Early Steps in Autophagy Depend on Direct Phosphorylation of Atg9 by the Atg1 Kinase

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"Autophagy suite": Atg9 cycling in the cytoplasm to vacuole targeting pathway

Macroautophagy continues to gather increasing attention because it is connected with a wide range of human pathophysiologies, developmental processes, and life span extension. It is also an interesting process from a basic cellular biology standpoint, as it involves dynamic membrane rearrangements and multiple protein-protein interactions. Although macroautophagy can be nonspecific, there are many examples of selective sequestration including pexophagy, mitophagy and the cytoplasm to vacuole targeting (Cvt) pathway. At present, the Cvt pathway is unique in that it is the only example of a biosynthetic use of macroautophagy. Most of the autophagy-related (Atg) proteins are involved in the Cvt pathway, and various types of analyses have placed these proteins at particular stages of the process. For example, Atg9 is the only characterized transmembrane protein that is absolutely required for Cvt vesicle formation, and it is proposed to carry membrane from peripheral donor sites to the phagophore assembly site where the vesicle forms. Additional proteins, including Atg11, Atg23 and Atg27 are involved in this anterograde movement, whereas Atg1-Atg13 and Atg2-Atg18 are required for the retrograde return to the peripheral sites. Even when we illustrate our understanding of these events in a schematic model, however, they are by necessity flat two-dimensional representations, lacking movement and sound. Yet the cell is a living entity that is not well served by this sole method of information display. Accordingly, we decided to present the Cvt pathway as a vibrant, dynamic process by combining science, music and illustration.     

An Atg1/Atg13 Complex with Multiple Roles in TOR-mediated Autophagy Regulation

The TOR kinases are conserved negative regulators of autophagy in response to nutrient conditions, but the signaling mechanisms are poorly understood. Here we describe a complex containing the protein kinase Atg1 and the phosphoprotein Atg13 that functions as a critical component of this regulation in Drosophila. We show that knockout of Atg1 or Atg13 results in a similar, selective defect in autophagy in response to TOR inactivation. Atg1 physically interacts with TOR and Atg13 in vivo, and both Atg1 and Atg13 are phosphorylated in a nutrient-, TOR- and Atg1 kinase-dependent manner. In contrast to yeast, phosphorylation of Atg13 is greatest under autophagic conditions and does not preclude Atg1-Atg13 association. Atg13 stimulates both the autophagic activity of Atg1 and its inhibition of cell growth and TOR signaling, in part by disrupting the normal trafficking of TOR. In contrast to the effects of normal Atg13 levels, increased expression of Atg13 inhibits autophagosome expansion and recruitment of Atg8/LC3, potentially by decreasing the stability of Atg1 and facilitating its inhibitory phosphorylation by TOR. Atg1-Atg13 complexes thus function at multiple levels to mediate and adjust nutrient-dependent autophagic signaling.     

Atg27 is required for autophagy-dependent cycling of Atg9

Autophagy is a catabolic pathway for the degradation of cytosolic proteins or organelles and is conserved among all eukaryotic cells. The hallmark of autophagy is the formation of double-membrane cytosolic vesicles, termed autophagosomes, which sequester cytoplasm; however, the mechanism of vesicle formation and the membrane source remain unclear. In the yeast Saccharomyces cerevisiae, selective autophagy mediates the delivery of specific cargos to the vacuole, the analog of the mammalian lysosome. The transmembrane protein Atg9 cycles between the mitochondria and the pre-autophagosomal structure, which is the site of autophagosome biogenesis. Atg9 is thought to mediate the delivery of membrane to the forming autophagosome. Here, we characterize a second transmembrane protein Atg27 that is required for specific autophagy in yeast. Atg27 is required for Atg9 cycling and shuttles between the pre-autophagosomal structure, mitochondria, and the Golgi complex. These data support a hypothesis that multiple membrane sources supply the lipids needed for autophagosome formation.     

