The two C. elegans ATG-16 homologs have partially redundant functions in the basal autophagy pathway

The presence of multiple homologs of the same yeast ATG genes endows an extra layer of complexity on the autophagic machinery in higher eukaryotes. The physiological function of individual homologs in the autophagy pathway remains poorly understood. Here we characterized the function of the two atg16 homologs, atg-16.1 and atg-16.2, in the autophagy pathway in C. elegans. We showed that atg-16.2 mutants exhibit a stronger autophagic defect than atg-16.1 mutants. atg-16.2; atg-16.1 double mutants display a much more severe defect than either single mutant. ATG-16.1 and ATG-16.2 interact with themselves and each other and also directly associate with ATG-5. atg-16.1 mutant embryos exhibit a wild-type expression and distribution pattern of LGG-1/Atg8, while LGG-1 puncta are markedly fewer in number and weaker in intensity in atg-16.2 mutants. In atg-16.2; atg-16.1 double mutants, the lipidated form of LGG-1 accumulates, but LGG-1 puncta are completely absent. ATG-16.2 ectopically expressed on the plasma membrane provides novel sites of LGG-1 puncta formation. We also demonstrated that the C-terminal WD repeats are dispensable for the role of atg-16.2 in aggrephagy (the degradation of protein aggregates by autophagy). Genetic epistasis analysis placed atg-16.2 upstream of atg-2, epg-6, and atg-18. Our study indicated that C. elegans ATG-16s are involved in specifying LGG-1 puncta formation and the two ATG-16 homologs have partially redundant yet distinct functions in the aggrephagy pathway.     

Autophagy genes promote apoptotic cell corpse clearance

Autophagy is a catabolic process through which damaged organelles and protein aggregates are delivered to lysosomes for degradation. Autophagy genes are reported to promote exposure of “eat me” signals on the surface of apoptotic cells, but whether they function in engulfing cells is not clear. Recently, we found that the autophagy mutants atg-18 and epg-5 are defective in removing apoptotic cells derived from the C. elegans Q neuroblast, a phenotype that can be fully rescued by expression of ATG-18 and EPG-5 in the engulfing cell. Loss of ATG-18 or EPG-5 does not affect cell corpse engulfment but causes defects in phagosomal recruitment of RAB-5 and RAB-7 and formation of phagolysosomes. EPG-5, ATG-18 and LGG-1 are sequentially recruited to phagosomes, suggesting that they function at different steps of phagosomal maturation. Our studies indicate that autophagy genes function sequentially to promote apoptotic cell corpse degradation in the engulfing cell.     

Human GABARAP can restore autophagosome biogenesis in a C. elegans lgg-1 mutant

We recently described in C. elegans embryos, the acquisition of specialized functions for orthologs of yeast Atg8 (e.g., mammalian MAP1LC3/LC3) in allophagy, a selective and developmentally regulated autophagic process. During the formation of double-membrane autophagosomes, the ubiquitin-like Atg8/LC3 proteins are recruited to the membrane through a lipidation process. While at least 6 orthologs and paralogs are present in mammals, C. elegans only possesses 2 orthologs, LGG-1 and LGG-2, corresponding to the GABARAP-GABARAPL2/GATE-16 and the MAP1LC3 families, respectively. During allophagy, LGG-1 acts upstream of LGG-2 and is essential for autophagosome biogenesis, whereas LGG-2 facilitates their maturation. We demonstrated that LGG-2 directly interacts with the HOPS complex subunit VPS-39, and mediates the tethering between autophagosomes and lysosomes, which also requires RAB-7. In the present addendum, we compared the localization of autophagosomes, endosomes, amphisomes, and lysosomes in vps-39, rab-7, and lgg-2 depleted embryos. Our results suggest that lysosomes interact with autophagosomes or endosomes through a similar mechanism. We also performed a functional complementation of an lgg-1 null mutant with human GABARAP, its closer homolog, and showed that it localizes to autophagosomes and can rescue LGG-1 functions in the early embryo.     

