The role of Tecpr1 in selective autophagy as a cargo receptor

Bacterial intrusion of host cells can be recognized by the innate immune system, including autophagy, via multiple cellular pathways. We have identified Tecpr1 as an Atg5-binding partner, and found that Tecpr1 interacts with the Atg12-Atg5-Atg16L1 complex via binding to Atg5. In Shigella infected cells, Tecpr1 colocalizes with Atg5 and LC3 at Shigella-containing phagophores. Tecpr1 activity is required for efficient autophagy to target bacteria, but a deficiency of Tecpr1 in host cells does not have a marked effect on canonical autophagy. Tecpr1 plays an important role in promoting selective autophagy via the WIPI-2-Tecpr1-Atg5 pathway in targeting bacteria, protein aggregates and damaged mitochondria.     

A Tecpr1-dependent selective autophagy pathway targets bacterial pathogens

Selective autophagy of bacterial pathogens represents a host innate immune mechanism. Selective autophagy has been characterized on the basis of distinct cargo receptors but the mechanisms by which different cargo receptors are targeted for autophagic degradation remain unclear. In this study we identified a highly conserved Tectonin domain-containing protein, Tecpr1, as an Atg5 binding partner that colocalized with Atg5 at Shigella-containing phagophores. Tecpr1 activity is necessary for efficient autophagic targeting of bacteria, but has no effect on rapamycin- or starvation-induced canonical autophagy. Tecpr1 interacts with WIPI-2, a yeast Atg18 homolog and PI(3)P-interacting protein required for phagophore formation, and they colocalize to phagophores. Although Tecpr1-deficient mice appear normal, Tecpr1-deficient MEFs were defective for selective autophagy and supported increased intracellular multiplication of Shigella. Further, depolarized mitochondria and misfolded protein aggregates accumulated in the Tecpr1-knockout MEFs. Thus, we identify a Tecpr1-dependent pathway as important in targeting bacterial pathogens for selective autophagy.     

Nondegradative role of Atg5-Atg12/ Atg16L1 autophagy protein complex in antiviral activity of interferon gamma

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.     

Atg17 functions in cooperation with Atg1 and Atg13 in yeast autophagy

In eukaryotic cells, nutrient starvation induces the bulk degradation of cellular materials; this process is called autophagy. In the yeast Saccharomyces cerevisiae, most of the ATG (autophagy) genes are involved in not only the process of degradative autophagy, but also a biosynthetic process, the cytoplasm to vacuole (Cvt) pathway. In contrast, the ATG17 gene is required specifically in autophagy. To better understand the function of Atg17, we have performed a biochemical characterization of the Atg17 protein. We found that the atg17delta mutant under starvation condition was largely impaired in autophagosome formation and only rarely contained small autophagosomes, whose size was less than one-half of normal autophagosomes in diameter. Two-hybrid analyses and coimmunoprecipitation experiments demonstrated that Atg17 physically associates with Atg1-Atg13 complex, and this binding was enhanced under starvation conditions. Atg17-Atg1 binding was not detected in atg13delta mutant cells, suggesting that Atg17 interacts with Atg1 through Atg13. A point mutant of Atg17, Atg17(C24R), showed reduced affinity for Atg13, resulting in impaired Atg1 kinase activity and significant defects in autophagy. Taken together, these results indicate that Atg17-Atg13 complex formation plays an important role in normal autophagosome formation via binding to and activating the Atg1 kinase.     

Beyond Atg8 binding: The role of AIM/LIR motifs in autophagy

Selective macroautophagy/autophagy mediates the selective delivery of cytoplasmic cargo material via autophagosomes into the lytic compartment for degradation. This selectivity is mediated by cargo receptor molecules that link the cargo to the phagophore (the precursor of the autophagosome) membrane via their simultaneous interaction with the cargo and Atg8 proteins on the membrane. Atg8 proteins are attached to membrane in a conjugation reaction and the cargo receptors bind them via short peptide motifs called Atg8-interacting motifs/LC3-interacting regions (AIMs/LIRs). We have recently shown for the yeast Atg19 cargo receptor that the AIM/LIR motifs also serve to recruit the Atg12–Atg5-Atg16 complex, which stimulates Atg8 conjugation, to the cargo. We could further show in a reconstituted system that the recruitment of the Atg12–Atg5-Atg16 complex is sufficient for cargo-directed Atg8 conjugation. Our results suggest that AIM/LIR motifs could have more general roles in autophagy.     

