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Autophagosome biogenesis is a complex process orchestrated by dynamic interactions between Atg (autophagy-related) proteins and characterized by the turnover of specific cargoes, which can differ over time and depending on how autophagy is stimulated. Proteomic analyses are central to uncover protein-protein interaction networks and when combined with proximity-dependent biotinylation or proximity labeling (PL) approaches, they also permit to detect transient and weak interactions. However, current PL procedures for yeast Saccharomyces cerevisiae , one of the leading models for the study of autophagy, do not allow to keep temporal specificity and thus identify interactions and cargoes at a precise time point upon autophagy induction. Here, we present a new ascorbate peroxidase 2 (APEX2)-based PL protocol adapted to yeast that preserves temporal specificity and allows uncovering neighbor proteins by either western blot or proteomics. As a proof of concept, we applied this new method to identify Atg8 and Atg9 interactors and detected known binding partners as well as potential uncharacterized ones in rich and nitrogen starvation conditions. Also, as a proof of concept, we confirmed the spatial proximity interaction between Atg8 and Faa1. We believe that this protocol will be a new important experimental tool for all those researchers studying the mechanism and roles of autophagy in yeast, but also other cellular pathways in this model organism. Abbreviations : APEX2, ascorbate peroxidase 2, Atg, autophagy-related; BP, biotin phenol; Cvt, cytoplasm-to-vacuole targeting; ER, endoplasmic reticulum; LN2, liquid nitrogen; MS, mass spectrometry; PAS, phagophore assembly site; PL, proximity labeling; PE, phosphatidylethanolamine; PPINs, protein-protein interaction networks; PPIs, protein-protein interactions; RT, room temperature; SARs, selective autophagy receptors; WT, wild-type.

Among the MAP1LC3/LC3 subfamily of Atg8 proteins, LC3B and LC3C constitute the most and least studied members, respectively, LC3B being generally considered as an autophagosomal marker and a canonical representative of the LC3 subfamily. In several recent studies, LC3C has emerged as an important modulator in various processes of cell homeostasis. Our own research data demonstrate that LC3C induces higher levels of tethering and of intervesicular lipid mixing than LC3B. LC3C contains a peculiar N-terminal region, different from the other Atg8-family protein members. Using a series of mutants, we have shown that the N-terminal region of LC3C is responsible for the enhanced vesicle tethering, membrane perturbation and vesicle-vesicle fusion activities of LC3C as compared to LC3B. Abbreviations: ATG: autophagy related; GABARAP: gamma-aminobutyric acid receptor associated protein; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PC: phosphatidyl choline; PE: phosphatidylethanolamine; PEmal: maleimide-derivatized PE; PtdIns: phosphatidylinositol.

The noncanonical ubiquitin-like conjugation cascade involving the E1 (Atg7), E2 (Atg3, Atg10), and E3 (Atg12-Atg5-Atg16 complex) enzymes is essential for incorporation of Atg8 into the growing phagophore via covalent linkage to PE. This process is an indispensable step in autophagy. Atg8 and E1-E3 enzymes are the first subset from the core autophagy protein machinery structures that were investigated in earlier studies by crystallographic analyses of globular domains. However, research over the past decade shows that many important functions in the conjugation machinery are mediated by intrinsically disordered protein regions (IDPRs) - parts of the protein that do not adopt a stable secondary or tertiary structure, which are inherently dynamic and well suited for protein-membrane interactions but are invisible in protein crystals. Here, we summarize earlier and recent findings on the autophagy conjugation machinery by focusing on the IDPRs. This summary reveals that IDPRs, originally considered dispensable, are in fact major players and a driving force in the function of the autophagy conjugation system. Abbreviation : AD, activation domain of Atg7; AH, amphipathic helix; AIM, Atg8-family interacting motif; CL, catalytic loop (of Atg7); CTD, C-terminal domain; FR, flexible region (of Atg3 or Atg10); GUV, giant unilammelar vesicles; HR, handle region (of Atg3); IDPR, intrinsically disordered protein region; IDPs: intrinsically disordered proteins; LIR, LC3-interacting region; NHD: N-terminal helical domain; NMR, nuclear magnetic resonance; PE, phosphatidylethanolamine; UBL, ubiquitin like.

Macroautophagy is often quantified by live imaging of autophagosomes labeled with fluorescently tagged ATG8 protein (FP-ATG8) in Arabidopsis thaliana. The labeled particles are then counted in single focal planes. This approach may lead to inaccurate results as the actual 3D distribution of autophagosomes is not taken into account and appropriate sampling in the Z-direction is not performed. To overcome this issue, we developed a workflow consisting of immunolabeling of autophagosomes with an anti-ATG8 antibody followed by stereological image analysis using the optical disector and the Cavalieri principle. Our protocol specifically recognized autophagosomes in epidermal cells of Arabidopsis root. Since the anti-ATG8 antibody recognizes multiple AtATG8 isoforms, we were able to detect a higher number of immunolabeled autophagosomes than with the FP-AtATG8e marker, that most probably does not recognize all autophagosomes in a cell. The number of autophagosomes per tissue volume positively correlated with the intensity of autophagy induction. Compared with the quantification of autophagosomes in maximum intensity projections, stereological methods were able to detect the autophagosomes present in a given volume with higher accuracy. Our novel workflow provides a powerful toolkit for unbiased and reproducible quantification of autophagosomes and offers a convenient alternative to the standard of live imaging with FP-ATG8 markers., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Autophagy of glycogen (glycophagy) is crucial for the maintenance of cellular glucose homeostasis and physiology in mammals. STBD1 can serve as an autophagy receptor to mediate glycophagy by specifically recognizing glycogen and relevant key autophagic factors, but with poorly understood mechanisms. Here, we systematically characterize the interactions of STBD1 with glycogen and related saccharides, and determine the crystal structure of the STBD1 CBM20 domain with maltotetraose, uncovering a unique binding mode involving two different oligosaccharide-binding sites adopted by STBD1 CBM20 for recognizing glycogen. In addition, we demonstrate that the LC3-interacting region (LIR) motif of STBD1 can selectively bind to six mammalian ATG8 family members. We elucidate the detailed molecular mechanism underlying the selective interactions of STBD1 with ATG8 family proteins by solving the STBD1 LIR/GABARAPL1 complex structure. Importantly, our cell-based assays reveal that both the STBD1 LIR/GABARAPL1 interaction and the intact two oligosaccharide binding sites of STBD1 CBM20 are essential for the effective association of STBD1, GABARAPL1, and glycogen in cells. Finally, through mass spectrometry, biochemical, and structural modeling analyses, we unveil that STBD1 can directly bind to the Claw domain of RB1CC1 through its LIR, thereby recruiting the key autophagy initiation factor RB1CC1. In all, our findings provide mechanistic insights into the recognitions of glycogen, ATG8 family proteins, and RB1CC1 by STBD1 and shed light on the potential working mechanism of STBD1-mediated glycophagy., Competing Interests: Competing interests statement:The authors declare no competing interest.

