Cdc48/p97 and Shp1/p47 regulate autophagosome biogenesis in concert with ubiquitin-like Atg8

The molecular details of the biogenesis of double-membraned autophagosomes are poorly understood. We identify the Saccharomyces cerevisiae AAA–adenosine triphosphatase Cdc48 and its substrate-recruiting cofactor Shp1/Ubx1 as novel components needed for autophagosome biogenesis. In mammals, the Cdc48 homologue p97/VCP and the Shp1 homologue p47 mediate Golgi reassembly by extracting an unknown monoubiquitinated fusion regulator from a complex. We find no requirement of ubiquitination or the proteasome system for autophagosome biogenesis but detect interaction of Shp1 with the ubiquitin-fold autophagy protein Atg8. Atg8 coupled to phosphatidylethanolamine (PE) is crucial for autophagosome elongation and, in vitro, mediates tethering and hemifusion. Interaction with Shp1 requires an FK motif within the N-terminal non–ubiquitin-like Atg8 domain. Based on our data, we speculate that autophagosome formation, in contrast to Golgi reassembly, requires a complex in which Atg8 functionally substitutes ubiquitin. This, for the first time, would give a rationale for use of the ubiquitin-like Atg8 during macroautophagy and would explain why Atg8-PE delipidation is necessary for efficient macroautophagy.     

SNARE proteins are required for macroautophagy

Macroautophagy mediates the degradation of long-lived proteins and organelles via the de novo formation of double-membrane autophagosomes that sequester cytoplasm and deliver it to the vacuole/lysosome; however, relatively little is known about autophagosome biogenesis. Atg8, a phosphatidylethanolamine-conjugated protein, was previously proposed to function in autophagosome membrane expansion, based on the observation that it mediates liposome tethering and hemifusion in vitro. We show here that with physiological concentrations of phosphatidylethanolamine, Atg8 does not act as a fusogen. Rather, we provide evidence for the involvement of exocytic Q/t-SNAREs in autophagosome formation, acting in the recruitment of key autophagy components to the site of autophagosome formation, and in regulating the organization of Atg9 into tubulovesicular clusters. Additionally, we 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 transport. Thus, multiple SNARE-mediated fusion events are likely to be involved in autophagosome biogenesis.     

Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion

Autophagy involves de novo formation of double membrane-bound structures called autophagosomes, which engulf material to be degraded in lytic compartments. Atg8 is a ubiquitin-like protein required for this process in Saccharomyces cerevisiae that can be conjugated to the lipid phosphatidylethanolamine by a ubiquitin-like system. Here, we show using an in vitro system that Atg8 mediates the tethering and hemifusion of membranes, which are evoked by the lipidation of the protein and reversibly modulated by the deconjugation enzyme Atg4. Mutational analyses suggest that membrane tethering and hemifusion observed in vitro represent an authentic function of Atg8 in autophagosome formation in vivo. In addition, electron microscopic analyses indicate that these functions of Atg8 are involved in the expansion of autophagosomal membranes. Our results provide further insights into the mechanisms underlying the unique membrane dynamics of autophagy and also indicate the functional versatility of ubiquitin-like proteins.     

Mutation at the cargo-receptor binding site of Atg8 also affects its general autophagy regulation function

Autophagy is a highly conserved degradation pathway for intracellular macromolecules and organelles. Among those characterized autophagy regulators, the ubiquitin-like protein Atg8 is found to be a membrane modifier that both regulates biogenesis of transport vesicles and interacts with the cargo receptor Atg19 for selective autophagic transport of the vacuolar enzyme prApe1 in budding yeast. The role of Atg8 in the enlargement of vesicle membrane during autophagosome biogenesis has been well documented, but how Atg8 coordinates vesicle formation and sorting of selective cargo is largely unknown. Identification of the cargo-receptor binding site of Atg8 would provide information to solve this issue. Here we characterized Atg8 mutants that were defective in interaction with the prApe1 receptor Atg19 and found that the vesicle formation function of these Atg8 mutants was also compromised to different extents. Atg8 mutants with single-residue substitution at the Atg19-binding site were defective in lipid conjugation and/or subcellular localization. Additional Atg8 mutants were found defective in autophagosome formation without affecting their interaction with Atg19, suggesting partially overlapping of the cargo-sorting site and its domains critical for autophagy control. Our observation paves the road for a more comprehensive understanding on how Atg8 coordinates cargo sorting and vesicle formation in selective autophagic pathways.     