Structure of Atg5.Atg16, a complex essential for autophagy

Atg5 is covalently modified with a ubiquitin-like modifier, Atg12, and the Atg12-Atg5 conjugate further forms a complex with the multimeric protein Atg16. The Atg12-Atg5.Atg16 multimeric complex plays an essential role in autophagy, the bulk degradation system conserved in all eukaryotes. We have reported here the crystal structure of Atg5 complexed with the N-terminal region of Atg16 at 1.97A resolution. Atg5 comprises two ubiquitin-like domains that flank a helix-rich domain. The N-terminal region of Atg16 has a helical structure and is bound to the groove formed by these three domains. In vitro analysis showed that Arg-35 and Phe-46 of Atg16 are crucial for the interaction. Atg16, with a mutation at these residues, failed to localize to the pre-autophagosomal structure and could not restore autophagy in Atg16-deficient yeast strains. Furthermore, these Atg16 mutants could not restore a severe reduction in the formation of the Atg8-phosphatidylethanolamine conjugate, another essential factor for autophagy, in Atg16-deficient strains under starvation conditions. These results taken together suggest that the direct interaction between Atg5 and Atg16 is crucial to the performance of their roles in autophagy.     

Structure-function relationship of Atg12, a ubiquitin-like modifier essential for autophagy

Atg12, a post-translational modifier, is activated and conjugated to Atg5 by a ubiquitin-like conjugation system, though it has no obvious sequence homology to ubiquitin. The Atg12-Atg5 conjugate is essential for autophagy, an intracellular bulk degradation process. Here, we show that the carboxyl-terminal region of Atg12 that is predicted to fold into a ubiquitin-like structure is necessary and sufficient for both conjugation and autophagy, which indicates that the domain essential for autophagy resides in the ubiquitin-fold region. We further show that two hydrophobic residues within the ubiquitin-fold region are important for autophagy: mutation at Y149 affects conjugate formation catalyzed by Atg10, an E2-like enzyme, while mutation at F154 has no effect on Atg12-Atg5 conjugate formation but its hydrophobic nature is essential for autophagy. In response to the F154 mutation, Atg8-PE conjugation, the other ubiquitin-like conjugation in autophagy, is severely reduced and autophagosome formation fails. Gel filtration analysis suggests that F154 plays a critical role in the assembly of a functional Atg12-Atg5.Atg16 complex that is requisite for autophagosome formation.     

Structure-Function Relationship of Atg12, a Ubiquitin-Like Modifier Essential for Autophagy

Atg12, a post-translational modifier, is activated and conjugated to Atg5 by a ubiquitin-like conjugation system, though it has no obvious sequence homology to ubiquitin. The Atg12-Atg5 conjugate is essential for autophagy, an intracellular bulk degradation process. Here, we show that the carboxyl-terminal region of Atg12 that is predicted to fold into a ubiquitin-like structure is necessary and sufficient for both conjugation and autophagy, which indicates that the domain essential for autophagy resides in the ubiquitin-fold region. We further show that two hydrophobic residues within the ubiquitin-fold region are important for autophagy: mutation at Y149 affects conjugate formation catalyzed by Atg10, an E2-like enzyme, while mutation at F154 has no effect on Atg12-Atg5 conjugate formation but its hydrophobic nature is essential for autophagy. In response to the F154 mutation, Atg8-PE conjugation, the other ubiquitin-like conjugation in autophagy, is severely reduced and autophagosome formation fails. Gel filtration analysis suggests that F154 plays a critical role in the assembly of a functional Atg12-Atg5?Atg16 complex that is requisite for autophagosome formation.     

The role of Atg29 phosphorylation in PAS assembly

Macroautophagy (hereafter autophagy) initiates at the phagophore assembly site (PAS), where most of the AuTophaGy-related (Atg) proteins are at least transiently localized. As the first protein complex targeted to the PAS, the Atg17-Atg31-Atg29 complex serves as the scaffold for other Atg proteins and plays a critical role for the organization of the PAS, and in autophagy initiation. We recently showed that this complex is constitutively formed and activated by the phosphorylation of Atg29 when autophagy is induced. Phosphorylation of Atg29 is required for its interaction with Atg11, another scaffold protein, and its function for promoting the proper assembly of the PAS. Single-particle electron microscopy analysis of the Atg17-Atg31-Atg29 complex reveals an elongated structure with Atg29 located at the opposing ends. This structural arrangement allows Atg29 to interact with Atg11, and is critical in the organization of the intact Atg1 complex.     

