Insights into autophagosome maturation revealed by the structures of ATG5 with its interacting partners

Autophagy is a bulky catabolic process that responds to nutrient homeostasis and extracellular stress signals and is a conserved mechanism in all eukaryotes. When autophagy is induced, cellular components are sequestered within an autophagosome and finally degraded by subsequent fusion with a lysosome. During this process, the ATG12–ATG5 conjugate requires 2 different binding partners, ATG16L1 for autophagosome elongation and TECPR1 for lysosomal fusion. In our current study, we describe the crystal structures of human ATG5 in complex with an N-terminal domain of ATG16L1 as well as an internal AIR domain of TECPR1. Both binding partners exhibit a similar α-helical structure containing a conserved binding motif termed AFIM. Furthermore, we characterize the critical role of the C-terminal unstructured region of the AIR domain of TECPR1. These findings are further confirmed by biochemical and cell biological analyses. These results provide new insights into the molecular details of the autophagosome maturation process, from its elongation to its fusion with a lysosome.     

Swapping of interaction partners with ATG5 for autophagosome maturation

Autophagy is a tightly regulated lysosome-mediated catabolic process in eukaryotes that maintains cellular homeostasis. A distinguishable feature of autophagy is the formation of double-membrane structures, autophagosome, which envelopes the intracellular cargoes and finally degrades them by fusion with lysosomes. So far, many structures of Atg proteins working on the autophagosome formation have been reported, however those involved in autophagosome maturation, a fusion with lysosome, are relatively unknown. One of the molecules in autophagosome maturation, TECPR1, has been identified and recently, structural studies on both ATG5-TECPR1 and ATG5-ATG16L1 complexes revealed that TECPR1 and ATG16L1 share the same binding site on ATG5. These results, in combination with supporting biochemical and cellular biological data, provide an insight into a model for swapping ATG5 partners for autophagosome maturation. [BMB Reports 2015; 48(3): 129-130]     

A tethering coherent protein in autophagosome maturation

Autophagy is a cellular pathway that degrades damaged organelles, cytosol and microorganisms, thereby maintaining human health by preventing various diseases including cancers, neurodegenerative disorders and diabetes. In autophagy, autophagosomes carrying cellular cargoes fuse with lysosomes for degradation. The proper autophagosome-lysosome fusion is pivotal for efficient autophagy activity. However, the molecular mechanism that specifically directs the fusion process is not clear. Our study reported that lysosome-localized TECPR1 (TECtonin β-Propeller Repeat containing 1) binds the autophagosome-localized ATG12–ATG5 conjugate and recruits it to autolysosomes. TECPR1 also binds PtdIns3P in an ATG12–ATG5-dependent manner. Consequently, depletion of TECPR1 leads to a severe defect in autophagosome maturation. We propose that the interaction between TECPR1 and ATG12–ATG5 initiates the fusion between the autophagosome and lysosome, and TECPR1 is a TEthering Coherent PRotein in autophagosome maturation.     

A tethering coherent protein in autophagosome maturation

Autophagy is a cellular pathway that degrades damaged organelles, cytosol and microorganisms, thereby maintaining human health by preventing various diseases including cancers, neurodegenerative disorders and diabetes. In autophagy, autophagosomes carrying cellular cargoes fuse with lysosomes for degradation. The proper autophagosome-lysosome fusion is pivotal for efficient autophagy activity. However, the molecular mechanism that specifically directs the fusion process is not clear. Our study reported that lysosome-localized TECPR1 (TECtonin β-Propeller Repeat containing 1) binds the autophagosome-localized ATG12-ATG5 conjugate and recruits it to autolysosomes. TECPR1 also binds PtdIns3P in an ATG12-ATG5-dependent manner. Consequently, depletion of TECPR1 leads to a severe defect in autophagosome maturation. We propose that the interaction between TECPR1 and ATG12-ATG5 initiates the fusion between the autophagosome and lysosome, and TECPR1 is a TEthering Coherent PRotein in autophagosome maturation.     

