A Revised Assay for Monitoring Autophagic Flux in Arabidopsis thaliana Reveals Involvement of AUTOPHAGY-RELATED9 in Autophagy

Autophagy targets cytoplasmic cargo to a lytic compartment for degradation. Autophagy-related (Atg) proteins, including the transmembrane protein Atg9, are involved in different steps of autophagy in yeast and mammalian cells. Functional classification of core Atg proteins in plants has not been clearly confirmed, partly because of the limited availability of reliable assays for monitoring autophagic flux. By using proUBQ10-GFP-ATG8a as an autophagic marker, we showed that autophagic flux is reduced but not completely compromised in Arabidopsis thaliana atg9 mutants. In contrast, we confirmed full inhibition of auto-phagic flux in atg7 and that the difference in autophagy was consistent with the differences in mutant phenotypes such as hypersensitivity to nutrient stress and selective autophagy. Autophagic flux is also reduced by an inhibitor of phosphatidylinositol kinase. Our data indicated that atg9 is phenotypically distinct from atg7 and atg2 in Arabidopsis, and we proposed that ATG9 and phosphatidylinositol kinase activity contribute to efficient autophagy in Arabidopsis.     

Evidence for autophagy-dependent pathways of rRNA turnover in Arabidopsis

Ribosomes account for a majority of the cell''s RNA and much of its protein and represent a significant investment of cellular resources. The turnover and degradation of ribosomes has been proposed to play a role in homeostasis and during stress conditions. Mechanisms for the turnover of rRNA and ribosomal proteins have not been fully elucidated. We show here that the RNS2 ribonuclease and autophagy participate in RNA turnover in Arabidopsis thaliana under normal growth conditions. An increase in autophagosome formation was seen in an rns2–2 mutant, and this increase was dependent on the core autophagy genes ATG9 and ATG5. Autophagosomes and autophagic bodies in rns2–2 mutants contain RNA and ribosomes, suggesting that autophagy is activated as an attempt to compensate for loss of rRNA degradation. Total RNA accumulates in rns2–2, atg9–4, atg5–1, rns2–2 atg9–4, and rns2–2 atg5–1 mutants, suggesting a parallel role for autophagy and RNS2 in RNA turnover. rRNA accumulates in the vacuole in rns2–2 mutants. Vacuolar accumulation of rRNA was blocked by disrupting autophagy via an rns2–2 atg5–1 double mutant but not by an rns2–2 atg9–4 double mutant, indicating that ATG5 and ATG9 function differently in this process. Our results suggest that autophagy and RNS2 are both involved in homeostatic degradation of rRNA in the vacuole.     

ATG8 lipidation and ATG8‐mediated autophagy in Arabidopsis require ATG12 expressed from the differentially controlled ATG12A AND ATG12B loci

Autophagic recycling of intracellular plant constituents is maintained at a basal level under normal growth conditions but can be induced in response to nutritional demand, biotic stress, and senescence. One route requires the ubiquitin‐fold proteins Autophagy‐related (ATG)‐8 and ATG12, which become attached to the lipid phosphatidylethanolamine (PE) and the ATG5 protein, respectively, during formation of the engulfing vesicle and delivery of its cargo to the vacuole for breakdown. Here, we genetically analyzed the conjugation machinery required for ATG8/12 modification in Arabidopsis thaliana with a focus on the two loci encoding ATG12. Whereas single atg12a and atg12b mutants lack phenotypic consequences, atg12a atg12b double mutants senesce prematurely, are hypersensitive to nitrogen and fixed carbon starvation, and fail to accumulate autophagic bodies in the vacuole. By combining mutants eliminating ATG12a/b, ATG5, or the ATG10 E2 required for their condensation with a method that unequivocally detects the ATG8‐PE adduct, we also show that ATG8 lipidation requires the ATG12–ATG5 conjugate. Unlike ATG8, ATG12 does not associate with autophagic bodies, implying that its role(s) during autophagy is restricted to events before the vacuolar deposition of vesicles. The expression patterns of the ATG12a and ATG12b genes and the effects of single atg12a and atg12b mutants on forming the ATG12–ATG5 conjugate reveal that the ATG12b locus is more important during basal autophagy while the ATG12a locus is more important during induced autophagy. Taken together, we conclude that the formation of the ATG12–ATG5 adduct is essential for ATG8‐mediated autophagy in plants by promoting ATG8 lipidation.     

