Atg14L and Rubicon: Yin and yang of Beclin 1-mediated autophagy control

Emerging evidence suggests that Beclin 1, the mammalian ortholog of yeast Atg6/Vps30, functions to coordinate two important cellular pathways: autophagy and apoptosis. Beclin 1 is a component of the Vps34/class III phosphatidylinositol 3-kinase (PtdIns3K) protein complex. However, the Beclin 1-Vps34/PtdIns3K protein complex formation and its function in autophagy regulation remain to be elucidated. Through an integrated approach that combines mouse genetics and biochemistry, we identified two novel Beclin 1 interacting proteins, Atg14L and Rubicon. We found that Atg14L and Rubicon play opposing roles in autophagy regulation by forming distinct complexes with Beclin 1, modulating the Vps34/PtdIns3K activity and targeting distinct steps of the autophagic process.     

A novel ER-localized transmembrane protein,EMC6, interacts with RAB5A and regulates cell autophagy

Autophagy is mediated by a unique organelle, the autophagosome, which encloses a portion of the cytoplasm for delivery to the lysosome. Phosphatidylinositol 3-phosphate (PtdIns3P) produced by the class III phosphatidylinositol 3-kinase (PtdIns3K) complex is essential for canonical autophagosome formation. RAB5A, a small GTPase localized to early endosomes, has been shown to associate with the class III PtdIns3K complex, regulate its activity and promote autophagosome formation. However, little is known about how endosome-localized RAB5A functions with the class III PtdIns3K complex. Here we identified a novel endoplasmic reticulum (ER)-localized transmembrane protein, ER membrane protein complex subunit 6 (EMC6), which interacted with both RAB5A and BECN1/Beclin 1 and colocalized with the omegasome marker ZFYVE1/DFCP1. It was shown to regulate autophagosome formation, and its deficiency caused the accumulation of autophagosomal precursor structures and impaired autophagy. Our study showed for the first time that EMC6 is a novel regulator involved in autophagy.     

Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation

Canonical autophagy is positively regulated by the Beclin 1/phosphatidylinositol 3-kinase class III (PtdIns3KC3) complex that generates an essential phospholipid, phosphatidylinositol 3-phosphate (PtdIns(3)P), for the formation of autophagosomes. Previously, we identified the human WIPI protein family and found that WIPI-1 specifically binds PtdIns(3)P, accumulates at the phagophore and becomes a membrane protein of generated autophagosomes. Combining siRNA-mediated protein downregulation with automated high through-put analysis of PtdIns(3)P-dependent autophagosomal membrane localization of WIPI-1, we found that WIPI-1 functions upstream of both Atg7 and Atg5, and stimulates an increase of LC3-II upon nutrient starvation. Resveratrol-mediated autophagy was shown to enter autophagic degradation in a noncanonical manner, independent of Beclin 1 but dependent on Atg7 and Atg5. By using electron microscopy, LC3 lipidation and GFP-LC3 puncta-formation assays we confirmed these results and found that this effect is partially wortmannin-insensitive. In line with this, resveratrol did not promote phagophore localization of WIPI-1, WIPI-2 or the Atg16L complex above basal level. In fact, the presence of resveratrol in nutrient-free conditions inhibited phagophore localization of WIPI-1. Nevertheless, we found that resveratrol-mediated autophagy functionally depends on canonical-driven LC3-II production, as shown by siRNA-mediated downregulation of WIPI-1 or WIPI-2. From this it is tempting to speculate that resveratrol promotes noncanonical autophagic degradation downstream of the PtdIns(3)P-WIPI-Atg7-Atg5 pathway, by engaging a distinct subset of LC3-II that might be generated at membrane origins apart from canonical phagophore structures.     

Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation

Canonical autophagy is positively regulated by the Beclin 1/phosphatidylinositol 3-kinase class III (PtdIns3KC3) complex that generates an essential phospholipid, phosphatidylinositol 3-phosphate (PtdIns(3)P), for the formation of autophagosomes. Previously, we identified the human WIPI protein family and found that WIPI-1 specifically binds PtdIns(3)P, accumulates at the phagophore and becomes a membrane protein of generated autophagosomes. Combining siRNA-mediated protein downregulation with automated high through-put analysis of PtdIns(3)P-dependent autophagosomal membrane localization of WIPI-1, we found that WIPI-1 functions upstream of both Atg7 and Atg5, and stimulates an increase of LC3-II upon nutrient starvation. Resveratrol-mediated autophagy was shown to enter autophagic degradation in a noncanonical manner, independent of Beclin 1 but dependent on Atg7 and Atg5. By using electron microscopy, LC3 lipidation and GFP-LC3 puncta-formation assays we confirmed these results and found that this effect is partially wortmannin-insensitive. In line with this, resveratrol did not promote phagophore localization of WIPI-1, WIPI-2 or the Atg16L complex above basal level. In fact, the presence of resveratrol in nutrient-free conditions inhibited phagophore localization of WIPI-1. Nevertheless, we found that resveratrol-mediated autophagy functionally depends on canonical-driven LC3-II production, as shown by siRNA-mediated downregulation of WIPI-1 or WIPI-2. From this it is tempting to speculate that resveratrol promotes noncanonical autophagic degradation downstream of the PtdIns(3)P-WIPI-Atg7-Atg5 pathway, by engaging a distinct subset of LC3-II that might be generated at membrane origins apart from canonical phagophore structures.     

Negative regulation of phosphatidylinositol 3-phosphate levels in early-to-late endosome conversion

Phosphatidylinositol 3-phosphate (PtdIns3P) plays a central role in endosome fusion, recycling, sorting, and early-to-late endosome conversion, but the mechanisms that determine how the correct endosomal PtdIns3P level is achieved remain largely elusive. Here we identify two new factors, SORF-1 and SORF-2, as essential PtdIns3P regulators in Caenorhabditis elegans. Loss of sorf-1 or sorf-2 leads to greatly elevated endosomal PtdIns3P, which drives excessive fusion of early endosomes. sorf-1 and sorf-2 function coordinately with Rab switching genes to inhibit synthesis of PtdIns3P, allowing its turnover for endosome conversion. SORF-1 and SORF-2 act in a complex with BEC-1/Beclin1, and their loss causes elevated activity of the phosphatidylinositol 3-kinase (PI3K) complex. In mammalian cells, inactivation of WDR91 and WDR81, the homologs of SORF-1 and SORF-2, induces Beclin1-dependent enlargement of PtdIns3P-enriched endosomes and defective degradation of epidermal growth factor receptor. WDR91 and WDR81 interact with Beclin1 and inhibit PI3K complex activity. These findings reveal a conserved mechanism that controls appropriate PtdIns3P levels in early-to-late endosome conversion.     

综述: Ⅲ型磷脂酰肌醇3-激酶复合物与自噬的研究进展

Ⅲ型磷脂酰肌醇3-激酶(class Ⅲ PI3K)是以磷脂酰肌醇(PtdIns)为底物催化产生PtdIns3 P的激酶,与多种不同的调节蛋白结合形成Ⅲ型PI3K(PI3KC3)复合物,在自噬及膜泡运输中起重要作用．PI3KC3复合物组成成员PI(3)KC3、p150、Beclin 1、ATG14L、UVRAG、Bif-1和Rubicon在进化上大多具有高度的同源性和保守性,并且与神经系统发育、胸腹腔内脏反位及肿瘤等多种疾病的发生和发展密切相关．     

BAC-mediated transgenic expression of fluorescent autophagic protein Beclin 1 reveals a role for Beclin 1 in lymphocyte development

Beclin 1/Atg6 is an essential component of the evolutionary conserved PtdIns(3)-kinase (Vps34) protein complex that regulates macroautophagy (autophagy) in eukaryotic cells and also interacts with antiapoptotic Bcl-2 family members, Bcl-2, and Bcl-x(L). To elucidate the physiological function of Beclin 1, we generated transgenic mice producing a green fluorescent Beclin 1 protein (Beclin 1-GFP) under Beclin 1 endogenous regulation. The beclin 1-GFP transgene is functional because it completely rescues early embryonic lethality in beclin 1-deficient mice. The transgenic mice appear normal, with undetected change in basal autophagy levels in different tissues, despite the additional expression of functional Beclin 1-GFP. Staining of Beclin 1-GFP shows mostly diffuse cytoplasmic distribution in various tissues. Detailed analysis of the transgene expression by flow cytometry reveals a Bcl-2-like biphasic expression pattern in developing T and B cells, as well as differential regulation of expression in mature versus immature thymocytes following in vitro stimulation. Moreover, thymocytes expressing high Beclin 1-GFP levels appear increasingly sensitive to glucocorticoid-induced apoptosis in vitro. Our results, therefore, support a role for Beclin 1 in lymphocyte development involving cross talk between autophagy and apoptosis.     

