Significance: Autophagy is a highly conserved eukaryotic cellular recycling process. Through the degradation of cytoplasmic organelles, proteins, and macromolecules, and the recycling of the breakdown products, autophagy plays important roles in cell survival and maintenance. Accordingly, dysfunction of this process contributes to the pathologies of many human diseases. Recent Advances: Extensive research is currently being done to better understand the process of autophagy. In this review, we describe current knowledge of the morphology, molecular mechanism, and regulation of mammalian autophagy. Critical Issues: At the mechanistic and regulatory levels, there are still many unanswered questions and points of confusion that have yet to be resolved. Future Directions: Through further research, a more complete and accurate picture of the molecular mechanism and regulation of autophagy will not only strengthen our understanding of this significant cellular process, but will aid in the development of new treatments for human diseases in which autophagy is not functioning properly. Antioxid. Redox Signal. 20, 460–473.
Macroautophagy (hereafter autophagy) initiates at the phagophore assembly site (PAS), where most of the AuTophaGy-related (Atg) proteins are at least transiently localized. As the first protein complex targeted to the PAS, the Atg17-Atg31-Atg29 complex serves as the scaffold for other Atg proteins and plays a critical role for the organization of the PAS, and in autophagy initiation. We recently showed that this complex is constitutively formed and activated by the phosphorylation of Atg29 when autophagy is induced. Phosphorylation of Atg29 is required for its interaction with Atg11, another scaffold protein, and its function for promoting the proper assembly of the PAS. Single-particle electron microscopy analysis of the Atg17-Atg31-Atg29 complex reveals an elongated structure with Atg29 located at the opposing ends. This structural arrangement allows Atg29 to interact with Atg11, and is critical in the organization of the intact Atg1 complex.
autophagy; PAS; scaffold; vacuole; yeast
Mitophagy, the autophagic removal of mitochondria, occurs through a highly selective mechanism. In the yeast Saccharomyces cerevisiae, the mitochondrial outer membrane protein Atg32 confers selectivity for mitochondria sequestration as a cargo by the autophagic machinery through its interaction with Atg11, a scaffold protein for selective types of autophagy. The activity of mitophagy in vivo must be tightly regulated considering that mitochondria are essential organelles that produce most of the cellular energy, but also generate reactive oxygen species that can be harmful to cell physiology. We found that Atg32 was proteolytically processed at its C terminus upon mitophagy induction. Adding an epitope tag to the C terminus of Atg32 interfered with its processing and caused a mitophagy defect, suggesting the processing is required for efficient mitophagy. Furthermore, we determined that the mitochondrial i-AAA protease Yme1 mediated Atg32 processing and was required for mitophagy. Finally, we found that the interaction between Atg32 and Atg11 was significantly weakened in yme1∆ cells. We propose that the processing of Atg32 by Yme1 acts as an important regulatory mechanism of cellular mitophagy activity.
mitochondrial protease; mitophagy; starvation; vacuole; yeast
As a highly dynamic organelle, mitochondria undergo constitutive fusion and fission as well as biogenesis and degradation. Mitophagy, selective mitochondrial degradation through autophagy, is a conserved cellular process used for the elimination of excessive and damaged mitochondria in eukaryotes. Despite the significance of mitophagy in cellular physiology and pathophysiologies, the underlying mechanism of this process is far from clear. In this report, we studied the role of mitochondrial fission during mitophagy, and uncover a direct link between the fission complex and mitophagy machinery in Saccharomyces cerevisiae.
mitophagy; phagophore; stress; vacuole; yeast
Science informs art, and art informs science. Both processes involve creativity and imagination, and collaboration between scientists and artists often leads to new insights in both fields. We took advantage of the power of artistic imagery to demonstrate a dynamic cellular process, autophagy. In particular, we depicted the cytoplasm to vacuole targeting pathway, which involves dynamic membrane rearrangements to sequester a specific cargo via an autophagy-related process. By depicting this event in the context of a crowded cellular milieu, we hoped to stimulate researchers to consider aspects of the process that might be overlooked in the overly simplistic schematic drawing that typify most scientific models.