The scaffold protein EPG-7 links cargo–receptor complexes with the autophagic assembly machinery

The mechanism by which protein aggregates are selectively degraded by autophagy is poorly understood. Previous studies show that a family of Atg8-interacting proteins function as receptors linking specific cargoes to the autophagic machinery. Here we demonstrate that during Caenorhabditis elegans embryogenesis, epg-7 functions as a scaffold protein mediating autophagic degradation of several protein aggregates, including aggregates of the p62 homologue SQST-1, but has little effect on other autophagy-regulated processes. EPG-7 self-oligomerizes and is degraded by autophagy independently of SQST-1. SQST-1 directly interacts with EPG-7 and colocalizes with EPG-7 aggregates in autophagy mutants. Mutations in epg-7 impair association of SQST-1 aggregates with LGG-1/Atg8 puncta. EPG-7 interacts with multiple ATG proteins and colocalizes with ATG-9 puncta in various autophagy mutants. Unlike core autophagy genes, epg-7 is dispensable for starvation-induced autophagic degradation of substrate aggregates. Our results indicate that under physiological conditions a scaffold protein endows cargo specificity and also elevates degradation efficiency by linking the cargo–receptor complex with the autophagic machinery.     

Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways

Autophagy is an important mechanism for nonselective intracellular breakdown whereby cytosol and organelles are encapsulated in vesicles, which are then engulfed and digested by lytic vacuoles/lysosomes. In yeast, this encapsulation employs a set of autophagy (ATG) proteins that direct the conjugation of two ubiquitin-like protein tags, ATG8 and ATG12, to phosphatidylethanolamine and the ATG5 protein, respectively. Using an Arabidopsis (Arabidopsis thaliana) atg7 mutant unable to ligate either tag, we previously showed that the ATG8/12 conjugation system is important for survival under nitrogen-limiting growth conditions. By reverse-genetic analyses of the single Arabidopsis gene encoding ATG5, we show here that the subpathway that forms the ATG12-ATG5 conjugate also has an essential role in plant nutrient recycling. Similar to plants missing ATG7, those missing ATG5 display early senescence and are hypersensitive to either nitrogen or carbon starvation, which is accompanied by a more rapid loss of organellar and cytoplasmic proteins. Multiple ATG8 isoforms could be detected immunologically in seedling extracts. Their abundance was substantially elevated in both the atg5 and atg7 mutants, caused in part by an increase in abundance of several ATG8 mRNAs. Using a green fluorescent protein-ATG8a fusion in combination with concanamycin A, we also detected the accumulation of autophagic bodies inside the vacuole. This accumulation was substantially enhanced by starvation but blocked in the atg7 background. The use of this fusion in conjunction with atg mutants now provides an important marker to track autophagic vesicles in planta.     

The phenotypes of ATG9, ATG16 and ATG9/16 knock-out mutants imply autophagy-dependent and -independent functions

Macroautophagy is a highly conserved intracellular bulk degradation system of all eukaryotic cells. It is governed by a large number of autophagy proteins (ATGs) and is crucial for many cellular processes. Here, we describe the phenotypes of Dictyostelium discoideum ATG16⁻ and ATG9⁻/16⁻ cells and compare them to the previously reported ATG9⁻ mutant. ATG16 deficiency caused an increase in the expression of several core autophagy genes, among them atg9 and the two atg8 paralogues. The single and double ATG9 and ATG16 knock-out mutants had complex phenotypes and displayed severe and comparable defects in pinocytosis and phagocytosis. Uptake of Legionella pneumophila was reduced. In addition, ATG9⁻ and ATG16⁻ cells had dramatic defects in autophagy, development and proteasomal activity which were much more severe in the ATG9⁻/16⁻ double mutant. Mutant cells showed an increase in poly-ubiquitinated proteins and contained large ubiquitin-positive protein aggregates which partially co-localized with ATG16-GFP in ATG9⁻/16⁻ cells. The more severe autophagic, developmental and proteasomal phenotypes of ATG9⁻/16⁻ cells imply that ATG9 and ATG16 probably function in parallel in autophagy and have in addition autophagy-independent functions in further cellular processes.     

