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.     

A novel mammalian trans-membrane protein reveals an alternative initiation pathway for autophagy

Autophagy is an early cellular event during acute pancreatitis, a disease defined as pancreas self-digestion. The Vacuole Membrane Protein 1 (VMP1) is a trans-membrane protein highly activated in acinar cells early during pancreatitis-induced autophagy and it remains in the autophagosomal membrane. We have shown that VMP1 expression is able to trigger autophagy in mammalian cells, even under nutrient-replete conditions. VMP1 is induced by autophagy stimuli and its expression is required for autophagosome development. VMP1 interacts with Beclin 1 through its hydrophilic C-terminal region, which we named Atg domain, as it is essential for autophagy. Remarkably, VMP1 pancreas-specific transgenic expression in mice promotes autophagosome formation. Most of the autophagy-related proteins were described in yeast or have a yeast homologue. VMP1 does not have any known homologue in yeast but its expression is required to start the autophagic process in mammalian cells. These findings support the hypothesis that mammalian cells may regulate autophagy in a different way. We propose that VMP1 is a novel autophagy related trans-membrane protein, which may lead the way in the search for alternative mechanisms of autophagosome formation.     

HMGB1: a novel Beclin 1-binding protein active in autophagy

The autophagosome delivers damaged cytoplasmic constituents and proteins to the lysosome or to the extracellular space. Beclin 1, an essential: autophagic protein, is a BH3-only protein that binds Bcl-2 anti-apoptotic family members and has a critical role in the initiation of autophagy. How the Beclin 1 complex specifically promotes autophagy remains largely unknown. We have found that high mobility group box 1 (HMGB1), a chromatin-associated nuclear protein and extracellular damage associated molecular pattern molecule (DAMP), is a novel Beclin 1-binding protein important in sustaining autophagy. HMGB1 shares considerable sequence homology with Beclin 1 in yeast, mice and human, representing an evolutionarily conserved regulatory step in early autophagosome formation. Endogenous HMGB1 competes with Bcl-2 for interaction with Beclin 1, and orients Beclin 1 to autophagosomes. Moreover, the intramolecular disulfide bridge (C23/45) of HMGB1 is required for binding to Beclin 1 and sustaining autophagy. Taken together, these findings indicate that endogenous HMGB1 functions as an autophagy effector by regulation of autophagosome formation.     

HMGB1: A novel Beclin 1-binding protein active in autophagy

The autophagosome delivers damaged cytoplasmic constituents and proteins to the lysosome or to the extracellular space. Beclin 1, an essential

autophagic protein, is a BH3-only protein that binds Bcl-2 anti-apoptotic family members and has a critical role in the initiation of autophagy. How the Beclin 1 complex specifically promotes autophagy remains largely unknown. We have found that high mobility group box 1 (HMGB1), a chromatin-associated nuclear protein and extracellular damage associated molecular pattern molecule (DAMP), is a novel Beclin 1-binding protein important in sustaining autophagy. HMGB1 shares considerable sequence homology with Beclin 1 in yeast, mice and human, representing an evolutionarily conserved regulatory step in early autophagosome formation. Endogenous HMGB1 competes with Bcl-2 for interaction with Beclin 1, and orients Beclin 1 to autophagosomes. Moreover, the intramolecular disulfide bridge (C23/45) of HMGB1 is required for binding to Beclin 1 and sustaining autophagy. Taken together, these findings indicate that endogenous HMGB1 functions as an autophagy effector by regulation of autophagosome formation.     

Hrr25: An emerging major player in selective autophagy regulation in Saccharomyces cerevisiae

As with the case of the mechanism of autophagosome formation, studies in yeast have taken a leading role in elucidating the molecular basis of target recognition during selective autophagy. Degradation targets are recognized by receptor proteins, which also bind to Atg8 homologs on growing phagophore membranes, leading to the loading of the targets into autophagosomes. However, it remains to be elucidated how these processes are regulated. In yeast, receptors also interact with the scaffold/adaptor protein Atg11, which subsequently recruits core Atg proteins onto receptor-target complexes to initiate autophagosome formation. Recently, we found that Hrr25, a homolog of CSNK1D/casein kinase 1δ, regulates 3 of 4 selective autophagy-related pathways in the budding yeast Saccharomyces cerevisiae by a uniform mechanism: phosphoregulation of the receptor-scaffold interaction.     

