Autophagy and p62/sequestosome 1 generate neo-antimicrobial peptides (cryptides) from cytosolic proteins

In a manifestation of the immunological autophagy termed xenophagy, autophagic adapter proteins such as p62 and NDP52 directly capture microbes for delivery to autophagosomal organelles where they are eliminated. In a mirror image phenomenon, which is also an immunological variant of the process termed decryption, p62 and autophagy contribute to the elimination of Mycobacterium tuberculosis. During decryption, p62 sequesters cytosolic proteins into autophagosomes where they are proteolytically converted into peptides termed cryptides. A subset of cryptides possesses antimicrobial peptide properties exhibited upon their delivery to parasitophorous vacuoles where they kill intracellular microbes.     

ERADication of EDEM1 occurs by selective autophagy and requires deglycosylation by cytoplasmic peptide N-glycanase

ER degradation-enhancing α-mannosidase-like 1 protein (EDEM1) is involved in the routing of misfolded glycoproteins for degradation in the cytoplasm. Previously, we reported that EDEM1 leaves the endoplasmic reticulum via non-COPII vesicles (Zuber et al. in Proc Natl Acad Sci USA 104:4407–4412, 2007) and becomes degraded by basal autophagy (Le Fourn et al. in Cell Mol Life Sci 66:1434–1445, 2009). However, it is unknown which type of autophagy is involved. Likewise, how EDEM1 is targeted to autophagosomes remains elusive. We now show that EDEM1 is degraded by selective autophagy. It colocalizes with the selective autophagy cargo receptors p62/SQSTM1, neighbor of BRCA1 gene 1 (NBR1) and autophagy-linked FYVE (Alfy) protein, and becomes engulfed by autophagic isolation membranes. The interaction with p62/SQSTM1 and NBR1 is required for routing of EDEM1 to autophagosomes since it can be blocked by short inhibitory RNA knockdown of the cargo receptors. Furthermore, p62/SQSTM1 interacts only with deglycosylated EDEM1 that is also ubiquitinated. The deglycosylation of EDEM1 occurs by the cytosolic peptide N-glycanase and is a prerequisite for interaction and aggregate formation with p62/SQSTM1 as demonstrated by the effect of peptide N-glycanase inhibitors on the formation of protein aggregates. Conversely, aggregation of p62/SQSTM1 and EDEM1 occurs independent of cytoplasmic histone deacetylase. These data provide novel insight into the mechanism of autophagic degradation of the ER-associated protein degradation (ERAD) component EDEM1 and disclose hitherto unknown parallels with the clearance of cytoplasmic aggregates of misfolded proteins by selective autophagy.     

Mycobacterium tuberculosis activates the DNA-dependent cytosolic surveillance pathway within macrophages

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Highlights? M. tuberculosis permeabilizes phagosomes and activates cytosolic signaling pathways ? Host cytoplasmic DNA receptors sense M. tuberculosis extracellular DNA ? DNA recognition activates the STING/TBK1/IRF3 pathway ? Cytosolic sensing promotes M. tuberculosis infection     

Early endosomes and endosomal coatomer are required for autophagy

Autophagy, an intracellular degradative pathway, maintains cell homeostasis under normal and stress conditions. Nascent double-membrane autophagosomes sequester and enclose cytosolic components and organelles, and subsequently fuse with the endosomal pathway allowing content degradation. Autophagy requires fusion of autophagosomes with late endosomes, but it is not known if fusion with early endosomes is essential. We show that fusion of AVs with functional early endosomes is required for autophagy. Inhibition of early endosome function by loss of COPI subunits (β′, β, or α) results in accumulation of autophagosomes, but not an increased autophagic flux. COPI is required for ER-Golgi transport and early endosome maturation. Although loss of COPI results in the fragmentation of the Golgi, this does not induce the formation of autophagosomes. Loss of COPI causes defects in early endosome function, as both transferrin recycling and EGF internalization and degradation are impaired, and this loss of function causes an inhibition of autophagy, an accumulation of p62/SQSTM-1, and ubiquitinated proteins in autophagosomes.     

Phospholipase D1 regulates autophagic flux and clearance of α-synuclein aggregates

Many neurodegenerative diseases, such as Alzheimer''s disease and Parkinson''s disease, are characterized by abnormal accumulations of aggregated proteins. Brains in these diseases also show accumulation of autophagic vesicles in the neuronal cytoplasm, suggesting impairment of the autophagic process. As autophagy involves de novo membrane production and vesicle fusion, extensive changes in lipid molecules are necessary. However, the involvement of signaling lipid-modifying enzymes in autophagy and their roles in neurodegenerative diseases are not clear. Using specific inhibitor, we show that loss of phospholipase D1 (PLD1) activity resulted in an accumulation of microtubule-associated protein light chain 3 (LC3), p62, and polyubiquitinated proteins, signs representing malfunction in autophagic flux. Fluorescence and electron microscopic analyses demonstrated impaired fusion of autophagosomes with lysosomes, resulting in accumulation of autophagosomes. Within the cells with impaired autophagic flux, α-synuclein aggregates accumulated in autophagosomes. Knockdown of PLD1 expression using small interfering RNA also resulted in impaired autophagic flux and accumulation of α-synuclein aggregates in autophagosomes. Neuronal toxicity caused by α-synuclein accumulation was rescued by overexpression of PLD1; however, expression of activity-deficient mutant, PLD1-KRM, showed reduced rescue effects. Finally, we demonstrated that both PLD activity and expression levels were reduced in brain tissues of dementia with Lewy bodies (DLB) patients, whereas the amounts of α-synuclein and p62 were increased in the same tissue samples. Collectively, these results suggest that insufficient PLD activity, and therefore, the changes in phospholipid compositions within membranes, might be an important contributor to impaired autophagic process and protein accumulation in Lewy body diseases.Macroautophagy is the best-characterized autophagy pathway that mediates the lysosomal degradation of the cytoplasmic organelles and proteins.¹ In this paper, macroautophagy will be simply referred to as autophagy. Autophagy may be characterized by nonspecific sequestration and degradation of the bulk cytoplasm, a process that recycles essential building blocks for production of macromolecules under conditions where nutrients are limited. Autophagy may also occur to selectively degrade polyubiquitinated targets, and this is often referred to as the quality control autophagy.² Many long-lived proteins and perhaps protein aggregates may be the substrates of the quality control autophagy.As being an essential process for macromolecular metabolism, perturbation of autophagy has been linked to various human diseases, such as neurodegenerative diseases, cancer, and infectious diseases.³ Autophagic dysfunction in neurons, in particular, causes accumulation of aggregation-prone proteins and neurodegeneration that are associated with various neurodegenerative diseases, including Alzheimer''s disease (AD), Parkinson''s disease (PD), and Huntington''s disease (HD).⁴ Recently, genetic mutations that are linked to some of the major neurodegenerative diseases have turned out to reside in the genes that are involved in multiple steps in the autophagic pathways,⁴ implicating the therapeutic potential of controlling autophagy.Autophagy involves sequestration of cytoplasmic substrates by a double-membraned compartment known as the autophagosome (AP).¹ Autophagy process initiates with the formation of a distinct structure referred to as the phagophore that extends its ends and seals in circle to form the AP. APs can fuse with various endosomal vesicles, forming amphisomes, and eventually fuse with lysosomes to form autolysosomes, where degradation of contents takes place. Autophagy involves a wide range of changes in membrane structures, such as de novo membrane biogenesis and membrane fusion. Therefore, lipid molecules and lipid-metabolizing enzymes must play essential roles in the autophagic process. A well-characterized such lipid enzyme is the class III phosphatidylinositol-3 kinase (PI3K), Vps34, that is essential for biogenesis of APs through interactions with various proteins.⁵ Other than Vps34, little has been known about the roles of lipid enzymes in autophagic process.In the current study, we explored the role of phospholipase D1 (PLD1) in autophagy and clearance of α-synuclein aggregates, suspected culprit of PD.⁶ PLD1 generates phosphatidic acid (PA) from phosphatidylcholine and has been known to be involved in intracellular vesicle trafficking.⁷ Our results suggest that PLD1 is an important player for maintaining autophagic flux via regulating autolysosome formation. We also showed that enzymatic inhibition and reduction in expression of PLD1 resulted in impaired clearance of α-synuclein aggregates. Finally, our data showed that reduced expression, and thus activity, of PLD1 was associated with Lewy body diseases.     