ATG14 controls SNARE-mediated autophagosome fusion with a lysosome

Autophagosome fusion with a lysosome constitutes the last barrier for autophagic degradation. It is speculated that this fusion process is precisely and tightly regulated. Recent genetic evidence suggests that a set of SNARE proteins, including STX17, SNAP29, and VAMP8, are essential for the fusion between autophagosomes and lysosomes. However, it remains unclear whether these SNAREs are fusion competent and how their fusogenic activity is specifically regulated during autophagy. Using a combination of biochemical, cell biology, and genetic approaches, we demonstrated that fusogenic activity of the autophagic SNARE complex is temporally and spatially controlled by ATG14/Barkor/Atg14L, an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex (PtdIns3K). ATG14 directly binds to the STX17-SNAP29 binary complex on autophagosomes and promotes STX17-SNAP29-VAMP8-mediated autophagosome fusion with lysosomes. ATG14 homo-oligomerization is required for SNARE binding and fusion promotion, but is dispensable for PtdIns3K stimulation and autophagosome biogenesis. Consequently, ATG14 homo-oligomerization is required for autophagosome fusion with a lysosome, but is dispensable for autophagosome biogenesis. These data support a key role of ATG14 in controlling autophagosome fusion with a lysosome.     

HS1BP3 inhibits autophagy by regulation of PLD1

Macroautophagy/autophagy is a membrane trafficking and intracellular degradation process involving the formation of double-membrane autophagosomes and their ultimate fusion with lysosomes. Much is yet to be learned about the regulation of this process, especially at the level of the membranes and lipids involved. We have recently found that the PX domain protein HS1BP3 (HCLS1 binding protein 3) is a negative regulator of autophagosome formation. HS1BP3 depletion increases the formation of LC3-positive autophagosomes both in human cells and zebrafish. HS1BP3 localizes to ATG16L1- and ATG9-positive autophagosome precursors deriving from recycling endosomes, which appear to fuse with LC3-positive phagophores. The HS1BP3 PX domain interacts with phosphatidic acid (PA) and 3’-phosphorylated phosphoinositides. When HS1BP3 is depleted, the total cellular PA content is upregulated stemming from increased activity of the PA-producing enzyme PLD (phospholipase D) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 negatively regulates autophagy by decreasing the PA content of the ATG16L1-positive autophagosome precursor membranes through inhibition of PLD1 activity and localization.     

Transiently expressed ATG16L1 inhibits autophagosome biogenesis and aberrantly targets RAB11-positive recycling endosomes

The membrane source for autophagosome biogenesis is an unsolved mystery in the study of autophagy. ATG16L1 forms a complex with ATG12–ATG5 (the ATG16L1 complex). The ATG16L1 complex is recruited to autophagic membranes to convert MAP1LC3B-I to MAP1LC3B-II. The ATG16L1 complex dissociates from the phagophore before autophagosome membrane closure. Thus, ATG16L1 can be used as an early event marker for the study of autophagosome biogenesis. We found that among 3 proteins in the ATG16L1 complex, only ATG16L1 formed puncta-like structures when transiently overexpressed. ATG16L1⁺ puncta formed by transient expression could represent autophagic membrane structures. We thoroughly characterized the transiently expressed ATG16L1 in several mammalian cell lines. We found that transient expression of ATG16L1 not only inhibited autophagosome biogenesis, but also aberrantly targeted RAB11-positive recycling endosomes, resulting in recycling endosome aggregates. We conclude that transient expression of ATG16L1 is not a physiological model for the study of autophagy. Caution is warranted when reviewing findings derived from a transient expression model of ATG16L1.     

WIPI2B links PtdIns3P to LC3 lipidation through binding ATG16L1

WIPI proteins, phosphatidylinositol 3-phosphate (PtdIns3P) binding proteins with β-propeller folds, are recruited to the omegasome following PtdIns3P production. The functions of the WIPI proteins in autophagosome formation are poorly understood. In a recent study, we reported that WIPI2B directly binds ATG16L1 and functions by recruiting the ATG12–ATG5-ATG16L1 complex to forming autophagosomes during starvation- or pathogen-induced autophagy. Our model of WIPI2 function provides an explanation for the PtdIns3P-dependent recruitment of the ATG12–ATG5-ATG16L1 complex during initiation of autophagy.     

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.     

