Atg8 Lipidation Research Articles

Biomolecular condensation of ERC1 recruits ATG8 and NBR1 to drive autophagosome formation for plant heat tolerance.

Macroautophagy (hereafter autophagy) is essential for cells to respond to nutrient stress by delivering cytosolic contents to vacuoles for degradation via the formation of a multi-layer vesicle named autophagosome. A set of autophagy-related (ATG) regulators are recruited to the phagophore assembly site for the initiation of phagophore, as well as its expansion and closure and subsequent delivery into the vacuole. However, it remains elusive that how the phagophore assembly is regulated under different stress conditions. Here, we described an unknown Arabidopsis (Arabidopsis thaliana) cytosolic ATG8-interaction protein family (ERC1/2), that binds ATG8 and NBR1 to promote autophagy. ERC1 proteins translocate to the phagophore membrane and develop into classical ring-like autophagosomes upon autophagic induction. However, ERC1 proteins form large droplets together with ATG8e proteins when in the absence of ATG8 lipidation activity. We described the property of these structures as phase-separated membraneless condensates by solving the in vivo organization with spatial and temporal resolution. Moreover, ERC1 condensates elicits a strong recruitment of the autophagic receptor NBR1. Loss of ERC1 suppressed NBR1 turnover and attenuated plant tolerance to heat stress condition. This work provides novel insights into the mechanical principle of phagophore initiation via an unreported ERC1-mediated biomolecular condensation for heat tolerance in Arabidopsis .

ATG7(2) Interacts With Metabolic Proteins and Regulates Central Energy Metabolism.

Macroautophagy/autophagy is an essential catabolic process that targets a wide variety of cellular components including proteins, organelles, and pathogens. ATG7, a protein involved in the autophagy process, plays a crucial role in maintaining cellular homeostasis and can contribute to the development of diseases such as cancer. ATG7 initiates autophagy by facilitating the lipidation of the ATG8 proteins in the growing autophagosome membrane. The noncanonical isoform ATG7(2) is unable to perform ATG8 lipidation; however, its cellular regulation and function are unknown. Here, we uncovered a distinct regulation and function of ATG7(2) in contrast with ATG7(1), the canonical isoform. First, affinity-purification mass spectrometry analysis revealed that ATG7(2) establishes direct protein-protein interactions (PPIs) with metabolic proteins, whereas ATG7(1) primarily interacts with autophagy machinery proteins. Furthermore, we identified that ATG7(2) mediates a decrease in metabolic activity, highlighting a novel splice-dependent function of this important autophagy protein. Then, we found a divergent expression pattern of ATG7(1) and ATG7(2) across human tissues. Conclusively, our work uncovers the divergent patterns of expression, protein interactions, and function of ATG7(2) in contrast to ATG7(1). These findings suggest a molecular switch between main catabolic processes through isoform-dependent expression of a key autophagy gene.

The V-ATPase-ATG16L1 axis recruits LRRK2 to facilitate the lysosomal stress response.

Leucine-rich repeat kinase 2 (LRRK2), a Rab kinase associated with Parkinson's disease and several inflammatory diseases, has been shown to localize to stressed lysosomes and get activated to regulate lysosomal homeostasis. However, the mechanisms of LRRK2 recruitment and activation have not been well understood. Here, we found that the ATG8 conjugation system regulates the recruitment of LRRK2 as well as LC3 onto single membranes of stressed lysosomes/phagosomes. This recruitment did not require FIP200-containing autophagy initiation complex, nor did it occur on double-membrane autophagosomes, suggesting independence from canonical autophagy. Consistently, LRRK2 recruitment was regulated by the V-ATPase-ATG16L1 axis, which requires the WD40 domain of ATG16L1 and specifically mediates ATG8 lipidation on single membranes. This mechanism was also responsible for the lysosomal stress-induced activation of LRRK2 and the resultant regulation of lysosomal secretion and enlargement. These results indicate that the V-ATPase-ATG16L1 axis serves a novel non-autophagic role in the maintenance of lysosomal homeostasis by recruiting LRRK2.