Mitophagy in yeast occurs through a selective mechanism

The regulation of mitochondrial degradation through autophagy is expected to be a tightly controlled process, considering the significant role of this organelle in many processes ranging from energy production to cell death. However, very little is known about the specific nature of the degradation process. We developed a new method to detect mitochondrial autophagy (mitophagy) by fusing the green fluorescent protein at the C terminus of two endogenous mitochondrial proteins and monitored vacuolar release of green fluorescent protein. Using this method, we screened several atg mutants and found that ATG11, a gene that is essential only for selective autophagy, is also essential for mitophagy. In addition, we found that mitophagy is blocked even under severe starvation conditions, if the carbon source makes mitochondria essential for metabolism. These findings suggest that the degradation of mitochondria is a tightly regulated process and that these organelles are largely protected from nonspecific autophagic degradation.     

ATG5-knockout mutants of Physcomitrella provide a platform for analyzing the involvement of autophagy in senescence processes in plant cells

Autophagy is a pathway in which a cell degrades part of its cytoplasm in vacuoles or lysosomes. To identify the physiological functions of autophagy in plants, we disrupted ATG5, an autophagy-related gene, in Physcomitrella, and confirmed that atg5 mutants are deficient in the process of autophagy. On carbon or nitrogen starvation medium, atg5 colonies turned yellow earlier than the wild-type (WT) colonies, showing that Physcomitrella atg5 mutants, like yeast and Arabidopsis, are sensitive to nutrient starvation. In the dark, even under nutrient-sufficient conditions, colonies turned yellow and the net degradation of chlorophyll and Rubisco protein occurred together with the upregulation of several senescence-associated genes. Yellowing reactions were inhibited by the protein synthesis inhibitor cycloheximide, suggesting that protonemal colonies undergo dark-induced senescence like the green leaves of higher plants. Such senescence responses in the dark occurred earlier in atg5 colonies than WT colonies. The sugar content was almost the same between WT and atg5 colonies, indicating that the early-senescence phenotype of atg5 is not explained by sugar deficiency. However, the levels of 7 amino acids showed significantly different alteration between atg5 and WT in the dark: 6 amino acids, particularly arginine and alanine, were much more deficient in the atg5 mutants, irrespective of the early degradation of Rubisco protein. On nutrient-sufficient medium supplemented with casamino acids, the early-senescence phenotype was slightly moderated. We propose that the early-senescence phenotype in atg5 mutants is partly explained by amino acid imbalance because of the lack of cytoplasmic degradation by autophagy in Physcomitrella.     

Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis

Autophagy is an evolutionarily conserved pathway responsible for degradation of cytoplasmic material via the lysosome. Although autophagy has been reported to contribute to cell death, the underlying mechanisms remain largely unknown. In this study, we show that autophagy controls DNA fragmentation during late oogenesis in Drosophila melanogaster. Inhibition of autophagy by genetically removing the function of the autophagy genes atg1, atg13, and vps34 resulted in late stage egg chambers that contained persisting nurse cell nuclei without fragmented DNA and attenuation of caspase-3 cleavage. The Drosophila inhibitor of apoptosis (IAP) dBruce was found to colocalize with the autophagic marker GFP-Atg8a and accumulated in autophagy mutants. Nurse cells lacking Atg1 or Vps34 in addition to dBruce contained persisting nurse cell nuclei with fragmented DNA. This indicates that autophagic degradation of dBruce controls DNA fragmentation in nurse cells. Our results reveal autophagic degradation of an IAP as a novel mechanism of triggering cell death and thereby provide a mechanistic link between autophagy and cell death.     

