Autophagy is an intracellular degradation process that is mediated by autophagosomes. Mammalian Atg2 proteins Atg2A and Atg2B are identified and characterized as essential for autophagy. They are also present on lipid droplets and are involved in regulation of lipid droplet volume and distribution.
Macroautophagy is an intracellular degradation system by which cytoplasmic materials are enclosed by the autophagosome and delivered to the lysosome. Autophagosome formation is considered to take place on the endoplasmic reticulum and involves functions of autophagy-related (Atg) proteins. Here, we report the identification and characterization of mammalian Atg2 homologues Atg2A and Atg2B. Simultaneous silencing of Atg2A and Atg2B causes a block in autophagic flux and accumulation of unclosed autophagic structures containing most Atg proteins. Atg2A localizes on the autophagic membrane, as well as on the surface of lipid droplets. The Atg2A region containing amino acids 1723–1829, which shows relatively high conservation among species, is required for localization to both the autophagic membrane and lipid droplet and is also essential for autophagy. Depletion of both Atg2A and Atg2B causes clustering of enlarged lipid droplets in an autophagy-independent manner. These data suggest that mammalian Atg2 proteins function both in autophagosome formation and regulation of lipid droplet morphology and dispersion.
Autophagy induced by nutrient depletion is involved in survival during starvation conditions. In addition to starvation-induced autophagy, the yeast Saccharomyces cerevisiae also has a constitutive autophagy-like system, the Cvt pathway. Among 31 autophagy-related (Atg) proteins, the function of Atg17, Atg29, and Atg31 is required specifically for autophagy. In this study, we investigated the role of autophagy-specific (i.e., non-Cvt) proteins under autophagy-inducing conditions. For this purpose, we used atg11Δ cells in which the Cvt pathway is abrogated. The autophagy-unique proteins are required for the localization of Atg proteins to the pre-autophagosomal structure (PAS), the putative site for autophagosome formation, under starvation condition. It is likely that these Atg proteins function as a ternary complex, because Atg29 and Atg31 bind to Atg17. The Atg1 kinase complex (Atg1–Atg13) is also essential for recruitment of Atg proteins to the PAS. The assembly of Atg proteins to the PAS is observed only under autophagy-inducing conditions, indicating that this structure is specifically involved in autophagosome formation. Our results suggest that Atg1 complex and the autophagy-unique Atg proteins cooperatively organize the PAS in response to starvation signals.
The relative balance between the quantity of white and brown adipose tissue can profoundly affect lipid storage and whole-body energy homeostasis. However, the mechanisms regulating the formation, expansion, and interconversion of these 2 distinct types of fat remain unknown. Recently, the lysosomal degradative pathway of macroautophagy has been identified as a regulator of cellular differentiation, suggesting that autophagy may modulate this process in adipocytes. The function of autophagy in adipose differentiation was therefore examined in the current study by genetic inhibition of the critical macroautophagy gene autophagy-related 7 (Atg7). Knockdown of Atg7 in 3T3-L1 preadipocytes inhibited lipid accumulation and decreased protein levels of adipocyte differentiation factors. Knockdown of Atg5 or pharmacological inhibition of autophagy or lysosome function also had similar effects. An adipocyte-specific mouse knockout of Atg7 generated lean mice with decreased white adipose mass and enhanced insulin sensitivity. White adipose tissue in knockout mice had increased features of brown adipocytes, which, along with an increase in normal brown adipose tissue, led to an elevated rate of fatty acid, β-oxidation, and a lean body mass. Autophagy therefore functions to regulate body lipid accumulation by controlling adipocyte differentiation and determining the balance between white and brown fat.
