PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (993923)

Clipboard (0)
None

Related Articles

1.  A role for Atg8–PE deconjugation in autophagosome biogenesis 
Autophagy  2012;8(5):780-793.
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.
doi:10.4161/auto.19385
PMCID: PMC3378420  PMID: 22622160
2.  A high-throughput FRET-based assay for determination of Atg4 activity 
Autophagy  2012;8(3):401-412.
Atg4 is required for cleaving Atg8, allowing it to be conjugated to phosphatidylethanolamine on phagophore membranes, a key step in autophagosome biogenesis. Deconjugation of Atg8 from autophagosomal membranes could be also a regulatory step in controlling autophagy. Therefore, the activity of Atg4 is important for autophagy and could be a target for therapeutic intervention. In this study, a sensitive and specific method to measure the activity of two Atg4 homologs in mammalian cells, Atg4A and Atg4B, was developed using a fluorescence resonance energy transfer (FRET)-based approach. Thus LC3B and GATE-16, two substrates that could be differentially cleaved by Atg4A and Atg4B, were fused with CFP and YFP at the N- and C-terminus, respectively, allowing FRET to occur. The FRET signals decreased in proportion to the Atg4-mediated cleavage, which separated the two fluorescent proteins. This method is highly efficient for measuring the enzymatic activity and kinetics of Atg4A and Atg4B under in vitro conditions. Applications of the assay indicated that the activity of Atg4B was dependent on its catalytic cysteine and expression level, but showed little changes under several common autophagy conditions. In addition, the assays displayed excellent performance in high throughput format and are suitable for screening and analysis of potential modulators. In summary, the FRET-based assay is simple and easy to use, is sensitive and specific, and is suitable for both routine measurement of Atg4 activity and high-throughput screening.
doi:10.4161/auto.18777
PMCID: PMC3337841  PMID: 22302004
Atg4; Atg8; FRET assay; GATE-16; LC3B
3.  A PCR analysis of the ubiquitin-like conjugation systems in macroautophagy 
Autophagy  2011;7(12):1410-1414.
A central part of the core macroauto-phagy (hereafter autophagy) machinery includes the two ubiquitin-like (Ubl) conjugation systems that involve the Ubl proteins Atg8 and Atg12.1 Although the functions of these proteins have not been fully elucidated, they play critical roles in autophagosome formation. For example, Atg8 is involved in cargo recognition,2,3 and the amount of Atg8 in part determines the size of the autophagosome,4 whereas Atg12 is part of a trimer that may function as an E3 ligase to facilitate Atg8 conjugation to phosphatidylethanolamine and determine, in part, the site of the conjugation reaction.5 Thus, fully functional autophagy requires both the Atg8 and Atg12 conjugation systems. Dysfunctional autophagy is associated with various human pathophysiologies including cancer, neurodegeneration, gastrointestinal disorders and heart disease. So, if you are wondering whether autophagy is operating properly in your own body, what can you do? The problem is that there are relatively few methods for analyzing autophagy in vivo.6-11 Minimally, you might want to find out if the relevant genes are intact and have the correct sequence. Considering the rapid advances being made in DNA sequencing technology, it is likely only a matter of time before people can submit a DNA sample and obtain a rapid readout of particular genes, or their entire genome. Thus, anticipating the future, we decided to analyze a select set of autophagy-related (ATG) genes, with a focus on those encoding components of the Ubl conjugation systems, by a polymerase chain reaction (PCR)-based method that combines science with art.
doi:10.4161/auto.7.12.16991
PMCID: PMC3288015  PMID: 22024756
autophagy; collaboration; gel electrophoresis; membrane; primer
4.  Characterisation of unusual families of ATG8-like proteins and ATG12 in the protozoan parasite Leishmania major 
Autophagy  2009;5(2):159-172.
Leishmania major possesses, apparently uniquely, four families of ATG8-like genes, designated ATG8, ATG8A, ATG8B and ATG8C, and 25 genes in total. L. major ATG8 and examples from the ATG8A, ATG8B and ATG8C families are able to complement a Saccharomyces cerevisiae ATG8-deficient strain, indicating functional conservation. Whereas ATG8 has been shown to form putative autophagosomes during differentiation and starvation of L. major, ATG8A primarily form puncta in response to starvation - indicating a role for ATG8A in starvation-induced autophagy. Recombinant ATG8A was processed at the scissile glycine by recombinant ATG4.2 but not ATG4.1 cysteine peptidases of L. major and, consistent with this, ATG4.2-deficient L. major mutants were unable to process ATG8A and were less able to withstand starvation than wild type cells. GFP-ATG8-containing puncta were less abundant in ATG4.2 over-expression lines, in which unlipidated ATG8 predominated, which is consistent with ATG4.2 being an ATG8-deconjugating enzyme as well as an ATG8A-processing enzyme. In contrast, recombinant ATG8, ATG8B and ATG8C were all processed by ATG4.1, but not by ATG4.2. ATG8B and ATG8C both have a distinct subcellular location close to the flagellar pocket, but the occurrence of the GFP-labelled puncta suggest that they do not have a role in autophagy. L. major genes encoding possible ATG5, ATG10 and ATG12 homologues were found to complement their respective S. cerevisiae mutants, and ATG12 localised in part to ATG8-containing puncta, suggestive of a functional ATG5-ATG12 conjugation pathway in the parasite. L. major ATG12 is unusual as it requires C-terminal processing by an as yet unidentified peptidase.
