A Role for Macro-ER-Phagy in ER Quality Control

The endoplasmic-reticulum quality-control (ERQC) system shuttles misfolded proteins for degradation by the proteasome through the well-defined ER-associated degradation (ERAD) pathway. In contrast, very little is known about the role of autophagy in ERQC. Macro-autophagy, a collection of pathways that deliver proteins through autophagosomes (APs) for degradation in the lysosome (vacuole in yeast), is mediated by autophagy-specific proteins, Atgs, and regulated by Ypt/Rab GTPases. Until recently, the term ER-phagy was used to describe degradation of ER membrane and proteins in the lysosome under stress: either ER stress induced by drugs or whole-cell stress induced by starvation. These two types of stresses induce micro-ER-phagy, which does not use autophagic organelles and machinery, and non-selective autophagy. Here, we characterize the macro-ER-phagy pathway and uncover its role in ERQC. This pathway delivers 20–50% of certain ER-resident membrane proteins to the vacuole and is further induced to >90% by overexpression of a single integral-membrane protein. Even though such overexpression in cells defective in macro-ER-phagy induces the unfolded-protein response (UPR), UPR is not needed for macro-ER-phagy. We show that macro-ER-phagy is dependent on Atgs and Ypt GTPases and its cargo passes through APs. Moreover, for the first time the role of Atg9, the only integral-membrane core Atg, is uncoupled from that of other core Atgs. Finally, three sequential steps of this pathway are delineated: Atg9-dependent exit from the ER en route to autophagy, Ypt1- and core Atgs-mediated pre-autophagsomal-structure organization, and Ypt51-mediated delivery of APs to the vacuole.     

Untangling knots between autophagic targets and candidate drugs,in cancer therapy

Autophagy is an evolutionarily conserved lysosomal mechanism implicated in a wide variety of pathological processes, such as cancer. Autophagy can be regulated by a limited number of autophagy‐related genes (Atgs) such as oncogenic Bcl‐2/Bcl‐X_L, mTORC1, Akt and PI3KCI, and tumour suppressive proteins PI3KCIII, Beclin‐1, Bif‐1, p53, DAPKs, PTEN and UVRAG, which play their crucial roles in regulating autophagy‐related cancer. As autophagy has a dual role in cancer cells, with tumour‐promoting and tumour‐suppressing properties, it has become an attractive target for a series of emerging small molecule drugs. In this review, we reveal new discoveries of related small molecules or chemical compounds that can regulate autophagic pathways and lead to pro‐death or pro‐survival autophagy, in different types of cancer. We discuss the knots between autophagic targets and candidate drugs, in the hope of shedding new light on exploiting new anti‐tumour small molecule drugs for future cancer therapy.     

自噬的相关分子机制

细胞自噬是真核生物中高度保守的一类生物学途径,它通过降解细胞浆内不同组分,维持细胞自身平衡并帮助细胞在应激情况下生存。自噬在生物体生长发育、免疫防御、肿瘤抑制及神经退行性疾病中都有重大的意义。哺乳动物细胞中,自噬过程主要由自噬相关蛋白（Atg）所形成的一系列复合物所调控,这些蛋白质分别在自噬的启动、自噬泡的形成、延伸及成熟和降解过程中发挥重要的作用。在此,本文针对一些重要的自噬相关蛋白质对近年来自噬分子机制的研究进展做一总结。     

Trs130 Participates in Autophagy Through GTPases Ypt31/32 in Saccharomyces cerevisiae

Trs130 is a specific component of the transport protein particle II complex, which functions as a guanine nucleotide exchange factor (GEF) for Rab GTPases Ypt31/32. Ypt31/32 is known to be involved in autophagy, although the precise mechanism has not been thoroughly studied. In this study, we investigated the potential involvement of Trs130 in autophagy and found that both the cytoplasm‐to‐vacuole targeting (Cvt) pathway and starvation‐induced autophagy were defective in a trs130ts (trs130 temperature‐sensitive) mutant. Mutant cells could not transport Atg8 and Atg9 to the pre‐autophagosomal structure/phagophore assembly site (PAS) properly, resulting in multiple Atg8 dots and Atg9 dots dispersed in the cytoplasm. Some dots were trapped in the trans‐Golgi. Genetic studies showed that the effect of the Trs130 mutation was downstream of Atg5 and upstream of Atg1, Atg13, Atg9 and Atg14 on the autophagic pathway. Furthermore, overexpression of Ypt31 or Ypt32, but not of Ypt1, rescued autophagy defects in trs130ts and trs65ts (Trs130‐HA Trs120‐myc trs65Δ) mutants. Our data provide mechanistic insight into how Trs130 participates in autophagy and suggest that vesicular trafficking regulated by GTPases/GEFs is important in the transport of autophagy proteins from the trans‐Golgi to the PAS.     