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.     

牦牛Atg5基因克隆及其在输卵管、卵巢和子宫中的表达定位

自噬是一种保守的细胞内降解过程,在多种生物体内证实自噬有重要的生物学意义。但是自噬在牦牛这一高原特色物种体内的研究未见报道。因此为探索正常生理条件下自噬相关基因在牦牛生殖过程中的表达,本研究分别采集牦牛不同繁殖周期（卵泡期、黄体期及妊娠期）的主要生殖器官（输卵管、卵巢、子宫）,克隆牦牛自噬相关基因5（Autophagy related 5,Atg5）基因并进行生物信息学分析;利用qRT-PCR检测Atg5 基因在组织中的相对表达量;并采用蛋白质免疫印迹（Western-blot, WB）检测Atg5和Atg5-Atg12（Autophagy related 12,自噬相关基因12）复合体在不同组织中的表达水平;免疫组织化学方法分析Atg5在各生殖器官中的分布特征。结果显示,成功克隆牦牛Atg5基因在进化过程中高度保守,编码的蛋白质为可溶性的非跨膜蛋白;qRT-PCR和WB检测结果显示Atg5和Atg5-Atg12复合体在牦牛输卵管、卵巢和子宫中均有表达。其中卵泡期卵巢Atg5-Atg12表达显著高于黄体期和妊娠期,卵泡期Atg5的表达却显著低于黄体期和妊娠期;黄体期输卵管Atg5-Atg12表达量显著高于卵泡期和妊娠期,妊娠期输卵管中Atg5的表达量显著高于卵泡期和黄体期;卵泡期和妊娠期子宫中Atg5-Atg12的表达量显著高于黄体期,而黄体期子宫中的Atg5的表达量又显著高于妊娠期和卵泡期,在蛋白水平上Atg5和Atg5-Atg12的表达呈负相关。免疫组织化学结果显示Atg5在输卵管黏膜上皮,卵巢卵泡膜、颗粒层、生殖上皮、黄体细胞,子宫内膜和子宫腺体均有表达。研究结果表明自噬在牦牛体内与其他物种相似具有保守性,通过检测Atg5-Atg12复合体在牦牛生殖器官中的表达,推测自噬可能参与牦牛生殖生理过程的调控。该研究结果对自噬在其他大型哺乳动物以及高寒低氧环境中动物的研究具有借鉴意义,有助于自噬参与其他动物生殖生理作用机制的研究。       

Phosphorylation of Serine 114 on Atg32 mediates mitophagy

Mitophagy, which selectively degrades mitochondria via autophagy, has a significant role in mitochondrial quality control. When mitophagy is induced in yeast, mitochondrial residential protein Atg32 binds Atg11, an adaptor protein for selective types of autophagy, and it is recruited into the vacuole along with mitochondria. The Atg11-Atg32 interaction is believed to be the initial molecular step in which the autophagic machinery recognizes mitochondria as a cargo, although how this interaction is mediated is poorly understood. Therefore, we studied the Atg11-Atg32 interaction in detail. We found that the C-terminus region of Atg11, which included the fourth coiled-coil domain, interacted with the N-terminus region of Atg32 (residues 100-120). When mitophagy was induced, Ser-114 and Ser-119 on Atg32 were phosphorylated, and then the phosphorylation of Atg32, especially phosphorylation of Ser-114 on Atg32, mediated the Atg11-Atg32 interaction and mitophagy. These findings suggest that cells can regulate the amount of mitochondria, or select specific mitochondria (damaged or aged) that are degraded by mitophagy, by controlling the activity and/or localization of the kinase that phosphorylates Atg32. We also found that Hog1 and Pbs2, which are involved in the osmoregulatory signal transduction cascade, are related to Atg32 phosphorylation and mitophagy.     

The non-canonical role of Atg family members as suppressors of innate antiviral immune signaling

Recent research on autophagy clearly demonstrates that the autophagosome-lysosome pathway plays essential roles in elimination of certain pathogens such as group A Streptococcus, Mycobacterium tuberculosis, Listeria monocytogenes, and herpesvirus. (1-4) We have recently found that a key regulator of the autophagic process, the Atg12-Atg5 conjugate, associates with the signaling molecules retinoic acid-inducible gene I (RIG-I) and interferon-beta promoter stimulator 1 (IPS-1), which are essential for recognition of RNA virus infection and which transmit signals to upregulate type I interferons (IFNs). Interestingly, the Atg12-Atg5 conjugate seemed to negatively regulate the type I IFN modulating pathway through direct interaction with caspase recruitment domains (CARDs) presented by RIG-1 and IPS-1.(5) Thus, in contrast to the bactericidal properties of autophagic processes, the autophagy regulator (the Atg12-Atg5 conjugate) appeared to promote RNA virus replication by inhibiting innate anti-virus immune responses. In this addendum, we discuss the non-canonical role of the Atg12-Atg5 conjugate as a suppressor of innate immune responses.     

