Autophagosome biogenesis requires SNAREs

We recently showed that phagophore biogenesis requires SNAREs. Our data indicate that the exocytic Q/t-SNAREs Sso1/2 and Sec9 are required for one of the earliest steps in autophagosome biogenesis, the homotypic fusion of Atg9-containing vesicles. We propose that this step precedes the formation of Atg9-containing tubulovesicular clusters (TVCs) that is a key step in perivacuolar, phagophore assembly. We also found that the endosomal Q/t-SNARE Tlg2 and the R/v-SNAREs Sec22 and Ykt6 interact with Sso1-Sec9, and are required for normal Atg9 trafficking. Thus, autophagosome biogenesis appears to involve multiple SNARE-mediated fusion events. These findings provide novel insights into the mechanism of autophagosome construction.     

The yeast autophagy protease Atg4 is regulated by thioredoxin

Autophagy is a membrane-trafficking process whereby double-membrane vesicles called autophagosomes engulf and deliver intracellular material to the vacuole for degradation. Atg4 is a cysteine protease with an essential function in autophagosome formation. Mounting evidence suggests that reactive oxygen species may play a role in the control of autophagy and could regulate Atg4 activity but the precise mechanisms remain unclear. In this study, we showed that reactive oxygen species activate autophagy in the model yeast Saccharomyces cerevisiae and unraveled the molecular mechanism by which redox balance controls Atg4 activity. A combination of biochemical assays, redox titrations, and site-directed mutagenesis revealed that Atg4 is regulated by oxidoreduction of a single disulfide bond between Cys338 and Cys394. This disulfide has a low redox potential and is very efficiently reduced by thioredoxin, suggesting that this oxidoreductase plays an important role in Atg4 regulation. Accordingly, we found that autophagy activation by rapamycin was more pronounced in a thioredoxin mutant compared with wild-type cells. Moreover, in vivo studies indicated that Cys338 and Cys394 are required for the proper regulation of autophagosome biogenesis, since mutation of these cysteines resulted in increased recruitment of Atg8 to the phagophore assembly site. Thus, we propose that the fine-tuning of Atg4 activity depending on the intracellular redox state may regulate autophagosome formation.     

The yeast autophagy protease Atg4 is regulated by thioredoxin

Autophagy is a membrane-trafficking process whereby double-membrane vesicles called autophagosomes engulf and deliver intracellular material to the vacuole for degradation. Atg4 is a cysteine protease with an essential function in autophagosome formation. Mounting evidence suggests that reactive oxygen species may play a role in the control of autophagy and could regulate Atg4 activity but the precise mechanisms remain unclear. In this study, we showed that reactive oxygen species activate autophagy in the model yeast Saccharomyces cerevisiae and unraveled the molecular mechanism by which redox balance controls Atg4 activity. A combination of biochemical assays, redox titrations, and site-directed mutagenesis revealed that Atg4 is regulated by oxidoreduction of a single disulfide bond between Cys338 and Cys394. This disulfide has a low redox potential and is very efficiently reduced by thioredoxin, suggesting that this oxidoreductase plays an important role in Atg4 regulation. Accordingly, we found that autophagy activation by rapamycin was more pronounced in a thioredoxin mutant compared with wild-type cells. Moreover, in vivo studies indicated that Cys338 and Cys394 are required for the proper regulation of autophagosome biogenesis, since mutation of these cysteines resulted in increased recruitment of Atg8 to the phagophore assembly site. Thus, we propose that the fine-tuning of Atg4 activity depending on the intracellular redox state may regulate autophagosome formation.     

Autophagosome biogenesis in plants: Roles of SH3P2

The autophagy core machinery is essentially conserved in eukaryotic cells for autophagy regulation. However, the underlying mechanisms for autophagosome formation in plant cells remain elusive. We have recently demonstrated that SH3 domain-containing protein 2 (SH3P2), a BAR (Bin-Amphiphysin-Rvs) domain protein, functions as a novel regulator for autophagosome biogenesis in Arabidopsis thaliana. Using SH3P2 and its GFP fusion as probes, we have characterized the dynamics and structures of autophagosome formation in plant cells. The phagophore assembly site, marked by SH3P2, is identified as having a close connection with the ER. SH3P2 also binds to phosphatidylinositol 3-phosphate (PtdIns3P) and functions downstream of the phosphatidylinositol 3-kinase (PtdIns3K) complex. Thus, SH3P2 serves as a novel membrane-associated protein in regulating autophagosome formation in Arabidopsis thaliana.     