ATG3 is subjected to redox regulation to quarantee ATG8 lipidation under ROS-generating stresses

ATG3 is subjected to redox regulation to quarantee ATG8 lipidation under ROS-generating stresses

Real-Time Monitoring of ATG8 Lipidation in vitro Using Fluorescence Spectroscopy.

Autophagy is an essential catabolic pathway used to sequester and engulf cytosolic substrates via a unique double-membrane structure, called an autophagosome. The ubiquitin-like ATG8 proteins play an important role in mediating autophagosome membrane expansion. They are covalently conjugated to phosphatidylethanolamine (PE) on the autophagosomes via a ubiquitin-like conjugation system called ATG8 lipidation. In vitro reconstitution of ATG8 lipidation with synthetic liposomes has been previously established and used widely to characterise the function of the E1 ATG7, the E2 ATG3, and the E3 complex ATG12-ATG5-ATG16L1. However, there is still a lack of a tool to provide kinetic measurements of this enzymatic reaction. In this protocol, we describe a real-time lipidation assay using NBD-labelled ATG8. This real-time assay can distinguish the formation of ATG8 intermediates (ATG7~ATG8 and/or ATG3~ATG8) and, finally, ATG8-PE conjugation. It allows kinetic characterisation of the activity of ATG7, ATG3, and the E3 complex during ATG8 lipidation. Furthermore, this protocol can be adapted to characterise the upstream regulators that may affect protein activity in ATG8 lipidation reaction with a kinetic readout. Key features • Preparation of ATG7 E1 from insect cells (Sf9 cells). • Preparation of ATG3 E2 from bacteria (E. coli). • Preparation of LC3B S3C from bacteria (E. coli). • Preparation of liposomes to monitor the kinetics of ATG8 lipidation in a real-time manner.

Complete set of the Atg8-E1-E2-E3 conjugation machinery forms an interaction web that mediates membrane shaping.

Atg8, a ubiquitin-like protein, is conjugated with phosphatidylethanolamine (PE) via Atg7 (E1), Atg3 (E2) and Atg12-Atg5-Atg16 (E3) enzymatic cascade and mediates autophagy. However, its molecular roles in autophagosome formation are still unclear. Here we show that Saccharomyces cerevisiae Atg8-PE and E1-E2-E3 enzymes together construct a stable, mobile membrane scaffold. The complete scaffold formation induces an in-bud in prolate-shaped giant liposomes, transforming their morphology into one reminiscent of isolation membranes before sealing. In addition to their enzymatic roles in Atg8 lipidation, all three proteins contribute nonenzymatically to membrane scaffolding and shaping. Nuclear magnetic resonance analyses revealed that Atg8, E1, E2 and E3 together form an interaction web through multivalent weak interactions, where the intrinsically disordered regions in Atg3 play a central role. These data suggest that all six Atg proteins in the Atg8 conjugation machinery control membrane shaping during autophagosome formation.

Structural view on autophagosome formation.

Autophagy is a conserved intracellular degradation system in eukaryotes, involving the sequestration of degradation targets into autophagosomes, which are subsequently delivered to lysosomes (or vacuoles in yeasts and plants) for degradation. In budding yeast, starvation-induced autophagosome formation relies on approximately 20 core Atg proteins, grouped into six functional categories: the Atg1/ULK complex, the phosphatidylinositol-3 kinase complex, the Atg9 transmembrane protein, the Atg2-Atg18/WIPI complex, the Atg8 lipidation system, and the Atg12-Atg5 conjugation system. Additionally, selective autophagy requires cargo receptors and other factors, including a fission factor, for specific sequestration. This review covers the 30-year history of structural studies on core Atg proteins and factors involved in selective autophagy, examining X-ray crystallography, NMR, and cryo-EM techniques. The molecular mechanisms of autophagy are explored based on protein structures, and future directions in the structural biology of autophagy are discussed, considering the advancements in the era of AlphaFold.

Redox-mediated activation of ATG3 promotes ATG8 lipidation and autophagy progression in Chlamydomonas reinhardtii.