Don't forget to be picky – selective autophagy of protein aggregates in neurodegenerative diseases

The homeostasis of cells depends on the selective degradation of damaged or superfluous cellular components. Autophagy is the major pathway that recognizes such components, sequesters them in de novo formed autophagosomes and delivers them to lysosomes for degradation. The recognition of specific cargo and the biogenesis of autophagosomes involve a dedicated machinery of autophagy related (ATG) proteins. Intense research over the past decades has revealed insights into the function of autophagy proteins and mechanisms that govern cargo recognition. Other aspects including the molecular mechanisms involved in the onset of human diseases are less well understood. However, autophagic dysfunctions, caused by age related decline in autophagy or mutations in ATG proteins, are directly related to a large number of human pathologies including neurodegenerative disorders. Here, we review most recent discoveries and breakthroughs in selective autophagy and its relationship to neurodegeneration.     

The Endosomal Protein CHARGED MULTIVESICULAR BODY PROTEIN1 Regulates the Autophagic Turnover of Plastids in Arabidopsis

Endosomal Sorting Complex Required for Transport (ESCRT)-III proteins mediate membrane remodeling and the release of endosomal intraluminal vesicles into multivesicular bodies. Here, we show that the ESCRT-III subunit paralogs CHARGED MULTIVESICULAR BODY PROTEIN1 (CHMP1A) and CHMP1B are required for autophagic degradation of plastid proteins in Arabidopsis thaliana. Similar to autophagy mutants, chmp1a chmp1b (chmp1) plants hyperaccumulated plastid components, including proteins involved in plastid division. The autophagy machinery directed the release of bodies containing plastid material into the cytoplasm, whereas CHMP1A and B were required for delivery of these bodies to the vacuole. Autophagy was upregulated in chmp1 as indicated by an increase in vacuolar green fluorescent protein (GFP) cleavage from the autophagic reporter GFP-ATG8. However, autophagic degradation of the stromal cargo RECA-GFP was drastically reduced in the chmp1 plants upon starvation, suggesting that CHMP1 mediates the efficient delivery of autophagic plastid cargo to the vacuole. Consistent with the compromised degradation of plastid proteins, chmp1 plastids show severe morphological defects and aberrant division. We propose that CHMP1 plays a direct role in the autophagic turnover of plastid constituents.     

ATG16 mediates the autophagic degradation of the 19S proteasomal subunits PSMD1 and PSMD2

Autophagy and the ubiquitin proteasome system are the two major cellular processes for protein and organelle recycling and clearance in eukaryotic cells. Evidence is accumulating that these two pathways are interrelated through adaptor proteins. Here, we found that PSMD1 and PSMD2, both components of the 19S regulatory particle of the proteasome, directly interact with Dictyostelium discoideum autophagy 16 (ATG16), a core autophagosomal protein. ATG16 is composed of an N-terminal domain, which is responsible for homo-dimerization and binding to ATG5 and a C-terminal β-propeller structure. Deletion analysis of ATG16 showed that the N-terminal half of ATG16 interacted directly only with PSMD1, while the C-terminal half interacted with both, PSMD1 and PSMD2. RFP-tagged PSMD1 as well as PSMD2 were enriched in large puncta, reminiscent of autophagosomes, in wild-type cells. These puncta were absent in atg16￣ and atg9￣/16￣ cells and weaker and less frequent in atg9￣ cells, showing that ATG16 was crucial and the autophagic process important for their formation. Co-expression of ATG16-GFP or GFP-ATG8a(LC3) with RFP-PSMD1 or RFP-PSMD2, respectively, in atg16￣ or wild-type cells revealed many instances of co-localization, suggesting that RFP-PSMD1 or RFP-PSMD2 positive puncta constitute autophagosomes. LysoTracker^® labeling and a proteolytic cleavage assay confirmed that PSMD1 and PSMD2 were present in lysosomes in wild-type cells. In vivo, ATG16 is required for their enrichment in ATG8a positive puncta, which mature into autolysosomes. We propose that ATG16 links autophagy and the ubiquitin proteasome system.     