Cells must regulate both biosynthesis and degradation to ensure proper homeostasis of cellular organelles and proteins. This balance is demonstrated in a unique way in the yeast Saccharomyces cerevisiae, which possesses two distinct, yet mechanistically related trafficking routes mediating the delivery of proteins from the cytoplasm to the vacuole: the biosynthetic cytoplasm to vacuole targeting (Cvt) and the degradative autophagy pathways. Several components employed by these two transport routes have been identified, but their mechanistic interactions remain largely unknown. Here we report a novel gene involved in these pathways, which we have named ATG23. Atg23 localizes to the pre-autophagosomal structure but also to other cytosolic punctate compartments. Our characterization of the Atg23 protein indicates that it is required for the Cvt pathway and efficient autophagy but not pexophagy. In the absence of Atg23, cargo molecules such as prApe1 are correctly recruited to a pre-autophagosomal structure that is unable to give rise to Cvt vesicles. We also demonstrate that Atg23 is a peripheral membrane protein that requires the presence of Atg9/Apg9 to be specifically targeted to lipid bilayers. Atg9 transiently interacts with Atg23 suggesting that it participates in the recruitment of this protein.
Autophagy is a fundamental cellular process required for organelle degradation and removal of invasive pathogens. Autophagosome formation involves the recruitment of, and interaction between, multiple proteins produced from autophagy-related (ATG) genes. One of the key complexes in autophagosome formation is the ATG12-ATG5-ATG16L1 complex. ATG16L1 functions as a molecular scaffold mediating protein-protein interactions necessary for formation of the autophagosome in response to both classical and pathogen-related autophagy stimuli. The coiled-coil domain of the yeast ortholog, ATG16, exists as a homodimer both in solution and in the crystal form. The yeast and human orthologs show poor sequence identity. Here we have sought to determine the minimal boundaries of the human ATG16L1 coiled-coil domain and ascertain its oligomeric status in solution. Using a range of biochemical and biophysical techniques we show that the secondary structure of the human ATG16L1 coiled-coil has the expected helical composition and that the domain forms a homodimer in solution. We also observe extensive sequence conservation across vertebrates providing strong support for the crucial functional role of the ATG16L1 coiled-coil.
Autophagy is an evolutionarily conserved bulk-protein degradation pathway in which isolation membranes engulf the cytoplasmic constituents, and the resulting autophagosomes transport them to lysosomes. Two ubiquitin-like conjugation systems, termed Atg12 and Atg8 systems, are essential for autophagosomal formation. In addition to the pathophysiological roles of autophagy in mammals, recent mouse genetic studies have shown that the Atg8 system is predominantly under the control of the Atg12 system. To clarify the roles of the Atg8 system in mammalian autophagosome formation, we generated mice deficient in Atg3 gene encoding specific E2 enzyme for Atg8. Atg3-deficient mice were born but died within 1 d after birth. Conjugate formation of mammalian Atg8 homologues was completely defective in the mutant mice. Intriguingly, Atg12–Atg5 conjugation was markedly decreased in Atg3-deficient mice, and its dissociation from isolation membranes was significantly delayed. Furthermore, loss of Atg3 was associated with defective process of autophagosome formation, including the elongation and complete closure of the isolation membranes, resulting in malformation of the autophagosomes. The results indicate the essential role of the Atg8 system in the proper development of autophagic isolation membranes in mice.
The Atg12–Atg5 conjugate was prepared by in vivo reconstitution and was crystallized with Atg16 using the free-interface diffusion method.
Autophagy mediates the bulk degradation of cytoplasmic components in lysosomes/vacuoles. Five autophagy-related (Atg) proteins are involved in a ubiquitin-like protein conjugation system. Atg12 is conjugated to its sole target, Atg5, by two enzymes, Atg7 and Atg10. The Atg12–Atg5 conjugates form a multimeric complex with Atg16. Formation of the Atg12–Atg5–Atg16 ternary complex is crucial for the functions of these proteins on autophagy. Here, the expression, purification and crystallization of the Atg12–Atg5 conjugate bound to the N-terminal region of Atg16 (Atg16N) are reported. The Atg12–Atg5 conjugates were formed by co-expressing Atg5, Atg7, Atg10 and Atg12 in Eschericia coli. The Atg12–Atg5–Atg16N ternary complex was formed by mixing purified Atg12–Atg5 conjugates and Atg16N, and was further purified by gel-filtration chromatography. Crystallization screening was performed by the free-interface diffusion method. Using obtained microcrystals as seeds, large crystals for diffraction data collection were obtained by the sitting-drop vapour-diffusion method. The crystal contained one ternary complex per asymmetric unit, and diffracted to 2.6 Å resolution.
