Autophagy in Yeast: Mechanistic Insights and Physiological Function

Unicellular eukaryotic organisms must be capable of rapid adaptation to changing environments. While such changes do not normally occur in the tissues of multicellular organisms, developmental and pathological changes in the environment of cells often require adaptation mechanisms not dissimilar from those found in simpler cells. Autophagy is a catabolic membrane-trafficking phenomenon that occurs in response to dramatic changes in the nutrients available to yeast cells, for example during starvation or after challenge with rapamycin, a macrolide antibiotic whose effects mimic starvation. Autophagy also occurs in animal cells that are serum starved or challenged with specific hormonal stimuli. In macroautophagy, the form of autophagy commonly observed, cytoplasmic material is sequestered in double-membrane vesicles called autophagosomes and is then delivered to a lytic compartment such as the yeast vacuole or mammalian lysosome. In this fashion, autophagy allows the degradation and recycling of a wide spectrum of biological macromolecules. While autophagy is induced only under specific conditions, salient mechanistic aspects of autophagy are functional in a constitutive fashion. In Saccharomyces cerevisiae, induction of autophagy subverts a constitutive membrane-trafficking mechanism called the cytoplasm-to-vacuole targeting pathway from a specific mode, in which it carries the resident vacuolar hydrolase, aminopeptidase I, to a nonspecific bulk mode in which significant amounts of cytoplasmic material are also sequestered and recycled in the vacuole. The general aim of this review is to focus on insights gained into the mechanism of autophagy in yeast and also to review our understanding of the physiological significance of autophagy in both yeast and higher organisms.     

The molecular mechanism of autophagy

Autophagy is a conserved trafficking pathway that is highly regulated by environmental conditions. During autophagy, portions of cytoplasm are sequestered into a double-membrane autophagosome and delivered to a degradative organelle, the vacuole in yeast and the lysosome in mammalian cells, for breakdown and recycling. Autophagy is induced under starvation conditions and in mammalian cells is also invoked in response to specific hormones. In yeast, under nutrient-rich conditions, a constitutive biosynthetic pathway, termed the cytoplasm to vacuole targeting (Cvt) pathway, utilizes most of the same molecular machinery and topologically similar vesicles for the delivery of the resident hydrolase aminopeptidase I to the vacuole. Both autophagy and the Cvt pathway have been extensively studied and comprehensively reviewed in the past few years. In this review, we focus on the yeast system, which has provided most of the insight into the molecular mechanism of autophagy and the Cvt pathway, and highlight the most recent additions to our current knowledge of both pathways.     

Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice

Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation. This mechanism is involved in the preservation of nutrients under starvation condition as well as the normal turnover of cytoplasmic component. Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies. Here, we generated conditional knockout mice of Atg7, an essential gene for autophagy in yeast. Atg7 was essential for ATG conjugation systems and autophagosome formation, amino acid supply in neonates, and starvation-induced bulk degradation of proteins and organelles in mice. Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates. Our results indicate the important role of autophagy in starvation response and the quality control of proteins and organelles in quiescent cells.     

The actin cytoskeleton is required for selective types of autophagy, but not nonspecific autophagy, in the yeast Saccharomyces cerevisiae

Autophagy is a catabolic multitask transport route that takes place in all eukaryotic cells. During starvation, cytoplasmic components are randomly sequestered into huge double-membrane vesicles called autophagosomes and delivered into the lysosome/vacuole where they are destroyed. Cells are able to modulate autophagy in response to their needs, and under certain circumstances, cargoes such as aberrant protein aggregates, organelles and bacteria can be selectively and exclusively incorporated into autophagosomes. In the yeast Saccharomyces cerevisiae, for example, double-membrane vesicles are used to transport the Ape1 protease into the vacuole, or for the elimination of superfluous peroxisomes. In the present study we reveal that in this organism, actin plays a role in these two types of selective autophagy but not in the nonselective, bulk process. In particular, we show that precursor Ape1 is not correctly recruited to the PAS, the putative site of double-membrane vesicle biogenesis, and superfluous peroxisomes are not degraded in a conditional actin mutant. These phenomena correlate with a defect in Atg9 trafficking from the mitochondria to the PAS.     

Harpooning the Cvt complex to the phagophore assembly site

Autophagy is a catabolic process employed by eukaryotes to degrade and recycle intracellular components. When this pathway is induced by starvation conditions, part of the cytoplasm and organelles are sequestered into double-membrane vesicles called autophagosomes, and delivered into the lysosome/vacuole for degradation. In addition to the random bulk elimination of cytoplasmic contents, the selective removal of specific cargo molecules has also been described. These selective types of autophagy are characterized by the recruitment of the cargo destined for degradation in close proximity to the forming double-membrane vesicle that results in an exclusive incorporation (that is, without bulk cytoplasm). A number of factors required for selective types of autophagy have been identified. In particular, we have recently shown that actin and the actin-binding Arp2/3 protein complex are involved in the cytoplasm to vacuole targeting (Cvt) pathway, a yeast selective type of autophagy. The contribution at a molecular level of these factors, however, remains unknown. In this addendum, we present mechanistic models that take into account possible roles of actin and the Arp2/3 complex in the Cvt pathway.     

