The machinery of macroautophagy

Terms and structures

The most readily identifiable feature of macroautophagy is the sequestration of cargo within cytosolic double-membrane vesicles, and many researchers still consider the analysis of macroautophagy by electron microscopy to be the “gold standard” for verifying macroautophagy activity. Below, we present some of the most common terms describing the principle structures of macroautophagy (Figure 1).

Open in a separate window

Figure 1

Schematic depiction of the two main types of yeast autophagy. In macroautophagy, random cytoplasm and dysfunctional organelles are sequestered by the expanding phagophore, leading to the formation of the autophagosome. The autophagosome subsequently fuses with the vacuole membrane, releasing the autophagic body into the vacuole lumen. Eventually, the sequestered cargos are degraded or processed by vacuolar hydrolases. In microautophagy, cargos are directly taken up by the invagination of the vacuole membrane, followed by scission, and subsequent lysis, exposing the cargo to vacuolar hydrolases for degradation.

Autophagic body: The inner-membrane-bound structure released into the vacuole lumen after the outer membrane of the autophagosome fuses with vacuolar membrane⁴. Note that autophagic bodies only appear in yeast and plants due to the large size of the vacuole, and are not found in mammalian lysosomes.

Autophagosome: The completed double-membrane-bound compartment, the product of phagophore expansion and closure, which sequesters cytoplasmic cargos during macroautophagy^5,6.

PAS: The phagophore assembly site; a peri-vacuolar location or compartment where the nucleation of the phagophore initiates. Most components of the macroautophagy core machinery locate at least transiently at the PAS in yeast; however, a mammalian equivalent of the PAS has not been identified^7,8,9.

Phagophore: The double-membrane structure that functions in the initial sequestering of cargo, and thus the active compartment of macroautophagy. The phagophore further elongates/expands and ultimately closes, generating a completed autophagosome¹⁰.

The history of discovery

The term “autophagy” was coined by Christian de Duve at the CIBA Foundation Symposium on Lysosomes in 1963. This was based on his discovery of lysosomes in 1955, which won him the Nobel Prize in Physiology or Medicine in 1974¹¹. Autophagy was named morphologically by the observations from electron microscopy of rat hepatic cell lysosomes, where single membrane vesicles, containing cytoplasm or organelles such as mitochondria and endoplasmic reticulum (ER) were observed^12,13,14,15. Based on Thomas Ashford's and Keith Porter's early findings in 1962 that revealed the presence of sequestered organelles in rat hepatocytes following their exposure to glucagon¹², de Duve and his colleagues confirmed that this hormone can induce autophagy¹⁶. Subsequently, other researchers further examined the hormonal and enzymatic regulation of autophagy. For instance, Per Seglen and Paul Gordon discovered the inhibitory role of 3-methyladenine¹⁷. As de Duve mentioned in 1966 that autophagy could be selective¹⁴, the specific sequestration of organelles including ER¹⁸, mitochondria¹⁹ and peroxisomes²⁰ by autophagy was demonstrated in the following decade. Although autophagy was initially revealed in mammalian systems, the molecular understanding of it was largely expanded and facilitated through genetic studies in yeast. Genetic screens of macroautophagy-defective mutants in Saccharomyces cerevisiae were carried out by the Ohsumi and Thumm laboratories^21,22. Shortly thereafter, the first autophagy-specific gene, APG1 (now ATG1²³), was identified, and the corresponding gene product, Apg1/Atg1, was characterized as a Ser/Thr protein kinase²⁴. Multiple laboratories working on autophagy primarily in S. cerevisiae, Pichia pastoris and Hansenula polymorpha subsequently discovered more than thirty autophagy-related (ATG) genes. The molecular basis of autophagy was first connected to human diseases by Beth Levine's laboratory through the identification of BECN1/VPS30/ATG6 as a tumor suppressor gene²⁵. Subsequently, a series of studies uncovered the connections between autophagy and pathophysiological conditions, such as pathogen infection^26,27,28,29 and neurodegeneration³⁰, and its dual role in cell growth and death^31,32.