autophagy; ubiquitin-like conjugation; crystallization; free-interface diffusion method
In eukaryotic cells, nutrient starvation induces the bulk degradation of cellular materials; this process is called autophagy. In the yeast Saccharomyces cerevisiae, most of the ATG (autophagy) genes are involved in not only the process of degradative autophagy, but also a biosynthetic process, the cytoplasm to vacuole (Cvt) pathway. In contrast, the ATG17 gene is required specifically in autophagy. To better understand the function of Atg17, we have performed a biochemical characterization of the Atg17 protein. We found that the atg17Δ mutant under starvation condition was largely impaired in autophagosome formation and only rarely contained small autophagosomes, whose size was less than one-half of normal autophagosomes in diameter. Two-hybrid analyses and coimmunoprecipitation experiments demonstrated that Atg17 physically associates with Atg1-Atg13 complex, and this binding was enhanced under starvation conditions. Atg17-Atg1 binding was not detected in atg13Δ mutant cells, suggesting that Atg17 interacts with Atg1 through Atg13. A point mutant of Atg17, Atg17C24R, showed reduced affinity for Atg13, resulting in impaired Atg1 kinase activity and significant defects in autophagy. Taken together, these results indicate that Atg17-Atg13 complex formation plays an important role in normal autophagosome formation via binding to and activating the Atg1 kinase.
Progression of development has to be insulated from the damaging impacts of environmental and genetic perturbations to produce highly predictable phenotypes. Molecular chaperones, such as the heat shock proteins (HSPs), are known to buffer various environmental stresses, and are deeply involved in protein homeostasis. These characteristics of HSPs imply that they might affect developmental buffering and canalization.
We examined the role of nine Hsp genes using the GAL4/UAS-RNAi system on phenotypic variation of various morphological traits in Drosophila melanogaster. The stability of bristle number, wing size and wing shape was characterized through fluctuating asymmetry (FA) and the coefficient of variation (CV), or among-individual variation. Progeny of the GAL4/Hsp-RNAi crosses tended to have reduced trait means for both wing size and wing shape. Transcriptional knockdown of Hsp67Bc and Hsp22 significantly increased FA of bristle number, while knockdown of Hsp67Ba significantly increased FA and among-individual variation of wing shape but only in males. Suppression of Hsp67Bb expression significantly increased among-individual variation of bristle number. The knockdown of gene expression was confirmed for Hsp67Ba, Hsp67Bc, Hsp22, and Hsp67Bb. Correlation between FA and CV or among-individual variation of each trait is weak and not significant except for the case of male wing shape.
Four small Hsp genes (Hsp22, Hsp67Ba, Hsp67Bb and Hsp67Bc) showed involvement in the processes of morphogenesis and developmental stability. Due to possible different functions in terms of developmental buffering of these small Hsps, phenotypic stability of an organism is probably maintained by multiple mechanisms triggered by different environmental and genetic stresses on different traits. This novel finding may lead to a better understanding of non-Hsp90 molecular mechanisms controlling variability in morphological traits.
