AUT1, a gene essential for autophagocytosis in the yeast Saccharomyces cerevisiae.

Autophagocytosis is a starvation-induced process responsible for transport of cytoplasmic proteins to the vacuole. In Saccharomyces cerevisiae, autophagy is characterized by the phenotypic appearance of autophagic vesicles inside the vacuole of strains deficient in proteinase yscB. The AUT1 gene, essential for autophagy, was isolated by complementation of the sporulation deficiency of a diploid aut1-1 mutant strain by a yeast genomic library and characterized. AUT1 is located on the right arm of chromosome XIV, 10 kb from the centromere, and encodes a protein of 310 amino acids, with an estimated molecular weight of 36 kDa. Cells carrying a chromosomal deletion of AUT1 are defective in the starvation-induced bulk flow transport of cytoplasmic proteins to the vacuole. aut1 null mutant strains are completely viable but show decreased survival rates during starvation. Homozygous delta aut1 diploid cells fail to sporulate. The selective cytoplasm-to-vacuole transport of aminopeptidase I is blocked in logarithmically growing and in starved delta autl cells. Deletion of the AUT1 gene had no obvious influence on secretion, fluid phase endocytosis, or vacuolar protein sorting. This supports the idea of autophagocytosis as being a novel route transporting proteins from the cytoplasm to the vacuole.     

Autophagy and the cvt pathway both depend on AUT9

In growing cells of the yeast Saccharomyces cerevisiae, proaminopeptidase I reaches the vacuole via the selective cytoplasm-to-vacuole targeting (cvt) pathway. During nutrient limitation, autophagy is also responsible for the transport of proaminopeptidase I. These two nonclassical protein transport pathways to the vacuole are distinct in their characteristics but in large part use identical components. We expanded our initial screen for aut(-) mutants and isolated aut9-1 cells, which show a defect in both pathways, the vacuolar targeting of proaminopeptidase I and autophagy. By complementation of the sporulation defect of homocygous diploid aut9-1 mutant cells with a genomic library, in this study we identified and characterized the AUT9 gene, which is allelic with CVT7. aut9-deficient cells have no obvious defects in growth on rich media, vacuolar biogenesis, and acidification, but like other mutant cells with a defect in autophagy, they exhibit a reduced survival rate and reduced total protein turnover during starvation. Aut9p is the first putative integral membrane protein essential for autophagy. A biologically active green fluorescent protein-Aut9 fusion protein was visualized at punctate structures in the cytosol of growing cells.     

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.     

Aut2p and Aut7p, two novel microtubule-associated proteins are essential for delivery of autophagic vesicles to the vacuole.

AUT2 and AUT7, two novel genes essential for autophagocytosis in the yeast Saccharomyces cerevisiae were isolated. AUT7 was identified as a low copy suppressor of autophagic defects in aut2-1 cells. Aut7p is a homologue of the rat microtubule-associated protein (MAP) light chain 3 (LC3). Aut2p and Aut7p interact physically. Aut7p is attached to microtubules via Aut2p, which interacts with tubulins Tub1p and Tub2p. aut2- and aut7-deleted cells are unable to deliver autophagic vesicles and the precursor of aminopeptidase I to the vacuole. Double membrane-layered autophagosome-like vesicles accumulate in the cytoplasm of these cells. Our findings suggest that microtubules and an attached protein complex of Aut2p and Aut7p are involved in the delivery of autophagic vesicles to the vacuole.     

Autophagy and the cytoplasm to vacuole targeting pathway both require Aut10p.

We here report the identification of AUT10 as a novel gene required for both the cytoplasm to vacuole targeting of proaminopeptidase I and starvation-induced autophagy. aut10Delta cells are impaired in maturation of proaminopeptidase I under starvation and non-starvation conditions. A lack of Aut10p causes a defect in autophagy prior to vacuolar uptake of autophagosomes. Homozygous aut10Delta diploids do not sporulate. Vacuolar acidification indicated by accumulation of quinacrine is normal in aut10Delta cells and mature vacuolar proteinases are present. A biologically active Ha-tagged Aut10p, chromosomally expressed from its endogenous promoter, localizes in indirect immunofluorescence microscopy in the cytosol and on granulated structures, which appear clustered around the vacuolar membrane. This localization differs from known autophagy proteins.     

