The expanding role of Atg8.

It would be quite convenient if every protein had one distinct function, one distinct role in just a single cellular process. In the field of macroautophagy/autophagy, however, we are increasingly finding that this is not the case; several autophagy proteins have two or more roles within the process of autophagy and many even "moonlight" as functional members of entirely different cellular processes. This is perhaps best exemplified by the Atg8-family proteins. These dynamic proteins have already been reported to serve several functions both within autophagy (membrane tethering, membrane fusion, binding to cargo receptors, binding to autophagy machinery) and beyond (LC3-associated phagocytosis, formation of EDEMosomes, immune signaling) but as Maruyama and colleagues suggest in their recent report, this list of functions may not yet be complete.

Mammalian Atg8 proteins and the autophagy factor IRGM control mTOR and TFEB at a regulatory node critical for responses to pathogens.

Autophagy is a homeostatic process with multiple functions in mammalian cells. Here, we show that mammalian Atg8 proteins (mAtg8s) and the autophagy regulator IRGM control TFEB, a transcriptional activator of the lysosomal system. IRGM directly interacted with TFEB and promoted the nuclear translocation of TFEB. An mAtg8 partner of IRGM, GABARAP, interacted with TFEB. Deletion of all mAtg8s or GABARAPs affected the global transcriptional response to starvation and downregulated subsets of TFEB targets. IRGM and GABARAPs countered the action of mTOR as a negative regulator of TFEB. This was suppressed by constitutively active RagB, an activator of mTOR. Infection of macrophages with the membrane-permeabilizing microbe Mycobacterium tuberculosis or infection of target cells by HIV elicited TFEB activation in an IRGM-dependent manner. Thus, IRGM and its interactors mAtg8s close a loop between the autophagosomal pathway and the control of lysosomal biogenesis by TFEB, thus ensuring coordinated activation of the two systems that eventually merge during autophagy.

Biological functions of the autophagy-related proteins Atg4 and Atg8 in Cryptococcus neoformans.

Autophagy is a mechanism responsible for intracellular degradation and recycling of macromolecules and organelles, essential for cell survival in adverse conditions. More than 40 autophagy-related (ATG) genes have been identified and characterized in fungi, among them ATG4 and ATG8. ATG4 encodes a cysteine protease (Atg4) that plays an important role in autophagy by initially processing Atg8 at its C-terminus region. Atg8 is a ubiquitin-like protein essential for the synthesis of the double-layer membrane that constitutes the autophagosome vesicle, responsible for delivering the cargo from the cytoplasm to the vacuole lumen. The contributions of Atg-related proteins in the pathogenic yeast in the genus Cryptococcus remain to be explored, to elucidate the molecular basis of the autophagy pathway. In this context, we aimed to investigate the role of autophagy-related proteins 4 and 8 (Atg4 and Atg8) during autophagy induction and their contribution with non-autophagic events in C. neoformans. We found that Atg4 and Atg8 are conserved proteins and that they interact physically with each other. ATG gene deletions resulted in cells sensitive to nitrogen starvation. ATG4 gene disruption affects Atg8 degradation and its translocation to the vacuole lumen, after autophagy induction. Both atg4 and atg8 mutants are more resistant to oxidative stress, have an impaired growth in the presence of the cell wall-perturbing agent Congo Red, and are sensitive to the proteasome inhibitor bortezomib (BTZ). By that, we conclude that in C. neoformans the autophagy-related proteins Atg4 and Atg8 play an important role in the autophagy pathway; which are required for autophagy regulation, maintenance of amino acid levels and cell adaptation to stressful conditions.

Overexpression of ATG8 in Arabidopsis Stimulates Autophagic Activity and Increases Nitrogen Remobilization Efficiency and Grain Filling.

Autophagy knock-out mutants in maize and in Arabidopsis are impaired in nitrogen (N) recycling and exhibit reduced levels of N remobilization to their seeds. It is thus impoortant to determine whether higher autophagy activity could, conversely, improve N remobilization efficiency and seed protein content, and under what circumstances. As the autophagy machinery involves many genes amongst which 18 are important for the core machinery, the choice of which AUTOPHAGY (ATG) gene to manipulate to increase autophagy was examined. We choose ATG8 overexpression since it has been shown that this gene could increase autophagosome size and autophagic activity in yeast. The results we report here are original as they show for the first time that increasing ATG8 gene expression in plants increases autophagosome number and promotes autophagy activity. More importantly, our data demonstrate that, when cultivated under full nitrate conditions, known to repress N remobilization due to sufficient N uptake from the soil, N remobilization efficiency can nevertheless be sharply and significantly increased by overexpressing ATG8 genomic sequences under the control of the ubiquitin promoter. We show that overexpressors have improved seed N% and at the same time reduced N waste in their dry remains. In addition, we show that overexpressing ATG8 does not modify vegetative biomass or harvest index, and thus does not affect plant development.

Coordination of Autophagosome-Lysosome Fusion by Atg8 Family Members.