Structure of the WD40‐domain of human ATG16L1

Autophagy‐related protein ATG16L1 is a component of the mammalian ATG12～ATG5/ATG16L1 complex, which acts as E3‐ligase to catalyze lipidation of LC3 during autophagosome biogenesis. The N‐terminal part of ATG16L1 comprises the ATG5‐binding site and coiled‐coil dimerization domain, both also present in yeast ATG16 and essential for bulk and starvation induced autophagy. While absent in yeast ATG16, mammalian ATG16L1 further contains a predicted C‐terminal WD40‐domain, which has been shown to be involved in mediating interaction with diverse factors in the context of alternative functions of autophagy, such as inflammatory control and xenophagy. In this work, we provide detailed information on the domain boundaries of the WD40‐domain of human ATG16L1 and present its crystal structure at a resolution of 1.55 Å.     

ATG16L1 meets ATG9 in recycling endosomes

Autophagosomes are formed by double-membraned structures, which engulf portions of cytoplasm. Autophagosomes ultimately fuse with lysosomes, where their contents are degraded. The origin of the autophagosome membrane may involve different sources, such as mitochondria, Golgi, endoplasmic reticulum, plasma membrane, and recycling endosomes. We recently observed that ATG9 localizes on the plasma membrane in clathrin-coated structures and is internalized following a classical endocytic pathway through early and then recycling endosomes. By contrast, ATG16L1 is also internalized by clathrin-mediated endocytosis but via different clathrin-coated pits, and appears to follow a different route to the recycling endosomes. The R-SNARE VAMP3 mediates the coalescence of the 2 different pools of vesicles (containing ATG16L1 or ATG9) in recycling endosomes. The heterotypic fusion between ATG16L1- and ATG9-containing vesicles strongly correlates with subsequent autophagosome formation. Thus, ATG9 and ATG16L1 both traffic from the plasma membrane to autophagic precursor structures and provide 2 routes from the plasma membrane to autophagosomes.     

SNX18 tubulates recycling endosomes for autophagosome biogenesis

The role of membrane remodeling and phosphoinositide-binding proteins in autophagy remains elusive. PX domain proteins bind phosphoinositides and participate in membrane remodeling and trafficking events and we therefore hypothesized that one or several PX domain proteins are involved in autophagy. Indeed, the PX-BAR protein SNX18 was identified as a positive regulator of autophagosome formation using an image-based siRNA screen. We show that SNX18 interacts with ATG16L1 and LC3, and functions downstream of ATG14 and the class III PtdIns3K complex in autophagosome formation. SNX18 facilitates recruitment of ATG16L1 to perinuclear recycling endosomes, and its overexpression leads to tubulation of ATG16L1- and LC3-positive membranes. We propose that SNX18 promotes LC3 lipidation and tubulation of recycling endosomes to provide membrane for phagophore expansion.     

Commentary: ATG16L1 meets ATG9 in recycling endosomes: Additional roles for the plasma membrane and endocytosis in autophagosome biogenesis

Autophagosomes are formed by double-membraned structures, which engulf portions of cytoplasm. Autophagosomes ultimately fuse with lysosomes, where their contents are degraded. The origin of the autophagosome membrane may involve different sources, such as mitochondria, Golgi, endoplasmic reticulum, plasma membrane, and recycling endosomes. We recently observed that ATG9 localizes on the plasma membrane in clathrin-coated structures and is internalized following a classical endocytic pathway through early and then recycling endosomes. By contrast, ATG16L1 is also internalized by clathrin-mediated endocytosis but via different clathrin-coated pits, and appears to follow a different route to the recycling endosomes. The R-SNARE VAMP3 mediates the coalescence of the 2 different pools of vesicles (containing ATG16L1 or ATG9) in recycling endosomes. The heterotypic fusion between ATG16L1- and ATG9-containing vesicles strongly correlates with subsequent autophagosome formation. Thus, ATG9 and ATG16L1 both traffic from the plasma membrane to autophagic precursor structures and provide 2 routes from the plasma membrane to autophagosomes.     

A mammalian autophagosome maturation mechanism mediated by TECPR1 and the Atg12-Atg5 conjugate

Autophagy is a major catabolic pathway in eukaryotes associated with a broad spectrum of human diseases. In autophagy, autophagosomes carrying cellular cargoes fuse with lysosomes for degradation. However, the molecular mechanism underlying autophagosome maturation is largely unknown. Here we report that TECPR1 binds to the Atg12-Atg5 conjugate and phosphatidylinositol 3-phosphate (PtdIns[3]P) to promote autophagosome-lysosome fusion. TECPR1 and Atg16 form mutually exclusive complexes with the Atg12-Atg5 conjugate, and TECPR1 binds PtdIns(3)P upon association with the Atg12-Atg5 conjugate. Strikingly, TECPR1 localizes to and recruits Atg5 to autolysosome membrane. Consequently, elimination of TECPR1 leads to accumulation of autophagosomes and blocks autophagic degradation of LC3-II and p62. Finally, autophagosome maturation marked by GFP-mRFP-LC3 is defective in TECPR1-deficient cells. Thus, we propose that the concerted interactions among TECPR1, Atg12-Atg5, and PtdIns(3)P provide the fusion specificity between autophagosomes and lysosomes and that the assembly of this complex initiates the autophagosome maturation process.     