UNC51‐like kinase 1, autophagic regulator and cancer therapeutic target

Autophagy, the cell process of self‐digestion, plays a pivotal role in maintaining energy homoeostasis and protein synthesis. When required, it causes degradation of long‐lived proteins and damaged organelles, indicating that it may play a dual role in cancer, by both protecting against and promoting cell death. The autophagy‐related gene (Atg) family, with more than 35 members, regulates multiple stages of the process. Serine/threonine protein kinase Atg1 in yeast, for example, can interact with other ATG gene products, functioning in autophagosome formation. One mammalian homologue of Atg1, UNC‐51‐like kinase 1 (ULK1) and its related complex ULK1–mAtg13–FIP200 can mediate autophagy under nutrient‐deprived conditions, by protein–protein interactions and post‐translational modifications. Although specific mechanisms of how ULK1 and its complex transduces upstream signals to the downstream central autophagy pathways is not fully understood, past studies have indicated that ULK1 can both suppress and promote tumour growth under different conditions. Here, we summarize some properties of ULK1 which can regulate autophagy in cancer, which may shed new light on future cancer therapy strategies, utilizing ULK1 as a potential new target.     

Atg8: an autophagy-related ubiquitin-like protein family

Autophagy-related (Atg) proteins are eukaryotic factors participating in various stages of the autophagic process. Thus far 34 Atgs have been identified in yeast, including the key autophagic protein Atg8. The Atg8 gene family encodes ubiquitin-like proteins that share a similar structure consisting of two amino-terminal α helices and a ubiquitin-like core. Atg8 family members are expressed in various tissues, where they participate in multiple cellular processes, such as intracellular membrane trafficking and autophagy. Their role in autophagy has been intensively studied. Atg8 proteins undergo a unique ubiquitin-like conjugation to phosphatidylethanolamine on the autophagic membrane, a process essential for autophagosome formation. Whereas yeast has a single Atg8 gene, many other eukaryotes contain multiple Atg8 orthologs. Atg8 genes of multicellular animals can be divided, by sequence similarities, into three subfamilies: microtubule-associated protein 1 light chain 3 (MAP1LC3 or LC3), γ-aminobutyric acid receptor-associated protein (GABARAP) and Golgi-associated ATPase enhancer of 16 kDa (GATE-16), which are present in sponges, cnidarians (such as sea anemones, corals and hydras) and bilateral animals. Although genes from all three subfamilies are found in vertebrates, some invertebrate lineages have lost the genes from one or two subfamilies. The amino terminus of Atg8 proteins varies between the subfamilies and has a regulatory role in their various functions. Here we discuss the evolution of Atg8 proteins and summarize the current view of their function in intracellular trafficking and autophagy from a structural perspective.     

Autophagy mediates cytotoxicity of human colorectal cancer cells treated with garcinielliptone FC

