Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation.
ABSTRACT: Macroautophagy is primarily a degradative process that cells use to break down their own components to recycle macromolecules and provide energy under stress conditions, and defects in macroautophagy lead to a wide range of diseases. Atg9, conserved from yeast to mammals, is the only identified transmembrane protein in the yeast core macroautophagy machinery required for formation of the sequestering compartment termed the autophagosome. This protein undergoes dynamic movement between the phagophore assembly site (PAS), where the autophagosome precursor is nucleated, and peripheral sites that may provide donor membrane for expansion of the phagophore. Atg9 is a phosphoprotein that is regulated by the Atg1 kinase. We used stable isotope labeling by amino acids in cell culture (SILAC) to identify phosphorylation sites on this protein and identified an Atg1-independent phosphorylation site at serine 122. A nonphosphorylatable Atg9 mutant showed decreased autophagy activity, whereas the phosphomimetic mutant enhanced activity. Electron microscopy analysis suggests that the different levels of autophagy activity reflect differences in autophagosome formation, correlating with the delivery of Atg9 to the PAS. Finally, this phosphorylation regulates Atg9 interaction with Atg23 and Atg27.
Project description: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.
Project description:Autophagy is the degradation of a cell's own components within lysosomes (or the analogous yeast vacuole), and its malfunction contributes to a variety of human diseases. Atg9 is the sole integral membrane protein required in formation of the initial sequestering compartment, the phagophore, and is proposed to play a key role in membrane transport; the phagophore presumably expands by vesicular addition to form a complete autophagosome. It is not clear through what mechanism Atg9 functions at the phagophore assembly site (PAS). Here we report that Atg9 molecules self-associate independently of other known autophagy proteins in both nutrient-rich and starvation conditions. Mutational analyses reveal that self-interaction is critical for anterograde transport of Atg9 to the PAS. The ability of Atg9 to self-interact is required for both selective and nonselective autophagy at the step of phagophore expansion at the PAS. Our results support a model in which Atg9 multimerization facilitates membrane flow to the PAS for phagophore formation.
Project description:Yeast macroautophagy begins with the de novo formation of a double-membrane phagophore at the preautophagosomal structure/phagophore assembly site (PAS), followed by its expansion into the autophagosome responsible for cargo engulfment. The kinase Atg1 is recruited to the PAS by Atg13 through interactions between the EAT domain of the former and the tMIM motif of the latter. Mass-spectrometry data have shown that, in the absence of Atg13, the EAT domain structure is strikingly dynamic, but the function of this Atg13-free dynamic state has been unclear. We used structure-based mutational analysis and quantitative and superresolution microscopy to show that Atg1 is present on autophagic puncta at, on average, twice the stoichiometry of Atg13. Moreover, Atg1 colocalizes with the expanding autophagosome in a manner dependent on Atg8 but not Atg13. We used isothermal titration calorimetry and crystal structure information to design an EAT domain mutant allele ATG1DD that selectively perturbs the function of the Atg13-free state. Atg1DD shows reduced PAS formation and does not support phagophore expansion, showing that the EAT domain has an essential function that is separate from its Atg13-dependent role in autophagy initiation.
Project description:During autophagosome formation, autophagosome-related (Atg) proteins are recruited hierarchically to organize the preautophagosomal structure (PAS). Atg13, which plays a central role in the initial step of PAS formation, consists of two structural regions, the N-terminal HORMA (from Hop1, Rev7, and Mad2) domain and the C-terminal disordered region. The C-terminal disordered region of Atg13, which contains the binding sites for Atg1 and Atg17, is essential for the initiation step in which the Atg1 complex is formed to serve as a scaffold for the PAS. The N-terminal HORMA domain of Atg13 is also essential for autophagy, but its molecular function has not been established. In this study, we searched for interaction partners of the Atg13 HORMA domain and found that it binds Atg9, a multispanning membrane protein that exists on specific cytoplasmic vesicles (Atg9 vesicles). After the Atg1 complex is formed, Atg9 vesicles are recruited to the PAS and become part of the autophagosomal membrane. HORMA domain mutants, which are unable to interact with Atg9, impaired the PAS localization of Atg9 vesicles and exhibited severe defects in starvation-induced autophagy. Thus, Atg9 vesicles are recruited to the PAS via the interaction with the Atg13 HORMA domain. Based on these findings, we propose that the two distinct regions of Atg13 play crucial roles in distinct steps of autophagosome formation: In the first step, Atg13 forms a scaffold for the PAS via its C-terminal disordered region, and subsequently it recruits Atg9 vesicles via its N-terminal HORMA domain.
