In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker

Macroautophagy mediates the bulk degradation of cytoplasmic components. It accounts for the degradation of most long-lived proteins: cytoplasmic constituents, including organelles, are sequestered into autophagosomes, which subsequently fuse with lysosomes, where degradation occurs. Although the possible involvement of autophagy in homeostasis, development, cell death, and pathogenesis has been repeatedly pointed out, systematic in vivo analysis has not been performed in mammals, mainly because of a limitation of monitoring methods. To understand where and when autophagy occurs in vivo, we have generated transgenic mice systemically expressing GFP fused to LC3, which is a mammalian homologue of yeast Atg8 (Aut7/Apg8) and serves as a marker protein for autophagosomes. Fluorescence microscopic analyses revealed that autophagy is differently induced by nutrient starvation in most tissues. In some tissues, autophagy even occurs actively without starvation treatments. Our results suggest that the regulation of autophagy is organ dependent and the role of autophagy is not restricted to the starvation response. This transgenic mouse model is a useful tool to study mammalian autophagy.     

Characterization of a novel autophagy-specific gene, ATG29

Autophagy is a process whereby cytoplasmic proteins and organelles are sequestered for bulk degradation in the vacuole/lysosome. At present, 16 ATG genes have been found that are essential for autophagosome formation in the yeast Saccharomyces cerevisiae. Most of these genes are also involved in the cytoplasm to vacuole transport pathway, which shares machinery with autophagy. Most Atg proteins are colocalized at the pre-autophagosomal structure (PAS), from which the autophagosome is thought to originate, but the precise mechanism of autophagy remains poorly understood. During a genetic screen aimed to obtain novel gene(s) required for autophagy, we identified a novel ORF, ATG29/YPL166w. atg29Delta cells were sensitive to starvation and induction of autophagy was severely retarded. However, the Cvt pathway operated normally. Therefore, ATG29 is an ATG gene specifically required for autophagy. Additionally, an Atg29-GFP fusion protein was observed to localize to the PAS. From these results, we propose that Atg29 functions in autophagosome formation at the PAS in collaboration with other Atg proteins.     

Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells

Autophagy is a lysosomal bulk degradation pathway for cytoplasmic cargo, such as long-lived proteins, lipids, and organelles. Induced upon nutrient starvation, autophagic degradation is accomplished by the concerted actions of autophagy-related (ATG) proteins. Here we demonstrate that two ATGs, human Atg2A and Atg14L, colocalize at cytoplasmic lipid droplets (LDs) and are functionally involved in controlling the number and size of LDs in human tumor cell lines. We show that Atg2A is targeted to cytoplasmic ADRP-positive LDs that migrate bidirectionally along microtubules. The LD localization of Atg2A was found to be independent of the autophagic status. Further, Atg2A colocalized with Atg14L under nutrient-rich conditions when autophagy was not induced. Upon nutrient starvation and dependent on phosphatidylinositol 3-phosphate [PtdIns(3)P] generation, both Atg2A and Atg14L were also specifically targeted to endoplasmic reticulum-associated early autophagosomal membranes, marked by the PtdIns(3)P effectors double-FYVE containing protein 1 (DFCP1) and WD-repeat protein interacting with phosphoinositides 1 (WIPI-1), both of which function at the onset of autophagy. These data provide evidence for additional roles of Atg2A and Atg14L in the formation of early autophagosomal membranes and also in lipid metabolism.     

Organelle degradation during the lens and erythroid differentiation is independent of autophagy

Autophagy is a bulk degradation system within cells through which cytoplasmic components are degraded within lysosomes. Primary roles of autophagy are starvation adaptation and intracellular protein quality control. In contrast to the ubiquitin-proteasome system, autophagy can also degrade organelles. Here we examined a possible role of autophagy in organelle degradation during lens and erythroid differentiation. We observed that autophagy occurs in embryonic lens cells. However, organelle degradation in lens and erythroid cells occurred normally in autophagy-deficient Atg5(-/-) mice. Our data suggest that degradation system(s) other than autophagy play major roles in organelle degradation during these processes.     

