Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG

Class III phosphatidylinositol 3-kinase (PI3-kinase) regulates multiple membrane trafficking. In yeast, two distinct PI3-kinase complexes are known: complex I (Vps34, Vps15, Vps30/Atg6, and Atg14) is involved in autophagy, and complex II (Vps34, Vps15, Vps30/Atg6, and Vps38) functions in the vacuolar protein sorting pathway. Atg14 and Vps38 are important in inducing both complexes to exert distinct functions. In mammals, the counterparts of Vps34, Vps15, and Vps30/Atg6 have been identified as Vps34, p150, and Beclin 1, respectively. However, orthologues of Atg14 and Vps38 remain unknown. We identified putative mammalian homologues of Atg14 and Vps38. The Vps38 candidate is identical to UV irradiation resistance-associated gene (UVRAG), which has been reported as a Beclin 1-interacting protein. Although both human Atg14 and UVRAG interact with Beclin 1 and Vps34, Atg14, and UVRAG are not present in the same complex. Although Atg14 is present on autophagic isolation membranes, UVRAG primarily associates with Rab9-positive endosomes. Silencing of human Atg14 in HeLa cells suppresses autophagosome formation. The coiled-coil region of Atg14 required for binding with Vps34 and Beclin 1 is essential for autophagy. These results suggest that mammalian cells have at least two distinct class III PI3-kinase complexes, which may function in different membrane trafficking pathways.     

Binding Rubicon to cross the Rubicon

Beclin 1 is an antitumor protein, required for mammalian autophagy, but its precise molecular function is poorly understood. Mass spectrometry analysis reveals that two novel proteins, Atg14L and Rubicon, associate with Beclin 1, together with a known Beclin 1-binding protein, UVRAG. The interactions of Atg14L and UVRAG with the Beclin 1-Vps34 (class III PI3-kinase)-Vps15 core complex are mutually exclusive; Rubicon associates with a subpopulation of UVRAG-containing complexes. The Atg14L complex, which positively regulates autophagy at an early step, localizes to the phagophore/isolation membrane, autophagosome and endoplasmic reticulum. In contrast, the Rubicon-UVRAG complex localizes to the late endosome/lysosome and negatively regulates both autophagy at a later step and the endocytic pathway. Thus, the Beclin 1-Vps34-Vps15 complex functions in autophagy and the endocytic pathway, but its function in a given context depends on the identity of its interacting subunits.     

Nrbf2 Protein Suppresses Autophagy by Modulating Atg14L Protein-containing Beclin 1-Vps34 Complex Architecture and Reducing Intracellular Phosphatidylinositol-3 Phosphate Levels

Autophagy is a tightly regulated lysosomal degradation pathway for maintaining cellular homeostasis and responding to stresses. Beclin 1 and its interacting proteins, including the class III phosphatidylinositol-3 kinase Vps34, play crucial roles in autophagy regulation in mammals. We identified nuclear receptor binding factor 2 (Nrbf2) as a Beclin 1-interacting protein from Becn1^−/−;Becn1-EGFP/+ mouse liver and brain. We also found that Nrbf2-Beclin 1 interaction required the N terminus of Nrbf2. We next used the human retinal pigment epithelial cell line RPE-1 as a model system and showed that transiently knocking down Nrbf2 by siRNA increased autophagic flux under both nutrient-rich and starvation conditions. To investigate the mechanism by which Nrbf2 regulates autophagy, we demonstrated that Nrbf2 interacted and colocalized with Atg14L, suggesting that Nrbf2 is a component of the Atg14L-containing Beclin 1-Vps34 complex. Moreover, ectopically expressed Nrbf2 formed cytosolic puncta that were positive for isolation membrane markers. These results suggest that Nrbf2 is involved in autophagosome biogenesis. Furthermore, we showed that Nrbf2 deficiency led to increased intracellular phosphatidylinositol-3 phosphate levels and diminished Atg14L-Vps34/Vps15 interactions, suggesting that Nrbf2-mediated Atg14L-Vps34/Vps15 interactions likely inhibit Vps34 activity. Therefore, we propose that Nrbf2 may interact with the Atg14L-containing Beclin 1-Vps34 protein complex to modulate protein-protein interactions within the complex, leading to suppression of Vps34 activity, autophagosome biogenesis, and autophagic flux. This work reveals a novel aspect of the intricate mechanism for the Beclin 1-Vps34 protein-protein interaction network to achieve precise control of autophagy.     