PMCID: PMC2642932  PMID: 19066473
autophagy; Leishmania; protozoan parasite; ATG4; ATG8; ATG12
5.  Dissecting the role of the Atg12–Atg5-Atg16 complex during autophagosome formation 
Autophagy  2013;9(3):424-425.
The activity of the conserved Atg12–Atg5-Atg16 complex is essential for autophagosome formation. However, little is known about its mechanism of action during this process. In our study we employed in vitro systems consisting of purified proteins and giant unilamellar vesicles (GUVs) or small liposomes to investigate membrane binding by the Atg12–Atg5-Atg16 complex and its interplay with the Atg8 conjugation system. We showed that Atg5 directly binds membranes and that this membrane binding is negatively regulated by Atg12 conjugation but activated by Atg16. Membrane binding by the Atg12–Atg5-Atg16 complex is required for efficient promotion of Atg8 lipidation. Additionally, we found that the Atg12–Atg5-Atg16 complex tethered vesicles in an Atg8-independent manner. In yeast, membrane binding by Atg5 is not required for its recruitment to the phagophore assembly site (PAS) but is essential for efficient promotion of autophagy and the cytoplasm-to-vacuole targeting (Cvt) pathway at a stage preceding Atg8 lipidation and autophagosome closure. Our findings provide new insights into the role of the Atg12–Atg5-Atg16 complex during autophagosome formation.
doi:10.4161/auto.22931
PMCID: PMC3590266  PMID: 23321721
Atg12; Atg16; Atg5; Atg8; autophagosome; cytoplasm-to-vacuole targeting; phagophore assembly site
6.  Non-degradative Role of Atg5-Atg12/Atg16L1 Autophagy Protein Complex in Antiviral Activity of Interferon gamma 
Cell host & microbe  2012;11(4):397-409.
SUMMARY
Host resistance to viral infection requires Type-I (α/β) and -II (γ) interferon (IFN) production. Another important defense mechanism is the degradative activity of macroautophagy (herein autophagy), mediated by the coordinated action of evolutionarily conserved autophagy proteins (Atg). We show that the Atg5-Atg12/Atg16L1 protein complex, whose prior known function is in autophagosome formation, is required for IFNγ-mediated host defense against murine norovirus (MNV) infection. Importantly, the direct antiviral activity of IFNγ against MNV in macrophages required Atg5-Atg12, Atg7, and Atg16L1, but not induction of autophagy, the degradative activity of lysosomal proteases, fusion of autophagosomes and lysosomes, or the Atg8 processing protein Atg4B. IFNγ, via Atg5-Atg12/Atg16L1, inhibited formation of the membranous cytoplasmic MNV replication complex, where Atg16L1 localized. Thus, the Atg5-Atg12/Atg16L1 complex performs a pivotal, nondegradative role in IFNγ-mediated antiviral defense, establishing that multicellular organisms have evolved to use portions of the autophagy pathway machinery in a cassette-like fashion for host defense.
doi:10.1016/j.chom.2012.03.002
PMCID: PMC3348177  PMID: 22520467
7.  Mechanism and functions of membrane binding by the Atg5-Atg12/Atg16 complex during autophagosome formation 
The EMBO journal  2012;31(22):4304-4317.