Yeast Atg1 kinase complex

The Atg1 kinase complex regulates the magnitude of autophagy^{58,59,60,61,62}. This complex also plays a role in mediating the retrieval of Atg9 from the PAS⁶³. The Atg1 kinase complex is extensively regulated, receiving input from several signaling pathways. In yeast, much of the regulation centers around nutrient signaling and involves kinases that sense the nutritional status of the cell including the target of rapamycin (TOR), protein kinase A (PKA), Gcn2 and Snf1. The yeast Atg1 kinase complex includes Atg1, a Ser/Thr protein kinase, Atg13, a regulatory subunit, and the Atg17-Atg31-Atg29 complex, which may function in part as a scaffold (Figure 2). Atg1 is the only kinase of the core autophagy machinery; however, despite the fact that it was the first Atg protein to be identified, the key physiological substrate of yeast Atg1 is not known. Atg1 kinase activity is required for autophagosome formation and the Cvt pathway, although there are conflicting data concerning the changes in kinase activity when yeast cells shift from vegetative growth to autophagy-inducing conditions^24,64,65. Atg1 recruitment to the PAS may be regulated by PKA-dependent phosphorylation⁶⁶. TOR also phosphorylates Atg1, and autophosphorylation is important in regulating Atg1 kinase activity^67,68.

Open in a separate window

Figure 2

Yeast Atg1 kinase complex. The yeast Atg1 kinase complex includes Atg1, a Ser/Thr protein kinase, Atg13, a regulatory subunit required for Atg1 kinase activity, and the Atg17-Atg31-Atg29 complex, which may function in part as a scaffold. Atg31 bridges Atg17 and Atg29, Atg17 binds Atg13, and Atg29 binds Atg11.

Atg13 is required for Atg1 kinase activity. Atg13 is hyperphosphorylated under nutrient-rich conditions, and its phosphorylation is regulated by TOR complex 1 (TORC1)⁶⁹ and/or PKA⁷⁰. Atg13 is rapidly, but only partially, dephosphorylated upon autophagy induction⁷¹. These data, coupled with affinity isolation studies, led to a model suggesting that the interaction between Atg1 and Atg13 is controlled by Atg13 phosphorylation — dephosphorylation would lead to the formation of an Atg1-Atg13 complex, and subsequent activation of Atg1 kinase activity⁶¹. Recent data, however, suggest that Atg1 and Atg13 interact in a constitutive manner⁷², which is similar to the situation in other organisms.

Atg17-Atg31-Atg29 exists as a stable complex under both vegetative and starvation conditions, suggesting that formation of the complex is not involved in autophagy induction. Atg31 bridges Atg17 and Atg29, Atg17 binds Atg13, and Atg29 binds Atg11; the latter is a scaffold protein that may also be considered part of the Atg1 kinase complex because it binds Atg1 and is involved in the transition of the PAS that occurs during the switch from vegetative to starvation conditions^{40,41,58,73,74,75}. Both Atg29 and Atg31 are phosphoproteins. The phosphorylation of Atg29 appears to be involved in regulation and is proposed to alter the conformation of an inhibitory C-terminal peptide to allow the protein to become active⁴¹.

Mammalian ULK1/2 Complex

The mammalian ULK1/2 complex includes ULK1/2 (mammalian homologs of Atg1), ATG13 (a homolog of yeast Atg13), RB1CC1/FIP200 (a putative Atg17 homolog) and C12orf44/ATG101 (the latter component is not conserved in S. cerevisiae)^{76,77,78,79,80}. ULK1/2 interact with ATG13, which directly binds RB1CC1 and mediates the latter's interaction with the ULKs^76,81. In mammalian cells, ULK1 kinase can be activated by both AMP-activated protein kinase (AMPK)-dependent (glucose starvation) and -independent (amino acid starvation) processes⁸². The ULK1/2-ATG13-RB1CC1 interaction is nutrient independent, forming a complex even in nutrient-rich conditions⁸⁰. In this situation, MTORC1 phosphorylates and inhibits ULK1/2 and ATG13, disrupting the interaction between ULK1 and AMPK⁸². In contrast, under autophagy-inducing conditions, MTOR is released from the complex, resulting in activation of ULK1/2, which phosphorylates, and presumably activates, ATG13 and RB1CC1. AMBRA1 and BECN1, components of the autophagy-promoting PtdIns3K complexes, are also phosphorylated by ULK1; these modifications result in localization of the lipid kinase complex to the ER and its activation^83,84.