Autophagy and acid phosphatase activity in the corpora allata of adult mated females of Diploptera punctata

The corpora allata exbibit cycles of synchronous cell growth and atrophy during ovarian cycles in adult females of the cockroach Diploptera punctata. In the present report, the process of synchronous autophagy of organelles which results in cellular atrophy was investigated. In general, unwanted organelles were sequentially sequestered by several different mechanisms and then targeted for destruction. Autophagy was initiated on day 4 when corpus allatum cells were largest and most actively synthesizing juvenile hormone. The first sign of the initiation of autophagy was aggregation of ribosomes in an isolation membrane. By day 5, many organelles were isolated in the autophagic vacuoles. The ribosomecontaining vacuoles were wrapped by flattened stacks of Golgi cisternae to form conspicuous whorl-like autophagosomes. This is a previously undescribed type of autophagic vacuole with the entire complex of Golgi cisternae forming part of the autophagic membranes. Smooth endoplasmic reticulum was wrapped into membranous autophagic vacuoles with concentric arrays of doubel membranes. Plasma membrane was invaginated and then isolated in a multivesicular body. These three different types of isolated vacuoles did not show acid phosphatase activity as indicated by histochemical staining with -glycerophosphate as substrate. Subsequently, these autophagosomes fused with each other and with 1° or 2° lysosomes to form giant autophagolysosomes. Some mitochondria appeared to have coalesced directly into autophagolysosomes. Golgi complexes were evident during this period; they actively participated in making lysosomal enzymes. Cytoskeletons were frequently observed in the vicinity of autophagic vacuoles and were presumably involved in the transport of the vacuoles. As a result of lysosomal degradation lipofuscins and dense bodies were frequently observed by days 9–12 indicating atrophy of corpus allatum cells. Structural parameters, especially those present early in autophagy, such as the isolation membrane, ribosome-containing vacuoles and whorl-like autophagosomes, can be used to search for potential growth regulators responsible for the induction of autophagy, of the corpora allata, and the subsequent termination in juvenile hormone synthesis.     

DRAM1 promotes the targeting of mycobacteria to selective autophagy

Autophagy provides an important defense mechanism against intracellular bacteria, such as Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis disease (TB). We recently reported that pathogen recognition and antibacterial autophagy are connected by the induction of the DNA damage-regulated autophagy modulator DRAM1 via the toll-like receptor (TLR)-MYD88-NFKB innate immunity signaling pathway. Having shown that DRAM1 colocalizes with Mtb in human macrophages, we took advantage of a zebrafish model for TB to investigate the function of DRAM1 in autophagic host defense in vivo. We found that DRAM1 protects the zebrafish host from infection with Mycobacterium marinum (Mm), a close relative of Mtb. Overexpression of DRAM1 increases autophagosome formation and promotes autophagic flux by a mechanism dependent on the cytosolic DNA sensor TMEM173/STING and the ubiquitin receptor SQSTM1/p62. Here we summarize and discuss the implications of these findings.     