Inducible disruption of autophagy in the lung causes airway hyper-responsiveness

Autophagy is a highly conserved process primarily known for its role in cellular adaptation to nutritional stress. This bulk protein degradation pathway relocates nutrients during starvation. Recent studies, however, have revealed essential roles of autophagy in various organs under normal conditions. Especially, autophagy is now recognized as the pathway responsible for the elimination of damaged proteins resulting from environmental stress. Lungs are constantly exposed to high oxygen tension and environmental chemicals. To investigate the importance of autophagy in lung physiology, we used an inducible system to ablate Atg7 expression, which is a protein essential for autophagy, in the respiratory epithelial cells of adult mice. We found that Atg7 deficiency caused swelling of bronchiolar epithelial cells and accumulation of p62, which links substrate proteins to the autophagy machinery. Bronchiolar epithelial cells, isolated by micro-dissection of lung tissues, had elevated expression of cytoprotective genes that are typically activated by Nrf2. Interestingly, Atg7-deficient lungs displayed hyper-responsiveness to cholinergic stimuli without apparent inflammatory signs. Swollen bronchiolar epithelial cells may have lead to mechanical airway constriction and lowered the threshold for the increase of airway resistance. This study demonstrates the critical role of autophagy in the lungs for the maintenance of pulmonary homeostasis.     

Loss of autophagy in pro-opiomelanocortin neurons perturbs axon growth and causes metabolic dysregulation

The hypothalamic melanocortin system, which includes neurons that produce pro-opiomelanocortin (POMC)-derived peptides, is a major negative regulator of energy balance. POMC neurons begin to acquire their unique properties during neonatal life. The formation of functional neural systems requires massive cytoplasmic remodeling that may involve autophagy, an important intracellular mechanism for the degradation of damaged proteins and organelles. Here we investigated the functional and structural effects of the deletion of an essential autophagy gene, Atg7, in POMC neurons. Lack of Atg7 in POMC neurons caused higher postweaning body weight, increased adiposity, and glucose intolerance. These metabolic impairments were associated with an age-dependent accumulation of ubiquitin/p62-positive aggregates in the hypothalamus and a disruption in the maturation of POMC-containing axonal projections. Together, these data provide direct genetic evidence that Atg7 in POMC neurons is required for normal metabolic regulation and neural development, and they implicate hypothalamic autophagy deficiency in the pathogenesis of obesity.     

A novel,human Atg13 binding protein,Atg101, interacts with ULK1 and is essential for macroautophagy

Macroautophagy is an intracellular, vesicle-mediated mechanism for the sequestration and ultimate lysosomal degradation of cytoplasmic proteins, organelles and macromolecules. The macroautophagy process and many of the autophagy specific (Atg) proteins are remarkably well conserved in higher eukaryotes. In yeast, the Atg1 kinase complex includes Atg1, Atg13, Atg17, and at least four other interacting proteins, some of which are phosphorylated in a TOR-dependent manner, placing the Atg1 signaling complex downstream of a major nutrient-sensing pathway. Atg1 orthologs, including mammalian unc-51-like kinase 1 (ULK1), have been identified in higher eukaryotes and have been functionally linked to autophagy. This suggests that other components of the Atg1 complex exist in higher eukaryotes. Recently, a putative human Atg13 ortholog, FLJ20698, was identified by gapped-BLAST analysis. We show here that FLJ20698 (Atg13) is a ULK1-interacting phosphoprotein that is essential for macroautophagy. Furthermore, we identify a novel, human Atg13-interacting protein, FLJ11773, which we have termed Atg101. Atg101 is essential for autophagy and interacts with ULK1 in an Atg13-dependent manner. Additionally, we present evidence that intracellular localization of the ULK1 complex is regulated by nutrient conditions. Finally, we demonstrate that Atg101 stabilizes the expression of Atg13 in the cell, suggesting that Atg101 contributes to Atg13 function by protecting Atg13 from proteasomal degradation. Therefore, the identification of the novel protein, Atg101, and the validation of Atg13 and Atg101 as ULK1-interacting proteins, suggests an Atg1 complex is involved in the induction of macroautophagy in mammalian cells.     

