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ABSTRACTATG5 plays a pivotal role in membrane Atg8ylation, influencing downstream processes encompassing canonical autophagy and noncanonical processes. Remarkably, genetic ablation of ATG5 in myeloid cells leads to an exacerbated pathological state in murine models of tuberculosis, characterized by an early surge in mortality much more severe when compared to the depletion of other components involved in Atg8ylation or canonical autophagy. This study shows that in the absence of ATG5, but not other core canonical autophagy factors, endolysosomal organelles display a lysosomal hypersensitivity phenotype when subjected to damage. This is in part due to a compromised recruitment of ESCRT proteins to lysosomes in need of repair. Mechanistically, in the absence of ATG5, the ESCRT protein PDCD6IP/ALIX is sequestered by the alternative conjugate ATG12–ATG3, contributing to excessive exocytic processes while not being available for lysosomal repair. Specifically, this condition increases secretion of extracellular vesicles and particles, and leads to excessive degranulation in neutrophils. Our findings uncover unique functions of ATG5 outside of the autophagy and Atg8ylation paradigm. This finding is of in vivo relevance for tuberculosis pathogenesis as modeled in mice.Abbreviations:Atg5: autophagy related 5; ESCRT: endosomal sorting complex required for transport; EVPs: extracellular vesicles and particles; FPR1: formyl peptide receptor 1; LyHYP: lysosomal hypersensitivity phenotype; LysoIP: lysosome immunopurification; Mtb: Mycobacterium tuberculosis; ORF3a: open reading frame 3a protein; PDCD6IP/ALIX: programmed cell death 6 interacting protein; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2, TFEB: transcription factor EB.

ABSTRACTThe functions of mammalian Atg8 proteins (mATG8s) expand beyond canonical autophagy and include processes collectively referred to as Atg8ylation. Global modulation of protein synthesis under stress conditions is governed by MTOR and liquid-liquid phase separated condensates containing ribonucleoprotein particles known as stress granules (SGs). We report that lysosomal damage induces SGs acting as a hitherto unappreciated inhibitor of protein translation via EIF2A/eIF2α phosphorylation while favoring an ATF4-dependent integrated stress response. SGs are induced by lysosome-damaging agents, SARS-CoV-2 open reading frame 3a protein (ORF3a) expression, Mycobacterium tuberculosisinfection, and exposure to proteopathic MAPT/tau. Proteomic studies revealed recruitment to damaged lysosomes of the core SG proteins NUFIP2 and G3BP1 along with the GABARAPs of the mATG8 family. The recruitment of these proteins is independent of SG condensates or canonical autophagy. GABARAPs interact directly with NUFIP2 and G3BP1 whereas Atg8ylation is needed for their recruitment to damaged lysosomes. At the lysosome, NUFIP2 contributes to MTOR inactivation together with LGALS8 (galectin 8) via the Ragulator-RRAGA-RRAGB complex. The separable functions of NUFIP2 and G3BP1 in SG formation vis-a-vis their role in MTOR inactivation are governed by GABARAP and Atg8ylation. Thus, cells employ membrane Atg8ylation to control and coordinate SG and MTOR responses to lysosomal damage.Abbreviations:Atg8: autophagy related 8; ATG: autophagy related; ATF4: activating transcription factor 4; EIF2A/eIF2α: eukaryotic translation initiation factor 2A; GABARAP: GABA type A receptor-associated protein; G3BP1: G3BP stress granule assembly factor 1; LLOMe: L-leucyl-L-leucine methyl ester; LysoIP: lysosome immunopurification; mRNA: messenger ribonucleic acid; MTOR: mechanistic target of rapamycin kinase; NUFIP2: nuclear FMR1 interacting protein 2; ORF3a: open reading frame 3a protein; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SG: stress granule; TIA1: TIA1 cytotoxic granule associated RNA binding protein

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 tuberculosisor 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.