Atg14 and UVRAG: Mutually exclusive subunits of mammalian Beclin 1-PI3K complexes

Vps34, a Class III phosphatidylinositol 3-kinase (PI3-kinase), produces phosphatidylinositol 3 phosphate (PI3P) and functions in various membrane traffic pathways including endocytosis, multivesicular body formation and autophagy. In mammalian cells, Vps34 forms a complex with Beclin 1, but it remains unclear how this Vps34 complex exerts its specific function on each membrane trafficking pathway. We recently identified mammalian Atg14, a new binding partner of the Vps34-Beclin 1 complex, using a computational approach. The Atg14 complex consists of Vps34, Beclin 1 and p150, but lacks UVRAG, which was previously reported to bind the Vps34-Beclin 1 complex. Atg14 localizes to isolation membrane/phagophore during starvation and is essential for autophagosome formation. In contrast, UVRAG primarily localizes to late endosomes. Since UVRAG shows homology with yeast Vps38, we speculate that it could be a mammalian Vps38 ortholog. These findings indicate that the Vps34-Beclin 1 complex has at least two distinct functions, which can be promoted by its binding partners Atg14 and UVRAG.     

The evolutionarily conserved domain of Beclin 1 is required for Vps34 binding, autophagy and tumor suppressor function

Atg6/Beclin 1 is an evolutionarily conserved protein family that has been shown to function in vacuolar protein sorting (VPS) in yeast; in autophagy in yeast, Drosophila, Dictyostelium, C.elegans, and mammals; and in tumor suppression in mice. Atg6/Beclin 1 is thought to function as a VPS and autophagy protein as part of a complex with Class III phosphatidylinositol 3'-kinase (PI3K)/Vps34. However, nothing is known about which domains of Atg6/Beclin 1 are required for its functional activity and binding to Vps34. We hypothesized that the most highly conserved region of human Beclin 1 spanning from amino acids 244-337 is essential for Vps34 binding, autophagy, and tumor suppressor function. To investigate this hypothesis, we evaluated the effects of wild-type and mutant beclin 1 gene transfer in autophagy-deficient MCF7 human breast carcinoma cells. We found that, unlike wild-type Beclin 1, a Beclin 1 mutant lacking aa 244-337 (Beclin 1DeltaECD), is unable to enhance starvation-induced autophagy in low Beclin 1-expressing MCF7 human breast carcinoma cells. In contrast to wild-type Beclin 1, mutant Beclin 1DeltaECD is unable to immunoprecipitate Vps34, has no Beclin 1-associated Vps34 kinase activity, and lacks tumor suppressor function in an MCF7 scid mouse xenograft tumor model. The maturation of cathepsin D, which requires intact Vps34-dependent VPS function, is comparable in autophagy-deficient low-Beclin 1 expressing MCF7 cells, autophagy-deficient MCF7 cells transfected with Beclin 1DeltaECD, and autophagy-competent MCF7 cells transfected with wild-type Beclin 1. These findings identify an evolutionarily conserved domain of Beclin 1 that is essential for Vps34 interaction, autophagy function, and tumor suppressor function. Furthermore, they suggest a connection between Beclin 1-associated Class III PI3K/Vps34-dependent autophagy, but not VPS, function and the mechanism of Beclin 1 tumor suppressor action in human breast cancer cells.     

Dapper1 promotes autophagy by enhancing the Beclin1-Vps34-Atg14L complex formation

Autophagy is an intracellular degradation process to clear up aggregated proteins or aged and damaged organelles. The Beclin1-Vps34-Atg14L complex is essential for autophagosome formation. However, how the complex formation is regulated is unclear. Here, we show that Dapper1 (Dpr1) acts as a critical regulator of the Beclin1-Vps34-Atg14L complex to promote autophagy. Dpr1 ablation in the central nervous system results in motor coordination defect and accumulation of p62 and ubiquitinated proteins. Dpr1 increases autophagosome formation as indicated by elevated puncta formation of LC3, Atg14L and DFCP1 (Double FYVE-containing protein 1). Conversely, loss of Dpr1 impairs LC3 lipidation and causes p62/SQSTM1 accumulation. Dpr1 directly interacts with Beclin1 and Atg14L and enhances the Beclin1-Vps34 interaction and Vps34 activity. Together, our findings suggest that Dpr1 enhances the Atg14L-Beclin1-Vps34 complex formation to drive autophagy.     