Hsp90 inhibition results in autophagy-mediated proteasome-independent degradation of IκB kinase （IKK）

Autophagic and proteasomal proteolysis are two major pathways for degradation of cellular constituents. Current models suggest that autophagy is responsible for the nonselective bulk degradation of long-lived proteins and organelles while the proteasome specifically degrades short-lived proteins including misfolded proteins caused by the absence of Hsp90 function. Here, we show that the IκB kinase （IKK）, an essential activator of NF-κB, is selectively degraded by autophagy when Hsp90 is inhibited by geldanamycin （GA）, a specific Hsp90 inhibitor showing highly effective anti-tumor activity. We find that in this case inactivation of ubiquitination or proteasome fails to block IKK degradation. However, inhibition of autophagy by an autophagy inhibitor or knockout of Atg5, a key component of the autophagy pathway, significantly rescues IKK from GA-induced degradation. These findings provide the first evidence that an Hsp90 client may be degraded by a mechanism different from the proteasome pathway and establish a molecular link among Hsp90, NF-κB and autophagy     

Intracellular Quality Control by Autophagy: How Does Autophagy Prevent Neurodegeneration?

Autophagy is an intracellular bulk degradation process, through which a portion of cytoplasm is delivered to lysosomes to be degraded. In many organisms, the primary role of autophagy is adaptation to starvation. However, we have found that autophagy is also important for intracellular protein quality control. Atg5^-/- mice die shortly after birth due, at least in part, to nutrient deficiency. These mice also exhibit an intracellular accumulation of protein aggregates in neurons and hepatocytes. We now report the generation of neural cell-specific Atg5-deficient mice. Atg5^flox/flox;Nestin-Cre mice show progressive deficits in motor function and degeneration of some neural cells. In autophagy-deficient cells, diffuse accumulation of abnormal proteins occurs, followed by the generation of aggregates and inclusions. This study emphasizes the point that basal autophagy is important even in individuals who do not express neurodegenerative disease-associated mutant proteins. Furthermore, the primary targets of autophagy are diffuse cytosolic proteins, not protein aggregates themselves.     

Purification and characterization of a ubiquitin-like system for autophagosome formation

Autophagy refers to the bulk degradation of cellular proteins and organelles through an autophagosome and plays a pivotal role in the development, cellular differentiation, aging, and elimination of aberrant structures. A failure of autophagy has been implicated in a growing list of mammalian disease states, including cancer and cardiomyopathy. Two ubiquitin-like systems are highly involved in autophagy, especially in the formation of autophagosomes. Here, we purified and characterized Atg7 (an E1-like enzyme), and Atg3 and Atg10 (E2-like enzymes) in order to gain an insight into the role played by ubiquitin-like systems in the formation of autophagosomes. Interestingly, we observed that Atg7 forms a homodimer to construct an active conformation, unlike other E1-like enzymes. Although Atg3 was detected as a monomer under physiological conditions, Atg10 existed in an oligomeric form, indicating that the mechanism by which Atg10 functions may differ from that of Atg3.     

Intracellular quality control by autophagy: how does autophagy prevent neurodegeneration?

Autophagy is an intracellular bulk degradation process, through which a portion of cytoplasm is delivered to lysosomes to be degraded. In many organisms, the primary role of autophagy is adaptation to starvation. However, we have found that autophagy is also important for intracellular protein quality control. Atg5(-/-) mice die shortly after birth due, at least in part, to nutrient deficiency. These mice also exhibit an intracellular accumulation of protein aggregates in neurons and hepatocytes. We now report the generation of neural cell-specific Atg5-deficient mice. Atg5( flox/flox);Nestin-Cre mice show progressive deficits in motor function and degeneration of some neural cells. In autophagy-deficient cells, diffuse accumulation of abnormal proteins occurs, followed by the generation of aggregates and inclusions. This study emphasizes the point that basal autophagy is important even in individuals who do not express neurodegenerative disease-associated mutant proteins. Furthermore, the primary targets of autophagy are diffuse cytosolic proteins, not protein aggregates themselves.     

Targeting autophagy for drug-induced hepatotoxicity

Autophagy is a lysosomal degradation pathway for bulk cytosolic proteins and damaged organelles, and is well known to act as a cell survival mechanism. Acetaminophen (APAP) overdose can cause liver injury in animals and humans by inducing necrosis due to mitochondrial damage. We recently found that pharmacological induction of autophagy by rapamycin protects against, whereas pharmacological suppression of autophagy by chloroquine exacerbates, APAP-induced liver injury in mice. Autophagy is induced to remove APAP-induced damaged mitochondria and thus attenuates APAP-induced hepatocyte necrosis. To our surprise, we found that liver-specific Atg5 knockout mice are not more susceptible, but are resistant to APAP-induced liver injury due to compensatory effects. Our work suggests that pharmacological modulation of autophagy is a novel therapeutic approach to ameliorate APAP-induced liver injury. Moreover, our work also suggests that caution needs to be exercised when using genetic autophagy gene knockout mice for pathophysiological studies.     

