InteractiveFly: GeneBrief

Autophagy-related 2: Biological Overview | References

Gene name - Autophagy-related 2

Synonyms -

Cytological map position - 62F3-62F4

Function - lipid transport

Keywords - autophagy - lipid transport function - transports short fatty acyl chain phosphatidylethanolamine species - a potential tether between ER and phagophores - transports lipids from the ER to promote autophagosome biogenesis - promotes mitochondrial health and muscle and heart function - a candidate genetic modifier of the eye pigmentation phenotype of carmine mutants

Symbol - Atg2

FlyBase ID: FBgn0044452

Genetic map position - chrX:10,764,878-10,768,338

Cellular location - cytoplasmic

NCBI links: EntrezGene, Nucleotide, Protein

Atg-2 orthologs: Biolitmine

Autophagy is mediated by membrane-bound organelles and it is an intrinsic catabolic and recycling process of the cell, which is very important for the health of organisms. The biogenesis of autophagic membranes is still incompletely understood. In vitro studies suggest that Atg2 protein transports lipids presumably from the ER to the expanding autophagic structures. Autophagy research has focused heavily on proteins and very little is known about the lipid composition of autophagic membranes. This study describes a method for immunopurification of autophagic structures from Drosophila melanogaster (an excellent model to study autophagy in a complete organism) for subsequent lipidomic analysis. Western blots of several organelle markers indicate the high purity of the isolated autophagic vesicles, visualized by various microscopy techniques. Mass spectrometry results show that phosphatidylethanolamine (PE) is the dominant lipid class in wild type (control) membranes. In Atg2 mutants (Atg2-), phosphatidylinositol (PI), negatively charged phosphatidylserine (PS), and phosphatidic acid (PA) with longer fatty acyl chains accumulate on stalled, negatively charged phagophores. Tandem mass spectrometry analysis of lipid species composing the lipid classes reveal the enrichment of unsaturated PE and phosphatidylcholine (PC) in controls versus PI, PS and PA species in Atg2-. Significant differences in the lipid profiles of control and Atg2- flies suggest that the lipid composition of autophagic membranes dynamically changes during their maturation. These lipidomic results also point to the in vivo lipid transport function of the Atg2 protein, pointing to its specific role in the transport of short fatty acyl chain PE species (Laczko-Dobos, 2021).

Macroautophagy (autophagy hereafter) is a 'self-eating' process of eukaryotic cells, during which damaged or obsolete cytoplasmic components (organelles, proteins, lipids, sugars etc.) are removed and degraded in lysosomes. Misregulation of autophagy contributes to neurodegenarative diseases, cancer, accelerated aging, etc. This fundamental catabolic process relies on the biogenesis of unique, very dynamic membranes and membrane-bound autophagic vesicles. The pathway initiates with the nucleation of a double-membrane structure called phagophore (formerly also known as isolation membrane), which expands and engulfs a portion of the cytoplasm. After closure and sealing, it will form the autophagosome, a double membrane vesicle. In the last step, autophagosomes will fuse with lysosomes to form autolysosomes, where the sequestered cytoplasmic material will be degraded and recycled back to the cytosol (Laczko-Dobos, 2021).

Fluorescence and electron microscopy are widely used to study these specific organelles. Autophagic structures can be isolated for example from human cell lines, yeast, mouse and rat tissues by applying subcellular fractionation, immunoprecipitation or combination of these two methods. Fruit flies are an excellent model organism to study autophagy in a complete animal as they can be genetically manipulated very easily, and about 75% of their genes show homology with disease associated human genes, so they can serve as models for various human diseases (Laczko-Dobos, 2021).

Autophagosomes are unique organelles regarding their lipid and protein composition, morphology and biogenesis. Autophagosome formation occurs very rapidly upon induction of autophagy, which requires a tremendous amount of membrane source(s). De novo synthesis of autophagic membranes is the most enigmatic field of autophagy; almost every compartment of the endomembrane system has been implicated in this process. Interestingly, also the contact sites between organelles such as ER and mitochondria may play an important role in this biogenesis event. During phagophore expansion, the synthesis or delivery of lipids must be distinctly controlled. Formation of these specific membranes relies on a collaborative work between proteins encoded by autophagy-related (Atg) genes and membrane lipids (Laczko-Dobos, 2021).