Autophagy is a conserved process for the bulk degradation of cytoplasmic material. Triggering of autophagy results in the formation of double membrane-bound vesicles termed autophagosomes. The conserved Atg5-Atg12/Atg16 complex is essential for autophagosome formation. Here we show that the yeast Atg5-Atg12/Atg16 complex directly binds membranes. Membrane binding is mediated by Atg5, inhibited by Atg12 and activated by Atg16. In a fully reconstituted system using giant unilamellar vesicles and recombinant proteins we reveal that all components of the complex are required for efficient promotion of Atg8 conjugation to phosphatidylethanolamine and are able to assign precise functions to all of its components during this process. In addition, we report that in vitro the Atg5-Atg12/Atg16 complex is able to tether membranes independently of Atg8. Furthermore, we show that membrane binding by Atg5 is downstream of its recruitment to the preautophagosomal structure but is essential for autophagy and cytoplasm-to-vacuole transport at a stage preceding Atg8 conjugation and vesicle closure. Our findings provide important insights into the mechanism of action of the Atg5-Atg12/Atg16 complex during autophagosome formation.
doi:10.1038/emboj.2012.278
PMCID: PMC3501226  PMID: 23064152
autophagy; autophagosome; Atg5; Atg8; Atg16
8.  Cdc48/p97 and Shp1/p47 regulate autophagosome biogenesis in concert with ubiquitin-like Atg8 
The Journal of Cell Biology  2010;190(6):965-973.
Cdc48/p97/VCP plays a ubiquitin-independent role during autophagosome formation in S. cerevisiae.
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.
doi:10.1083/jcb.201002075
PMCID: PMC3101598  PMID: 20855502
9.  The Atg8 Conjugation System Is Indispensable for Proper Development of Autophagic Isolation Membranes in Mice 
Molecular Biology of the Cell  2008;19(11):4762-4775.
Autophagy is an evolutionarily conserved bulk-protein degradation pathway in which isolation membranes engulf the cytoplasmic constituents, and the resulting autophagosomes transport them to lysosomes. Two ubiquitin-like conjugation systems, termed Atg12 and Atg8 systems, are essential for autophagosomal formation. In addition to the pathophysiological roles of autophagy in mammals, recent mouse genetic studies have shown that the Atg8 system is predominantly under the control of the Atg12 system. To clarify the roles of the Atg8 system in mammalian autophagosome formation, we generated mice deficient in Atg3 gene encoding specific E2 enzyme for Atg8. Atg3-deficient mice were born but died within 1 d after birth. Conjugate formation of mammalian Atg8 homologues was completely defective in the mutant mice. Intriguingly, Atg12–Atg5 conjugation was markedly decreased in Atg3-deficient mice, and its dissociation from isolation membranes was significantly delayed. Furthermore, loss of Atg3 was associated with defective process of autophagosome formation, including the elongation and complete closure of the isolation membranes, resulting in malformation of the autophagosomes. The results indicate the essential role of the Atg8 system in the proper development of autophagic isolation membranes in mice.
doi:10.1091/mbc.E08-03-0309
PMCID: PMC2575156  PMID: 18768753
10.  ATG12 Conjugation to ATG3 Regulates Mitochondrial Homeostasis and Cell Death 
Cell  2010;142(4):590-600.
SUMMARY
ATG12, an ubiquitin-like modifier required for macroautophagy, has a single known conjugation target, another autophagy regulator called ATG5. Here, we identify ATG3 as a substrate for ATG12 conjugation. ATG3 is the E2-like enzyme necessary for ATG8/LC3 lipidation during autophagy. ATG12-ATG3 complex formation requires ATG7 as the E1 enzyme and ATG3 autocatalytic activity as the E2, resulting in the covalent linkage of ATG12 onto a single lysine on ATG3. Surprisingly, disrupting ATG12 conjugation to ATG3 does not affect starvation-induced autophagy. Rather, the lack of ATG12-ATG3 complex formation produces an expansion in mitochondrial mass and inhibits cell death mediated by mitochondrial pathways. Overall, these results unveil a role for ATG12-ATG3 in mitochondrial homeostasis, and implicate the ATG12 conjugation system in cellular functions distinct from the early steps of autophagosome formation.
doi:10.1016/j.cell.2010.07.018
PMCID: PMC2925044  PMID: 20723759
11.  The Atg16L Complex Specifies the Site of LC3 Lipidation for Membrane Biogenesis in Autophagy 
Molecular Biology of the Cell  2008;19(5):2092-2100.
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.
doi:10.1091/mbc.E07-12-1257
PMCID: PMC2366860  PMID: 18321988
12.  OATL1, a novel autophagosome-resident Rab33B-GAP, regulates autophagosomal maturation 
The Journal of Cell Biology  2011;192(5):839-853.
The GAP activity of OATL1, which is recruited to autophagosomes by Atg8, regulates autophagosome–lysosome fusion.