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.     

Atg11 directs autophagosome cargoes to the PAS along actin cables

For more than 40 years, autophagy has been almost exclusively studied as a cellular response that allows adaptation to starvation situations. In nutrient-deprived conditions, cytoplasmic components and organelles are randomly sequestered into double-membrane vesicles called autophagosomes, creating the notion that this pathway is a nonselective process (reviewed in Refs 1, 2). Recent results, however, have demonstrated that under certain circumstances, cargoes such as protein complexes, organelles and bacteria can be selectively and exclusively incorporated into double-membrane vesicles.(1) We have recently shown that actin plays an essential role in two selective types of autophagy in the yeast Saccharomyces cerevisiae, the cytoplasm to vacuole targeting (Cvt) pathway and pexophagy, raising the possibility that the structures formed by polymers of this protein helps autophagosomes in recognizing the cargoes that must be delivered to the vacuole.(3) In this addendum, we discuss the possible central role of Atg11 as a molecule connecting cargoes, actin and pre-utophagosomal structure (PAS) elements.     

The Cvt pathway as a model for selective autophagy

Autophagy is a highly conserved, ubiquitous process that is responsible for the degradation of cytosolic components in response to starvation. Autophagy is generally considered to be non-selective; however, there are selective types of autophagy that use receptor and adaptor proteins to specifically isolate a cargo. One type of selective autophagy in yeast is the cytoplasm to vacuole targeting (Cvt) pathway. The Cvt pathway is responsible for the delivery of the hydrolase aminopeptidase I to the vacuole; as such, it is the only known biosynthetic pathway that utilizes the core machinery of autophagy. Nonetheless, it serves as a model for the study of selective autophagy in other organisms.     

From signal transduction to autophagy of plant cell organelles: lessons from yeast and mammals and plant-specific features

Autophagy is an evolutionarily conserved intracellular process for the vacuolar degradation of cytoplasmic constituents. The central structures of this pathway are newly formed double-membrane vesicles (autophagosomes) that deliver excess or damaged cell components into the vacuole or lysosome for proteolytic degradation and monomer recycling. Cellular remodeling by autophagy allows organisms to survive extensive phases of nutrient starvation and exposure to abiotic and biotic stress. Autophagy was initially studied by electron microscopy in diverse organisms, followed by molecular and genetic analyses first in yeast and subsequently in mammals and plants. Experimental data demonstrate that the basic principles, mechanisms, and components characterized in yeast are conserved in mammals and plants to a large extent. However, distinct autophagy pathways appear to differ between kingdoms. Even though direct information remains scarce particularly for plants, the picture is emerging that the signal transduction cascades triggering autophagy and the mechanisms of organelle turnover evolved further in higher eukaryotes for optimization of nutrient recycling. Here, we summarize new research data on nitrogen starvation-induced signal transduction and organelle autophagy and integrate this knowledge into plant physiology.     

Harpooning the Cvt complex to the phagophore assembly site

Autophagy is a catabolic process employed by eukaryotes to degrade and recycle intracellular components. When this pathway is induced by starvation conditions, part of the cytoplasm and organelles are sequestered into double-membrane vesicles called autophagosomes, and delivered into the lysosome/vacuole for degradation. In addition to the random bulk elimination of cytoplasmic contents, the selective removal of specific cargo molecules has also been described. These selective types of autophagy are characterized by the recruitment of the cargo destined for degradation in close proximity to the forming double-membrane vesicle that results in an exclusive incorporation (that is, without bulk cytoplasm). A number of factors required for selective types of autophagy have been identified. In particular, we have recently shown that actin and the actin-binding Arp2/3 protein complex are involved in the cytoplasm to vacuole targeting (Cvt) pathway, a yeast selective type of autophagy. The contribution at a molecular level of these factors, however, remains unknown. In this addendum, we present mechanistic models that take into account possible roles of actin and the Arp2/3 complex in the Cvt pathway.

Addendum to: Monastyrska I, He C, Geng J, Hoppe D, Li Z, Klionsky DJ.Arp2 links autophagic machinery with the actin cytoskeleton. Mol Biol Cell 2008; 19:1962-75.     