The tautomeric pair of garcinielliptone FC (GFC) is a novel tautomeric pair of polyprenyl benzophenonoid isolated from the pericarps of Garcinia subelliptica Merr. (G. subelliptica, Clusiaceae), a tree with abundant sources of polyphenols. Our previous report demonstrated that GFC induced apoptosis on various types of human cancer cell lines including chemoresistant human colorectal cancer HT‐29 cells. In the present study, we observed that many autophagy‐related genes in GFC‐treated HT‐29 cells were up‐ and down‐regulated using a cDNA microarray containing oncogenes and kinase genes. GFC‐induced autophagy of HT‐29 cells was confirmed by observing the formation of acidic vesicular organelles, LC3 puncta, and double‐membrane autophagic vesicles using flow cytometry, confocal microscopy, and transmission electron microscopy, respectively. Inhibition of AKT/mTOR/P70S6K signaling as well as formation of Atg5‐Atg12 and PI3K/Beclin‐1 complexes were observed using Western blot. Administration of autophagy inhibitor (3‐methyladenine and shRNA Atg5) and apoptosis inhibitor Z‐VAD showed that the GFC‐induced autophagy was cytotoxic form and GFC‐induced apoptosis enhanced GFC‐induced autophagy. Our data suggest the involvement of autophagy and apoptosis in GFC‐induced anticancer mechanisms of human colorectal cancer.     

Vrl1 relies on its VPS9‐domain to play a role in autophagy in Saccharomyces cerevisiae

Autophagy is an intracellular degradation process involving many Atg proteins, which are recruited hierarchically to regulate this process. Rab/Ypt GTPases and their activators, guanine nucleotide exchange factors (GEFs), which are critical for regulating vesicle trafficking, are also involved in autophagy. Previously, we reported that yeast Vps21 and its GEF Vps9 are required for autophagy. Later, a third yeast VPS9‐domain‐containing protein, V AR P‐l ike 1 (Vrl1), which was identified as a mutant in major laboratory strains, had partially overlapping functions with Vps9 in trafficking. In this study, we showed that Vrl1 performed roles in autophagy, and its VPS9‐domain was crucial for its role in autophagy. We found that localization of Vrl1 differed from the other two VPS9‐domain‐containing proteins, Vps9 and Muk1, and only Vrl1 changed from multipoint to diffusion after starvation. Like Vps9, Vrl1 suppressed autophagic defects caused by the VPS9 deletion. We further showed that these VPS9‐domain‐containing proteins, Vps9, Muk1, and Vrl1, all co‐localized with Atg8 on autophagosomes in cells blocked in any late step of starvation‐induced autophagy, with Vrl1 most often co‐localizing with Atg8. A small portion (<25%) of these VPS9‐domain‐containing proteins were degraded through autophagy. However, a large portion (>60%) of Vrl1 decreased independently of autophagy. We propose that Vrl1 may regulate autophagy in a similar way as Vps9, and the level of Vrl1 partly decreases through both autophagy‐dependent and ‐independent routes.     

Systematic prediction of autophagy-related proteins using Arabidopsis thaliana interactome data

Autophagy is a self-degradative process that is crucial for maintaining cellular homeostasis by removing damaged cytoplasmic components and recycling nutrients. Such an evolutionary conserved proteolysis process is regulated by the autophagy-related (Atg) proteins. The incomplete understanding of plant autophagy proteome and the importance of a proteome-wide understanding of the autophagy pathway prompted us to predict Atg proteins and regulators in Arabidopsis. Here, we developed a systems-level algorithm to identify autophagy-related modules (ARMs) based on protein subcellular localization, protein–protein interactions, and known Atg proteins. This generates a detailed landscape of the autophagic modules in Arabidopsis. We found that the newly identified genes in each ARM tend to be upregulated and coexpressed during the senescence stage of Arabidopsis. We also demonstrated that the Golgi apparatus ARM, ARM13, functions in the autophagy process by module clustering and functional analysis. To verify the in silico analysis, the Atg candidates in ARM13 that are functionally similar to the core Atg proteins were selected for experimental validation. Interestingly, two of the previously uncharacterized proteins identified from the ARM analysis, AGD1 and Sec14, exhibited bona fide association with the autophagy protein complex in plant cells, which provides evidence for a cross-talk between intracellular pathways and autophagy. Thus, the computational framework has facilitated the identification and characterization of plant-specific autophagy-related proteins and novel autophagy proteins/regulators in higher eukaryotes.     

