Pre-autophagosomal Structure Research Articles

Phosphatidylinositol-3-phosphate in the regulation of autophagy membrane dynamics.

Phosphatidylinositol-3-phosphate (PI3P) is a key player in membrane dynamics and trafficking regulation. Most PI3P is associated with endosomal membranes and with the autophagosome preassembly machinery, presumably at the endoplasmic reticulum. The enzyme responsible for most PI3P synthesis, VPS34 and proteins such as Beclin1 and ATG14L that regulate PI3P levels are positive modulators of autophagy initiation. It had been assumed that a local PI3P pool was present at autophagosomes and preautophagosomal structures, such as the omegasome and the phagophore. This was recently confirmed by the demonstration that PI3P-binding proteins participate in the complex sequence of signalling that results in autophagosome assembly and activity. Here we summarize the historical discoveries of PI3P lipid kinase involvement in autophagy, and we discuss the proposed role of PI3P during autophagy, notably during the autophagosome biogenesis sequence.

ATG9 regulates autophagosome progression from the endoplasmic reticulum in Arabidopsis

Autophagy is a conserved pathway for bulk degradation of cytoplasmic material by a double-membrane structure named the autophagosome. The initiation of autophagosome formation requires the recruitment of autophagy-related protein 9 (ATG9) vesicles to the preautophagosomal structure. However, the functional relationship between ATG9 vesicles and the phagophore is controversial in different systems, and the molecular function of ATG9 remains unknown in plants. Here, we demonstrate that ATG9 is essential for endoplasmic reticulum (ER)-derived autophagosome formation in plants. Through a combination of genetic, in vivo imaging and electron tomography approaches, we show that Arabidopsis ATG9 deficiency leads to a drastic accumulation of autophagosome-related tubular structures in direct membrane continuity with the ER upon autophagic induction. Dynamic analyses demonstrate a transient membrane association between ATG9 vesicles and the autophagosomal membrane during autophagy. Furthermore, trafficking of ATG18a is compromised in atg9 mutants during autophagy by forming extended tubules in a phosphatidylinositol 3-phosphate-dependent manner. Taken together, this study provides evidence for a pivotal role of ATG9 in regulating autophagosome progression from the ER membrane in Arabidopsis.

Orchestrated Action of PP2A Antagonizes Atg13 Phosphorylation and Promotes Autophagy after the Inactivation of TORC1.

Target of rapamycin complex 1 (TORC1) phosphorylates autophagy-related Atg13 and represses autophagy under nutrient-rich conditions. However, when TORC1 becomes inactive upon nutrient depletion or treatment with the TORC1 inhibitor rapamycin, Atg13 dephosphorylation occurs rapidly, and autophagy is induced. At present, the phosphatases involved in Atg13 dephosphorylation remain unknown. Here, we show that two protein phosphatase 2A (PP2A) phosphatases, PP2A-Cdc55 and PP2A-Rts1, which are activated by inactivation of TORC1, are required for sufficient Atg13 dephosphorylation and autophagy induction after TORC1 inactivation in budding yeast. After rapamycin treatment, dephosphorylation of Atg13, activation of Atg1 kinase, pre-autophagosomal structure (PAS) formation and autophagy induction are all impaired in PP2A-deleted cells. Conversely, overexpression of non-phosphorylatable Atg13 suppressed defects in autophagy in PP2A mutant. This study revealed that the orchestrated action of PP2A antagonizes Atg13 phosphorylation and promotes autophagy after the inactivation of TORC1.

Trs33-Containing TRAPP IV: A Novel Autophagy-Specific Ypt1 GEF.