Divergent roles of BECN1 in LC3 lipidation and autophagosomal function

BECN1/Beclin 1 is regarded as a critical component in the class III phosphatidylinositol 3-kinase (PtdIns3K) complex to trigger autophagy in mammalian cells. Despite its significant role in a number of cellular and physiological processes, the exact function of BECN1 in autophagy remains controversial. Here we created a BECN1 knockout human cell line using the TALEN technique. Surprisingly, the complete loss of BECN1 had little effect on LC3 (MAP1LC3B/LC3B) lipidation, and LC3B puncta resembling autophagosomes by fluorescence microscopy were still evident albeit significantly smaller than those in the wild-type cells. Electron microscopy (EM) analysis revealed that BECN1 deficiency led to malformed autophagosome-like structures containing multiple layers of membranes under amino acid starvation. We further confirmed that the PtdIns3K complex activity and autophagy flux were disrupted in BECN1^−/− cells. Our results demonstrate the essential role of BECN1 in the functional formation of autophagosomes, but not in LC3B lipidation.     

AMPK regulates autophagy by phosphorylating BECN1 at threonine 388

Macroautophagy/autophagy is a conserved catabolic process that recycles cytoplasmic material during low energy conditions. BECN1/Beclin1 (Beclin 1, autophagy related) is an essential protein for function of the class 3 phosphatidylinositol 3-kinase (PtdIns3K) complexes that play a key role in autophagy nucleation and elongation. Here, we show that AMP-activated protein kinase (AMPK) regulates autophagy by phosphorylating BECN1 at Thr388. Phosphorylation of BECN1 is required for autophagy upon glucose withdrawal. BECN1^T388A, a phosphorylation defective mutant, suppresses autophagy through decreasing the interaction between PIK3C3 (phosphatidylinositol 3-kinase catalytic subunit type 3) and ATG14 (autophagy-related 14). The BECN1^T388A mutant has a higher affinity for BCL2 than its wild-type counterpart; the mutant is more prone to dimer formation. Conversely, a BECN1 phosphorylation mimic mutant, T388D, has stronger binding to PIK3C3 and ATG14, and promotes higher autophagy activity than the wild-type control. These findings uncover a novel mechanism of autophagy regulation.     

Autophagy and Akt promote survival in glioma

Signaling through phosphatidylinositol 3-kinase (PtdIns3K)-Akt-mTOR is frequently activated in cancers including glioblastoma multiforme (GBM), where this kinase network regulates survival. It is thus surprising that inhibitors of these pathways induce minimal cell death in glioma. We showed that the dual PtdIns3K-mTOR inhibitor PI-103 induces autophagy in therapy-resistant, PTEN-mutant glioma, with blockade of mTOR complex 1 (mTORC1) and complex 2 (mTORC2) contributing independently to autophagy. Inhibition of autophagosome maturation synergizes with PI-103 to induce apoptosis through the Bax-dependent intrinsic mitochondrial pathway, indicating that PI-103 induces autophagy as a survival pathway in this setting. Not all inhibitors of PtdIns3K-Akt-mTOR signaling synergize with inhibitors of autophagy. The allosteric mTORC1 inhibitor rapamycin fails to induce apoptosis in conjunction with blockade of autophagy, due to feedback-activation of Akt. Apoptosis in the setting of rapamycin therapy requires concurrent inhibition of both autophagy and of PtdIns3K-Akt. Moreover, the clinical PtdIns3K-mTOR inhibitor NVP-BEZ235 cooperates with the clinical lysosomotropic autophagy inhibitor chloroquine to induce apoptosis in PTEN-mutant glioma xenografts in vivo, offering a therapeutic approach translatable to patients.     

Autophagy and Akt promote survival in glioma

Signaling through phosphatidylinositol 3-kinase (PtdIns3K)-Akt-mTOR is frequently activated in cancers including glioblastoma multiforme (GBM), where this kinase network regulates survival. It is thus surprising that inhibitors of these pathways induce minimal cell death in glioma. We showed that the dual PtdIns3K-mTOR inhibitor PI-103 induces autophagy in therapy-resistant, PTEN-mutant glioma, with blockade of mTOR complex 1 (mTORC1) and complex 2 (mTORC2) contributing independently to autophagy. Inhibition of autophagosome maturation synergizes with PI-103 to induce apoptosis through the Bax-dependent intrinsic mitochondrial pathway, indicating that PI-103 induces autophagy as a survival pathway in this setting. Not all inhibitors of PtdIns3K-Akt-mTOR signaling synergize with inhibitors of autophagy. The allosteric mTORC1 inhibitor rapamycin fails to induce apoptosis in conjunction with blockade of autophagy, due to feedback-activation of Akt. Apoptosis in the setting of rapamycin therapy requires concurrent inhibition of both autophagy and of PtdIns3K-Akt. Moreover, the clinical PtdIns3K-mTOR inhibitor NVP-BEZ235 cooperates with the clinical lysosomotropic autophagy inhibitor chloroquine to induce apoptosis in PTEN-mutant glioma xenografts in vivo, offering a therapeutic approach translatable to patients.     