ABSTRACTMembrane integrity is essential for cellular survival and function. The spectrum of mechanisms protecting cellular and intracellular membranes is not fully known. Our recent work has uncovered a cellular system termed MERIT for lysosomal membrane repair, removal and replacement. Specifically, lysosomal membrane damage induces, in succession, ESCRT-dependent membrane repair, macroautophagy/autophagy-dominant removal of damaged lysosomes, and initiation of lysosomal biogenesis via transcriptional programs. The MERIT system is governed by galectins, a family of cytosolically synthesized lectins recognizing β-galactoside glycans. We found in this study that LGALS3 (galectin 3) detects membrane damage by detecting exposed lumenal glycosyl groups, recruits and organizes ESCRT components PDCD6IP/ALIX, CHMP4A, and CHMPB at damaged sites on the lysosomes, and facilitates ESCRT-driven repair of lysosomal membrane. At later stages, LGALS3 cooperates with TRIM16, an autophagy receptor-regulator, to engage autophagy machinery in removal of excessively damaged lysosomes. In the absence of LGALS3, repair and autophagy are less efficient, whereas TFEB nuclear translocation increases to compensate lysosomal deficiency via de novo lysosomal biogenesis. The MERIT system protects endomembrane integrity against a broad spectrum of agents damaging the endolysosomal network including lysosomotropic drugs, Mycobacterium tuberculosis, or neurotoxic MAPT/tau.AbbreviationsAMPK: AMP-activated protein kinase; APEX2: engineered ascorbate peroxidase 2; ATG13: autophagy related 13; ATG16L1: autophagy related 16 like 1; BMMs: bone marrow-derived macrophages; ESCRT: endosomal sorting complexes required for transport; GPN: glycyl-L-phenylalanine 2-naphthylamide; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MERIT: membrane repair, removal and replacement; MTOR: mechanistic target of rapamycin kinase; TFEB: transcription factor EB; TFRC: transferrin receptor; TRIM16: tripartite motif-containing 16

Autophagy is conventionally described as a degradative, catabolic pathway and a tributary to the lysosomal system where the cytoplasmic material sequestered by autophagosomes gets degraded. However, autophagosomes or autophagosome-related organelles do not always follow this route. It has recently come to light that autophagy can terminate in cytosolic protein secretion or release of sequestered material from the cells, rather than in their degradation. In this review, we address this relatively new but growing aspect of autophagy as a complex pathway, which is far more versatile than originally anticipated.

ABSTRACTThe Ser/Thr protein kinase MTOR (mechanistic target of rapamycin kinase) regulates cellular metabolism and controls macroautophagy/autophagy. Autophagy has both metabolic and quality control functions, including recycling nutrients at times of starvation and removing dysfunctional intracellular organelles. Lysosomal damage is one of the strongest inducers of autophagy, and yet mechanisms of its activation in response to lysosomal membrane damage are not fully understood. Our recent study has uncovered a new signal transduction system based on cytosolic galectins that elicits autophagy by controlling master regulators of metabolism and autophagy, MTOR and AMPK, in response to lysosomal damage. Thus, intracellular galectins are not, as previously thought, passive tags recognizing damage to guide selective autophagy receptors, but control the activation state of AMPK and MTOR in response to endomembrane damage.Abbreviations:MTOR: mechanistic target of rapamycin kinase; AMPK: AMP-activated protein kinase / Protein Kinase AMP-Activated; SLC38A9: Solute Carrier Family 38 Member 9; APEX2: engineered ascorbate peroxidase 2; RRAGA/B: Ras Related GTP Binding A or B; LAMTOR1: Late Endosomal/Lysosomal Adaptor, MAPK and MTOR Activator 1; LGALS8: Lectin, Galactoside-Binding, Soluble, 8 / Galectin 8; LGALS9: Lectin, Galactoside-Binding, Soluble, 9 / Galectin 9; TAK1: TGF-Beta Activated Kinase 1 / Mitogen-Activated Protein Kinase Kinase Kinase 7 (MAP3K7); STK11/LKB1: Serine/Threonine Kinase 11 / Liver Kinase B1; ULK1: Unc-51 Like Autophagy Activating Kinase 1.