Autophagy is one of the main degradative pathways used by eukaryotic organisms to eliminate useless or damaged intracellular material to maintain cellular homeostasis under stress conditions. Mounting evidence indicates a strong interplay between the generation of reactive oxygen species and the activation of autophagy. Although a tight redox regulation of autophagy has been shown in several organisms, including microalgae, the molecular mechanisms underlying this control remain poorly understood. In this study, we have performed an in-depth in vitro and in vivo redox characterization of ATG3, an E2-activating enzyme involved in ATG8 lipidation and autophagosome formation, from 2 evolutionary distant unicellular model organisms: the green microalga Chlamydomonas (Chlamydomonas reinhardtii) and the budding yeast Saccharomyces cerevisiae. Our results indicated that ATG3 activity from both organisms is subjected to redox regulation since these proteins require reducing equivalents to transfer ATG8 to the phospholipid phosphatidylethanolamine. We established the catalytic Cys of ATG3 as a redox target in algal and yeast proteins and showed that the oxidoreductase thioredoxin efficiently reduces ATG3. Moreover, in vivo studies revealed that the redox state of ATG3 from Chlamydomonas undergoes profound changes under autophagy-activating stress conditions, such as the absence of photoprotective carotenoids, the inhibition of fatty acid synthesis, or high light irradiance. Thus, our results indicate that the redox-mediated activation of ATG3 regulates ATG8 lipidation under oxidative stress conditions in this model microalga.

TECPR1 is activated by damage‐induced sphingomyelin exposure to mediate noncanonical autophagy

Cells use noncanonical autophagy, also called conjugation of ATG8 to single membranes (CASM), to label damaged intracellular compartments with ubiquitin‐like ATG8 family proteins in order to signal danger caused by pathogens or toxic compounds. CASM relies on E3 complexes to sense membrane damage, but so far, only the mechanism to activate ATG16L1‐containing E3 complexes, associated with proton gradient loss, has been described. Here, we show that TECPR1‐containing E3 complexes are key mediators of CASM in cells treated with a variety of pharmacological drugs, including clinically relevant nanoparticles, transfection reagents, antihistamines, lysosomotropic compounds, and detergents. Interestingly, TECPR1 retains E3 activity when ATG16L1 CASM activity is obstructed by the Salmonella Typhimurium pathogenicity factor SopF. Mechanistically, TECPR1 is recruited by damage‐induced sphingomyelin (SM) exposure using two DysF domains, resulting in its activation and ATG8 lipidation. In vitro assays using purified human TECPR1‐ATG5‐ATG12 complex show direct activation of its E3 activity by SM, whereas SM has no effect on ATG16L1‐ATG5‐ATG12. We conclude that TECPR1 is a key activator of CASM downstream of SM exposure.

Redox partner interactions in the ATG8 lipidation system in microalgae.

Autophagy is a catabolic pathway that functions as a degradative and recycling process to maintain cellular homeostasis in most eukaryotic cells, including photosynthetic organisms such as microalgae. This process involves the formation of double-membrane vesicles called autophagosomes, which engulf the material to be degraded and recycled in lytic compartments. Autophagy is mediated by a set of highly conserved autophagy-related (ATG) proteins that play a fundamental role in the formation of the autophagosome. The ATG8 ubiquitin-like system catalyzes the conjugation of ATG8 to the lipid phosphatidylethanolamine, an essential reaction in the autophagy process. Several studies identified the ATG8 system and other core ATG proteins in photosynthetic eukaryotes. However, how ATG8 lipidation is driven and regulated in these organisms is not fully understood yet. A detailed analysis of representative genomes from the entire microalgal lineage revealed a high conservation of ATG proteins in these organisms with the remarkable exception of red algae, which likely lost ATG genes before diversification. Here, we examine in silico the mechanisms and dynamic interactions between different components of the ATG8 lipidation system in plants and algae. Moreover, we also discuss the role of redox post-translational modifications in the regulation of ATG proteins and the activation of autophagy in these organisms by reactive oxygen species.

Role of autophagy-related proteins ATG8f and ATG8h in the maintenance of autophagic activity in Arabidopsis roots under phosphate starvation.