Phosphorylation of mitophagy and pexophagy receptors coordinates their interaction with Atg8 and Atg11

The selective autophagy receptors Atg19 and Atg32 interact with two proteins of the core autophagic machinery: the scaffold protein Atg11 and the ubiquitin‐like protein Atg8. We found that the Pichia pastoris pexophagy receptor, Atg30, also interacts with Atg8. Both Atg30 and Atg32 interactions are regulated by phosphorylation close to Atg8‐interaction motifs. Extending this finding to Saccharomyces cerevisiae, we confirmed phosphoregulation for the mitophagy and pexophagy receptors, Atg32 and Atg36. Each Atg30 molecule must interact with both Atg8 and Atg11 for full functionality, and these interactions occur independently and not simultaneously, but rather in random order. We present a common model for the phosphoregulation of selective autophagy receptors.     

Atg27 Tyrosine Sorting Motif is Important for Its Trafficking and Atg9 Localization

During autophagy, the transmembrane protein Atg27 facilitates transport of the major autophagy membrane protein Atg9 to the preautophagosomal structure (PAS). To better understand the function of Atg27 and its relationship with Atg9, Atg27 trafficking and localization were examined. Atg27 localized to endosomes and the vacuolar membrane, in addition to previously described PAS, Golgi and Atg9‐positive structures. Atg27 vacuolar membrane localization was dependent on the adaptor AP‐3, which mediates direct transport from the trans‐Golgi to the vacuole. The four C‐terminal amino acids (YSAV) of Atg27 comprise a tyrosine sorting motif. Mutation of the YSAV abrogated Atg27 transport to the vacuolar membrane and affected its distribution in TGN/endosomal compartments, while PAS localization was normal. Also, in atg27(ΔYSAV) or AP‐3 mutants, accumulation of Atg9 in the vacuolar lumen was observed upon autophagy induction. Nevertheless, PAS localization of Atg9 was normal in atg27(ΔYSAV) cells. The vacuole lumen localization of Atg9 was dependent on transport through the multivesicular body, as Atg9 accumulated in the class E compartment and vacuole membrane in atg27(ΔYSAV) vps4Δ but not in ATG27 vps4Δ cells. We suggest that Atg27 has an additional role to retain Atg9 in endosomal reservoirs that can be mobilized during autophagy.      

Combination Erlotinib-Cisplatin and Atg3-Mediated Autophagy in Erlotinib Resistant Lung Cancer

Tyrosine kinase inhibitors such as erlotinib are commonly used as a therapeutic agent against cancer due to its relatively low side-effect profile and, at times, greater efficacy. However, erlotinib resistance (ER) in non-small cell lung cancer is being recognized as a major problem. Therefore, understanding the mechanism behind ER and developing effective regimens are needed. Autophagy’s role in cancer has been controversial and remains unclear. In this study, we examined the effectiveness of low dose erlotinib-cisplatin combination in erlotinib resistant lung adenocarcinoma (ERPC9) cells and the role of autophagy in ER. ERPC9 cells were established from erlotinib sensitive PC9 cells. Appropriate treatments were done over two days and cell survival was quantified with Alamar Blue assay. LC3II and regulatory proteins of autophagy were measured by western blot. Small interfering RNA (siRNA) was utilized to inhibit translation of the protein of interest. In ERPC9 cells, combination treatment induced synergistic cell death and a significant decrease in autophagy. At baseline, ERPC9 cells had a significantly higher LC3II and lower p-mTOR levels compared to PC9 cells. The addition of rapamycin increased resistance and 3-methyladenine sensitized ERPC9 cells, indicating autophagy may be acting as a protective mechanism. Further examination revealed that ERPC9 cells harbored high baseline Atg3 levels. The high basal Atg3 was targeted and significantly lowered with combination treatment. siRNA transfection of Atg3 resulted in the reversal of ER; 42.0% more cells died in erlotinib-alone treatment with transfection compared to non-transfected ERPC9 cells. We reveal a novel role for Atg3 in the promotion of ER as the inhibition of Atg3 translation was able to result in the re-sensitization of ERPC9 cells to erlotinib-alone treatment. Also, we demonstrate that combination erlotinib-cisplatin is an effective treatment against erlotinib resistant cancer by targeting (down-regulating) Atg3 mediated autophagy and induction of apoptotic cell death.     

