The mechanisms and roles of selective autophagy in mammals.

Autophagy is a process that targets various intracellular elements for degradation. Autophagy can be non-selective - associated with the indiscriminate engulfment of cytosolic components - occurring in response to nutrient starvation and is commonly referred to as bulk autophagy. By contrast, selective autophagy degrades specific targets, such as damaged organelles (mitophagy, lysophagy, ER-phagy, ribophagy), aggregated proteins (aggrephagy) or invading bacteria (xenophagy), thereby being importantly involved in cellular quality control. Hence, not surprisingly, aberrant selective autophagy has been associated with various human pathologies, prominently including neurodegeneration and infection. In recent years, considerable progress has been made in understanding mechanisms governing selective cargo engulfment in mammals, including the identification of ubiquitin-dependent selective autophagy receptors such as p62, NBR1, OPTN and NDP52, which can bind cargo and ubiquitin simultaneously to initiate pathways leading to autophagy initiation and membrane recruitment. This progress opens the prospects for enhancing selective autophagy pathways to boost cellular quality control capabilities and alleviate pathology.

Essential role for GABARAP autophagy proteins in interferon-inducible GTPase-mediated host defense.

Mammalian autophagy-related 8 (Atg8) homologs consist of LC3 proteins and GABARAPs, all of which are known to be involved in canonical autophagy. In contrast, the roles of Atg8 homologs in noncanonical autophagic processes are not fully understood. Here we show a unique role of GABARAPs, in particular gamma-aminobutyric acid (GABA)-A-receptor-associated protein-like 2 (Gabarapl2; also known as Gate-16), in interferon-γ (IFN-γ)-mediated antimicrobial responses. Cells that lacked GABARAPs but not LC3 proteins and mice that lacked Gate-16 alone were defective in the IFN-γ-induced clearance of vacuolar pathogens such as Toxoplasma. Gate-16 but not LC3b specifically associated with the small GTPase ADP-ribosylation factor 1 (Arf1) to mediate uniform distribution of interferon-inducible GTPases. The lack of GABARAPs reduced Arf1 activation, which led to formation of interferon-inducible GTPase-containing aggregates and hampered recruitment of interferon-inducible GTPases to vacuolar pathogens. Thus, GABARAPs are uniquely required for antimicrobial host defense through cytosolic distribution of interferon-inducible GTPases.

YIPF3 and YIPF4 regulate autophagic turnover of the Golgi apparatus.

The degradation of organelles by autophagy is essential for cellular homeostasis. The Golgi apparatus has recently been demonstrated to be degraded by autophagy, but little is known about how the Golgi is recognized by the forming autophagosome. Using quantitative proteomic analysis and two novel Golgiphagy reporter systems, we found that the five-pass transmembrane Golgi-resident proteins YIPF3 and YIPF4 constitute a Golgiphagy receptor. The interaction of this complex with LC3B, GABARAP, and GABARAPL1 is dependent on a LIR motif within YIPF3 and putative phosphorylation sites immediately upstream; the stability of the complex is governed by YIPF4. Expression of a YIPF3 protein containing a mutated LIR motif caused an elongated Golgi morphology, indicating the importance of Golgi turnover via selective autophagy. The reporter assays reported here may be readily adapted to different experimental contexts to help deepen our understanding of Golgiphagy.

Autophagy and autophagy-related proteins in the immune system.

Autophagy is an intracellular bulk degradation system that is highly conserved in eukaryotes. The discovery of autophagy-related ('ATG') proteins in the 1990s greatly advanced the mechanistic understanding of autophagy and clarified the fact that autophagy serves important roles in various biological processes. In addition, studies have revealed other roles for the autophagic machinery beyond autophagy. In this Review, we introduce advances in the knowledge of the roles of autophagy and its components in immunity, including innate immunity, inflammatory responses and adaptive immunity.

Bidirectional Control of Autophagy by BECN1 BARA Domain Dynamics.