The WD40 repeat PtdIns(3)P-binding protein EPG-6 regulates progression of omegasomes to autophagosomes

PtdIns(3)P plays critical roles in the autophagy pathway. However, little is known about how PtdIns(3)P effectors act with autophagy proteins in autophagosome formation. Here we identified an essential autophagy gene in C.?elegans, epg-6, which encodes a WD40 repeat-containing protein with PtdIns(3)P-binding activity. EPG-6 directly interacts with ATG-2. epg-6 and atg-2 regulate progression of omegasomes to autophagosomes, and their loss of function?causes accumulation of enlarged early autophagic structures. Another WD40 repeat PtdIns(3)P effector, ATG-18, plays a distinct role in autophagosome formation. We also established the hierarchical relationship of autophagy genes in degradation of?protein aggregates and revealed that the UNC-51/Atg1 complex, EPG-8/Atg14, and binding of lipidated LGG-1 to protein aggregates are required for?omegasome formation. Our study demonstrates that autophagic PtdIns(3)P effectors play distinct roles in autophagosome formation and also provides?a framework for understanding the concerted action of autophagy genes in protein aggregate degradation.     

Differential Function of the Two Atg4 Homologues in the Aggrephagy Pathway in Caenorhabditis elegans

The presence of multiple homologues of the same yeast Atg protein endows an additional layer of complexity on the autophagy pathway in higher eukaryotes. The physiological function of the individual genes, however, remains largely unknown. Here we investigated the role of the two Caenorhabditis elegans homologues of the cysteine protease Atg4 in the pathway responsible for degradation of protein aggregates. Loss of atg-4.1 activity causes defective degradation of a variety of protein aggregates, whereas atg-4.2 mutants remove these substrates normally. LGG-1 precursors accumulate in atg-4.1 mutants, but not atg-4.2 mutants. LGG-1 puncta, formation of which depends on lipidation of LGG-1, are present in atg-4.1 and atg-4.2 single mutants, but are completely absent in atg-4.1; atg-4.2 double mutants. In vitro enzymatic analysis revealed that ATG-4.1 processes LGG-1 precursors about 100-fold more efficiently than ATG-4.2. Expression of a mutant form LGG-1, which mimics the processed precursor, rescues the defective autophagic degradation of protein aggregates in atg-4.1 mutants and, to a lesser extent, in atg-4.1; atg-4.2 double mutants. Our study reveals that ATG-4.1 and ATG-4.2 are functionally redundant yet display differential LGG-1 processing and deconjugating activity in the aggrephagy pathway in C. elegans.     

Human GABARAP can restore autophagosome biogenesis in a C. elegans lgg-1 mutant

We recently described in C. elegans embryos, the acquisition of specialized functions for orthologs of yeast Atg8 (e.g., mammalian MAP1LC3/LC3) in allophagy, a selective and developmentally regulated autophagic process. During the formation of double-membrane autophagosomes, the ubiquitin-like Atg8/LC3 proteins are recruited to the membrane through a lipidation process. While at least 6 orthologs and paralogs are present in mammals, C. elegans only possesses 2 orthologs, LGG-1 and LGG-2, corresponding to the GABARAP-GABARAPL2/GATE-16 and the MAP1LC3 families, respectively. During allophagy, LGG-1 acts upstream of LGG-2 and is essential for autophagosome biogenesis, whereas LGG-2 facilitates their maturation. We demonstrated that LGG-2 directly interacts with the HOPS complex subunit VPS-39, and mediates the tethering between autophagosomes and lysosomes, which also requires RAB-7. In the present addendum, we compared the localization of autophagosomes, endosomes, amphisomes, and lysosomes in vps-39, rab-7, and lgg-2 depleted embryos. Our results suggest that lysosomes interact with autophagosomes or endosomes through a similar mechanism. We also performed a functional complementation of an lgg-1 null mutant with human GABARAP, its closer homolog, and showed that it localizes to autophagosomes and can rescue LGG-1 functions in the early embryo.     

The scaffold protein EPG-7 links cargo–receptor complexes with the autophagic assembly machinery

The mechanism by which protein aggregates are selectively degraded by autophagy is poorly understood. Previous studies show that a family of Atg8-interacting proteins function as receptors linking specific cargoes to the autophagic machinery. Here we demonstrate that during Caenorhabditis elegans embryogenesis, epg-7 functions as a scaffold protein mediating autophagic degradation of several protein aggregates, including aggregates of the p62 homologue SQST-1, but has little effect on other autophagy-regulated processes. EPG-7 self-oligomerizes and is degraded by autophagy independently of SQST-1. SQST-1 directly interacts with EPG-7 and colocalizes with EPG-7 aggregates in autophagy mutants. Mutations in epg-7 impair association of SQST-1 aggregates with LGG-1/Atg8 puncta. EPG-7 interacts with multiple ATG proteins and colocalizes with ATG-9 puncta in various autophagy mutants. Unlike core autophagy genes, epg-7 is dispensable for starvation-induced autophagic degradation of substrate aggregates. Our results indicate that under physiological conditions a scaffold protein endows cargo specificity and also elevates degradation efficiency by linking the cargo–receptor complex with the autophagic machinery.     