Plant autophagy plays an important role in delaying senescence, nutrient recycling, and stress responses. Functional analysis of plant autophagy has almost exclusively focused on the proteins required for the core process of autophagosome assembly, but little is known about the proteins involved in other important processes of autophagy, including autophagy cargo recognition and sequestration. In this study, we report functional genetic analysis of Arabidopsis NBR1, a homolog of mammalian autophagy cargo adaptors P62 and NBR1. We isolated two nbr1 knockout mutants and discovered that they displayed some but not all of the phenotypes of autophagy-deficient atg5 and atg7 mutants. Like ATG5 and ATG7, NBR1 is important for plant tolerance to heat, oxidative, salt, and drought stresses. The role of NBR1 in plant tolerance to these abiotic stresses is dependent on its interaction with ATG8. Unlike ATG5 and ATG7, however, NBR1 is dispensable in age- and darkness-induced senescence and in resistance to a necrotrophic pathogen. A selective role of NBR1 in plant responses to specific abiotic stresses suggest that plant autophagy in diverse biological processes operates through multiple cargo recognition and delivery systems. The compromised heat tolerance of atg5, atg7, and nbr1 mutants was associated with increased accumulation of insoluble, detergent-resistant proteins that were highly ubiquitinated under heat stress. NBR1, which contains an ubiquitin-binding domain, also accumulated to high levels with an increasing enrichment in the insoluble protein fraction in the autophagy-deficient mutants under heat stress. These results suggest that NBR1-mediated autophagy targets ubiquitinated protein aggregates most likely derived from denatured or otherwise damaged nonnative proteins generated under stress conditions.
Autophagy is an evolutionarily conserved process that sequestrates and delivers cytoplasmic macromolecules and organelles to the vacuoles or lysosomes for degradation. In plants, autophagy is involved in supplying internal nutrients during starvation and in promoting cell survival during senescence and during biotic and abiotic stresses. Arabidopsis NBR1 is a homolog of mammalian autophagy cargo adaptors P62 and NBR1. Disruption of Arabidopsis NBR1 caused increased sensitivity to a spectrum of abiotic stresses but had no significant effect on plant senescence, responses to carbon starvation, or resistance to a necrotrophic pathogen. NBR1 contains an ubiquitin-binding domain, and the compromised stress tolerance of autophagy mutants was associated with increased accumulation of NBR1 and ubiquitin-positive cellular protein aggregates in the insoluble protein fraction under stress conditions. Based on these results, we propose that NBR1 targets ubiquitinated protein aggregates most likely derived from denatured and otherwise damaged nonnative proteins for autophagic clearance under stress conditions.
In yeast, ∼31 autophagy-related (Atg) proteins have been identified. Most of them reside at the phagophore assembly site (PAS), although the function of the PAS mostly remains unclear. One reason for the latter is the lack of stoichiometric information regarding the Atg proteins at this site. We report the application of fluorescence microscopy to study the amount of Atg proteins at the PAS. We find that an increase in the amount of Atg11 at the PAS enhances the recruitment of Atg8 and Atg9 to this site and facilitates the formation of more cytoplasm-to-vacuole targeting vesicles. In response to autophagy induction, the amount of most Atg proteins remains unchanged at the PAS, whereas we see an enhanced recruitment of Atg8 and 9 at this site. During autophagy, the amount of Atg8 at the PAS showed a periodic change, indicating the formation of autophagosomes. Application of this method and further analysis will provide more insight into the functions of Atg proteins.
A central part of the core macroauto-phagy (hereafter autophagy) machinery includes the two ubiquitin-like (Ubl) conjugation systems that involve the Ubl proteins Atg8 and Atg12.1 Although the functions of these proteins have not been fully elucidated, they play critical roles in autophagosome formation. For example, Atg8 is involved in cargo recognition,2,3 and the amount of Atg8 in part determines the size of the autophagosome,4 whereas Atg12 is part of a trimer that may function as an E3 ligase to facilitate Atg8 conjugation to phosphatidylethanolamine and determine, in part, the site of the conjugation reaction.5 Thus, fully functional autophagy requires both the Atg8 and Atg12 conjugation systems. Dysfunctional autophagy is associated with various human pathophysiologies including cancer, neurodegeneration, gastrointestinal disorders and heart disease. So, if you are wondering whether autophagy is operating properly in your own body, what can you do? The problem is that there are relatively few methods for analyzing autophagy in vivo.6-11 Minimally, you might want to find out if the relevant genes are intact and have the correct sequence. Considering the rapid advances being made in DNA sequencing technology, it is likely only a matter of time before people can submit a DNA sample and obtain a rapid readout of particular genes, or their entire genome. Thus, anticipating the future, we decided to analyze a select set of autophagy-related (ATG) genes, with a focus on those encoding components of the Ubl conjugation systems, by a polymerase chain reaction (PCR)-based method that combines science with art.