Membrane targeting of the BECN1-containing class III PI 3-kinase (PI3KC3) complexes is pivotal to the regulation of autophagy. The interaction of PI3KC3 complex II and its ubiquitously expressed inhibitor, Rubicon, was mapped to the first ß sheet of the BECN1 BARA domain and the UVRAG BARA2 domain by hydrogen-deuterium exchange and cryo-EM. These data suggest that the BARA ß sheet 1 unfolds to directly engage the membrane. This mechanism was confirmed using protein engineering, giant unilamellar vesicle assays, and molecular simulations. Using this mechanism, a BECN1 ß sheet-1 derived peptide activates both PI3KC3 complexes I and II, while HIV-1 Nef inhibits complex II. These data reveal how BECN1 switches on and off PI3KC3 binding to membranes. The observations explain how PI3KC3 inhibition by Rubicon, activation by autophagy-inducing BECN1 peptides, and inhibition by HIV-1 Nef are mediated by the switchable ability of the BECN1 BARA domain to partially unfold and insert into membranes.

Lysophagy protects against propagation of α-synuclein aggregation through ruptured lysosomal vesicles.

The neuron-to-neuron propagation of misfolded α-synuclein (αSyn) aggregates is thought to be key to the pathogenesis of synucleinopathies. Recent studies have shown that extracellular αSyn aggregates taken up by the endosomal-lysosomal system can rupture the lysosomal vesicular membrane; however, it remains unclear whether lysosomal rupture leads to the transmission of αSyn aggregation. Here, we applied cell-based αSyn propagation models to show that ruptured lysosomes are the pathway through which exogenous αSyn aggregates transmit aggregation, and furthermore, this process was prevented by lysophagy, i.e., selective autophagy of damaged lysosomes. αSyn aggregates accumulated predominantly in lysosomes, causing their rupture, and seeded the aggregation of endogenous αSyn, initially around damaged lysosomes. Exogenous αSyn aggregates induced the accumulation of LC3 on lysosomes. This LC3 accumulation was not observed in cells in which a key regulator of autophagy, RB1CC1/FIP200, was knocked out and was confirmed as lysophagy by transmission electron microscopy. Importantly, RB1CC1/FIP200-deficient cells treated with αSyn aggregates had increased numbers of ruptured lysosomes and enhanced propagation of αSyn aggregation. Furthermore, various types of lysosomal damage induced using lysosomotropic reagents, depletion of lysosomal enzymes, or more toxic species of αSyn fibrils also exacerbated the propagation of αSyn aggregation, and impaired lysophagy and lysosomal membrane damage synergistically enhanced propagation. These results indicate that lysophagy prevents exogenous αSyn aggregates from escaping the endosomal-lysosomal system and transmitting aggregation to endogenous cytosolic αSyn via ruptured lysosomal vesicles. Our findings suggest that the progression and severity of synucleinopathies are associated with damage to lysosomal membranes and impaired lysophagy.

HKDC1, a target of TFEB, is essential to maintain both mitochondrial and lysosomal homeostasis, preventing cellular senescence.

Mitochondrial and lysosomal functions are intimately linked and are critical for cellular homeostasis, as evidenced by the fact that cellular senescence, aging, and multiple prominent diseases are associated with concomitant dysfunction of both organelles. However, it is not well understood how the two important organelles are regulated. Transcription factor EB (TFEB) is the master regulator of lysosomal function and is also implicated in regulating mitochondrial function; however, the mechanism underlying the maintenance of both organelles remains to be fully elucidated. Here, by comprehensive transcriptome analysis and subsequent chromatin immunoprecipitation-qPCR, we identified hexokinase domain containing 1 (HKDC1), which is known to function in the glycolysis pathway as a direct TFEB target. Moreover, HKDC1 was upregulated in both mitochondrial and lysosomal stress in a TFEB-dependent manner, and its function was critical for the maintenance of both organelles under stress conditions. Mechanistically, the TFEB-HKDC1 axis was essential for PINK1 (PTEN-induced kinase 1)/Parkin-dependent mitophagy via its initial step, PINK1 stabilization. In addition, the functions of HKDC1 and voltage-dependent anion channels, with which HKDC1 interacts, were essential for the clearance of damaged lysosomes and maintaining mitochondria-lysosome contact. Interestingly, HKDC1 regulated mitophagy and lysosomal repair independently of its prospective function in glycolysis. Furthermore, loss function of HKDC1 accelerated DNA damage-induced cellular senescence with the accumulation of hyperfused mitochondria and damaged lysosomes. Our results show that HKDC1, a factor downstream of TFEB, maintains both mitochondrial and lysosomal homeostasis, which is critical to prevent cellular senescence.

Neuronal MML-1/MXL-2 regulates systemic aging via glutamate transporter and cell nonautonomous autophagic and peroxidase activity.