Autophagy is a universal degradation system in eukaryotic cells. In plants, although autophagosome biogenesis has been extensively studied, the mechanism of how autophagosomes are transported to the vacuole for degradation remains largely unexplored. In this study, we demonstrated that upon autophagy induction, Arabidopsis homotypic fusion and protein sorting (HOPS) subunit VPS41 converts first from condensates to puncta, then to ring-like structures, termed VPS41-associated phagic vacuoles (VAPVs), which enclose autophagy-related gene (ATG)8s for vacuolar degradation. This process is initiated by ADP ribosylation factor (ARF)-like GTPases ARLA1s and occurs concurrently with autophagy progression through coupling with the synaptic-soluble N-ethylmaleimide-sensitive factor attachment protein rmleceptor (SNARE) proteins. Unlike in other eukaryotes, autophagy degradation in Arabidopsis is largely independent of the RAB7 pathway. By contrast, dysfunction in the condensates-to-VAPVs conversion process impairs autophagosome structure and disrupts their vacuolar transport, leading to a significant reduction in autophagic flux and plant survival rate. Our findings suggest that the conversion pathway might be an integral part of the autophagy program unique to plants., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Most autophagy-related genes, or ATG genes, have been identified through studies using budding yeast. Although the functions of the ATG genes are well understood, the contributions of individual genes to non-selective and various types of selective autophagy remain to be fully elucidated. In this study, we quantified the activity of non-selective autophagy, the cytoplasm-to-vacuole targeting (Cvt) pathway, mitophagy, endoplasmic reticulum (ER)-phagy and pexophagy in all Saccharomyces cerevisiae atg mutants. Among the mutants of the core autophagy genes considered essential for autophagy, the atg13 mutant and mutants of the genes involved in the two ubiquitin-like conjugation systems retained residual autophagic functionality. In particular, mutants of the Atg8 ubiquitin-like conjugation system (the Atg8 system) exhibited substantial levels of non-selective autophagy, the Cvt pathway and pexophagy, although mitophagy and ER-phagy were undetectable. Atg8-system mutants also displayed intravacuolar vesicles resembling autophagic bodies, albeit at significantly reduced size and frequency. Thus, our data suggest that membranous sequestration and vacuolar delivery of autophagic cargo can occur in the absence of the Atg8 system. Alongside these findings, the comprehensive analysis conducted here provides valuable datasets for future autophagy research., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The Atg8-family proteins (MAP1LC3/LC3A, LC3B, LC3C, GABARAP, GABARAPL1 and GABARAPL2) play a pivotal role in macroautophagy/autophagy through their ability to help form autophagosomes. Although autophagosomes form in the cytoplasm, nuclear levels of the Atg8-family proteins are significant. Recently, the nuclear/cytoplasmic shuttling of LC3B was shown to require deacetylation of two Lys residues (K49 and K51 in LC3B), which are conserved in Atg8-family proteins. To exit the nucleus, deacetylated LC3B must bind TP53INP2/DOR (tumor protein p53 inducible nuclear protein 2) through interaction with the LC3-interacting region (LIR) of TP53INP2 (TP53INP2LIR). To examine their selectivity for TP53INP2 and the role of the conserved Lys residues in Atg8-family proteins, we prepared the six human Atg8-family proteins and acetylated variants of LC3A and GABARAP for biophysical and structural characterization of their interactions with the TP53INP2LIR. Isothermal titration calorimetry (ITC) experiments demonstrate that this LIR binds preferentially to GABARAP subfamily proteins, and that only acetylation of the second Lys residue reduces binding to GABARAP and LC3A. Crystal structures of complexes with GABARAP and LC3A (acetylated and deacetylated) define a β-sheet in the TP53INP2LIR that determines the GABARAP selectivity and establishes the importance of acetylation at the second Lys. The in vitro results were confirmed in cells using acetyl-mimetic variants of GABARAP and LC3A to examine nuclear/cytoplasmic shuttling and colocalization with TP53INP2. Together, the results demonstrate that TP53INP2 shows selectivity to the GABARAP subfamily and acetylation at the second Lys of GABARAP and LC3A disrupts key interactions with TP53INP2 required for their nuclear/cytoplasmic shuttling.

Defects in organellar acidification indicate compromised or infected compartments. Recruitment of the autophagy-related ATG16L1 complex to pathologically neutralized organelles targets ubiquitin-like ATG8 molecules to perturbed membranes. How this process is coupled to proton gradient disruption is unclear. Here, we reveal that the V 1 H subunit of the vacuolar ATPase (V-ATPase) proton pump binds directly to ATG16L1. The V 1 H/ATG16L1 interaction only occurs within fully assembled V-ATPases, allowing ATG16L1 recruitment to be coupled to increased V-ATPase assembly following organelle neutralization. Cells lacking V 1 H fail to target ATG8s during influenza infection or after activation of the immune receptor stimulator of interferon genes (STING). We identify a loop within V 1 H that mediates ATG16L1 binding. A neuronal V 1 H isoform lacks this loop and is associated with attenuated ATG8 targeting in response to ionophores in primary murine and human iPSC-derived neurons. Thus, V 1 H controls ATG16L1 recruitment following proton gradient dissipation, suggesting that the V-ATPase acts as a cell-intrinsic damage sensor., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Organisms experience constant nutritional flux. Mechanisms at the interface of opposing nutritional states-scarcity and surplus-enable organismal energy homeostasis. Contingent on nutritional stores, adipocytes secrete adipokines, such as the fat hormone leptin, to signal nutrient status to the central brain. Increased leptin secretion underlies metabolic dysregulation during common obesity, but the molecular mechanisms regulating leptin secretion from human adipocytes are poorly understood. Here, we report that Atg8/LC3 family proteins, best known for their role in autophagy during nutrient scarcity, play an evolutionarily conserved role during nutrient surplus by promoting adipokine secretion. We show that in a well-fed state, Atg8/LC3 promotes the secretion of the Drosophila functional leptin ortholog unpaired 2 (Upd2) and leptin from human adipocytes. Proteomic analyses reveal that LC3 directs leptin to a secretory pathway in human cells. We identified LC3-dependent extracellular vesicle (EV) loading and secretion (LDELS) as a required step for leptin release, highlighting a unique secretory route adopted by leptin in human adipocytes. In Drosophila, mutations to Upd2's Atg8 interaction motif (AIM) result in constitutive adipokine retention. Atg8-mediated Upd2 retention alters lipid storage and hunger response and rewires the bulk organismal transcriptome in a manner conducive to starvation survival. Thus, Atg8/LC3's bidirectional role in nutrient sensing-conveying nutrient surplus and responding to nutrient deprivation-enables organisms to manage nutrient flux effectively. We posit that decoding how bidirectional molecular switches-such as Atg8/LC3-operate at the nexus of nutritional scarcity and surplus will inform therapeutic strategies to tackle chronic metabolic disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The hormone leptin is critical for regulation of food intake, energy expenditure and overall metabolism. However, the mechanisms that promote leptin secretion from adipocytes in response to nutrient surplus and limit its secretion during nutrient scarcity are unclear. New work reveals that the autophagy protein Atg8/LC3 has a bidirectional role in leptin secretion, both facilitating and limiting its release., Competing Interests: Declaration of interests The author declares no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Membrane atg8ylation is a homeostatic process responding to membrane remodeling and stress signals. Membranes are atg8ylated by mammalian ATG8 ubiquitin-like proteins through a ubiquitylation-like cascade. A model has recently been put forward which posits that atg8ylation of membranes is conceptually equivalent to ubiquitylation of proteins. Like ubiquitylation, membrane atg8ylation involves E1, E2 and E3 enzymes. The E3 ligases catalyze the final step of atg8ylation of aminophospholipids in membranes. Until recently, the only known E3 ligase for membrane atg8ylation was ATG16L1 in a noncovalent complex with the ATG12-ATG5 conjugate. ATG16L1 was first identified as a factor in canonical autophagy. During canonical autophagy, the ATG16L1-based E3 ligase complex includes WIPI2, which in turn recognizes phosphatidylinositiol 3-phosphate and directs atg8ylation of autophagic phagophores. As an alternative to WIPIs, binding of ATG16L1 to the proton pump V-ATPase guides atg8ylation of endolysosomal and phagosomal membranes in response to lumenal pH changes. Recently, a new E3 complex containing TECPR1 instead of ATG16L1, has been identified that responds to sphingomyelin's presence on the cytofacial side of perturbed endolysosomal membranes. In present review, we cover the principles of membrane atg8ylation, catalog its various presentations, and provide a perspective on the growing repertoire of E3 ligase complexes directing membrane atg8ylation at diverse locations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

