Atg35, a micropexophagy-specific protein that regulates micropexophagic apparatus formation in Pichia pastoris

Autophagy-related (Atg) pathways deliver cytosol and organelles to the vacuole in double-membrane vesicles called autophagosomes, which are formed at the phagophore assembly site (PAS), where most of the core Atg proteins assemble. Atg28 is a component of the core autophagic machinery partially required for all Atg pathways in Pichia pastoris. This coiled-coil protein interacts with Atg17 and is essential for micropexophagy. However, the role of Atg28 in micropexophagy was unknown. We used the yeast two-hybrid system to search for Atg28 interaction partners from P. pastoris and identified a new Atg protein, named Atg35. The atg35? mutant was not affected in macropexophagy, cytoplasm-to-vacuole targeting or general autophagy. However, both Atg28 and Atg35 were required for micropexophagy and for the formation of the micropexophagic apparatus (MIPA). This requirement correlated with a stronger expression of both proteins on methanol and glucose. Atg28 mediated the interaction of Atg35 with Atg17. Trafficking of overexpressed Atg17 from the peripheral ER to the nuclear envelope was required to organize a peri-nuclear structure (PNS), the site of Atg35 colocalization during micropexophagy. In summary, Atg35 is a new Atg protein that relocates to the PNS and specifically regulates MIPA formation during micropexophagy.     

Atg35, a micropexophagy-specific protein that regulates micropexophagic apparatus formation in Pichia pastoris

Autophagy-related (Atg) pathways deliver cytosol and organelles to the vacuole in double-membrane vesicles called autophagosomes, which are formed at the phagophore assembly site (PAS), where most of the core Atg proteins assemble. Atg28 is a component of the core autophagic machinery partially required for all Atg pathways in Pichia pastoris. This coiled-coil protein interacts with Atg17 and is essential for micropexophagy. However, the role of Atg28 in micropexophagy was unknown. We used the yeast two-hybrid system to search for Atg28 interaction partners from P. pastoris and identified a new Atg protein, named Atg35. The atg35Δ mutant was not affected in macropexophagy, cytoplasm-to-vacuole targeting or general autophagy. However, both Atg28 and Atg35 were required for micropexophagy and for the formation of the micropexophagic apparatus (MIPA). This requirement correlated with a stronger expression of both proteins on methanol and glucose. Atg28 mediated the interaction of Atg35 with Atg17. Trafficking of overexpressed Atg17 from the peripheral ER to the nuclear envelope was required to organize a peri-nuclear structure (PNS), the site of Atg35 colocalization during micropexophagy. In summary, Atg35 is a new Atg protein that relocates to the PNS and specifically regulates MIPA formation during micropexophagy.Key words: Atg protein, peroxisome, micropexophagy, MIPA, nucleus     

PpATG9 encodes a novel membrane protein that traffics to vacuolar membranes, which sequester peroxisomes during pexophagy in Pichia pastoris

When Pichia pastoris adapts from methanol to glucose growth, peroxisomes are rapidly sequestered and degraded within the vacuole by micropexophagy. During micropexophagy, sequestering membranes arise from the vacuole to engulf the peroxisomes. Fusion of the sequestering membranes and incorporation of the peroxisomes into the vacuole is mediated by the micropexophagy-specific membrane apparatus (MIPA). In this study, we show the P. pastoris ortholog of Atg9, a novel membrane protein is essential for the formation of the sequestering membranes and assembly of MIPA. During methanol growth, GFP-PpAtg9 localizes to multiple structures situated near the plasma membrane referred as the peripheral compartment (Atg9-PC). On glucose-induced micropexophagy, PpAtg9 traffics from the Atg9-PC to unique perivacuolar structures (PVS) that contain PpAtg11, but lack PpAtg2 and PpAtg8. Afterward, PpAtg9 distributes to the vacuole surface and sequestering membranes. Movement of the PpAtg9 from the Atg9-PC to the PVS requires PpAtg11 and PpVps15. PpAtg2 and PpAtg7 are essential for PpAtg9 trafficking from the PVS to the vacuole and sequestering membranes, whereas trafficking of PpAtg9 proceeds independent of PpAtg1, PpAtg18, and PpVac8. In summary, our data suggest that PpAtg9 transits from the Atg9-PC to the PVS and then to the sequestering membranes that engulf the peroxisomes for degradation.     