collaboration; Cvt complex; membrane; molecular model; organelle; science
In the course of my work as Autophagy editor, I try to gauge the overall patterns of interest in autophagy research. Not surprisingly, the number of papers associated with this topic has increased steadily. However, that trend provides only one glimpse into the way interest in this field has been changing—that the number of people working on autophagy has expanded. Perhaps not surprisingly, the number of different research areas that now include autophagy studies is also increasing. Thus, I decided to carry out an informal, imprecise analysis of the number of different journals (presumably reflecting in part the number of topics) that include papers on autophagy.
autophagy; black hole; gravity; lysosome; stress; vacuole
Molecular biology holds the promise not only of increasing our understanding of basic cell biology, but also of advancing our ability to design targeted therapeutic methods for treating a range of diseases. One example that seems to hold tremendous potential is gene therapy, the use of exogenous DNA to replace or suppress a mutant gene in the patient’s genome, or to boost the activity of a normal gene. A recent report (highlighted in a punctum in this issue of the journal) has brought autophagy into the gene therapy realm.
TFEB; lysosome; macroautophagy; α1-antitrypsin deficiency
Macroautophagy mediates recycling of intracellular material by a multistep pathway, ultimately leading to the fusion of closed double-membrane structures, called autophagosomes, with the lysosome. This event ensures the degradation of the autophagosome content by lysosomal proteases followed by the release of macromolecules by permeases and, thus, it accomplishes the purpose of macroautophagy (hereafter referred to as autophagy). Because fusion of unclosed autophagosomes (i.e., phagophores) with the lysosome would fail to degrade the autophagic cargo, this critical step has to be tightly controlled. Yet, until recently, little was known about the regulation of this event and the factors orchestrating it. A punctum in this issue highlights the recent paper by Noboru Mizushima and his collaborators that answered the question of how premature fusion of phagophores with the lysosome is prevented prior to completion of autophagosome closure.
autophagosome; fusion; glycine zipper; lysosome; SNARE
These days, when we talk about the origin of a protein, or even a pathway, we are typically referring to evolutionary lineages based on nucleotide sequences. For example, is a particular protein’s function conserved? How far back did it first appear? Are there homologs in higher eukaryotes? However, a simpler question (or perhaps I should say, a non-molecular biology question) is when was the process first detected in the paleontological record? Of course I assumed that macroautophagy was ancient, but a new finding (see p. 632 in this issue of the journal) provides an unexpected—and exciting—piece of information for our field. For the first time, scientists have discovered fossil evidence for an actual subcellular pathway—and it looks like it might actually be autophagy (I admit I am biased, but you can decide for yourself).
autophagy; fossil; lysosome; stress; vacuole
To tell the truth, I find it difficult to work when flying, or even when sitting in an airport for an extended period of time. So, typically I take along a book to read. And when I truly cannot concentrate, for example when a flight is considerably delayed, I have even been known to resort to word puzzles. Depending on the type, they do not require much attention (that is, you can pick up right where you left off after you glance at the flight status screen for the twentieth or so time, even though you know nothing has changed), or effort (although you need to use a pen or pencil, not a keyboard), but nonetheless they can keep your mind somewhat occupied. I even rationalize doing them based on the assumption that they are sharpening my observational/pattern-finding skills. One type of word puzzle that is particularly mindless, but for that very reason I still enjoy in the above circumstances, is a word search; you are given a grid with letters and/or numbers, and a list of “hidden” terms, and you circle them within the grid, crossing them off the list as you go along. I do admit that the categories of terms used in the typical word searches can become rather mundane (breeds of dog, types of food, words that are followed by “stone,” words associated with a famous movie star, words from a particular television show, etc.). Therefore, on one of my last seminar trips I decided to generate my own word search, using the category of autophagy.
autophagy; lysosome; stress; vacuole; word search
The current working definition of autophagy is the following: all processes in which intracellular material is degraded within the lysosome/vacuole and where the macromolecular constituents are recycled. There are several ways to classify the different types of autophagy. For example, we can separate autophagy into two primary types, based on the initial site of cargo sequestration. In particular, during microautophagy and chaperone-mediated autophagy, uptake occurs directly at the limiting membrane of the lysosome or vacuole. In contrast, macroautophagy—whether selective or nonselective—and endosomal microautophagy involve sequestration within an autophagosome or an omegasome, or late endosomes/multivesicular bodies, respectively; the key point being that in these types of autophagy the initial sequestration event does not occur at the limiting membrane of the degradative organelle. In any case, the cargo is ultimately delivered into the lysosome or vacuole lumen for subsequent degradation. Thus, I think most autophagy researchers view the degradative organelle as the ultimate destination of the pathway. Indeed, this fits with the general concept that organelles allow reactions to be compartmentalized. With regard to the lysosome or vacuole, this also confers a level of safety by keeping the lytic contents away from the remainder of the cell. If we are willing to slightly modify our definition of autophagy, with a focus on “degradation of a cell’s own components through the lysosomal/vacuolar machinery,” we can include a newly documented process, programmed nuclear destruction (PND).