Dual function of CALCOCO2/NDP52 during xenophagy

During xenophagy, pathogens are selectively targeted by autophagy receptors to the autophagy machinery for their subsequent degradation. In infected cells, the autophagy receptor CALCOCO2/NDP52 targets Salmonella Typhimurium to the phagophore membrane by concomitantly interacting with LC3C and binding to ubiquitinated cytosolic bacteria or to LGALS8/GALECTIN 8 adsorbed on damaged vacuoles that contain bacteria. We recently reported that in addition, CALCOCO2 is also necessary for the maturation step of Salmonella Typhimurium-containing autophagosomes. Interestingly, the role of CALCOCO2 in maturation is independent of its role in targeting, as these functions rely on distinct binding domains and protein partners. Indeed, to mediate autophagosome maturation CALCOCO2 binds on the one hand to LC3A, LC3B, or GABARAPL2, and on the other hand to MYO6/MYOSIN VI, whereas the interaction with LC3C is dispensable. Therefore, the autophagy receptor CALCOCO2 plays a dual function during xenophagy first by targeting bacteria to nascent autophagosomes and then by promoting autophagosome maturation in order to destroy bacteria.Xenophagy is the process referring to the selective degradation of intracellular microorganisms by autophagy. Xenophagy is a very potent intrinsic cellular line of defense to fight pathogens and requires first the detection and targeting of microorganisms to growing phagophores prior to autophagosome maturation leading to microbial destruction. The targeting step can be achieved by cytosolic autophagy receptors, which bind on the one hand to the pathogen and on the other hand to LC3, a phagophore membrane-anchored protein. Once entrapped within an autophagosome, bacteria can survive or escape, unless they are rapidly destroyed. Therefore, autophagosome maturation allows the discharge of lysosomal enzymes in autolysosomes, allowing destruction of the bacteria. It is, however, not well known how autophagosomes mature, especially in the context of xenophagy. Recently, the endosomal membrane-bound protein TOM1 and the dynein motor MYO6 have been both shown to be implicated in the transport of endosomes into the vicinity of autophagosomes in order to ensure fusion of autophagosomes with vesicles of the endo/lysosomal pathway. Moreover, the concomitant absence of 3 autophagy receptors, CALCOCO2, TAX1BP1/T6BP, and OPTN/OPTINEURIN, impairs autophagosome biogenesis and maturation. As CALCOCO2 was already shown to have a MYO6 binding domain, we wondered whether CALCOCO2 could also be implicated in autophagosome maturation per se to promote bacterial degradation.We first observed that the binding site of CALCOCO2 to MYO6 was required for cells to control Salmonella Typhimurium intracellular growth. Nevertheless, when the binding of CALCOCO2 to MYO6 was abolished, bacteria were still efficiently targeted to autophagosomes, but yet still able to replicate to levels similar to the one observed in CALCOCO2-depleted cells. Strikingly, in noninfected cells the absence of CALCOCO2 perturbs the autophagy flux, resulting in a strong accumulation of autophagosomes, suggesting a positive role for CALCOCO2 in the autophagosome-lysosome fusion process. Surprisingly, we found that CALCOCO2 binding to LC3C, through its noncanonical LC3 interacting region (CLIR), is not involved in the maturation of autophagosomes. Instead, we identified another motif in the primary sequence of CALCOCO2, which mediates binding to at least LC3A, LC3B, and GABARAPL2 (but not LC3C). We referred to this motif as “LIR-like” as it differs from the canonical LIR motif by the absence of a hydrophobic residue in position X₃. This LIR-like motif was necessary for autophagosome maturation, along with the domain of CALCOCO2 responsible for its binding to MYO6. Eventually, mutation of this LIR-like motif also resulted in an increased Salmonella Typhimurium intracellular proliferation, whereas bacteria were still efficiently targeted within nondegradative autophagosomes. Interestingly, the absence of the autophagy receptor OPTN also led to the accumulation of nondegradative autophagosomes, suggesting that other autophagy receptors could share CALCOCO2 dual functions in xenophagy.Having autophagy receptors ensuring both targeting and degradation of pathogens could be an important evolutionary advantage against infections. Indeed, this mechanism could help to reduce the delay necessary for maturation, thus avoiding adaptation of the pathogen to its new environment (as proposed for Coxiella burnetti, Listeria monocytogenes, and Legionella pneumophila) or its escape from the autophagosome. Conversely, pathogens could avoid autophagy entrapment or autophagic degradation by targeting CALCOCO2 or any other autophagy receptors, which could play similar roles. For instance Chikungunya virus was reported to target CALCOCO2 in human cells leading to increased virus replication. Nevertheless, redundancy among autophagy receptors could also ensure a selective immune advantage against pathogens targeting any one of these receptors.Our results and those from others suggest for now that CALCOCO2 serves as a docking platform for MYO6-bound endosomes, thus facilitating autophagosome maturation (Fig. 1). How this action is coordinated with CALCOCO2 directing pathogens to the phagophore membranes remains unclear. During xenophagy against Salmonella Typhimurium, CALCOCO2 interaction first with LC3C is necessary to further recruit other ATG8 orthologs and ensure the final degradation of bacteria. Since the LIR-like motifs bind several ATG8s, whereas the CLIR motif only mediates binding to LC3C, it is possible that binding of CALCOCO2 to LC3C induces conformational changes and uncovers the LIR-like motif that can be then engaged with other ATG8 orthologs to trigger autophagosome maturation. Moreover, it is still unclear whether the action of CALCOCO2 in autophagosome maturation is coordinated with other partners, such as STX17/SYNTAXIN 17, which is recruited on the external membrane of autophagosomes and regulate fusion with lysosomes. Open in a separate windowFigure 1.Schematic model for the dual role of CALCOCO2 in xenophagy. CALCOCO2 targets bacteria to the phagophore through its LC3C binding site (CLIR motif), and, independently, regulates autophagosome maturation through its LC3A, LC3B, or GABARAPL2 binding site (LIR-like motif) and its MYO6 interacting region.Our findings reveal a new role for the autophagy receptor CALCOCO2 in autophagosome maturation, unravelling another function for CALCOCO2 in cell autonomous defense against pathogens: CALCOCO2 not only targets pathogens to phagophore membranes, but also regulates subsequent maturation of pathogen-containing autophagosomes, thus assuring efficient degradation of autophagy-targeted pathogens.     

Amphisomes: out of the autophagosome shadow?