While the molecular mechanism of autophagy is well studied, the cargoes delivered by autophagy remain incompletely characterized. To examine the selectivity of autophagy cargo, we conducted proteomics on isolated yeast autophagic bodies, which are intermediate structures in the autophagy process. We identify a protein, Hab1, that is highly preferentially delivered to vacuoles. The N-terminal 42 amino acid region of Hab1 contains an amphipathic helix and an Atg8-family interacting motif, both of which are necessary and sufficient for the preferential delivery of Hab1 by autophagy. We find that fusion of this region with a cytosolic protein results in preferential delivery of this protein to the vacuole. Furthermore, attachment of this region to an organelle allows for autophagic delivery in a manner independent of canonical autophagy receptor or scaffold proteins. We propose a novel mode of selective autophagy in which a receptor, in this case Hab1, binds directly to forming isolation membranes during bulk autophagy., (© 2024. The Author(s).)

In the realm of parasitology, autophagy has emerged as a critical focal point, particularly in combating Leishmaniasis. Central to this endeavour is the recognition of the protein ATG8 as pivotal for the survival and infectivity of the parasitic organism Leishmania major, thereby making it a potential target for therapeutic intervention. Consequently, there is a pressing need to delve into the structural characteristics of ATG8 to facilitate the design of effective drugs. In this study, our efforts centered on the purification of ATG8 from Leishmania major, which enabled novel insights into its structural features through meticulous spectroscopic analysis. We aimed to comprehensively assess the stability and behaviour of ATG8 in the presence of various denaturants, including urea, guanidinium chloride, and SDS-based chemicals. Methodically, our approach included secondary structural analysis utilizing CD spectroscopy, which not only validated but also augmented computationally predicted structures of ATG8 reported in previous investigations. Remarkably, our findings unveiled that the purified ATG8 protein retained its folded conformation, exhibiting the anticipated secondary structure. Moreover, our exploration extended to the influence of lipids on ATG8 stability, yielding intriguing revelations. We uncovered a nuanced perspective suggesting that targeting both the lipid composition of Leishmania major and ATG8 could offer a promising strategy for future therapeutic approaches in combating leishmaniasis. Collectively, our study underscores the importance of understanding the structural intricacies of ATG8 in driving advancements towards the development of targeted therapies against Leishmaniasis, thereby providing a foundation for future investigations in this field., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

HIV-1 entry into CD4 + T lymphocytes relies on the viral and cellular membranes' fusion, leading to viral capsid delivery in the target cell cytoplasm. Atg8/LC3B conjugation to lipids, process named Atg8ylation mainly studied in the context of macroautophagy/autophagy, occurs transiently in the early stages of HIV-1 replication in CD4 + T lymphocytes. Despite numerous studies investigating the HIV-1-autophagy interplays, the Atg8ylation impact in these early stages of infection remains unknown. Here we found that HIV-1 exposure leads to the rapid LC3B enrichment toward the target cell plasma membrane, in close proximity with the incoming viral particles. Furthermore, we demonstrated that Atg8ylation is a key event facilitating HIV-1 entry in target CD4 + T cells. Interestingly, this effect is independent of canonical autophagy as ATG13 silencing does not prevent HIV-1 entry. Together, our results provide an unconventional role of LC3B conjugation subverted by HIV-1 to achieve a critical step of its replication cycle. Abbreviations : BafA1: bafilomycin A1; BlaM: beta-lactamase; CD4 + TL: CD4 + T lymphocytes; PtdIns3K-BECN1 complex: BECN1-containing class III phosphatidylinositol 3-kinase complex; Env: HIV-1 envelope glycoproteins; HIV-1: type 1 human immunodeficiency virus; PM: plasma membrane; PtdIns3P: phosphatidylinositol-3-phosphate; VLP: virus-like particle.

ATG8 proteins form a family of small ubiquitin-like modifiers, well-known for their importance in both macroautophagy and autophagy-independent processes. A unique feature of this protein family is their conjugation to membrane lipids through the covalent attachment of a glycine residue at the C-terminus of ATG8 proteins. Notably, most ATG8 proteins are expressed with additional amino acids at their C-terminus, shielding the key glycine residue. Consequently, lipidation requires the activation of the ATG8 precursors through proteolytic cleavage, known as priming. ATG4 proteases catalyze this priming process, and under physiological conditions, unprimed forms of ATG8 are not detected. This raises the question about the purpose of the C-terminal extension of ATG8 proteins. While the roles of lipidated and free, primed ATG8 proteins have been extensively studied, the potential function of their precursor form or the priming process itself remains largely unexplored. Here, we summarize information from existing literature and our own experiments to contribute to the understanding of these neglected amino acids., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Autophagy refers to a set of degradative mechanisms whereby cytoplasmic contents are targeted to the lysosome. This is best described for macroautophagy, where a double-membrane compartment (autophagosome) is generated to engulf cytoplasmic contents. Autophagosomes are decorated with ubiquitin-like ATG8 molecules (ATG8s), which are recruited through covalent lipidation, catalysed by the E3-ligase-like ATG16L1 complex. LC3 proteins are ATG8 family members that are often used as a marker for autophagosomes. In contrast to canonical macroautophagy, conjugation of ATG8s to single membranes (CASM) describes a group of non-canonical autophagy processes in which ATG8s are targeted to pre-existing single-membrane compartments. CASM occurs in response to disrupted intracellular pH gradients, when the V-ATPase proton pump recruits ATG16L1 in a process called V-ATPase-ATG16L1-induced LC3 lipidation (VAIL). Recent work has demonstrated a parallel, alternative axis for CASM induction, triggered when the membrane recruitment factor TECPR1 recognises sphingomyelin exposed on the cytosolic face of a membrane and forms an alternative E3-ligase-like complex. This sphingomyelin-TECPR1-induced LC3 lipidation (STIL) is independent of the V-ATPase and ATG16L1. In light of these discoveries, this Cell Science at a Glance article summarises these two mechanisms of CASM to highlight how they differ from canonical macroautophagy, and from each other., Competing Interests: Competing interests The authors declare no competing or financial interests. High-resolution poster and poster panels A high-resolution version of the poster and individual poster panels are available for downloading at https://journals.biologists.com/jcs/article-lookup/doi/10.1242/jcs.261031#supplementary-data., (© 2024. Published by The Company of Biologists Ltd.)