Yeast Atg9

In yeast cells, Atg9 is a transmembrane protein that transits between the PAS and peripheral sites (Figure 3); the latter have been termed Atg9 reservoirs or tubulovesicular clusters, and are proposed to represent sites from which membrane is delivered to the forming phagophore^49,63,85, although the exact role of Atg9 in this process is not clear. Atg9 is proposed to consist of six transmembrane domains, with both the amino and carboxyl termini exposed in the cytosol. Atg9 self-interacts and may exist in a complex^85,86.

Open in a separate window

Figure 3

Trafficking of yeast Atg9. In yeast cells, Atg9 is a transmembrane protein that transits between the PAS and peripheral sites (Atg9 reservoirs). Atg9 localization to the PAS from the peripheral sites depends on Atg11, Atg23 and Atg27. Return to the peripheral sites requires Atg1-Atg13, Atg2 and Atg18; PAS recruitment of the latter involves binding to PtdIns3P, the product of the class III PtdIns3K complex.

Atg9 localization to the PAS from the peripheral sites (referred to as anterograde transport) depends on Atg11, Atg23 and Atg27^63,87,88,89. Return to the peripheral sites (retrograde movement) requires Atg1-Atg13, Atg2 and Atg18; PAS recruitment of the latter involves binding to PtdIns3P and thus necessitates the function of Atg14 and the class III PtdIns3K complex⁶³. Atg27 is a type I integral transmembrane protein, while the other components involved in Atg9 movement are soluble and/or peripherally associated with membranes.

Mammalian ATG9

The mammalian Atg9 homolog ATG9A localizes to the trans-Golgi network and late endosomes in nutrient-rich conditions⁴⁴; another Atg9 ortholog, ATG9B, displays a similar localization, but is expressed only in the placenta and pituitary gland, whereas ATG9A is expressed ubiquitously⁴³. ATG9 appears to have a conserved role in coordinating membrane transport from donor sources to the phagophore and displays a cycling pattern similar to that seen in yeast^44,90. ATG9 and ATG16L1 also appear to traffic at least in part on vesicles derived from the plasma membrane, which are delivered to the recycling endosome; the two proteins are initially present in separate vesicular populations that subsequently fuse as part of the process of autophagosome formation⁹¹. ATG9 movement is dependent on the activity of the ULK1 and PIK3C3/VPS34 kinases, as well as WIPI2 (a homolog of yeast Atg18).

Yeast PtdIns3K complexes I and II

In yeast, Vps15 (a putative regulatory protein kinase that is required for Vps34 membrane association), Vps30/Atg6, Vps34 (the PtdIns3K), Atg14 and Atg38, form the autophagy-specific class III PtdIns3K complex I (Figure 4), which localizes at the PAS^92,93,94. One key role of the PtdIns3K complex, the generation of PtdIns3P, is presumably to recruit PtdIns3P-binding proteins such as Atg18 to the PAS. Vps15, Vps34 and Vps30 are present in a second complex that includes Vps38 instead of Atg14. Thus, the latter protein appears to determine the specificity of the PtdIns3K complex I for macroautophagy.

Open in a separate window

Figure 4

Yeast PtdIns3K complex I. In yeast, Vps15 (a putative regulatory protein kinase that is required for Vps34 membrane association), Vps30/Atg6, Vps34 (the PtdIns3K), Atg14 and Atg38 form the autophagy-specific class III PtdIns3K complex, which localizes at the PAS. Vps15, Vps34 and Vps30 are present in the second class III PtdIns3K complex that includes Vps38 instead of Atg14.