MTORC1-mediated NRBF2 phosphorylation functions as a switch for the class III PtdIns3K and autophagy

NRBF2/Atg38 has been identified as the fifth subunit of the macroautophagic/autophagic class III phosphatidylinositol 3-kinase (PtdIns3K) complex, along with ATG14/Barkor, BECN1/Vps30, PIK3R4/p150/Vps15 and PIK3C3/Vps34. However, its functional mechanism and regulation are not fully understood. Here, we report that NRBF2 is a fine tuning regulator of PtdIns3K controlled by phosphorylation. Human NRBF2 is phosphorylated by MTORC1 at S113 and S120. Upon nutrient starvation or MTORC1 inhibition, NRBF2 phosphorylation is diminished. Phosphorylated NRBF2 preferentially interacts with PIK3C3/PIK3R4. Suppression of NRBF2 phosphorylation by MTORC1 inhibition alters its binding preference from PIK3C3/PIK3R4 to ATG14/BECN1, leading to increased autophagic PtdIns3K complex assembly, as well as enhancement of ULK1 protein complex association. Consequently, NRBF2 in its unphosphorylated form promotes PtdIns3K lipid kinase activity and autophagy flux, whereas its phosphorylated form blocks them. This study reveals NRBF2 as a critical molecular switch of PtdIns3K and autophagy activation, and its on/off state is precisely controlled by MTORC1 through phosphorylation.     

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.     

Structure of the novel C-terminal domain of vacuolar protein sorting 30/autophagy-related protein 6 and its specific role in autophagy

Vacuolar protein sorting 30 (Vps30)/autophagy-related protein 6 (Atg6) is a common component of two distinct phosphatidylinositol 3-kinase complexes. In complex I, Atg14 links Vps30 to Vps34 lipid kinase and exerts its specific role in autophagy, whereas in complex II, Vps38 links Vps30 to Vps34 and plays a crucial role in vacuolar protein sorting. However, the molecular role of Vps30 in each pathway remains unclear. Here, we report the crystal structure of the carboxyl-terminal domain of Vps30. The structure is a novel globular fold comprised of three β-sheet-α-helix repeats. Truncation analyses showed that the domain is dispensable for the construction of both complexes, but is specifically required for autophagy through the targeting of complex I to the pre-autophagosomal structure. Thus, the domain is named the β-α repeated, autophagy-specific (BARA) domain. On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar protein sorting. These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this protein family in various biological processes.     

