ER-phagy: mechanisms, regulation, and diseases connected to the lysosomal clearance of the endoplasmic reticulum.

ER-phagy (reticulophagy) defines the degradation of portions of the endoplasmic reticulum (ER) within lysosomes or vacuoles. It is part of the self-digestion (i.e., autophagic) programs recycling cytoplasmic material and organelles, which rapidly mobilize metabolites in cells confronted with nutrient shortage. Moreover, selective clearance of ER subdomains participates in the control of ER size and activity during ER stress, the reestablishment of ER homeostasis after ER stress resolution, and the removal of ER parts in which aberrant and potentially cytotoxic material has been segregated. ER-phagy relies on the individual and/or concerted activation of the ER-phagy receptors, ER peripheral or integral membrane proteins that share the presence of LC3/Atg8-binding motifs in their cytosolic domains. ER-phagy involves the physical separation of portions of the ER from the bulk ER network and their delivery to the endolysosomal/vacuolar catabolic district. This last step is accomplished by a variety of mechanisms including macro-ER-phagy (in which ER fragments are sequestered by double-membrane autophagosomes that eventually fuse with lysosomes/vacuoles), micro-ER-phagy (in which ER fragments are directly engulfed by endosomes/lysosomes/vacuoles), or direct fusion of ER-derived vesicles with lysosomes/vacuoles. ER-phagy is dysfunctional in specific human diseases, and its regulators are subverted by pathogens, highlighting its crucial role for cell and organism life.

Heteromeric clusters of ubiquitinated ER-shaping proteins drive ER-phagy.

Membrane-shaping proteins characterized by reticulon homology domains play an important part in the dynamic remodelling of the endoplasmic reticulum (ER). An example of such a protein is FAM134B, which can bind LC3 proteins and mediate the degradation of ER sheets through selective autophagy (ER-phagy)1. Mutations in FAM134B result in a neurodegenerative disorder in humans that mainly affects sensory and autonomic neurons2. Here we report that ARL6IP1, another ER-shaping protein that contains a reticulon homology domain and is associated with sensory loss3, interacts with FAM134B and participates in the formation of heteromeric multi-protein clusters required for ER-phagy. Moreover, ubiquitination of ARL6IP1 promotes this process. Accordingly, disruption of Arl6ip1 in mice causes an expansion of ER sheets in sensory neurons that degenerate over time. Primary cells obtained from Arl6ip1-deficient mice or from patients display incomplete budding of ER membranes and severe impairment of ER-phagy flux. Therefore, we propose that the clustering of ubiquitinated ER-shaping proteins facilitates the dynamic remodelling of the ER during ER-phagy and is important for neuronal maintenance.

Digest it all: the lysosomal turnover of cytoplasmic aggregates.

Aggrephagy describes the selective lysosomal transport and turnover of cytoplasmic protein aggregates by macro-autophagy. In this process, protein aggregates and conglomerates are polyubiquitinated and then sequestered by autophagosomes. Soluble selective autophagy receptors (SARs) are central to aggrephagy and physically bind to both ubiquitin and the autophagy machinery, thus linking the cargo to the forming autophagosomal membrane. Because the accumulation of protein aggregates is associated with cytotoxicity in several diseases, a better molecular understanding of aggrephagy might provide a conceptual framework to develop therapeutic strategies aimed at delaying the onset of these pathologies by preventing the buildup of potentially toxic aggregates. We review recent advances in our knowledge about the mechanism of aggrephagy.

Mammalian ATG8 proteins maintain autophagosomal membrane integrity through ESCRTs.

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.

A dimer to bridge early autophagosomal membranes.

The Atg1/ULK complex plays a key role in the early stages of autophagosome assembly. In this issue, Ragusa et al. reveal the molecular basis for some interactions within this complex, finding that the crescent-shaped Atg17 dimer is critical for autophagy, whereas Atg1 may have the ability to cluster membranes.

Phospholipid imbalance impairs autophagosome completion.

