Lap3 is a selective target of autophagy in yeast, Saccharomyces cerevisiae

Autophagy is a primarily non-selective degradation system of cytoplasmic constituents in lysosomes/vacuoles during starvation. In yeast, autophagy is also involved in the selective transport of Ape1, a vacuolar hydrolase, as a biosynthetic route. Ald6, a soluble cytoplasmic enzyme, is preferentially eliminated from cytoplasm via autophagy. However, little is known about the mechanisms of Ald6 targeting to autophagosomes. Here, we show that Lap3, a soluble cytosolic cysteine protease, is spatially associated with Ape1 and selectively transported to the vacuole during nitrogen starvation. The rate of Lap3 transport is much higher than that of Ald6 and is similar to that of Ape1. Moreover, ATG11 and ATG19, essential factors for Ape1 transport, are important for Lap3 transport. Most Lap3 is degraded within a couple of hours in the vacuole in contrast to Ape1; therefore, we conclude that the machinery required for Ape1 biosynthesis is used for selective degradation of Lap3.     

You are what you eat: multifaceted functions of autophagy during C. elegans development

Autophagy involves the sequestration of a portion of the cytosolic contents in an enclosed double-membrane autophagosomal structure and its subsequent delivery to lysosomes for degradation. Autophagy activity functions in multiple biological processes during Caenorhabditis elegans development. The basal level of autophagy in embryos removes aggregate-prone proteins, paternal mitochondria and spermatid-specific membranous organelles (MOs). Autophagy also contributes to the efficient removal of embryonic apoptotic cell corpses by promoting phagosome maturation. During larval development, autophagy modulates miRNA-mediated gene silencing by selectively degrading AIN-1, a component of miRNA-induced silencing complex, and thus participates in the specification of multiple cell fates controlled by miRNAs. During development of the hermaphrodite germline, autophagy acts coordinately with the core apoptotic machinery to execute genotoxic stress-induced germline cell death and also cell death when caspase activity is partially compromised. Autophagy is also involved in the utilization of lipid droplets in the aging process in adult animals. Studies in C. elegans provide valuable insights into the physiological functions of autophagy in the development of multicellular organisms.     

Regulation of ATG4B Stability by RNF5 Limits Basal Levels of Autophagy and Influences Susceptibility to Bacterial Infection

Autophagy is the mechanism by which cytoplasmic components and organelles are degraded by the lysosomal machinery in response to diverse stimuli including nutrient deprivation, intracellular pathogens, and multiple forms of cellular stress. Here, we show that the membrane-associated E3 ligase RNF5 regulates basal levels of autophagy by controlling the stability of a select pool of the cysteine protease ATG4B. RNF5 controls the membranal fraction of ATG4B and limits LC3 (ATG8) processing, which is required for phagophore and autophagosome formation. The association of ATG4B with—and regulation of its ubiquitination and stability by—RNF5 is seen primarily under normal growth conditions. Processing of LC3 forms, appearance of LC3-positive puncta, and p62 expression are higher in RNF5^−/− MEF. RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 puncta. Further, increased puncta seen in RNF5^−/− using WT but not LC3 mutant, which bypasses ATG4B processing, substantiates the role of RNF5 in early phases of LC3 processing and autophagy. Similarly, RNF-5 inactivation in Caenorhabditis elegans increases the level of LGG-1/LC3::GFP puncta. RNF5^−/− mice are more resistant to group A Streptococcus infection, associated with increased autophagosomes and more efficient bacterial clearance by RNF5^−/− macrophages. Collectively, the RNF5-mediated control of membranalATG4B reveals a novel layer in the regulation of LC3 processing and autophagy.     

Plant cell remodeling by autophagy: Switching peroxisomes for green life

Plant seedlings are not photoautotrophs until they are equipped with photosynthetic machinery. Some plant cells are remodeled after being exposed to light, and a group of peroxisomal proteins are degraded during the remodeling. Autophagy was proposed as one of the mechanisms for the degradation of peroxisomal proteins. We recently showed that ATG7-dependent autophagy is partially responsible for the degradation of obsolete peroxisomal proteins during Arabidopsis seedling growth.     