A novel type of autophagy occurs together with vacuole genesis in miniprotoplasts prepared from tobacco culture cells

Mature plant cells have large vacuoles. But how these vacuoles are formed has not been fully understood. It has been reported that autophagy is involved in the genesis of plant vacuoles. Thus we examined whether autophagy occurs in the vacuole genesis of a plant cell model called miniprotoplasts, in which preexisting large vacuoles have been removed. We prepared miniprotoplasts from tobacco culture cells (BY-2) and observed the formation of vacuoles by light and electron microscopy. The miniprotoplasts had few vacuoles immediately after preparation, but had large vacuoles after 1 to 2 d. When the cysteine protease inhibitor E-64c or E-64d was added to culture media, almost all vacuoles formed contained materials of cytoplasmic origin. This result suggests that autophagy occurs together with the genesis of the vacuoles in miniprotoplasts. 3-Methyladenine and phosphatidylinositol 3-kinase inhibitors such as wortmannin and LY294002, all of which block starvation?induced autophagy in tobacco culture cells and constitutive autophagy in Arabidopsis root cells, did not affect the autophagy in miniprotoplasts. Thus the form of autophagy in miniprotoplasts is probably different from the form of autophagy that arises as a result of sucrose starvation and constitutive autophagy in root tip cells. The causal connection between autophagy and vacuole genesis in miniprotoplasts was not clarified in this study.     

Development by self-digestion: molecular mechanisms and biological functions of autophagy

Autophagy is the major cellular pathway for the degradation of long-lived proteins and cytoplasmic organelles. It involves the rearrangement of subcellular membranes to sequester cargo for delivery to the lysosome where the sequestered material is degraded and recycled. For many decades, it has been known that autophagy occurs in a wide range of eukaryotic organisms and in multiple different cell types during starvation, cellular and tissue remodeling, and cell death. However, until recently, the functions of autophagy in normal development were largely unknown. The identification of a set of evolutionarily conserved genes that are essential for autophagy has opened up new frontiers for deciphering the role of autophagy in diverse biological processes. In this review, we summarize our current knowledge about the molecular machinery of autophagy and the role of the autophagic machinery in eukaryotic development.     

Visualization of autophagy in Arabidopsis using the fluorescent dye monodansylcadaverine and a GFP-AtATG8e fusion protein

Autophagy is a process that is thought to occur in all eukaryotes in which cells recycle cytoplasmic contents when subjected to environmental stress conditions or during certain stages of development. Upon induction of autophagy, double membrane-bound structures called autophagosomes engulf portions of the cytoplasm and transfer them to the vacuole or lysosome for degradation. In this study, we have characterized two potential markers for autophagy in plants, the fluorescent dye monodansylcadaverine (MDC) and a green fluorescent protein (GFP)-AtATG8e fusion protein, and propose that they both label autophagosomes in Arabidopsis. Both markers label the same small, apparently membrane-bound structures found in cells under conditions that are known to induce autophagy such as starvation and senescence. They are usually seen in the cytoplasm, but occasionally can be observed within the vacuole, consistent with a function in the transfer of cytoplasmic material into the vacuole for degradation. MDC-staining and the GFP-AtATG8e fusion protein can now be used as very effective tools to complement biochemical and genetic approaches to the study of autophagy in plant systems.     

Bulk RNA degradation by nitrogen starvation‐induced autophagy in yeast

Autophagy is a catabolic process conserved among eukaryotes. Under nutrient starvation, a portion of the cytoplasm is non‐selectively sequestered into autophagosomes. Consequently, ribosomes are delivered to the vacuole/lysosome for destruction, but the precise mechanism of autophagic RNA degradation and its physiological implications for cellular metabolism remain unknown. We characterized autophagy‐dependent RNA catabolism using a combination of metabolome and molecular biological analyses in yeast. RNA delivered to the vacuole was processed by Rny1, a T2‐type ribonuclease, generating 3′‐NMPs that were immediately converted to nucleosides by the vacuolar non‐specific phosphatase Pho8. In the cytoplasm, these nucleosides were broken down by the nucleosidases Pnp1 and Urh1. Most of the resultant bases were not re‐assimilated, but excreted from the cell. Bulk non‐selective autophagy causes drastic perturbation of metabolism, which must be minimized to maintain intracellular homeostasis.     

Autophagy is required for maintenance of amino acid levels and protein synthesis under nitrogen starvation

Autophagy is a transport system of cytoplasmic components to the lysosome/vacuole for degradation well conserved in eukaryotes. Autophagy is strongly induced by nutrient starvation. Several specific proteins, including amino acid synthesis enzymes and vacuolar enzymes, are increased during nitrogen starvation in wild-type cells but not in autophagy-defective delta atg7 cells despite similar mRNA levels. We further examined deficiencies in these cells. Bulk protein synthesis was substantially reduced in delta atg7 cells under nitrogen starvation compared with wild-type cells. The total intracellular amino acid pool was reduced in delta atg7 cells, and the levels of several amino acids fell below critical values. In contrast, wild-type cells maintained amino acid levels compatible with life. Autophagy-defective cells fail to maintain physiologic amino acid levels, and their inability to synthesize new proteins may explain most phenotypes associated with autophagy mutants at least partly.     