PFKP facilitates ATG4B phosphorylation during amino acid deprivation-induced autophagy

ATG4B facilitates autophagy by promoting autophagosome maturation through the reversible lipidation and delipidation of LC3. Recent reports have shown that phosphorylation of ATG4B regulates its activity and LC3 processing, leading to modulate autophagy activity. However, the mechanism about how ATG4B phosphorylation is involved in amino acid deprivation-induced autophagy is unclear. Here, we combined the tandem affinity purification with mass spectrometry (MS) and identified the ATG4B-interacting proteins including its well-known partner gamma-aminobutyric acid receptor-associated protein (GABARAP, a homolog of LC3) and phosphofructokinase 1 platelet isoform (PFKP). Further immunoprecipitation assays showed that amino acid deprivation strengthened the interaction between ATG4B and PFKP. By genetic depletion of PFKP using CRISPR/Cas9, we uncovered that PFKP loss reduced the degradation of LC3-II and p62 due to a partial block in autophagic flux. Furthermore, MS analysis of Flag-tagged ATG4B immunoprecipitates identified phosphorylation of ATG4B serine 34 residue (S34) and PFKP serine 386 residue (S386) under amino acid deprivation condition. In vitro kinase assay validated that PFKP functioning as a protein kinase phosphorylated ATG4B at S34. This phosphorylation could enhance ATG4B activity and p62 degradation. In addition, PFKP S386 phosphorylation was important to ATG4B S34 phosphorylation and autophagy in HEK293T cells. In brief, our findings describe that PFKP, a rate-limiting enzyme in the glycolytic pathway, functions as a protein kinase for ATG4B to regulate ATG4B activity and autophagy under amino acid deprivation condition.     

AUTOPHAGY-RELATED11 Plays a Critical Role in General Autophagy- and Senescence-Induced Mitophagy in Arabidopsis

Autophagy-mediated turnover removes damaged organelles and unwanted cytoplasmic constituents and thus plays critical roles in cellular housekeeping and nutrient recycling. This “self eating” is tightly regulated by the AUTOPHAGY-RELATED1/13 (ATG1/13) kinase complex, which connects metabolic and environmental cues to the vacuolar delivery of autophagic vesicles. Here, we describe the Arabidopsis thaliana accessory proteins ATG11 and ATG101, which help link the ATG1/13 complex to autophagic membranes. ATG11 promotes vesicle delivery to the vacuole but is not essential for synthesizing the ATG12-ATG5 and ATG8-phosphatidylethanolamine adducts that are central to autophagic vesicle assembly. ATG11, ATG101, ATG1, and ATG13 colocalize with each other and with ATG8, with ATG1 tethered to ATG8 via a canonical ATG8-interacting motif. Also, the presence of ATG11 encourages starvation-induced phosphorylation of ATG1 and turnover of ATG1 and ATG13. Like other atg mutants, ATG11-deficient plants senesce prematurely and are hypersensitive to nitrogen and fixed-carbon limitations. Additionally, we discovered that the senescence-induced breakdown of mitochondria-resident proteins and mitochondrial vesicles occurs via an autophagic process requiring ATG11 and other ATG components. Together, our data indicate that ATG11 (and possibly ATG101) provides important scaffolds connecting the ATG1/13 complex to both general autophagy and selective mitophagy.     

ATG4B/autophagin-1 regulates intestinal homeostasis and protects mice from experimental colitis

The identification of inflammatory bowel disease (IBD) susceptibility genes by genome-wide association has linked this pathology to autophagy, a lysosomal degradation pathway that is crucial for cell and tissue homeostasis. Here, we describe autophagy-related 4B, cysteine peptidase/autophagin-1 (ATG4B) as an essential protein in the control of inflammatory response during experimental colitis. In this pathological condition, ATG4B protein levels increase in parallel with the induction of autophagy. Moreover, ATG4B expression is significantly reduced in affected areas of the colon from IBD patients. Consistently, atg4b^−/− mice present Paneth cell abnormalities, as well as an increased susceptibility to DSS-induced colitis. atg4b-deficient mice exhibit significant alterations in proinflammatory cytokines and mediators of the immune response to bacterial infections, which are reminiscent of those found in patients with Crohn disease or ulcerative colitis. Additionally, antibiotic treatments and bone marrow transplantation from wild-type mice reduced colitis in atg4b^−/− mice. Taken together, these results provided additional evidence for the importance of autophagy in intestinal pathologies and describe ATG4B as a novel protective protein in inflammatory colitis. Finally, we propose that atg4b-null mice are a suitable model for in vivo studies aimed at testing new therapeutic strategies for intestinal diseases associated with autophagy deficiency.     