Accumulating evidence has demonstrated the presence of intertissue-communication regulating systemic aging, but the underlying molecular network has not been fully explored. We and others previously showed that two basic helix-loop-helix transcription factors, MML-1 and HLH-30, are required for lifespan extension in several longevity paradigms, including germlineless Caenorhabditis elegans. However, it is unknown what tissues these factors target to promote longevity. Here, using tissue-specific knockdown experiments, we found that MML-1 and its heterodimer partners MXL-2 and HLH-30 act primarily in neurons to extend longevity in germlineless animals. Interestingly, however, the downstream cascades of MML-1 in neurons were distinct from those of HLH-30. Neuronal RNA interference (RNAi)-based transcriptome analysis revealed that the glutamate transporter GLT-5 is a downstream target of MML-1 but not HLH-30. Furthermore, the MML-1-GTL-5 axis in neurons is critical to prevent an age-dependent collapse of proteostasis and increased oxidative stress through autophagy and peroxidase MLT-7, respectively, in long-lived animals. Collectively, our study revealed that systemic aging is regulated by a molecular network involving neuronal MML-1 function in both neural and peripheral tissues.

Microautophagy regulated by STK38 and GABARAPs is essential to repair lysosomes and prevent aging.

Lysosomes are degradative organelles and signaling hubs that maintain cell and tissue homeostasis, and lysosomal dysfunction is implicated in aging and reduced longevity. Lysosomes are frequently damaged, but their repair mechanisms remain unclear. Here, we demonstrate that damaged lysosomal membranes are repaired by microautophagy (a process termed "microlysophagy") and identify key regulators of the first and last steps. We reveal the AGC kinase STK38 as a novel microlysophagy regulator. Through phosphorylation of the scaffold protein DOK1, STK38 is specifically required for the lysosomal recruitment of the AAA+ ATPase VPS4, which terminates microlysophagy by promoting the disassembly of ESCRT components. By contrast, microlysophagy initiation involves non-canonical lipidation of ATG8s, especially the GABARAP subfamily, which is required for ESCRT assembly through interaction with ALIX. Depletion of STK38 and GABARAPs accelerates DNA damage-induced cellular senescence in human cells and curtails lifespan in C. elegans, respectively. Thus, microlysophagy is regulated by STK38 and GABARAPs and could be essential for maintaining lysosomal integrity and preventing aging.

The P4-ATPase Drs2 interacts with and stabilizes the multisubunit tethering complex TRAPPIII in yeast.

Multisubunit Tethering Complexes (MTCs) are a set of conserved protein complexes that tether vesicles at the acceptor membrane. Interactions with other components of the trafficking machinery regulate MTCs through mechanisms that are partially understood. Here, we systematically investigate the interactome that regulates MTCs. We report that P4-ATPases, a family of lipid flippases, interact with MTCs that participate in the anterograde and retrograde transport at the Golgi, such as TRAPPIII. We use the P4-ATPase Drs2 as a paradigm to investigate the mechanism and biological relevance of this interplay during transport of Atg9 vesicles. Binding of Trs85, the sole-specific subunit of TRAPPIII, to the N-terminal tail of Drs2 stabilizes TRAPPIII on membranes loaded with Atg9 and is required for Atg9 delivery during selective autophagy, a role that is independent of P4-ATPase canonical functions. This mechanism requires a conserved I(S/R)TTK motif that also mediates the interaction of the P4-ATPases Dnf1 and Dnf2 with MTCs, suggesting a broader role of P4-ATPases in MTC regulation.

The autophagosome: origins unknown, biogenesis complex.

Healthy cells use autophagy as a general 'housekeeping' mechanism and to survive stress, including stress induced by nutrient deprivation. Autophagy is initiated at the isolation membrane (originally termed the phagophore), and the coordinated action of ATG (autophagy-related) proteins results in the expansion of this membrane to form the autophagosome. Although the biogenesis of the isolation membrane and the autophagosome is complex and incompletely understood, insight has been gained into the molecular processes involved in initiating the isolation membrane, the source from which this originates (for example, it was recently proposed that the isolation membrane forms from the mitochondria-associated endoplasmic reticulum (ER) membrane (MAM)) and the role of ATG proteins and the vesicular trafficking machinery in autophagosome formation.

Methods in mammalian autophagy research.