The yeast autophagy protease Atg4 is regulated by thioredoxin

Autophagy is a membrane-trafficking process whereby double-membrane vesicles called autophagosomes engulf and deliver intracellular material to the vacuole for degradation. Atg4 is a cysteine protease with an essential function in autophagosome formation. Mounting evidence suggests that reactive oxygen species may play a role in the control of autophagy and could regulate Atg4 activity but the precise mechanisms remain unclear. In this study, we showed that reactive oxygen species activate autophagy in the model yeast Saccharomyces cerevisiae and unraveled the molecular mechanism by which redox balance controls Atg4 activity. A combination of biochemical assays, redox titrations, and site-directed mutagenesis revealed that Atg4 is regulated by oxidoreduction of a single disulfide bond between Cys338 and Cys394. This disulfide has a low redox potential and is very efficiently reduced by thioredoxin, suggesting that this oxidoreductase plays an important role in Atg4 regulation. Accordingly, we found that autophagy activation by rapamycin was more pronounced in a thioredoxin mutant compared with wild-type cells. Moreover, in vivo studies indicated that Cys338 and Cys394 are required for the proper regulation of autophagosome biogenesis, since mutation of these cysteines resulted in increased recruitment of Atg8 to the phagophore assembly site. Thus, we propose that the fine-tuning of Atg4 activity depending on the intracellular redox state may regulate autophagosome formation.     

Conserved Atg8 recognition sites mediate Atg4 association with autophagosomal membranes and Atg8 deconjugation

Deconjugation of the Atg8/LC3 protein family members from phosphatidylethanolamine (PE) by Atg4 proteases is essential for autophagy progression, but how this event is regulated remains to be understood. Here, we show that yeast Atg4 is recruited onto autophagosomal membranes by direct binding to Atg8 via two evolutionarily conserved Atg8 recognition sites, a classical LC3‐interacting region (LIR) at the C‐terminus of the protein and a novel motif at the N‐terminus. Although both sites are important for Atg4–Atg8 interaction in vivo, only the new N‐terminal motif, close to the catalytic center, plays a key role in Atg4 recruitment to autophagosomal membranes and specific Atg8 deconjugation. We thus propose a model where Atg4 activity on autophagosomal membranes depends on the cooperative action of at least two sites within Atg4, in which one functions as a constitutive Atg8 binding module, while the other has a preference toward PE‐bound Atg8.     

Atg9 Trafficking in Yeast Saccharomyces cerevisiae

Autophagy can be divided into selective and non-selective modes. This process is considered selective when a precise cargo is specifically and exclusively incorporated into autophagosomes, the double-membrane vesicles that are the hallmark of autophagy. In contrast, during nonselective, bulk autophagy, cytoplasmic components are randomly enwrapped into autophagosomes. To date, approximately 30 autophagy-related genes called ATG have been identified. Sixteen of them compose the general basic machinery catalyzing the formation of double-membrane vesicles in all eukaryotic cells. The rest of them are often not conserved between species and cooperate with the basic Atg proteins during either selective or nonselective autophagy. Atg9 is the only integral membrane component of the conserved Atg machinery and appears to be a crucial organizational element.5 Recent studies in the S. cerevisiae have shown that Atg9 transport is differentially regulated depending on the autophagy mode. In this addendum, we will review and discuss what has recently been unveiled about yeast S. cerevisiae Atg9 trafficking, its modulators and its potential role in double-membrane vesicle biogenesis.

Addendum to:

Atg9 Sorting from Mitochondria is Impaired in Early Secretion and VFT Complex Mutants in Saccharomyces cerevisiae

F. Reggiori and D.J. Klionsky

J Cell Sci 2006: 119:2903-11     

β‐Guanidinopropionic acid extends the lifespan of Drosophila melanogaster via an AMP‐activated protein kinase‐dependent increase in autophagy