Nutrient-driven O-GlcNAc cycling influences autophagic flux and neurodegenerative proteotoxicity

O-GlcNAcylation is an abundant post-translational modification implicated in human neurodegenerative diseases. We showed that loss-of-function of OGT (O-linked GlcNAc transferase) alleviated, while loss of OGA (O-GlcNAc selective β-N-acetyl-D-glucosaminidase) enhanced, the proteotoxicity of C. elegans neurodegenerative disease models including tauopathy, β-amyloid peptide and polyglutamine expansion. The O-GlcNAc cycling mutants act, in part, by altering insulin signaling, proteasome activity and autophagy. In mutants lacking either of these enzymes of O-GlcNAc cycling, there is a striking accumulation of GFP::LGG-1 (C. elegans homolog of Atg8 and LC3) and increased phosphatidylethanolamine (PE)-modified GFP::LGG-1 upon starvation. We speculate that O-GlcNAc cycling is a key nutrient-responsive regulator of autophagic flux acting at multiple levels including direct modification of BECN1 and BCL2.     

Small differences make a big impact: Structural insights into the differential function of the 2 Atg8 homologs in C. elegans

The 2 C. elegans homologs of Atg8, LGG-1 and LGG-2, show differential function in the degradation of protein aggregates during embryogenesis. LGG-1 is essential for the degradation of various protein aggregates, while LGG-2 has cargo-specific and developmental stage-specific roles. LGG-1 and LGG-2 differentially interact with autophagy substrates and ATG proteins. LGG-1 and LGG-2 possess 2 hydrophobic pockets, the W-site and the L-site, which recognize the LIR motif in Atg8-binding proteins. The plasticity of the W-site and the size and shape of the L-site differ between LGG-1 and LGG-2, thus determining their preferences for distinct LIR motifs. The N-terminal tails of LGG-1 and LGG-2 adopt unique closed and open conformations, respectively, which may result in distinct membrane tethering and fusion activities. LGG-1 and LGG-2 have different affinities for ATG-7 and ATG-3, and lipidation of LGG-2 is regulated by levels of lipidated LGG-1. Taken together, the structural differences between LGG-1 and LGG-2 provide insights into their differential functions in the aggrephagy pathway.     

Age-associated and tissue-specific decline in autophagic activity in the nematode C. elegans

Macroautophagy/autophagy is a cellular recycling process that is required for the extended life span observed in many longevity paradigms, including in the nematode C. elegans. However, little is known regarding the spatiotemporal changes in autophagic activity in such long-lived mutants as well as in wild-type animals during normal aging. In a recent study, we report that autophagic activity decreases with age in several major tissues of wild-type C. elegans, including the intestine, body-wall muscle, pharynx, and nerve-ring neurons. Moreover, long-lived daf-2/insulin-signaling mutants and glp-1/Notch receptor mutants display increased autophagic activity, yet with different time- and tissue-specific differences. Notably, the intestine appears to be a critical tissue in which autophagy contributes to longevity in glp-1, but not in daf-2 mutants. Our findings indicate that autophagic degradation is reduced with age, possibly with distinct kinetics in different tissues, and that long-lived mutants increase autophagy in a tissue-specific manner, resulting in increased life span.     

Decreased Insulin-Receptor Signaling Promotes the Autophagic Degradation of β-Amyloid Peptide in C. elegans

Autophagy is a conserved membrane trafficking pathway that mediates the delivery of cytoplasmic substrates to the lysosome for degradation. Impaired autophagic function is implicated in the pathology of various neurodegenerative diseases. We have generated transgenic C. elegans that express human β-amyloid peptide (Aβ) in order to examine the mechanism(s) of Aβ-toxicity. In this model, Aβ expression causes autophagosome accumulation, thereby mimicking a pathology found in brains of Alzheimer’s disease patients. Furthermore, we demonstrate that decreased insulin-receptor signaling [using the daf-2(e1370) mutation] suppresses Aβ-induced paralysis by a mechanism that requires autophagy. Surprisingly, the daf-2 mutation also decreases Aβ-induced autophagosome accumulation. These observations can be explained by a model in which decreased insulin-receptor signaling promotes the maturation of autophagosomes into degradative autolysosomes, whereas Aβ impairs this process. Consistent with this model, we find that RNAi-mediated knock-down of lysosomal components results in enhanced Aβ-toxicity and autophagosome accumulation. Also, Aβ; daf-2(e1370) nematodes contain more lysosomes than either Aβ or control strains. Finally, we demonstrate that decreased insulin-receptor signaling promotes the autophagic degradation of Aβ.     