Autophagy has been implicated in many physiological and pathological processes. Accordingly, there is a growing scientific need to accurately identify, quantify, and manipulate the process of autophagy. However, as autophagy involves dynamic and complicated processes, it is often analyzed incorrectly. In this Primer, we discuss methods to monitor autophagy and to modulate autophagic activity, with a primary focus on mammalian macroautophagy.

The role of Atg proteins in autophagosome formation.

Macroautophagy is mediated by a unique organelle, the autophagosome, which encloses a portion of cytoplasm for delivery to the lysosome. Autophagosome formation is dynamically regulated by starvation and other stresses and involves complicated membrane reorganization. Since the discovery of yeast Atg-related proteins, autophagosome formation has been dissected at the molecular level. In this review we describe the molecular mechanism of autophagosome formation with particular focus on the function of Atg proteins and the long-standing discussion regarding the origin of the autophagosome membrane.

PACSIN1 is indispensable for amphisome-lysosome fusion during basal autophagy and subsets of selective autophagy.

Autophagy is an indispensable process that degrades cytoplasmic materials to maintain cellular homeostasis. During autophagy, double-membrane autophagosomes surround cytoplasmic materials and either fuse with endosomes (called amphisomes) and then lysosomes, or directly fuse with lysosomes, in both cases generating autolysosomes that degrade their contents by lysosomal hydrolases. However, it remains unclear if there are specific mechanisms and/or conditions which distinguish these alternate routes. Here, we identified PACSIN1 as a novel autophagy regulator. PACSIN1 deletion markedly decreased autophagic activity under basal nutrient-rich conditions but not starvation conditions, and led to amphisome accumulation as demonstrated by electron microscopic and co-localization analysis, indicating inhibition of lysosome fusion. PACSIN1 interacted with SNAP29, an autophagic SNARE, and was required for proper assembly of the STX17 and YKT6 complexes. Moreover, PACSIN1 was required for lysophagy, aggrephagy but not mitophagy, suggesting cargo-specific fusion mechanisms. In C. elegans, deletion of sdpn-1, a homolog of PACSINs, inhibited basal autophagy and impaired clearance of aggregated protein, implying a conserved role of PACSIN1. Taken together, our results demonstrate the amphisome-lysosome fusion process is preferentially regulated in response to nutrient state and stress, and PACSIN1 is a key to specificity during autophagy.

Rubicon prevents autophagic degradation of GATA4 to promote Sertoli cell function.

Autophagy degrades unnecessary proteins or damaged organelles to maintain cellular function. Therefore, autophagy has a preventive role against various diseases including hepatic disorders, neurodegenerative diseases, and cancer. Although autophagy in germ cells or Sertoli cells is known to be required for spermatogenesis and male fertility, it remains poorly understood how autophagy participates in spermatogenesis. We found that systemic knockout mice of Rubicon, a negative regulator of autophagy, exhibited a substantial reduction in testicular weight, spermatogenesis, and male fertility, associated with upregulation of autophagy. Rubicon-null mice also had lower levels of mRNAs of Sertoli cell-related genes in testis. Importantly, Rubicon knockout in Sertoli cells, but not in germ cells, caused a defect in spermatogenesis and germline stem cell maintenance in mice, indicating a critical role of Rubicon in Sertoli cells. In mechanistic terms, genetic loss of Rubicon promoted autophagic degradation of GATA4, a transcription factor that is essential for Sertoli cell function. Furthermore, androgen antagonists caused a significant decrease in the levels of Rubicon and GATA4 in testis, accompanied by elevated autophagy. Collectively, we propose that Rubicon promotes Sertoli cell function by preventing autophagic degradation of GATA4, and that this mechanism could be regulated by androgens.

Vacuolar protein Tag1 and Atg1-Atg13 regulate autophagy termination during persistent starvation in S. cerevisiae.

Under starvation conditions, cells degrade their own components via autophagy in order to provide sufficient nutrients to ensure their survival. However, even if starvation persists, the cell is not completely degraded through autophagy, implying the existence of some kind of termination mechanism. In the yeast Saccharomyces cerevisiae, autophagy is terminated after 10-12 h of nitrogen starvation. In this study, we found that termination is mediated by re-phosphorylation of Atg13 by the Atg1 protein kinase, which is also affected by PP2C phosphatases, and the eventual dispersion of the pre-autophagosomal structure, also known as the phagophore assembly site (PAS). In a genetic screen, we identified an uncharacterized vacuolar membrane protein, Tag1, as a factor responsible for the termination of autophagy. Re-phosphorylation of Atg13 and eventual PAS dispersal were defective in the Δtag1 mutant. The vacuolar luminal domain of Tag1 and autophagic progression are important for the behaviors of Tag1. Together, our findings reveal the mechanism and factors responsible for termination of autophagy in yeast.