Autophagy regulates the formation of primary cilia, which in turn affects autophagy. The relationship between autophagy and cilia is known to be bidirectional although the specific mechanisms involved have yet to be elucidated. In this study, we found for the first time that ATG8 protein localizes in the basal body of the dorsal kineties and the base of the ventral cirri in Euplotes amieti. ATG8 protein maintains the structural integrity of cilia and plays a role in the construction of the cortical ciliature and microtubule cytoskeleton associated with cilia. ATG8 gene interference leads to the degradation of IFT88, the transport protein in cilia, thus inhibiting the generation of cilia, and affecting the swing of cilia. This influences the swimming speed and cilia pattern, leading to death in Euplotes amieti., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Autophagy is relevant for diverse processes in eukaryotic cells, making its regulation of fundamental importance. The formation and maturation of autophagosomes require a complex choreography of numerous factors. The endosomal sorting complex required for transport (ESCRT) is implicated in the final step of autophagosomal maturation by sealing of the phagophore membrane. ESCRT-III components were shown to mediate membrane scission by forming filaments that interact with cellular membranes. However, the molecular mechanisms underlying the recruitment of ESCRTs to non-endosomal membranes remain largely unknown. Here we focus on the ESCRT-associated protein ALG2-interacting protein X (ALIX) and identify Ca 2+ -dependent lipid binding protein 1 (CaLB1) as its interactor. Our findings demonstrate that CaLB1 interacts with AUTOPHAGY8 (ATG8) and PI(3)P, a phospholipid found in autophagosomal membranes. Moreover, CaLB1 and ALIX localize with ATG8 on autophagosomes upon salt treatment and assemble together into condensates. The depletion of CaLB1 impacts the maturation of salt-induced autophagosomes and leads to reduced delivery of autophagosomes to the vacuole. Here, we propose a crucial role of CaLB1 in augmenting phase separation of ALIX, facilitating the recruitment of ESCRT-III to the site of phagophore closure thereby ensuring efficient maturation of autophagosomes., (© 2024. The Author(s).)

Atg8 paralogs, consisting of LC3A/B/C and GBRP/GBRPL1/GATE16, function in canonical autophagy; however, their function is controversial because of functional redundancy. In innate immunity, xenophagy and non-canonical single membranous autophagy called "conjugation of Atg8s to single membranes" (CASM) eliminate bacteria in various cells. Previously, we reported that intracellular Streptococcus pneumoniae can induce unique hierarchical autophagy comprised of CASM induction, shedding, and subsequent xenophagy. However, the molecular mechanisms underlying these processes and the biological significance of transient CASM induction remain unknown. Herein, we profile the relationship between Atg8s, autophagy receptors, poly-ubiquitin, and Atg4 paralogs during pneumococcal infection to understand the driving principles of hierarchical autophagy and find that GATE16 and GBRP sequentially play a pivotal role in CASM shedding and subsequent xenophagy induction, respectively, and LC3A and GBRPL1 are involved in CASM/xenophagy induction. Moreover, we reveal ingenious bacterial tactics to gain intracellular survival niches by manipulating CASM-xenophagy progression by generating intracellular pneumococci-derived H 2 O 2 ., Competing Interests: Declaration of interests Authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Plants rely on autophagy and membrane trafficking to tolerate stress, combat infections, and maintain cellular homeostasis. However, the molecular interplay between autophagy and membrane trafficking is poorly understood. Using an AI-assisted approach, we identified Rab3GAP-like (Rab3GAPL) as a key membrane trafficking node that controls plant autophagy negatively. Rab3GAPL suppresses autophagy by binding to ATG8, the core autophagy adaptor, and deactivating Rab8a, a small GTPase essential for autophagosome formation and defense-related secretion. Rab3GAPL reduces autophagic flux in three model plant species, suggesting that its negative regulatory role in autophagy is conserved in land plants. Beyond autophagy regulation, Rab3GAPL modulates focal immunity against the oomycete pathogen Phytophthora infestans by preventing defense-related secretion. Altogether, our results suggest that Rab3GAPL acts as a molecular rheostat to coordinate autophagic flux and defense-related secretion by restraining Rab8a-mediated trafficking. This unprecedented interplay between a RabGAP-Rab pair and ATG8 sheds new light on the intricate membrane transport mechanisms underlying plant autophagy and immunity., Competing Interests: Declaration of interests T.B. and C.D. receive funding from industry on NLR biology. T.B. and C.D. are founders and shareholders at Resurrect Bio Ltd., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Autophagy is a eukaryote-specific cellular process that can engulf unwanted targets with double-membrane autophagosomes and subject them to the vacuole or lysosome for breaking down and recycling, playing dual roles in plant growth and environmental adaptions. However, perception of specific environmental signals for autophagy induction is largely unknown, limiting its application in agricultural usage. Identification of plant-unique DUF641 family COST1 (Constitutively Stressed 1) protein directly links drought perception and autophagy induction, shedding light on manipulating autophagy for breeding stress tolerant crops. In this study, we performed a genome-wide analysis of DUF641/COST family in tomato, and identified five SlCOST genes SlCOST1, -2, -3, -4, and -5. SlCOST genes show both overlapping and distinct expression patterns in plant growth and stress responding. In addition, SlCOST1, -3, -4, -5 proteins demonstrate co-localization with autophagy adaptor protein ATG8e, and all five SlCOST proteins show interactions ATG8e in planta. However, only SlCOST1, the closest ortholog of Arabidopsis AtCOST1, can restore cost1 mutant to WT level, suggesting conserved role of COST1 and functional diversification of SlCOST family in tomato. Our study provides clues for future investigation of autophagy-related COST family and its promising implementations in breeding crops with robust environmental plasticity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Atg8 family proteins play crucial roles in autophagy to maintain cellular homeostasis. However, the physiological roles of Atg8 family proteins have not been systematically determined. In this study, we generated Atg8a and Atg8b (homologs of Atg8 in Drosophila melanogaster) knockout flies. We found that the loss of Atg8a affected autophagy and resulted in partial lethality, abnormal wings, decreased lifespan, and decreased climbing ability in flies. Furthermore, the loss of Atg8a resulted in reduced muscle integrity and the progressive degeneration of the neuron system. We also found that the phosphorylation at Ser88 of Atg8a is important for autophagy and neuronal integrity. The loss of Atg8b did not affect autophagy but induced male sterility in flies. Here, we take full advantage of the fly system to elucidate the physiological function of Atg8a and Atg8b in Drosophila., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Plant pathogens secrete effector proteins into host cells to suppress immune responses and manipulate fundamental cellular processes. One of these processes is autophagy, an essential recycling mechanism in eukaryotic cells that coordinates the turnover of cellular components and contributes to the decision on cell death or survival., Results: We report the characterization of AVH195, an effector from the broad-spectrum oomycete plant pathogen, Phytophthora parasitica. We show that P. parasitica expresses AVH195 during the biotrophic phase of plant infection, i.e., the initial phase in which host cells are maintained alive. In tobacco, the effector prevents the initiation of cell death, which is caused by two pathogen-derived effectors and the proapoptotic BAX protein. AVH195 associates with the plant vacuolar membrane system and interacts with Autophagy-related protein 8 (ATG8) isoforms/paralogs. When expressed in cells from the green alga, Chlamydomonas reinhardtii, the effector delays vacuolar fusion and cargo turnover upon stimulation of autophagy, but does not affect algal viability. In Arabidopsis thaliana, AVH195 delays the turnover of ATG8 from endomembranes and promotes plant susceptibility to P. parasitica and the obligate biotrophic oomycete pathogen Hyaloperonospora arabidopsidis., Conclusions: Taken together, our observations suggest that AVH195 targets ATG8 to attenuate autophagy and prevent associated host cell death, thereby favoring biotrophy during the early stages of the infection process., (© 2024. The Author(s).)