The role of autophagy in neonatal tissues: just a response to amino acid starvation?

Autophagy is activated soon after birth in neonatal tissues and is essential for survival because mice deficient in Atg5 or Atg7 autophagy genes die within 1 day after birth. Amino acid starvation has been considered as a major deleterious effect of autophagy deficiency, since the concentration of amino acids in plasma was decreased by 20% in the two knockout models, whereas blood glucose and fatty acid levels were apparently not affected. However, autophagy may have other important functions in neonatal physiology, including glycogen degradation, programmed cell remodeling and response to oxidative stress.     

The Crystal Structure of Plant ATG12 and its Biological Implication in Autophagy

Atg12 is a post-translational modifier that is activated and conjugated to its single target, Atg5, by a ubiquitin-like conjugation system. The Atg12-Atg5 conjugate is essential for autophagy, the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. Here, we demonstrate that the Atg12 conjugation system exists in Arabidopsis and is essential for plant autophagy as well as in yeast and mammals. We also report the crystal structure of Arabidopsis thaliana (At) ATG12 at 1.8 Å resolution. Despite no obvious sequence homology with ubiquitin, the structure of AtATG12 shows a ubiquitin fold strikingly similar to those of mammalian homologs of Atg8, the other ubiquitin-like modifier essential for autophagy, which is conjugated to phosphatidylethanolamine. Two types of hydrophobic patches are present on the surface of AtATG12: one is conserved in both Atg12 and Atg8 orthologs, while the other is unique to Atg12 orthologs. Considering that they share Atg7 as an E1-like enzyme, we suggest that the first hydrophobic patch is responsible for the conjugation reaction, while the latter is involved in Atg12-specific functions.     

Cis1/Atg31 is required for autophagosome formation in Saccharomyces cerevisiae

Autophagy is the bulk degradation of cytosolic materials in lysosomes/vacuoles of eukaryotic cells. In the yeast Saccharomyces cerevisiae, 17 Atg proteins are known to be involved in autophagosome formation. Genome wide analyses have shown that Atg17 interacts with numerous proteins. Further studies on these interacting proteins may provide further insights into membrane dynamics during autophagy. Here, we identify Cis1/Atg31 as a protein that exhibits similar phenotypes to Atg17. ATG31 null cells were defective in autophagy and lost viability under starvation conditions. Localization of Atg31 to pre-autophagosomal structures (PAS) was dependent on Atg17. Coimmunoprecipitation experiments indicated that Atg31 interacts with Atg17. Together, Atg31 is a novel protein that, in concert with Atg17, is required for proper autophagosome formation.     

The crystal structure of plant ATG12 and its biological implication in autophagy

Atg12 is a post-translational modifier that is activated and conjugated to its single target, Atg5, by a ubiquitin-like conjugation system. The Atg12-Atg5 conjugate is essential for autophagy, the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. Here, we demonstrate that the Atg12 conjugation system exists in Arabidopsis and is essential for plant autophagy as well as in yeast and mammals. We also report the crystal structure of Arabidopsis thaliana (At) ATG12 at 1.8 A resolution. Despite no obvious sequence homology with ubiquitin, the structure of AtATG12 shows a ubiquitin fold strikingly similar to those of mammalian homologs of Atg8, the other ubiquitin-like modifier essential for autophagy, which is conjugated to phosphatidylethanolamine. Two types of hydrophobic patches are present on the surface of AtATG12: one is conserved in both Atg12 and Atg8 orthologs, while the other is unique to Atg12 orthologs. Considering that they share Atg7 as an E1-like enzyme, we suggest that the first hydrophobic patch is responsible for the conjugation reaction, while the latter is involved in Atg12-specific functions.     