Macroautophagy is a bulk degradation system conserved in all eukaryotic cells. A ubiquitin-like protein, Atg8, and its homologues are essential for autophagosome formation and act as a landmark for selective autophagy of aggregated proteins and damaged organelles. In this study, we report evidence demonstrating that OATL1, a putative Rab guanosine triphosphatase–activating protein (GAP), is a novel binding partner of Atg8 homologues in mammalian cells. OATL1 is recruited to isolation membranes and autophagosomes through direct interaction with Atg8 homologues and is involved in the fusion between autophagosomes and lysosomes through its GAP activity. We further provide evidence that Rab33B, an Atg16L1-binding protein, is a target substrate of OATL1 and is involved in the fusion between autophagosomes and lysosomes, the same as OATL1. Because both its GAP activity and its Atg8 homologue–binding activity are required for OATL1 to function, we propose a model that OATL1 uses Atg8 homologues as a scaffold to exert its GAP activity and to regulate autophagosomal maturation.
doi:10.1083/jcb.201008107
PMCID: PMC3051816  PMID: 21383079
13.  ATG5 Is Essential for ATG8-Dependent Autophagy and Mitochondrial Homeostasis in Leishmania major 
PLoS Pathogens  2012;8(5):e1002695.
Macroautophagy has been shown to be important for the cellular remodelling required for Leishmania differentiation. We now demonstrate that L. major contains a functional ATG12-ATG5 conjugation system, which is required for ATG8-dependent autophagosome formation. Nascent autophagosomes were found commonly associated with the mitochondrion. L. major mutants lacking ATG5 (Δatg5) were viable as promastigotes but were unable to form autophagosomes, had morphological abnormalities including a much reduced flagellum, were less able to differentiate and had greatly reduced virulence to macrophages and mice. Analyses of the lipid metabolome of Δatg5 revealed marked elevation of phosphatidylethanolamines (PE) in comparison to wild type parasites. The Δatg5 mutants also had increased mitochondrial mass but reduced mitochondrial membrane potential and higher levels of reactive oxygen species. These findings indicate that the lack of ATG5 and autophagy leads to perturbation of the phospholipid balance in the mitochondrion, possibly through ablation of membrane use and conjugation of mitochondrial PE to ATG8 for autophagosome biogenesis, resulting in a dysfunctional mitochondrion with impaired oxidative ability and energy generation. The overall result of this is reduced virulence.
Author Summary
Leishmaniasis is a disease of humans that is of major significance throughout many parts of the world. It is caused by the protozoan parasite Leishmania and mammals are infected through the bite of a sand fly in which the parasite develops. Parasite remodelling crucial for generation of the human-infective forms is aided by the catabolic process known as autophagy in which cell material is packaged within organelles called autophagosomes and subsequently broken down in the digestive lysosomal compartment. Here we show that autophagy in Leishmania requires the coordinated actions of two pathways, one of which involves a protein called ATG5. We have generated parasite mutants lacking this protein and shown that ATG5 is required for both autophagosome formation and also maintenance of a fully functional mitochondrion. The mutants lacking ATG5 have increased mitochondrial mass and phospholipid content, high levels of oxidants and reduced membrane potential, all being hallmarks of a dysfunctional mitochondrion with impaired ability for energy generation. Our results have thus revealed that a functional autophagic pathway is crucial for phospholipid homeostasis and mitochondrial function in the parasite and important for the parasite's differentiation, infectivity and virulence to its mammalian host.
doi:10.1371/journal.ppat.1002695
PMCID: PMC3355087  PMID: 22615560
14.  Atg3-Mediated Lipidation of Atg8 Is Involved in Encystation of Acanthamoeba 
Autophagy is a catabolic process involved in the degradation of a cell's own components for cell growth, development, homeostasis, and the recycling of cellular products. Autophagosome is an essential component in the protozoan parasite during differentiation and encystation. The present study identified and characterized autophagy-related protein (Atg) 3, a member of Atg8 conjugation system, in Acanthamoeba castellanii (AcAtg3). AcAtg3 encoding a 304 amino acid protein showed high similarity with the catalytic cysteine site of other E2 like enzymes of ubiquitin system. Predicted 3D structure of AcAtg3 revealed a hammer-like shape, which is the characteristic structure of E2-like enzymes. The expression level of AcAtg3 did not increase during encystation. However, the formation of mature cysts was significantly reduced in Atg3-siRNA transfected cells in which the production of Atg8-phosphatidylethanolamine conjugate was inhibited. Fluorescent microscopic analysis revealed that dispersed AcAtg3-EGFP fusion protein gathered around autophagosomal membranes during encystation. These results provide important information for understanding autophagic machinery through the lipidation reaction mediated by Atg3 in Acanthamoeba.
doi:10.3347/kjp.2011.49.2.103
PMCID: PMC3121065  PMID: 21738264
Acanthamoeba; encystation; autophagosome; Atg3
15.  Early Steps in Autophagy Depend on Direct Phosphorylation of Atg9 by the Atg1 Kinase 
Molecular Cell  2014;53(3):471-483.