Members of the ATG8 (autophagy-related protein 8) protein family can be non-canonically conjugated to single membrane-bound organelles. The exact function of ATG8 on these single membranes remains poorly understood. Recently, using Arabidopsis thaliana as a model system, we identified a non-canonical conjugation of ATG8 pathway involved in the reconstruction of the Golgi apparatus upon heat stress. Short acute heat stress resulted in rapid vesiculation of the Golgi, which was accompanied with the translocation of ATG8 proteins (ATG8a to ATG8i) to the dilated cisternae. More importantly, we found that ATG8 proteins can recruit clathrin to facilitate Golgi reassembly by stimulating the budding of ATG8-positive vesicles from dilated cisternae. These findings provide new insight into one of the possible functions of ATG8 translocation onto single membrane organelles, and will contribute to a better understanding of non-canonical conjugation of ATG8 in eukaryotic cells. Abbreviations: ADS, AIMs docking site; AIM, ATG8-interacting motif; ATG, autophagy-related; CLC2, Clathrin light chain 2; ConcA, concanamycin A; HS, heat stress; PE, phosphatidylethanolamine; PM, plasma membrane; PS, phosphatidylserine; TGN, trans-Golgi network; V-ATPase, vacuolar-type ATPase.

Parkinson disease (PD) is caused by the loss of ventral midbrain dopaminergic neurons (mDANs) in the substantia nigra pars compacta (SNpc). These cells are especially vulnerable to stress but can be protected by autophagy enhancement strategies in vitro and in vivo. In our recent study, we focused on the LIM (Lin11, Isl-1, and Mec-3)-domain homeobox transcription factors LMX1A (LIM homeobox transcription factor 1 alpha) and LMX1B (LIM homeobox transcription factor 1 beta), crucial drivers of mDAN differentiation with roles in autophagy gene expression for stress protection in the developed brain. Using human induced pluripotent stem cell (hiPSC)-derived mDANs and transformed human cell lines, we found that these autophagy gene transcription factors are themselves regulated by autophagy-mediated turnover. LMX1B possesses a non-canonical LC3-interacting region (LIR) in its C-terminus through which it interacts with ATG8 family members. The LMX1B LIR-like domain enables binding to ATG8 proteins in the nucleus, where ATG8 proteins act as co-factors for robust transcription of LMX1B target genes. Thus, we propose a novel role for ATG8 proteins as autophagy gene transcriptional co-factors for mDAN stress protection in PD.

Autophagy supports cell survival under different stress conditions, where ATG8-family proteins are required for autophagosome biogenesis/maturation and selective autophagy. Here, we present a protocol for studying ATG8-family protein phosphorylation using Phos-tag gel, a modified SDS-PAGE system, when the related phosphorylation site information and/or specific phospho-antibody are unavailable. We describe steps for generating GST-ATG8 proteins in bacteria, purifying S protein-Flag-SBP protein (SFB)-tagged kinasefrom cells, preparing gel, and an in vitro kinase assay. We then detail procedures for western blotting and image processing. For complete details on the use and execution of this protocol, please refer to Seo et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Autophagy is a catabolic process in which a double-membrane organelle, the autophagosome (AP), engulfs cellular components that will be degraded in the lysosomes. ATG8 protein family members participate at various stages of AP formation. The present study compares the capacity to induce lipid-vesicle tethering and fusion of two ATG8 family members, LC3B and LC3C, with model membranes. LC3B is the most thoroughly studied ATG8 protein. It is generally considered as an autophagosomal marker and a canonical representative of the LC3 subfamily. LC3C is less studied, but recent data have reported its implication in various processes, crucial to cellular homeostasis. The results in this paper show that LC3C induces higher levels of tethering and of intervesicular lipid mixing than LC3B. As the N-terminus of LC3C is different from that of the other family members, various mutants of the N-terminal region of both LC3B and LC3C were designed, and their activities compared. It was concluded that the N-terminal region of LC3C was responsible for the enhanced vesicle tethering, membrane perturbation and vesicle-vesicle fusion activities of LC3C as compared to LC3B. The results suggest a specialized function of LC3C in the AP expansion process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Ksp1 is a casein II-like kinase whose activity prevents aberrant macroautophagy/autophagy induction in nutrient-rich conditions in yeast. Here, we describe a kinase-independent role of Ksp1 as a novel autophagic receptor protein for Ssn2/Med13, a known cargo of Snx4-assisted autophagy of transcription factors. In this pathway, a subset of conserved transcriptional regulators, Ssn2/Med13, Rim15, and Msn2, are selectively targeted for vacuolar proteolysis following nitrogen starvation, assisted by the sorting nexin heterodimer Snx4-Atg20. Here we show that phagophores also engulf Ksp1 alongside its cargo for vacuolar proteolysis. Ksp1 directly associates with Atg8 following nitrogen starvation at the interface of an Atg8-family interacting motif (AIM)/LC3-interacting region (LIR) in Ksp1 and the LIR/AIM docking site (LDS) in Atg8. Mutating the LDS site prevents the autophagic degradation of Ksp1. However, deletion of the C terminal canonical AIM still permitted Ssn2/Med13 proteolysis, suggesting that additional non-canonical AIMs may mediate the Ksp1-Atg8 interaction. Ksp1 is recruited to the perivacuolar phagophore assembly site by Atg29, a member of the trimeric scaffold complex. This interaction is independent of Atg8 and Snx4, suggesting that Ksp1 is recruited early to phagophores, with Snx4 delivering Ssn2/Med13 thereafter. Finally, normal cell survival following prolonged nitrogen starvation requires Ksp1. Together, these studies define a kinase-independent role for Ksp1 as an autophagic receptor protein mediating Ssn2/Med13 degradation. They also suggest that phagophores built by the trimeric scaffold complex are capable of receptor-mediated autophagy. These results demonstrate the dual functionality of Ksp1, whose kinase activity prevents autophagy while it plays a scaffolding role supporting autophagic degradation. Abbreviations: 3-AT: 3-aminotriazole; 17C: Atg17-Atg31-Atg29 trimeric scaffold complex; AIM: Atg8-family interacting motif; ATG: autophagy related; CKM: CDK8 kinase module; Cvt: cytoplasm-to-vacuole targeting; IDR: intrinsically disordered region; LIR: LC3-interacting region; LDS: LIR/AIM docking site; MoRF: molecular recognition feature; NPC: nuclear pore complex; PAS: phagophore assembly site; PKA: protein kinase A; RBP: RNA-binding protein; UPS: ubiquitin-proteasome system. SAA-TF: Snx4-assisted autophagy of transcription factors; Y2H: yeast two-hybrid.