Ypt/Rab GTPases, key regulators of intracellular trafficking pathways, are activated by guanine-nucleotide exchange factors (GEFs). Here, we identify a novel GEF complex, TRAPP IV, which regulates Ypt1-mediated autophagy. In the yeast Saccharomyces cerevisiae, Ypt1 GTPase is required for the initiation of secretion and autophagy, suggesting that it regulates these two distinct pathways. However, whether these pathways are coordinated by Ypt1 and by what mechanism is still unknown. TRAPP is a conserved modular complex that acts as a Ypt/Rab GEF. Two different TRAPP complexes, TRAPP I and the Trs85-containing TRAPP III, activate Ypt1 in the secretory and autophagic pathways, respectively. Importantly, whereas TRAPP I depletion copies Ypt1 deficiency in secretion, depletion of TRAPP III does not fully copy the autophagy phenotypes of autophagy-specific ypt1 mutations. If GEFs are required for Ypt/Rab function, this discrepancy implies the existence of an additional GEF that activates Ypt1 in autophagy. Trs33, a nonessential TRAPP subunit, was assigned to TRAPP I without functional evidence. We show that in the absence of Trs85, Trs33 is required for Ypt1-mediated autophagy and for the recruitment of core-TRAPP and Ypt1 to the preautophagosomal structure, which marks the onset of autophagy. In addition, Trs33 and Trs85 assemble into distinct TRAPP complexes, and we term the Trs33-containing autophagy-specific complex TRAPP IV. Because TRAPP I is required for Ypt1-mediated secretion, and either TRAPP III or TRAPP IV is required for Ypt1-mediated autophagy, we propose that pathway-specific GEFs activate Ypt1 in secretion and autophagy.

A conserved glycine residue in the C-terminal region of human ATG9A is required for its transport from the endoplasmic reticulum to the Golgi apparatus

ATG9A is the only polytopic protein of the mammalian autophagy-related protein family whose members regulate autophagosome formation during macroautophagy. At steady state, ATG9A localizes to several intracellular sites, including the Golgi apparatus, endosomes and the plasma membrane, and it redistributes towards autophagosomes upon autophagy induction. Interestingly, the transport of yeast Atg9 to the pre-autophagosomal structure depends on its self-association, which is mediated by a short amino acid motif located in the C-terminal region of the protein. Here, we investigated whether the residues that align with this motif in human ATG9A (V515-C519) are also required for its trafficking in mammalian cells. Interestingly, our findings support that human ATG9A self-interacts as well, and that this process promotes transport of ATG9A molecules through the Golgi apparatus. Furthermore, our data reveal that the transport of ATG9A out of the ER is severely impacted after mutation of the conserved V515-C519 motif. Nevertheless, the mutated ATG9A molecules could still interact with each other, indicating that the molecular mechanism of self-interaction differs in mammalian cells compared to yeast. Using sequential amino acid substitutions of glycine 516 and cysteine 519, we found that the stability of ATG9A relies on both of these residues, but that only the former is required for efficient transport of human ATG9A from the endoplasmic reticulum to the Golgi apparatus.

Dynamics and architecture of the NRBF2-containing phosphatidylinositol 3-kinase complex I of autophagy

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is central to autophagy initiation. We previously reported the V-shaped architecture of the four-subunit version of PI3KC3-C1 consisting of VPS (vacuolar protein sorting) 34, VPS15, BECN1 (Beclin 1), and ATG (autophagy-related) 14. Here we show that a putative fifth subunit, nuclear receptor binding factor 2 (NRBF2), is a tightly bound component of the complex that profoundly affects its activity and architecture. NRBF2 enhances the lipid kinase activity of the catalytic subunit, VPS34, by roughly 10-fold. We used hydrogen-deuterium exchange coupled to mass spectrometry and negative-stain electron microscopy to map NRBF2 to the base of the V-shaped complex. NRBF2 interacts primarily with the N termini of ATG14 and BECN1. We show that NRBF2 is a homodimer and drives the dimerization of the larger PI3KC3-C1 complex, with implications for the higher-order organization of the preautophagosomal structure.

The Intrinsically Disordered Protein Atg13 Mediates Supramolecular Assembly of Autophagy Initiation Complexes

Autophagosome formation in yeast entails starvation-induced assembly of the pre-autophagosomal structure (PAS), in which multiple Atg1 complexes (composed of Atg1, Atg13, and the Atg17-Atg29-Atg31 subcomplex) are initially engaged. However, the molecular mechanisms underlying the multimeric assembly of these complexes remain unclear. Using structural and biological techniques, we herein demonstrate that Atg13 has a large intrinsically disordered region (IDR) and interacts with two distinct Atg17 molecules using two binding regions in the IDR. We further reveal that these two binding regions are essential not only for Atg1 complex assembly invitro, but also for PAS organization invivo. These findings underscore the structural and functional significance of the IDR of Atg13 in autophagy initiation: Atg13 provides intercomplex linkages between Atg17-Atg29-Atg31 complexes, thereby leading to supramolecular self-assembly of Atg1 complexes, in turn accelerating the initial events of autophagy, including autophosphorylation of Atg1, recruitment of Atg9 vesicles, and phosphorylation of Atg9 by Atg1.