Summary
Bulk degradation of cytoplasmic material is mediated by a highly conserved intracellular trafficking pathway termed autophagy. This pathway is characterized by the formation of double-membrane vesicles termed autophagosomes engulfing the substrate and transporting it to the vacuole/lysosome for breakdown and recycling. The Atg1/ULK1 kinase is essential for this process; however, little is known about its targets and the means by which it controls autophagy. Here we have screened for Atg1 kinase substrates using consensus peptide arrays and identified three components of the autophagy machinery. The multimembrane-spanning protein Atg9 is a direct target of this kinase essential for autophagy. Phosphorylated Atg9 is then required for the efficient recruitment of Atg8 and Atg18 to the site of autophagosome formation and subsequent expansion of the isolation membrane, a prerequisite for a functioning autophagy pathway. These findings show that the Atg1 kinase acts early in autophagy by regulating the outgrowth of autophagosomal membranes.
Graphical Abstract
Highlights
•The Atg1 kinase phosphorylation consensus was identified on peptide arrays•Atg9 is a direct target of the Atg1/ULK1 kinase in vitro and in vivo•Atg9 phosphorylation recruits Atg18 and Atg8 to the PAS•Atg9 phosphorylation is required for isolation membrane expansion/autophagy function
Autophagy function is pivotal to cell health. Papinski et al. identify the phosphorylation consensus of the central kinase in this pathway, Atg1. The autophagy-related protein Atg9 is a direct target of Atg1. Atg9 phosphorylation by Atg1 is required for autophagosome formation. This finding sheds light on how Atg1 controls autophagy.
doi:10.1016/j.molcel.2013.12.011
PMCID: PMC3978657  PMID: 24440502
16.  Autophagy Protein Atg3 is Essential for Maintaining Mitochondrial Integrity and for Normal Intracellular Development of Toxoplasma gondii Tachyzoites 
PLoS Pathogens  2011;7(12):e1002416.
Autophagy is a cellular process that is highly conserved among eukaryotes and permits the degradation of cellular material. Autophagy is involved in multiple survival-promoting processes. It not only facilitates the maintenance of cell homeostasis by degrading long-lived proteins and damaged organelles, but it also plays a role in cell differentiation and cell development. Equally important is its function for survival in stress-related conditions such as recycling of proteins and organelles during nutrient starvation. Protozoan parasites have complex life cycles and face dramatically changing environmental conditions; whether autophagy represents a critical coping mechanism throughout these changes remains poorly documented. To investigate this in Toxoplasma gondii, we have used TgAtg8 as an autophagosome marker and showed that autophagy and the associated cellular machinery are present and functional in the parasite. In extracellular T. gondii tachyzoites, autophagosomes were induced in response to amino acid starvation, but they could also be observed in culture during the normal intracellular development of the parasites. Moreover, we generated a conditional T. gondii mutant lacking the orthologue of Atg3, a key autophagy protein. TgAtg3-depleted parasites were unable to regulate the conjugation of TgAtg8 to the autophagosomal membrane. The mutant parasites also exhibited a pronounced fragmentation of their mitochondrion and a drastic growth phenotype. Overall, our results show that TgAtg3-dependent autophagy might be regulating mitochondrial homeostasis during cell division and is essential for the normal development of T. gondii tachyzoites.
Author Summary
Autophagy is a catabolic process involved in maintaining cellular homeostasis in eukaryotic cells, while coping with their changing environmental conditions. Mechanistically, it is also a process of considerable complexity involving multiple protein factors and implying numerous protein-protein and protein-membrane interactions. The cellular material to be degraded by autophagy is contained in a membrane-bound compartment called the autophagosome. We have characterised the formation of autophagosomes in the protozoan parasite Toxoplasma gondii by following the relocalisation of autophagosome-bound TgAtg8. Thus, exploiting GFP-TgAtg8 as a marker, we showed that it is a process that is regulated and can be induced artificially by amino acid starvation. Autophagic vesicles were also observed in normally dividing intracellular parasites. Depleting Toxoplasma of the TgAtg3 autophagy protein led to an impairment of TgAtg8 conjugation to the autophagosomal membrane and, at the cellular level, to a fragmentation of the single mitochondrion of the parasite and to a severe growth arrest. We have thus found that TgAtg3-dependent autophagy is essential for normal intracellular development of T. gondii tachyzoites.
doi:10.1371/journal.ppat.1002416
PMCID: PMC3228817  PMID: 22144900
17.  Global Analysis of Fission Yeast Mating Genes Reveals New Autophagy Factors 
PLoS Genetics  2013;9(8):e1003715.