Atg8 is essential for macropexophagy in Hansenula polymorpha

We have isolated a peroxisome-degradation-deficient (pdd) mutant of the methylotrophic yeast Hansenula polymorpha via gene tagging mutagenesis. Sequencing revealed that the mutant was affected in the HpATG8 gene. HpAtg8 is a protein with high sequence similarity to both Pichia pastoris and Saccharomyces cerevisiae Atg8 and appeared to be essential for selective peroxisome degradation (macropexophagy) and nitrogen-limitation induced microautophagy. Fluorescence microscopy revealed that a GFP.Atg8 fusion protein was located close to the vacuole. After induction of macropexophagy, the GFP.Atg8 containing spot extended to engulf an individual peroxisome. In cells of a constructed deletion strain, sequestration of individual organelles was never completed; analysis of series of serial sections revealed that invariably a minor diaphragm-like opening remained. We hypothesize that H. polymorpha Atg8 facilitates sealing of the sequestering membranes during selective peroxisome degradation.     

The requirement of sterol glucoside for pexophagy in yeast is dependent on the species and nature of peroxisome inducers

Sterol glucosyltransferase, Ugt51/Atg26, is essential for both micropexophagy and macropexophagy of methanol-induced peroxisomes in Pichia pastoris. However, the role of this protein in pexophagy in other yeast remained unclear. We show that oleate- and amine-induced peroxisomes in Yarrowia lipolytica are degraded by Atg26-independent macropexophagy. Surprisingly, Atg26 was also not essential for macropexophagy of oleate- and amine-induced peroxisomes in P. pastoris, suggesting that the function of sterol glucoside (SG) in pexophagy is both species and peroxisome inducer specific. However, the rates of degradation of oleate- and amine-induced peroxisomes in P. pastoris were reduced in the absence of SG, indicating that P. pastoris specifically uses sterol conversion by Atg26 to enhance selective degradation of peroxisomes. However, methanol-induced peroxisomes apparently have lost the redundant ability to be degraded without SG. We also show that the P. pastoris Vac8 armadillo repeat protein is not essential for macropexophagy of methanol-, oleate-, or amine-induced peroxisomes, which makes PpVac8 the first known protein required for the micropexophagy, but not for the macropexophagy, machinery. The uniqueness of Atg26 and Vac8 functions under different pexophagy conditions demonstrates that not only pexophagy inducers, such as glucose or ethanol, but also the inducers of peroxisomes, such as methanol, oleate, or primary amines, determine the requirements for subsequent pexophagy in yeast.     

Pexophagy: autophagic degradation of peroxisomes

The abundance of peroxisomes within a cell can rapidly decrease by selective autophagic degradation (also designated pexophagy). Studies in yeast species have shown that at least two modes of peroxisome degradation are employed, namely macropexophagy and micropexophagy. During macropexophagy, peroxisomes are individually sequestered by membranes, thus forming a pexophagosome. This structure fuses with the vacuolar membrane, resulting in exposure of the incorporated peroxisome to vacuolar hydrolases. During micropexophagy, a cluster of peroxisomes is enclosed by vacuolar membrane protrusions and/or segmented vacuoles as well as a newly formed membrane structure, the micropexophagy-specific membrane apparatus (MIPA), which mediates the enclosement of the vacuolar membrane. Subsequently, the engulfed peroxisome cluster is degraded. This review discusses the current state of knowledge of pexophagy with emphasis on studies on methylotrophic yeast species.     