autophagy; collaboration; gel electrophoresis; membrane; primer
Autophagy is a catabolic process involved in the degradation of a cell's own components for cell growth, development, homeostasis, and the recycling of cellular products. Autophagosome is an essential component in the protozoan parasite during differentiation and encystation. The present study identified and characterized autophagy-related protein (Atg) 3, a member of Atg8 conjugation system, in Acanthamoeba castellanii (AcAtg3). AcAtg3 encoding a 304 amino acid protein showed high similarity with the catalytic cysteine site of other E2 like enzymes of ubiquitin system. Predicted 3D structure of AcAtg3 revealed a hammer-like shape, which is the characteristic structure of E2-like enzymes. The expression level of AcAtg3 did not increase during encystation. However, the formation of mature cysts was significantly reduced in Atg3-siRNA transfected cells in which the production of Atg8-phosphatidylethanolamine conjugate was inhibited. Fluorescent microscopic analysis revealed that dispersed AcAtg3-EGFP fusion protein gathered around autophagosomal membranes during encystation. These results provide important information for understanding autophagic machinery through the lipidation reaction mediated by Atg3 in Acanthamoeba.
Acanthamoeba; encystation; autophagosome; Atg3
The ubiquitin-proteasome and lysosome-autophagy pathways are the two major intracellular protein degradation systems that work cooperatively to maintain homeostasis. Proteasome inhibitors (PIs) have clinical activity in hematological tumors, and inhibitors of autophagy are also being evaluated as potential antitumor therapies. In the current study we found that chemical proteasome inhibitors and siRNA-mediated knockdown of the proteasome's enzymatic subunits promoted autophagosome formation, stimulated autophagic flux, and upregulated expression of the autophagy specific genes (ATGs) (ATG5 and ATG7) in some human prostate cancer cells and immortalized mouse embryonic fibroblasts (MEFs). Upregulation of ATG5 and ATG7 only occurred in cells displaying PIs-induced phosphorylation of the eukaryotic translation initiation factor 2alpha (eIF2α), an important component of the unfolded protein responses. Furthermore, PIs did not induce autophagy or upregulate ATG5 in MEFs expressing a phosphorylation-deficient mutant form of eIF2α. Combined inhibition of autophagy and the proteasome induced an accumulation of intracellular protein aggregates reminiscent of neuronal inclusion bodies and caused more cancer cell death than blocking either degradation pathway alone. Overall, our data demonstrate that proteasome inhibition activates autophagy via a phospho-eIF2α-dependent mechanism to eliminate protein aggregates and alleviate proteotoxic stress.
Bortezomib; NPI-0052; autophagy; unfolded protein response; prostate cancer
Formation of the autophagosome is likely the most complex step of macroautophagy, and indeed it is the morphological and functional hallmark of this process; accordingly, it is critical to understand the corresponding molecular mechanism. Atg8 is the only known autophagy-related (Atg) protein required for autophagosome formation that remains associated with the completed sequestering vesicle. Approximately one-fourth of all of the characterized Atg proteins that participate in autophagosome biogenesis affect Atg8, regulating its conjugation to phosphatidylethanolamine (PE), localization to the phagophore assembly site and/or subsequent deconjugation. An unanswered question in the field regards the physiological role of the deconjugation of Atg8–PE. Using an Atg8 mutant that bypasses the initial Atg4-dependent processing, we demonstrate that Atg8 deconjugation is an important step required to facilitate multiple events during macroautophagy. The inability to deconjugate Atg8–PE results in the mislocalization of this protein to the vacuolar membrane. We also show that the deconjugation of Atg8–PE is required for efficient autophagosome biogenesis, the assembly of Atg9-containing tubulovesicular clusters into phagophores/autophagosomes, and for the disassembly of PAS-associated Atg components.