自体吞噬在植物生长发育中的功能

自体吞噬是一种细胞内自我降解系统,它能将植物细胞内溶物运输至液泡并降解.自体吞噬可划分为内溶物的包裹、运输至液泡、内溶物的降解和降解产物的重新利用几个连续步骤.关于细胞自体吞噬的认识主要来源于酵母、人类、小鼠、果蝇和线虫等生物,以拟南芥等为代表的植物细胞自体吞噬的研究虽然刚刚开始,但也取得了一些标志性的成果,且近十几年来已迅速成为植物研究领域的热点之一.自体吞噬在植物体内具有多种生理和病理作用,如对饥饿的适应、细胞内蛋白质和细胞器的清除、种子中贮藏蛋白的积累、抵制微生物、细胞死亡和胁迫响应等.本文在介绍自体吞噬形成过程的基础上,着重探讨了自体吞噬在植物生长发育中的功能,并对植物中自体吞噬的研究方向进行了展望.     

Physiological role of autophagy as an intracellular recycling system: With an emphasis on nutrient metabolism

Autophagy is a major intracellular degradation system in which the cytoplasmic contents are degraded in the lysosome. Its fundamental and evolutionarily conserved role is adaptation to starvation. Recent studies using autophagy-defective mutants of various organisms including mammals have indeed demonstrated the importance of autophagy during starvation; however, the exact mechanism underlying this beneficial effect remains unclear. In addition, it is now apparent that autophagy is also important for cellular homeostasis even under non-starvation conditions, and both non-selective and selective types of autophagy appear to be critical for this function. Here, we discuss the role of this catabolic pathway in recycling intracellular components, with particular reference to nutrient metabolism.     

Carotenoid deficiency triggers autophagy in the model green alga Chlamydomonas reinhardtii

All aerobic organisms have developed sophisticated mechanisms to prevent, detect and respond to cell damage caused by the unavoidable production of reactive oxygen species (ROS). Plants and algae are able to synthesize specific pigments in the chloroplast called carotenoids to prevent photo-oxidative damage caused by highly reactive by-products of photosynthesis. In this study we used the unicellular green alga Chlamydomonas reinhardtii to demonstrate that defects in carotenoid biosynthesis lead to the activation of autophagy, a membrane-trafficking process that participates in the recycling and degradation of damaged or toxic cellular components. Carotenoid depletion caused by either the mutation of phytoene synthase or the inhibition of phytoene desaturase by the herbicide norflurazon, resulted in a strong induction of autophagy. We found that high light transiently activates autophagy in wild-type Chlamydomonas cells as part of an adaptation response to this stress. Our results showed that a Chlamydomonas mutant defective in the synthesis of specific carotenoids that accumulate during high light stress exhibits constitutive autophagy. Moreover, inhibition of the ROS-generating NADPH oxidase partially reduced the autophagy induction associated to carotenoid deficiency, which revealed a link between photo-oxidative damage, ROS accumulation and autophagy activation in Chlamydomonas cells with a reduced carotenoid content.     

Autophagosomes are formed at a distinct cellular structure

Autophagy is characterized by the formation of double-membrane vesicles called autophagosomes, which deliver bulk cytoplasmic material to the lytic compartment of the cell for degradation. Autophagosome formation is initiated by assembly and recruitment of the core autophagy machinery to distinct cellular sites, referred to as phagophore assembly sites (PAS) in yeast or autophagosome formation sites in other organisms. A large number of autophagy proteins involved in the formation of autophagosomes has been identified; however, how the individual components of the PAS are assembled and how they function to generate autophagosomes remains a fundamental question. Here, we highlight recent studies that provide molecular insights into PAS organization and the role of the endoplasmic reticulum and the vacuole in autophagosome formation.     

Liver autophagy: physiology and pathology

Autophagy has long been thought of as a bulk degradation system in which cytoplasmic components are sequestered by double-membrane structures called autophagosomes, and the contents are then degraded after autophagosomes fuse with lysosomes. Genetic experiments in yeast identified a set of Autophagy-related (ATG) genes that are essential for autophagy. We have since elucidated many of the molecular underpinnings of autophagy and the physiologic roles of these processes in various systems. This review summarizes the physiologic roles of autophagy with a particular focus on liver autophagy based on analyses of knockout mice lacking Atg genes.