autophagy; lysosome; meiosis; spore; stress; vacuole
The vast majority of studies addressing the induction of autophagy have focused upon cytoplasmic aspects of its regulation. Recently, we have started to expand our knowledge regarding the nuclear events of autophagic induction. Many autophagy-related genes are transcriptionally upregulated upon induction of autophagy, but only in a limited number of cases do we know the pathways leading to this upregulation. Few transcription factors have been implicated in controlling autophagy genes in yeast. However, many of the ATG genes show some level of transcriptional induction upon starvation. Now, we show that transcription of ATG8 is repressed under growing conditions by the Ume6-Sin3-Rpd3 complex.
autophagosome; lysosome; phagophore; stress; vacuole
In autophagic processes a variety of cargos is delivered to the degradative compartment of cells. Recent progress in autophagy research has provided support for the notion that when autophagic processes are operating in selective mode, a receptor protein complex will process the cargo. Here we present a concept of receptor protein complexes as comprising a functional tetrad of components: a ligand, a receptor, a scaffold and an Atg8 family protein. Our current understanding of each of the four components and their interaction in the context of cargo selection are considered in turn.
autophagic cargo; ligand; receptor; scaffold protein; Atg8 family protein; phagophore
fission complex; mitophagy; scaffold; phagophore; vacuole; yeast
autophagosome; lysosome; phagophore; stress; vacuole
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.
autophagosome; autophagy; degradation; lysosome; phagophore; stress; vacuole
Due in part to the increasing number of links between autophagy malfunction and human diseases, this field has gained tremendous attention over the past decade. Our increased understanding of the molecular machinery involved in macroautophagy (hereafter autophagy) seems to indicate that the most complex step, or at least the stage of the process where the majority of the autophagy-related (Atg) proteins participate, is in the formation of the double-membrane sequestering vesicle. Thus, it is important to establish reliable approaches to monitor this specific process. One of the most commonly used methods is morphological analysis by electron microscopy of the cytosolic vesicles used in the cytoplasm-to-vacuole targeting (Cvt) pathway and autophagy, or the single-membrane intralumenal products, termed Cvt or autophagic bodies, that are formed after the fusion of these vesicles with the yeast vacuole. This method, however, can be costly and time consuming, and reliable analysis requires expert input. Furthermore, it is extremely difficult to detect an incomplete autophagosome by electron microscopy because of the difficulty of obtaining a section that randomly cuts through the open portion of the phagophore. The primary Cvt pathway cargo, precursor amminopeptidase I (prApe1), is enwrapped within either a Cvt vesicle or autophagosome depending on the nutritional conditions. The proteolytic sensitivity of the prApe1 propeptide can therefore serve as a useful tool to determine the completion status of double-membrane Cvt vesicles/autophagosomes in the presence of exogenously added proteinase. Here, we describe an assay that examines the proteinase protection of prApe1 for determining the completion of Cvt vesicles/autophagosomes.
autophagy; lysosome; stress; vacuole; yeast
Selective macroautophagy uses double-membrane vesicles, termed autophagosomes, to transport cytoplasmic pathogens, organelles and protein complexes to the vacuole for degradation. Autophagosomes are formed de novo by membrane fusion events at the phagophore assembly site (PAS). Therefore, precursor membrane material must be targeted and transported to the PAS. While some autophagy-related (Atg) proteins, such as Atg9 and Atg11, are known to be involved in this process, most of the mechanistic details are not understood. Previous work has also implicated the small Rab-family GTPase Ypt1 in the process, identifying Trs85 as a unique subunit of the TRAPPIII targeting complex and showing that it plays a macroautophagy-specific role; however, the relationship between Ypt1, Atg9 and Atg11 was not clear. Now, a recent report shows that Atg11 is a Trs85-specific effector of the Rab Ypt1, and may act as a classic coiled-coil membrane tether that targets Atg9-containing membranes to the PAS. Here, we review this finding in the context of what is known about Atg11, other Rab-dependent coiled-coil tethers, and other tethering complexes involved in autophagosome formation.
