Atg3 promotes Atg8 lipidation via altering lipid diffusion and rearrangement.
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ABSTRACT:
SUBMITTER: Wang S
PROVIDER: S-EPMC7255502 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Acetylation of Atg3 regulates the lipidation of the protein Atg8 in autophagy. The molecular mechanism behind this important biochemical event remains to be elucidated. We describe the first semi-synthesis of homogeneous K19/K48-diacetylated Atg3 through sequential hydrazide-based native chemical ligation. In vitro reconstitution experiments with the semi-synthetic proteins confirm that Atg3 acetylation can promote the lipidation of Atg8. We find that acetylation of Atg3 enhances its binding to phosphatidylethanolamine-containing liposomes and to endoplasmic reticulum, through which it promotes the lipidation process.
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Project description:Autophagy is a catabolic process involved in the degradation of a cell's own components for cell growth, development, homeostasis, and the recycling of cellular products. Autophagosome is an essential component in the protozoan parasite during differentiation and encystation. The present study identified and characterized autophagy-related protein (Atg) 3, a member of Atg8 conjugation system, in Acanthamoeba castellanii (AcAtg3). AcAtg3 encoding a 304 amino acid protein showed high similarity with the catalytic cysteine site of other E2 like enzymes of ubiquitin system. Predicted 3D structure of AcAtg3 revealed a hammer-like shape, which is the characteristic structure of E2-like enzymes. The expression level of AcAtg3 did not increase during encystation. However, the formation of mature cysts was significantly reduced in Atg3-siRNA transfected cells in which the production of Atg8-phosphatidylethanolamine conjugate was inhibited. Fluorescent microscopic analysis revealed that dispersed AcAtg3-EGFP fusion protein gathered around autophagosomal membranes during encystation. These results provide important information for understanding autophagic machinery through the lipidation reaction mediated by Atg3 in Acanthamoeba.
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Project description:ATG8 family proteins are evolutionary conserved ubiquitin-like modifiers, which become attached to the headgroup of the membrane lipid phosphatidylethanolamine in a process referred to as lipidation. This reaction is carried out analogous to the conjugation of ubiquitin to its target proteins, involving the E1-like ATG7, the E2-like ATG3 and the E3-like ATG12-ATG5-ATG16 complex, which determines the site of lipidation. ATG8 lipidation is a hallmark of autophagy where these proteins are involved in autophagosome formation, the fusion of autophagosomes with lysosomes and cargo selection. However, it has become evident that ATG8 lipidation also occurs in processes that are not directly related to autophagy. Here we discuss recent insights into the targeting of ATG8 lipidation in autophagy and other pathways with special emphasis on the recruitment and activation of the E3-like complex.
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Project description:Autophagosome biogenesis requires two ubiquitin-like conjugation systems. One couples ubiquitin-like Atg8 to phosphatidylethanolamine, and the other couples ubiquitin-like Atg12 to Atg5. Atg12~Atg5 then forms a heterodimer with Atg16. Membrane recruitment of the Atg12~Atg5/Atg16 complex defines the Atg8 lipidation site. Lipidation requires a PI3P-containing precursor. How PI3P is sensed and used to coordinate the conjugation systems remained unclear. Here, we show that Atg21, a WD40 ?-propeller, binds via PI3P to the preautophagosomal structure (PAS). Atg21 directly interacts with the coiled-coil domain of Atg16 and with Atg8. This latter interaction requires the conserved F5K6-motif in the N-terminal helical domain of Atg8, but not its AIM-binding site. Accordingly, the Atg8 AIM-binding site remains free to mediate interaction with its E2 enzyme Atg3. Atg21 thus defines PI3P-dependently the lipidation site by linking and organising the E3 ligase complex and Atg8 at the PAS.