Atg1 kinase organizes autophagosome formation by phosphorylating Atg9

The conserved Ser/Thr kinase Atg1/ULK1 plays a crucial role in the regulation of autophagy. However, only very few Atg1 targets have been identified, impeding elucidation of the mechanisms by which Atg1 regulates autophagy. In our study, we determined the Saccharomyces cerevisiae Atg1 consensus phosphorylation sequence using a peptide array-based approach. Among proteins containing this sequence we identified Atg9, another essential component of the autophagic machinery. We showed that phosphorylation of Atg9 by Atg1 is required for phagophore elongation, shedding light on the mechanism by which Atg1 regulates early steps of autophagy.     

Atg9 Trafficking in Mammalian Cells

The molecular mechanisms of autophagy have been best characterized in the yeast Saccharomyces cerevisiae, where a number of proteins have been identified to be essential for this degradative pathway. ATG (autophagy-related) proteins localize to a unique compartment, the pre-autophagosomal structure (PAS). Isolation membranes are suggested to originate from the PAS, enwrapping cytoplasmic components to form a double membrane autophagosome, which then fuses with the vacuole. Although many Atg proteins have been identified, the source of the PAS membrane in yeast is unknown. Identification of the source of the PAS in yeast has been hindered due to the transient association of Atg proteins with forming autophagosomes. Likewise, in mammalian cells, it is not known if a PAS equivalent exists or if the formation of autophagosomes occurs from numerous membrane sources. The identification of stably associated markers would allow us to address this question further. Thus, characterization of the only transmembrane autophagy protein so far identified, Atg9, may aid in the search for the source of the PAS. Recent data from our lab suggests that mammalian Atg9 (mAtg9) traffics between the Golgi and endosomes, and suggests an involvement of the Golgi complex in the autophagic pathway. Here we address the implications of our model with regard to membrane trafficking events in mammalian cells after starvation.

Addendum to:

Starvation and ULK1-Dependent Cycling of Mammalian Atg9 Between the TGN and Andosomes

A.R.J. Young, E.Y.W. Chan, X.W. Hu, R. Köchl, S.G. Crawshaw, S. High, D.W. Hailey, J. Lippincott-Schwartz and S.A. Tooze

J Cell Sci 2006; 119:3888-900     

Atg9 trafficking in autophagy-related pathways

The origin of the autophagosomal membrane and the lipid delivery mechanism during autophagy remain unsolved mysteries. Some important hints to these questions come from Atg9, which is the only integral membrane protein required for autophagosome formation and considered a membrane carrier in autophagy-related pathways. In S. cerevisiae, Atg9 cycles between peripheral sites and the pre-autophagosomal structure/phagophore assembly site (PAS), the nucleating site for formation of the sequestering vesicle. We recently identified a peripheral membrane protein, Atg11, as a binding partner of Atg9, in a yeast two-hybrid screen. Based on our analysis we propose a model for Atg9 cycling. Our model suggests that a pool of Atg11 mediates the anterograde transport of Atg9 to the PAS along the actin cytoskeleton, and that this delivery process may serve as a membrane shuttle for vesicle assembly during yeast selective autophagy. Here, we discuss the implications of the model and present additional evidence that extends it with regard to membrane trafficking modes during pexophagy.     

Atg9 Trafficking in Autophagy-Related Pathways

The origin of the autophagosomal membrane and the lipid delivery mechanism during autophagy remain unsolved mysteries. Some important hints to these questions come from Atg9, which is the only integral membrane protein required for autophagosome formation and considered a membrane carrier in autophagy-related pathways. In S. cerevisiae, Atg9 cycles between peripheral sites and the preautophagosomal structure/phagophore assembly site (PAS), the nucleating site for formation of the sequestering vesicle. We recently identified a peripheral membrane protein, Atg11, as a binding partner of Atg9, in a yeast two-hybrid screen. Based on our analysis we propose a model for Atg9 cycling. Our model suggests that a pool of Atg11 mediates the anterograde transport of Atg9 to the PAS along the actin cytoskeleton, and that this delivery process may serve as a membrane shuttle for vesicle assembly during yeast selective autophagy. Here, we discuss the implications of the model and present additional evidence that extends it with regard to membrane trafficking modes during pexophagy.