Macroautophagy is a conserved intracellular lysosomal degradative pathway, vital for the maintenance of cellular homeostasis. It is characterized by double-membrane vesicles called autophagosomes, which sequester the cytoplasmic material destined for lysosomal turnover. In a final step, autophagosomes fuse with lysosomes to release their cargo into the acidic and hydrolytic lumen of these organelles. In recent years, numerous new insights into this fusion event have been gained. Notably, many proteins implicated in autophagosome-lysosome fusion interact with members of the Atg8 protein family. Moreover, Atg8 proteins are described to have intrinsic membrane tethering and fusogenic properties themselves. Here, we summarize the current knowledge about the members of this intriguing protein family, which highlights them as possible hubs for the coordination of the final fusion stages of autophagy.

MeWRKY20 and its interacting and activating autophagy-related protein 8 (MeATG8) regulate plant disease resistance in cassava.

As a highly conserved mechanism, autophagy is responsible for the transport of cytoplasmic constituents in the vacuoles or lysosomes. Moreover, autophagy is essential for plant development and various stress responses. In this study, 34 MeATGs were systematically identified in cassava, and their transcripts were commonly regulated by Xanthomonas axonopodis pv manihotis (Xam). Through transient expression in Nicotiana benthamiana, the subcellular locations of 4 MeATG8s were revealed. Notably, MeWRKY20 was identified as physical interacting protein of MeATG8a/8f/8h and upstream transcriptional activator of MeATG8a. Through virus-induced gene silencing (VIGS) in cassava, we found that MeATG8-silenced and MeWRKY20-silenced plants resulted in disease sensitive, with less callose depositions and lower autophagic activity. This study may facilitate our understanding of the upstream MeWRKY20 and underlying target as well as interacting proteins of MeATG8s in immune response. Taken together, MeWRKY20 and MeATG8a/8f/8h are essential for disease resistance against bacterial blight by forming various transcriptional modules and interacting complex in cassava.

[Research progress on mechanism of Nix-mediated mitophagy].

Autophagy is fundamental to maintain cellular homeostasis. As one kind of the most well-studied selective autophagy, autophagy of mitochondria (mitophagy)is crucial for the clearance of damaged mitochondria. Mitophagy dysfunction has been proved to be closely associated with many human diseases. Nix is a key protein for mitophagy during the maturation of reticulocytes. However, the detailed molecular mechanisms underlying Nix-mediated mitophagy are not fully understood. This article summarizes three possible working models of Nix in mitophagy induction. Firstly, Nix can interplay with Parkin, another important protein for mitophagy, to initiate mitophagy. Secondly, Nix can serve as a receptor for autophagy machinery by interacting with Atg8 family through its LIR motif. Finally, as a BH3-only protein, Nix can compete with Beclin-1 to bind other members of Bcl-2 family resulting in increased free Beclin-1 in cytosol, which further promotes autophagy flux.

Ubiquitin-like Atg8 protein is expressed during autophagy and the encystation process in Naegleria gruberi.

Members of the Naegleria genus are free-living amoebae, and the only pathogenic specie described to date for humans is N. fowleri. However, as the complete genome of this specie has not been reported, non-pathogenic N. gruberi is employed to describe molecular pathways in N. fowleri. Regardless, certain mechanisms, such as autophagy, have not yet been characterized in N. gruberi. Autophagy is involved in different cellular processes in some protozoa, including the recycling of unnecessary organelles, development, and cell differentiation. In this work, we characterized autophagy in N. gruberi using the specific inducer rapamycin. The formation of autophagy vacuoles in treated trophozoites was observed by ultrastructural analysis, and real time quantitative PCR demonstrated overexpression of the atg8 gene. In addition, we detected an increase in the vacuolar acidification of treated amoebae using the LysoTracker. Finally, confocal microscopy was utilized to identify Atg8 protein signal in the cytoplasm of N. gruberi trophozoites induced with rapamycin and even in trophozoites induced to encyst. In conclusion, N. gruberi possesses an Atg8 protein homolog that is overexpressed during the autophagic mechanism induced by rapamycin and also during encystation of this free-living amoeba.

White spot syndrome virus entry is dependent on multiple endocytic routes and strongly facilitated by Cq-GABARAP in a CME-dependent manner.

White spot syndrome virus (WSSV) is a lethal pathogen of shrimp and many other crustaceans, including crayfish. However, the molecular mechanism underlying its cellular entry remains elusive due to the lack of shrimp cell lines for viral propagation. Crayfish hematopoietic tissue (Hpt) cell culture was recently established as a good model for WSSV infection study. Here, we showed that multiple endocytic routes, including clathrin-mediated endocytosis (CME), macropinocytosis and caveolae-mediated endocytosis, were indispensably employed for the viral entry into Hpt cell of the crayfish Cherax quadricarinatus. Intriguingly, cellular autophagic activity was positively correlated with efficient viral entry, in which a key autophagy-related protein, γ-aminobutyric acid receptor-associated protein (Cq-GABARAP), that not only localized but also co-localized with WSSV on the Hpt cell membrane, strongly facilitated WSSV entry by binding to the viral envelope VP28 in a CME-dependent manner that was negatively regulated by Cq-Rac1. Furthermore, cytoskeletal components, including Cq-ß-tubulin and Cq-ß-actin, bound to both recombinant rCq-GABARAP and WSSV envelope proteins, which likely led to viral entry promotion via cooperation with rCq-GABARAP. Even under conditions that promoted viral entry, rCq-GABARAP significantly reduced viral replication at an early stage of infection, which was probably caused by the formation of WSSV aggregates in the cytoplasm.