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Project description:The autophagy-related protein 8 (Atg8) conjugation system is essential for the formation of double-membrane vesicles called autophagosomes during autophagy, a bulk degradation process conserved among most eukaryotes. It is also important in yeast for recognizing target vacuolar enzymes through the receptor protein Atg19 during the cytoplasm-to-vacuole targeting (Cvt) pathway, a selective type of autophagy. Atg3 is an E2-like enzyme that conjugates Atg8 with phosphatidylethanolamine. Here, we show that Atg3 directly interacts with Atg8 through the WEDL sequence, which is distinct from canonical interaction between E2 and ubiquitin-like modifiers. Moreover, NMR experiments suggest that the mode of interaction between Atg8 and Atg3 is quite similar to that between Atg8/LC3 and the Atg8 family interacting motif (AIM) conserved in autophagic receptors, such as Atg19 and p62. Thus, the WEDL sequence in Atg3 is a canonical AIM. In vitro analyses showed that Atg3 AIM is crucial for the transfer of Atg8 from the Atg8?Atg3 thioester intermediate to phosphatidylethanolamine but not for the formation of the intermediate. Intriguingly, in vivo experiments showed that it is necessary for the Cvt pathway but not for starvation-induced autophagy. Atg3 AIM attenuated the inhibitory effect of Atg19 on Atg8 lipidation in vitro, suggesting that Atg3 AIM may be important for the lipidation of Atg19-bound Atg8 during the Cvt pathway.
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Project description:The autophagy-related proteins are thought to serve multiple functions in Plasmodium and are considered essential to parasite survival and development. We have studied two key interacting proteins, Atg8 and Atg3, of the autophagy pathway in Plasmodium falciparum. These proteins are vital for the formation and elongation of the autophagosome and essential to the process of macroautophagy. Autophagy may be required for conversion of the sporozoite into erythrocytic-infective merozoites and may be crucial for other functions during asexual blood stages. Here we describe the identification of an Atg8 family interacting motif (AIM) in Plasmodium Atg3, which binds Plasmodium Atg8. We determined the co-crystal structure of PfAtg8 with a short Atg3¹?³?¹¹? peptide, corresponding to this motif, to 2.2 Å resolution. Our in vitro interaction studies are in agreement with our X-ray crystal structure. Furthermore they suggest an important role for a unique Apicomplexan loop absent from human Atg8 homologues. Prevention of the protein-protein interaction of full length PfAtg8 with PfAtg3 was achieved at low micromolar concentrations with a small molecule, 1,2,3-trihydroxybenzene. Together our structural and interaction studies represent a starting point for future antimalarial drug discovery and design for this novel protein-protein interaction.
| S-EPMC3496014 | biostudies-literature
Project description:Yeast Atg8 and its homologs are involved in autophagosome biogenesis in all eukaryotes. These are the most widely used markers for autophagy thanks to the association of their lipidated forms with autophagic membranes. The Atg8 protein family expanded in animals and plants, with most Drosophila species having two Atg8 homologs. In this Brief Report, we use clear-cut genetic analysis in Drosophila melanogaster to show that lipidated Atg8a is required for autophagy, while its non-lipidated form is essential for developmentally programmed larval midgut elimination and viability. In contrast, expression of Atg8b is restricted to the male germline and its loss causes male sterility without affecting autophagy. We find that high expression of non-lipidated Atg8b in the male germline is required for fertility. Consistent with these non-canonical functions of Atg8 proteins, loss of Atg genes required for Atg8 lipidation lead to autophagy defects but do not cause lethality or male sterility.