The characterization of interactions between autophagy modifiers (Atg8-family proteins) and their natural ligands (peptides and proteins) or small molecules is important for a detailed understanding of selective autophagy mechanisms and for the design of potential Atg8 inhibitors that affect the autophagy processes in cells. The fluorescence polarization (FP) assay is a rapid, cost-effective, and robust method that provides affinity and selectivity information for small molecules and peptide ligands targeting human Atg8 proteins.This chapter introduces the basic principles of FP assays. In addition, a case study on peptide interaction with human Atg8 proteins (LC3/GABARAPs) is described. Finally, data analysis and quality control of FP assays are discussed for the proper calculation of K i values for the measured compounds., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Establishing the hATG8 binding selectivity of AIM (autophagy-interacting motif) sequences found within autophagy system proteins provides insights into their biological roles, and in the case of disease-associated AIM mutations, potential pathophysiological mechanisms. Given the sometimes small differences in affinity for an individual AIM amongst the six hATG8 proteins, establishing AIM preferences can be experimentally challenging. We describe a native mass spectrometry method that is suitable for detecting such differences, using synthetic AIM peptides and recombinant hATG8 proteins, to probe hATG8-AIM interactions in the gas phase. Binding preferences of a single AIM peptide against multiple hATG8s, or two AIM peptides against a single hATG8 (e.g., wild-type versus mutant AIM), may be determined., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Selective autophagic degradation of cellular components has been shown to be mediated by the interaction of LIR motif-containing proteins with ATG8-family proteins. Here, we present a detailed methodology for the in silico evaluation of potential binding between LIR motif-containing proteins and ATG8-family proteins. We visualize AlphaFold-predicted protein complexes using PyMOL to assess potential interactions, providing an effective computational tool for this purpose., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Autophagy is a highly conserved intracellular pathway that is essential for survival in all eukaryotes. In healthy cells, autophagy is used to remove damaged intracellular components, which can be as simple as unfolded proteins or as complex as whole mitochondria. Once the damaged component is captured, the autophagosome engulfs it and closes, isolating the content from the cytoplasm. The autophagosome then fuses with the late endosome and/or lysosome to deliver its content to the lysosome for degradation. Formation of the autophagosome, sequestration or capture of content, and closure all require the ATG proteins, which constitute the essential core autophagy protein machinery. This brief 'nutshell' will highlight recent data revealing the importance of small membrane-associated domains in the ATG proteins. In particular, recent findings from two parallel studies reveal the unexpected key role of α-helical structures in the ATG8 conjugation machinery and ATG8s. These studies illustrate how unique membrane association modules can control the formation of autophagosomes., (© 2023 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Cells harness multiple pathways to maintain lysosome integrity, a central homeostatic process. Damaged lysosomes can be repaired or targeted for degradation by lysophagy, a selective autophagy process involving ATG8/LC3. Here, we describe a parallel ATG8/LC3 response to lysosome damage, mechanistically distinct from lysophagy. Using a comprehensive series of biochemical, pharmacological, and genetic approaches, we show that lysosome damage induces non-canonical autophagy and Conjugation of ATG8s to Single Membranes (CASM). Following damage, ATG8s are rapidly and directly conjugated onto lysosome membranes, independently of ATG13/WIPI2, lipidating to PS (and PE), a molecular hallmark of CASM. Lysosome damage drives V-ATPase V0-V1 association, direct recruitment of ATG16L1 via its WD40-domain/K490A, and is sensitive to Salmonella SopF. Lysosome damage-induced CASM is associated with formation of dynamic, LC3A-positive tubules, and promotes robust LC3A engagement with ATG2, a lipid transfer protein central to lysosome repair. Together, our data identify direct ATG8 conjugation as a rapid response to lysosome damage, with important links to lipid transfer and dynamics., (© 2023 Cross et al.)

Macroautophagy/autophagy and lipid droplet (LD) biology are intricately linked, with autophagosome-dependent degradation of LDs in response to different signals. LDs play crucial roles in forming autophagosomes possibly by providing essential lipids and serving as a supportive autophagosome assembly platform at the endoplasmic reticulum (ER)-LD interface. LDs and autophagosomes share common proteins, such as VPS13, ATG2, ZFYVE1/DFCP1, and ATG14, but their dual functions remain poorly understood. In our recent study, we found that prolonged starvation leads to ATG3 localizing to large LDs and lipidating LC3B, revealing a non-canonical autophagic role on LDs. In vitro, ATG3 associates with purified and artificial LDs, and conjugated Atg8-family proteins. In long-term starved cells, only LC3B is found on the specific large LDs, positioned near LC3B-positive membranes that undergo lysosome-mediated acidification. This implies that LD-lipidated LC3B acts as a tethering factor, connecting phagophores to LDs and promoting degradation. Our data also support the notion that certain LD surfaces may function as lipidation stations for LC3B, which may move to nearby sites of autophagosome formation. Overall, our study unveils an unknown non-canonical implication of LDs in autophagy processes. Abbreviation: ATG: autophagy-related enzyme, ATP: adenosine triphosphate, E2 enzyme: ubiquitin-conjugating enzyme, ER: endoplasmic reticulum, LD: lipid droplet, LIR motif: LC3-interacting region, MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta, PE: phosphatidylethanolamine, PLIN1: perilipin 1, PNPLA2/ATGL: patatin-like phospholipase domain containing 2, SQSTM1/p62: sequestosome 1, VSP13: vacuolar protein sorting 13, ZFYVE1/DFCP1: zinc finger, FYVE domain containing 1.

ER-phagy is a selective autophagy process that targets specific regions of the endoplasmic reticulum (ER) for removal via lysosomal degradation. During cellular stress induced by starvation, cargo receptors concentrate at distinct ER-phagy sites (ERPHS) to recruit core autophagy proteins and initiate ER-phagy. However, the molecular mechanism responsible for ERPHS formation remains unclear. In our study, we discovered that the autophagy regulator UV radiation Resistance-Associated Gene (UVRAG) plays a crucial role in orchestrating the assembly of ERPHS. Upon starvation, UVRAG localizes to ERPHS and interacts with specific ER-phagy cargo receptors, such as FAM134B, ATL3, and RTN3L. UVRAG regulates the oligomerization of cargo receptors and facilitates the recruitment of Atg8 family proteins. Consequently, UVRAG promotes efficient ERPHS assembly and turnover of both ER sheets and tubules. Importantly, UVRAG-mediated ER-phagy contributes to the clearance of pathogenic proinsulin aggregates. Remarkably, the involvement of UVRAG in ER-phagy initiation is independent of its canonical function as a subunit of class III phosphatidylinositol 3-kinase complex II., (© 2023 The Authors.)

