Macroautophagy/autophagy is a key catabolic process in which different cellular components are sequestered inside double-membrane vesicles called autophagosomes for subsequent degradation. In yeast, autophagosome formation occurs at the phagophore assembly site (PAS), a specific perivacuolar location that works as an organizing center for the recruitment of different autophagy-related (Atg) proteins. How the PAS is localized to the vacuolar periphery is not well understood. Here we show that the vacuolar membrane protein Vac8 is required for correct vacuolar localization of the PAS. We provide evidence that Vac8 anchors the PAS to the vacuolar membrane by binding Atg13 and recruiting the Atg1 initiation complex. VAC8 deletion or mislocalization of the protein reduce autophagy activity, highlighting the importance of both the PAS and the correct vacuolar localization of the Atg1 initiation complex for efficient and robust autophagy.Abbreviations: AID: auxin-inducible degradation; Atg: autophagy-related; Cvt: cytoplasm-to-vacuole targeting; DMSO: dimethyl sulfoxide; ER: endoplasmic reticulum; GFP: green fluorescent protein; IAA: 3-indole acetic acid; PAS: phagophore assembly site; RFP: red fluorescent protein.

Author(s): Klionsky, DJ; Abdelmohsen, K; Abe, A; Abedin, MJ; Abeliovich, H; Arozena, AA; Adachi, H; Adams, CM; Adams, PD; Adeli, K; Adhihetty, PJ; Adler, SG; Agam, G; Agarwal, R; Aghi, MK; Agnello, M; Agostinis, P; Aguilar, PV; Aguirre-Ghiso, J; Airoldi, EM; Ait-Si-Ali, S; Akematsu, T; Akporiaye, ET; Al-Rubeai, M; Albaiceta, GM; Albanese, C; Albani, D; Albert, ML; Aldudo, J; Algul, H; Alirezaei, M; Alloza, I; Almasan, A; Almonte-Beceril, M; Alnemri, ES; Alonso, C; Altan-Bonnet, N; Altieri, DC; Alvarez, S; Alvarez-Erviti, L; Alves, S; Amadoro, G; Amano, A; Amantini, C; Ambrosio, S; Amelio, I; Amer, AO; Amessou, M; Amon, A; An, Z; Anania, FA; Andersen, SU; Andley, UP; Andreadi, CK; Andrieu-Abadie, N; Anel, A; Ann, DK; Anoopkumar-Dukie, S; Antonioli, M; Aoki, H; Apostolova, N; Aquila, S; Aquilano, K; Araki, K; Arama, E; Aranda, A; Araya, J; Arcaro, A; Arias, E; Arimoto, H; Ariosa, AR; Armstrong, JL; Arnould, T; Arsov, I; Asanuma, K; Askanas, V; Asselin, E; Atarashi, R; Atherton, SS; Atkin, JD; Attardi, LD; Auberger, P; Auburger, G; Aurelian, L; Autelli, R

Anticancer therapeutics aimed at the inhibition of mTORC1 activity shift metabolism to favor the degradation of extracellular proteins. Recently Thompson and colleagues demonstrated a novel regulatory mechanism whereby mTORC1 plays a distinct role as a key regulator of metabolism depending on the environmental nutrient status.

The proteins that comprise the Atg1 kinase complex constitute a key set of components that participate in macroautophagy (hereafter autophagy). Among these proteins, Atg13 plays a particularly important, although as yet undefined role, in that it is critical for the proper localization of Atg1 to the phagophore assembly site (PAS) and its efficient kinase activity. Atg13 is hyperphosphorylated in vegetative conditions when autophagy occurs at a basal level, and is largely dephosphorylated upon the induction of autophagy. Inhibitory phosphorylation of Atg13 reflects the activity of TOR complex 1 (TORC1) and protein kinase A. Accordingly, monitoring the phosphorylation state of Atg13 provides a convenient way to follow early steps of autophagy induction as well as the activity of some of the upstream nutrient-sensing kinases. However, the detection of Atg13 by western blot can be problematic. Here, we present a detailed protocol for sample preparation and detection of the Atg13 protein from yeast.

The proteins that comprise the Atg1 kinase complex constitute a key set of components that participate in macroautophagy (hereafter autophagy). Among these proteins, Atg13 plays a particularly important, although as yet undefined role, in that it is critical for the proper localization of Atg1 to the phagophore assembly site (PAS) and its efficient kinase activity. Atg13 is hyperphosphorylated in vegetative conditions when autophagy occurs at a basal level, and is largely dephosphorylated upon the induction of autophagy. Inhibitory phosphorylation of Atg13 reflects the activity of TOR complex 1 (TORC1) and protein kinase A. Accordingly, monitoring the phosphorylation state of Atg13 provides a convenient way to follow early steps of autophagy induction as well as the activity of some of the upstream nutrient-sensing kinases. However, the detection of Atg13 by western blot can be problematic. Here, we present a detailed protocol for sample preparation and detection of the Atg13 protein from yeast.