Atg27 is required for autophagy-dependent cycling of Atg9

Autophagy is a catabolic pathway for the degradation of cytosolic proteins or organelles and is conserved among all eukaryotic cells. The hallmark of autophagy is the formation of double-membrane cytosolic vesicles, termed autophagosomes, which sequester cytoplasm; however, the mechanism of vesicle formation and the membrane source remain unclear. In the yeast Saccharomyces cerevisiae, selective autophagy mediates the delivery of specific cargos to the vacuole, the analog of the mammalian lysosome. The transmembrane protein Atg9 cycles between the mitochondria and the pre-autophagosomal structure, which is the site of autophagosome biogenesis. Atg9 is thought to mediate the delivery of membrane to the forming autophagosome. Here, we characterize a second transmembrane protein Atg27 that is required for specific autophagy in yeast. Atg27 is required for Atg9 cycling and shuttles between the pre-autophagosomal structure, mitochondria, and the Golgi complex. These data support a hypothesis that multiple membrane sources supply the lipids needed for autophagosome formation.     

The Atg17-Atg31-Atg29 complex and Atg11 regulate autophagosome-vacuole fusion

The macroautophagy (hereafter autophagy) process involves de novo formation of double-membrane autophagosomes; after sequestering cytoplasm these transient organelles fuse with the vacuole/lysosome. Genetic studies in yeasts have characterized more than 40 autophagy-related (Atg) proteins required for autophagy, and the majority of these proteins play roles in autophagosome formation. The fusion of autophagosomes with the vacuole is mediated by the Rab GTPase Ypt7, its guanine nucleotide exchange factor Mon1-Ccz1, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. However, these factors are not autophagosome-vacuole fusion specific. We recently showed that 2 autophagy scaffold proteins, the Atg17-Atg31-Atg29 complex and Atg11, regulate autophagosome-vacuole fusion by recruiting the vacuolar SNARE Vam7 to the phagophore assembly site (PAS), where an autophagosome forms in yeast.     

Autophagosome Requires Specific Early Sec Proteins for Its Formation and NSF/SNARE for Vacuolar Fusion

Double membrane structure, autophagosome, is formed de novo in the process of autophagy in the yeast Saccharomyces cerevisiae, and many Apg proteins participate in this process. To further understand autophagy, we analyzed the involvement of factors engaged in the secretory pathway. First, we showed that Sec18p (N-ethylmaleimide-sensitive fusion protein, NSF) and Vti1p (soluble N-ethylmaleimide-sensitive fusion protein attachment protein, SNARE), and soluble N-ethylmaleimide-sensitive fusion protein receptor are required for fusion of the autophagosome to the vacuole but are not involved in autophagosome formation. Second, Sec12p was shown to be essential for autophagy but not for the cytoplasm to vacuole-targeting (Cvt) (pathway, which shares mostly the same machinery with autophagy. Subcellular fractionation and electron microscopic analyses showed that Cvt vesicles, but not autophagosomes, can be formed in sec12 cells. Three other coatmer protein (COPII) mutants, sec16, sec23, and sec24, were also defective in autophagy. The blockage of autophagy in these mutants was not dependent on transport from endoplasmic reticulum-to-Golgi, because mutations in two other COPII genes, SEC13 and SEC31, did not affect autophagy. These results demonstrate the requirement for subgroup of COPII proteins in autophagy. This evidence demonstrating the involvement of Sec proteins in the mechanism of autophagosome formation is crucial for understanding membrane flow during the process.     

Membrane Delivery to the Yeast Autophagosome from the Golgi�CEndosomal System

While many of the proteins required for autophagy have been identified, the source of the membrane of the autophagosome is still unresolved with the endoplasmic reticulum (ER), endosomes, and mitochondria all having been evoked. The integral membrane protein Atg9 is delivered to the autophagosome during starvation and in the related cytoplasm-to-vacuole (Cvt) pathway that occurs constitutively in yeast. We have examined the requirements for delivery of Atg9-containing membrane to the yeast autophagosome. Atg9 does not appear to originate from mitochondria, and Atg9 cannot reach the forming autophagosome directly from the ER or early Golgi. Components of traffic between Golgi and endosomes are known to be required for the Cvt pathway but do not appear required for autophagy in starved cells. However, we find that pairwise combinations of mutations in Golgi-endosomal traffic components apparently only required for the Cvt pathway can cause profound defects in Atg9 delivery and autophagy in starved cells. Thus it appears that membrane that contains Atg9 is delivered to the autophagosome from the Golgi-endosomal system rather than from the ER or mitochondria. This is underestimated by examination of single mutants, providing a possible explanation for discrepancies between yeast and mammalian studies on Atg9 localization and autophagosome formation.     