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.     

The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy

Two ubiquitin-like molecules, Atg12 and LC3/Atg8, are involved in autophagosome biogenesis. Atg12 is conjugated to Atg5 and forms an ~800-kDa protein complex with Atg16L (referred to as Atg16L complex). LC3/Atg8 is conjugated to phosphatidylethanolamine and is associated with autophagosome formation, perhaps by enabling membrane elongation. Although the Atg16L complex is required for efficient LC3 lipidation, its role is unknown. Here, we show that overexpression of Atg12 or Atg16L inhibits autophagosome formation. Mechanistically, the site of LC3 lipidation is determined by the membrane localization of the Atg16L complex as well as the interaction of Atg12 with Atg3, the E2 enzyme for the LC3 lipidation process. Forced localization of Atg16L to the plasma membrane enabled ectopic LC3 lipidation at that site. We propose that the Atg16L complex is a new type of E3-like enzyme that functions as a scaffold for LC3 lipidation by dynamically localizing to the putative source membranes for autophagosome formation.     

The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. 'Protein modifications: beyond the usual suspects' review series

As a lysosomal/vacuolar degradative pathway that is conserved in eukaryotic organisms, autophagy mediates the turnover of long-lived proteins and excess or aberrant organelles. The main characteristic of autophagy is the formation of a double-membrane vesicle, the autophagosome, which envelops part of the cytoplasm and delivers it to the lysosome/vacuole for breakdown and eventual recycling of the degradation products. Among the approximately 30 autophagy-related (Atg) genes identified so far, there are two ubiquitin-like proteins, Atg12 and Atg8. Analogous to ubiquitination, Atg12 is conjugated to Atg5 by Atg7--an E1-like protein--and Atg10--an E2-like protein. Similarly, Atg7 and Atg3 are the respective E1-like and E2-like proteins that mediate the conjugation of Atg8 to phosphatidylethanolamine. Both Atg12-Atg5 and Atg8 localize to the developing autophagosome. The Atg12-Atg5 conjugate facilitates the lipidation of Atg8 and directs its correct subcellular localization. Atg8-phosphatidylethanolamine is probably a scaffold protein that supports membrane expansion and the amount present correlates with the size of autophagosomes.     

Atg4 recycles inappropriately lipidated Atg8 to promote autophagosome biogenesis

Atg8 is a ubiquitin-like protein required for autophagy in the budding yeast Saccharomyces cerevisiae. A ubiquitin-like system mediates the conjugation of the C terminus of Atg8 to the lipid phosphatidylethanolamine (PE), and this conjugate (Atg8-PE) plays a crucial role in autophagosome formation at the phagophore assembly site/pre-autophagosomal structure (PAS). The cysteine protease Atg4 processes the C terminus of newly synthesized Atg8 and also delipidates Atg8 to release the protein from membranes. While the former is a prerequisite for lipidation of Atg8, the significance of the latter in autophagy has remained unclear. Here, we show that autophagosome formation is significantly retarded in cells deficient for Atg4-mediated delipidation of Atg8. We find that Atg8-PE accumulates on various organelle membranes including the vacuole, the endosome and the ER in these cells, which depletes unlipidated Atg8 and thereby attenuates its localization to the PAS. Our results suggest that the Atg8-PE that accumulates on organelle membranes is erroneously produced by lipidation system components independently of the normal autophagic process. It is also suggested that delipidation of Atg8 by Atg4 on different organelle membranes promotes autophagosome formation. Considered together with other results, we propose that Atg4 acts to compensate for the intrinsic defect in the lipidation system; it recycles Atg8-PE generated on inappropriate membranes to maintain a reservoir of unlipidated Atg8 that is required for autophagosome formation at the PAS.     