Autophagosome closure requires membrane scission

During the intracellular process of macroautophagy (hereafter autophagy), a membrane-bound organelle, the autophagosome, is generated de novo. The remodeling of the autophagic membrane during the life cycle of the organelle is a complex multistep process and involves several changes in the topology of the autophagic membrane. Here, we focus on the final step of autophagosome formation, the closure of the phagophore, during which the inner and outer autophagic membranes become separate entities. We argue that this topological membrane transformation is a membrane scission event. Surprisingly, not a single recent review describes this substep as membrane scission (or membrane fission). In contrast, a number of publications imply that membrane fusion is involved. We discuss the potential sources for misinterpretation and recommend to consistent use of the unambiguous term “membrane scission.”     

GFP-Atg8 protease protection as a tool to monitor autophagosome biogenesis

Perhaps the most complex step of macroautophagy is the formation of the double-membrane autophagosome. The majority of the autophagy-related (Atg) proteins are thought to participate in nucleation and expansion of the phagophore, and/or the completion of this compartment. Monitoring this part of the process is difficult, and typically involves electron microscopy analysis; however, unless three-dimensional tomography is performed, even this method cannot be used to easily determine if the phagophore is completely enclosed. Accordingly, a complementary approach is to examine the accessibility of sequestered cargo to exogenously added protease. This type of protease protection analysis has been used to monitor the formation of cytoplasm-to-vacuole targeting (Cvt) vesicles and autophagosomes by examining the protease sensitivity of precursor aminopeptidase I (prApe1). For determining the status of autophagosomes formed during nonselective autophagy, however, prApe1 is not the best marker protein. Here, we describe an alternative method for examining autophagosome completion using GFP-Atg8 as a marker for protease protection.     

GFP-Atg8 protease protection as a tool to monitor autophagosome biogenesis

Perhaps the most complex step of macroautophagy is the formation of the double-membrane autophagosome. The majority of the autophagy-related (Atg) proteins are thought to participate in nucleation and expansion of the phagophore, and/or the completion of this compartment. Monitoring this part of the process is difficult, and typically involves electron microscopy analysis; however, unless three-dimensional tomography is performed, even this method cannot be used to easily determine if the phagophore is completely enclosed. Accordingly, a complementary approach is to examine the accessibility of sequestered cargo to exogenously added protease. This type of protease protection analysis has been used to monitor the formation of cytoplasm-to-vacuole targeting (Cvt) vesicles and autophagosomes by examining the protease sensitivity of precursor aminopeptidase I (prApe1). For determining the status of autophagosomes formed during nonselective autophagy, however, prApe1 is not the best marker protein. Here, we describe an alternative method for examining autophagosome completion using GFP-Atg8 as a marker for protease protection.     

The machinery of macroautophagy

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.     

Proteinase protection of prApe1 as a tool to monitor Cvt vesicle/autophagosome biogenesis

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.     

A current perspective of autophagosome biogenesis

Autophagy is a bulk degradation system induced by cellular stresses such as nutrient starvation. Its function relies on the formation of double-membrane vesicles called autophagosomes. Unlike other organelles that appear to stably exist in the cell, autophagosomes are formed on demand, and once their formation is initiated, it proceeds surprisingly rapidly. How and where this dynamic autophagosome formation takes place has been a long-standing question, but the discovery of Atg proteins in the 1990''s significantly accelerated our understanding of autophagosome biogenesis. In this review, we will briefly introduce each Atg functional unit in relation to autophagosome biogenesis, and then discuss the origin of the autophagosomal membrane with an introduction to selected recent studies addressing this problem.     

The progression of peroxisomal degradation through autophagy requires peroxisomal division

Peroxisomes are highly dynamic organelles that have multiple functions in cellular metabolism. To adapt the intracellular conditions to the changing extracellular environment, peroxisomes undergo constitutive segregation and degradation. The segregation of peroxisomes is mediated by 2 dynamin-related GTPases, Dnm1 and Vps1, whereas, the degradation of peroxisomes is accomplished through pexophagy, a selective type of autophagy. During pexophagy, the size of the organelle is always a challenging factor for the efficiency of engulfment by the sequestering compartment, the phagophore, which implies a potential role for peroxisomal fission in the degradation process, similar to the situation with selective mitochondria degradation. In this study, we report that peroxisomal fission is indeed critical for the efficient elimination of the organelle. When pexophagy is induced, both Dnm1 and Vps1 are recruited to the degrading peroxisomes through interactions with Atg11 and Atg36. In addition, we found that specific peroxisomal fission, which is only needed for pexophagy, occurs at mitochondria-peroxisome contact sites.