Ubiquitination and phosphorylation of Beclin 1 and its binding partners: Tuning class III phosphatidylinositol 3-kinase activity and tumor suppression

The class III phosphatidylinositol 3-kinase (PI3K-III) complex and its phosphorylated lipid product phosphatidylinositol 3-phosphate (PtdIns3P) control the three topologically related membrane-involution processes autophagy, endocytosis, and cytokinesis. The activity of the catalytic unit of PI3K-III complex, the Vacuolar sorting protein 34 (VPS34), depends on the membrane targeting unit Vacuolar sorting protein 15 (VPS15), and the tumor suppressor protein Beclin 1. It is established that the overall activity of VPS34 is positively regulated by Beclin 1, whose positive influence is further controlled through the association with a set of Beclin1 interacting components, which stimulate or inhibit VPS34. The interaction between Beclin 1 and Beclin 1-associated components are controllable and is regulated by phosphorylation in a context-dependent manner. Here, we focus on an emerging concept whereby the activity of the PI3K-III complex is controlled by ubiquitination of Beclin 1 or Beclin 1-associated molecules. In summary, at least three different ubiquitin ligases can affect the positive regulatory function of Beclin 1 towards VPS34, suggesting that ubiquitination is an important and physiologically relevant event in tuning the tumor suppressor function of Beclin 1.     

Association of a phosphatidylinositol-specific 3-kinase with a human trans-Golgi network resident protein

The eukaryotic trans-Golgi network (TGN) is a key site for the formation of transport vesicles destined for different intracellular compartments [1]. A key marker for the mammalian TGN is TGN38/46 [2]. This integral membrane glycoprotein cycles between the TGN and the cell surface and is implicated in recruitment of cytosolic factors and regulation of at least one type of vesicle formation at the mammalian TGN [2] and [3]. In this study, we have identified a phosphatidylinositol (PtdIns)-specific 3-kinase activity associated with the human orthologue (TGN46), which is sensitive to lipid kinase inhibitors. Treatment of HeLa cells with low levels of these inhibitors reveals subtle morphological changes in TGN46-positive compartments. Our findings suggest a role for PtdIns 3-kinases and presumably for the product, PtdIns 3-phosphate (PtdIns3P), in the formation of secretory transport vesicles by mechanisms conserved in yeast and mammals.     

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

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

Dynamics and function of PtdIns(3)P in autophagy

Phosphorylation of phosphatidylinositol (PtdIns) by PtdIns 3-kinase is essential for autophagy. However, the distribution and function of the enzymatic product, PtdIns 3-phosphate (PtdIns(3)P), has been unknown. We monitored PtdIns(3)P distribution during autophagy by live imaging, biochemistry, and electron microscopy, and found that PtdIns(3)P is massively delivered into the vacuole via autophagy. PtdIns(3)P is highly enriched as a membrane component of the elongating isolation membranes and autophagosome membranes rather than as an enclosed cargo, implying direct involvement of PtdIns(3)P in autophagosome formation. This observation also provides important basic information on the nature of the autophagosome membrane, which is still poorly understood. Notably, PtdIns(3)P is highly enriched on the inner (concave) surfaces of the isolation membrane and autophagosome compared to the outer surfaces. PtdIns(3)P is also enriched on ambiguous structures juxtaposed to the elongating tips of isolation membranes. We also investigated the function of PtdIns(3)P in autophagy, and show that PtdIns(3)P recruits the Atg18-Atg2 complex to autophagic membranes through an Atg18-PtdIns(3)P interaction. Interestingly, PtdIns(3)P is required only for the association of the Atg18-Atg2 complex to autophagic membranes but not for any subsequent functional activity of the Atg18-Atg2 complex, suggesting that PtdIns(3)P does not act allosterically on Atg18. Based on these results we discuss the function of PtdIns(3)P in autophagy.     