Mammalian class III PtdIns3K

Mammalian cells have both a class I phosphoinositide 3-kinase and a class III PtdIns3K; the class III enzyme complexes consist of homologs of Vps34 (PIK3C3), Vps15 (PIK3R4) and Vps30 (BECN1)⁹⁵. These core components are part of at least three different complexes^96,97. The ATG14 complex contains ATG14 and is regulated by the binding of BECN1 to AMBRA1 and BCL2, which stimulate and inhibit the complex, respectively. This complex functions similarly to the yeast PtdIns3K complex I. The UVRAG complex replaces ATG14 and AMBRA1 with UVRAG and its positive regulator SH3GLB1, and participates in both endocytosis and macroautophagy. In the third complex, SH3GLB1 is replaced with KIAA0226/Rubicon, which inhibits UVRAG; this complex acts to negatively regulate macroautophagy.

Yeast Ubl conjugation systems

There are two Ubl protein conjugation systems that participate in macroautophagy⁴⁸. These include two Ubl proteins, Atg8 and Atg12, which are used to generate the conjugation products Atg8–PE and Atg12–Atg5, respectively (Figure 5). The Ubl conjugation systems participate in phagophore expansion, although their functions are not fully understood. The Atg12–Atg5 conjugate, along with a third component, Atg16, may act in part as an E3 ligase to facilitate the conjugation of Atg8 to PE, and the amount of Atg8 can regulate the size of autophagosomes⁹⁸; however, Atg8 also functions in cargo binding during selective autophagy⁹⁹.

Open in a separate window

Figure 5

Yeast Ubl conjugation systems. There are two Ubl protein conjugations systems, involving Atg8 and Atg12, which are used to generate the conjugation products Atg8–PE and Atg12–Atg5, respectively. Atg8 is initially processed by Atg4, a cysteine protease, and during autophagy, Atg8 is released from Atg8–PE by a second Atg4-dependent cleavage. The Atg12–Atg5 conjugate, along with Atg16, facilitates the conjugation of Atg8 to PE. Both of the Ubl protein conjugation systems share a single activating enzyme, Atg7. The conjugating enzymes are Atg3 for Atg8, and Atg10 for Atg12.

Yeast Atg8 is initially synthesized with a C-terminal arginine residue that is removed by Atg4, a cysteine protease^38,100. During autophagy, Atg8 is released from Atg8–PE by a second Atg4-dependent cleavage referred to as deconjugation¹⁰¹, whereas there is no similar cleavage that separates Atg12 from Atg5. Both of the Ubl protein conjugation systems share a single E1-like activating enzyme, Atg7^{100,102,103,104}. The E2-like conjugating enzymes are Atg3 for Atg8, and Atg10 for Atg12^102,105.

Mammalian Ubl protein conjugation systems

The Ubl protein conjugation systems are highly conserved from yeast to mammals^{106,107,108,109}. One of the primary differences is the existence of multiple homologs of yeast Atg8, which are divided into two subfamilies, LC3 and GABARAP. LC3 functions at the stage of phagophore elongation, whereas GABARAP proteins act at a later stage of maturation¹¹⁰. There are four mammalian ATG4 homologs, with ATG4B carrying out the primary role in macroautophagy¹¹¹. As in yeast, this enzyme cleaves after the C-terminal glycine of proLC3 to form cytosolic LC3-I, followed by its subsequent conjugation to PE to generate the membrane-associated LC3-II form¹¹². The GABARAP proteins undergo a similar type of posttranslational modification.