Effects of neuronal PIK3C3/Vps34 deletion on autophagy and beyond

PIK3C3/Vps34 plays important roles in the endocytic and autophagic pathways, both of which are essential for maintaining neuronal integrity. However, it is unclear how inactivating PIK3C3 may affect neuronal endosomal versus autophagic processes in vivo. We generated a conditional null allele of the Pik3c3 gene in mouse, and specifically deleted it in postmitotic sensory neurons. Subsequent analyses reveal several interesting and surprising findings.Key words: PIK3C3/Vps34, ATG7, sensory neurons, neurodegeneration, autophagy, abnormal endosomePIK3C3 (commonly known as Vps34) is the class III phosphatidylinositol 3-kinase (PtdIns3K) that specifically catalyzes the formation of phosphatidylinositol-3-phosphate (PtdIns3P). It is the only PtdIns3K that is conserved from lower eukaryotes to mammals, and represents the most ancient form of PtdIns3Ks. Studies in invertebrate organisms as well as mammalian cell lines show that PIK3C3/Vps34 regulates multiple aspects of both the endocytic and the autophagic pathways. On one hand, PIK3C3 is important for the progression of early endosome to late endosome, and the biogenesis of multivesicular bodies. On the other hand, PIK3C3 is critical for the initiation of autophagosome formation. A chemical inhibitor of PIK3C3, 3-MA, has been commonly used as a specific inhibitor for autophagy. The distinct functions of PIK3C3 are thought to be carried out by at least two different PIK3C3 complexes. In yeast, complex I (Vps34, Vps15, Atg6 and Atg14) is involved in autophagy, whereas complex II (Vps34, Vps15, Atg6 and Vps38) functions in the vacuolar protein sorting process. In mammals, the homologue of complex I (PIK3C3, p150, Beclin 1 and Atg14L) activates autophagy, whereas the homologue of complex II (PIK3C3, p150, Beclin 1 and UVRAG/Vps38) regulates endocytic trafficking.To characterize the in vivo function of PIK3C3 in mammals, we generated a conditional allele of the Pik3c3 gene in mouse and specifically deleted it in postmitotic sensory neurons (Pik3c3-cKO mouse). We focused our analyses on sensory neurons because Pik3c3 is most abundantly expressed in these neurons. Detailed analyses of the sensory ganglia in the knockout mice reveal rapid but differential neurodegenerations of different types of sensory neurons within a few days after birth. Large-diameter myelinated mechanosensory and proprioceptive neurons undergo fast degeneration, whereas mutant small-diameter unmyelinated nociceptive neurons degenerate slower and survive longer.Interestingly, the large-diameter Pik3c3-deleted neurons rapidly accumulate ubiquitin-positive aggregates as well as numerous enlarged vesicles, which are likely abnormal endosomes. The accumulation of enlarged vesicles not only sequesters the cellular membrane source, but also could create trafficking jams that block the transport of prosurvival signals and/or material and organelles, and thus may underlie the rapid demise of large neurons. By contrast, the small-diameter Pik3c3-deleted neurons contain a limited number of vacuoles but gradually build up lysosome- like organelles. The marked increase of lysosomes seems to be more tolerable by neurons, but the mechanism underlying this phenotype is unclear. It could represent a protective and homeostatic response of neurons challenged with stress and insults to their endomembrane system. Alternatively, since sorting of many lysosomal proteins requires PtdIns3P, this phenotype may also result from a build-up of nonfunctional lysosomes as was the case in cathepsin B and L knockout mice. It is also unclear why two types of sensory neurons respond differently to a universal insult. One speculative explanation is that the large-diameter neurons are constantly activated under normal physiological conditions by touch and body movement and thus they contain more active endocytic and membrane trafficking processes; whereas small-diameter pain-sensing neurons are normally not activated and have less endocytic events. These differences might allow the two types of neurons to respond differently to PIK3C3 deletion.We further show that the fast and differential degeneration phenotypes in the Pik3c3-cKO mice are caused primarily by a disruption in the endosomal but not the autophagic pathway. This is validated by comparing the neuronal phenotypes of Pik3c3-cKO mice with those of Atg7-cKO mice, in which the autophagy-specific gene Atg7 is deleted using the same sensory neuron-specific cre driver. Disrupting autophagy leads to a slow degeneration of all types of sensory neurons over a period of several months, and formation of very large intracellular inclusion bodies in all sensory neurons. No increase of lysosomes or accumulation of enlarged vesicles is observed. The completely distinct phenotypes observed in Atg7-cKO versus Pik3c3-cKO mice suggest that inactivation of PIK3C3 primarily disrupts the endosomal pathway rather than inhibiting autophagy (at least in neurons). It calls into attention that care needs to be taken to interpret the results of using PIK3C3 inhibitors such as 3-MA as autophagy-specific inhibitors.The most surprising finding is the existence and activation of a noncanonical, PIK3C3-independent macroautophagy pathway in small-diameter Pik3c3-mutant neurons. Although PIK3C3 is traditionally viewed as indispensable for autophagy initiation, several recent studies suggest a possible PIK3C3-independent autophagy pathway in various cell lines and in Drosophila. We show that this noncanonical autophagy pathway can occur in sensory neurons in vivo using three different assays: crossing Pik3c3-cKO mice to the GFP-LC3 reporter line, western blot analyses of LC3 isoforms, and performing autophagy flux experiments. Interestingly, analyses of Pik3c3/Atg7 double-mutant neurons indicate that this alternative autophagosome initiation pathway still requires ATG7 and hence the conventional conjugation systems. Therefore, this non-canonical autophagy is distinct from the newly reported ATG5/ATG7-independent but PIK3C3-dependent autophagy. We speculate that activation of this PIK3C3-independent autophagy in small-diameter mutant neurons is part of the reason for their longer survival period.The molecular mechanism underlying the PIK3C3-independent autophagosome formation is unknown. It is possible that PtdIns3P can be generated at a low level on the membrane of pre-autophagosomes/phagophores by salvage pathways using other lipid kinases or phosphatases. Alternatively, other mechanisms may direct the formation of the crescent-shaped double membrane structures. For instance, asymmetric insertion into the membrane of proteins with amphipathic helices can induce membrane curvature; BAR domain-containing proteins can also detect and facilitate the formation of curved membrane structures. Thus, these types of proteins might potentially be recruited to nucleate the formation of pre-autophagosomes in the absence of PIK3C3. Finally, the role of this PIK3C3-independent autophagy under normal physiological conditions in vivo needs to be explored.     