EMBO J (2013) 32: 3130–3144 doi: 10.1038/emboj.2013.233; published online November012013Amphisomes are intermediate organelles, formed during autophagy through the fusion between autophagosomes and endosomes. Complex multivesicular vacuoles that resemble amphisomes have been observed in various cell types, but whether they have cellular roles other than being a precursor structure is still enigmatic. While autophagy-related (ATG) proteins interact with the endocytic pathways in other processes different from autophagy, Patel and colleagues now report that these factors come together to generate amphisome-like compartments that regulate mucin secretion in goblet cells.ATG and endosomal proteins have been linked to secretion, and the specific loss of them impairs the function of different secretory cell types (Jung et al, 2008; DeSelm et al, 2011; Ushio et al, 2011; Sasidharan et al, 2012). ATG proteins have also been shown to interact with the endocytic pathway in few situations that do not involve autophagy. For example in phagocytic cells, the surface of bacteria-containing phagosomes acquires LC3/Atg8 through the concerted action of a subpopulation of ATG proteins. This process, which has been termed LC3-associated phagocytosis (LAP), promotes the fusion of phagosomes with lysosomes (Sanjuan et al, 2007). Something similar occurs during entotic cell death, an engulfment programme leading to the elimination of cells into lysosomes. The entotic vacuole membranes surrounding the internalized cells also recruit LC3 through a mechanism that depends on several ATG proteins, but not on autophagosome formation (Florey et al, 2011).In their work aimed to understand the function of ATG proteins in goblet cells, Patel et al (2013) show that the autophagy and endocytic machinery converge at the amphisomes to promote the secretion of mucins. In the gastrointestinal tract, secretory cells have a crucial role in providing the mucus barrier that protects against intestinal pathogens. Mucins, the main components of the mucus, are produced in goblet cells where large polymers of these highly glycosylated proteins are packed into secretory granules that accumulate at the apical surface. The release of these mucin granules relies on a series of cellular events that are tightly coordinated. Patel et al (2013) show that knockout mice lacking ATG5 in the intestinal epithelium, that is, Atg5VC mice, exhibit both a dramatic accumulation of mucin granules in goblet cells and a diminished mucus secretion. Taking advantage of a newly developed in vitro system to culture and differentiate intestinal epithelial stem cells into secretory goblet cells, the authors also demonstrate that the ablation of other ATG proteins causes the same phenotype showing that the autophagy machinery is required for mucin secretion in these specialized cells (Patel et al, 2013). Interestingly, ATG proteins affect the functionality of another gastrointestinal secretory lineage, the Paneth cells. Paneth cells homozygous for the atg16L1 risk allele, associated with Crohn disease, produce less secretory granules than in controls (Cadwell et al, 2008). This suggests that although ATG proteins regulate secretion in the two most abundant secretory lineages in the intestinal tract, two different mechanisms are probably involved.A microarray analysis of mRNA from Atg5VC mouse colonic epithelial cells revealed a possible alteration in the endocytic pathway. Indeed, blocking endocytosis also provoked an accumulation of mucin granules. While LC3B has been previously found on the surface of secretory granules (Ushio et al, 2011; Ishibashi et al, 2012), immuno-electron microscopy of wild-type mouse intestinal tissue revealed a distribution of LC3B not on mucin granules, but on multivesicular vacuoles positive for several endosomal proteins (Patel et al, 2013). Because of the morphological and molecular characteristics of these compartments, it appears that the ATG proteins together with the endocytic pathway regulate secretion in goblet cells by converging in what could be a new amphisome-like organelle (Figure 1).Open in a separate windowFigure 1Schematic representation for the regulated secretion of mucin granules by amphisome-like structures in goblet cells. ROS generated by NADPH oxidases promote the fusion of LC3-positive vesicles with endosomes marked by Rab5 and containing the NADPH oxidase subunit p22phox. The resulting amphisomes-like organelles are decorated with LC3, endosomal proteins (Rab5, Rab7 and EEA1) and p22phox and localize near the mucin granules. The formation of these copartments probably prolong and/or enhance the production of ROS by the NADPH oxidase, which in turn increases the levels of cytoplasmic calcium through an unknown mechanism leading to the release of the mucin granules.NADPH oxidases are known to be present in endosomes, and NADPH oxidase-generated reactive oxygen species (ROS) are necessary for LC3 recruitment to phagosomes.(Huang et al, 2009). Patel et al (2013) thus explored whether these enzymes played a role in mucin granule secretion in goblet cells. Indeed, expression of a mutant form of p22phox, a transmembrane subunit of several NADPH oxidase complexes, altered the exocytosis of these carriers. Moreover, p22phox was found to localize to Rab5-positive endosomes and also with the observed amphisome-like structures (Figure 1). Because a mutant form of p22phox also caused a misslocalization of both LC3 and the early-endosomal marker protein EEA1, the obvious conclusion was that ROS production by endosomes is necessary to trigger the formation of the amphisome-like organelles via the acquisition of the ATG machinery (Figure 1). Interestingly, addition of H₂O₂ that mimics ROS generation was able to induce mucin granule exocytosis in the p22phox mutant cells, showing that ROS was also required to regulate secretion in goblet cells (Patel et al, 2013). Furthermore, H₂O₂ bypassed as well the mucin granule secretion defect in autophagy and endocytosis-deficient goblet cells through an increase of cytosolic calcium levels (Patel et al, 2013). This, together with the observation that the loss of ATG5 and the block of the endocytic pathway impair the production of ROS has led Patel et al (2013) to propose that amphisome-like organelles are a signalling platform, where NADPH oxidase-driven ROS production promotes the release of the mucin granules.Amphisomes have been characterized and defined as autophagic vacuoles formed upon fusion between autophagosomes and endosomes. Given that ATG and endosomal proteins converge in multivesicular and/or vacuolar compartments resembling amphisomes in cellular processes independent of autophagy, one could consider to use the term amphisomes to describe a more heterogenous and ampler population of unnamed compartments where part of the autophagy and endosomal machineries co-localize. Based on this consideration, the study by Patel et al (2013) has identified an amphisome-like structure where molecular events interconnect to trigger granule secretion. While their work adds to the still limited number of non-degradative roles of the autophagic pathway, which include unconventional secretion (Subramani and Malhotra, 2013), it is one of the first reports highlighting that amphisomes (or any autophagosomal intermediate structure) could be more than just a transport intermediate, and at least in goblet cells, they could act as a platform where signals integrating some aspects of the cell physiology are elicited.Though it remains to be establish whether the organelles described by Patel et al (2013) are indeed amphisomes, especially as they are formed by fusion of endosomes with LC3-positive single-membrane vesicles rather than LC3-positive double-membrane autophagosomes, their study raises some intriguing questions. Are these compartments persistent or will they eventually fuse with lysosomes? Why has the cell opted to signal from amphisomes and not from endosomes, where the NADPH oxidases are normally present? Maybe the answer to these questions is hidden in the transient life of amphisomes. In the most classical signalling pathways, the transduction cascade amplifies the initial cue but it also turn it off subsequently through negative feedback loops. This permits to precisely modulate the signal output temporally (and locally). The amphisome-like structures observed in goblet cells could also act as the molecular switch for the signal-stimulating mucin granule secretion. The ROS generated initially from endosomes would trigger the recruitment of LC3 through vesicle fusion events, and the production of this second messenger will be prolonged and/or enhanced in the resulting amphisomes-like structure, leading to a stimulation of mucin granule exocytosis (Figure 1). The subsequent fusion of the amphisomes with lysosomes could lead to the termination of the signal. Other scenarios, however, cannot be excluded like, for example, the delivery of a protein enhancing the NADPH oxidase activity to the endosomes by the LC3-positive vesicles.While these are just hypotheses, it is clear that Patel et al (2013) have opened a window on a new and unexplored area of the autophagy field. Future investigations will tell us whether what observed in goblet cells is a unique situation or the intermediate organelles characterizing autophagy can carry out cellular functions different from the one delivering unwanted structures into the lysosome interior for degradation, including to serve as signalling platforms.     