It has been shown recently that Atg2 protein is not only a potential tether between ER and phagophores, but it may also transport lipids from the ER to promote autophagosome biogenesis (Osawa, 2019a; Osawa, 2019b). Several in vitro studies on yeast (and human) Atg2 (Atg2A and B) showed the lipid transport activity of this special protein, which is able to transport several lipids at once using its hydrophobic cavity (Osawa, 2019b; Valverde, 2019; Maeda, 2019; Osawa, 2020). A very recent discovery is that the fast expansion of autophagic membranes after autophagy induction relies on localized 'on-demand' de novo phospholipid synthesis, by the aid of Acyl-CoA synthetase Faa1 enzymes identified on nucleated phagophores in yeast, and the Atg2-Atg18 protein complex may also be involved in this process. The P-element induced Drosophila mutant (Atg2EP3697), bearing the transposon insertion at the 5' non-translated region of the Atg2 gene that resulted in a strong hypomorphic allele, showed an autophagy defect similar to mammalian and C. elegans Atg2 mutants [26,27]. Microscopy and biochemical investigations support that loss of Atg2 protein function in Drosophila, worms, and also in mammalian cells causes a sealing defect of phagophores, leading to accumulation of enlarged phagophore-like structures. Other key players of autophagy are Atg8 proteins together with their lipid conjugation (including the E3-like Atg5-Atg12-Atg16 complex) and deconjugation machinery. They have important roles in the biogenesis of autophagic membranes, and they are reversebly conjugated to the PE head groups present in the membranes of autophagic structures. The two distinct forms of Atg8 proteins: the non-lipidated Atg8 (Atg8-I) and lipidated Atg8 (Atg8-II) are the most widely used autophagic markers, including Atg8a in Drosophila (Laczko-Dobos, 2021).

Lipids are largely unexplored players of the phagophore biogenesis machinery. The major structural lipids in eukaryotic membranes are glycerophospholipids, sphingolipids and sterols. The most abundant glycerophospholipid classes of Drosophila are: phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI) and less abundant lipid classes are phosphatidylglycerol (PG) and phosphatidic acid (PA). PC accounts for more than 50% of the phospholipids in most eukaryotic membranes. Interestingly, in Drosophila, PE is the most abundant structural lipid and also a component of the lipoproteins (like Atg8), in contrast with more PC-centric lipidome of mammalian cells. These main lipid classes differ in the chemical composition of their head groups and in length and saturation level of their fatty acyl chains. This determines the shape and physicochemical behavior of these lipid molecules, such as phase transition properties and thickness of the membranes. All these properties influence membrane curvature and fusion events (Laczko-Dobos, 2021).

Lipids are not only structural components of the membranes but they also play a regulatory role in cellular processes, such as autophagy. Inactivation of Desat1 (desaturase coding gene, responsible for double bond generation) in Drosophila resulted in an autophagic defect: autolysosomes could not form properly. Glycerolipids play important roles in the initiation of autophagy, elongation of phagophores, autophagosome maturation as well as in autophagosome-lysosome fusion. Different phosphorylated forms of PI including PI3P, PI4P, PI(4,5)P2, PI(3,5)P2 are also found in autophagic membranes, as different types of kinases present at these membranes are responsible for their generation. Although they are minor lipids (representing less than 1% of total lipids), together with PE they play important roles in autophagosome biogenesis by influencing the recruitment of specific proteins to the membrane. Interestingly, phosphatidic acid (PA) molecules may directly affect the physicochemical properties of lipid bilayers independently of protein effectors (Laczko-Dobos, 2021).

This study established a method for isolating autophagic structures from adult Drosophila melanogaster, which was optimized for subsequent lipidomic investigations. Deciphering the lipid composition of autophagic membranes is crucial to fully understand the mechanism of autophagy. Using powerful Drosophila genetics and the advantage of high-throughput mass spectrometry, this study points to the important in vivo lipid transport function of Atg2 protein (Laczko-Dobos, 2021).

Atg2, Atg9 and Atg18 in mitochondrial integrity, cardiac function and healthspan in Drosophila

In yeast, the Atg2-Atg18 complex regulates Atg9 recycling from phagophore assembly site during autophagy; their function in higher eukaryotes remains largely unknown. In a targeted screening in Drosophila melanogaster, this study shows that Mef2-GAL4-RNAi-mediated knockdown of Atg2, Atg9 or Atg18 in the heart and indirect flight muscles led to shortened healthspan (declined locomotive function) and lifespan. These flies displayed an accelerated age-dependent loss of cardiac function along with cardiac hypertrophy (increased heart tube wall thickness) and structural abnormality (distortion of the lumen surface). Using the Mef2-GAL4-MitoTimer mitochondrial reporter system and transmission electron microscopy, significant elongation of mitochondria and reduced number of lysosome-targeted autophagosomes containing mitochondria were observed in the heart tube but exaggerated mitochondrial fragmentation and reduced mitochondrial density in indirect flight muscles. These findings provide the first direct evidence of the importance of Atg2-Atg18/Atg9 autophagy complex in the maintenance of mitochondrial integrity and, regulation of heart and muscle functions in Drosophila, raising the possibility of augmenting Atg2-Atg18/Atg9 activity in promoting mitochondrial health and, muscle and heart function (Xu, 2018).