Project description:During autophagy, the transmembrane protein Atg27 facilitates transport of the major autophagy membrane protein Atg9 to the preautophagosomal structure (PAS). To better understand the function of Atg27 and its relationship with Atg9, Atg27 trafficking and localization were examined. Atg27 localized to endosomes and the vacuolar membrane, in addition to previously described PAS, Golgi and Atg9-positive structures. Atg27 vacuolar membrane localization was dependent on the adaptor AP-3, which mediates direct transport from the trans-Golgi to the vacuole. The four C-terminal amino acids (YSAV) of Atg27 comprise a tyrosine sorting motif. Mutation of the YSAV abrogated Atg27 transport to the vacuolar membrane and affected its distribution in TGN/endosomal compartments, while PAS localization was normal. Also, in atg27(?YSAV) or AP-3 mutants, accumulation of Atg9 in the vacuolar lumen was observed upon autophagy induction. Nevertheless, PAS localization of Atg9 was normal in atg27(?YSAV) cells. The vacuole lumen localization of Atg9 was dependent on transport through the multivesicular body, as Atg9 accumulated in the class E compartment and vacuole membrane in atg27(?YSAV) vps4? but not in ATG27 vps4? cells. We suggest that Atg27 has an additional role to retain Atg9 in endosomal reservoirs that can be mobilized during autophagy.
Project description:Autophagosomes are double-membrane vesicles that sequester cytoplasmic material for lysosomal degradation. Their biogenesis is initiated by recruitment of Atg9-vesicles to the phagophore assembly site. This process depends on the regulated activation of the Atg1-kinase complex. However, the underlying molecular mechanism remains unclear. Here we reconstitute this early step in autophagy from purified components in vitro. We find that on assembly from its cytoplasmic subcomplexes, the Atg1-kinase complex becomes activated, enabling it to recruit and tether Atg9-vesicles. The scaffolding protein Atg17 targets the Atg1-kinase complex to autophagic membranes by specifically recognizing the membrane protein Atg9. This interaction is inhibited by the two regulatory subunits Atg31 and Atg29. Engagement of the Atg1-Atg13 subcomplex restores the Atg9-binding and membrane-tethering activity of Atg17. Our data help to unravel the mechanism that controls Atg17-mediated tethering of Atg9-vesicles, providing the molecular basis to understand initiation of autophagosome-biogenesis.
Project description:Macroautophagy is a bulk clearance mechanism in which the double-membraned phagophore grows and engulfs cytosolic material. In yeast, the phagophore nucleates from a cluster of 20-30 nm diameter Atg9-containing vesicles located at a multiprotein assembly known as the preautophagosomal structure (PAS). The crystal structure of a 2:2:2 complex of the earliest acting PAS proteins, Atg17, Atg29, and Atg31, was solved at 3.05 Å resolution. Atg17 is crescent shaped with a 10 nm radius of curvature. Dimerization of the Atg17-Atg31-Atg29 complex is critical for both PAS formation and autophagy, and each dimer contains two separate and complete crescents. Upon induction of autophagy, Atg17-Atg31-Atg29 assembles with Atg1 and Atg13, which in turn initiates the formation of the phagophore. The C-terminal EAT domain of Atg1 was shown to sense membrane curvature, dimerize, and tether lipid vesicles. These data suggest a structural mechanism for the organization of Atg9 vesicles into the early phagophore.