Autophagy, a conserved eukaryotic intracellular catabolic pathway, maintains cell homeostasis by lysosomal degradation of cytosolic material engulfed in double membrane vesicles termed autophagosomes, which form upon sealing of single-membrane cisternae called phagophores. While the role of phosphatidylinositol 3-phosphate (PI3P) and phosphatidylethanolamine (PE) in autophagosome biogenesis is well-studied, the roles of other phospholipids in autophagy remain rather obscure. Here we utilized budding yeast to study the contribution of phosphatidylcholine (PC) to autophagy. We reveal for the first time that genetic loss of PC biosynthesis via the CDP-DAG pathway leads to changes in lipid composition of autophagic membranes, specifically replacement of PC by phosphatidylserine (PS). This impairs closure of the autophagic membrane and autophagic flux. Consequently, we show that choline-dependent recovery of de novo PC biosynthesis via the CDP-choline pathway restores autophagosome formation and autophagic flux in PC-deficient cells. Our findings therefore implicate phospholipid metabolism in autophagosome biogenesis.

Decoding the function of Atg13 phosphorylation reveals a role of Atg11 in bulk autophagy initiation.

Autophagy is initiated by the assembly of multiple autophagy-related proteins that form the phagophore assembly site where autophagosomes are formed. Atg13 is essential early in this process, and a hub of extensive phosphorylation. How these multiple phosphorylations contribute to autophagy initiation, however, is not well understood. Here we comprehensively analyze the role of phosphorylation events on Atg13 during nutrient-rich conditions and nitrogen starvation. We identify and functionally characterize 48 in vivo phosphorylation sites on Atg13. By generating reciprocal mutants, which mimic the dephosphorylated active and phosphorylated inactive state of Atg13, we observe that disrupting the dynamic regulation of Atg13 leads to insufficient or excessive autophagy, which are both detrimental to cell survival. We furthermore demonstrate an involvement of Atg11 in bulk autophagy even during nitrogen starvation, where it contributes together with Atg1 to the multivalency that drives phase separation of the phagophore assembly site. These findings reveal the importance of post-translational regulation on Atg13 early during autophagy initiation, which provides additional layers of regulation to control bulk autophagy activity and integrate cellular signals.

SNARE proteins are required for macroautophagy.

Macroautophagy mediates the degradation of long-lived proteins and organelles via the de novo formation of double-membrane autophagosomes that sequester cytoplasm and deliver it to the vacuole/lysosome; however, relatively little is known about autophagosome biogenesis. Atg8, a phosphatidylethanolamine-conjugated protein, was previously proposed to function in autophagosome membrane expansion, based on the observation that it mediates liposome tethering and hemifusion in vitro. We show here that with physiological concentrations of phosphatidylethanolamine, Atg8 does not act as a fusogen. Rather, we provide evidence for the involvement of exocytic Q/t-SNAREs in autophagosome formation, acting in the recruitment of key autophagy components to the site of autophagosome formation, and in regulating the organization of Atg9 into tubulovesicular clusters. Additionally, we found that the endosomal Q/t-SNARE Tlg2 and the R/v-SNAREs Sec22 and Ykt6 interact with Sso1-Sec9, and are required for normal Atg9 transport. Thus, multiple SNARE-mediated fusion events are likely to be involved in autophagosome biogenesis.

ER-Phagy, ER Homeostasis, and ER Quality Control: Implications for Disease.

Lysosomal degradation of endoplasmic reticulum (ER) fragments by autophagy, termed ER-phagy or reticulophagy, occurs under normal as well as stress conditions. The recent discovery of multiple ER-phagy receptors has stimulated studies on the roles of ER-phagy. We discuss how the ER-phagy receptors and the cellular components that work with these receptors mediate two important functions: ER homeostasis and ER quality control. We highlight that ER-phagy plays an important role in alleviating ER expansion induced by ER stress, and acts as an alternative disposal pathway for misfolded proteins. We suggest that the latter function explains the emerging connection between ER-phagy and disease. Additional ER-phagy-associated functions and important unanswered questions are also discussed.