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．     

The Beclin 1 network regulates autophagy and apoptosis

Beclin 1, the mammalian orthologue of yeast Atg6, has a central role in autophagy, a process of programmed cell survival, which is increased during periods of cell stress and extinguished during the cell cycle. It interacts with several cofactors (Atg14L, UVRAG, Bif-1, Rubicon, Ambra1, HMGB1, nPIST, VMP1, SLAM, IP(3)R, PINK and survivin) to regulate the lipid kinase Vps-34 protein and promote formation of Beclin 1-Vps34-Vps15 core complexes, thereby inducing autophagy. In contrast, the BH3 domain of Beclin 1 is bound to, and inhibited by Bcl-2 or Bcl-XL. This interaction can be disrupted by phosphorylation of Bcl-2 and Beclin 1, or ubiquitination of Beclin 1. Interestingly, caspase-mediated cleavage of Beclin 1 promotes crosstalk between apoptosis and autophagy. Beclin 1 dysfunction has been implicated in many disorders, including cancer and neurodegeneration. Here, we summarize new findings regarding the organization and function of the Beclin 1 network in cellular homeostasis, focusing on the cross-regulation between apoptosis and autophagy.     

Assortment of phosphatidylinositol 3-kinase complexes--Atg14p directs association of complex I to the pre-autophagosomal structure in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, two similar phosphatidylinositol 3-kinase complexes (complexes I and II) function in distinct biological processes, complex I in autophagy and complex II in the vacuolar protein sorting via endosomes. Atg14p is only integrated into complex I, likely facilitating the function of complex I in autophagy. Deletion analysis of Atg14p revealed that N-terminal region containing the coiled-coil structures was essential and sufficient for autophagy. Atg14p localized to pre-autophagosomal structure (PAS) and vacuolar membranes, whereas Vps38p, a component specific to complex II, localized to endosomes and vacuolar membranes. Vps34p and Vps30p, components shared by the two complexes, localized to the PAS, vacuolar membranes, and several punctate structures that included endosomes. The localization of these components to the PAS was Atg14p dependent but not dependent on Vps38p. Conversely, localization of these proteins to endosomes required Vps38p but not Atg14p. Vps15p, regulatory subunit of the Vps34p complexes, localized to the PAS, vacuolar membranes, and punctate structures independent of both Atg14p and Vps38p. Together, these results indicate that complexes I and II function in distinct biological processes by localizing to specific compartments in a manner mediated by specific components of each complex, Atg14p and Vps38p, respectively.     

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.     