Macroautophagy (autophagy) is crucial for cell survival during starvation and plays important roles in animal development and human diseases. Molecular understanding of autophagy has mainly come from the budding yeast Saccharomyces cerevisiae, and it remains unclear to what extent the mechanisms are the same in other organisms. Here, through screening the mating phenotype of a genome-wide deletion collection of the fission yeast Schizosaccharomyces pombe, we obtained a comprehensive catalog of autophagy genes in this highly tractable organism, including genes encoding three heretofore unidentified core Atg proteins, Atg10, Atg14, and Atg16, and two novel factors, Ctl1 and Fsc1. We systematically examined the subcellular localization of fission yeast autophagy factors for the first time and characterized the phenotypes of their mutants, thereby uncovering both similarities and differences between the two yeasts. Unlike budding yeast, all three Atg18/WIPI proteins in fission yeast are essential for autophagy, and we found that they play different roles, with Atg18a uniquely required for the targeting of the Atg12–Atg5·Atg16 complex. Our investigation of the two novel factors revealed unforeseen autophagy mechanisms. The choline transporter-like protein Ctl1 interacts with Atg9 and is required for autophagosome formation. The fasciclin domain protein Fsc1 localizes to the vacuole membrane and is required for autophagosome-vacuole fusion but not other vacuolar fusion events. Our study sheds new light on the evolutionary diversity of the autophagy machinery and establishes the fission yeast as a useful model for dissecting the mechanisms of autophagy.
Author Summary
Autophagy is a eukaryotic cellular process that transports cytoplasmic contents into lysosomes/vacuoles for degradation. It has been linked to multiple human diseases, including cancer and neurodegenerative disorders. The molecular machinery of autophagy was first identified and has been best characterized in the budding yeast Saccharomyces cerevisiae, but little is known about the autophagy machinery in another important unicellular model organism, the fission yeast Schizosaccharomyces pombe. In this study, we performed an unbiased and comprehensive screening of the fission yeast autophagy genes by profiling the mating phenotypes of nearly 3000 deletion strains. Following up on the screening results, we systematically characterized both previously known and newly identified fission yeast autophagy factors by examining their localization and the phenotype of their mutants. Our analysis increased the number of experimentally defined fission yeast autophagy factors from 14 to 23, including two novel factors that act in ways different from all previously known autophagy proteins. Together, our data reveal unexpected evolutionary divergence of autophagy mechanisms and establish a new model system for unraveling the molecular details of the autophagy process.
doi:10.1371/journal.pgen.1003715
PMCID: PMC3738441  PMID: 23950735
18.  Atg21 Is a Phosphoinositide Binding Protein Required for Efficient Lipidation and Localization of Atg8 during Uptake of Aminopeptidase I by Selective Autophagy 
Molecular Biology of the Cell  2004;15(8):3553-3566.
Delivery of proteins and organelles to the vacuole by autophagy and the cytoplasm to vacuole targeting (Cvt) pathway involves novel rearrangements of membrane resulting in the formation of vesicles that fuse with the vacuole. The mechanism of vesicle formation and the origin of the membrane are complex issues still to be resolved. Atg18 and Atg21 are proteins essential to vesicle formation and together with Ygr223c form a novel family of phosphoinositide binding proteins that are associated with the vacuole and perivacuolar structures. Their localization requires the activity of Vps34, suggesting that phosphatidylinositol(3)phosphate may be essential for their function. The activity of Atg18 is vital for all forms of autophagy, whereas Atg21 is required for the Cvt pathway but not for nitrogen starvation-induced autophagy. The loss of Atg21 results in the absence of Atg8 from the pre-autophagosomal structure (PAS), which may be ascribed to a reduced rate of conjugation of Atg8 to phosphatidylethanolamine. A similar defect in localization of a second ubiquitin-like conjugate, Atg12-Atg5, suggests that Atg21 may be involved in the recruitment of membrane to the PAS.
doi:10.1091/mbc.E04-02-0147
PMCID: PMC491818  PMID: 15155809
19.  WIPI2 Links LC3 Conjugation with PI3P, Autophagosome Formation, and Pathogen Clearance by Recruiting Atg12–5-16L1 
Molecular Cell  2014;55(2):238-252.