Autophagy is an intracellular degradation process, through which cytosolic materials are delivered to the lysosome. Despite recent identification of many autophagy-related genes, how autophagosomes are generated remains unclear. Here, we examined the hierarchical relationships among mammalian Atg proteins. Under starvation conditions, ULK1, Atg14, WIPI-1, LC3 and Atg16L1 target to the same compartment, whereas DFCP1 localizes adjacently to these Atg proteins. In terms of puncta formation, the protein complex including ULK1 and FIP200 is the most upstream unit and is required for puncta formation of the Atg14-containing PI3-kinase complex. Puncta formation of both DFCP1 and WIPI-1 requires FIP200 and Atg14. The Atg12-Atg5-Atg16L1 complex and LC3 are downstream units among these factors. The punctate structures containing upstream Atg proteins such as ULK1 and Atg14 tightly associate with the ER, where the ER protein vacuole membrane protein 1 (VMP1) also transiently localizes. These structures are formed even when cells are treated with wortmannin to suppress autophagosome formation. These hierarchical analyses suggest that ULK1, Atg14 and VMP1 localize to the ER-associated autophagosome formation sites in a PI3-kinase activity-independent manner.
autophagosome; PI3-kinase; isolation membrane; endoplasmic reticulum; ULK
Autophagy is the chief machinery for bulk degradation of superfluous or aberrant cytoplasmic components. This study used the rat moderate fluid percussion injury model to investigate whether the autophagy pathway plays a key role after traumatic brain injury (TBI). Induction of autophagy is manifested by accumulation of autophagosomes (APs), observable under transmission electron microscopy (EM). Two hallmarks of autophagy, i.e., the microtubule-associated protein light chain 3 (LC3)-II and the autophagy-related gene (ATG)12-ATG5 conjugates, were explored by biochemical and confocal microscopic analyses of brain tissues. Under EM, both APs and autolysosomes were markedly accumulated in neurons from 4 h onward after TBI. Western blot analysis showed that ATG12-ATG5 conjugate was markedly redistributed during 5 to 15 days in brain tissues after TBI. LC3-II conjugate was initially unchanged but was drastically upregulated from 24 h onward in the pre-AP-containing fraction after TBI. LC-3 immunostaining was mainly located in living neurons under confocal microscopy. These results clearly show that the autophagy pathway is persistently activated after TBI. Because the autophagy pathway is the chief machinery for bulk elimination of aberrant cell components, we propose that activation of this pathway serves as a protective mechanism for maintaining cellular homeostasis after TBI.
ATG12-ATG5; autophagy; LC3-ATG8-APG8; lysosome; traumatic brain injury; ubiquitin
Macroautophagy (autophagy) is a bulk cytoplasmic degradation process that is conserved from yeast to mammals. Autophagy is an important cellular response to starvation and stress, and plays important roles in development, cell death, aging, immunity, and cancer. The fruit fly Drosophila melanogaster provides an excellent model system to study autophagy in vivo, in the context of a developing organism. Autophagy (atg) genes and their regulators are conserved in Drosophila, and autophagy is induced in response to nutrient starvation and hormones during development. In this review we provide an overview of how Drosophila research has contributed to our understanding of the role and regulation of autophagy in cell survival, growth, nutrient utilization, and cell death. Recent Drosophila research has also provided important mechanistic information about the role of autophagy in protein aggregation disorders, neurodegeneration, aging, and innate immunity. Differences in the role of autophagy in specific contexts and/or cell types suggest that there may be cell-context-specific regulators of autophagy, and studies in Drosophila are well-suited to yield discoveries about this specificity.