Cvt pathway

With regard to the details of cargo recognition, the Cvt pathway was the first well-studied type of selective autophagy in yeast; it is also the primary example of a biosynthetic rather than degradative use of autophagy^{119,120,121,122}. The double-membrane sequestering vesicle is termed a Cvt vesicle rather than an autophagosome; as noted above, this vesicle contains selective cargos of the Cvt pathway, but excludes bulk cytoplasm, in accordance with the biosynthetic and constitutive nature of the Cvt pathway compared to starvation-induced autophagy. The Cvt pathway delivers precursor forms of hydrolases, such as Ape1 (aminopeptidase I), Ape4 and Ams1 (α-mannosidase), from the cytoplasm to the vacuole, where they carry out their functions. The formation of the Cvt vesicle at the PAS is driven by the formation of a cargo complex, composed primarily of oligomerized precursor (prApe1) along with a few other resident vacuolar hydrolases^123,124,125. Atg19 acts as a cargo receptor that binds the ligand (the prApe1 propeptide) and is thus recruited to the complex¹¹³. Subsequently, the interaction between Atg19 and Atg11, a key scaffold that functions in most or all types of selective autophagy in yeast, mediates the delivery of the cargo complex to the PAS^99,126. Atg19 also interacts with Atg8, which might play a role in tethering the Cvt complex to the phagophore¹²⁷ and in directing the selective sequestration of the cargo during vesicle formation⁹⁹.

Mitophagy

Mitophagy, the selective degradation of mitochondria by autophagy, occurs through both macro- and microautophagic processes. When yeast cells are shifted from a nonfermentable carbon source such as glycerol to a fermentable/preferred carbon source such as glucose, a part of the mitochondria population that is now superfluous is subjected to degradation through mitophagy, and this is particularly enhanced when cells are simultaneously starved for nitrogen. The selective recognition of the cargo is mediated through the mitochondria outer-membrane protein Atg32, which functions as a receptor; this protein is not required for the Cvt pathway or nonselective autophagy¹²⁸. Similar to the situation with Atg19, Atg32 interacts with Atg11 to recruit mitochondria to the PAS, and also binds to Atg8 to complete vesicle formation^116,128 (Figure 6). Atg33 is another protein that is specifically involved in mitophagy^115,129. Mitophagy can also be induced by growth in a nonfermentable carbon source when yeast cells grow past the logarithmic phase. Though post-logarithmic-induced mitophagy does not completely share the same mechanism with starvation-induced mitophagy, they are both controlled by two mitogen-activated protein kinases (MAPKs), Slt2 and Hog1^129,130. Slt2 affects the recruitment of mitochondria to the PAS, and is also required in pexophagy, whereas Hog1 is mitophagy-specific¹³⁰. In addition, the absence of the mitochondrial outer membrane protein Uth1 impairs the induction of autophagy by rapamycin treatment or starvation¹³¹, suggesting its potential role as a ligand¹¹⁸. Ptc6/Aup1, a yeast mitochondrial protein phosphatase homolog, is required for post-logarithmic phase mitophagy¹³², and the function of Ptc6 in mitophagy may be based on its regulation of Rtg3-dependent transcription¹³³.

Specific degradation of obsolete mitochondria is also observed in mammalian cells, although Atg32 is not conserved in higher eukaryotes^134,135,136. Instead, FUNDC1 and BNIP3, BNIP3L/NIX and SQSTM1/p62 function as receptors in mammalian mitophagy, depending on the inducing conditions, during hypoxia, erythrocyte maturation or damage-induced mitophagy, respectively^136,137,138. Moreover, mutations of PARK2/Parkin and PINK1 are linked to Parkinson^136,139,140 and Gaucher diseases¹⁴¹. An elegant model for PINK1 stabilization on, and PINK1-dependent recruitment of PARK2 to, the mitochondrial outer membrane, that relies on the E3 ubiquitin ligase activity of PARK2 to induce mitophagy, has been proposed in mammalian cells¹³⁶.