Atg38 is required for autophagy-specific phosphatidylinositol 3-kinase complex integrity

Autophagy is a conserved eukaryotic process of protein and organelle self-degradation within the vacuole/lysosome. Autophagy is characterized by the formation of an autophagosome, for which Vps34-dervied phosphatidylinositol 3-phosphate (PI3P) is essential. In yeast, Vps34 forms two distinct protein complexes: complex I, which functions in autophagy, and complex II, which is involved in protein sorting to the vacuole. Here we identify and characterize Atg38 as a stably associated subunit of complex I. In atg38Δ cells, autophagic activity was significantly reduced and PI3-kinase complex I dissociated into the Vps15–Vps34 and Atg14–Vps30 subcomplexes. We find that Atg38 physically interacted with Atg14 and Vps34 via its N terminus. Further biochemical analyses revealed that Atg38 homodimerizes through its C terminus and that this homodimer formation is indispensable for the integrity of complex I. These data suggest that the homodimer of Atg38 functions as a physical linkage between the Vps15–Vps34 and Atg14–Vps30 subcomplexes to facilitate complex I formation.     

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.     

Two distinct Vps34 phosphatidylinositol 3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces cerevisiae

Vps30p/Apg6p is required for both autophagy and sorting of carboxypeptidase Y (CPY). Although Vps30p is known to interact with Apg14p, its precise role remains unclear. We found that two proteins copurify with Vps30p. They were identified by mass spectrometry to be Vps38p and Vps34p, a phosphatidylinositol (PtdIns) 3-kinase. Vps34p, Vps38p, Apg14p, and Vps15p, an activator of Vps34p, were coimmunoprecipitated with Vps30p. These results indicate that Vps30p functions as a subunit of a Vps34 PtdIns 3-kinase complex(es). Phenotypic analyses indicated that Apg14p and Vps38p are each required for autophagy and CPY sorting, respectively, whereas Vps30p, Vps34p, and Vps15p are required for both processes. Coimmunoprecipitation using anti-Apg14p and anti-Vps38p antibodies and pull-down experiments showed that two distinct Vps34 PtdIns 3-kinase complexes exist: one, containing Vps15p, Vps30p, and Apg14p, functions in autophagy and the other containing Vps15p, Vps30p, and Vps38p functions in CPY sorting. The vps34 and vps15 mutants displayed additional phenotypes such as defects in transport of proteinase A and proteinase B, implying the existence of another PtdIns 3-kinase complex(es). We propose that multiple Vps34p-Vps15p complexes associated with specific regulatory proteins might fulfill their membrane trafficking events at different sites.     