Summary
Mammalian cell homeostasis during starvation depends on initiation of autophagy by endoplasmic reticulum-localized phosphatidylinositol 3-phosphate (PtdIns(3)P) synthesis. Formation of double-membrane autophagosomes that engulf cytosolic components requires the LC3-conjugating Atg12–5-16L1 complex. The molecular mechanisms of Atg12–5-16L1 recruitment and significance of PtdIns(3)P synthesis at autophagosome formation sites are unknown. By identifying interacting partners of WIPIs, WD-repeat PtdIns(3)P effector proteins, we found that Atg16L1 directly binds WIPI2b. Mutation experiments and ectopic localization of WIPI2b to plasma membrane show that WIPI2b is a PtdIns(3)P effector upstream of Atg16L1 and is required for LC3 conjugation and starvation-induced autophagy through recruitment of the Atg12–5-16L1 complex. Atg16L1 mutants, which do not bind WIPI2b but bind FIP200, cannot rescue starvation-induced autophagy in Atg16L1-deficient MEFs. WIPI2b is also required for autophagic clearance of pathogenic bacteria. WIPI2b binds the membrane surrounding Salmonella and recruits the Atg12–5-16L1 complex, initiating LC3 conjugation, autophagosomal membrane formation, and engulfment of Salmonella.
Graphical Abstract
Highlights
•WIPI2 binds Atg16L1 directly and recruits the Atg12–5-16L1 complex•WIPI2 binding to Atg16L1 is required for LC3 lipidation and autophagosome formation•WIPI2-Atg16L1 function requires PI3P binding by WIPI2 and is independent of FIP200•Autophagosomal engulfment of Salmonella requires the WIPI2-Atg16L1 complex
Starvation-induced autophagy requires PtdIns(3)P locally produced on ER-derived membranes. Dooley et al. demonstrate that the PtdIns(3)P effector WIPI2b binds Atg16L1 to recruit Atg12–5-16L1 to PtdIns(3)P-positive omegasomes, resulting in LC3 lipidation and starvation-induced autophagy. These findings suggest that WIPI2b senses increases in PtdIns(3)P and directs LC3 conjugation to developing autophagosomes.
doi:10.1016/j.molcel.2014.05.021
PMCID: PMC4104028  PMID: 24954904
20.  SNARE proteins are required for macroautophagy 
Cell  2011;146(2):290-302.
SUMMARY
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.
doi:10.1016/j.cell.2011.06.022
PMCID: PMC3143362  PMID: 21784249
Atg9; fusion; lysosome; membrane biogenesis; protein targeting; secretory pathway; stress; tubulovesicular clusters; vacuole; yeast
21.  Phosphatidylinositol-3-Phosphate Clearance Plays a Key Role in Autophagosome Completion 
Current biology : CB  2012;22(17):1545-1553.
Summary
Background
The biogenesis of autophagosomes, the hallmark of autophagy, depends on the function of the autophagy-related (Atg) proteins and the generation of phosphatidylinositol-3-phosphate (PtdIns3P) at the phagophore assembly site (PAS), the location where autophagosomes arise. The current model is that PtdIns3P is involved primarily in the recruitment of Atg proteins to the PAS and that once an autophagosome is complete, the Atg machinery is released from its surface back into the cytoplasm and reused for the formation of new vesicles.
Results
We have identified a PtdIns3P phosphatase, Ymr1, that is essential for the normal progression of both bulk and selective types of autophagy. This protein is recruited to the PAS at an early stage of formation of this structure through a process that requires both its GRAM domain and its catalytic activity. In the absence of Ymr1, Atg proteins fail to dissociate from the limiting membrane of autophagosomes, and these vesicles accumulate in the cytoplasm.
Conclusions
Our data thus reveal a key role for PtdIns3P turnover in the regulation of the late steps of autophagosome biogenesis and indicate that the disassembly of the Atg machinery from the surface of autophagosomes is a requisite for their fusion with the vacuole.
doi:10.1016/j.cub.2012.06.029
PMCID: PMC3615650  PMID: 22771041
22.  Distinct Roles in Autophagy and Importance in Infectivity of the Two ATG4 Cysteine Peptidases of Leishmania major* 
The Journal of Biological Chemistry  2012;288(5):3678-3690.
Background: ATG4 is a cysteine peptidase crucial for macroautophagy.
Results: Gene deletion mutants show that the two ATG4s of Leishmania perform distinct roles, although there is some redundancy.
Conclusion: ATG4s are not individually essential but macroautophagy, a process important in the virulence of the parasite, requires one.
Significance: Highlights the distinct roles of ATG4 isoforms and their importance for autophagy and parasite infectivity.