Getting on the right track: Interactions between viruses and the cytoskeletal motor proteins.

The cytoskeleton is an essential component of the cell and it is involved in multiple physiological functions, including intracellular organization and transport. It is composed of three main families of proteinaceous filaments; microtubules, actin filaments and intermediate filaments and their accessory proteins. Motor proteins, which comprise the dynein, kinesin and myosin superfamilies, are a remarkable group of accessory proteins that mainly mediate the intracellular transport of cargoes along with the cytoskeleton. Like other cellular structures and pathways, viruses can exploit the cytoskeleton to promote different steps of their life cycle through associations with motor proteins. The complexity of the cytoskeleton and the differences among viruses, however, has led to a wide diversity of interactions, which in most cases remain poorly understood. Unveiling the details of these interactions is necessary not only for a better comprehension of specific infections, but may also reveal new potential drug targets to fight dreadful diseases such as rabies disease and acquired immunodeficiency syndrome (AIDS). In this review, we describe a few examples of the mechanisms that some human viruses, that is, rabies virus, adenovirus, herpes simplex virus, human immunodeficiency virus, influenza A virus and papillomavirus, have developed to hijack dyneins, kinesins and myosins.

Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

Genetic Coding Variant in GPR65 Alters Lysosomal pH and Links Lysosomal Dysfunction with Colitis Risk.

Although numerous polymorphisms have been associated with inflammatory bowel disease (IBD), identifying the function of these genetic factors has proved challenging. Here we identified a role for nine genes in IBD susceptibility loci in antibacterial autophagy and characterized a role for one of these genes, GPR65, in maintaining lysosome function. Mice lacking Gpr65, a proton-sensing G protein-coupled receptor, showed increased susceptibly to bacteria-induced colitis. Epithelial cells and macrophages lacking GPR65 exhibited impaired clearance of intracellular bacteria and accumulation of aberrant lysosomes. Similarly, IBD patient cells and epithelial cells expressing an IBD-associated missense variant, GPR65 I231L, displayed aberrant lysosomal pH resulting in lysosomal dysfunction, impaired bacterial restriction, and altered lipid droplet formation. The GPR65 I231L polymorphism was sufficient to confer decreased GPR65 signaling. Collectively, these data establish a role for GPR65 in IBD susceptibility and identify lysosomal dysfunction as a potentially causative element in IBD pathogenesis with effects on cellular homeostasis and defense.

ER-phagy requires the assembly of actin at sites of contact between the cortical ER and endocytic pits.

Fragments of the endoplasmic reticulum (ER) are selectively delivered to the lysosome (mammals) or vacuole (yeast) in response to starvation or the accumulation of misfolded proteins through an autophagic process known as ER-phagy. A screen of the Saccharomyces cerevisiae deletion library identified end3Δ as a candidate knockout strain that is defective in ER-phagy during starvation conditions, but not bulk autophagy. We find that loss of End3 and its stable binding partner Pan1, or inhibition of the Arp2/3 complex that is coupled by the End3-Pan1 complex to endocytic pits, blocks the association of the cortical ER autophagy receptor, Atg40, with the autophagosomal assembly scaffold protein Atg11. The membrane contact site module linking the rim of cortical ER sheets and endocytic pits, consisting of Scs2 or Scs22, Osh2 or Osh3, and Myo3 or Myo5, is also needed for ER-phagy. Both Atg40 and Scs2 are concentrated at the edges of ER sheets and can be cross-linked to each other. Our results are consistent with a model in which actin assembly at sites of contact between the cortical ER and endocytic pits contributes to ER sequestration into autophagosomes.

The dynamin Vps1 mediates Atg9 transport to the sites of autophagosome formation.