Atg20- and Atg24-family proteins promote organelle autophagy in fission yeast.

Autophagy cargos include not only soluble cytosolic materials but also bulky organelles, such as ER and mitochondria. In budding yeast, two proteins that contain the PX domain and the BAR domain, Atg20 and Atg24 (also known as Snx42 and Snx4, respectively) are required for organelle autophagy and contribute to general autophagy in a way that can be masked by compensatory mechanisms. It remains unclear why these proteins are important for organelle autophagy. Here, we show that in a distantly related fungal organism, the fission yeast Schizosaccharomyces pombe, autophagy of ER and mitochondria is induced by nitrogen starvation and is promoted by three Atg20- and Atg24-family proteins - Atg20, Atg24 and SPBC1711.11 (named here as Atg24b). These proteins localize at the pre-autophagosomal structure, or phagophore assembly site (PAS), during starvation. S. pombe Atg24 forms a homo-oligomer and acts redundantly with Atg20 and Atg24b, and the latter two proteins can form a hetero-oligomer. The organelle autophagy defect caused by the loss of these proteins is associated with a reduction of autophagosome size and a decrease in Atg8 accumulation at the PAS. These results provide new insights into the autophagic function of Atg20- and Atg24-family proteins.

Ole1, fatty acid desaturase, is required for Atg9 delivery and isolation membrane expansion during autophagy in Saccharomyces cerevisiae.

ABSTRACTMacroautophagy, a major degradation pathway of cytoplasmic components, is carried out through formation of a double-membrane structure, the autophagosome. Although the involvement of specific lipid species in the formation process remains largely obscure, we recently showed that mono-unsaturated fatty acids (MUFA) generated by stearoyl-CoA desaturase 1 (SCD1) are required for autophagosome formation in mammalian cells. To obtain further insight into the role of MUFA in autophagy, in this study we analyzed the autophagic phenotypes of the yeast mutant of OLE1, an orthologue of SCD1. Δole1 cells were defective in nitrogen starvation-induced autophagy, and the Cvt pathway, when oleic acid was not supplied. Defects in elongation of the isolation membrane led to a defect in autophagosome formation. In the absence of Ole1, the transmembrane protein Atg9 was not able to reach the pre-autophagosomal structure (PAS), the site of autophagosome formation. Thus, autophagosome formation requires Ole1 during the delivery of Atg9 to the PAS/autophagosome from its cellular reservoir.

Yvh1 protein phosphatase is required for pre-autophagosomal structure formation after TORC1 inactivation.

The pre-autophagosomal structure (PAS) is a putative site for autophagosome formation in budding yeast. Upon nutrient depletion or rapamycin treatment, target of rapamycin complex 1 (TORC1) becomes inactive, inducing PAS formation and autophagy. Here, we show that Yvh1 phosphatase is critical for PAS formation, but not autophagy induction, after TORC1 inactivation.

The Legionella Anti-autophagy Effector RavZ Targets the Autophagosome via PI3P- and Curvature-Sensing Motifs

Autophagy is a conserved membrane transport pathway used to destroy pathogenic microbes that access the cytosol of cells. The intracellular pathogen Legionella pneumophila interferes with autophagy by delivering an effector protein, RavZ, into the host cytosol. RavZ acts by cleaving membrane-conjugated Atg8/LC3 proteins from pre-autophagosomal structures. Its remarkable efficiency allows minute quantities of RavZ to block autophagy throughout the cell. To understand how RavZ targets pre-autophagosomes and specifically acts only on membrane-associated Atg8 proteins, we elucidated its structure. Revealed is a catalytic domain related in fold to Ulp family deubiquitinase-like enzymes and a C-terminal PI3P-binding module. RavZ targets the autophagosome via the PI3P-binding module and a catalytic domain helix, and it preferentially binds high-curvature membranes, intimating localization to highly curved domains in autophagosome intermediate membranes. RavZ-membrane interactions enhance substrate affinity, providing a mechanism for interfacial activation that also may be used by host autophagy proteins engaging only lipidated Atg8 proteins.