The loss of Drosophila APG4/AUT2 function modifies the phenotypes of cut and Notch signaling pathway mutants

A P-element line ( P0997) of Drosophila melanogaster in which the P element disrupts the Drosophila homolog of the Saccharomyces cerevisiae gene APG4/AUT2 was identified during the course of screening for cut ( ct) modifiers. The yeast gene APG4/AUT2 encodes a cysteine endoprotease directed against Apg8/Aut7 and is necessary for autophagy. The P0997 mutation enhances the wing margin loss associated with ct mutations, and also modifies the wing and eye phenotypes of Notch (N), Serrate (Ser), Delta (Dl), Hairless (H), deltex (dx), vestigial (vg) and strawberry notch (sno) mutants. These results therefore suggest an unexpected link between autophagy and the Notch signaling pathway.     

AUT3, a serine/threonine kinase gene, is essential for autophagocytosis in Saccharomyces cerevisiae.

Autophagocytosis is a starvation-induced process, carrying proteins destined for degradation to the lysosome. In the yeast Saccharomyces cerevisiae, the autophagic process is visualized by the appearance of autophagic vesicles in the vacuoles of proteinase yscB-deficient strains during starvation. aut3-1 mutant cells which exhibit a block in the autophagic process have been isolated previously. By using the drastically reduced sporulation frequency of homozygous aut3-1 diploid cells, the AUT3 gene was cloned by complementation. The Aut3 protein consists of 897 amino acids. The amino-terminal part of the protein shows significant homologies to serine/threonine kinases. aut3 null mutant cells are fully viable on rich media but show a reduced survival rate upon starvation. They are unable to accumulate autophagic vesicles in the vacuole during starvation. Starvation-induced vacuolar protein breakdown is almost completely impaired in aut3-deficient cells. Vacuolar morphology and acidification are not influenced in aut3-deficient cells. Also, secretion of invertase, endocytic uptake of Lucifer Yellow, and vacuolar protein sorting appear wild type like in aut3-deficient cells, suggesting autophagocytosis as a novel route for the transport of proteins from the cytosol to the vacuole. By using a fusion of Aut3p with green-fluorescent protein, Aut3p was localized to the cytosol.     

Autophagy in filamentous fungi

Autophagy is a ubiquitous, non-selective degradation process in eukaryotic cells that is conserved from yeast to man. Autophagy research has increased significantly in the last ten years, as autophagy has been connected with cancer, neurodegenerative disease and various human developmental processes. Autophagy also appears to play an important role in filamentous fungi, impacting growth, morphology and development. In this review, an autophagy model developed for the yeast Saccharomyces cerevisiae is used as an intellectual framework to discuss autophagy in filamentous fungi. Studies imply that, similar to yeast, fungal autophagy is characterized by the presence of autophagosomes and controlled by Tor kinase. In addition, fungal autophagy is apparently involved in protection against cell death and has significant effects on cellular growth and development. However, the only putative autophagy proteins characterized in filamentous fungi are Atg1 and Atg8. We discuss various strategies used to study and monitor fungal autophagy as well as the possible relationship between autophagy, physiology, and morphological development.     

Arabidopsis homologues of the autophagy protein Atg8 are a novel family of microtubule binding proteins

Autophagy is the non-selective transport of proteins and superfluous organelles destined for degradation to the vacuole in fungae, or the lysosome in animal cells. Some of the genes encoding components of the autophagy pathway are conserved in plants, and here we show that Arabidopsis homologues of yeast Atg8 (Apg8/Aut7) and Atg4 (Apg4/Aut2) partially complement the yeast deletion strains. The yeast double mutant, a deletion strain with respect to both Atg8 and Atg4, could not be complemented by Arabidopsis Atg8, indicating that Arabidopsis Atg8 requires Atg4 for its function. Moreover, Arabidopsis Atg8 and Arabidopsis Atg4 interact directly in a two-hybrid assay. Interestingly, Atg8 shows significant homology with the microtubule binding light chain 3 of MAP1A and B, and here we show that Arabidopsis Atg8 binds microtubules. Our results demonstrate that a principle component of the autophagic pathway in plants is similar to that in yeast and we suggest that microtubule binding plays a role in this process.     