Finding a fitting shoe for Cinderella: Searching for an autophagy inhibitor

Vps34 is the ancestral phosphatidylinositol 3-kinase (PtdIns3K) isoform and is essential for endosomal trafficking of proteins to the vacuole/lysosome, autophagy and phagocytosis. Vps34-containing complexes associate with specific cellular compartments to produce PtdIns(3)P. Understanding the roles of Vps34 has been hampered by the lack of potent, specific inhibitors. To boost development of Vps34 inhibitors, we determined the crystal structures of Vps34 alone and in complexes with multitargeted PtdIns3K inhibitors. These structures provided a first glimpse into the uniquely constricted ATP-binding site of Vps34 and enabled us to model Vps34 regulation. We showed that the substrate-binding “activation” loop and the flexibly attached amphipathic C-terminal helix are crucial for catalysis on membranes. The C-terminal helix also suppresses ATP hydrolysis in the absence of membranes. We propose that membrane binding shifts the C-terminal helix to orient the enzyme for catalysis, and the Vps15 regulatory subunit, which binds to this and the preceding helix, may facilitate this process. This C-terminal region may also represent a target for specific, non-ATP-competitive PtdIns3K inhibitors.Key words: Vps34, PI 3-kinase, structure, inhibitor, enzyme, autophagy, Vps15, PtdIns3P, phosphoinositidePtdIns3Ks phosphorylate their lipid substrates at the 3-hydroxyl position of the inositol headgroup. Vps34 is the primordial PtdIns3K present in all eukaryotes and the only PtdIns3K in fungi and plants. This Cinderella of the PtdIns3Ks is responsible for much of a cell''s cleaning and self-feeding: It is essential for multivesicular body formation, autophagy and phagocytosis. It associates with endosomes, omegasomes and phagosomes producing PtdIns(3)P, the most abundant 3-phosphoinositide in resting mammalian cells, which is essential for recruiting a range of complexes to intracellular membranes, including the autophagy machinery, ESCRTs, the retromer, motor proteins and components necessary for abscission in cytokinesis. In cells, Vps34 is at the core of larger complexes that also contain two regulatory proteins, Vps15 and Beclin 1, which bind directly to Vps34. The N-terminally myristoylated putative Ser/Thr protein kinase p150/Vps15 increases the lipid kinase activity of Vps34 and facilitates its translocation to endosomal membranes and the phagophore assembly site (PAS) or phagophore (Fig. 1A).Open in a separate windowFigure 1(A) Domain organization of Vps34, its regulatory subunit Vps15 and the adaptor proteins required for autophagy induction in mammalian cells, Beclin 1 and Atg14L/Barkor (Beclin1-associated autophagy-related key regulator). (B) Structure of Drosophila Vps34 helical (green) and catalytic (red/yellow) domains. A PtdIns substrate molecule has been modeled between the activation loop (magenta) and the catalytic loop (black) and ATP was modeled based on the p110γ/ATP structure (PDB ID 1E8X). The C2 domain (cyan) was also modeled from the p110γ/ATP structure. The enzyme is oriented so that the C2 domain and C-terminal helix interact with the membrane. Two regulatory proteins bind directly to Vps34: Vps15 binds to helices kα11 and kα12 (orange), and Beclin 1 binds to the C2 domain. Both Vps15 and Beclin 1 stimulate Vps34 activity. (C) A schematic representation of the Vps34 domains and the putative change in conformation of the kα12 helix. In solution (right), the helix is closed and interacts with residues in the substrate-binding and catalytic loops to exclude water. At the membrane (left), the kα12 helix undergoes a conformational change and interacts with the membrane, enabling productive substrate binding and catalysis.We have determined the structure of the catalytic core of Vps34 (PDB ID 2X6H) (Fig. 1B), which consists of a helical solenoid domain forming an extensive interface with a bilobal catalytic domain. The catalytic domain reveals key features that are important for the catalytic mechanism of all PtdIns3Ks: A phosphate-binding loop (P-loop) that interacts with the phosphates of ATP, a substrate-binding loop or “activation” loop that recognizes the PtdIns substrate, and a catalytic loop that is required for the transfer of the ATP γ-phosphate to the 3-hydroxyl of PtdIns. For the first time in any PtdIns3K structure, all three of these elements are completely ordered. The C-terminal helix (kα12) was previously shown to be required for Vps34 catalytic activity. However, the molecular basis for its function was unknown. The Vps34 structure suggests that the C-terminal helix closely associates with the substrate-binding loop and catalytic loop in the closed conformation. Site-specific mutagenesis guided by the crystal structure provides key insights into mechanisms of enzymatic regulation of Vps34 by this C-terminal helix. Deletion of the last 10 residues or point mutations within this helix, dramatically impairs lipid kinase activity in the presence of substrate lipids, but increases basal ATPase activity in the absence of substrate. These results suggest that in the closed form of the enzyme, the amphipathic C-terminal helix acts as a lid on the catalytic site to suppress activity in the absence of substrate lipid. Hydrophobic residues in this helix are also important for membrane interaction. Enzymatic activity and membrane binding measurements are consistent with a model whereby the C-terminal helix shifts to facilitate membrane interaction and orientation of the enzyme on the membrane interface for optimal catalysis (Fig. 1C). The amphipathic character of the C-terminal region is conserved in all of the PtdIns3Ks, and it probably represents a common regulatory element in the entire family of enzymes. This may also extend to the PtdIns3Krelated enzymes such as TOR where the equivalent region has been denoted as the “FATC” domain, which also associates with membranes.Early reports showed that methylated adenosine derivatives can inhibit autophagy. It was later demonstrated that the autophagy inhibitor 3-methyladenine (3-MA) inhibits PtdIns3Ks and that other general PtdIns3K inhibitors, such as wortmannin also inhibit autophagy. Although 3-MA shows some limited Vps34 preference in vitro, with an IC₅₀ of 25 µM for Vps34 as compared with 60 µM for PtdIns3Kγ it is typically employed in cellular studies at a concentration of 10 mM, which can inhibit all PtdIns3Ks. Specific, potent inhibitors of Vps34 are acutely needed. All current PtdIns3K inhibitors are ATP-competitive, i.e., they target the ATP-binding site that is conserved among various PtdIns3K isotypes. The Vps34 structure suggests that the lack of potent Vps34 inhibitors could be accounted for by the uniquely constricted conformation of the Vps34 ATP-binding site in comparison with other PtdIns3Ks. Our structures of Vps34 in complexes with 3-MA and multitargeted PtdIns3K inhibitors (PIK-90, PIK-93 and PI-103) have provided insight into how this enzyme might be specifically inhibited. The slight preference for Vps34 inhibition by 3-MA probably arises from a hydrophobic ring specific to Vps34, which encircles the 3-methyl group of 3-MA. The insights arising from these structures have enabled us to develop a first generation of inhibitors with improved potency and Vps34 selectivity, e.g., the compound PT210 that has an IC₅₀ of 0.45 µM as compared with 4.5 µM for PtdIns3Kγ. Further development of inhibitors guided by structures could lead to a new generation of improved inhibitors with applications as chemical tools to investigate PtdIns(3) P-dependendent pathways and as therapeutic agents.     