Alternaria extract activates autophagy that induces IL-18 release from airway epithelial cells

Alternaria alternata is a major outdoor allergen that causes allergic airway diseases. Alternaria extract (ALT-E) has been shown to induce airway epithelial cells to release IL-18 and thereby initiate Th2-type responses. We investigated the underlying mechanisms involved in IL-18 release from ALT-E-stimulated airway epithelial cells. Normal human bronchial epithelial cells and A549 human lung adenocarcinoma cells were stimulated with ALT-E in the presence of different inhibitors of autophagy or caspases. IL-18 levels in culture supernatants were measured by ELISA. The numbers of autophagosomes, an LC3-I to LC3-II conversion, and p62 degradation were determined by immunofluorescence staining and immunoblotting. 3-methyladenine and bafilomycin, which inhibit the formation of preautophagosomal structures and autolysosomes, respectively, suppressed ALT-E-induced IL-18 release by cells, whereas caspase 1 and 8 inhibitors did not. ALT-E-stimulation increased autophagosome formation, LC-3 conversion, and p62 degradation in airway epithelial cells. LPS-stimulation induced the LC3 conversion in A549 cells, but did not induce IL-18 release or p62 degradation. Unlike LPS, ALT-E induced airway epithelial cells to release IL-18 via an autophagy dependent, caspase 1 and 8 independent pathway. Although autophagy has been shown to negatively regulate canonical inflammasome activity in TLR-stimulated macrophages, our data indicates that this process is an unconventional mechanism of IL-18 secretion by airway epithelial cells.

PP2A phosphatase regulates autophagy in budding yeast

Autophagy is a highly conserved, degradative process in eukaryotic cells. It maintains homeostasis by degradation of damaged or unnecessary proteins and intracellular organelles, and supplied nutrients as degraded products. Dysfunction of autophagy leads to cellular disorder, such as cancer and neurodegenerative diseases including Alzheimer's, Parkinson's and Huntington's diseases. In budding yeast Saccharomyces cerevisiae, TORC1 phosphorylates Atg13 to prevent complex formation between Atg13 and other ATG proteins including the protein kinase Atg1. When TORC1 is inactivated by nutrient depletion, Atg13 is rapidly dephosphorylated and forms the Atg1‐13‐17‐29‐31 complex that triggers autophagy. In addition, protein kinase A (PKA) is involved in autophagy. Thus, various protein kinases involve in autophagy regulation are discovered, whereas protein phosphatases (PPs) involved autophagy are largely unknown. To find out protein phosphatases involved autophagy regulation, we assessed various autophagy profiles in 36 budding yeast PP‐knockout mutants and found that cells lacking PP2A are defective in autophagy and assembly of the pre‐autophagosomal structure (PAS). This study reveals that PP2A promotes autophagy after TORC1 inactivation.

Wheat homologs of yeast ATG6 function in autophagy and are implicated in powdery mildew immunity

BackgroundAutophagy-related ATG6 proteins are pleiotropic proteins functioning in autophagy and the phosphatidylinositol 3-phosphate-signaling pathways. Arabidopsis ATG6 regulates normal plant growth, pollen development and germination, and plant responses to biotic/abiotic stresses. However, the ATG6 functions in wheat (Triticum aestivum L.), an important food crop, are lacking.ResultsWe identified three members, TaATG6a-6c, of the ATG6 family from common wheat. TaATG6a, 6b and 6c were localized on homeologous chromosomes 3DL, 3BL and 3AL, respectively, of the allo-hexaploid wheat genome, and evidence was provided for their essential role in autophagy. The TaATG6a-GFP fusion protein was found in punctate pre-autophagosomal structures. The expression of each TaATG6 gene restored the accumulation of autophagic bodies in atg6-mutant yeast. Additionally, TaATG6 knockdown plants showed impaired constitutive and pathogen-induced autophagy and growth abnormalities under normal conditions. We also examined the expression patterns of wheat ATG6s for clues to their physiological roles, and found that their expression was induced by the fungus Blumeria graminis f. sp. tritici (Bgt), the causal agent of powdery mildew, and by abiotic stress factors. A role for TaATG6s in wheat immunity to powdery mildew was further implied when knockdowns of TaATG6s weakly compromised the broad-spectrum powdery mildew resistance gene Pm21-triggered resistance response and, conversely and significantly, enhanced the basal resistance of susceptible plants. In addition, leaf cell death was sometimes induced by growth-retarded small Bgt mycelia on susceptible TaATG6 knockdown plants after a long period of interaction. Thus, we provide an important extension of the previous characterization of plant ATG6 genes in wheat, and observed a role for autophagy genes in wheat immune responses to fungal pathogens.ConclusionsThree wheat ATG6s were identified and shown to be essential for autophagy biogenesis. Wheat ATG6s are implicated in immunity to powdery mildew, playing a weak, positive role in the Pm21-triggered resistance response and a negative role in the basal resistance of susceptible plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0472-y) contains supplementary material, which is available to authorized users.