Hierarchal Autophagic Divergence of Hematopoietic System

Autophagy is integral to hematopoiesis and protects against leukemogenesis. However, the fundamentals of the required molecular machinery have yet to be fully explored. Using conditional mouse models to create strategic defects in the hematopoietic hierarchy, we have shown that recovery capacities in stem cells and somatic cells differ if autophagy is impaired or flawed. An in vivo Atg7 deletion in hematopoietic stem cells completely ablates the autophagic response, leading to irreversible and ultimately lethal hematopoiesis. However, while no adverse phenotype is manifested in vivo by Atg7-deficient myeloid cells, they maintain active autophagy that is sensitive to brefeldin A, an inhibitor targeting Golgi-derived membranes destined for autophagosome formation in alternative autophagy. Removing Rab9, a key regulatory protein, in alternative autophagy, disables autophagy altogether in Atg7-deficient macrophages. Functional analysis indicates that ATG7-dependent canonical autophagy is physiologically active in both hematopoietic stem cells and in terminally differentiated hematopoietic cells; however, only terminally differentiated cells such as macrophages are rescued by alternative autophagy if canonical autophagy is ineffective. Thus, it appears that hematopoietic stem cells rely solely on ATG7-dependent canonical autophagy, whereas terminally differentiated or somatic cells are capable of alternative autophagy in the event that ATG7-mediated autophagy is dysfunctional. These findings offer new insight into the transformational trajectory of hematopoietic stem cells, which in our view renders the autophagic machinery in stem cells more vulnerable to disruption.     

Nutrient-regulated Phosphorylation of ATG13 Inhibits Starvation-induced Autophagy

Autophagy is a conserved catabolic process that utilizes a defined series of membrane trafficking events to generate a de novo double-membrane vesicle termed the autophagosome, which matures by fusing to the lysosome. Subsequently, the lysosome facilitates the degradation and recycling of the cytoplasmic cargo. In yeast, the upstream signals that regulate the induction of starvation-induced autophagy are clearly defined. The nutrient-sensing kinase Tor inhibits the activation of autophagy by regulating the formation of the Atg1-Atg13-Atg17 complex, through hyperphosphorylation of Atg13. However, in mammals, the ortholog complex ULK1-ATG13-FIP200 is constitutively formed. As such, the molecular mechanism by which mTOR regulates mammalian autophagy is unknown. Here we report the identification and characterization of novel nutrient-regulated phosphorylation sites on ATG13: Ser-224 and Ser-258. mTOR directly phosphorylates ATG13 on Ser-258 while Ser-224 is modulated by the AMPK pathway. In ATG13 knock-out cells reconstituted with an unphosphorylatable mutant of ATG13, ULK1 kinase activity is more potent, and amino acid starvation induced more rapid ATG13 and ULK1 translocation. These events culminated in a more rapid starvation-induced autophagy response. Therefore, ATG13 phosphorylation plays a crucial role in autophagy regulation.     

FGFR3/fibroblast growth factor receptor 3 inhibits autophagy through decreasing the ATG12–ATG5 conjugate,leading to the delay of cartilage development in achondroplasia

FGFR3 (fibroblast growth factor receptor 3) is a negative regulator of endochondral ossification. Gain-of-function mutations in FGFR3 are responsible for achondroplasia, the most common genetic form of dwarfism in humans. Autophagy, an evolutionarily conserved catabolic process, maintains chondrocyte viability in the growth plate under stress conditions, such as hypoxia and nutritional deficiencies. However, the role of autophagy and its underlying molecular mechanisms in achondroplasia remain elusive. In this study, we found activated FGFR3 signaling inhibited autophagic activity in chondrocytes, both in vivo and in vitro. By employing an embryonic bone culture system, we demonstrated that treatment with autophagy inhibitor 3-MA or chloroquine led to cartilage growth retardation, which mimics the effect of activated-FGFR3 signaling on chondrogenesis. Furthermore, we found that FGFR3 interacted with ATG12–ATG5 conjugate by binding to ATG5. More intriguingly, FGFR3 signaling was found to decrease the protein level of ATG12–ATG5 conjugate. Consistently, using in vitro chondrogenic differentiation assay system, we showed that the ATG12–ATG5 conjugate was essential for the viability and differentiation of chondrocytes. Transient transfection of ATG5 partially rescued FGFR3-mediated inhibition on chondrocyte viability and differentiation. Our findings reveal that FGFR3 inhibits the autophagic activity by decreasing the ATG12–ATG5 conjugate level, which may play an essential role in the pathogenesis of achondroplasia.