Macroautophagy in Leishmania, which is important for the cellular remodeling required during differentiation, relies upon the hydrolytic activity of two ATG4 cysteine peptidases (ATG4.1 and ATG4.2). We have investigated the individual contributions of each ATG4 to Leishmania major by generating individual gene deletion mutants (Δatg4.1 and Δatg4.2); double mutants could not be generated, indicating that ATG4 activity is required for parasite viability. Both mutants were viable as promastigotes and infected macrophages in vitro and mice, but Δatg4.2 survived poorly irrespective of infection with promastigotes or amastigotes, whereas this was the case only when promastigotes of Δatg4.1 were used. Promastigotes of Δatg4.2 but not Δatg4.1 were more susceptible than wild type promastigotes to starvation and oxidative stresses, which correlated with increased reactive oxygen species levels and oxidatively damaged proteins in the cells as well as impaired mitochondrial function. The antioxidant N-acetylcysteine reversed this phenotype, reducing both basal and induced autophagy and restoring mitochondrial function, indicating a relationship between reactive oxygen species levels and autophagy. Deletion of ATG4.2 had a more dramatic effect upon autophagy than did deletion of ATG4.1. This phenotype is consistent with a reduced efficiency in the autophagic process in Δatg4.2, possibly due to ATG4.2 having a key role in removal of ATG8 from mature autophagosomes and thus facilitating delivery to the lysosomal network. These findings show that there is a level of functional redundancy between the two ATG4s, and that ATG4.2 appears to be the more important. Moreover, the low infectivity of Δatg4.2 demonstrates that autophagy is important for the virulence of the parasite.
doi:10.1074/jbc.M112.415372
PMCID: PMC3561585  PMID: 23166325
Autophagy; Cysteine Protease; Leishmania; Parasite; Parasite Metabolism; Peptidases; Protease; ATG4
23.  Crystallization of the Atg12–Atg5 conjugate bound to Atg16 by the free-interface diffusion method 
Journal of Synchrotron Radiation  2008;15(Pt 3):266-268.
The Atg12–Atg5 conjugate was prepared by in vivo reconstitution and was crystallized with Atg16 using the free-interface diffusion method.
Autophagy mediates the bulk degradation of cytoplasmic components in lysosomes/vacuoles. Five autophagy-related (Atg) proteins are involved in a ubiquitin-like protein conjugation system. Atg12 is conjugated to its sole target, Atg5, by two enzymes, Atg7 and Atg10. The Atg12–Atg5 conjugates form a multimeric complex with Atg16. Formation of the Atg12–Atg5–Atg16 ternary complex is crucial for the functions of these proteins on autophagy. Here, the expression, purification and crystallization of the Atg12–Atg5 conjugate bound to the N-terminal region of Atg16 (Atg16N) are reported. The Atg12–Atg5 conjugates were formed by co-expressing Atg5, Atg7, Atg10 and Atg12 in Eschericia coli. The Atg12–Atg5–Atg16N ternary complex was formed by mixing purified Atg12–Atg5 conjugates and Atg16N, and was further purified by gel-filtration chromatography. Crystallization screening was performed by the free-interface diffusion method. Using obtained microcrystals as seeds, large crystals for diffraction data collection were obtained by the sitting-drop vapour-diffusion method. The crystal contained one ternary complex per asymmetric unit, and diffracted to 2.6 Å resolution.
doi:10.1107/S0909049507054799
PMCID: PMC2394807  PMID: 18421155
autophagy; ubiquitin-like conjugation; crystallization; free-interface diffusion method
24.  Mammalian Atg2 proteins are essential for autophagosome formation and important for regulation of size and distribution of lipid droplets 
Molecular Biology of the Cell  2012;23(5):896-909.
Autophagy is an intracellular degradation process that is mediated by autophagosomes. Mammalian Atg2 proteins Atg2A and Atg2B are identified and characterized as essential for autophagy. They are also present on lipid droplets and are involved in regulation of lipid droplet volume and distribution.
Macroautophagy is an intracellular degradation system by which cytoplasmic materials are enclosed by the autophagosome and delivered to the lysosome. Autophagosome formation is considered to take place on the endoplasmic reticulum and involves functions of autophagy-related (Atg) proteins. Here, we report the identification and characterization of mammalian Atg2 homologues Atg2A and Atg2B. Simultaneous silencing of Atg2A and Atg2B causes a block in autophagic flux and accumulation of unclosed autophagic structures containing most Atg proteins. Atg2A localizes on the autophagic membrane, as well as on the surface of lipid droplets. The Atg2A region containing amino acids 1723–1829, which shows relatively high conservation among species, is required for localization to both the autophagic membrane and lipid droplet and is also essential for autophagy. Depletion of both Atg2A and Atg2B causes clustering of enlarged lipid droplets in an autophagy-independent manner. These data suggest that mammalian Atg2 proteins function both in autophagosome formation and regulation of lipid droplet morphology and dispersion.
doi:10.1091/mbc.E11-09-0785
PMCID: PMC3290647  PMID: 22219374
25.  Atg8 Controls Phagophore Expansion during Autophagosome Formation 
Molecular Biology of the Cell  2008;19(8):3290-3298.
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.
doi:10.1091/mbc.E07-12-1292
PMCID: PMC2488302  PMID: 18508918

Results 1-25 (993923)