Plasma membrane helps autophagosomes grow

The membrane origin of autophagosomes has long been a mystery and it may involve multiple sources. In this punctum, we discuss our recent finding that the plasma membrane contributes to the formation of pre-autophagic structures via clathrin-mediated endocytosis. Our study suggests that Atg16L1 interacts with clathrin heavy-chain/AP2 and is also localized on vesicles (positive for clathrin or cholera toxin B) close to the plasma membrane. Live-cell imaging studies revealed that the plasma membrane contributes to Atg16L1-positive structures and that this process and autophagosome formation are impaired by knockdowns of genes regulating clathrin-mediated endocytosis.Key words: autophagy, plasma membrane, endocytosis, phagophore, originWhere do autophagosomes get their membrane from? Although the field of autophagy has grown tremendously since its discovery a few decades ago, the origin(s) of the membranes that contribute to autophagosome biogenesis has been a mystery among autophagy researchers until recently. Mammalian autophagosomes are formed randomly throughout the cytoplasm via a process that involves elongation and fusion of phagophores to form double-membraned autophagosomes. This process involves two ubiquitin-like conjugation systems: conjugation of Atg12 to Atg5 that later forms a macromolecular complex with Atg16L1, and conjugation of phosphatidylethanolamine (PE) with Atg8/LC3-I. The Atg12-Atg5-Atg16L1 complex is targeted to the preautophagic structures, which then acquire Atg8. Atg12-Atg5-Atg16L1 dissociates from completed autophagosomes, while LC3-PE (LC3-II) is associated both with pre-autophagic structures and completed autophagosomes.Some recent studies have explored the contribution of membranes from different organelles supporting the general idea that autophagosomes derive membranes from pre-existing organelles. It is quite possible that there may be multiple membrane sources involved. A few groups have revisited the hypothesis that the endoplasmic reticulum (ER) may be one of the membrane donors. High-resolution 2D electron microscopy (EM) and 3D EM-tomography studies have revealed connections between the ER and the growing autophagosomes. Whether the ER contributes to general autophagy or a specific form of autophagy, reticulophagy, remains to be determined. In addition, it has not been shown if ER membrane is required for autophagosome formation. Recently another study has reported that autophagosomes receive lipids from the outer mitochondrial membrane, but only under starvation conditions, again fueling the multiple-membrane source hypothesis.We have now found evidence for plasma membrane contribution to pre-autophagic structures via endocytosis. Unlike the previous studies, which have focused on LC3- positive structures, we looked specifically at the Atg5-, Atg12- and Atg16-positive pre-autophagic structures, an idea that stemmed from our finding that clathrin heavy-chain immunoprecipitates with Atg16L1. We think that this interaction is partly mediated by the adaptor protein AP2, since knockdown of AP2 decreases the clathrin heavy-chain-Atg16L1 interaction. Immunogold EM also shows clathrin localization on Atg16L1-labeled vesicles close to the plasma membrane.These findings led us to test whether knockdown of proteins involved in clathrin-mediated endocytosis affected Atg16L1-positive pre-autophagic structures. Indeed, knockdown of key proteins in the clathrin-mediated endocytic pathway results in a decrease in the formation of Atg16L1-positive structures both under basal or autophagy-induced conditions (starvation or trehalose treatment). This correlates with a decrease in the number of LC3-labeled autophagosomes. When we directly analyzed vesicle fusion by livecell microscopy, we observed that vesicles endocytosed from the plasma membrane fuse to the Atg16L1-positive vesicles close to the plasma membrane. This was confirmed by immuno-EM when we found cholera toxin B-labeling (used to label plasma membrane that is subsequently internalized by endocytosis) on Atg16L1-vesicles. We noticed that overexpression of an Atg16L1 mutant that does not bind clathrin heavy-chain does not form Atg16L1-vesicular structures in the way we see with wild-type Atg16L1, suggesting that the binding of Atg16L1 to AP2/clathrin is required for the subsequent formation of the Atg16L1 vesicles.When we blocked endocytic vesicle scission (using both genetic and chemical inhibitors) we found that Atg16L1 strongly immunoprecipitates with clathrin-heavy chain probably due to the accumulation of clathrin-Atg16L1 structures at the plasma membrane that failed to pinch off. This was strongly supported by our fluorescence microscopy and immuno-EM studies that showed what we predicted—accumulation of Atg16L1 at the plasma membrane. This suggests that Atg16L1 in a complex with AP2/clathrin is targeted to the plasma membrane and subsequently internalized as Atg16L1-positive structures. Thus, our data strongly suggest that plasma membrane contributes to early autophagic precursors that subsequently mature to form phagophores (Fig. 1).Open in a separate windowFigure 1Plasma membrane contributes to the formation of early autophagic precursors. Previous studies show that delivery of fully formed autophagosomes to lysosomes requires fusion of such autophagosomes with early or late endosomes to form amphisomes, which are Atg16L1-negative, LC3-positive and are also positive for endosomal markers. We show that blocking clathrin-mediated endocytosis inhibits formation of Atg16L1-positive structures that mature to form phagophores and later autophagosomes. These Atg16L1-vesicles are positive for other early autophagosomal markers like Atg5 and Atg12, but are negative for early endosomal markers like EEA1, suggesting that they are high up in the autophagosome biogenesis cascade. Inhibition of dynamin with Dynsasore or the use of a dominant negative K44A mutant blocks scission and results in Atg16L1 accumulation on the plasma membrane, suggesting that endosomal scission is critical for this process.Although previous studies suggest that completely formed autophagosomes need to fuse with early or late endosomes in order for subsequent autophagosomelysosome fusion to occur, they did not look at the formation of pre-autophagic structures. Our study shows that active endocytosis is required both for the formation of autophagosomes, when very early endocytic intermediates immediately pinching off the plasma membrane (not early endosomes) fuse with Atg16L1-positive structures to form phagophores, and also for maturation of autophagosomes when early or late endosomes fuse with Atg16L1-negative but LC3-positive autophagosomes to form amphisomes. Since blocking clathrin-mediated endocytosis does not completely abrogate autophagosome formation, we believe that other endocytic pathways may have a similar role. Depending on the cell type or the physiological conditions, the contributions from the different endocytic pathways may vary accordingly. It will be interesting to know if the endocytic pathway continuously delivers membrane for early steps in autophagy as the preautophagic structures grow and mature to form autophagosomes, deriving membrane from other sources.     

Impaired autophagy flux is associated with neuronal cell death after traumatic brain injury

Dysregulation of autophagy contributes to neuronal cell death in several neurodegenerative and lysosomal storage diseases. Markers of autophagy are also increased after traumatic brain injury (TBI), but its mechanisms and function are not known. Following controlled cortical impact (CCI) brain injury in GFP-Lc3 (green fluorescent protein-LC3) transgenic mice, we observed accumulation of autophagosomes in ipsilateral cortex and hippocampus between 1 and 7 d. This accumulation was not due to increased initiation of autophagy but rather to a decrease in clearance of autophagosomes, as reflected by accumulation of the autophagic substrate SQSTM1/p62 (sequestosome 1). This was confirmed by ex vivo studies, which demonstrated impaired autophagic flux in brain slices from injured as compared to control animals. Increased SQSTM1 peaked at d 1–3 but resolved by d 7, suggesting that the defect in autophagy flux is temporary. The early impairment of autophagy is at least in part caused by lysosomal dysfunction, as evidenced by lower protein levels and enzymatic activity of CTSD (cathepsin D). Furthermore, immediately after injury both autophagosomes and SQSTM1 accumulated predominantly in neurons. This was accompanied by appearance of SQSTM1 and ubiquitin-positive puncta in the affected cells, suggesting that, similar to the situation observed in neurodegenerative diseases, impaired autophagy may contribute to neuronal injury. Consistently, GFP-LC3 and SQSTM1 colocalized with markers of both caspase-dependent and caspase-independent cell death in neuronal cells proximal to the injury site. Taken together, our data indicated for the first time that autophagic clearance is impaired early after TBI due to lysosomal dysfunction, and correlates with neuronal cell death.     