The autophagosomal protein LGG-2 acts synergistically with LGG-1 in dauer formation and longevity in C. elegans

Autophagy has an important function in degrading cytoplasmic components to maintain cellular homeostasis, but is also required during development. The formation of the autophagic vesicles requires the recruitment of the Atg8 ubiquitin-like proteins to the membrane of the nascent autophagosomes. Atg8 is a highly conserved gene which has been duplicated during metazoan evolution. In this report we have investigated, in the nematode C. elegans, the functions and localizations of the two Atg8p homologues LGG-2 and LGG-1. Phylogenetic analyses suggest that LGG-2 is more closely related to the human protein LC3 than LGG-1. LGG-1 but not LGG-2 is able to functionally complement the atg8 mutant yeast. The C-terminal glycine residue of LGG-2 is essential for post-translational modification and localization to the autophagosomes. During C. elegans development the two proteins share a similar expression pattern and localization but LGG-2 is more abundant in the neurons. Using genetic tools to either reduce or increase the autophagic flux we show that both LGG-2 and LGG-1 are addressed to the autophagosomal/lysosomal degradative system. We also demonstrate that the localization of both proteins is modified in several physiological processes when autophagy is induced, namely during diapause “dauer” larval formation, starvation and aging. Finally, we demonstrate that both LGG-2 and LGG-1 act synergistically and are involved in dauer formation and longevity of the worm.     

Myostatin induces autophagy in skeletal muscle in vitro

Myostatin is an important regulator of muscle mass that contributes to the loss of muscle mass in a number of chronic diseases. Myostatin is known to activate the expression of components of the ubiquitin-proteosomal pathway but its effect on the autophagic pathway is not known. We therefore analysed the effect of myostatin and TGF-β on autophagy in C2C12 cells by determining the effect of these proteins on LC3 processing, autophagosome formation and autophagy gene expression. Both myostatin and TGF-β increased LC3II expression and turnover as well as autophagosome formation (marked by the formation of puncta in LC3-GFP transfected cells). Myostatin also significantly increased the expression of ATG-4B and ULK-2 mRNA while TGF-β caused a trend towards an increase in these genes. We conclude that myostatin and TGF-β increase autophagy in skeletal muscle cells.     

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.     

Highlights from this issue

Germline P granules are specialized protein/RNA aggregates that are found exclusively in germ cells in C. elegans. During the early embryonic divisions that generate germ blastomeres, aggregate-prone P granule components PGL-1 and PGL-3 that remain in the cytoplasm destined for somatic daughters are selectively removed by autophagy. Loss-of-function of components of the autophagy pathway, including the VPS-34/BEC-1 complex, causes accumulation of PGL-1 and PGL-3 into aggregates in somatic cells (termed PGL granules). Formation of PGL granules depends on SEPA-1, which is an integral component of these granules. SEPA-1 is preferentially degraded by autophagy and is also required for the autophagic degradation of PGL-1 and PGL-3. SEPA-1 functions as a bridging molecule in mediating degradation of P granule components by directly interacting with PGL-3 and also with the autophagy protein LGG-1/Atg8. The defect in embryonic development in autophagy mutants is suppressed by mutation of sepa-1, suggesting that autophagic degradation of PGL granule components may provide nutrients for embryogenesis and/or also prevent the formation of aggregates that could be toxic for animal development. Our study reveals a specific physiological function of selective autophagic degradation during C. elegans development.     

The C. elegans ATG101 homolog EPG-9 directly interacts with EPG-1/Atg13 and is essential for autophagy

Autophagy is an evolutionarily conserved catabolic process that involves the engulfment of cytoplasmic contents in a closed double-membrane structure, called the autophagosome, and their subsequent delivery to the vacuole/lysosomes for degradation. Genetic screens in Saccharomyces cerevisiae have identified more than 30 autophagy-related (Atg) genes that are essential for autophagosome formation. Here we isolated a novel autophagy gene, epg-9, whose loss of function causes defective autophagic degradation of a variety of protein aggregates during C. elegans embryogenesis. Mutations in epg-9 also reduce survival of animals under food depletion conditions. epg-9 mutants exhibit autophagy phenotypes characteristic of those associated with loss of function of unc-51/Atg1 and epg-1/Atg13. epg-9 encodes a protein with significant homology to mammalian ATG101. EPG-9 directly interacts with EPG-1/Atg13. Our study indicates that EPG-9 forms a complex with EPG-1 in the aggrephagy pathway in C. elegans.     