Phosphorylation of Atg31 is required for autophagy.

Autophagy is an evolutionarily conserved cellular process which degrades intracellular contents. The Atg17-Atg31-Atg29 complex plays a key role in autophagy induction by various stimuli. In yeast, autophagy occurs with autophagosome formation at a special site near the vacuole named the pre-autophagosomal structure (PAS). The Atg17-Atg31-Atg29 complex forms a scaffold for PAS organization, and recruits other autophagy-related (Atg) proteins to the PAS. Here, we show that Atg31 is a phosphorylated protein. The phosphorylation sites on Atg31 were identified by mass spectrometry. Analysis of mutants in which the phosphorylated amino acids were replaced by alanine, either individually or in various combinations, identified S174 as the functional phosphorylation site. An S174A mutant showed a similar degree of autophagy impairment as an Atg31 deletion mutant. S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment. Mass spectrometry analysis showed that S174 is phosphorylated constitutively, and expression of a phosphorylation-mimic mutant (S174D) in the Atg31 deletion strain restores autophagy. In the S174A mutant, Atg9-positive vesicles accumulate at the PAS. Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS. Our data demonstrate the role of phosphorylation of Atg31 in autophagy.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0138-4) contains supplementary material, which is available to authorized users.

Functional analysis of AoAtg11 in selective autophagy in the filamentous fungus Aspergillus oryzae

Autophagy is a highly conserved cellular degradation process in eukaryotes and consists of both non-selective and selective types. Selective autophagic processes include pexophagy, mitophagy, and the cytoplasm-to-vacuole targeting (Cvt) pathway of yeast, in which particular vacuolar proteins, such as aminopeptidase I (Ape1), are selectively transported to vacuoles. Although selective autophagy has been mainly studied in the yeasts Saccharomyces cerevisiae and Pichia pastoris, there is evidence for selective autophagy in filamentous fungi; however, the details are poorly understood. In S. cerevisiae, Atg11 is a selective autophagy-specific protein that recognizes and transports substrates to the pre-autophagosomal structure (PAS). Here, we first identified an ATG11 homologue in the filamentous fungus Aspergillus oryzae and analyzed the localization of the corresponding protein, designated AoAtg11, fused to enhanced green fluorescent protein (EGFP). Imaging analysis revealed that AoAtg11–EGFP was localized to PAS-like structures. We next constructed an Aoatg11 disruptant of A. oryzae and showed that AoAtg11 is involved in pexophagy and mitophagy. In addition, AoAtg11 was found to be dispensable for non-selective autophagy and for transporting AoApe1 to vacuoles. Taken together, these results suggest that AoAtg11 is a selective autophagy-specific protein in A. oryzae, and has distinct molecular functions from that of S. cerevisiae Atg11.