Project description:When macroautophagy, a catabolic process that rids the cells of unwanted proteins, is initiated, 30-60 nm Atg9 vesicles move from the Golgi to the preautophagosomal structure (PAS) to initiate autophagosome formation. The Rab GTPase Ypt1 and its mammalian homolog Rab1 regulate macroautophagy and two other trafficking events: endoplasmic reticulum-Golgi and intra-Golgi traffic. How a Rab, which localizes to three distinct cellular locations, achieves specificity is unknown. Here we show that transport protein particle III (TRAPPIII), a conserved autophagy-specific guanine nucleotide exchange factor for Ypt1/Rab1, is recruited to the PAS by Atg17. We also show that activated Ypt1 recruits the putative membrane curvature sensor Atg1 to the PAS, bringing it into proximity to its binding partner Atg17. Since Atg17 resides at the PAS, these events ensure that Atg1 will specifically localize to the PAS and not to the other compartments where Ypt1 resides. We propose that Ypt1 regulates Atg9 vesicle tethering by modulating the delivery of Atg1 to the PAS. These events appear to be conserved in higher cells.
Project description:Macroautophagy delivers cytoplasmic material to lysosomal/vacuolar compartments for degradation. Conserved multisubunit complexes, composed of autophagy-related (Atg) proteins, initiate the formation of membrane precursors, termed phagophores. Under physiological conditions these cup-shaped structures can capture cytoplasmic material highly selectively. Starvation or cytotoxic stresses, however, initiate the formation of much larger phagophores to enclose cytoplasm nonselectively. The biogenesis of nonselective autophagosomes is initiated by the hierarchical assembly of the Atg1 kinase complex and the recruitment of Atg9 vesicles at the phagophore assembly site (PAS). In this punctum we summarize our recent findings regarding tethering of Atg9 vesicles by the Atg1 kinase complex. We discuss membrane tethering by and activation of its central subunit Atg17 in the context of other canonical membrane tethering factors. Our results show that Atg17 suffices to bind and tether Atg9 vesicles. The Atg31-Atg29 subcomplex inhibits Atg17 activity, and activation of Atg17 depends on the formation of the Atg1 kinase complex that involves recruiting Atg1-Atg13. Our studies lead to a model of unconventional membrane tethering in autophagy.
Project description:Autophagy is a physiological process for the recycling and degradation of cellular materials. Forming the autophagosome from the phagophore, a cup-shaped double-membrane vesicle, is a critical step in autophagy. The origin of the cup shape of the phagophore is poorly understood. In yeast, fusion of a small number of Atg9-containing vesicles is considered a key step in autophagosome biogenesis, aided by Atg1 complexes (ULK1 in mammals) localized at the preautophagosomal structure (PAS). In particular, the S-shaped Atg17-Atg31-Atg29 subcomplex of Atg1 is critical for phagophore nucleation at the PAS. To study this process, we simulated membrane remodeling processes in the presence and absence of membrane associated Atg17. We show that at least three vesicles need to fuse to induce the phagophore shape, consistent with experimental observations. However, fusion alone is not sufficient. Interactions with 34-nm long, S-shaped Atg17 complexes are required to overcome a substantial kinetic barrier in the transition to the cup-shaped phagophore. Our finding rationalizes the recruitment of Atg17 complexes to the yeast PAS, and their unusual shape. In control simulations without Atg17, with weakly binding Atg17, or with straight instead of S-shaped Atg17, the membrane shape transition did not occur. We confirm the critical role of Atg17-membrane interactions experimentally by showing that mutations of putative membrane interaction sites result in reduction or loss of autophagic activity in yeast. Fusion of a small number of vesicles followed by Atg17-guided membrane shape-remodeling thus emerges as a viable route to phagophore formation.