Nutrient starvation-induced autophagy is a conserved process in eukaryotes. Plants defective in autophagy show hypersensitivity to carbon and nitrogen limitation. However, the role of autophagy in plant phosphate (Pi) starvation response is relatively less explored. Among the core autophagy-related (ATG) genes, ATG8 encodes a ubiquitin-like protein involved in autophagosome formation and selective cargo recruitment. The Arabidopsis thaliana ATG8 genes, AtATG8f and AtATG8h, are notably induced in roots under low Pi. In this study, we show that such upregulation correlates with their promoter activities and can be suppressed in the phosphate response 1 (phr1) mutant. Yeast one-hybrid analysis failed to attest the binding of the AtPHR1 transcription factor to the promoter regions of AtATG8f and AtATG8h. Dual luciferase reporter assays in Arabidopsis mesophyll protoplasts also indicated that AtPHR1 could not transactivate the expression of both genes. Loss of AtATG8f and AtATG8h leads to decreased root microsomal-enriched ATG8 but increased ATG8 lipidation. Moreover, atg8f/atg8h mutants exhibit reduced autophagic flux estimated by the vacuolar degradation of ATG8 in the Pi-limited root but maintain normal cellular Pi homeostasis with reduced number of lateral roots. While the expression patterns of AtATG8f and AtATG8h overlap in the root stele, AtATG8f is more strongly expressed in the root apex and root hair and remarkably at sites where lateral root primordia develop. We hypothesize that Pi starvation-induction of AtATG8f and AtATG8h may not directly contribute to Pi recycling but rely on a second wave of transcriptional activation triggered by PHR1 that fine-tunes cell type-specific autophagic activity.

Targeting of biomolecular condensates to the autophagy pathway.

Biomolecular condensates are membraneless compartments formed by liquid-liquid phase separation. They can phase transit into gel-like and solid states. The amount and state of biomolecular condensates must be tightly regulated to maintain normal cellular function. Autophagy targets biomolecular condensates to the lysosome for degradation or other purposes, which we term biocondensophagy. In biocondensophagy, autophagy receptors recognize biomolecular condensates and target them to the autophagosome, the vesicle carrier of autophagy. Multiple types of autophagy receptors have been identified and they are specifically involved in targeting biomolecular condensates with different phase transition states. The receptors also organize the phase transition of biomolecular condensate to facilitate biocondensophagy. Here, we briefly discuss the latest discoveries regarding how biomolecular condensates are recognized by autophagy receptors.

GAPDH mediates plant reovirus-induced incomplete autophagy for persistent viral infection in leafhopper vector

ABSTRACT Macroautophagy/autophagy is a conserved mechanism launched by host organisms to fight against virus infection. Double-membraned autophagosomes in arthropod vectors can be remodeled by arboviruses to accommodate virions and facilitate persistent viral propagation, but the underlying mechanism is unknown. Rice gall dwarf virus (RGDV), a plant nonenveloped double-stranded RNA virus, induces the formation of virus-containing double-membraned autophagosomes to benefit persistent viral propagation in leafhopper vectors. In this study, it was found that the capsid protein P2 of RGDV alone induced autophagy. P2 specifically interacted with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and ATG4B both in vitro and in vivo. Furthermore, the GAPDH-ATG4B complex could be recruited to virus-induced autophagosomes. Silencing of GAPDH or ATG4B expression suppressed ATG8 lipidation, autophagosome formation, and efficient viral propagation. Thus, P2 could directly recruit the GAPDH-ATG4B complex to induce the formation of initial autophagosomes. Furthermore, such autophagosomes were modified to evade fusion with lysosomes for degradation, and thus could be persistently exploited by viruses to facilitate efficient propagation. GAPDH bound to ATG14 and inhibited the interaction of ATG14 with SNAP29, thereby preventing ATG14-SNARE proteins from mediating autophagosome-lysosome fusion. Taken together, these results highlight how RGDV activates GAPDH to initiate autophagosome formation and block autophagosome degradation, finally facilitating persistent viral propagation in insect vectors. The findings reveal a positive regulation of immune response in insect vectors during viral infection.

Yeast phospholipase D, Spo14, is not required for macroautophagy.