Ald6p is a preferred target for autophagy in yeast, Saccharomyces cerevisiae

Macroautophagy is the process of intracellular bulk protein degradation induced by nutrient starvation and is generally considered to be a nonselective degradation of cytosolic enzymes and organelles. However, it remains a possibility that some proteins may be preferentially degraded by autophagy. In this study, we have performed a systematic analysis on the substrate selectivity of autophagy in yeast, Saccharomyces cerevisiae, using two-dimensional PAGE. We performed a differential screen on wild-type and Deltaatg7/apg7 autophagy-deficient cells and found that cytosolic acetaldehyde dehydrogenase (Ald6p) decreased under nitrogen starvation. As assessed by immunoblot, Ald6p was reduced by greater than 82% after 24 h of nitrogen starvation. This reduction was dependent on Atg/Apg proteins and vacuolar proteases but was not dependent on the proteasome or the cytoplasm to vacuole targetting (Cvt) pathway. Using pulse-chase and subcellular fractionation, we have also demonstrated that Ald6p was preferentially transported to vacuoles via autophagosomes. Deltaatg7 Deltaald6 double mutant cells were able to maintain higher rates of viability than Deltaatg7 cells under nitrogen starvation, and Ald6p-overexpressing cells were not able to maintain high rates of viability. Furthermore, the Ald6p(C306S) mutant, which lacks enzymatic activity, had viability rates similar to Deltaald6 cells. Ald6p enzymatic activity may be disadvantageous for survival under nitrogen starvation; therefore, yeast cells may preferentially eliminate Ald6p via autophagy.     

Atg22 recycles amino acids to link the degradative and recycling functions of autophagy

In response to stress conditions (such as nutrient limitation or accumulation of damaged organelles) and certain pathological situations, eukaryotic cells use autophagy as a survival mechanism. During nutrient stress the main purpose of autophagy is to degrade cytoplasmic materials within the lysosome/vacuole lumen and generate an internal nutrient pool that is recycled back to the cytosol. This study elucidates a molecular mechanism for linking the degradative and recycling roles of autophagy. We show that in contrast to published studies, Atg22 is not directly required for the breakdown of autophagic bodies within the lysosome/vacuole. Instead, we demonstrate that Atg22, Avt3, and Avt4 are partially redundant vacuolar effluxers, which mediate the efflux of leucine and other amino acids resulting from autophagic degradation. The release of autophagic amino acids allows the maintenance of protein synthesis and viability during nitrogen starvation. We propose a "recycling" model that includes the efflux of macromolecules from the lysosome/vacuole as the final step of autophagy.     

Ordered organelle degradation during starvation-induced autophagy

Upon starvation cells undergo autophagy, a cellular degradation pathway important in the turnover of whole organelles and long lived proteins. Starvation-induced protein degradation has been regarded as an unspecific bulk degradation process. We studied global protein dynamics during amino acid starvation-induced autophagy by quantitative mass spectrometry and were able to record nearly 1500 protein profiles during 36 h of starvation. Cluster analysis of the recorded protein profiles revealed that cytosolic proteins were degraded rapidly, whereas proteins annotated to various complexes and organelles were degraded later at different time periods. Inhibition of protein degradation pathways identified the lysosomal/autophagosomal system as the main degradative route. Thus, starvation induces degradation via autophagy, which appears to be selective and to degrade proteins in an ordered fashion and not completely arbitrarily as anticipated so far.     

The Atg1 kinase complex is involved in the regulation of protein recruitment to initiate sequestering vesicle formation for nonspecific autophagy in Saccharomyces cerevisiae

Autophagy is the major degradative process for recycling cytoplasmic constituents and eliminating unnecessary organelles in eukaryotic cells. Most autophagy-related (Atg) proteins are recruited to the phagophore assembly site (PAS), a proposed site for vesicle formation during either nonspecific or specific types of autophagy. Therefore, appropriate recruitment of Atg proteins to this site is critical for their function in autophagy. Atg11 facilitates PAS recruitment for the cytoplasm-to-vacuole targeting pathway, which is a specific, autophagy-like process that occurs under vegetative conditions. In contrast, it is not known how Atg proteins are recruited to the PAS, nor which components are involved in PAS formation under nonspecific autophagy-inducing, starvation conditions. Here, we studied PAS assembly during nonspecific autophagy, using an atg11Delta mutant background to eliminate the PAS formation that occurs during vegetative growth. We found that protein complexes containing the Atg1 kinase have two roles for PAS formation during nonspecific autophagy. The Atg1 C terminus mediates an interaction with Atg13 and Atg17, facilitating a structural role of Atg1 that is needed to efficiently organize an initial step of PAS assembly, whereas Atg1 kinase activity affects the dynamics of protein movement at the PAS involved in Atg protein cycling.