Cis1/Atg31 is required for autophagosome formation in Saccharomyces cerevisiae

Autophagy is the bulk degradation of cytosolic materials in lysosomes/vacuoles of eukaryotic cells. In the yeast Saccharomyces cerevisiae, 17 Atg proteins are known to be involved in autophagosome formation. Genome wide analyses have shown that Atg17 interacts with numerous proteins. Further studies on these interacting proteins may provide further insights into membrane dynamics during autophagy. Here, we identify Cis1/Atg31 as a protein that exhibits similar phenotypes to Atg17. ATG31 null cells were defective in autophagy and lost viability under starvation conditions. Localization of Atg31 to pre-autophagosomal structures (PAS) was dependent on Atg17. Coimmunoprecipitation experiments indicated that Atg31 interacts with Atg17. Together, Atg31 is a novel protein that, in concert with Atg17, is required for proper autophagosome formation.     

Mammalian Atg8s: One is simply not enough

Yeast Atg8, a key factor in the autophagic process, is a ubiquitin-like protein that undergoes a unique conjugation to phosphatidylethanolamine (PE). Atg8 plays a dual role in early stages of autophagosome formation: It was implicated in recruitment of cargo proteins such as Atg19 and Atg32 for Cvt and mitophagy, respectively, and in autophagosome biogenesis, serving as an elongation factor by mediating membrane hemi-fusion. Similarly, the mammalian Atg8 proteins, LC3s and GABARAPs, recruit cargo into autophagosomes by binding to adaptor proteins such as p62, NBR1 and Nix. These functions, however, are not essential for bulk autophagic flux. Other studies in which the activity of the mammalian Atg8s was blocked either by knockout of the E2-like enzyme Atg3 or by using a dominant negative mutant of the promiscuous protease Atg4B revealed, in agreement with the yeast Atg8 data, that the mammalian factors are crucial for the formation of normal and mature autophagosomes. While it seems that the single yeast Atg8 and the mammalian Atg8s share similar roles, it is still unclear why the mammalian system employs several homologs. Recent publications demonstrated that the mammalian Atg8s differ in their cargo specificity, as Nix, for example, binds exclusively to GABARAP-L1. This may suggest that these proteins exhibit distinct activity also in autophagosome biogenesis. In our study we divided the mammalian Atg8s into two subfamilies of homologs based on amino acid similarity, the LC3 and GABARAP/GATE-16 subfamilies, and tested their essentiality and role in autophagy. In agreement with previous studies we found that the mammalian Atg8s are essential for autophagy but, more importantly, that each of these subfamilies has a distinct role in the process of autophagosome biogenesis.     

Open and closed HORMAs regulate autophagy initiation

The Atg1/ULK complex functions as the most upstream factor among Atg proteins to initiate autophagy. ATG101 is a constitutive component of the Atg1/ULK complex in most eukaryotes except for budding yeast, and plays an essential role in autophagy; however, the structure and functions of ATG101 were largely unknown. Recently, we determined the crystal structure of fission yeast Atg101 in complex with the closed HORMA domain of Atg13, revealing that Atg101 is also a HORMA protein with an open conformation. These 2 HORMA proteins play essential roles in autophagy initiation through recruiting downstream factors to the autophagosome formation site.     

The Autophagy-related Protein Kinase Atg1 Interacts with the Ubiquitin-like Protein Atg8 via the Atg8 Family Interacting Motif to Facilitate Autophagosome Formation

In autophagy, a cup-shaped membrane called the isolation membrane is formed, expanded, and sealed to complete a double membrane-bound vesicle called the autophagosome that encapsulates cellular constituents to be transported to and degraded in the lysosome/vacuole. The formation of the autophagosome requires autophagy-related (Atg) proteins. Atg8 is a ubiquitin-like protein that localizes to the isolation membrane; a subpopulation of this protein remains inside the autophagosome and is transported to the lysosome/vacuole. In the budding yeast Saccharomyces cerevisiae, Atg1 is a serine/threonine kinase that functions in the initial step of autophagosome formation and is also efficiently transported to the vacuole via autophagy. Here, we explore the mechanism and significance of this autophagic transport of Atg1. In selective types of autophagy, receptor proteins recognize degradation targets and also interact with Atg8, via the Atg8 family interacting motif (AIM), to link the targets to the isolation membrane. We find that Atg1 contains an AIM and directly interacts with Atg8. Mutations in the AIM disrupt this interaction and abolish vacuolar transport of Atg1. These results suggest that Atg1 associates with the isolation membrane by binding to Atg8, resulting in its incorporation into the autophagosome. We also show that mutations in the Atg1 AIM cause a significant defect in autophagy, without affecting the functions of Atg1 implicated in triggering autophagosome formation. We propose that in addition to its essential function in the initial stage, Atg1 also associates with the isolation membrane to promote its maturation into the autophagosome.     

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.     

Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis

Autophagy is an evolutionarily conserved 'self-eating' process. Although the genes essential for autophagy (named Atg) have been identified in yeast, the molecular mechanism of how Atg proteins control autophagosome formation in mammalian cells remains to be elucidated. Here, we demonstrate that Bif-1 (also known as Endophilin B1) interacts with Beclin 1 through ultraviolet irradiation resistance-associated gene (UVRAG) and functions as a positive mediator of the class III PI(3) kinase (PI(3)KC3). In response to nutrient deprivation, Bif-1 localizes to autophagosomes where it colocalizes with Atg5, as well as microtubule-associated protein light chain 3 (LC3). Furthermore, loss of Bif-1 suppresses autophagosome formation. Although the SH3 domain of Bif-1 is sufficient for binding to UVRAG, both the BAR and SH3 domains are required for Bif-1 to activate PI(3)KC3 and induce autophagosome formation. We also observed that Bif-1 ablation prolongs cell survival under starvation conditions. Moreover, knockout of Bif-1 significantly enhances the development of spontaneous tumours in mice. These findings suggest that Bif-1 joins the UVRAG-Beclin 1 complex as a potential activator of autophagy and tumour suppressor.     

Molecular machinery of autophagosome formation in yeast, Saccharomyces cerevisiae

Autophagy is a degradation process accompanied by dynamic membrane organization. In the yeast, Saccharomyces cerevisiae, about 30 ATG (autophagy-related) genes have been identified as important genes for autophagy. Among them, 17 are indispensable for formation of the autophagosome, an organelle enclosed by a double lipid bilayer during starvation-induced autophagy. Recently, a central structure for autophagosome generation, termed the pre-autophagosomal structure, was identified. Despite intensive study, many questions regarding the mechanisms underlying autophagosome formation remain unanswered. In this review, we will give an overview of recent studies on the mechanisms of autophagosome formation and discuss these unresolved questions.     

The coiled-coil domain protein EPG-8 plays an essential role in the autophagy pathway in C. elegans

Macroautophagy (hereafter referred to as autophagy) involves the formation of a closed, double membrane structure, called the autophagosome. Most of the Atg proteins that are essential for autophagosome formation are evolutionarily conserved between yeast and higher eukaryotes. The functions of some Atg proteins, however, are mediated by highly divergent proteins in mammalian cells. In this study, we identified a novel coiled-coil domain protein, EPG-8, that plays an essential role in the autophagy pathway in C. elegans. Mutations in epg-8 cause defects in degradation of various autophagy substrates and also compromise survival of animals under nutrient-depletion conditions. In epg-8 mutants, lipidated LGG-1 (the C. elegans Atg8 homolog) accumulates but does not form distinct punctate structures. EPG-8 directly interacts with the C. elegans Beclin 1 homolog, BEC-1. Our study demonstrates that epg-8 may function as a highly divergent homolog of the yeast autophagy gene Atg14.     

Regulation of macroautophagy by mTOR and Beclin 1 complexes

Macroautophagy or autophagy is a vacuolar degradative pathway terminating in the lysosomal compartment after forming a cytoplasmic vacuole or autophagosome that engulfs macromolecules and organelles. The original discovery that ATG (AuTophaGy related) genes in yeast are involved in the formation of autophagosomes has greatly increased our knowledge of the molecular basis of autophagy, and its role in cell function that extends far beyond non-selective degradation. The regulation of autophagy by signaling pathways overlaps the control of cell growth, proliferation, cell survival and death. The evolutionarily conserved TOR (Target of Rapamycin) kinase complex 1 plays an important role upstream of the Atg1 complex in the control of autophagy by growth factors, nutrients, calcium signaling and in response to stress situations, including hypoxia, oxidative stress and low energy. The Beclin 1 (Atg6) complex, which is involved in the initial step of autophagosome formation, is directly targeted by signaling pathways. Taken together, these data suggest that multiple signaling checkpoints are involved in regulating autophagosome formation.     

A landmark protein essential for mitophagy

Degradation of mitochondria is a fundamental process conserved from yeast to humans that utilizes the machinery of autophagy. In contrast to starvation-induced, nonselective autophagy responsible for nutrient recycling, selective autophagy, which involves particular cues and receptors required for induction and cargo recognition, respectively, mediates mitochondria-specific breakdown. Although numerous studies highlight that mitochondria autophagy (mitophagy) contributes to homeostatic control of mitochondria, the molecular mechanisms underlying this selective clearance process are poorly understood. Using a genome-wide visual screen, we identified Atg32, a protein essential for mitophagy in budding yeast. During respiratory growth, Atg32 is highly expressed, likely in response to oxidative stress, and anchored on the surface of mitochondria. We also demonstrate that Atg32 interacts with Atg8 and Atg11, proteins critical for recognition of cargo receptors. Notably, Atg32 contains WXXI/L/V, a conserved motif that serves as a binding site for the Atg8 family members. Our recent findings suggest that Atg32 is a transmembrane receptor that directs autophagosome formation to mitochondria.