Autophagy in Saccharomyces cerevisiae requires the monomeric GTP-binding proteins,Arl1 and Ypt6

Macroautophagy/autophagy is a cellular degradation process that sequesters organelles or proteins into a double-membrane structure called the phagophore; this transient compartment matures into an autophagosome, which then fuses with the lysosome or vacuole to allow hydrolysis of the cargo. Factors that control membrane traffic are also essential for each step of autophagy. Here we demonstrate that 2 monomeric GTP-binding proteins in Saccharomyces cerevisiae, Arl1 and Ypt6, which belong to the Arf/Arl/Sar protein family and the Rab family, respectively, and control endosome-trans-Golgi traffic, are also necessary for starvation-induced autophagy under high temperature stress. Using established autophagy-specific assays we found that cells lacking either ARL1 or YPT6, which exhibit synthetic lethality with one another, were unable to undergo autophagy at an elevated temperature, although autophagy proceeds normally at normal growth temperature; specifically, strains lacking one or the other of these genes are unable to construct the autophagosome because these 2 proteins are required for proper traffic of Atg9 to the phagophore assembly site (PAS) at the restrictive temperature. Using degron technology to construct an inducible arl1Δ ypt6Δ double mutant, we demonstrated that cells lacking both genes show defects in starvation-inducted autophagy at the permissive temperature. We also found Arl1 and Ypt6 participate in autophagy by targeting the Golgi-associated retrograde protein (GARP) complex to the PAS to regulate the anterograde trafficking of Atg9. Our data show that these 2 membrane traffic regulators have novel roles in autophagy.     

Autophagy genes function sequentially to promote apoptotic cell corpse degradation in the engulfing cell

Apoptotic cell degradation is a fundamental process for organism development, and impaired clearance causes inflammatory or autoimmune disease. Although autophagy genes were reported to be essential for exposing the engulfment signal on apoptotic cells, their roles in phagocytes for apoptotic cell removal are not well understood. In this paper, we develop live-cell imaging techniques to study apoptotic cell clearance in the Caenorhabditis elegans Q neuroblast lineage. We show that the autophagy proteins LGG-1/LC3, ATG-18, and EPG-5 were sequentially recruited to internalized apoptotic Q cells in the phagocyte. In atg-18 or epg-5 mutants, apoptotic Q cells were internalized but not properly degraded; this phenotype was fully rescued by the expression of autophagy genes in the phagocyte. Time-lapse analysis of autophagy mutants revealed that recruitment of the small guanosine triphosphatases RAB-5 and RAB-7 to the phagosome and the formation of phagolysosome were all significantly delayed. Thus, autophagy genes act within the phagocyte to promote apoptotic cell degradation.     

The tissue- and developmental stage-specific involvement of autophagy genes in aggrephagy

ABSTRACT

Genetic screens have identified two sets of genes that act at distinct steps of basal autophagy in higher eukaryotes: the pan-eukaryotic ATG genes and the metazoan-specific EPG genes. Very little is known about whether these core macroautophagy/autophagy genes are differentially employed during multicellular organism development. Here we analyzed the function of core autophagy genes in autophagic removal of SQST-1/SQSTM1 during C. elegans development. We found that loss of function of genes acting at distinct steps in the autophagy pathway causes different patterns of SQST-1 accumulation in different tissues and developmental stages. We also identified that the calpain protease clp-2 acts in a cell context-specific manner in SQST-1 degradation. clp-2 is required for degradation of SQST-1 in the hypodermis and neurons, but is dispensable in the body wall muscle and intestine. Our results indicate that autophagy genes are differentially employed in a tissue- and stage-specific manner during the development of multicellular organisms.

Abbreviations: ATG: autophagy related; CLP: calpain family; EPG: ectopic PGL granules; ER: endoplasmic reticulum; ESCRT: endosomal sorting complex required for transport; GFP: green fluorescent protein; LGG-1/LC3: LC3, GABARAP and GATE-16 family; MIT: microtubule interacting and transport; PGL: P granule abnormality protein; SQST-1: sequestosome-related; UPS: ubiquitin-proteasome system