Addendum to:

Recruitment of Atg9 to the Preautophagosomal Structure by Atg11 is Essential for Selective Autophagy in Budding Yeast

C. He, H. Song, T. Yorimitsu, I. Monastyrska, W.-L. Yen, J.E. Legakis and D.J. Klionsky

J Cell Biol 2006; 175:925-35     

Atg13 Is Essential for Autophagy and Cardiac Development in Mice

Autophagy is a major intracellular degradation system by which cytoplasmic components are enclosed by autophagosomes and delivered to lysosomes. Formation of the autophagosome requires a set of autophagy-related (Atg) proteins. Among these proteins, the ULK1 complex, which is composed of ULK1 (or ULK2), FIP200, Atg13, and Atg101, acts at an initial step. Previous studies showed that ULK1 and FIP200 also function in pathways other than autophagy. However, whether Atg13 and Atg101 act similarly to ULK1 and FIP200 remains unknown. In the present study, we generated Atg13 knockout mice. Like FIP200-deficient mice, Atg13-deficient mice die in utero, which is distinct from most other types of Atg-deficient mice. Atg13-deficient embryos show growth retardation and myocardial growth defects. In cultured fibroblasts, Atg13 deficiency blocks autophagosome formation at an upstream step. In addition, sensitivity to tumor necrosis factor alpha (TNF-α)-induced apoptosis is enhanced by deletion of Atg13 or FIP200, but not by other Atg proteins, as well as by simultaneous deletion of ULK1 and ULK2. These results suggest that Atg13 has both autophagic and nonautophagic functions and that the latter are essential for cardiac development and likely shared with FIP200 but not with ULK1/2.     

Atg9 trafficking in Mammalian cells

The molecular mechanisms of autophagy have been best characterized in the yeast Saccharomyces cerevisiae, where a number of proteins have been identified to be essential for this degradative pathway. ATG (autophagy-related) proteins(1) localize to a unique compartment, the pre-autophagosomal structure (PAS). Isolation membranes are suggested to originate from the PAS, enwrapping cytoplasmic components to form a double membrane autophagosome, which then fuses with the vacuole. Although many Atg proteins have been identified, the source of the PAS membrane in yeast is unknown. Identification of the source of the PAS in yeast has been hindered due to the transient association of Atg proteins with forming autophagosomes.(2) Likewise, in mammalian cells, it is not known if a PAS equivalent exists or if the formation of autophagosomes occurs from numerous membrane sources. The identification of stably associated markers would allow us to address this question further. Thus, characterization of the only transmembrane autophagy protein so far identified, Atg9, may aid in the search for the source of the PAS. Recent data from our lab suggests that mammalian Atg9 (mAtg9) traffics between the Golgi and endosomes, and suggests an involvement of the Golgi complex in the autophagic pathway.(3) Here we address the implications of our model with regard to membrane trafficking events in mammalian cells after starvation.     

The Atg1-Atg13 complex regulates Atg9 and Atg23 retrieval transport from the pre-autophagosomal structure

To survive extreme environmental conditions, and in response to certain developmental and pathological situations, eukaryotic organisms employ the catabolic process of autophagy. Structures targeted for destruction are enwrapped by double-membrane vesicles, then delivered into the interior of the lysosome/vacuole. Despite the identification of many specific components, the molecular mechanism that directs formation of the sequestering vesicles remains largely unknown. We analyzed the trafficking of Atg23 and the integral membrane protein Atg9 in the yeast Saccharomyces cerevisiae. These components localize both to the pre-autophagosomal structure (PAS) and other cytosolic punctate compartments. We show that Atg9 and Atg23 cycle through the PAS in a process governed by the Atg1-Atg13 signaling complex. Atg1 kinase activity is essential only for retrograde transport of Atg23, while recycling of Atg9 requires additional factors including Atg18 and Atg2. We postulate that Atg9 employs a recycling system mechanistically similar to that used at yeast early and late endosomes.     

Atg7 Activates an Autophagy-Essential Ubiquitin-like Protein Atg8 through Multi-Step Recognition

Atg8 is a unique ubiquitin-like protein that is covalently conjugated with a phosphatidylethanolamine through reactions similar to ubiquitination and plays essential roles in autophagy. Atg7 is the E1 enzyme for Atg8, and it activates the C-terminal Gly116 of Atg8 using ATP. Here, we report the crystal structure of Atg8 bound to the C-terminal domain of Atg7 in an unprecedented mode. Atg8 neither contacts with the central β-sheet nor binds to the catalytic site of Atg7, both of which were observed in previously reported Atg7–Atg8 structures. Instead, Atg8 binds to the C-terminal α-helix and crossover loop, thereby changing the autoinhibited conformation of the crossover loop observed in the free Atg7 structure into a short helix and a disordered loop. Mutational analyses suggested that this interaction mode is important for the activation reaction. We propose that Atg7 recognizes Atg8 through multiple steps, which would be necessary to induce a conformational change in Atg7 that is optimal for the activation reaction.     