Atg4 recycles inappropriately lipidated Atg8 to promote autophagosome biogenesis

Atg8 is a ubiquitin-like protein required for autophagy in the budding yeast Saccharomyces cerevisiae. A ubiquitin-like system mediates the conjugation of the C terminus of Atg8 to the lipid phosphatidylethanolamine (PE), and this conjugate (Atg8–PE) plays a crucial role in autophagosome formation at the phagophore assembly site/pre-autophagosomal structure (PAS). The cysteine protease Atg4 processes the C terminus of newly synthesized Atg8 and also delipidates Atg8 to release the protein from membranes. While the former is a prerequisite for lipidation of Atg8, the significance of the latter in autophagy has remained unclear. Here, we show that autophagosome formation is significantly retarded in cells deficient for Atg4-mediated delipidation of Atg8. We find that Atg8–PE accumulates on various organelle membranes including the vacuole, the endosome and the ER in these cells, which depletes unlipidated Atg8 and thereby attenuates its localization to the PAS. Our results suggest that the Atg8–PE that accumulates on organelle membranes is erroneously produced by lipidation system components independently of the normal autophagic process. It is also suggested that delipidation of Atg8 by Atg4 on different organelle membranes promotes autophagosome formation. Considered together with other results, we propose that Atg4 acts to compensate for the intrinsic defect in the lipidation system; it recycles Atg8–PE generated on inappropriate membranes to maintain a reservoir of unlipidated Atg8 that is required for autophagosome formation at the PAS.     

Atg8 controls phagophore expansion during autophagosome formation

Autophagy is a potent intracellular degradation process with pivotal roles in health and disease. Atg8, a lipid-conjugated ubiquitin-like protein, is required for the formation of autophagosomes, double-membrane vesicles responsible for the delivery of cytoplasmic material to lysosomes. How and when Atg8 functions in this process, however, is not clear. Here we show that Atg8 controls the expansion of the autophagosome precursor, the phagophore, and give the first real-time, observation-based temporal dissection of the autophagosome formation process. We demonstrate that the amount of Atg8 determines the size of autophagosomes. During autophagosome biogenesis, Atg8 forms an expanding structure and later dissociates from the site of vesicle formation. On the basis of the dynamics of Atg8, we present a multistage model of autophagosome formation. This model provides a foundation for future analyses of the functions and dynamics of known autophagy-related proteins and for screening new genes.     

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.     

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.     

Atg8: an autophagy-related ubiquitin-like protein family

Autophagy-related (Atg) proteins are eukaryotic factors participating in various stages of the autophagic process. Thus far 34 Atgs have been identified in yeast, including the key autophagic protein Atg8. The Atg8 gene family encodes ubiquitin-like proteins that share a similar structure consisting of two amino-terminal α helices and a ubiquitin-like core. Atg8 family members are expressed in various tissues, where they participate in multiple cellular processes, such as intracellular membrane trafficking and autophagy. Their role in autophagy has been intensively studied. Atg8 proteins undergo a unique ubiquitin-like conjugation to phosphatidylethanolamine on the autophagic membrane, a process essential for autophagosome formation. Whereas yeast has a single Atg8 gene, many other eukaryotes contain multiple Atg8 orthologs. Atg8 genes of multicellular animals can be divided, by sequence similarities, into three subfamilies: microtubule-associated protein 1 light chain 3 (MAP1LC3 or LC3), γ-aminobutyric acid receptor-associated protein (GABARAP) and Golgi-associated ATPase enhancer of 16 kDa (GATE-16), which are present in sponges, cnidarians (such as sea anemones, corals and hydras) and bilateral animals. Although genes from all three subfamilies are found in vertebrates, some invertebrate lineages have lost the genes from one or two subfamilies. The amino terminus of Atg8 proteins varies between the subfamilies and has a regulatory role in their various functions. Here we discuss the evolution of Atg8 proteins and summarize the current view of their function in intracellular trafficking and autophagy from a structural perspective.     

A role for Atg8–PE deconjugation in autophagosome biogenesis

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.     

A role for Atg8-PE deconjugation in autophagosome biogenesis

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.     

Small but mighty: Atg8s and Rabs in membrane dynamics during autophagy

Autophagy is a degradative pathway during which autophagosomes are formed that enwrap cytosolic material destined for turnover within the lytic compartment. Autophagosome biogenesis requires controlled lipid and membrane rearrangements to allow the formation of an autophagosomal seed and its subsequent elongation into a fully closed and fusion-competent double membrane vesicle. Different membrane remodeling events are required, which are orchestrated by the distinct autophagy machinery. An important player among these autophagy proteins is the small lipid-modifier Atg8. Atg8 proteins facilitate various aspects of autophagosome formation and serve as a binding platform for autophagy factors. Also Rab GTPases have been implicated in autophagosome biogenesis. As Atg8 proteins interact with several Rab GTPase regulators, they provide a possible link between autophagy progression and Rab GTPase activity. Here, we review central aspects in membrane dynamics during autophagosome biogenesis with a focus on Atg8 proteins and selected Rab GTPases.     