THOC4 regulates energy homeostasis by stabilizing TFEB mRNA during prolonged starvation.

TFEB, a basic helix-loop-helix transcription factor, is a master regulator of autophagy, lysosome biogenesis and lipid catabolism. Compared to posttranslational regulation of TFEB, the regulation of TFEB mRNA stability remains relatively uncharacterized. In this study, we identified the mRNA-binding protein THOC4 as a novel regulator of TFEB. In mammalian cells, siRNA-mediated knockdown of THOC4 decreased the level of TFEB protein to a greater extent than other bHLH transcription factors. THOC4 bound to TFEB mRNA and stabilized it after transcription by maintaining poly(A) tail length. We further found that this mode of regulation was conserved in Caenorhabditiselegans and was essential for TFEB-mediated lipid breakdown, which becomes over-represented during prolonged starvation. Taken together, our findings reveal the presence of an additional layer of TFEB regulation by THOC4 and provide novel insights into the function of TFEB in mediating autophagy and lipid metabolism.

The parasitophorous vacuole membrane of Toxoplasma gondii is targeted for disruption by ubiquitin-like conjugation systems of autophagy.

Autophagy is a lysosomal degradation pathway that is important in cellular homeostasis. Prior work showed a key role for the autophagy related 5 (Atg5) in resistance to Toxoplasma gondii. Here we show that the cassette of autophagy proteins involved in the conjugation of microtubule-associated protein 1 light chain 3 (LC3) to phosphatidylethanolamine, including Atg7, Atg3, and the Atg12-Atg5-Atg16L1 complex play crucial roles in the control of T. gondii in vitro and in vivo. In contrast, pharmacologic modulation of the degradative autophagy pathway or genetic deletion of other essential autophagy genes had no substantial effects. Rather the conjugation system was required for targeting of LC3 and interferon-γ effectors onto the vacuolar membrane of T. gondii and its consequent disruption. These data suggest that the ubiquitin-like conjugation systems that reorganize intracellular membranes during canonical autophagy are necessary for proper targeting of immune effectors to the intracellular vacuole membranes utilized by pathogens.

Structural basis for autophagy inhibition by the human Rubicon-Rab7 complex.

Rubicon is a potent negative regulator of autophagy and a potential target for autophagy-inducing therapeutics. Rubicon-mediated inhibition of autophagy requires the interaction of the C-terminal Rubicon homology (RH) domain of Rubicon with Rab7-GTP. Here we report the 2.8-Å crystal structure of the Rubicon RH domain in complex with Rab7-GTP. Our structure reveals a fold for the RH domain built around four zinc clusters. The switch regions of Rab7 insert into pockets on the surface of the RH domain in a mode that is distinct from those of other Rab-effector complexes. Rubicon residues at the dimer interface are required for Rubicon and Rab7 to colocalize in living cells. Mutation of Rubicon RH residues in the Rab7-binding site restores efficient autophagic flux in the presence of overexpressed Rubicon, validating the Rubicon RH domain as a promising therapeutic target.

Non-canonical roles of ATG8 for TFEB activation.

Autophagy is an evolutionally conserved cytoplasmic degradation pathway in which the double membrane structure, autophagosome sequesters cytoplasmic material and delivers them to lysosomes for degradation. Many autophagy related (ATG) proteins participate in the regulation of the several steps of autophagic process. Among ATGs, ubiquitin-like protein, ATG8 plays a pivotal role in autophagy. ATG8 is directly conjugated on lipid in autophagosome membrane upon induction of autophagy thus providing a good marker to monitor and analyze autophagy process. However, recent discoveries suggest that ATG8 has autophagy independent non-canonical functions and ATG8 positive structures are not always autophagosomes. This review briefly overviews canonical and non-canonical roles of ATG8 and introduce novel function of ATG8 to activate Transcriptional Factor EB(TFEB), a master transcription factor of autophagy and lysosome function during lysosomal damage.