PLEKHM1: Adapting to life at the lysosome

The endosomal system and autophagy are 2 intertwined pathways that share a number of common protein factors as well as a final destination, the lysosome. Identification of adaptor platforms that can link both pathways are of particular importance, as they serve as common nodes that can coordinate the different trafficking arms of the endolysosomal system. Using a mass spectrometry approach to identify interaction partners of active (GTP-bound) RAB7, the late endosome/lysosome GTPase, and yeast 2-hybrid screening to identify LC3/GABARAP interaction partners we discovered the multivalent adaptor protein PLEKHM1. We discovered a highly conserved LC3-interaction region (LIR) between 2 PH domains of PLEKHM1 that mediated direct binding to all LC3/GABARAP family members. Subsequent mass spectrometry analysis of PLEKHM1 precipitated from cells revealed the HOPS (homotypic fusion and protein sorting) complex as a prominent interaction partner. Functionally, depletion of PLEKHM1, HOPS, or RAB7 results in decreased autophagosome-lysosome fusion. In Plekhm1 knockout (KO) mouse embryonic fibroblasts (MEFs) we observed increased lipidated LC3B, decreased colocalization between LC3B and LAMP1 under amino acid starvation conditions and decreased autolysosome formation. Finally, PLEKHM1 binding to LC3-positive autophagosomes was also essential for selective autophagy pathways, as shown by clearance of puromycin-aggregates, in a PLEKHM1-LIR-dependent manner. Overall, we have identified PLEKHM1 as an endolysosomal adaptor platform that acts as a central hub to integrate endocytic and autophagic pathways at the lysosome.PLEKHM1 (pleckstrin homology domain containing, family M [with RUN domain] member 1) is a ubiquitously expressed protein involved in the regulation of osteoclast function and bone resorption. Recently, it was also described in the context of negatively regulating the endocytic pathway but not autophagy. However, in our recent studies, we show that PLEKHM1 positively regulates the terminal stages of both endocytic and autophagy pathways through direct interaction between PLEKHM1, RAB7, the HOPS complex, and mammalian Atg8 proteins (Fig. 1A). In addition, the PLEKHM1-RAB7-HOPS complex is a direct target for the Salmonella (Salmonella enterica Typhimurium) effector protein SifA (Salmonella-induced filament protein A) that together regulate the Salmonella-containing vacuole (Fig. 1B). Open in a separate windowFigure 1.Model of PLEKHM1 function in the endocytic and autophagic pathways. (A) Domain structure of PLEKHM1 and their interactions. RUN (RUNDC3A/RPIP8, UNC-14 and RUSC1/NESCA); PH1 and PH2 (Pleckstrin homology domain 1 and 2); C1/Zinc finger (C1); HOPS (homotypic fusion and protein sorting). (B) Proposed positioning of PLEKHM1 and its associated complexes in the autophagy and endocytic pathway. PLEKHM1 localizes to late endosomes and lysosomes in an RAB7-dependent manner. The interaction between PLEKHM1, RAB7, and HOPS on vesicles positions these vesicles for tethering and fusion with autophagosomes, through direct interaction with MAP1LC3/GABARAP proteins. The autophagosomes may also fuse with late endosomes/MVBs (multivesicular bodies) in a PLEKHM1-RAB7-HOPS-dependent manner to produce amphisomes, prior to fusion with the lysosome. PLEKHM1-RAB7-HOPS can also be subverted by the Salmonella effector SifA, for the proper maintenance of the Salmonella-containing vacuole (SCV) and Sif (Salmonella-induced filament) formation. mAtg8s, MAP1LC3/GABARAP proteins.Using a 2-pronged approach, we identified PLEKHM1 as an interaction partner of RAB7 in its GTP-bound active state, RAB7(GTP), and MAP1LC3/GABARAP proteins. PLEKHM1 interacts directly with all MAP1LC3/GABARAP proteins through a highly conserved LC3-interaction motif (LIR) located between the Pleckstrin homology domain 1 (PH1) and PH2 domains of PLEKHM1 (Fig. 1A). Endogenous PLEKHM1 colocalizes with LAMP1 at the cytosolic-facing membrane, but not the lumenal side, of LC3B-containing amphisomes/autolysosomes, indicating that PLEKHM1 is an autophagy adaptor protein rather than a selective cargo receptor.Using SILAC (stable isotope labeling of cells in culture)-labeled inducible PLEKHM1 cells, we identified the HOPS complex as a significant interaction partner. The hexameric HOPS complex is an essential component of the late endocytic fusion machinery and is required for autolysosome formation. PLEKHM1 interacts directly with the HOPS complex, mediated by the RUN domain of PLEKHM1 and the C terminus of VPS39 (Fig. 1A) Crucially, PLEKHM1 forms an endogenous complex with HOPS. In the context of vesicle fusion, the HOPS complex acts as a tether to anchor and position the vesicles prior to fusion that is driven by SNARE proteins. Multiple SNARE proteins, such as VAMP7, VAMP8, VTI1B, SNAP29, and STX17 have been described to be required for autophagosome-lysosome fusion. Upon autophagy induction, enhanced PLEKHM1 coprecipitation is detected with the HOPS complex and the autophagosome specific SNARE STX17, reinforcing a role for PLEKHM1 in autophagosome-lysosome fusion.Both RAB7 and the HOPS complex are integral components of the endocytic pathway and, as such, we wanted to test the effect of PLEKHM1 loss on EGFR (epidermal growth factor receptor) degradation. We used 2 epithelial cell lines, HeLa and Hke3. In both instances, loss of PLEKHM1 causes a marked decrease in the rate of EGFR degradation and increases retention in early endosomes. This is in stark contrast to previous reports that used A549 cells and showed that a lack of PLEKHM1 accelerates EGFR degradation. Clearly, cell lines and their background mutations will have to be considered for future studies.In addition to the endocytic pathway, RAB7 and the HOPS complex are essential for the autophagosome-to-autolysosome transition. Therefore, we also wanted to explore this facet of PLEKHM1 function. We generated Plekhm1 KO MEFs to analyze the effects of autophagy flux in the absence of PLEKHM1. Plekhm1 KO MEFs show a block in autophagy, with the accumulation of SQSTM1/p62 and LC3B-II and, using tandem-fluorescence-LC3B as a marker, a decrease in autolysosome formation. Taken together, these findings suggest that PLEKHM1 functions at the point of autophagosome-lysosome fusion (Fig. 1B).Finally, we were interested in testing the functional role of PLEKHM1, and in particular the LIR, during selective autophagy of protein aggregates. We treated control and PLEKHM1-depleted cells with puromycin and observed aggregate clearance over time after puromycin removal. Cells lacking PLEKHM1 and those reconstituted with a PLEKHM1-LIR mutant were unable to efficiently remove SQSTM1-ubiquitin-positive aggregates, compared to control or PLEKHM1-wild type reconstituted cells, indicating an important role for the final stages of endosome and autophagosome maturation (Fig. 1B).“No man is an island, entire of itself” seems of particular prudence when considering the intertwined nature of both autophagic and endocytic pathways. Indeed, it is interesting that there are multiple RAB7 effector proteins functioning at the late endocytic step that also contribute to autophagy, including FYCO1, KIAA0226/Rubicon, UVRAG and now PLEKHM1, where only PLEKHM1 and UVRAG have been shown to interact with the HOPS complex. All of which, when mutated or depleted, have effects on both the endocytic and autophagic pathways. Clearly the roles of these proteins in cell-type and tissue-specific settings have to be determined before we fully comprehend the complexities of how the endosomal and autophagic pathways integrate and communicate with each other.     