Several selective macroautophagy receptor and adaptor proteins bind members of the Atg8 (autophagy related 8) family using short linear motifs (SLiMs), most often referred to as Atg8-family interacting motifs (AIMs) or LC3-interacting regions (LIRs). AIM/LIR motifs have been extensively studied during the last fifteen years, since they can uncover the underlying biological mechanisms and possible substrates for this key catabolic process of eukaryotic cells. Prompted by the fact that experimental information regarding LIR motifs can be found scattered across heterogeneous literature resources, we have developed LIRcentral (https://lircentral.eu), a freely available online repository for user-friendly access to comprehensive, high-quality information regarding LIR motifs from manually curated publications. Herein, we describe the development of LIRcentral and showcase currently available data and features, along with our plans for the expansion of this resource. Information incorporated in LIRcentral is useful for accomplishing a variety of research tasks, including: (i) guiding wet biology researchers for the characterization of novel instances of LIR motifs, (ii) giving bioinformaticians/computational biologists access to high-quality LIR motifs for building novel prediction methods for LIR motifs and LIR containing proteins (LIRCPs) and (iii) performing analyses to better understand the biological importance/features of functional LIR motifs. We welcome feedback on the LIRcentral content and functionality by all interested researchers and anticipate this work to spearhead a community effort for sustaining this resource which will further promote progress in studying LIR motifs/LIRCPs. Abbreviations : AIM, Atg8-family interacting motif; Atg8, autophagy related 8; GABARAP, GABA type A receptor-associated protein; LIR, LC3-interacting region; LIRCP, LIR-containing protein; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; PMID, PubMed identifier; PPI, protein-protein interaction; SLiM, short linear motif.

Selective macroautophagy/autophagy is tightly regulated by cargo receptors that recruit specific substrates to the ATG8-family proteins for autophagic degradation. Therefore, identification of selective receptors and their new cargoes will improve our understanding of selective autophagy functions in plant development and stress responses. We have recently demonstrated that the small peptide VISP1 acts as a selective autophagy receptor to mediate degradation of suppressors of RNA silencing (VSRs) of several RNA and DNA viruses. Moreover, VISP1 induces symptom recovery through fine-tuning the balance of plant immunity and virus pathogenicity. Our findings provide new insights into the double-edged sword roles of selective autophagy in plant-virus interactions.

Nix is a membrane-anchored outer mitochondrial protein that induces mitophagy. While Nix has an LC3-interacting (LIR) motif that binds to ATG8 proteins, it also contains a minimal essential region (MER) that induces mitophagy through an unknown mechanism. We used chemically induced dimerization (CID) to probe the mechanism of Nix-mediated mitophagy and found that both the LIR and MER are required for robust mitophagy. We find that the Nix MER interacts with the autophagy effector WIPI2 and recruits WIPI2 to mitochondria. The Nix LIR motif is also required for robust mitophagy and converts a homogeneous WIPI2 distribution on the surface of the mitochondria into puncta, even in the absence of ATG8s. Together, this work reveals unanticipated mechanisms in Nix-induced mitophagy and the elusive role of the MER, while also describing an interesting example of autophagy induction that acts downstream of the canonical initiation complexes., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA.)

Selective macroautophagy of the endoplasmic reticulum (ER) and the nucleus, known as ER-phagy and nucleophagy, respectively, are processes whose mechanisms remain inadequately understood. Through an imaging-based screen, we find that in the fission yeast Schizosaccharomyces pombe, Yep1 (also known as Hva22 or Rop1), the ortholog of human REEP1-4, is essential for ER-phagy and nucleophagy but not for bulk autophagy. In the absence of Yep1, the initial phase of ER-phagy and nucleophagy proceeds normally, with the ER-phagy/nucleophagy receptor Epr1 coassembling with Atg8. However, ER-phagy/nucleophagy cargos fail to reach the vacuole. Instead, nucleus- and cortical-ER-derived membrane structures not enclosed within autophagosomes accumulate in the cytoplasm. Intriguingly, the outer membranes of nucleus-derived structures remain continuous with the nuclear envelope-ER network, suggesting a possible outer membrane fission defect during cargo separation from source compartments. We find that the ER-phagy role of Yep1 relies on its abilities to self-interact and shape membranes and requires its C-terminal amphipathic helices. Moreover, we show that human REEP1-4 and budding yeast Atg40 can functionally substitute for Yep1 in ER-phagy, and Atg40 is a divergent ortholog of Yep1 and REEP1-4. Our findings uncover an unexpected mechanism governing the autophagosomal enclosure of ER-phagy/nucleophagy cargos and shed new light on the functions and evolution of REEP family proteins., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Zou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

During nutrient stress, macroautophagy degrades cellular macromolecules, thereby providing biosynthetic building blocks while simultaneously remodelling the proteome 1,2 . Although the machinery responsible for initiation of macroautophagy has been well characterized 3,4 , our understanding of the extent to which individual proteins, protein complexes and organelles are selected for autophagic degradation, and the underlying targeting mechanisms, is limited. Here we use orthogonal proteomic strategies to provide a spatial proteome census of autophagic cargo during nutrient stress in mammalian cells. We find that macroautophagy has selectivity for recycling membrane-bound organelles (principally Golgi and endoplasmic reticulum). Through autophagic cargo prioritization, we identify a complex of membrane-embedded proteins, YIPF3 and YIPF4, as receptors for Golgiphagy. During nutrient stress, YIPF3 and YIPF4 interact with ATG8 proteins through LIR motifs and are mobilized into autophagosomes that traffic to lysosomes in a process that requires the canonical autophagic machinery. Cells lacking YIPF3 or YIPF4 are selectively defective in elimination of a specific cohort of Golgi membrane proteins during nutrient stress. Moreover, YIPF3 and YIPF4 play an analogous role in Golgi remodelling during programmed conversion of stem cells to the neuronal lineage in vitro. Collectively, the findings of this study reveal prioritization of membrane protein cargo during nutrient-stress-dependent proteome remodelling and identify a Golgi remodelling pathway that requires membrane-embedded receptors., (© 2023. The Author(s).)