Receptor‐mediated clustering of FIP200 bypasses the role of LC3 lipidation in autophagy

Autophagosome formation requires multiple autophagy‐related (ATG) factors. However, we find that a subset of autophagy substrates remains robustly targeted to the lysosome in the absence of several core ATGs, including the LC3 lipidation machinery. To address this unexpected result, we performed genome‐wide CRISPR screens identifying genes required for NBR1 flux in ATG7^KO cells. We find that ATG7‐independent autophagy still requires canonical ATG factors including FIP200. However, in the absence of LC3 lipidation, additional factors are required including TAX1BP1 and TBK1. TAX1BP1''s ability to cluster FIP200 around NBR1 cargo and induce local autophagosome formation enforces cargo specificity and replaces the requirement for lipidated LC3. In support of this model, we define a ubiquitin‐independent mode of TAX1BP1 recruitment to NBR1 puncta, highlighting that TAX1BP1 recruitment and clustering, rather than ubiquitin binding per se, is critical for function. Collectively, our data provide a mechanistic basis for reports of selective autophagy in cells lacking the lipidation machinery, wherein receptor‐mediated clustering of upstream autophagy factors drives continued autophagosome formation.     

RNA-binding protein,human antigen R regulates hypoxia-induced autophagy by targeting ATG7/ATG16L1 expressions and autophagosome formation

Autophagy, a prosurvival mechanism offers a protective role during acute kidney injury. We show novel findings on the functional role of RNA binding protein, HuR during hypoxia-induced autophagy in renal proximal tubular cells-2 (HK-2). HK-2 cells showed upregulated expressions of HuR and autophagy-related proteins such as autophagy related 7 (ATG7), autophagy related 16 like 1 (ATG16L1), and LC3II under hypoxia. Increased autophagosome formation was visualized as LC3 puncta in hypoxic cells. Further, short hairpin-RNA-mediated loss of HuR function in HK-2 cells significantly decreased ATG7 and ATG16L1 protein expressions. Bioinformatics prediction revealed HuR motif binding on the coding region of ATG7 and AU-rich element at 3′UTR ATG16L1 messnger RNA (mRNA). The RNA immunoprecipitation study showed that HuR was predominantly associated with ATG7 and ATG16L1 mRNAs under hypoxia. In addition, HuR enhanced autophagosome formation by regulating LC3II expressions. These results show that HuR regulates ATG7 and ATG16L1 expressions and thereby mediate autophagy in HK-2 cells. Importantly, HuR knockdown cells underwent apoptosis during hypoxia as observed through the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Collectively, these findings show the crucial role of HuR under hypoxia by regulating autophagy and suppressing apoptosis in renal tubular cells.     

SNX18 regulates ATG9A trafficking from recycling endosomes by recruiting Dynamin‐2

Trafficking of mammalian ATG9A between the Golgi apparatus, endosomes and peripheral ATG9A compartments is important for autophagosome biogenesis. Here, we show that the membrane remodelling protein SNX18, previously identified as a positive regulator of autophagy, regulates ATG9A trafficking from recycling endosomes. ATG9A is recruited to SNX18‐induced tubules generated from recycling endosomes and accumulates in juxtanuclear recycling endosomes in cells lacking SNX18. Binding of SNX18 to Dynamin‐2 is important for ATG9A trafficking from recycling endosomes and for formation of ATG16L1‐ and WIPI2‐positive autophagosome precursor membranes. We propose a model where upon autophagy induction, SNX18 recruits Dynamin‐2 to induce budding of ATG9A and ATG16L1 containing membranes from recycling endosomes that traffic to sites of autophagosome formation.     