Atg1 kinase complex

Atg1, together with the regulatory subunit Atg13, forms the core machinery of the Atg1 kinase complex. Structural data of the entire putative holo-complex (i.e., containing at least Atg1-Atg11-Atg13-Atg17-Atg31-Atg29, Figure 2) are still unavailable. Crystallization of the Atg13 N-terminal domain (NTD) shows a HORMA (Hop1, Rev1 and Mad2) fold¹⁴⁷. This domain is important for Atg14 recruitment but not for Atg13 localization or its interaction with Atg1.

In addition to the core machinery, a stable ternary complex composed of Atg17-Atg31-Atg29 (written in the order that reflects the known protein interactions) is also important for the induction of nonselective autophagy. Structural studies^41,75,148 reveal that Atg17 is a coiled-coil protein that is made up of four helices. The Atg17 monomer exhibits a crescent shape, which suggests the possibility that it binds curved membranes. The N terminus of Atg17 is involved in binding Atg13. The Atg31-Atg29 proteins are located toward the ends of the Atg17 crescent, where Atg31 serves as a connector bridging Atg17 and Atg29. Atg31 binds to Atg17 through its C-terminal α-helix and interacts with Atg29 via its N-terminal β sandwich. No direct interaction is detected between Atg17 and Atg29. The position of Atg31-Atg29 indicates that this subcomplex may function in regulating Atg17 membrane binding. The ternary complex needs dimerization to be functional, and this is essential for the initiation of autophagy. The dimerization is only mediated by Atg17 helix α4, which is located in the C-terminal region; thus, it does not involve Atg31-Atg29 and may be independent of ternary complex formation. Atg17 dimerization stabilizes the two crescents where they oppose each other, making a “wave” or “S” shape. This two-crescent conformation suggests that the dimer may be involved in both vesicle binding and tethering. Previous studies suggest that the C-terminal domain of Atg1 can also bind vesicles and contains the binding site for Atg13¹⁴⁹. The relationship between this domain and Atg17-Atg31-Atg29 has not been determined. Both Atg29 and Atg31 are phosphoproteins, and phosphorylation of Atg29 appears to alter the conformation of the protein to alleviate inhibition by the extreme C terminus as discussed above⁴¹. The structure of the C-terminal EAT (“early autophagy targeting/tethering”) domain of Atg1 has not been solved, but based on its predicted helical structure and the observation that it can bind highly curved vesicles, it may sense membrane curvature, dimerize and tether liposomes⁷⁵.

Atg9 complex

Atg9 was the first identified transmembrane protein in the Atg protein family, but detailed structural information is not available. Similarly, no structural information has been published for the Atg11, Atg23 and Atg27 proteins, which are involved in Atg9 localization to the PAS^63,87,88,89 (Figure 3). Atg18, together with Atg2, helps Atg9 cycle from the PAS to peripheral sites⁶³. Although the structure of Atg18 has not been solved, a paralog, Hsv2, which shares high sequence similarity with Atg18, has been studied in different yeast strains^150,151,152. Both proteins belong to the “PROPPINs” family, which are defined by β propeller structures and the existence of phosphoinositide binding sites^152,153. Hsv2 displays the typical propeller fold that consists of seven β blades. In addition, Hsv2 (and Atg18 along with a second paralog, Atg21) harbor an FRRG motif, which is required for phosphoinositide binding. Detailed analysis indicates that Hsv2 possesses two PtdIns3P binding sites, located in blade 5 and blade 6. Mutations in the corresponding sites in Atg18 result in loss of Atg18 localization. Moreover, the blade 6 β3-β4 loop of Hsv2 proves to be important for protein docking to the membrane. When several hydrophobic residues in this region, or the corresponding residues in Atg18, are mutated, the binding of the protein to liposomes is eliminated¹⁵². These results suggest a potential Atg18-docking model: the disk-like Atg18 touches the membrane with its edge; two binding sites interact with PtdIns3P on the membrane surface and the hydrophobic loop penetrates the membrane to stabilize the interaction.