Previous studies have demonstrated that AMP‐activated protein kinase (AMPK) controls autophagy through the mammalian target of rapamycin (mTOR) and Unc‐51 like kinase 1 (ULK1/Atg1) signaling, which augments the quality of cellular housekeeping, and that β‐guanidinopropionic acid (β‐GPA), a creatine analog, leads to a chronic activation of AMPK. However, the relationship between β‐GPA and aging remains elusive. In this study, we hypothesized that feeding β‐GPA to adult Drosophila produces the lifespan extension via activation of AMPK‐dependent autophagy. It was found that dietary administration of β‐GPA at a concentration higher than 900 mm induced a significant extension of the lifespan of Drosophila melanogaster in repeated experiments. Furthermore, we found that Atg8 protein, the homolog of microtubule‐associated protein 1A/1B‐light chain 3 (LC3) and a biomarker of autophagy in Drosophila, was significantly upregulated by β‐GPA treatment, indicating that autophagic activity plays a role in the effect of β‐GPA. On the other hand, when the expression of Atg5 protein, an essential protein for autophagy, was reduced by RNA interference (RNAi), the effect of β‐GPA on lifespan extension was abolished. Moreover, we found that AMPK was also involved in this process. β‐GPA treatment significantly elevated the expression of phospho‐T172‐AMPK levels, while inhibition of AMPK by either AMPK‐RNAi or compound C significantly attenuated the expression of autophagy‐related proteins and lifespan extension in Drosophila. Taken together, our results suggest that β‐GPA can induce an extension of the lifespan of Drosophila via AMPK‐Atg1‐autophagy signaling pathway.     

Golgi membrane‐associated degradation pathway in yeast and mammals

Autophagy is a cellular process that degrades subcellular constituents, and is conserved from yeast to mammals. Although autophagy is believed to be essential for living cells, cells lacking Atg5 or Atg7 are healthy, suggesting that a non‐canonical degradation pathway exists to compensate for the lack of autophagy. In this study, we show that the budding yeast Saccharomyces cerevisiae, which lacks Atg5, undergoes bulk protein degradation using Golgi‐mediated structures to compensate for autophagy when treated with amphotericin B1, a polyene antifungal drug. We named this mechanism Golgi membrane‐associated degradation (GOMED) pathway. This process is driven by the disruption of PI(4)P‐dependent anterograde trafficking from the Golgi, and it also exists in Atg5‐deficient mammalian cells. Biologically, when an Atg5‐deficient β‐cell line and Atg7‐deficient β‐cells were cultured in glucose‐deprived medium, a disruption in the secretion of insulin granules from the Golgi occurred, and GOMED was induced to digest these (pro)insulin granules. In conclusion, GOMED is activated by the disruption of PI(4)P‐dependent anterograde trafficking in autophagy‐deficient yeast and mammalian cells.     

Atg17/FIP200 localizes to perilysosomal Ref(2)P aggregates and promotes autophagy by activation of Atg1 in Drosophila

Phagophore-derived autophagosomes deliver cytoplasmic material to lysosomes for degradation and reuse. Autophagy mediated by the incompletely characterized actions of Atg proteins is involved in numerous physiological and pathological settings including stress resistance, immunity, aging, cancer, and neurodegenerative diseases. Here we characterized Atg17/FIP200, the Drosophila ortholog of mammalian RB1CC1/FIP200, a proposed functional equivalent of yeast Atg17. Atg17 disruption inhibits basal, starvation-induced and developmental autophagy, and interferes with the programmed elimination of larval salivary glands and midgut during metamorphosis. Upon starvation, Atg17-positive structures appear at aggregates of the selective cargo Ref(2)P/p62 near lysosomes. This location may be similar to the perivacuolar PAS (phagophore assembly site) described in yeast. Drosophila Atg17 is a member of the Atg1 kinase complex as in mammals, and we showed that it binds to the other subunits including Atg1, Atg13, and Atg101 (C12orf44 in humans, 9430023L20Rik in mice and RGD1359310 in rats). Atg17 is required for the kinase activity of endogenous Atg1 in vivo, as loss of Atg17 prevents the Atg1-dependent shift of endogenous Atg13 to hyperphosphorylated forms, and also blocks punctate Atg1 localization during starvation. Finally, we found that Atg1 overexpression induces autophagy and reduces cell size in Atg17-null mutant fat body cells, and that overexpression of Atg17 promotes endogenous Atg13 phosphorylation and enhances autophagy in an Atg1-dependent manner in the fat body. We propose a model according to which the relative activity of Atg1, estimated by the ratio of hyper- to hypophosphorylated Atg13, contributes to setting low (basal) vs. high (starvation-induced) autophagy levels in Drosophila.