COPII囊泡衣被蛋白SEC24A在巨自噬通路中的功能研究

细胞自噬是一种重要且保守的细胞内降解过程,通过形成双层膜的自噬体包裹细胞内容物进行降解。内质网来源的COPII囊泡被认为是饥饿诱导的应激过程中自噬体的膜源。探究了COPII囊泡衣被蛋白SEC24A在巨自噬通路中的作用。利用siRNA干扰技术敲低SEC24A的表达,EBSS饥饿处理对照组和SEC24A敲低组HeLa细胞2 h诱导自噬发生,经Western blot和免疫荧光实验检测自噬底物蛋白p62和自噬标志蛋白LC3-II的蛋白水平变化,以确定SEC24A是否参与自噬。通过RFP-GFP-LC3串联荧光检测自噬体和自噬溶酶体的数目,利用蛋白酶K保护实验验证自噬缺陷发生在自噬体闭合之前或者之后,利用免疫荧光实验检测敲低SEC24A对自噬通路上ATG复合物的影响,以确定SEC24A调控自噬通路的位点。通过免疫共沉淀实验验证SEC24A与自噬相关蛋白ATG9A是否存在相互作用。蛋白检测实验发现,饥饿条件下与对照细胞相比,敲低SEC24A细胞内自噬底物蛋白p62积累,而标志蛋白LC3-II减少。RFP-GFP-LC3串联荧光实验显示,敲低SEC24A后自噬体及自噬溶酶体的数目均减少。蛋白酶K保护实验显示,SEC24A敲低细胞中受膜结构保护的p62和GFP-LC3均减少,提示SEC24A作用位点在自噬体闭合之前。免疫荧光实验显示,敲低SEC24A的表达后ATG14L、ATG16L1点状结构减少,而ATG9A点状结构的数量没有明显变化,提示SEC24A作用于ATG14L、ATG16L1上游。免疫共沉淀实验显示SEC24A与ATG9A存在相互作用。研究结果不仅有助于深化对自噬体形成过程和分子机制的了解,也为全面解读COPII囊泡及其衣被蛋白在自噬中的重要作用提供了信息。     

The plasma membrane as a control center for autophagy

Autophagosomes may derive membrane from diverse sources, including the plasma membrane, Golgi, endoplasmic reticulum and mitochondria. The plasma membrane contributes membrane to ATG12–ATG5-ATG16L1-positive phagophore precursor vesicles (LC3-negative) by both clathrin-dependent and -independent routes. We recently observed that ARF6 regulates autophagy and that this could be explained, at least in part, by its role in the generation of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂], which influences endocytic uptake of plasma membrane into autophagosome precursors. The subsequent maturation of these small phagophore precursors into phagophores (ATG12–ATG5-ATG16L1-positive and LC3-positive), is assisted by SNARE-mediated homotypic fusion that increase their size and enhance their ability to acquire LC3-II. It appears that a plasma membrane-derived pool of VAMP7 is a key mediator of these fusion events. Thus, events at the plasma membrane may regulate distinct steps in the biogenesis of phagophores.     

The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14

ULK1 (unc-51 like autophagy activating kinase 1), the key mediator of MTORC1 signaling to autophagy, regulates early stages of autophagosome formation in response to starvation or MTORC1 inhibition. How ULK1 regulates the autophagy induction process remains elusive. Here, we identify that ATG13, a binding partner of ULK1, mediates interaction of ULK1 with the ATG14-containing PIK3C3/VPS34 complex, the key machinery for initiation of autophagosome formation. The interaction enables ULK1 to phosphorylate ATG14 in a manner dependent upon autophagy inducing conditions, such as nutrient starvation or MTORC1 inhibition. The ATG14 phosphorylation mimics nutrient deprivation through stimulating the kinase activity of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex and facilitates phagophore and autophagosome formation. By monitoring the ATG14 phosphorylation, we determined that the ULK1 activity requires BECN1/Beclin 1 but not the phosphatidylethanolamine (PE)-conjugation machinery and the PIK3C3 kinase activity. Monitoring the phosphorylation also allowed us to identify that ATG9A is required to suppress the ULK1 activity under nutrient-enriched conditions. Furthermore, we determined that ATG14 phosphorylation depends on ULK1 and dietary conditions in vivo. These results define a key molecular event for the starvation-induced activation of the ATG14-containing PtdIns3K complex by ULK1, and demonstrate hierarchical relations between the ULK1 activation and other autophagy proteins involved in phagophore formation.