Molecular interactions of the Saccharomyces cerevisiae Atg1 complex provide insights into assembly and regulatory mechanisms

The Atg1 complex, which contains 5 major subunits: Atg1, Atg13, Atg17, Atg29, and Atg31, regulates the induction of autophagy and autophagosome formation. To gain a better understanding of the overall architecture and assembly mechanism of this essential autophagy regulatory complex, we have reconstituted a core assembly of the Saccharomyces cerevisiae Atg1 complex composed of full-length Atg17, Atg29, and Atg31, along with the C-terminal domains of Atg1 (Atg1[CTD]) and Atg13 (Atg13[CTD]). Using chemical-crosslinking coupled with mass spectrometry (CXMS) analysis we systematically mapped the intersubunit interaction interfaces within this complex. Our data revealed that the intrinsically unstructured C-terminal domain of Atg29 interacts directly with Atg17, whereas Atg17 interacts with Atg13 in 2 distinct intrinsically unstructured regions, including a previously unknown motif that encompasses several putative phosphorylation sites. The Atg1[CTD] crosslinks exclusively to the Atg13[CTD] and does not appear to make direct contact with the Atg17-Atg31-Atg29 scaffold. Finally, single-particle electron microscopy analysis revealed that both the Atg13[CTD] and Atg1[CTD] localize to the tip regions of Atg17-Atg31-Atg29 and do not alter the distinct curvature of this scaffolding subcomplex. This work provides a comprehensive understanding of the subunit interactions in the fully assembled Atg1 core complex, and uncovers the potential role of intrinsically disordered regions in regulating complex integrity.     

ER stress (PERK/eIF2alpha phosphorylation) mediates the polyglutamine-induced LC3 conversion, an essential step for autophagy formation

Expanded polyglutamine 72 repeat (polyQ72) aggregates induce endoplasmic reticulum (ER) stress-mediated cell death with caspase-12 activation and vesicular formation (autophagy). We examined this relationship and the molecular mechanism of autophagy formation. Rapamycin, a stimulator of autophagy, inhibited the polyQ72-induced cell death with caspase-12 activation. PolyQ72, but not polyQ11, stimulated Atg5-Atg12-Atg16 complex-dependent microtubule-associated protein 1 (MAP1) light chain 3 (LC3) conversion from LC3-I to -II, which plays a key role in autophagy. The eucaryotic translation initiation factor 2 alpha (eIF2alpha) A/A mutation, a knock-in to replace a phosphorylatable Ser51 with Ala51, and dominant-negative PERK inhibited polyQ72-induced LC3 conversion. PolyQ72 as well as ER stress stimulators upregulated Atg12 mRNA and proteins via eIF2alpha phosphorylation. Furthermore, Atg5 deficiency as well as the eIF2alpha A/A mutation increased the number of cells showing polyQ72 aggregates and polyQ72-induced caspase-12 activation. Thus, autophagy formation is a cellular defense mechanism against polyQ72-induced ER-stress-mediated cell death by degrading polyQ72 aggregates, with PERK/eIF2alpha phosphorylation being involved in polyQ72-induced LC3 conversion.     

How the Atg1 complex assembles to initiate autophagy

The Atg1 complex, comprising Atg1, Atg13, Atg17, Atg29, and Atg31, is a key initiator of autophagy. The Atg17-Atg31-Atg29 subcomplex is constitutively present at the phagophore assembly site (PAS), while Atg1 and Atg13 join the complex when autophagy is triggered by starvation or other signals. We sought to understand the energetics and dynamics of assembly using isothermal titration calorimetry (ITC), sedimentation velocity analytical ultracentrifugation, and hydrogen-deuterium exchange (HDX). We showed that the membrane and Atg13-binding domain of Atg1, Atg1_EAT, is dynamic on its own, but is rigidified in its high-affinity (∼100 nM) complex with Atg13. Atg1_EAT and Atg13 form a 2:2 dimeric assembly and together associate with lower affinity (∼10 μM) with the 2:2:2 Atg17-Atg31-Atg29 complex. These results lead to an overall model for the assembly pathway of the Atg1 complex. The model highlights the Atg13-Atg17 binding event as the weakest link in the assembly process and thus as a natural regulatory checkpoint.     