Autophagy is a key process in eukaryotes to maintain cellular homeostasis by delivering cellular components to lysosomes/vacuoles for degradation and reuse of the resulting metabolites. Membrane rearrangements and trafficking events are mediated by the core machinery of autophagy-related (Atg) proteins, which carry out a variety of functions. How Atg9, a lipid scramblase and the only conserved transmembrane protein within this core Atg machinery, is trafficked during autophagy remained largely unclear. Here, we addressed this question in yeast Saccharomyces cerevisiae and found that retromer complex and dynamin Vps1 mutants alter Atg9 subcellular distribution and severely impair the autophagic flux by affecting two separate autophagy steps. We provide evidence that Vps1 interacts with Atg9 at Atg9 reservoirs. In the absence of Vps1, Atg9 fails to reach the sites of autophagosome formation, and this results in an autophagy defect. The function of Vps1 in autophagy requires its GTPase activity. Moreover, Vps1 point mutants associated with human diseases such as microcytic anemia and Charcot-Marie-Tooth are unable to sustain autophagy and affect Atg9 trafficking. Together, our data provide novel insights on the role of dynamins in Atg9 trafficking and suggest that a defect in this autophagy step could contribute to severe human pathologies.

The surface of lipid droplets constitutes a barrier for endoplasmic reticulum-resident integral membrane proteins.

Lipid droplets (LDs) are globular subcellular structures that store neutral lipids. LDs are closely associated with the endoplasmic reticulum (ER) and are limited by a phospholipid monolayer harboring a specific set of proteins. Most of these proteins associate with LDs through either an amphipathic helix or a membrane-embedded hairpin motif. Here, we address the question of whether integral membrane proteins can localize to the surface of LDs. To test this, we fused perilipin 3 (PLIN3), a mammalian LD-targeted protein, to ER-resident proteins. The resulting fusion proteins localized to the periphery of LDs in both yeast and mammalian cells. This peripheral LD localization of the fusion proteins, however, was due to a redistribution of the ER around LDs, as revealed by bimolecular fluorescence complementation between ER- and LD-localized partners. A LD-tethering function of PLIN3-containing membrane proteins was confirmed by fusing PLIN3 to the cytoplasmic domain of an outer mitochondrial membrane protein, OM14. Expression of OM14-PLIN3 induced a close apposition between LDs and mitochondria. These data indicate that the ER-LD junction constitutes a barrier for ER-resident integral membrane proteins.

The yeast LYST homolog Bph1 is a Rab5 effector and prevents Atg8 lipidation at endosomes.

Lysosomes mediate degradation of macromolecules to their precursors for cellular recycling. Additionally, lysosome-related organelles mediate cell type-specific functions. Chédiak-Higashi syndrome is an autosomal, recessive disease, in which loss of the protein LYST causes defects in lysosomes and lysosome-related organelles. The molecular function of LYST, however, is largely unknown. Here, we dissected the function of the yeast LYST homolog, Bph1. We show that Bph1 is an endosomal protein and an effector of the minor Rab5 isoform Ypt52. Strikingly, bph1Δ mutant cells have lipidated Atg8 on their endosomes, which is sorted via late endosomes into the vacuole lumen under non-autophagy-inducing conditions. In agreement with this, proteomic analysis of bph1Δ vacuoles reveals an accumulation of Atg8, reduced flux via selective autophagy, and defective endocytosis. Additionally, bph1Δ cells have reduced autophagic flux under starvation conditions. Our observations suggest that Bph1 is a novel Rab5 effector that maintains endosomal functioning. When Bph1 is lost, Atg8 is lipidated at endosomes even during normal growth and ends up in the vacuole lumen. Thus, our results contribute to the understanding of the role of LYST-related proteins and associated diseases.

Inhibition of IRGM establishes a robust antiviral immune state to restrict pathogenic viruses.