Identification of Atg2 and ArfGAP1 as candidate genetic modifiers of the eye pigmentation phenotype of Adaptor Protein-3 (AP-3) mutants in Drosophila melanogaster

The Adaptor Protein (AP)-3 complex is a molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. A forward screening was performed for genetic modifiers of the eye pigmentation AP-3 (carmine) gene in Drosophila. One modifier was the Atg2 gene, encoding a conserved protein involved in autophagy. Loss of one copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, Biogenesis of lysosome-related organelles complex 1, subunit 1 (Blos1) and lightoid (Rab32), and even increased the eye pigment content of wild-type flies. A second modifier was the ArfGAP1 gene, encoding a conserved GTPase-activating protein. Loss of a single copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants. Strikingly, loss of the second copy of the gene elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 or lightoid. These results provide evidence for functional links connecting the machinery for biogenesis of LROs with autophagy and small GTPase regulation (Rodriguez-Fernandez, 2015).

Tousled-like kinase mediated a new type of cell death pathway in Drosophila

Programmed cell death (PCD) has an important role in sculpting organisms during development. However, much remains to be learned about the molecular mechanism of PCD. This study found that ectopic expression of tousled-like kinase (tlk) in Drosophila initiated a new type of cell death. Furthermore, the TLK-induced cell death is likely to be independent of the canonical caspase pathway and other known caspase-independent pathways. Genetically, atg2 RNAi could rescue the TLK-induced cell death, and this function of atg2 is likely distinct from its role in autophagy. In the developing retina, loss of tlk resulted in reduced PCD in the interommatidial cells (IOCs). Similarly, an increased number of IOCs was present in the atg2 deletion mutant clones. However, double knockdown of tlk and atg2 by RNAi did not have a synergistic effect. These results suggested that ATG2 may function downstream of TLK. In addition to a role in development, tlk and atg2 RNAi could rescue calcium overload-induced cell death. Together, these results suggest that TLK mediates a new type of cell death pathway that occurs in both development and calcium cytotoxicity (Zhang, 2015).

Different effects of Atg2 and Atg18 mutations on Atg8a and Atg9 trafficking during starvation in Drosophila

The Atg2-Atg18 complex acts in parallel to Atg8 and regulates Atg9 recycling from phagophore assembly site (PAS) during autophagy in yeast. This study shows that in Drosophila, both Atg9 and Atg18 are required for Atg8a puncta formation, unlike Atg2. Selective autophagic degradation of ubiquitinated proteins is mediated by Ref(2)P/p62. The transmembrane protein Atg9 accumulates on refractory to Sigma P (Ref(2)P) aggregates in Atg7, Atg8a and Atg2 mutants. No accumulation of Atg9 is seen on Ref(2)P in cells lacking Atg18 or Vps34 lipid kinase function, while the Atg1 complex subunit FIP200 is recruited. The simultaneous interaction of Atg18 with both Atg9 and Ref(2)P raises the possibility that Atg18 may facilitate selective degradation of ubiquitinated protein aggregates by autophagy (Nagy, 2014).

Functions of Atg-2 orthologs in other species

Human ATG2B possesses a lipid transfer activity which is accelerated by negatively charged lipids and WIPI4

Atg2 is one of the essential factors for autophagy. Recent advance of structural and biochemical study on yeast Atg2 proposed that Atg2 tethers the edge of the isolation membrane (IM) to the endoplasmic reticulum and mediates direct lipid transfer (LT) from ER to IM for IM expansion. In mammals, two Atg2 orthologs, ATG2A and ATG2B, participate in autophagic process. This study showed that human ATG2B possesses the membrane tethering (MT) and LT activity that was promoted by negatively charged membranes and an Atg18 ortholog WIPI4. By contrast, negatively charged membranes reduced the yeast Atg2 activities in the absence of Atg18. These results suggest that the MT/LT activity of Atg2 is evolutionally conserved although their regulation differs among species (Osawa, 2020).