Autophagosome biogenesis in plants: Roles of SH3P2

The autophagy core machinery is essentially conserved in eukaryotic cells for autophagy regulation. However, the underlying mechanisms for autophagosome formation in plant cells remain elusive. We have recently demonstrated that SH3 domain-containing protein 2 (SH3P2), a BAR (Bin-Amphiphysin-Rvs) domain protein, functions as a novel regulator for autophagosome biogenesis in Arabidopsis thaliana. Using SH3P2 and its GFP fusion as probes, we have characterized the dynamics and structures of autophagosome formation in plant cells. The phagophore assembly site, marked by SH3P2, is identified as having a close connection with the ER. SH3P2 also binds to phosphatidylinositol 3-phosphate (PtdIns3P) and functions downstream of the phosphatidylinositol 3-kinase (PtdIns3K) complex. Thus, SH3P2 serves as a novel membrane-associated protein in regulating autophagosome formation in Arabidopsis thaliana.     

Ambra1

Mutations in the gene for the E3 ubiquitin ligase Parkin are the most prevalent cause of autosomal recessive Parkinson disease (PD), an incurable neurodegenerative disorder. Parkin surveys mitochondrial quality by translocating to depolarized mitochondria and inducing their selective macroautophagic removal (mitophagy). We recently reported that Parkin interacts with Ambra1 (activating molecule in Beclin 1-regulated autophagy), a protein that promotes autophagy in the vertebrate central nervous system. We discovered that prolonged mitochondrial depolarization strongly increases the interaction of Parkin with Ambra1. Ambra1 is recruited in a Parkin-dependent manner to perinuclear clusters of depolarized mitochondria, activates the class III phosphatidylinositol 3-kinase (PtdIns3K) complex around these mitochondria and contributes to their selective autophagic clearance. Here, we discuss these findings and suggest a model where translocated Parkin efficiently triggers mitophagy through combined recruitment of Ambra1 and ubiquitination of outer mitochondrial membrane proteins.     

Ambra1: a Parkin-binding protein involved in mitophagy

Mutations in the gene for the E3 ubiquitin ligase Parkin are the most prevalent cause of autosomal recessive Parkinson disease (PD), an incurable neurodegenerative disorder. Parkin surveys mitochondrial quality by translocating to depolarized mitochondria and inducing their selective macroautophagic removal (mitophagy). We recently reported that Parkin interacts with Ambra1 (activating molecule in Beclin 1-regulated autophagy), a protein that promotes autophagy in the vertebrate central nervous system. We discovered that prolonged mitochondrial depolarization strongly increases the interaction of Parkin with Ambra1. Ambra1 is recruited in a Parkin-dependent manner to perinuclear clusters of depolarized mitochondria, activates the class III phosphatidylinositol 3-kinase (PtdIns3K) complex around these mitochondria and contributes to their selective autophagic clearance. Here, we discuss these findings and suggest a model where translocated Parkin efficiently triggers mitophagy through combined recruitment of Ambra1 and ubiquitination of outer mitochondrial membrane proteins.     

Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.     

3-methyladenine inhibits autophagy in tobacco culture cells under sucrose starvation conditions

Tobacco (Nicotiana tabacum) culture cells perform autophagy and degrade cellular proteins in response to sucrose starvation. When protein degradation is blocked by the cysteine protease inhibitor E-64c, lysosomes containing particles of cytoplasm (autolysosomes) accumulate in the cells. Therefore, using light microscopy, we can determine whether cells have performed autophagy. In this study, we investigated whether or not 3-methyladenine (3-MA), which is a known inhibitor of autophagy in mammalian cells, blocks autophagy in tobacco culture cells. The accumulation of autolysosomes was blocked by the addition to the culture media of 5 mM 3-MA together with E-64c. We did not detect autolysosomes or structures thought to be involved with autophagy, such as autophagosomes, accumulating in these cells, as observed by electron microscopy. 3-MA blocked cellular protein degradation without any effect on cellular protease activity. In mammalian cells, phosphatidylinositol 3-kinase (PtdIns 3-kinase) is a putative target of 3-MA. The PtdIns 3-kinase inhibitors wortmannin and LY294002 also inhibited the accumulation of autolysosomes in tobacco culture cells. These results suggest that (1) 3-MA inhibits autophagy by blocking the formation of autophagosomes in tobacco culture cells, and (2) PtdIns 3-kinase is essential for autophagy in tobacco cells.