During macroautophagy, the Atg8 protein is conjugated to phosphatidylethanolamine (PE) in autophagic membranes. In Apicomplexan parasites, two cysteine proteases, Atg4 and ovarian tumor unit (Otu), have been identified to delipidate Atg8 to release this protein from membranes. Here, we investigated the role of cysteine proteases in Atg8 conjugation and deconjugation and found that the Plasmodium parasite consists of both activities. We successfully disrupted the genes individually; however, simultaneously, they were refractory to deletion and essential for parasite survival. Mutants lacking Atg4 and Otu showed normal blood and mosquito stage development. All mice infected with Otu KO sporozoites became patent; however, Atg4 KO sporozoites either failed to establish blood infection or showed delayed patency. Through in vitro and in vivo analysis, we found that Atg4 KO sporozoites invade and normally develop into early liver stages. However, nuclear and organelle differentiation was severely hampered during late stages and failed to mature into hepatic merozoites. We found a higher level of Atg8 in Atg4 KO parasites, and the deconjugation of Atg8 was hampered. We confirmed Otu localization on the apicoplast; however, parasites lacking Otu showed no visible developmental defects. Our data suggest that Atg4 is the primary deconjugating enzyme and that Otu cannot replace its function completely because it cleaves the peptide bond at the N-terminal side of glycine, thereby irreversibly inactivating Atg8 during its recycling. These findings highlight a role for the Atg8 deconjugation pathway in organelle biogenesis and maintenance of the homeostatic cellular balance., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Autophagy is a lysosomal degradation process known as autophagic flux, involving the engulfment of damaged proteins and organelles by double-membrane autophagosomes. It comprises microautophagy, chaperone-mediated autophagy (CMA), and macroautophagy. Macroautophagy consists of three stages: induction, autophagosome formation, and autolysosome formation. Atg8-family proteins are valuable for tracking autophagic structures and have been widely utilized for monitoring autophagy. The conversion of LC3 to its lipidated form, LC3-II, served as an indicator of autophagy. Autophagy is implicated in human pathophysiology, such as neurodegeneration, cancer, and immune disorders. Moreover, autophagy impacts urological diseases, such as interstitial cystitis /bladder pain syndrome (IC/BPS), ketamine-induced ulcerative cystitis (KIC), chemotherapy-induced cystitis (CIC), radiation cystitis (RC), erectile dysfunction (ED), bladder outlet obstruction (BOO), prostate cancer, bladder cancer, renal cancer, testicular cancer, and penile cancer. Autophagy plays a dual role in the management of urologic diseases, and the identification of potential biomarkers associated with autophagy is a crucial step towards a deeper understanding of its role in these diseases. Methods for monitoring autophagy include TEM, Western blot, immunofluorescence, flow cytometry, and genetic tools. Autophagosome and autolysosome structures are discerned via TEM. Western blot, immunofluorescence, northern blot, and RT-PCR assess protein/mRNA levels. Luciferase assay tracks flux; GFP-LC3 transgenic mice aid study. Knockdown methods (miRNA and RNAi) offer insights. This article extensively examines autophagy's molecular mechanism, pharmacological regulation, and therapeutic application involvement in urological diseases.

The inflammatory repressor TNIP1/ABIN-1 is important for keeping in check inflammatory and cell-death pathways to avoid potentially dangerous sustained activation of these pathways. We have now found that TNIP1 is rapidly degraded by selective macroautophagy/autophagy early (0-4 h) after activation of TLR3 by poly(I:C)-treatment to allow expression of pro-inflammatory genes and proteins. A few hours later (6 h), TNIP1 levels rise again to counteract sustained inflammatory signaling. TBK1-mediated phosphorylation of a TNIP1 LIR motif regulates selective autophagy of TNIP1 by stimulating interaction with Atg8-family proteins. This is a novel level of regulation of TNIP1, whose protein level is crucial for controlling inflammatory signaling.

The (macro)autophagy field is facing a paradigm shift after the recent discovery that cytosolic cargoes can still be selectively targeted to phagophores (the precursors to autophagosomes) even in the absence of LC3 or other Atg8-protein family members. Several in vitro studies have indeed reported on the existence of an unconventional selective autophagic pathway that involves the in-situ formation of an autophagosome around the cargo through the direct selective autophagy receptor-mediated recruitment of RB1CC1/FIP200, thereby bypassing the requirement of LC3. In an article recently published in Science , we demonstrate the physiological importance of this unconventional autophagic pathway in the context of TNF (tumor necrosis factor) signaling. We show that it promotes the degradation of the cytotoxic TNFRSF1A/TNFR1 (TNF receptor superfamily member 1A) complex II that assembles upon TNF sensing and thereby protects mice from TNFRSF1A-driven embryonic lethality and skin inflammation. Abbreviations: ATG: autophagy related; CASP: caspase; FIR: RB1CC1/FIP200-interacting region; LIR: LC3-interacting region; M1: linear; PAS: phagophore assembly site; PtdIns3K: phosphatidylinositol 3-kinase; TNF: tumor necrosis factor; TNFRSF1A: TNF receptor superfamily member 1A.

The selectivity in selective macroautophagy/autophagy pathways is achieved via selective autophagy receptors (SARs) - proteins that bind a ligand on the substrate to be degraded and an Atg8-family protein on the growing autophagic membrane, phagophore, effectively bridging them. In mammals, the most common ligand of SARs is ubiquitin, a small protein modifier that tags substrates for their preferential degradation by autophagy. Consequently, most common SARs are ubiquitin-binding SARs, such as SQSTM1/p62 (sequestosome 1). Surprisingly, there is only one SAR of this type in yeast - Cue5, which acts as the receptor for aggrephagy and proteaphagy - pathways that remove ubiquitinated protein aggregates and proteasomes, respectively. However, recent studies described ubiquitin-dependent autophagic pathways that do not require Cue5, e.g. the stationary phase lipophagy for lipid droplets or nitrogen starvation-induced mitophagy for mitochondria. What is the role of ubiquitin in these pathways? Here, we propose that ubiquitinated lipid droplets and mitochondria are recognized by alternative ubiquitin-binding SARs. Our analysis identifies proteins that could potentially fulfill this role in yeast. We think that matching of ubiquitin-dependent (but Cue5-independent) autophagic pathways with ubiquitin- and Atg8-binding proteins enlisted here might uncover novel ubiquitin-binding SARs in yeast. Abbreviations: AIM: Atg8-family interacting motif; CUE: coupling of ubiquitin conjugation to ER degradation; ERMES: endoplasmic reticulum-mitochondria encounter structure; HECT: homologous to the E6-AP carboxyl terminus; LD: lipid droplet; SAR: selective autophagy receptor; SGD: Saccharomyces Genome Database; UBA: ubiquitin-associated; UBX: ubiquitin regulatory X; UIM: ubiquitin-interacting motif.

The canonical autophagy pathway in mammalian cells sequesters diverse cytoplasmic cargo within the double membrane autophagosomes that eventually convert into degradative compartments via fusion with endolysosomal intermediates. Here, we report that autophagosomal membranes show permeability in cells lacking principal ATG8 proteins (mATG8s) and are unable to mature into autolysosomes. Using a combination of methods including a novel in vitro assay to measure membrane sealing, we uncovered a previously unappreciated function of mATG8s to maintain autophagosomal membranes in a sealed state. The mATG8 proteins GABARAP and LC3A bind to key ESCRT-I components contributing, along with other ESCRTs, to the integrity and imperviousness of autophagic membranes. Autophagic organelles in cells lacking mATG8s are permeant, are arrested as amphisomes, and do not progress to functional autolysosomes. Thus, autophagosomal organelles need to be maintained in a sealed state in order to become lytic autolysosomes., (© 2023 The Authors.)