Visualization of Atg3 during Autophagosome Formation in Saccharomyces cerevisiae

Macroautophagy (autophagy) is a highly conserved cellular recycling process involved in degradation of eukaryotic cellular components. During autophagy, macromolecules and organelles are sequestered into the double-membrane autophagosome and degraded in the vacuole/lysosome. Autophagy-related 8 (Atg8), a core Atg protein essential for autophagosome formation, is a marker of several autophagic structures: the pre-autophagosomal structure (PAS), isolation membrane (IM), and autophagosome. Atg8 is conjugated to phosphatidylethanolamine (PE) through a ubiquitin-like conjugation system to yield Atg8-PE; this reaction is called Atg8 lipidation. Although the mechanisms of Atg8 lipidation have been well studied in vitro, the cellular locale of Atg8 lipidation remains enigmatic. Atg3 is an E2-like enzyme that catalyzes the conjugation reaction between Atg8 and PE. Therefore, we hypothesized that the localization of Atg3 would provide insights about the site of the lipidation reaction. To explore this idea, we constructed functional GFP-tagged Atg3 (Atg3-GFP) by inserting the GFP portion immediately after the handle region of Atg3. During autophagy, Atg3-GFP transiently formed a single dot per cell on the vacuolar membrane. This Atg3-GFP dot colocalized with 2× mCherry-tagged Atg8, demonstrating that Atg3 is localized to autophagic structures. Furthermore, we found that Atg3-GFP is localized to the IM by fine-localization analysis. The localization of Atg3 suggests that Atg3 plays an important role in autophagosome formation at the IM.

Atg27 tyrosine sorting motif is important for its trafficking and Atg9 localization.

During autophagy, the transmembrane protein Atg27 facilitates transport of the major autophagy membrane protein Atg9 to the preautophagosomal structure (PAS). To better understand the function of Atg27 and its relationship with Atg9, Atg27 trafficking and localization were examined. Atg27 localized to endosomes and the vacuolar membrane, in addition to previously described PAS, Golgi and Atg9-positive structures. Atg27 vacuolar membrane localization was dependent on the adaptor AP-3, which mediates direct transport from the trans-Golgi to the vacuole. The four C-terminal amino acids (YSAV) of Atg27 comprise a tyrosine sorting motif. Mutation of the YSAV abrogated Atg27 transport to the vacuolar membrane and affected its distribution in TGN/endosomal compartments, while PAS localization was normal. Also, in atg27(ΔYSAV) or AP-3 mutants, accumulation of Atg9 in the vacuolar lumen was observed upon autophagy induction. Nevertheless, PAS localization of Atg9 was normal in atg27(ΔYSAV) cells. The vacuole lumen localization of Atg9 was dependent on transport through the multivesicular body, as Atg9 accumulated in the class E compartment and vacuole membrane in atg27(ΔYSAV) vps4Δ but not in ATG27 vps4Δ cells. We suggest that Atg27 has an additional role to retain Atg9 in endosomal reservoirs that can be mobilized during autophagy.

PI3P binding by Atg21 organises Atg8 lipidation.

Autophagosome biogenesis requires two ubiquitin-like conjugation systems. One couples ubiquitin-like Atg8 to phosphatidylethanolamine, and the other couples ubiquitin-like Atg12 to Atg5. Atg12~Atg5 then forms a heterodimer with Atg16. Membrane recruitment of the Atg12~Atg5/Atg16 complex defines the Atg8 lipidation site. Lipidation requires a PI3P-containing precursor. How PI3P is sensed and used to coordinate the conjugation systems remained unclear. Here, we show that Atg21, a WD40 β-propeller, binds via PI3P to the preautophagosomal structure (PAS). Atg21 directly interacts with the coiled-coil domain of Atg16 and with Atg8. This latter interaction requires the conserved F5K6-motif in the N-terminal helical domain of Atg8, but not its AIM-binding site. Accordingly, the Atg8 AIM-binding site remains free to mediate interaction with its E2 enzyme Atg3. Atg21 thus defines PI3P-dependently the lipidation site by linking and organising the E3 ligase complex and Atg8 at the PAS.

Localization of Atg3 to autophagy-related membranes and its enhancement by the Atg8-family interacting motif to promote expansion of the membranes

The E2 enzyme Atg3 conjugates the ubiquitin-like protein Atg8 to phosphatidylethanolamine (PE) to drive autophagosome formation in Saccharomyces cerevisiae. In this study, we show that Atg3 localizes to the pre-autophagosomal structure (PAS) and the isolation membrane (IM), providing crucial evidence that Atg8-PE conjugates are produced on these structures. We also find that mutations in the Atg8-family interacting motif (AIM) of Atg3 significantly impairs the PAS/IM localization of Atg3, resulting in inefficient IM expansion. It is suggested that the AIM-mediated PAS/IM localization of Atg3 facilitates membrane expansion in these structures probably by ensuring active production of Atg8-PE on the membranes.