The Atg18 β1-β2 loop and β2-β3 loop in blade 2 are important for PAS targeting¹⁵¹. Blade 2 is located at the opposite position to blade 5 and blade 6, and thus is not involved in membrane docking. Additional studies indicate that the loops in blade 2 are important for Atg18-Atg2 interaction^151,154, allowing a refinement of the model for Atg18 membrane binding. While Atg18 binds to the membrane through blades 5 and 6, blade 2, which is located on the opposite side of the protein, interacts with Atg2. The structural data further suggest that the Atg18-Atg2 interaction is independent of membrane docking. Detailed information about the Atg18-Atg2 interaction awaits progress on the crystallization of Atg2, which may also provide insight into their interdependent localization to the PAS.

Two Ubl protein conjugation systems

By far, the most structural information for the Atg proteins concerns those involved in the two Ubl protein conjugation systems (Figure 5). Atg8 is the first identified Ubl Atg protein, and there are several mammalian homologs, comprising the LC3 and GABARAP subfamilies. The crystal structure reveals that the major part of LC3 resembles a ubiquitin core, which consists of a five-stranded β sheet and two α-helices¹⁵⁵. Besides the ubiquitin fold, LC3 also contains two N-terminal α-helices and an extended C-terminal tail. As noted above, Atg4 is a cysteine protease that is responsible for removal of the C-terminal residues that follow Gly of Atg8/LC3. The structural analysis of human ATG4B, the homolog of yeast Atg4 that appears to be the most relevant isoform for autophagy, shows that the catalytic residue Cys74 is located in the center of the unique papain-like domain^156,157. The catalytic site is masked by a regulatory loop of ATG4B when it is not interacting with its substrate. When LC3 binds ATG4B through its ubiquitin core, both proteins undergo large conformational changes. Trp142 of ATG4B is spatially replaced by Phe119 of LC3, which generates an open conformation of the ATG4B regulatory loop, resulting in exposure of its catalytic site. The opening of the active site is so limited that only the small Gly120 of LC3 can gain access to Cys74, which then attacks the covalent bond between Gly120 and the neighboring residue. At this moment, the open regulatory loop, together with Trp142 of ATG4B, serves as a clamp to stabilize the conformation of LC3-ATG4B¹⁵⁷. After the C-terminal amino acid residues are removed, processed LC3 with an exposed Gly120 is released and ATG4B goes back to its auto-inhibited conformation.

LC3/Atg8 is then sent to a non-canonical E1-like enzyme, ATG7/Atg7. The Atg7 monomer is composed of three parts: NTD, adenylation domain (AD) and C-terminal Atg7-specific domain (ECTD)¹⁵⁸. The NTD of Atg7 is highly basic, and Atg3, the cognate E2 enzyme, recognizes this positively charged domain. The AD is the key functional domain in Atg7. This domain possesses a long loop region containing the catalytic residue Cys507. ATP, which is important for activation of Atg8, also binds to this domain. The C-terminal tail of the ECTD shows no secondary structure and can interact with Atg8. Biochemical data further show that this interaction is important for recognition of Atg8 by Atg7^158,159. Based on the structural and biochemical data, Atg8 is first recognized by the ECTD C-terminal tail of Atg7. Next, the long loop in the Atg7 AD undergoes a large conformational change, which allows Atg8 binding to the AD through its ubiquitin core. At the same time, ATP is also bound to the Atg7 AD. The C-terminal Gly of Atg8 then becomes adenylated. Subsequently, the catalytic Cys507 of Atg7 forms a thioester bond with Atg8. The crystal structure data reveal that Atg7 functions as a homodimer, in which the monomers interact with each other through their AD¹⁵⁸. Considering that the NTD of Atg7 is responsible for Atg3 binding, this suggests that Atg8 is activated by one Atg7 and then transferred to Atg3 that is bound to another Atg7¹⁶⁰. This trans-transfer model displays higher efficiency than a cis-model and may explain why Atg7 forms a homodimer.