Autophagy-related gene (Atg) proteins are key players in autophagy. Some proteins that function in vesicle trafficking and lipid metabolism are also involved in autophagy. The SPO14 in yeast, which encodes phospholipase D (PLD), is involved in membrane trafficking and plays a vital role in sporulation during meiosis. Crosstalk has been identified between autophagy and sporulation. Although the PLD is required for macroautophagy in mammals, its role in yeast macroautophagy remains unclear. We observed that Spo14 is not required for macroautophagy in yeast and that it is dispensable for Atg8 lipidation, which plays an important role in phagophore extension. Our results also revealed that green fluorescent protein (GFP)-Atg8 degradation is not completely blocked in atg1Δ/atg1Δ cells under sporulation condition. Therefore, Spo14 is not required for macroautophagy in yeast.

A nonstructural protein encoded by a rice reovirus induces an incomplete autophagy to promote viral spread in insect vectors.

Viruses can hijack autophagosomes as the nonlytic release vehicles in cultured host cells. However, how autophagosome-mediated viral spread occurs in infected host tissues or organs in vivo remains poorly understood. Here, we report that an important rice reovirus, rice gall dwarf virus (RGDV) hijacks autophagosomes to traverse multiple insect membrane barriers in the midgut and salivary gland of leafhopper vector to enhance viral spread. Such virus-containing double-membraned autophagosomes are prevented from degradation, resulting in increased viral propagation. Mechanistically, viral nonstructural protein Pns11 induces autophagy and embeds itself in the autophagosome membranes. The autophagy-related protein 5 (ATG5)-ATG12 conjugation is essential for initial autophagosome membrane biogenesis. RGDV Pns11 specifically interacts with ATG5, both in vitro and in vivo. Silencing of ATG5 or Pns11 expression suppresses ATG8 lipidation, autophagosome formation, and efficient viral propagation. Thus, Pns11 could directly recruit ATG5-ATG12 conjugation to induce the formation of autophagosomes, facilitating viral spread within the insect bodies. Furthermore, Pns11 potentially blocks autophagosome degradation by directly targeting and mediating the reduced expression of N-glycosylated Lamp1 on lysosomal membranes. Taken together, these results highlight how RGDV remodels autophagosomes to benefit viral propagation in its insect vector.

The yeast LYST homolog Bph1 is a Rab5 effector and prevents Atg8 lipidation at endosomes.

Lysosomes mediate degradation of macromolecules to their precursors for cellular recycling. Additionally, lysosome-related organelles mediate cell type-specific functions. Chédiak-Higashi syndrome is an autosomal, recessive disease, in which loss of the protein LYST causes defects in lysosomes and lysosome-related organelles. The molecular function of LYST, however, is largely unknown. Here, we dissected the function of the yeast LYST homolog, Bph1. We show that Bph1 is an endosomal protein and an effector of the minor Rab5 isoform Ypt52. Strikingly, bph1Δ mutant cells have lipidated Atg8 on their endosomes, which is sorted via late endosomes into the vacuole lumen under non-autophagy-inducing conditions. In agreement with this, proteomic analysis of bph1Δ vacuoles reveals an accumulation of Atg8, reduced flux via selective autophagy, and defective endocytosis. Additionally, bph1Δ cells have reduced autophagic flux under starvation conditions. Our observations suggest that Bph1 is a novel Rab5 effector that maintains endosomal functioning. When Bph1 is lost, Atg8 is lipidated at endosomes even during normal growth and ends up in the vacuole lumen. Thus, our results contribute to the understanding of the role of LYST-related proteins and associated diseases.

Monitoring of ATG4 Protease Activity During Autophagy in the Model Microalga Chlamydomonas reinhardtii.

Deciphering the molecular mechanisms underlying the regulation of the ATG4 protease is essential to understand the regulation of ATG8 lipidation, a key step in the biogenesis of the autophagosome and hence in autophagy progression. Here, we describe two complementary approaches to monitor ATG4 proteolytic activity in the model green alga Chlamydomonas reinhardtii: an in vitro assay using recombinant ATG4 and recombinant ATG8 as substrate, and a cell-free assay using soluble total protein extract from Chlamydomonas and recombinant Chlamydomonas ATG8 as substrate. Both assays are followed by non-reducing SDS-PAGE and immuno-blot analysis. Given the high evolutionary conservation of the ATG8 maturation process, these assays have also been validated to monitor ATG4 activity in yeast using Chlamydomonas ATG8 as substrate.