Atg9; membrane trafficking; stress; Trs85; vacuole; Ypt1
There are various definitions of community. A definition that I found in one of my dictionaries is the following: “A social, religious, occupational, or other group sharing common characteristics or interests and perceived or perceiving itself as distinct in some respect from the larger society within which it exists.” Thus, I think it is fair to say that there is a worldwide autophagy community. That is, there is a group of researchers (our occupation), whose members share an interest in autophagy (our common characteristic), and that group is distinct from the larger society (I do not want to begin describing the many ways this applies). But do we feel like a community, and do we need a community? I suggest that a community is indeed beneficial, and I propose one mechanism for enhancing the development of the autophagy community.
lysosome; methods; people; stress; vacuole
The transparency, external development and simple drug administration of zebrafish embryos makes them a useful model for studying autophagy during embryonic development in vivo. Cloning of zebrafish lc3 and generation of a transgenic GFP-Lc3 fish line provide excellent tools to monitor autophagy in this organism.1 This protocol discusses several convenient autophagy assays in zebrafish, including immunoblotting of Lc3 lipidation, microscopy imaging of GFP-Lc3 and lysosomal staining.
lysosome; protein degradation; protein targeting; stress; vacuole
Autophagy is a rapidly expanding field in the sense that our knowledge about the molecular mechanism and its connections to a wide range of physiological processes has increased substantially in the past decade. Similarly, the vocabulary associated with autophagy has grown concomitantly. This fact makes it difficult for readers, even those who work in the field, to keep up with the ever-expanding terminology associated with the various autophagy-related processes. Accordingly, we have developed a comprehensive glossary of autophagy-related terms that is meant to provide a quick reference for researchers who need a brief reminder of the regulatory effects of transcription factors or chemical agents that induce or inhibit autophagy, the function of the autophagy-related proteins, or the role of accessory machinery or structures that are associated with autophagy.
autophagy; definitions; glossary; lexicon; terms
Formation of the autophagosome is likely the most complex step of macroautophagy, and indeed it is the morphological and functional hallmark of this process; accordingly, it is critical to understand the corresponding molecular mechanism. Atg8 is the only known autophagy-related (Atg) protein required for autophagosome formation that remains associated with the completed sequestering vesicle. Approximately one-fourth of all of the characterized Atg proteins that participate in autophagosome biogenesis affect Atg8, regulating its conjugation to phosphatidylethanolamine (PE), localization to the phagophore assembly site and/or subsequent deconjugation. An unanswered question in the field regards the physiological role of the deconjugation of Atg8–PE. Using an Atg8 mutant that bypasses the initial Atg4-dependent processing, we demonstrate that Atg8 deconjugation is an important step required to facilitate multiple events during macroautophagy. The inability to deconjugate Atg8–PE results in the mislocalization of this protein to the vacuolar membrane. We also show that the deconjugation of Atg8–PE is required for efficient autophagosome biogenesis, the assembly of Atg9-containing tubulovesicular clusters into phagophores/autophagosomes, and for the disassembly of PAS-associated Atg components.
Degradation in the lysosome/vacuole is not the final step of autophagy. In particular, for starvation-induced autophagy it is necessary to release the breakdown products back into the cytosol. However, some researchers ignore this last step and simply refer to the endpoint of autophagy as degradation, or perhaps even cargo delivery. In many cases this is not a serious issue; however, the analysis of autophagy’s role in certain diseases makes clear that this can be a significant error.
autophagy; cholesterol; lipids; lysosome; stress
In the August 2009 issue of Autophagy, I indicated that we were launching a new category of article, Protocols. At that time, I noted that we would ultimately be placing these articles on a new site online. Well, that time has finally arrived (see www.landesbioscience.com/journals/autophagy/protocols/ for links to these papers). Therefore, it seems appropriate for me to briefly distinguish among three types of community-oriented papers, Protocol, Toolbox and Resource.
autophagy; lysosome; methods; stress; vacuole