The TOR kinases are conserved negative regulators of autophagy in response to nutrient conditions, but the signaling mechanisms are poorly understood. Here we describe a complex containing the protein kinase Atg1 and the phosphoprotein Atg13 that functions as a critical component of this regulation in Drosophila. We show that knockout of Atg1 or Atg13 results in a similar, selective defect in autophagy in response to TOR inactivation. Atg1 physically interacts with TOR and Atg13 in vivo, and both Atg1 and Atg13 are phosphorylated in a nutrient-, TOR- and Atg1 kinase-dependent manner. In contrast to yeast, phosphorylation of Atg13 is greatest under autophagic conditions and does not preclude Atg1-Atg13 association. Atg13 stimulates both the autophagic activity of Atg1 and its inhibition of cell growth and TOR signaling, in part by disrupting the normal trafficking of TOR. In contrast to the effects of normal Atg13 levels, increased expression of Atg13 inhibits autophagosome expansion and recruitment of Atg8/LC3, potentially by decreasing the stability of Atg1 and facilitating its inhibitory phosphorylation by TOR. Atg1-Atg13 complexes thus function at multiple levels to mediate and adjust nutrient-dependent autophagic signaling.
Mitochondrial quality control is important to maintain proper cellular homeostasis. Although selective mitochondrial degradation by autophagy (mitophagy) is suggested to have an important role for quality control and there is evidence for a direct relation between mitophagy and neurodegenerative diseases, the molecular mechanism of mitophagy is poorly understood. Using a screen for mitophagy-deficient mutants, we found that YIL146C/ECM37 is essential for mitophagy. This gene is not required for other types of selective autophagy or for nonspecific macroautophagy. We designated this autophagy-related (ATG) gene as ATG32. The Atg32 protein localizes on mitochondria. Following the induction of mitophagy, Atg32 binds Atg11, an adaptor protein for selective types of autophagy, and then Atg32 is recruited to and imported into the vacuole along with mitochondria. Therefore, Atg32 confers selectivity for mitochondrial sequestration as a cargo and is necessary for recruitment of this organelle by the autophagy machinery for mitophagy.
autophagy; ECM37; lysosome; mitochondria; stress; vacuole; yeast
Atg16L1, a protein essential for autophagy, is localized on dense-core vesicles in PC12 cells, and knockdown of Atg16L1 inhibits hormone secretion independently of autophagy. In addition, Atg16L1 interacts with the small GTPase Rab33A, and this interaction is required for the dense-core vesicle localization of Atg16L1.
Autophagy is a bulk degradation system in all eukaryotic cells and regulates a variety of biological activities in higher eukaryotes. Recently involvement of autophagy in the regulation of the secretory pathway has also been reported, but the molecular mechanism linking autophagy with the secretory pathway remains largely unknown. Here we show that Atg16L1, an essential protein for canonical autophagy, is localized on hormone-containing dense-core vesicles in neuroendocrine PC12 cells and that knockdown of Atg16L1 causes a dramatic reduction in the level of hormone secretion independently of autophagic activity. We also find that Atg16L1 interacts with the small GTPase Rab33A and that this interaction is required for the dense-core vesicle localization of Atg16L1 in PC12 cells. Our findings indicate that Atg16L1 regulates not only autophagy in all cell types, but also secretion from dense-core vesicles, presumably by acting as a Rab33A effector, in particular cell types.
Autophagy has paradoxical and complex functions in cancer development, and autophagy-related genes (ATG) are key regulators in autophagy. Until now, more than 30 different ATG proteins have been identified in yeast, and their mammalian counterparts also have been reported. Although the roles of a few ATG proteins in cancer have been characterized, the role of ATG10 is almost completely unknown.
To investigate the clinicopathological role of ATG10 in colorectal cancer, we analyzed ATG10 expression in colorectal cancer tissues and cell lines. Protein expression analysis showed that ATG10 is highly increased in colorectal cancer (tissue - 18/37 cases, 48%; cell line –8/12 cell lines, 66%). Immunohistochemical analysis with clinicopathological features indicated a strong association of the up-regulation of ATG10 with tumor lymph node metastasis (p = 0.005) and invasion (p<0.001). Moreover, both 5-year disease free survival and overall survival rates of patients bearing tumors that did not express ATG10 were significantly higher than those of patients bearing ATG10-expressing tumors (p = 0.012).