Dissecting autophagosome formation: The missing pieces

The formation of autophagosomes is the central part of the macroautophagy pathway. Little is known, however, about how the participants in this process affect the membrane dynamics at the phagophore assembly site (PAS). Recently, we demonstrated that Atg8, a lipid-conjugated ubiquitin-like protein, controls the expansion of the phagophore. In addition, we showed that the autophagosome formation process can be traced and dissected by time-lapse fluorescence microscopy observation of GFP-Atg8. These findings constitute one step further in our understanding of autophagosome formation. Key questions remain open, however, on how the actions of other proteins at the PAS are coordinated with that of Atg8 and on the precise role of Atg8.

Addendum to: Xie Z, Nair U, Klionsky DJ. Atg8 controls phagophore expansion during autophagosome formation. Mol Biol Cell 2008; 19:3290-8.     

Autophagosome precursor maturation requires homotypic fusion

Autophagy is a catabolic process in which lysosomes degrade intracytoplasmic contents transported in double-membraned autophagosomes. Autophagosomes are formed by the elongation and fusion of phagophores, which can be derived from preautophagosomal structures coming from the plasma membrane and other sites like the endoplasmic reticulum and mitochondria. The mechanisms by which preautophagosomal structures elongate their membranes and mature toward fully formed autophagosomes still remain unknown. Here, we show that the maturation of the early Atg16L1 precursors requires homotypic fusion, which is essential for subsequent autophagosome formation. Atg16L1 precursor homotypic fusion depends on the SNARE protein VAMP7 together with partner SNAREs. Atg16L1 precursor homotypic fusion is a critical event in the early phases of autophagy that couples membrane acquisition and autophagosome biogenesis, as this step regulates the size of the vesicles, which in turn appears to influence their subsequent maturation into LC3-positive autophagosomes.     

Membrane remodeling by the PX-BAR protein SNX18 promotes autophagosome formation

The membrane remodeling events required for autophagosome biogenesis are still poorly understood. Because PX domain proteins mediate membrane remodeling and trafficking, we conducted an imaging-based siRNA screen for autophagosome formation targeting human PX proteins. The PX-BAR protein SNX18 was identified as a positive regulator of autophagosome formation, and its Drosophila melanogaster homologue SH3PX1 was found to be required for efficient autophagosome formation in the larval fat body. We show that SNX18 is required for recruitment of Atg16L1-positive recycling endosomes to a perinuclear area and for delivery of Atg16L1- and LC3-positive membranes to autophagosome precursors. We identify a direct interaction of SNX18 with LC3 and show that the pro-autophagic activity of SNX18 depends on its membrane binding and tubulation capacity. We also show that the function of SNX18 in membrane tubulation and autophagy is negatively regulated by phosphorylation of S233. We conclude that SNX18 promotes autophagosome formation by virtue of its ability to remodel membranes and provide membrane to forming autophagosomes.     

Mitochondria: One of the origins for autophagosomal membranes?

Macroautophagy is a transport pathway to the lysosome/vacuole that contributes to the degradation of numerous intracellular components. Despite the recent advances achieved in the understanding of the molecular mechanism underlying macroautophagy, the membrane origin of autophagosomes, the hallmark of this process is still a mystery. It has been suggested that mitochondria may be one of the lipid sources for autophagosome formation and that possibly this organelle provides the phosphatidylethanolamine (PE) that covalently links to the members of the ubiquitin-like Atg8/microtubule-associated protein 1 light chain 3 (LC3) protein family. These lipidated proteins are inserted into the outer and inner surface of autophagosomes and are essential for the biogenesis of these large double-membrane vesicles. However, because PE is an integral component of all cellular membranes, designing appropriate experiments to determine the origin of the autophagosomal PE is not easy. In this review, we discuss the idea that mitochondria provide the pool of PE necessary for the autophagosome biogenesis and we propose some possible experimental approaches aimed to explore this possibility.