEC-1 forms a complex with the antiapoptotic protein CED-9/Bcl-2, and its depletion triggers CED-3/Caspase-dependent PCD. Based on our results, we propose that bec-1 represents a link between autophagy and apoptosis, thus supporting the view that the two processes act in concerted manner in the cell death machinery.     

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.     

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.      

Wingless secretion requires endosome-to-Golgi retrieval of Wntless/Evi/Sprinter by the retromer complex

The glycolipoproteins of the Wnt family raise interesting trafficking issues, especially with respect to spreading within tissues. Recently, the retromer complex has been suggested to participate in packaging Wnts into long-range transport vehicles. Our analysis of a Drosophila mutant in Vps35 show that, instead, the retromer complex is required for efficient progression of Wingless (a Drosophila Wnt) through the secretory pathway. Indeed expression of senseless, a short-range target gene, is lost in Vps35-deficient imaginal discs. In contrast, Vps35 is not required for Hedgehog secretion, suggesting specificity. Overexpression of Wntless, a transmembrane protein known to be specifically required for Wingless secretion overcomes the secretion block of Vps35-mutant cells. Furthermore, biochemical evidence confirms that Wntless engages with the retromer complex. We propose that Wntless accompanies Wingless to the plasma membrane where the two proteins dissociate. Following dissociation from Wingless, Wntless is internalized and returns to the Golgi apparatus in a retromer-dependent manner. Without the retromer-dependent recycling route, Wingless secretion is impaired and, as electron microscopy suggests, Wntless is diverted to a degradative compartment.     

Protein kinase CK2 is required for Wntless internalization and Wnt secretion

Wnt proteins are lipid modified signaling molecules that have essential functions in development and adult tissue homeostasis. Secretion of Wnt is mediated by the transmembrane protein Wntless, which binds Wnt and transports it from the endoplasmic reticulum to the cell surface for release. To maintain efficient Wnt secretion, Wntless is recycled back to the Golgi and the endoplasmic reticulum through endocytosis and retromer dependent endosome to Golgi transport. We have previously identified protein kinase CK2 (CK2) in a genome-wide screen for regulators of Wnt signaling in Caenorhabditis elegans. Here, we show that CK2 function is required in Wnt producing cells for Wnt secretion. This function is evolutionarily conserved, as inhibition of CK2 activity interferes with Wnt5a secretion from mammalian cells. Mechanistically, we show that inhibition of CK2 function results in enhanced plasma membrane localization of Wls in C. elegans and mammalian cells, consistent with the notion that CK2 is involved in the regulation of Wls internalization.     

Retromer Ensures the Degradation of Autophagic Cargo by Maintaining Lysosome Function in Drosophila

The retromer is an evolutionarily conserved coat complex that consists of Vps26, Vps29, Vps35 and a heterodimer of sorting nexin (Snx) proteins in yeast. Retromer mediates the recycling of transmembrane proteins from endosomes to the trans‐Golgi network, including receptors that are essential for the delivery of hydrolytic enzymes to lysosomes. Besides its function in lysosomal enzyme receptor recycling, involvement of retromer has also been proposed in a variety of vesicular trafficking events, including early steps of autophagy and endocytosis. Here we show that the late stages of autophagy and endocytosis are impaired in Vps26 and Vps35 deficient Drosophila larval fat body cells, but formation of autophagosomes and endosomes is not compromised. Accumulation of aberrant autolysosomes and amphisomes in the absence of retromer function appears to be the consequence of decreased degradative capacity, as they contain undigested cytoplasmic material. Accordingly, we show that retromer is required for proper cathepsin L trafficking mainly independent of LERP, the Drosophila homolog of the cation‐independent mannose 6‐phosphate receptor. Finally, we find that Snx3 and Snx6 are also required for proper autolysosomal degradation in Drosophila larval fat body cells.      

The long and the short of Wnt signaling in C. elegans

The simplicity of C. elegans makes it an outstanding system to study the role of Wnt signaling in development. Many asymmetric cell divisions in C. elegans require the Wnt/beta-catenin asymmetry pathway. Recent studies confirm that SYS-1 is a structurally and functionally divergent beta-catenin, and implicate lipids and retrograde trafficking in maintenance of WRM-1/beta-catenin asymmetry. Wnts also regulate short-range events such as spindle rotation and gastrulation, and a PCP-like pathway regulates asymmetric divisions. Long-range, cell non-autonomous Wnt signals regulate vulval induction. Both short-range and long-range Wnt signal s are regulated by recycling of MIG-14/Wntless via the retromer complex. These studies indicate that C. elegans continues to be useful for identifying new, conserved mechanisms underlying Wnt signaling in metazoans.     