Dissecting the localization and function of Atg18, Atg21 and Ygr223c

Atg18p and Atg21p are two highly homologous yeast autophagy proteins. Atg18p functions in both autophagy and the selective Cvt-pathway, while the function of Atg21p is restricted to the Cvt-pathway. The yeast genome encodes with Ygr223cp (Hsv2p), a third member of this protein family. So far no function has been assigned to Ygr223cp. By colocalization with the endosomal marker Snf7-RFP and an RFP-tagged FYVE domain, we here identify the localization of a pool of Atg18p, Atg21p and Ygr223cp at endosomes. Endosomal recruitment of all three proteins depends on PtdIns3P generated by the Vps34-complex II containing Vps38p, but not on the function of the Vps34-complex I. Since only the Vps34-complex I is essential for autophagy, we expect that at endosomes Atg18p, Atg21p and Ygr223cp have a function distinct from autophagy. Some Vps Class D mutants involved in Golgi-to-endosome transport are required for the endosomal recruitment of GFP-Atg18p, -Atg21p and -Ygr223cp. These include the Qa-SNARE Pep12p, its SM protein Vps45p, the Rab GTPase Vps21p and the Rab effector Vac1p. Deletion of ATG18, ATG21 and YGR223c, alone or simultaneously has no obvious function on the MVB-pathway and CPY-sorting. However, overexpression of ATG21 leads to CPY secretion. We further show, to our knowledge for the first time, that Ygr223cp affects an autophagic process, namely micronucleophagy.     

Beclin 1-Independent Pathway of Damage-Induced Mitophagy and Autophagic Stress: Implications for Neurodegeneration and Cell Death

Growing evidence supports an active role for dysregulated macroautophagy (autophagic stress) in neuronal cell death and neurodegeneration. Alterations in mitochondrial function and dynamics are also strongly implicated in neurodegenerative diseases. Interestingly, whereas the core autophagy machinery is evolutionarily conserved and shared among constitutive and induced or selective autophagy, recent studies implicate distinct mechanisms regulating mitochondrial autophagy (mitophagy) in response to general autophagic stimuli. Little is known about pathways regulating selective, damage-induced mitophagy. We found that the parkinsonian neurotoxin MPP+ induces autophagy and mitochondrial degradation that is inhibited by siRNA knockdown of autophagy proteins Atg5, Atg7 and Atg8, but occurs independently of Beclin 1, a component of the class III (PIK3C3/Vps34) phosphoinositide 3-kinase (PI3K) complex. Instead, MPP+-induced mitophagy is dependent upon MAPK signaling. Interestingly, all treatments that inhibited autophagy also conferred protection from MPP+-induced cell death. A prior human tissue study further supports a role for ERK/MAPK-regulated autophagy in Parkinson's and Lewy body diseases. As competition for limiting amounts of Beclin 1 may serve to prevent harmful overactivation of autophagy, understanding mechanisms that bypass or complement a requirement for PI3K-Beclin 1 activity could lead to strategies to modulate harmful autophagic stress in injured or degenerating neurons.