A sorting nexin PpAtg24 regulates vacuolar membrane dynamics during pexophagy via binding to phosphatidylinositol-3-phosphate

Diverse cellular processes such as autophagic protein degradation require phosphoinositide signaling in eukaryotic cells. In the methylotrophic yeast Pichia pastoris, peroxisomes can be selectively degraded via two types of pexophagic pathways, macropexophagy and micropexophagy. Both involve membrane fusion events at the vacuolar surface that are characterized by internalization of the boundary domain of the fusion complex, indicating that fusion occurs at the vertex. Here, we show that PpAtg24, a molecule with a phosphatidylinositol 3-phosphate-binding module (PX domain) that is indispensable for pexophagy, functions in membrane fusion at the vacuolar surface. CFP-tagged PpAtg24 localized to the vertex and boundary region of the pexophagosome-vacuole fusion complex during macropexophagy. Depletion of PpAtg24 resulted in the blockage of macropexophagy after pexophagosome formation and before the fusion stage. These and other results suggest that PpAtg24 is involved in the spatiotemporal regulation of membrane fusion at the vacuolar surface during pexophagy via binding to phosphatidylinositol 3-phosphate, rather than the previously suggested function in formation of the pexophagosome.     

PpAtg30 tags peroxisomes for turnover by selective autophagy

Autophagy, an intrinsically nonselective process, can also target selective cargo for degradation. The mechanism of selective peroxisome turnover by autophagy-related processes (pexophagy), termed micropexophagy and macropexophagy, is unknown. We show how a Pichia pastoris protein, PpAtg30, mediates peroxisome selection during pexophagy. It is necessary for pexophagy, but not for other selective and nonselective autophagy-related processes. It localizes at the peroxisome membrane via interaction with peroxins, and during pexophagy it colocalizes transiently at the preautophagosomal structure (PAS) and interacts with the autophagy machinery. PpAtg30 is required for formation of pexophagy intermediates, such as the micropexophagy apparatus (MIPA) and the pexophagosome (Ppg). During pexophagy, PpAtg30 undergoes multiple phosphorylations, at least one of which is required for pexophagy. PpAtg30 overexpression stimulates pexophagy even under peroxisome-induction conditions, impairing peroxisome biogenesis. Therefore, PpAtg30 is a key player in the selection of peroxisomes as cargo and in their delivery to the autophagy machinery for pexophagy.     

Phosphatidylethanolamine, a limiting factor of autophagy in yeast strains bearing a defect in the carboxypeptidase Y pathway of vacuolar targeting

Vps4p and Vps36p of Saccharomyces cerevisiae are involved in the transport of proteins to the vacuole via the carboxypeptidase Y pathway. We found that deletion of VPS4 and VPS36 caused impaired maturation of the vacuolar proaminopeptidase I (pAPI) via autophagy or the cytosol to vacuole targeting pathway. Supplementation with ethanolamine rescued this defect, leading to an increase of the cellular amount of phosphatidylethanolamine (PtdEtn), an enhanced level of the PtdEtn-binding autophagy protein Atg8p and a balanced rate of autophagy. We also discovered that maturation of pAPI was generally affected by PtdEtn depletion in a psd1Delta psd2Delta mutant due to reduced recruitment of Atg8p to the preautophagosomal structure. Ethanolamine supplementation provided the necessary amounts of PtdEtn for complete maturation of pAPI. Since the expression level of Atg8p was not compromised in the psd1Delta psd2Delta strain, we concluded that the amount of available PtdEtn was limiting. Thus, PtdEtn appears to be a limiting factor for the balance of the carboxypeptidase Y pathway and autophagy/the cytosol to vacuole targeting pathway in the yeast.     

Peroxisomicine A1, a potential antineoplastic agent,causes micropexophagy in addition to macropexophagy

Peroxisomicine A1 (PA1) is a potential antineoplastic agent with high and selective toxicity toward peroxisomes of tumor cells. Pexophagy is a selective autophagy process that degrades damaged peroxisomes; this process has been studied mainly in methylotrophic yeasts. There are two main modes of pexophagy in yeast: macropexophagy and micropexophagy. Previous studies showed that peroxisomes damaged by a prolonged exposition to PA1 are eliminated by macropexophagy. In this work, Candida boidinii was grown in methanol‐containing media, and PA1 was added to the cultures at 2 µg/mL after they reached the mid‐exponential growth phase. Samples were taken at 5, 10, 15, 20, and 25 min after the addition of PA1 and processed for ultrastructural analysis. Typical morphological characteristics of micropexophagy were observed: the direct engulfment of peroxisomes by the vacuolar membrane and the presence of the micropexophagic membrane apparatus (MIPA), which mediates the fusion between the opposing tips of the vacuole to complete sequestration of peroxisomes from the cytosol. In conclusion, here we report that, in addition to macropexophagy, peroxisomes damaged by PA1 can be eliminated by micropexophagy. This information is useful to deepen the knowledge of the mechanism of action of PA1 and of that of pexophagy per se.     