Yeast Atg1 initiates autophagy in response to nutrient limitation. The Ulk gene family encompasses the mammalian orthologs of yeast ATG1. We created mice deficient for both Ulk1 and Ulk2 and found that the mice die within 24 h of birth. When found alive, pups exhibited signs of respiratory distress. Histological sections of lungs of the Ulk1/2 DKO pups showed reduced airspaces with thickened septae. A similar defect was seen in Atg5-deficient pups as both Ulk1/2 DKO and Atg5 KO lungs show numerous glycogen-laden alveolar type II cells by electron microscopy, PAS staining, and increased levels of glycogen in lung homogenates. No abnormalities were noted in expression of genes encoding surfactant proteins but the ability to incorporate exogenous choline into phosphatidylcholine, the major phospholipid component of surfactant, was increased in comparison to controls. Despite this, there was a trend for total phospholipid levels in lung tissue to be lower in Ulk1/2 DKO and Atg5 KO compared with controls. Autophagy was abundant in lung epithelial cells from wild-type mice, but lacking in Atg5 KO and Ulk1/2 DKO mice at P1. Analysis of the autophagy signaling pathway showed the existence of a negative feedback loop between the ULK1 and 2 and MTORC1 and 2, in lung tissue. In the absence of autophagy, alveolar epithelial cells are unable to mobilize internal glycogen stores independently of surfactant maturation. Together, the data suggested that autophagy plays a vital role in lung structural maturation in support of perinatal adaptation to air breathing.

Macroautophagy (hereafter autophagy) functions in the nonselective clearance of cytoplasm. This process participates in many aspects of cell physiology, and is conserved in all eukaryotes. Autophagy begins with the organization of the phagophore assembly site (PAS), where most of the AuTophaGy-related (Atg) proteins are at least transiently localized. Autophagy occurs at a basal level and can be induced by various types of stress; the process must be tightly regulated because insufficient or excessive autophagy can be deleterious. A complex composed of Atg17-Atg31-Atg29 is vital for PAS organization and autophagy induction, implying a significant role in autophagy regulation. In this study, we demonstrate that Atg29 is a phosphorylated protein and that this modification is critical to its function; alanine substitution at the phosphorylation sites blocks its interaction with the scaffold protein Atg11 and its ability to facilitate assembly of the PAS. Atg29 has the characteristics of an intrinsically disordered protein, suggesting that it undergoes dynamic conformational changes on interaction with a binding partner(s). Finally, single-particle electron microscopy analysis of the Atg17-Atg31-Atg29 complex reveals an elongated structure with Atg29 located at the opposing ends.

Autophagy plays an important role in the cellular response to a variety of metabolic stress conditions thus contributing to the maintenance of intracellular homeostasis. Studies in yeast have defined the genetic components involved in the initiation of autophagy as well as the progression through the autophagic cascade. The yeast kinase Atg1 initiates autophagy in response to nutrient limitation in a TOR-dependent manner. The ulk family of genes encodes the mammalian orthologue of yeast Atg1. Our recent work using mouse embryonic fibroblast (MEF) cell lines deficient for both ulk1 and ulk2, has revealed that autophagy induction is more complex in mammals than in yeast. Furthermore, these data confirm the surprising finding that a by-product of amino acid metabolism, ammonia, is a strong inducer of autophagy, as first shown by the Abraham laboratory.

The metabolism of cancer cells is reprogrammed both by oncogene signaling and by dysregulation of metabolic enzymes. The resulting altered metabolism supports cellular proliferation and survival but leaves cancer cells dependent on a continuous supply of nutrients. Thus, many metabolic enzymes have become targets for new cancer therapies. Recently, two processes—expression of specific isoforms of metabolic enzymes and autophagy—have been shown to be crucial for the adaptation of tumor cells to changes in nutrient availability. An increasing number of approved and experimental therapeutics target these two processes. A better understanding of the molecular basis of cancer-associated metabolic changes may lead to improved cancer therapies.