The type I interferon (IFN) response is the major host arsenal against invading viruses. IRGM is a negative regulator of IFN responses under basal conditions. However, the role of human IRGM during viral infection has remained unclear. In this study, we show that IRGM expression is increased upon viral infection. IFN responses induced by viral PAMPs are negatively regulated by IRGM. Conversely, IRGM depletion results in a robust induction of key viral restriction factors including IFITMs, APOBECs, SAMHD1, tetherin, viperin, and HERC5/6. Additionally, antiviral processes such as MHC-I antigen presentation and stress granule signaling are enhanced in IRGM-deficient cells, indicating a robust cell-intrinsic antiviral immune state. Consistently, IRGM-depleted cells are resistant to the infection with seven viruses from five different families, including Togaviridae, Herpesviridae, Flaviviverdae, Rhabdoviridae, and Coronaviridae. Moreover, we show that Irgm1 knockout mice are highly resistant to chikungunya virus (CHIKV) infection. Altogether, our work highlights IRGM as a broad therapeutic target to promote defense against a large number of human viruses, including SARS-CoV-2, CHIKV, and Zika virus.

Vps13 is required for the packaging of the ER into autophagosomes during ER-phagy.

Endoplasmic reticulum (ER) macroautophagy (hereafter called ER-phagy) uses autophagy receptors to selectively degrade ER domains in response to starvation or the accumulation of aggregation-prone proteins. Autophagy receptors package the ER into autophagosomes by binding to the ubiquitin-like yeast protein Atg8 (LC3 in mammals), which is needed for autophagosome formation. In budding yeast, cortical and cytoplasmic ER-phagy requires the autophagy receptor Atg40. While different ER autophagy receptors have been identified, little is known about other components of the ER-phagy machinery. In an effort to identify these components, we screened the genome-wide library of viable yeast deletion mutants for defects in the degradation of cortical ER following treatment with rapamycin, a drug that mimics starvation. Among the mutants we identified was vps13Δ. While yeast has one gene that encodes the phospholipid transporter VPS13, humans have four vacuolar protein-sorting (VPS) protein 13 isoforms. Mutations in all four human isoforms have been linked to different neurological disorders, including Parkinson's disease. Our findings have shown that Vps13 acts after Atg40 engages the autophagy machinery. Vps13 resides at contact sites between the ER and several organelles, including late endosomes. In the absence of Vps13, the cortical ER marker Rtn1 accumulated at late endosomes, and a dramatic decrease in ER packaging into autophagosomes was observed. Together, these studies suggest a role for Vps13 in the sequestration of the ER into autophagosomes at late endosomes. These observations may have important implications for understanding Parkinson's and other neurological diseases.

Manipulation of selective macroautophagy by pathogens at a glance.

Macroautophagy (hereafter autophagy) is a highly conserved catabolic pathway, which mediates the delivery of unwanted cytoplasmic structures and organelles to lysosomes for degradation. In numerous situations, autophagy is highly selective and exclusively targets specific intracellular components. Selective types of autophagy are a central element of our cell-autonomous innate immunity as they can mediate the turnover of viruses or bacteria, that gain access to the cytoplasm of the cell. Selective autophagy also modulates other aspects of our immunity by turning over specific immunoregulators. Throughout evolution, however, the continuous interaction between this fundamental cellular pathway and pathogens has led several pathogens to develop exquisite mechanisms to inhibit or subvert selective types of autophagy, to promote their intracellular multiplication. This Cell Science at a Glance article and the accompanying poster provides an overview of the selective autophagy of both pathogens, known as xenophagy, and of immunoregulators, and highlights a few archetypal examples that illustrate molecular strategies developed by viruses and bacteria to manipulate selective autophagy for their own benefit.

Molecular regulation of autophagosome formation.

Macroautophagy, hereafter autophagy, is a degradative process conserved among eukaryotes, which is essential to maintain cellular homeostasis. Defects in autophagy lead to numerous human diseases, including various types of cancer and neurodegenerative disorders. The hallmark of autophagy is the de novo formation of autophagosomes, which are double-membrane vesicles that sequester and deliver cytoplasmic materials to lysosomes/vacuoles for degradation. The mechanism of autophagosome biogenesis entered a molecular era with the identification of autophagy-related (ATG) proteins. Although there are many unanswered questions and aspects that have raised some controversies, enormous advances have been done in our understanding of the process of autophagy in recent years. In this review, we describe the current knowledge about the molecular regulation of autophagosome formation, with a particular focus on budding yeast and mammalian cells.