| S-EPMC8496532 | biostudies-literature
Project description:Coupling of Atg8 to phosphatidylethanolamine is crucial for the expansion of the crescent-shaped phagophore during cargo engulfment. Atg21, a PtdIns3P-binding beta-propeller protein, scaffolds Atg8 and its E3-like complex Atg12-Atg5-Atg16 during lipidation. The crystal structure of Atg21, in complex with the Atg16 coiled-coil domain, showed its binding at the bottom side of the Atg21 beta-propeller. Our structure allowed detailed analyses of the complex formation of Atg21 with Atg16 and uncovered the orientation of the Atg16 coiled-coil domain with respect to the membrane. We further found that Atg21 was restricted to the phagophore edge, near the vacuole, known as the vacuole isolation membrane contact site (VICS). We identified a specialized vacuolar subdomain at the VICS, typical of organellar contact sites, where the membrane protein Vph1 was excluded, while Vac8 was concentrated. Furthermore, Vac8 was required for VICS formation. Our results support a specialized organellar contact involved in controlling phagophore elongation. Abbreviations: FCCS: fluorescence cross correlation spectroscopy; NVJ: nucleus-vacuole junction; PAS: phagophore assembly site; PE: phosphatidylethanolamine; PROPPIN: beta-propeller that binds phosphoinositides; PtdIns3P: phosphatidylinositol- 3-phosphate; VICS: vacuole isolation membrane contact site.
| S-EPMC8205015 | biostudies-literature
Project description:Autophagy depends on the E2 enzyme, Atg3, functioning in a conserved E1-E2-E3 trienzyme cascade that catalyzes lipidation of Atg8-family ubiquitin-like proteins (UBLs). Molecular mechanisms underlying Atg8 lipidation remain poorly understood despite association of Atg3, the E1 Atg7, and the composite E3 Atg12-Atg5-Atg16 with pathologies including cancers, infections and neurodegeneration. Here, studying yeast enzymes, we report that an Atg3 element we term E123IR (E1, E2, and E3-interacting region) is an allosteric switch. NMR, biochemical, crystallographic and genetic data collectively indicate that in the absence of the enzymatic cascade, the Atg3E123IR makes intramolecular interactions restraining Atg3's catalytic loop, while E1 and E3 enzymes directly remove this brace to conformationally activate Atg3 and elicit Atg8 lipidation in vitro and in vivo. We propose that Atg3's E123IR protects the E2~UBL thioester bond from wayward reactivity toward errant nucleophiles, while Atg8 lipidation cascade enzymes induce E2 active site remodeling through an unprecedented mechanism to drive autophagy.
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Project description:Atg8 proteins fused with tags are commonly used to detect autophagy. The expression patterns of Lepidopteran insect Atg8 are relatively well documented. However, the influence of protein tags on characterization of Atg8 is still not very clear. Our results showed that endogenous Spodoptera litura Atg8 and HA tagged Atg8 driven by the baculovirus ie2 promoter were enriched in cytoplasm. The recombinant plasmid pEGFP-Atg8(EGFP) in which Atg8 contained a stop codon was constructed and expressed. Green fluorescence was accumulated in cytoplasm. However, red fluorescence was located in both cytoplasm and nucleoplasm in most cells transfected with the recombinant plasmid pmCherry-Atg8(EGFP). In contrast to pEGFP-Atg8(EGFP), green fluorescence was also located in both cytoplasm and nucleoplasm in most cells transfected with the recombinant plasmid pie2/EGFP-Atg8 driven by the baculovirus ie2 promoter in which the CMV promoter and EGFP nucleotide sequences were removed, and the high level of the EGFP-Atg8 expression significantly increased its abundance in nucleoplasm. HA-Atg8 expressed at high level through baculovirus under the control of polyherin promoter was also localized in cytoplasm and nucleoplasm. The cleavage of mCherry-Atg8 was different from that of EGFP-Atg8. Both the mutant mCherry-Atg8F77/79A resulting in non-cleavage of the Atg8 and the mutant mCherry-Atg8G exposing its glycine residue at the end of C-terminus were also localized in cytoplasm and nucleoplasm. The increase of autophagosomes decreased the abundance of mCherry-Atg8 in nucleoplasm. In addition, the ratio of HA-Atg8-PE/HA-Atg8 was less than that of endogenous Atg8-PE/Atg8. These results demonstrated that the Atg8 is located in both nucleus and cytoplasm when expressed at high level and exported to the cytoplasm when autophagy is activated, and the fusion tags of Atg8 might have influence on the processing of Atg8 fusion proteins.
| S-EPMC4008565 | biostudies-literature