Autophagic Processes in Yeast: Mechanism, Machinery and Regulation
TL;DR: Many aspects of autophagy are conserved from yeast to human; in particular, this applies to the gene products mediating these pathways as well as some of the signaling cascades regulating it, so that the information the authors relate is relevant to higher eukaryotes.
Abstract: Autophagy refers to a group of processes that involve degradation of cytoplasmic components including cytosol, macromolecular complexes, and organelles, within the vacuole or the lysosome of higher eukaryotes. The various types of autophagy have attracted increasing attention for at least two reasons. First, autophagy provides a compelling example of dynamic rearrangements of subcellular membranes involving issues of protein trafficking and organelle identity, and thus it is fascinating for researchers interested in questions pertinent to basic cell biology. Second, autophagy plays a central role in normal development and cell homeostasis, and, as a result, autophagic dysfunctions are associated with a range of illnesses including cancer, diabetes, myopathies, some types of neurodegeneration, and liver and heart diseases. That said, this review focuses on autophagy in yeast. Many aspects of autophagy are conserved from yeast to human; in particular, this applies to the gene products mediating these pathways as well as some of the signaling cascades regulating it, so that the information we relate is relevant to higher eukaryotes. Indeed, as with many cellular pathways, the initial molecular insights were made possible due to genetic studies in Saccharomyces cerevisiae and other fungi.
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TL;DR: This review focuses on macroautophagy, briefly describing the discovery of this process in mammalian cells, discussing the current views concerning the donor membrane that forms the phagophore, and characterizing the autophagy machinery including the available structural information.
Abstract: Autophagy is a primarily degradative pathway that takes place in all eukaryotic cells. It is used for recycling cytoplasm to generate macromolecular building blocks and energy under stress conditions, to remove superfluous and damaged organelles to adapt to changing nutrient conditions and to maintain cellular homeostasis. In addition, autophagy plays a critical role in cytoprotection by preventing the accumulation of toxic proteins and through its action in various aspects of immunity including the elimination of invasive microbes and its participation in antigen presentation. The most prevalent form of autophagy is macroautophagy, and during this process, the cell forms a double-membrane sequestering compartment termed the phagophore, which matures into an autophagosome. Following delivery to the vacuole or lysosome, the cargo is degraded and the resulting macromolecules are released back into the cytosol for reuse. The past two decades have resulted in a tremendous increase with regard to the molecular studies of autophagy being carried out in yeast and other eukaryotes. Part of the surge in interest in this topic is due to the connection of autophagy with a wide range of human pathophysiologies including cancer, myopathies, diabetes and neurodegenerative disease. However, there are still many aspects of autophagy that remain unclear, including the process of phagophore formation, the regulatory mechanisms that control its induction and the function of most of the autophagy-related proteins. In this review, we focus on macroautophagy, briefly describing the discovery of this process in mammalian cells, discussing the current views concerning the donor membrane that forms the phagophore, and characterizing the autophagy machinery including the available structural information.
1,568 citations
TL;DR: Regulation of autophagy can be used as effective interventional strategies for cancer therapy and contributes to the survival and growth of the established tumors and promotes aggressiveness of the cancers by facilitating metastasis.
Abstract: Autophagy, as a type II programmed cell death, plays crucial roles with autophagy-related (ATG) proteins in cancer. Up to now, the dual role of autophagy both in cancer progression and inhibition remains controversial, in which the numerous ATG proteins and their core complexes including ULK1/2 kinase core complex, autophagy-specific class III PI3K complex, ATG9A trafficking system, ATG12 and LC3 ubiquitin-like conjugation systems, give multiple activities of autophagy pathway and are involved in autophagy initiation, nucleation, elongation, maturation, fusion and degradation. Autophagy plays a dynamic tumor-suppressive or tumor-promoting role in different contexts and stages of cancer development. In the early tumorigenesis, autophagy, as a survival pathway and quality-control mechanism, prevents tumor initiation and suppresses cancer progression. Once the tumors progress to late stage and are established and subjected to the environmental stresses, autophagy, as a dynamic degradation and recycling system, contributes to the survival and growth of the established tumors and promotes aggressiveness of the cancers by facilitating metastasis. This indicates that regulation of autophagy can be used as effective interventional strategies for cancer therapy.