The Atg18-Atg2 complex is recruited to autophagic membranes via phosphatidylinositol 3-phosphate and exerts an essential function

Atg18 is essential for both autophagy and the regulation of vacuolar morphology. The latter process is mediated by phosphatidylinositol 3,5-bisphosphate binding, which is dispensable for autophagy. Atg18 also binds to phosphatidylinositol 3-phosphate (PtdIns(3)P) in vitro. Here, we investigate the relationship between PtdIns(3)P-binding of Atg18 and autophagy. Using an Atg18 variant, Atg18(FTTG), which is unable to bind phosphoinositides, we found that PtdIns(3)P binding of Atg18 is essential for full activity in both selective and nonselective autophagy. Atg18(FTTG) formed a complex with Atg2 in a normal manner, and Atg18-Atg2 complex formation occurred in cells in the absence of PtdIns(3)P, indicating that Atg18-Atg2 complex formation is independent of PtdIns(3)P-binding of Atg18. Atg18 localized to endosomes, the vacuolar membrane, and autophagic membranes, whereas Atg18(FTTG) did not localize to these structures. The localization of Atg2 to autophagic membranes was also lost in Atg18(FTTG) cells. These data indicate that PtdIns(3)P-binding of Atg18 is involved in directing the Atg18-Atg2 complex to autophagic membranes. Connection of a 2xFYVE domain, a specific PtdIns(3)P-binding domain, to the C terminus of Atg18(FTTG) restored the localization of Atg18-Atg2 to autophagic membranes and full autophagic activity, indicating that PtdIns(3)P-binding by Atg18 is dispensable for the function of the Atg18-Atg2 complex but is required for its localization. This also suggests that PtdIns(3)P does not act allosterically on Atg18. Taken together, Atg18 forms a complex with Atg2 irrespective of PtdIns(3)P binding, associates tightly to autophagic membranes by interacting with PtdIns(3)P, and plays an essential role.     

How the Atg1 complex assembles to initiate autophagy

The Atg1 complex, comprising Atg1, Atg13, Atg17, Atg29, and Atg31, is a key initiator of autophagy. The Atg17-Atg31-Atg29 subcomplex is constitutively present at the phagophore assembly site (PAS), while Atg1 and Atg13 join the complex when autophagy is triggered by starvation or other signals. We sought to understand the energetics and dynamics of assembly using isothermal titration calorimetry (ITC), sedimentation velocity analytical ultracentrifugation, and hydrogen-deuterium exchange (HDX). We showed that the membrane and Atg13-binding domain of Atg1, Atg1_EAT, is dynamic on its own, but is rigidified in its high-affinity (～100 nM) complex with Atg13. Atg1_EAT and Atg13 form a 2:2 dimeric assembly and together associate with lower affinity (～10 μM) with the 2:2:2 Atg17-Atg31-Atg29 complex. These results lead to an overall model for the assembly pathway of the Atg1 complex. The model highlights the Atg13-Atg17 binding event as the weakest link in the assembly process and thus as a natural regulatory checkpoint.     

Dimeric Coiled-coil Structure of Saccharomyces cerevisiae Atg16 and Its Functional Significance in Autophagy

Atg16 interacts with the Atg12-Atg5 protein conjugate through its N-terminal domain and self-assembles through its coiled-coil domain (CCD). Formation of the Atg12-Atg5·Atg16 complex is essential for autophagy, the bulk degradation process conserved among most eukaryotes. Here, we report the crystal structures of full-length Saccharomyces cerevisiae Atg16 at 2.8 Å resolution and its CCD at 2.5 Å resolution. The CCD and full-length Atg16 each exhibit an extended α-helix, 90 and 130 Å, respectively, and form a parallel coiled-coil dimer in the crystals. Although the apparent molecular weight of Atg16 observed by gel-filtration chromatography suggests that Atg16 is tetrameric, an analytical ultracentrifugation study showed Atg16 as a dimer in solution, consistent with the crystal structure. Evolutionary conserved surface residues clustered at the C-terminal half of Atg16 CCD were shown to be crucial for autophagy. These results will give a structural basis for understanding the molecular functions and significance of Atg16 in autophagy.