ABSTRACTMacroautophagy/autophagy delivers cytoplasmic cargo to lysosomes for degradation. In yeast, the single Atg8 protein plays a role in the formation of autophagosomes whereas in mammalian cells there are five to seven paralogs, referred to as mammalian Atg8s (mAtg8s: GABARAP, GABARAPL1, GABARAPL2, LC3A, LC3B, LC3B2 and LC3C) with incompletely defined functions. Here we show that a subset of mAtg8s directly control lysosomal biogenesis. This occurs at the level of TFEB, the principal regulator of the lysosomal transcriptional program. mAtg8s promote TFEB’s nuclear translocation in response to stimuli such as starvation. GABARAP interacts directly with TFEB, whereas RNA-Seq analyses reveal that knockout of six genes encoding mAtg8s, or a triple knockout of the genes encoding all GABARAPs, diminishes the TFEB transcriptional program. We furthermore show that GABARAPs in cooperation with other proteins, IRGM, a factor implicated in tuberculosis and Crohn disease, and STX17, are required during starvation for optimal inhibition of MTOR, an upstream kinase of TFEB, and activation of the PPP3/calcineurin phosphatase that dephosphorylates TFEB, thus promoting its nuclear translocation. In conclusion, mAtg8s, IRGM and STX17 control lysosomal biogenesis by their combined or individual effects on MTOR, TFEB, and PPP3/calcineurin, independently of their roles in the formation of autophagosomal membranes.Abbreviations: AMPK: AMP-activated protein kinase; IRGM: immunity related GTPase M; mAtg8s: mammalian Atg8 proteins; MTOR: mechanistic target of rapamycin kinase; PPP3CB: protein phosphatase 3 catalytic subunit beta; RRAGA: Ras related GTP binding A.; STX17: syntaxin 17; ULK1: unc-51 like autophagy activating kinase 1

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

ABSTRACTMacroautophagy/autophagy is a homeostatic process delivering cytoplasmic targets, including damaged organelles, to lysosomes for degradation; however, it is not completely understood how compromised endomembranes are recognized by the autophagic apparatus. We have described previously that the TRIM family of proteins act as receptors for selective autophagy. In this study we uncovered the property of TRIMs to directly interact with members of the family of cytosolic lectins termed galectins. Galectins patrol the cytoplasm and recognize compromised membranes. We show that TRIM16 uses LGALS3 (galectin 3) to detect damaged lysosomes and phagosomes. TRIM16 assembles the core autophagic machinery and is found in protein complexes with MTOR and TFEB, thus regulating their activity to set in motion endomembrane quality control. The TRIM16-LGALS3 system plays a key role in autophagic homeostasis of lysosomes and in the control of Mycobacterium tuberculosis in vivo.

ABSTRACTMacroautophagy/autophagy plays a role in unconventional secretion of leaderless cytosolic proteins. Whether and how secretory autophagy diverges from conventional degradative autophagy is unclear. We have shown that the prototypical secretory autophagy cargo IL1B/IL-1β (interleukin 1 β) is recognized by TRIM16, and that this first to be identified secretory autophagy receptor interacts with the R-SNARE SEC22B to jointly deliver cargo to the MAP1LC3B-II-positive sequestration membranes. Cargo secretion is unaffected by knockdowns of STX17, a SNARE catalyzing autophagosome-lysosome fusion as a prelude to cargo degradation. Instead, SEC22B in combination with plasma membrane syntaxins completes cargo secretion. Thus, secretory autophagy diverges from degradative autophagy by using specialized receptors and a dedicated SNARE machinery to bypass fusion with lysosomes.