ATG2 transports lipids to promote autophagosome biogenesis

During macroautophagic stress, autophagosomes can be produced continuously and in high numbers. Many different organelles have been reported as potential donor membranes for this sustained autophagosome growth, but specific machinery to support the delivery of lipid to the growing autophagosome membrane has remained unknown. This study shows that the autophagy protein, ATG2, without a clear function since its discovery over 20 yr ago, is in fact a lipid-transfer protein likely operating at the ER-autophagosome interface. ATG2A can bind tens of glycerophospholipids at once and transfers lipids robustly in vitro. An N-terminal fragment of ATG2A that supports lipid transfer in vitro is both necessary and fully sufficient to rescue blocked autophagosome biogenesis in ATG2A/ATG2B KO cells, implying that regulation of lipid homeostasis is the major autophagy-dependent activity of this protein and, by extension, that protein-mediated lipid transfer across contact sites is a principal contributor to autophagosome formation (Valverde, 2019).

Regulation of phagocyte triglyceride by a STAT-ATG2 pathway controls mycobacterial infection

Mycobacterium tuberculosis remains a global threat to human health, yet the molecular mechanisms regulating immunity remain poorly understood. Cytokines can promote or inhibit mycobacterial survival inside macrophages and the underlying mechanisms represent potential targets for host-directed therapies. This study shows that cytokine-STAT signalling promotes mycobacterial survival within macrophages by deregulating lipid droplets via ATG2 repression. In Drosophila infected with Mycobacterium marinum, mycobacterium-induced STAT activity triggered by unpaired-family cytokines reduces Atg2 expression, permitting deregulation of lipid droplets. Increased Atg2 expression or reduced macrophage triglyceride biosynthesis, normalizes lipid deposition in infected phagocytes and reduces numbers of viable intracellular mycobacteria. In human macrophages, addition of IL-6 promotes mycobacterial survival and BCG-induced lipid accumulation by a similar, but probably not identical, mechanism. These results reveal Atg2 regulation as a mechanism by which cytokines can control lipid droplet homeostasis and consequently resistance to mycobacterial infection in Drosophila (Pean, 2017).


Search PubMed for articles about Drosophila Atg-2

Laczko-Dobos, H., Maddali, A. K., Jipa, A., Bhattacharjee, A., Vegh, A. G. and Juhasz, G. (2021). Lipid profiles of autophagic structures isolated from wild type and Atg2 mutant Drosophila. Biochim Biophys Acta Mol Cell Biol Lipids 1866(3): 158868. PubMed ID: 33333179

Nagy, P., Hegedus, K., Pircs, K., Varga, A. and Juhasz, G. (2014). Different effects of Atg2 and Atg18 mutations on Atg8a and Atg9 trafficking during starvation in Drosophila. FEBS Lett 588(3): 408-413. PubMed ID: 24374083

Pean, C. B., Schiebler, M., Tan, S. W., Sharrock, J. A., Kierdorf, K., Brown, K. P., Maserumule, M. C., Menezes, S., Pilatova, M., Bronda, K., Guermonprez, P., Stramer, B. M., Andres Floto, R. and Dionne, M. S. (2017). Regulation of phagocyte triglyceride by a STAT-ATG2 pathway controls mycobacterial infection. Nat Commun 8: 14642. PubMed ID: 28262681

Osawa, T., Ishii, Y. and Noda, N. N. (2020). Human ATG2B possesses a lipid transfer activity which is accelerated by negatively charged lipids and WIPI4. Genes Cells 25(1): 65-70. PubMed ID: 31721365

Dell'Angelica, E. C. (2015). Identification of Atg2 and ArfGAP1 as candidate genetic modifiers of the eye pigmentation phenotype of Adaptor Protein-3 (AP-3) mutants in Drosophila melanogaster. PLoS One 10: e0143026. PubMed ID: 26565960

Valverde, D. P., Yu, S., Boggavarapu, V., Kumar, N., Lees, J. A., Walz, T., Reinisch, K. M. and Melia, T. J. (2019). ATG2 transports lipids to promote autophagosome biogenesis. J Cell Biol 218(6): 1787-1798. PubMed ID: 30952800

Xu, P., Damschroder, D., Zhang, M., Ryall, K. A., Adler, P. N., Saucerman, J. J., Wessells, R. J. and Yan, Z. (2019). Atg2, Atg9 and Atg18 in mitochondrial integrity, cardiac function and healthspan in Drosophila. J Mol Cell Cardiol 127: 116-124. PubMed ID: 30571977

Zhang, Y., Cai, R., Zhou, R., Li, Y. and Liu, L. (2015). Tousled-like kinase mediated a new type of cell death pathway in Drosophila. Cell Death Differ [Epub ahead of print]. PubMed ID: 26088162

Biological Overview

date revised: 12 April 2023

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