Mechanism of Atg9 recruitment by Atg11 in the cytoplasm-to-vacuole targeting pathway

Autophagy is a lysosomal degradation pathway for the removal of damaged and superfluous cytoplasmic material. This is achieved by the sequestration of this cargo material within double-membrane vesicles termed autophagosomes. Autophagosome formation is mediated by the conserved autophagy machinery. In selective autophagy, this machinery including the transmembrane protein Atg9 is recruited to specific cargo material via cargo receptors and the Atg11/FIP200 scaffold protein. The molecular details of the interaction between Atg11 and Atg9 are unclear, and it is still unknown how the recruitment of Atg9 is regulated. Here we employ NMR spectroscopy of the N-terminal disordered domain of Atg9 (Atg9-NTD) to map its interaction with Atg11 revealing that it involves two short peptides both containing a PLF motif. We show that the Atg9-NTD binds to Atg11 with an affinity of about 1 μM and that both PLF motifs contribute to the interaction. Mutation of the PLF motifs abolishes the interaction of the Atg9-NTD with Atg11, reduces the recruitment of Atg9 to the precursor aminopeptidase 1 (prApe1) cargo, and blocks prApe1 transport into the vacuole by the selective autophagy-like cytoplasm-to-vacuole (Cvt) targeting pathway while not affecting bulk autophagy. Our results provide mechanistic insights into the interaction of the Atg11 scaffold with the Atg9 transmembrane protein in selective autophagy and suggest a model where only clustered Atg11 when bound to the prApe1 cargo is able to efficiently recruit Atg9 vesicles.     

Atg23 and Atg27 Act at the Early Stages of Atg9 Trafficking in S. cerevisiae

Atg9 is a conserved multipass transmembrane protein with an essential role in autophagy. In Saccharomyces cerevisiae, it travels through the secretory pathway to a unique compartment, the Atg9 peripheral structures. These structures are then targeted to the phagophore assembly site (PAS), where they are proposed to help deliver membrane to the forming autophagosome. We used ‘in vivo reconstitution’ of this process in a multiple‐knockout strain to define four proteins, Atg11, Atg19, Atg23 and Atg27, as the core minimal machinery necessary and sufficient for the trafficking of Atg9 to the PAS. Atg23 and Atg27 function in the formation of the Atg9 peripheral structures. Overexpression of Atg9 can bypass the need for Atg23, suggesting that the amount of Atg9 in each peripheral structure is a critical factor in their targeting to the PAS. In contrast, overexpression of Atg23 or Atg27 interferes with Atg9 trafficking, suggesting that these proteins must be present in the appropriate stoichiometry in order to function properly. These data allow us to resolve existing controversies regarding the role of Atg23 and Atg27, and propose a model that ties together previous observations regarding the role of Atg9 in autophagosome formation.      

Evolution of Atg1 function and regulation

The serine/threonine kinase Atg1 plays an essential role downstream of TOR for the regulation of autophagy. In yeast, where Atg1 was first identified, a complex regulatory mechanism has been described that includes at least seven other interacting proteins and a phosphorylation-dependent switch. Recent findings confirm that the mammalian Atg1 homologues ULK1 and 2 have autophagy regulatory roles. However, we and others have also demonstrated mechanistic differences with the yeast model and between these two Atg1 family members. Here, we elaborate on our growing understanding of Atg1 function, incorporating findings from yeast, C. elegans, D. melanogaster and mammalian cells. We propose that through evolution, Atg1 proteins have adopted additional cellular functions and regulatory mechanisms, which could involve multiple gene family isoforms working within multi-functional protein complexes. The gene family expansion observed in higher eukaryotes might reflect an increased functional diversity of Atg1 proteins in cell growth, differentiation and survival.