A novel requirement for C. elegans Alix/ALX-1 in RME-1-mediated membrane transport

BACKGROUND: Alix/Bro1p family proteins have recently been identified as important components of multivesicular endosomes (MVEs) and are involved in the sorting of endocytosed integral membrane proteins, interacting with components of the ESCRT complex, the unconventional phospholipid LBPA, and other known endocytosis regulators. During infection, Alix can be co-opted by enveloped retroviruses, including HIV, providing an important function during virus budding from the plasma membrane. In addition, Alix is associated with the actin cytoskeleton and might regulate cytoskeletal dynamics. RESULTS: Here we demonstrate a novel physical interaction between the only apparent Alix/Bro1p family protein in C. elegans, ALX-1, and a key regulator of receptor recycling from endosomes to the plasma membrane, called RME-1. The analysis of alx-1 mutants indicates that ALX-1 is required for the endocytic recycling of specific basolateral cargo in the C. elegans intestine, a pathway previously defined by the analysis of rme-1 mutants. The expression of truncated human Alix in HeLa cells disrupts the recycling of major histocompatibility complex class I, a known Ehd1/RME-1-dependent transport step, suggesting the phylogenetic conservation of this function. We show that the interaction of ALX-1 with RME-1 in C. elegans, mediated by RME-1/YPSL and ALX-1/NPF motifs, is required for this recycling process. In the C. elegans intestine, ALX-1 localizes to both recycling endosomes and MVEs, but the ALX-1/RME-1 interaction appears to be dispensable for ALX-1 function in MVEs and/or late endosomes. CONCLUSIONS: This work provides the first demonstration of a requirement for an Alix/Bro1p family member in the endocytic recycling pathway in association with the recycling regulator RME-1.     

Autophagy protects against hypoxic injury in C. elegans

Macroautophagy has been implicated in a variety of pathological processes. Hypoxic/ischemic cellular injury is one such process in which autophagy has emerged as an important regulator. In general, autophagy is induced after an hypoxic/ischemic insult; however, whether the induction of autophagy promotes cell death or recovery is controversial and appears to be context dependent. We have developed C. elegans as a genetically tractable model for the study of hypoxic cell injury. Both necrosis and apoptosis are mechanisms of cell death following hypoxia in C. elegans. However, the role of autophagy in hypoxic injury in C. elegans has not been examined. Here, we found that RNAi knockdown of the C. elegans homologs of beclin 1/Atg6 (bec-1) and LC3/Atg8 (lgg-1, lgg-2), and mutation of Atg1 (unc-51) decreased animal survival after a severe hypoxic insult. Acute inhibition of autophagy by the type III phosphatidylinositol 3-kinase inhibitors, 3-methyladenine and Wortmannin, also sensitized animals to hypoxic death. Hypoxia-induced neuronal and myocyte injury as well as necrotic cellular morphology were increased by RNAi knockdown of BEC-1. Hypoxia increased the expression of a marker of autophagosomes in a bec-1-dependent manner. Finally, we found that the hypoxia hypersensitive phenotype of bec-1(RNAi) animals could be blocked by loss-of-function mutations in either the apoptosis or necrosis pathway. These results argue that inhibition of autophagy sensitizes C. elegans and its cells to hypoxic injury and that this sensitization is blocked or circumvented when either of the two major cell death mechanisms is inhibited.     

Retromer Dependent Recycling of the Wnt Secretion Factor Wls Is Dispensable for Stem Cell Maintenance in the Mammalian Intestinal Epithelium

In C. elegans and Drosophila, retromer mediated retrograde transport of Wntless (Wls) from endosomes to the trans-Golgi network (TGN) is required for Wnt secretion. When this retrograde transport pathway is blocked, Wls is missorted to lysosomes and degraded, resulting in reduced Wnt secretion and various Wnt related phenotypes. In the mammalian intestine, Wnt signaling is essential to maintain stem cells. This prompted us to ask if retromer mediated Wls recycling is also important for Wnt signaling and stem cell maintenance in this system. To answer this question, we generated a conditional Vps35 ^fl allele. As Vps35 is an essential subunit of the retromer complex, this genetic tool allowed us to inducibly interfere with retromer function in the intestinal epithelium. Using a pan-intestinal epithelial Cre line (Villin-CreERT2), we did not observe defects in crypt or villus morphology after deletion of Vps35 from the intestinal epithelium. Wnt secreted from the mesenchyme of the intestine may compensate for a reduction in epithelial Wnt secretion. To exclude the effect of the mesenchyme, we generated intestinal organoid cultures. Loss of Vps35 in intestinal organoids did not affect the overall morphology of the organoids. We were able to culture Vps35 ^∆/∆ organoids for many passages without Wnt supplementation in the growth medium. However, Wls protein levels were reduced and we observed a subtle growth defect in the Vps35 ^∆/∆ organoids. These results confirm the role of retromer in the retrograde trafficking of Wls in the intestine, but show that retromer mediated Wls recycling is not essential to maintain Wnt signaling or stem cell proliferation in the intestinal epithelium.