The crystal structure of Atg3 shows a hammer-like conformation with an N-terminal head moiety and a C-terminal handle moiety¹⁶¹. The head moiety shares some similarities to canonical E2 enzymes, while the handle moiety, which is suggested to bind Atg7, is unique to Atg3. Cys234 in Atg3 forms the thioester bond with Atg8, generating the Atg3-Atg8 intermediate. The function of an E3 enzyme in the Atg8 Ubl conjugation system is not fully elucidated. One hypothesis is that the Atg12–Atg5-Atg16 complex, which is the product of the Atg12 Ubl conjugation system in autophagy, acts as the E3 enzyme¹⁶². Biochemical studies indicate that the unique catalytic site of Atg3 involves a Thr residue, instead of the more typical Arg, for the transfer of Atg8 to PE¹⁶³. Furthermore, interaction with the Atg12–Atg5 conjugate results in a change in the orientation of the catalytic cysteine, such that it moves closer to the Thr residue, facilitating the conjugation activity of Atg3.

ATG12 was first crystallized from plant¹⁶⁴. The topology of Arabidopsis thaliana ATG12 is similar to other ubiquitin-fold proteins, and ATG12 shares a high degree of structural similarity with LC3/Atg8 except for a unique hydrophobic patch. Similar to Atg8, Atg12 is activated by Atg7. Next, it is sent to another E2 enzyme, Atg10, forming an Atg12–Atg10 intermediate. It is unclear whether Atg3 will compete with Atg10 for interaction with Atg7. Previous studies show that Atg10 bound to Atg7 could be easily replaced by Atg3, but not the reverse, indicating that Atg3 has a higher affinity for binding Atg7¹⁵⁹. Atg10 possesses a conserved E2 core that is comprised of four β sheets and two α-helices. However, Atg10 contains two additional accessory α-helices and three additional β sheets, which are important for its normal function^165,166. Atg12 is finally conjugated to Lys149 of Atg5, and both structural and biochemical evidence indicate that Atg10 directly interacts with Atg5 through its accessory β5 and β6 sheets.

Atg5 has two Ubl domains, an NTD UblA and C-terminal domain UblB, with a helix-rich domain connecting them, and an extreme N-terminal α-helix^165,167. Analysis suggests that Atg5 β7 located in UblB is responsible for interacting with Atg10¹⁶⁵. After Atg12 is conjugated to Atg5, no major conformational change is detected¹⁶⁸. Atg12–Atg5 prefers to interact with Atg16, forming an Atg12–Atg5-Atg16 complex. Atg16 possesses a coiled-coil C-terminal region, which is responsible for Atg16 homodimer formation¹⁶⁹. The NTD of Atg16 is comprised of an α-helix and a loop moiety, and binds Atg5. Structural data reveal that the helix binds to the groove formed by UblA, UblB and the N-terminal α-helix of Atg5, while the loop moiety mainly binds to UblA¹⁶⁷.

As mentioned above, previous studies indicate that the Atg12–Atg5-Atg16 complex functions as an E3 ligase in Atg8 lipidation. The structural data of the ATG12–ATG5-ATG16L1N (containing the N-terminal part of ATG16L1) complex reveal that ATG16L1N binds to ATG5 at the opposite side to where ATG12 binds; no interaction is detected between ATG16L1N and ATG12¹⁷⁰. Human ATG12 can interact with ATG3 through its Phe108 residue¹⁶². Phe154 in plant ATG12 is equivalent to the human ATG12 Phe108, and the former is masked in the ATG12–ATG5 complex¹⁶⁸. This observation raises a possible explanation for the function of Atg12–Atg5-Atg16: The complex has two states; in the absence of Atg3–Atg8, the complex is in the closed state where Atg5 buries the Atg3-binding surface of Atg12. When Atg3–Atg8 forms, a conformational change is triggered that exposes the Atg3-binding surface in Atg12. Atg12 then interacts with Atg3 and brings the Atg3–Atg8 intermediate to the membrane, where the Atg12–Atg5-Atg16 complex facilitates Atg8–PE conjugation. Further studies are needed to support and/or refine this model.