Selective Transport of α-Mannosidase by Autophagic Pathways: IDENTIFICATION OF A NOVEL RECEPTOR,Atg34p*

In Saccharomyces cerevisiae, aminopeptidase I (Ape1p) and α-mannosidase (Ams1p) are known cargoes of selective autophagy. Atg19p has been identified as an Ape1p receptor and targets Ape1p to the preautophagosomal structure (PAS). Under nutrient-rich conditions, transport of Ams1p to the vacuole largely depends on Atg19p. Here, we show that Atg34p (Yol083wp), a homolog of Atg19p, is a receptor for Ams1p transport during autophagy. Atg34p interacted with Ams1p, Atg11p, and Atg8p using distinct domains. Homo-oligomerized Ams1p bound to the Ams1-binding domain of Atg34p; this binding was important for the formation of a higher order complex named the Ams1 complex. In the absence of the interaction of Atg34p with Atg8p, the Ams1 complex was targeted to the preautophagosomal structure but failed to transit to the vacuole, indicating that the interaction of Atg34p with Atg8p is crucial for the Ams1 complex to be enclosed by autophagosomes. Atg34p and Atg19p have similar domain structures and are important for Ams1p transport during autophagy.     

Knockdown of Atg7 suppresses Tumorigenesis in a murine model of liver cancer

Hepatocellular Carcinoma (HCC) is the most common type of primary liver cancer in adults and a leading cause of cancer-related deaths worldwide. Studies have shown that autophagy is significantly involved in carcinogenesis, in particular, driven by activated RAS signaling. Autophagy related 7 (Atg7) is a critical component for the formation of autophagosome and required for autophagy processes. We investigated the role of autophagy in RAS-driven tumorigenesis in the liver, via the knockdown of Atg7 in the model. Transposon vectors encoding short hairpin RNAs targeting Atg7 (Atg7 shRNA) were constructed. Inhibition of autophagy via Atg7 knockdown was tested in Hep3B cells cultured in nutrient-starved medium. Formation of autophagosome was suppressed in nutrient-starved Hep3B cells expressing Atg7 shRNA, demonstrating that it efficiently inhibited autophagy in HCC cells. Transposons encoding Atg7 shRNA were mixed with those expressing HRAS^G12V and p53 shRNA, and subsequently used for hydrodynamic injection to 5-week-old C57BL/6 mice. Tumorigenesis in livers induced by HRAS^G12V and p53 shRNA was significantly suppressed by Atg7 knockdown. The inhibition of autophagy led to a decreased proliferation of cancer cells, as determined by Ki-67 staining. Our data indicate that knockdown of Atg7 led to a significant decrease in tumorigenesis in a murine HCC model induced by activated RAS. Inhibition of autophagosome formation is expected to be a therapeutic option for liver cancer.     

Atg4 recycles inappropriately lipidated Atg8 to promote autophagosome biogenesis

Atg8 is a ubiquitin-like protein required for autophagy in the budding yeast Saccharomyces cerevisiae. A ubiquitin-like system mediates the conjugation of the C terminus of Atg8 to the lipid phosphatidylethanolamine (PE), and this conjugate (Atg8-PE) plays a crucial role in autophagosome formation at the phagophore assembly site/pre-autophagosomal structure (PAS). The cysteine protease Atg4 processes the C terminus of newly synthesized Atg8 and also delipidates Atg8 to release the protein from membranes. While the former is a prerequisite for lipidation of Atg8, the significance of the latter in autophagy has remained unclear. Here, we show that autophagosome formation is significantly retarded in cells deficient for Atg4-mediated delipidation of Atg8. We find that Atg8-PE accumulates on various organelle membranes including the vacuole, the endosome and the ER in these cells, which depletes unlipidated Atg8 and thereby attenuates its localization to the PAS. Our results suggest that the Atg8-PE that accumulates on organelle membranes is erroneously produced by lipidation system components independently of the normal autophagic process. It is also suggested that delipidation of Atg8 by Atg4 on different organelle membranes promotes autophagosome formation. Considered together with other results, we propose that Atg4 acts to compensate for the intrinsic defect in the lipidation system; it recycles Atg8-PE generated on inappropriate membranes to maintain a reservoir of unlipidated Atg8 that is required for autophagosome formation at the PAS.     