Increased expression of ATG10 in colorectal cancer is associated with lymphovascular invasion and lymph node metastasis indicating that ATG10 may be a potential prognostic maker in colorectal cancer.
Autophagy is a highly regulated program of self-degradation of the cytosolic constituents that has key roles during early development and in adult cell growth and homeostasis. To investigate the role of autophagy in otic neurogenesis, we studied the expression of autophagy genes in early stages of chicken (Gallus gallus) inner ear development and the consequences of inhibiting the autophagic pathway in organotypic cultures of explanted chicken otic vesicles (OVs). Here we show the expression of autophagy-related genes (Atg) Beclin-1 (Atg6), Atg5 and LC3B (Atg8) in the otocyst and the presence of autophagic vesicles by using transmission electron microscopy in the otic neurogenic zone. The inhibition of the transcription of LC3B by using antisense morpholinos and of class III phosphatidylinositol 3-kinase with 3-methyladenine causes an aberrant morphology of the OV with accumulation of apoptotic cells. Moreover, inhibition of autophagy provokes the misregulation of the cell cycle in the otic epithelium, impaired neurogenesis and poor axonal outgrowth. Finally, our results indicate that autophagy provides the energy required for the clearing of neuroepithelial dying cells and suggest that it is required for the migration of otic neuronal precursors. Taken together, our results show for the first time that autophagy is an active and essential process during early inner ear development.
apoptosis; autophagy; 3-MA; otic neurogenesis; IGF-I
Autophagy-related (Atg) pathways deliver cytosol and organelles to the vacuole in double-membrane vesicles called autophagosomes, which are formed at the phagophore assembly site (PAS), where most of the core Atg proteins assemble. Atg28 is a component of the core autophagic machinery partially required for all Atg pathways in Pichia pastoris. This coiled-coil protein interacts with Atg17 and is essential for micropexophagy. However, the role of Atg28 in micropexophagy was unknown. We used the yeast two-hybrid system to search for Atg28 interaction partners from P. pastoris and identified a new Atg protein, named Atg35. The atg35Δ mutant was not affected in macropexophagy, cytoplasm-to-vacuole targeting or general autophagy. However, both Atg28 and Atg35 were required for micropexophagy and for the formation of the micropexophagic apparatus (MIPA). This requirement correlated with a stronger expression of both proteins on methanol and glucose. Atg28 mediated the interaction of Atg35 with Atg17. Trafficking of overexpressed Atg17 from the peripheral ER to the nuclear envelope was required to organize a peri-nuclear structure (PNS), the site of Atg35 colocalization during micropexophagy. In summary, Atg35 is a new Atg protein that relocates to the PNS and specifically regulates MIPA formation during micropexophagy.
Atg protein; peroxisome; micropexophagy; MIPA; nucleus
Autophagy is a membrane-mediated degradation process, which is governed by sequential functions of Atg proteins. Although Atg proteins are highly conserved in eukaryotes, protozoa possess only a partial set of Atg proteins. Nonetheless, almost all protozoa have the complete factors belonging to the Atg8 conjugation system, namely, Atg3, Atg4, Atg7, and Atg8. Here, we report the biochemical properties and subcellular localization of the Atg8 protein of the human malaria parasite Plasmodium falciparum (PfAtg8). PfAtg8 is expressed during intra-erythrocytic development and associates with membranes likely as a lipid-conjugated form. Fluorescence microscopy and immunoelectron microscopy show that PfAtg8 localizes to the apicoplast, a four membrane-bound non-photosynthetic plastid. Autophagosome-like structures are not observed in the erythrocytic stages. These data suggest that, although Plasmodium parasites have lost most Atg proteins during evolution, they use the Atg8 conjugation system for the unique organelle, the apicoplast.