TMEM59 defines a novel ATG16L1‐binding motif that promotes local activation of LC3

Selective autophagy underlies many of the important physiological roles that autophagy plays in multicellular organisms, but the mechanisms involved in cargo selection are poorly understood. Here we describe a molecular mechanism that can target conventional endosomes for autophagic degradation. We show that the human transmembrane protein TMEM59 contains a minimal 19‐amino‐acid peptide in its intracellular domain that promotes LC3 labelling and lysosomal targeting of its own endosomal compartment. Interestingly, this peptide defines a novel protein motif that mediates interaction with the WD‐repeat domain of ATG16L1, thus providing a mechanistic basis for the activity. The motif is represented with the same ATG16L1‐binding ability in other molecules, suggesting a more general relevance. We propose that this motif may play an important role in targeting specific membranous compartments for autophagic degradation, and therefore it may facilitate the search for adaptor proteins that promote selective autophagy by engaging ATG16L1. Endogenous TMEM59 interacts with ATG16L1 and mediates autophagy in response to Staphylococcus aureus infection.     

Growth arrest and autophagy are required for salivary gland cell degradation in Drosophila

Autophagy is a catabolic process that is negatively regulated by growth and has been implicated in cell death. We find that autophagy is induced following growth arrest and precedes developmental autophagic cell death of Drosophila salivary glands. Maintaining growth by expression of either activated Ras or positive regulators of the class I phosphoinositide 3-kinase (PI3K) pathway inhibits autophagy and blocks salivary gland cell degradation. Developmental degradation of salivary glands is also inhibited in autophagy gene (atg) mutants. Caspases are active in PI3K-expressing and atg mutant salivary glands, and combined inhibition of both autophagy and caspases increases suppression of gland degradation. Further, induction of autophagy is sufficient to induce premature cell death in a caspase-independent manner. Our results provide in vivo evidence that growth arrest, autophagy, and atg genes are required for physiological autophagic cell death and that multiple degradation pathways cooperate in the efficient clearance of cells during development.     

Disrupting Autophagy Restores Peroxisome Function to an Arabidopsis lon2 Mutant and Reveals a Role for the LON2 Protease in Peroxisomal Matrix Protein Degradation

Peroxisomes house critical metabolic reactions that are essential for seedling development. As seedlings mature, metabolic requirements change, and peroxisomal contents are remodeled. The resident peroxisomal protease LON2 is positioned to degrade obsolete or damaged peroxisomal proteins, but data supporting such a role in plants have remained elusive. Arabidopsis thaliana lon2 mutants display defects in peroxisomal metabolism and matrix protein import but appear to degrade matrix proteins normally. To elucidate LON2 functions, we executed a forward-genetic screen for lon2 suppressors, which revealed multiple mutations in key autophagy genes. Disabling core autophagy-related gene (ATG) products prevents autophagy, a process through which cytosolic constituents, including organelles, can be targeted for vacuolar degradation. We found that atg2, atg3, and atg7 mutations suppressed lon2 defects in auxin metabolism and matrix protein processing and rescued the abnormally large size and small number of lon2 peroxisomes. Moreover, analysis of lon2 atg mutants uncovered an apparent role for LON2 in matrix protein turnover. Our data suggest that LON2 facilitates matrix protein degradation during peroxisome content remodeling, provide evidence for the existence of pexophagy in plants, and indicate that peroxisome destruction via autophagy is enhanced when LON2 is absent.