NBR1 and p62 as cargo receptors for selective autophagy of ubiquitinated targets

Autophagy is an evolutionary conserved cell survival process for degradation of long-lived proteins, damaged organelles and protein aggregates. The mammalian proteins p62 and NBR1 are selectively degraded by autophagy and can act as cargo receptors or adaptors for the autophagic degradation of ubiquitinated substrates. Despite differing in size and primary sequence, both proteins share a similar domain architecture containing an N-terminal PB1 domain, a LIR motif interacting with ATG8 family proteins, and a C-terminal UBA domain interacting with ubiquitin. The LIR motif is essential for their autophagic degradation, indicating that ATG8 family proteins are responsible for the docking of p62 and NBR1 to nucleating autophagosomes. p62 and NBR1 co-operate in the sequestration of misfolded and ubiquitinated proteins in p62 bodies and are both required for their degradation by autophagy. Here we discuss the role of p62 and NBR1 in degradation of ubiquitinated cargoes and the putative role of LIR as a general motif for docking of proteins to ATG8 family proteins.     

Endocytosis and Autophagy: Exploitation or Cooperation?

Autophagy is a lysosome-mediated degradative system that is a highly conserved pathway present in all eukaryotes. In all cells, double-membrane autophagosomes form and engulf cytoplasmic components, delivering them to the lysosome for degradation. Autophagy is essential for cell health and can be activated to function as a recycling pathway in the absence of nutrients or as a quality-control pathway to eliminate damaged organelles or even to eliminate invading pathogens. Autophagy was first identified as a pathway in mammalian cells using morphological techniques, but the Atg (autophagy-related) genes required for autophagy were identified in yeast genetic screens. Despite tremendous advances in elucidating the function of individual Atg proteins, our knowledge of how autophagosomes form and subsequently interact with the endosomal pathway has lagged behind. Recent progress toward understanding where and how both the endocytotic and autophagic pathways overlap is reviewed here.Autophagy is a lysosome-mediated pathway for the degradation of cytosolic proteins and organelles, which is essential for cell homeostasis, development, and for the prevention of several human diseases and infection (Choi et al. 2013). Importantly, autophagy cannot occur without an active lysosome. However, it is becoming increasingly recognized that the endosomal pathway plays a greater role than just providing the degradative enzymes found in the lysosome. Recent data suggest that in mammalian cells multiple contributions from several stages of the endocytic pathway are essential for efficient autophagy. Here we outline the autophagic pathway and then address the recent data on how different endosomal compartments contribute to autophagy, and the molecular machinery required for the interaction of the endosome and lysosome during the formation, and consumption of the autophagosome. Given the model emerging that the amino-acid-sensitive autophagic pathway originates from the endoplasmic reticulum (ER), several questions arise, including how do recognition and productive interaction occur between an ER-derived membrane and endosomes? How are these interactions mediated, and which are essential for efficient autophagy?     

Poliovirus induces autophagic signaling independent of the ULK1 complex

Poliovirus (PV), like many positive-strand RNA viruses, subverts the macroautophagy/autophagy pathway to promote its own replication. Here, we investigate whether the virus uses the canonical autophagic signaling complex, consisting of the ULK1/2 kinases, ATG13, RB1CC1, and ATG101, to activate autophagy. We find that the virus sends autophagic signals independent of the ULK1 complex, and that the members of the autophagic complex are not required for normal levels of viral replication. We also show that the SQSTM1/p62 receptor protein is not degraded in a conventional manner during infection, but is likely cleaved in a manner similar to that shown for coxsackievirus B3. This means that SQSTM1, normally used to monitor autophagic degradation, cannot be used to accurately monitor degradation during poliovirus infection. In fact, autophagic degradation may be affected by the loss of SQSTM1 at the same time as autophagic signals are being sent. Finally, we demonstrate that ULK1 and ULK2 protein levels are greatly reduced during PV infection, and ATG13, RB1CC1, and ATG101 protein levels are reduced as well. Surprisingly, autophagic signaling appears to increase as ULK1 levels decrease. Overexpression of wild-type or dominant-negative ULK1 constructs does not affect virus replication, indicating that ULK1 degradation may be a side effect of the ULK1-independent signaling mechanism used by PV, inducing complex instability. This demonstration of ULK1-independent autophagic signaling is novel and leads to a model by which the virus is signaling to generate autophagosomes downstream of ULK1, while at the same time, cleaving cargo receptors, which may affect cargo loading and autophagic degradative flux. Our data suggest that PV has a finely-tuned relationship with the autophagic machinery, generating autophagosomes without using the primary autophagy signaling pathway.

Abbreviations: ACTB - actin beta; ATG13 - autophagy related 13; ATG14 - autophagy related 14; ATG101 - autophagy related 101; BECN1 - beclin 1; CVB3 - coxsackievirus B3; DMV - double-membraned vesicles; EM - electron microscopy; EMCV - encephalomyocarditis virus; EV-71 - enterovirus 71; FMDV - foot and mouth disease virus; GFP - green fluorescent protein; MAP1LC3B/LC3B - microtubule associated protein 1 light chain 3 beta; MOI - multiplicity of infection; MTOR - mechanistic target of rapamycin kinase; PIK3C3 - phosphatidylinositol 3-kinase catalytic subunit type 3; PRKAA2 - protein kinase AMP-activated catalytic subunit alpha 2; PSMG1 - proteasome assembly chaperone 1; PSMG2 - proteasome assembly chaperone 2PV - poliovirus; RB1CC1 - RB1 inducible coiled-coil 1; SQSTM1 - sequestosome 1; ULK1 - unc-51 like autophagy activating kinase 1; ULK2 - unc-51 like autophagy activating kinase 2; WIPI1 - WD repeat domain, phosphoinositide interacting 1     

p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death

Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and co-immunoprecipitated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutant huntingtin. We suggest that p62 may, via LC3, be involved in linking polyubiquitinated protein aggregates to the autophagy machinery.     