591 citations
Cites background from "Autophagic Processes in Yeast: Mech..."
...starvation, hypoxia [27], some small molecular compounds [28], oxidation, and pathogen invasion [3, 29], a large number of autophagy is induced by the transduction of cellular signaling pathways, and many important autophagy-related proteins and their complex involved in the autophagic process [30]....
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TL;DR: The yeast Saccharomyces cerevisiae has been a favorite organism for pioneering studies on nutrient-sensing and signaling mechanisms, and its discovery of nutrient transceptors (transporter receptors) as nutrient sensors has led to important new concepts and insight into nutrient-controlled cellular regulation.
Abstract: The yeast Saccharomyces cerevisiae has been a favorite organism for pioneering studies on nutrient-sensing and signaling mechanisms. Many specific nutrient responses have been elucidated in great detail. This has led to important new concepts and insight into nutrient-controlled cellular regulation. Major highlights include the central role of the Snf1 protein kinase in the glucose repression pathway, galactose induction, the discovery of a G-protein-coupled receptor system, and role of Ras in glucose-induced cAMP signaling, the role of the protein synthesis initiation machinery in general control of nitrogen metabolism, the cyclin-controlled protein kinase Pho85 in phosphate regulation, nitrogen catabolite repression and the nitrogen-sensing target of rapamycin pathway, and the discovery of transporter-like proteins acting as nutrient sensors. In addition, a number of cellular targets, like carbohydrate stores, stress tolerance, and ribosomal gene expression, are controlled by the presence of multiple nutrients. The protein kinase A signaling pathway plays a major role in this general nutrient response. It has led to the discovery of nutrient transceptors (transporter receptors) as nutrient sensors. Major shortcomings in our knowledge are the relationship between rapid and steady-state nutrient signaling, the role of metabolic intermediates in intracellular nutrient sensing, and the identity of the nutrient sensors controlling cellular growth.
529 citations
TL;DR: In this article, the authors discuss canonical and non-canonical autophagy pathways and their current knowledge of antibacterial auto-pathway, with a focus on the interplay between bacterial factors and autophag components.
Abstract: Autophagy is a cellular process that targets proteins, lipids and organelles to lysosomes for degradation, but it has also been shown to combat infection with various pathogenic bacteria. In turn, bacteria have developed diverse strategies to avoid autophagy by interfering with autophagy signalling or the autophagy machinery and, in some cases, they even exploit autophagy for their growth. In this Review, we discuss canonical and non-canonical autophagy pathways and our current knowledge of antibacterial autophagy, with a focus on the interplay between bacterial factors and autophagy components.
449 citations
TL;DR: This is the first demonstration of lipid droplet turnover in yeast by microautophagy, which contributes to neutral lipid homeostasis by vacuolar lipolysis.
Abstract: Cytosolic lipid droplets (LDs) are ubiquitous organelles in prokaryotes and eukaryotes that play a key role in cellular and organismal lipid homeostasis. Triacylglycerols (TAGs) and steryl esters, which are stored in LDs, are typically mobilized in growing cells or upon hormonal stimulation by LD-associated lipases and steryl ester hydrolases. Here we show that in the yeast Saccharomyces cerevisiae, LDs can also be turned over in vacuoles/lysosomes by a process that morphologically resembles microautophagy. A distinct set of proteins involved in LD autophagy is identified, which includes the core autophagic machinery but not Atg11 or Atg20. Thus LD autophagy is distinct from endoplasmic reticulum–autophagy, pexophagy, or mitophagy, despite the close association between these organelles. Atg15 is responsible for TAG breakdown in vacuoles and is required to support growth when de novo fatty acid synthesis is compromised. Furthermore, none of the core autophagy proteins, including Atg1 and Atg8, is required for LD formation in yeast.
240 citations
Cites background from "Autophagic Processes in Yeast: Mech..."
...The autophagy machinery is highly conserved, and some 36 autophagy (Atg) proteins have been identified (Meijer et al., 2007; Reggiori and Klionsky, 2013)....