Genomic characterization of POS5, the Saccharomyces cerevisiae mitochondrial NADH kinase

Disruption of the Saccharomyces cerevisiae mitochondrial NADH kinase POS5 increases the mitochondrial mutation rate 50-fold. Whereas most multicellular eukaryotic genomes have one NADH kinase gene, the yeast genome contains three distinct genes encoding NAD/H kinase activity. To determine if all three genes are essential for viability we constructed combinations of gene knockouts. We show that only the pos5Deltautr1Delta combination is synthetically lethal, demonstrating an essential overlapping function, and showing that NAD/H kinase activity is essential for eukaryotic viability. The single human NAD/H kinase gene can rescue the lethality of the double knockout in yeast, demonstrating that the single human gene can fill the various functions provided by the three yeast genes. The human NAD/H kinase gene harbors very common sequence variants, but all of these equally complement the synthetic lethality in yeast, illustrating that each of these are functionally wild-type. To understand the molecular mechanism of the mitochondrial genome instability of pos5 mutation we performed gene expression analysis on the pos5Delta. The pos5Delta resulted in an increase in expression of most of the iron transport genes including key genes involved in iron-sulfur cluster assembly. Decreased expression occurred in many genes involved in the electron transport chain. We show that the pos5Delta expression pattern is similar to the frataxin homolog knockout (yfh1Delta), the yeast model for Friedreich's ataxia. These combined data show that the POS5 NAD/H kinase is an important protein required for a variety of essential cellular pathways and that deficient iron-sulfur cluster assembly may play a critical role in the mitochondrial mutator phenotype observed in the pos5Delta.     

Atg19 mediates a dual interaction cargo sorting mechanism in selective autophagy

Autophagy is a catabolic membrane-trafficking mechanism conserved in all eukaryotic cells. In addition to the nonselective transport of bulk cytosol, autophagy is responsible for efficient delivery of the vacuolar enzyme Ape1 precursor (prApe1) in the budding yeast Saccharomyces cerevisiae, suggesting the presence of a prApe1 sorting machinery. Sequential interactions between Atg19-Atg11 and Atg19-Atg8 pairs are thought responsible for targeting prApe1 to the vesicle formation site, the preautophagosomal structure (PAS), and loading it into transport vesicles, respectively. However, the different patterns of prApe1 transport defect seen in the atg11Delta and atg19Delta strains seem to be incompatible with this model. Here we report that prApe1 could not be targeted to the PAS and failed to be delivered into the vacuole in atg8Delta atg11Delta double knockout cells regardless of the nutrient conditions. We postulate that Atg19 mediates a dual interaction prApe1-sorting mechanism through independent, instead of sequential, interactions with Atg11 and Atg8. In addition, to efficiently deliver prApe1 to the vacuole, a proper interaction between Atg11 and Atg9 is indispensable. We speculate that Atg11 may elicit a cargo-loading signal and induce Atg9 shuttling to a specific PAS site, where Atg9 relays the signal and recruits other Atg proteins to induce vesicle formation.     

Induction of autophagy by second-fermentation yeasts during elaboration of sparkling wines

Autophagy is a transport system mediated by vesicles, ubiquitous in eukaryotic cells, by which bulk cytoplasm is targeted to a lysosome or vacuole for degradation. In the yeast Saccharomyces cerevisiae, autophagy is triggered by nutritional stress conditions (e.g., carbon- or nitrogen-depleted medium). In this study we showed that there is induction of autophagy in second-fermentation yeasts during sparkling wine making. Two methods were employed to detect autophagy: a biochemical approach based on depletion of the protein acetaldehyde dehydrogenase Ald6p and a morphological strategy consisting of visualization of autophagic bodies and autophagosomes, which are intermediate vesicles in the autophagic process, by transmission electron microscopy. This study provides the first demonstration of autophagy in second-fermentation yeasts under enological conditions. The correlation between autophagy and yeast autolysis during sparkling wine production is discussed, and genetic engineering of autophagy-related genes in order to accelerate the aging steps in wine making is proposed.     

Induction of Autophagy by Second-Fermentation Yeasts during Elaboration of Sparkling Wines

Autophagy is a transport system mediated by vesicles, ubiquitous in eukaryotic cells, by which bulk cytoplasm is targeted to a lysosome or vacuole for degradation. In the yeast Saccharomyces cerevisiae, autophagy is triggered by nutritional stress conditions (e.g., carbon- or nitrogen-depleted medium). In this study we showed that there is induction of autophagy in second-fermentation yeasts during sparkling wine making. Two methods were employed to detect autophagy: a biochemical approach based on depletion of the protein acetaldehyde dehydrogenase Ald6p and a morphological strategy consisting of visualization of autophagic bodies and autophagosomes, which are intermediate vesicles in the autophagic process, by transmission electron microscopy. This study provides the first demonstration of autophagy in second-fermentation yeasts under enological conditions. The correlation between autophagy and yeast autolysis during sparkling wine production is discussed, and genetic engineering of autophagy-related genes in order to accelerate the aging steps in wine making is proposed.     