Autophagy, a lysosome-mediated catabolic process, contributes to maintenance of intracellular homeostasis and cellular response to metabolic stress. In yeast, genes essential to the execution of autophagy have been defined, including autophagy-related gene 1 (ATG1), a kinase responsible for initiation of autophagy downstream of target of rapamycin. Here we investigate the role of the mammalian Atg1 homologs, uncoordinated family member (unc)-51-like kinase 1 and 2 (ULK1 and ULK2), in autophagy by generating mouse embryo fibroblasts (MEFs) doubly deficient for ULK1 and ULK2. We found that ULK1/2 are required in the autophagy response to amino acid deprivation but not for autophagy induced by deprivation of glucose or inhibition of glucose metabolism. This ULK1/2-independent autophagy was not the simple result of bioenergetic compromise and failed to be induced by AMP-activated protein kinase activators such as 5-aminoimidazole-4-carboxamide riboside and phenformin. Instead we found that autophagy induction upon glucose deprivation correlated with a rise in cellular ammonia levels caused by elevated amino acid catabolism. Even in complete medium, ammonia induced autophagy in WT and Ulk1/2(-/-) MEFs but not in Atg5-deficient MEFs. The autophagy response to ammonia is abrogated by a cell-permeable form of pyruvate resulting from the scavenging of excess ammonia through pyruvate conversion to alanine. Thus, although ULK1 and/or ULK2 are required for the autophagy response following deprivation of nitrogenous amino acids, the autophagy response to the enhanced amino acid catabolism induced by deprivation of glucose or direct exposure to ammonia does not require ULK1 and/or ULK2. Together, these data suggest that autophagy provides cells with a mechanism to adapt not only to nitrogen deprivation but also to nitrogen excess.

Autophagy is a major intracellular degradative pathway that is involved in various human diseases. The role of autophagy, however, is complex; although the process is generally considered to be cytoprotective, it can also contribute to cellular dysfunction and disease progression. Much progress has been made in our understanding of autophagy, aided in large part by the identification of the autophagy-related (ATG) genes. Nonetheless, our understanding of the molecular mechanism remains limited. In this study, we generated a Saccharomyces cerevisiae multiple-knockout strain with 24 ATG genes deleted, and we used it to carry out an in vivo reconstitution of the autophagy pathway. We determined minimum requirements for different aspects of autophagy and studied the initial protein assembly steps at the phagophore assembly site. In vivo reconstitution enables the study of autophagy within the context of the complex regulatory networks that control this process, an analysis that is not possible with an in vitro system.

An increasing number of reports have elucidated the importance of macroautophagy in cell physiology and pathology. Macroautophagy occurs at a basal level and participates in the turnover of cytoplasmic constituents including long-lived proteins to maintain cellular homeostasis, but it also serves as an adaptive response to protect cells from various intra- or extracellular stresses. In addition, macroautophagy plays a role in development and aging and acts to protect against cancer, microbial invasion, and neurodegeneration. The machinery involved in carrying out this process, the autophagy-related (Atg) proteins were identified and characterized in various fungal systems, in particular because of the powerful tools available for genetic manipulation and the relative abundance of good biochemical assays in these model organisms. The analysis of these Atg proteins has allowed us to begin to understand the molecular mechanism of this process. Furthermore, many of the autophagy genes are functionally conserved in higher eukaryotes, including mammals, allowing the findings in fungi to be applied to other systems. Here, we discuss three biochemical methods to measure autophagy-related activities and to examine individual steps of the corresponding process. These methods rely on the detection of different modification states of certain marker proteins. Processing of the precursor form of the resident vacuolar hydrolase aminopeptidase I (Ape1) is applicable to fungi, whereas cleavage of the GFP-Atg8 and Pex14-GFP chimeras can be used in a wide array of systems.

Autophagy is the major degradative process for recycling cytoplasmic constituents and eliminating unnecessary organelles in eukaryotic cells. Most autophagy-related (Atg) proteins are recruited to the phagophore assembly site (PAS), a proposed site for vesicle formation during either nonspecific or specific types of autophagy. Therefore, appropriate recruitment of Atg proteins to this site is critical for their function in autophagy. Atg11 facilitates PAS recruitment for the cytoplasm-to-vacuole targeting pathway, which is a specific, autophagy-like process that occurs under vegetative conditions. In contrast, it is not known how Atg proteins are recruited to the PAS, nor which components are involved in PAS formation under nonspecific autophagy-inducing, starvation conditions. Here, we studied PAS assembly during nonspecific autophagy, using an atg11Delta mutant background to eliminate the PAS formation that occurs during vegetative growth. We found that protein complexes containing the Atg1 kinase have two roles for PAS formation during nonspecific autophagy. The Atg1 C terminus mediates an interaction with Atg13 and Atg17, facilitating a structural role of Atg1 that is needed to efficiently organize an initial step of PAS assembly, whereas Atg1 kinase activity affects the dynamics of protein movement at the PAS involved in Atg protein cycling.