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References
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TL;DR: Identifying the autophagy genes in yeast and finding orthologs in other organisms reveals the conservation of the mechanism in eukaryotes and allows the use of molecular genetics and biology in different model systems to study this process.
Abstract: Autophagy, the process by which cells recycle cytoplasm and dispose of excess or defective organelles, has entered the research spotlight largely owing to the discovery of the protein components that drive this process. Identifying the autophagy genes in yeast and finding orthologs in other organisms reveals the conservation of the mechanism of autophagy in eukaryotes and allows the use of molecular genetics and biology in different model systems to study this process. By mostly morphological studies, autophagy has been linked to disease processes. Whether autophagy protects from or causes disease is unclear. Here, we summarize current knowledge about the role of autophagy in disease and health.
2,451 citations
"Autophagic Processes in Yeast: Mech..." refers background in this paper
...Autophagy can be divided into two main types, microautophagy and macroautophagy (Figure 1), and both of these include nonselective and selective processes (Shintani and Klionsky 2004a)....
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...In a wild-type strain only 30% of the cells have a detectable PAS (based on the localization of a fluorescent-tagged protein such as GFP– Atg8), whereas essentially the entire population displays a PAS when macroautophagy is blocked in an atg mutant (Shintani and Klionsky 2004b)....
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TL;DR: A new mode of protein lipidation is reported, in which Apg8 is covalently conjugated to phosphatidylethanolamine through an amide bond between the C-terminal glycine and the amino group of phosph atidyleanolamine, mediated by a ubiquitination-like system.
Abstract: Autophagy is a dynamic membrane phenomenon for bulk protein degradation in the lysosome/vacuole1,2. Apg8/Aut7 is an essential factor for autophagy in yeast3,4,5. We previously found that the carboxy-terminal arginine of nascent Apg8 is removed by Apg4/Aut2 protease, leaving a glycine residue at the C terminus6. Apg8 is then converted to a form (Apg8-X) that is tightly bound to the membrane6. Here we report a new mode of protein lipidation. Apg8 is covalently conjugated to phosphatidylethanolamine through an amide bond between the C-terminal glycine and the amino group of phosphatidylethanolamine. This lipidation is mediated by a ubiquitination-like system. Apg8 is a ubiquitin-like protein that is activated by an E1 protein, Apg7 (refs 7, 8), and is transferred subsequently to the E2 enzymes Apg3/Aut1 (ref. 9). Apg7 activates two different ubiquitin-like proteins, Apg12 (ref. 10) and Apg8, and assigns them to specific E2 enzymes, Apg10 (ref. 11) and Apg3, respectively. These reactions are necessary for the formation of Apg8-phosphatidylethanolamine. This lipidation has an essential role in membrane dynamics during autophagy6.
1,849 citations
"Autophagic Processes in Yeast: Mech..." refers background in this paper
...Atg3 forms a covalent bond between the now-exposed C-terminal glycine residue of Atg8 and PE (Ichimura et al. 2000)....
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TL;DR: It is shown here that a unique covalent-modification system is essential for autophagy to occur, the first report of a protein unrelated to ubiquitin that uses a ubiquitination-like conjugation system.
Abstract: Autophagy is a process for the bulk degradation of proteins, in which cytoplasmic components of the cell are enclosed by double-membrane structures known as autophagosomes for delivery to lysosomes or vacuoles for degradation1,2,3,4. This process is crucial for survival during starvation and cell differentiation. No molecules have been identified that are involved in autophagy in higher eukaryotes. We have isolated 14 autophagy-defective (apg) mutants of the yeast Saccharomyces cerevisiae5 and examined the autophagic process at the molecular level6,7,8,9. We show here that a unique covalent-modification system is essential for autophagy to occur. The carboxy-terminal glycine residue of Apg12, a 186-amino-acid protein, is conjugated to a lysine at residue 149 of Apg5, a 294-amino-acid protein. Of the apg mutants, we found that apg7 and apg10 were unable to form an Apg5/Apg12 conjugate. By cloning APG7, we discovered that Apg7 is a ubiquitin-E1-like enzyme. This conjugation can be reconstituted in vitro and depends on ATP. To our knowledge, this is the first report of a protein unrelated to ubiquitin that uses a ubiquitination-like conjugation system. Furthermore, Apg5 and Apg12 have mammalian homologues, suggesting that this new modification system is conserved from yeast to mammalian cells.