The Autophagy-related Protein Kinase Atg1 Interacts with the Ubiquitin-like Protein Atg8 via the Atg8 Family Interacting Motif to Facilitate Autophagosome Formation

In autophagy, a cup-shaped membrane called the isolation membrane is formed, expanded, and sealed to complete a double membrane-bound vesicle called the autophagosome that encapsulates cellular constituents to be transported to and degraded in the lysosome/vacuole. The formation of the autophagosome requires autophagy-related (Atg) proteins. Atg8 is a ubiquitin-like protein that localizes to the isolation membrane; a subpopulation of this protein remains inside the autophagosome and is transported to the lysosome/vacuole. In the budding yeast Saccharomyces cerevisiae, Atg1 is a serine/threonine kinase that functions in the initial step of autophagosome formation and is also efficiently transported to the vacuole via autophagy. Here, we explore the mechanism and significance of this autophagic transport of Atg1. In selective types of autophagy, receptor proteins recognize degradation targets and also interact with Atg8, via the Atg8 family interacting motif (AIM), to link the targets to the isolation membrane. We find that Atg1 contains an AIM and directly interacts with Atg8. Mutations in the AIM disrupt this interaction and abolish vacuolar transport of Atg1. These results suggest that Atg1 associates with the isolation membrane by binding to Atg8, resulting in its incorporation into the autophagosome. We also show that mutations in the Atg1 AIM cause a significant defect in autophagy, without affecting the functions of Atg1 implicated in triggering autophagosome formation. We propose that in addition to its essential function in the initial stage, Atg1 also associates with the isolation membrane to promote its maturation into the autophagosome.     

Hrr25 phosphorylates the autophagic receptor Atg34 to promote vacuolar transport of α-mannosidase under nitrogen starvation conditions

In Saccharomyces cerevisiae, under nitrogen-starvation conditions, the α-mannosidase Ams1 is recognized by the autophagic receptor Atg34 and transported into the vacuole, where it functions as an active enzyme. In this study, we identified Hrr25 as the kinase that phosphorylates Atg34 under these conditions. Hrr25-mediated phosphorylation does not affect the interaction of Atg34 with Ams1, but instead promotes Atg34 binding to the adaptor protein Atg11, which recruits the autophagy machinery to the Ams1–Atg34 complex, resulting in activation of the vacuolar transport of Ams1. Our findings reveal the regulatory mechanism of a biosynthetic pathway mediated by the autophagy machinery.     

Role of Vac8 in Cellular Degradation Pathways in Pichia pastoris

We have identified the Pichia pastoris Vac8 homolog, a 60-64 kDa armadillo repeat protein, and have examined the role of PpVac8 in the degradative pathways involving the yeast vacuole. We report here that PpVac8 is required for glucose-induced pexophagy and mitophagy, but not ethanol-induced pexophagy or starvation-induced autophagy. This has been demonstrated by the persistence of peroxisomal alcohol oxidase activity and GFP-labeled mitochondria in mutants lacking PpVac8 during glucose adaptation. During glucose-induced micropexophagy, in the absence of PpVac8, the vacuole was invaginated with arm-like “segmented” extensions that almost completely surrounded the adjacent peroxisomes. PpVac8-GFP was found at the vacuolar membrane and concentrated at the base of the sequestering membranes that extend from the vacuole to engulf the peroxisomes. The localization of PpVac8-GFP to the vacuolar membrane occurred independent of PpAtg1, PpAtg9 or PpAtg11. Mutagenesis of the palmitoylated cysteines to alanines or deletion of the myristoylation and palmitoylation sites of PpVac8, resulted in an impaired vacuolar association and decreased degradation of alcohol oxidase. Deletion of the central armadillo repeat domains of the PpVac8 did not alter its association with the vacuolar membrane, but resulted in a nonfunctional protein that suppressed the formation of the arm-like extensions from the vacuole to engulf the peroxisomes. PpVac8 is essential for the trafficking of PpAtg11, but not PpAtg1 or PpAtg18, to the vacuole membrane. Together, our results support a role for PpVac8 in early (formation of sequestering membranes) and late (post-MIPA membrane fusion) molecular events of glucose-induced pexophagy.     

Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure during the Cvt pathway

Atg21 and Atg18 are homologue yeast proteins. Whereas Atg18 is essential for the Cvt pathway and autophagy, a lack of Atg21 only blocks the Cvt pathway. Our proteinase protection experiments now demonstrate that growing atg21Delta cells fail to form proaminopeptidase I-containing Cvt vesicles. Quantitative measurement of autophagy in starving atg21Delta cells showed only 35% of the wild-type rate. This suggests that Atg21 plays a nonessential role in improving the fidelity of autophagy. The intracellular localization of Atg21 is unique among the Atg proteins. In cells containing multiple vacuoles, Atg21-yellow fluorescent protein clearly localizes to the vertices of the vacuole junctions. Cells with a single vacuole show most of the protein at few perivacuolar punctae. This distribution pattern is reminiscent to the Vps class C(HOPS) (homotypic fusion and vacuolar protein sorting) protein complex. In growing cells, Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure. Consistently, the covalent linkage of Atg8 to the lipid phosphatidylethanolamine is significantly retarded. Lipidated Atg8 is supposed to act during the elongation of autophagosome precursors. However, despite the reduced autophagic rate and the retardation of Atg8 lipidation, electron microscopy of starved atg21Delta ypt7Delta double mutant cells demonstrates the formation of normally sized autophagosomes with an average diameter of 450 nm.     

Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates

Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4’s specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes.     

Atg18 phosphoregulation controls organellar dynamics by modulating its phosphoinositide-binding activity

The PROPPIN family member Atg18 is a phosphoinositide-binding protein that is composed of a seven β-propeller motif and is part of the conserved autophagy machinery. Here, we report that the Atg18 phosphorylation in the loops in the propellar structure of blade 6 and blade 7 decreases its binding affinity to phosphatidylinositol 3,5-bisphosphate in the yeast Pichia pastoris. Dephosphorylation of Atg18 was necessary for its association with the vacuolar membrane and caused septation of the vacuole. Upon or after dissociation from the vacuolar membrane, Atg18 was rephosphorylated, and the vacuoles fused and formed a single rounded structure. Vacuolar dynamics were regulated according to osmotic changes, oxidative stresses, and nutrient conditions inducing micropexophagy via modulation of Atg18 phosphorylation. This study reveals how the phosphoinositide-binding activity of the PROPPIN family protein Atg18 is regulated at the membrane association domain and highlights the importance of such phosphoregulation in coordinated intracellular reorganization.     

Atg4 recycles inappropriately lipidated Atg8 to promote autophagosome biogenesis

Atg8 is a ubiquitin-like protein required for autophagy in the budding yeast Saccharomyces cerevisiae. A ubiquitin-like system mediates the conjugation of the C terminus of Atg8 to the lipid phosphatidylethanolamine (PE), and this conjugate (Atg8–PE) plays a crucial role in autophagosome formation at the phagophore assembly site/pre-autophagosomal structure (PAS). The cysteine protease Atg4 processes the C terminus of newly synthesized Atg8 and also delipidates Atg8 to release the protein from membranes. While the former is a prerequisite for lipidation of Atg8, the significance of the latter in autophagy has remained unclear. Here, we show that autophagosome formation is significantly retarded in cells deficient for Atg4-mediated delipidation of Atg8. We find that Atg8–PE accumulates on various organelle membranes including the vacuole, the endosome and the ER in these cells, which depletes unlipidated Atg8 and thereby attenuates its localization to the PAS. Our results suggest that the Atg8–PE that accumulates on organelle membranes is erroneously produced by lipidation system components independently of the normal autophagic process. It is also suggested that delipidation of Atg8 by Atg4 on different organelle membranes promotes autophagosome formation. Considered together with other results, we propose that Atg4 acts to compensate for the intrinsic defect in the lipidation system; it recycles Atg8–PE generated on inappropriate membranes to maintain a reservoir of unlipidated Atg8 that is required for autophagosome formation at the PAS.