Phosphatidylethanolamine, a limiting factor of autophagy in yeast strains bearing a defect in the carboxypeptidase Y pathway of vacuolar targeting

Vps4p and Vps36p of Saccharomyces cerevisiae are involved in the transport of proteins to the vacuole via the carboxypeptidase Y pathway. We found that deletion of VPS4 and VPS36 caused impaired maturation of the vacuolar proaminopeptidase I (pAPI) via autophagy or the cytosol to vacuole targeting pathway. Supplementation with ethanolamine rescued this defect, leading to an increase of the cellular amount of phosphatidylethanolamine (PtdEtn), an enhanced level of the PtdEtn-binding autophagy protein Atg8p and a balanced rate of autophagy. We also discovered that maturation of pAPI was generally affected by PtdEtn depletion in a psd1Delta psd2Delta mutant due to reduced recruitment of Atg8p to the preautophagosomal structure. Ethanolamine supplementation provided the necessary amounts of PtdEtn for complete maturation of pAPI. Since the expression level of Atg8p was not compromised in the psd1Delta psd2Delta strain, we concluded that the amount of available PtdEtn was limiting. Thus, PtdEtn appears to be a limiting factor for the balance of the carboxypeptidase Y pathway and autophagy/the cytosol to vacuole targeting pathway in the yeast.     

Autophagy in the fission yeast Schizosaccharomyces pombe

Autophagy is a non-selective degradation process in eukaryotic cells. The genome sequence of the fission yeast Schizosaccharomyces pombe has revealed that many of the genes required for autophagy are common between the fission yeast and budding yeast, suggesting that the basic machinery of autophagy is conserved between these species. Autophagy in fission yeast is specifically induced by nitrogen starvation based on monitoring a GFP-Atg8p marker. Upon nitrogen starvation, fission yeast cells exit the vegetative cell cycle and initiate sexual differentiation to produce spores. Most of the nitrogen used for de novo protein synthesis during sporulation derives from the autophagic protein degradation system. This review focuses on the recent advances in the role of autophagy in fission yeast.     

Autophagy is induced during cell death by incompatibility and is essential for differentiation in the filamentous fungus Podospora anserina

In filamentous fungi, a cell death reaction occurs when cells of unlike genotype fuse. This cell death reaction, known as incompatibility reaction, is genetically controlled by a set of loci termed het loci (for heterokaryon incompatibility loci). In Podospora anserina, genes induced during this cell death reaction (idi genes) have been identified. The idi-6/pspA gene encodes a serine protease that is the orthologue of the vacuolar protease B of Saccharomyces cerevisiae involved in autophagy. We report here that the PSPA protease participates in the degradative autophagic pathway in Podospora. We have identified the Podospora orthologue of the AUT7 gene of S. cerevisiae involved in the early steps of autophagy in yeast. This gene is induced during the development of the incompatibility reaction and was designated idi-7. We have used a GFP-IDI7 fusion protein as a cytological marker of the induction of autophagy. Relocalization of this fusion protein and detection of autophagic bodies inside the vacuoles during the development of the incompatibility reaction provide cytological evidence of induction of autophagy during this cell death reaction. Therefore, cell death by incompatibility in fungi appears to be related to type II programmed cell death in metazoans. In addition, we found that pspA and idi-7 null mutations confer differentiation defects such as the absence of female reproductive structures, indicating that autophagy is required for differentiation in Podospora.     

Global Analysis of Fission Yeast Mating Genes Reveals New Autophagy Factors

Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in animal development and human diseases. Molecular understanding of autophagy has mainly come from the budding yeast Saccharomyces cerevisiae, and it remains unclear to what extent the mechanisms are the same in other organisms. Here, through screening the mating phenotype of a genome-wide deletion collection of the fission yeast Schizosaccharomyces pombe, we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins, Atg10, Atg14, and Atg16, and two novel factors, Ctl1 and Fsc1. We systematically examined the subcellular localization of fission yeast autophagy factors for the first time and characterized the phenotypes of their mutants, thereby uncovering both similarities and differences between the two yeasts. Unlike budding yeast, all three Atg18/WIPI proteins in fission yeast are essential for autophagy, and we found that they play different roles, with Atg18a uniquely required for the targeting of the Atg12–Atg5·Atg16 complex. Our investigation of the two novel factors revealed unforeseen autophagy mechanisms. The choline transporter-like protein Ctl1 interacts with Atg9 and is required for autophagosome formation. The fasciclin domain protein Fsc1 localizes to the vacuole membrane and is required for autophagosome-vacuole fusion but not other vacuolar fusion events. Our study sheds new light on the evolutionary diversity of the autophagy machinery and establishes the fission yeast as a useful model for dissecting the mechanisms of autophagy.