Identifying specific receptors for cargo-mediated autophagy

Macroautophagy has been implicated in numerous diseases, yet our understanding of the proteins responsible for the turnover of specific cargo by autophagy is limited. In a recent paper published in Nature, Mancias et al. used quantitative proteomics to identify a cohort of autophagosome-enriched proteins, one of which, nuclear receptor coactivator 4 (NCOA4) was shown to be required for the selective delivery of ferritin to the lysosome, ultimately regulating intracellular iron by autophagic turnover of ferritin, or ferritinophagy.Autophagy is a cell survival process whereby double-membrane structures, autophagosomes, sequester bulk cytoplasm, damaged proteins, and organelles for delivery to the lysosome and turnover to maintain homeostasis. Autophagosomes are identified by ATG8 proteins (in mammalian cells these are LC3 and its family members) that have been shown to recruit selective receptors that deliver specific cargos for degradation; however, the full range of cargo proteins and their related receptor proteins are still largely unknown¹. Understanding which proteins are responsible for specific cargo degradation is needed to clarify the complicated roles of autophagy in human diseases, for example, to explain the dual roles that autophagy is thought to play in tumor suppression or in the survival and growth of tumors².A recent study from the Kimmelman and Harper labs has taken a crucial first step in robustly identifying proteins that are associated with autophagosomes and their turnover³. Although previous studies have identified proteins in autophagosome preparations, these studies had limitations and low protein overlap with one another^4,5,6. Most importantly, because autophagy non-selectively degrades cytosolic proteins as well as selectively targets specific cargos, the previous studies were bedeviled by the problem that many proteins targeted to autophagosomes might simply have been material whose likelihood of being in the autophagosome is not regulated but instead related to their overall abundance. In this paper, proteomics using stable isotope labeling by amino acids in cell culture (SILAC) was paired with a density gradient separation protocol for autophagosomes and a clever refinement where “hits” were filtered based on their abundance in the total proteome. This allows for a powerful association of proteins that are specific to autophagosomes. In addition, use of multiple human cell lines with differing reliance on autophagy (PANC-1, PA-TU-8988T, and MCF7) helped to strengthen the protein associations in regards to autophagy.Identification of specific autophagy-related proteins was achieved through the treatment of light isotope-labeled cells with wortmannin, a phosphoinositide 3-kinase inhibitor that prevents autophagosome formation, and heavy isotope-labeled cells treated with chloroquine, a lysosomal inhibitor to prevent autophagosome degradation and increase numbers. Autophagosomes were then isolated from the differentially labeled isotope samples which allowed for the identification of specific autophagy proteins (heavy) from those proteins that were isolated at the same density upon gradient centrifugation (light) (Figure 1A). A total of 50 high-stringency proteins were selected based on an equal or greater than one log₂ increase in the heavy:light ratio, protein overlaps between cell replicates, and protein overlap between the different cell types utilized. Several known autophagy proteins and cargo receptor proteins were identified, as well as plasma membrane and endocytosis-related proteins, which is consistent with previous findings and the intermixing of membranes during autophagosome maturation. Of the proteins not previously shown to associate with autophagy, NCOA4 was the highest and most consistently enriched protein identified. NCOA4 had previously been suggested to interact with androgen receptor^7,8; however, the new study describes an unrecognized role of NCOA4 as a specific cargo receptor for autophagy, which interacts with LC3 proteins to deliver selective cargo to the autophagosome. For example, characterization of GFP-labeled NCOA4 showed puncta accumulation that tended to localize with LC3B-positive puncta in response to chloroquine treatment.Open in a separate windowFigure 1Identification of autophagy-associated proteins and protein interaction of top HCIP: NCOA4. (A) Workflow of autophagy-associated protein identification. (B) Different complex interactions of NCOA4 with HERC2, NEURL4, and the ferritin complex. Arrows depict the directionality of interaction with line weight indicating the weighted and normalized D score (WDN). Dotted lines represent data from the STRING database. Numbers in parentheses are the log₂(H:L) ratio obtained from A.Further experiments were performed to understand how NCOA4 functions as a selective autophagy cargo receptor. Affinity purification-mass spectrometry was used to identify high-confidence interacting proteins (HCIP) associating with NCOA4. Among the HCIPs identified, ferritin heavy chain (FTH1), ferritin light chain (FTL), HERC2, and NEURL4 were verified to associate with NCOA4 by immunopreciptation followed by immunoblotting. As HERC2 and NEURL4 are not found in the autophagosome fraction and do not associate with ferritin immune complexes, it is believed that NCOA4 creates separate distinct complexes with ferritin and HERC2-NEURL4 (Figure 1B). This result indicates a further level of control whereby NCOA4 does not just deliver everything that it binds to the autophagosome. Instead, NCOA4 must have some mechanism by which it “knows” to only deliver cargo such as ferritin to the autophagosome.In previous studies, it was shown that ferritin can concentrate in the lysosome and upon iron chelation, ferritin is transported to the lysosome for degradation^9,10, thus allowing release of iron to the cell. Here co-localization of NCOA4, LC3B, and ferritin into puncta was shown to occur upon stimulation of ferritin expression with ferric ammonium citrate, reflecting the targeting of ferritin into autophagosomes for degradation through the lysosome. This process has been termed “ferritinophagy” by the authors. It was confirmed that NCOA4 and autophagy play a central role in ferritin degradation by the prevention of ferritin turnover through genetic inhibition of either ATG5 or NCOA4 by RNA interference (RNAi). On the other hand, RNAi against HERC2 did not prevent ferritin turnover, further confirming that the separate, apparently autophagy-independent complex NCOA4 forms with HERC2 that has no relationship to the turnover of ferritin. This provides a molecular explanation for how bioavailability of iron is controlled – when iron is needed, ferritin is shuttled to the autophagosome by NCOA4 and degraded, allowing release of iron to the cytoplasm.In summary, this study describes not only a comprehensive technique to identify autophagy-specific cargo proteins and a valuable list of autophagosome-associated proteins that the autophagy research community can start to mine, but also the first mechanistic understanding of ferritin degradation through autophagy. More generally, the study reveals an example of how sophisticated proteomic analysis can provide a much needed understanding of how particular proteins, organelles, and nutrients are turned over through autophagy, ultimately identifying targets for therapeutically directed strategies designed to manipulate these mechanisms in disease processes.