1,564 citations
"Autophagic Processes in Yeast: Mech..." refers background in this paper
...Recruitment of the components of the Atg8 conjugation system, i.e., Atg7 and Atg3, onto membranes depends on the Atg12—Atg5–Atg16 complex being able to associate with lipid bilayers (Romanov et al. 2012)....
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...Autophagy 349 Thus, the function of the Atg12—Atg5–Atg16 complex remains unclear, but recent structural studies have revealed that it is probably acting as a platform to bring into close proximity the activated Atg8 in the Atg8–Atg3 conjugate to the acceptor PE (Kaiser et al. 2012; Noda et al. 2013; Otomo et al. 2013)....
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...Noda, N. N., Y. Fujioka, T. Hanada, Y. Ohsumi, and F. Inagaki, 2013 Structure of the Atg12—Atg5 conjugate reveals a platform for stimulating Atg8—PE conjugation....
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...The activated Atg12 is then transferred to the Atg10-conjugating enzyme (Shintani et al. 1999), which catalyzes the formation of a covalent bond between the Cterminal glycine of Atg12 and an internal lysine of Atg5 (Mizushima et al. 1998), a protein that also contains two ubiquitin-like structural domains (Matsushita et al. 2007)....
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...Atg5, and preferentially the Atg12—Atg5 conjugate, noncovalently binds Atg16, promoting Atg16 selfinteraction (Mizushima et al. 1999), generating a dimer of the Atg12—Atg5–Atg16 complex (Kuma et al. 2002; Fujioka et al. 2010)....
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TL;DR: The term mitophagy is used to refer to mitochondrial degradation by autophagy, and the possible role of the mitochondrial permeability transition in mitophagic delivery to lysosomes is the major degradative pathway in mitochondrial turnover.
1,492 citations
"Autophagic Processes in Yeast: Mech..." refers methods in this paper
...The terms mitophagosome and pexophagosome have similarly been used when referring to mitophagy and pexophagy, respectively (Ano et al. 2005b; Kim et al. 2007)....
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TL;DR: It is shown that mouse Apg12-Apg5 conjugate localizes to the isolation membranes in mouse embryonic stem cells, and the covalent modification of Apg5 with Apg 12 is not required for its membrane targeting, but is essential for involvement of ApG5 in elongation of the isolation membrane.
Abstract: In macroautophagy, cytoplasmic components are delivered to lysosomes for degradation via autophagosomes that are formed by closure of cup-shaped isolation membranes. However, how the isolation membranes are formed is poorly understood. We recently found in yeast that a novel ubiquitin-like system, the Apg12-Apg5 conjugation system, is essential for autophagy. Here we show that mouse Apg12-Apg5 conjugate localizes to the isolation membranes in mouse embryonic stem cells. Using green fluorescent protein–tagged Apg5, we revealed that the cup-shaped isolation membrane is developed from a small crescent-shaped compartment. Apg5 localizes on the isolation membrane throughout its elongation process. To examine the role of Apg5, we generated Apg5-deficient embryonic stem cells, which showed defects in autophagosome formation. The covalent modification of Apg5 with Apg12 is not required for its membrane targeting, but is essential for involvement of Apg5 in elongation of the isolation membranes. We also show that Apg12-Apg5 is required for targeting of a mammalian Aut7/Apg8 homologue, LC3, to the isolation membranes. These results suggest that the Apg12-Apg5 conjugate plays essential roles in isolation membrane development.
1,372 citations
"Autophagic Processes in Yeast: Mech..." refers background in this paper
...In contrast, monitoring GFP–Atg8 fluorescence at the PAS suggests that a cycle of autophagosome formation and fusion with the vacuole occurs in 10 min (Xie et al. 2008), which is somewhat faster than the proposed 10 min half-life of mammalian autophagosomes (Mizushima et al. 2001)....
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