Atg5 Regulates Selective Autophagy of the Parental Macronucleus during Tetrahymena Sexual Reproduction.

Nuclear autophagy is an important selective autophagy process. The selective autophagy of sexual development micronuclei (MICs) and the programmed nuclear degradation of parental macronucleus (paMAC) occur during sexual reproduction in Tetrahymena thermophila. The molecular regulatory mechanism of nuclear selective autophagy is unclear. In this study, the autophagy-related protein Atg5 was identified from T. thermophila. Atg5 was localized in the cytoplasm in the early sexual-development stage and was localized in the paMAC in the late sexual-development stage. During this stage, the degradation of meiotic products of MIC was delayed in atg5i mutants. Furthermore, paMAC was abnormally enlarged and delayed or failed to degrade. The expression level and lipidation of Atg8.2 significantly decreased in the mutants. All these results indicated that Atg5 was involved in the regulation of the selective autophagy of paMAC by regulating Atg8.2 in Tetrahymena.

Structural basis for membrane recruitment of ATG16L1 by WIPI2 in autophagy.

Autophagy is a cellular process that degrades cytoplasmic cargo by engulfing it in a double-membrane vesicle, known as the autophagosome, and delivering it to the lysosome. The ATG12-5-16L1 complex is responsible for conjugating members of the ubiquitin-like ATG8 protein family to phosphatidylethanolamine in the growing autophagosomal membrane, known as the phagophore. ATG12-5-16L1 is recruited to the phagophore by a subset of the phosphatidylinositol 3-phosphate-binding seven-bladedß -propeller WIPI proteins. We determined the crystal structure of WIPI2d in complex with the WIPI2 interacting region (W2IR) of ATG16L1 comprising residues 207-230 at 1.85 Å resolution. The structure shows that the ATG16L1 W2IR adopts an alpha helical conformation and binds in an electropositive and hydrophobic groove between WIPI2 ß-propeller blades 2 and 3. Mutation of residues at the interface reduces or blocks the recruitment of ATG12-5-16 L1 and the conjugation of the ATG8 protein LC3B to synthetic membranes. Interface mutants show a decrease in starvation-induced autophagy. Comparisons across the four human WIPIs suggest that WIPI1 and 2 belong to a W2IR-binding subclass responsible for localizing ATG12-5-16 L1 and driving ATG8 lipidation, whilst WIPI3 and 4 belong to a second W34IR-binding subclass responsible for localizing ATG2, and so directing lipid supply to the nascent phagophore. The structure provides a framework for understanding the regulatory node connecting two central events in autophagy initiation, the action of the autophagic PI 3-kinase complex on the one hand and ATG8 lipidation on the other.

Atg12-Interacting Motif Is Crucial for E2-E3 Interaction in Plant Atg8 System.

Autophagy is an intracellular degradation system regulating cellular homeostasis. The two ubiquitin-like modification systems named the Atg8 system and the Atg12 system are essential for autophagy. Atg8 and Atg12 are ubiquitin-like proteins covalently conjugated with a phosphatidylethanolamine (PE) and Atg5, respectively, via enzymatic reactions. The Atg8-PE conjugate binds to autophagic membranes and recruits various proteins through direct interaction, whereas the Atg12-Atg5 conjugate recognizes Atg3, the E2 enzyme for Atg8, and facilitates Atg8-PE conjugation by functioning as the E3 enzyme. Although structural and biochemical analyses have well established the Atg8-family interacting motif (AIM), studies on the interacting sequence for Atg12 are rare (only one example for human ATG12-ATG3), thereby making it challenging to define a binding motif. Here we determined the crystal structure of the plant ATG12b as a complex with the ATG12b-binding region of ATG3 and revealed that ATG12b recognizes the aspartic acid (Asp)-methionine (Met) motif in ATG3 via a hydrophobic pocket and a basic residue, which we confirmed critical for the complex formation by mutational analysis. This recognition mode is similar to that reported between human ATG12 and ATG3, suggesting that the Asp-Met sequence is a conserved Atg12-interacting motif (AIM12). These data suggest that AIM12 mediates E2-E3 interaction during Atg8 lipidation and provide structural basis for developing chemicals that regulate autophagy by targeting Atg12-family proteins.