Atg9 trafficking in the yeast Saccharomyces cerevisiae

Autophagy can be divided into selective and nonselective modes. This process is considered selective when a precise cargo is specifically and exclusively incorporated into autophagosomes, the double-membrane vesicles that are the hallmark of autophagy. In contrast, during nonselective, bulk autophagy, cytoplasmic components are randomly enwrapped into autophagosomes. To date, approximately 30 autophagy-related genes called ATG have been identified. Sixteen of them compose the general basic machinery catalyzing the formation of double-membrane vesicles in all eukaryotic cells. The rest of them are often not conserved between species and cooperate with the basic Atg proteins during either selective or nonselective autophagy. Atg9 is the only integral membrane component of the conserved Atg machinery and appears to be a crucial organizational element. Recent studies in the S. cerevisiae have shown that Atg9 transport is differentially regulated depending on the autophagy mode. In this addendum, we will review and discuss what has recently been unveiled about yeast S. cerevisiae Atg9 trafficking, its modulators and its potential role in double-membrane vesicle biogenesis.     

Atg27 is a Second Transmembrane Cycling Protein

Autophagy is a degradative pathway conserved among all eukaryotic cells, and is responsible for the turnover of damaged organelles and long-lived proteins. The primary morphological feature of autophagy is the sequestration of cargo within a double-membrane cytosolic vesicle called an autophagosome. More than 25 AuTophaGy-related (ATG) genes that are essential for autophagy have been identified from the yeast Saccharomyces cerevisiae. Despite the identification and characterization of Atg proteins, it remains a mystery how the double-membrane vesicle is made, what the membrane source(s) are, and how the lipid is transported to the forming vesicle. Among Atg proteins, Atg9 was the only characterized transmembrane protein required for the formation of double-membrane vesicles. Evidence has been obtained in yeast and mammalian cells for Atg9 cycling between different peripheral compartments and the phagophore assembly site/pre-autophagosomal structure (PAS), the proposed site of organization for autophagosome formation. This cycling feature makes Atg9 a potential membrane carrier to deliver lipids that are used in the vesicle formation process.2 Recently, in our lab we characterized a second transmembrane protein, Atg27. The unique localization and cycling features of Atg27 suggest the involvement of the Golgi complex in the autophagy pathway. In this addendum, we discuss the trafficking of Atg27 in yeast and compare it with that of Atg9, and consider the possible meaning of Atg27 Golgi localization.

Addendum to:

Atg27 is Required for Autophagy-Dependent Cycling of Atg9

W.-L. Yen, J.E. Legakis, U. Nair and D.J. Klionsky

Mol Biol Cell 2006; In press     

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.     

Atg9 Cycles Between Mitochondria and the Pre-Autophagosomal Structure in Yeasts

Autophagy is a degradative process conserved among eukaryotic cells. It allows the elimination of cytoplasm including aberrant protein aggregates and damaged organelles. Accordingly, it is implicated in normal developmental processes and also serves a protective role in tumor suppression and elimination of invading pathogens, whereas defects in autophagy are associated with various human diseases including cancer and neurodegeneration. Atg proteins mediate the sequestration event that occurs at the preautophagosomal structure (PAS) by catalyzing the formation of double-membrane vesicles, termed autophagosomes. In Saccharomyces cerevisiae, the integral membrane protein Atg9 that is required for autophagy cycles through the PAS. Here, we demonstrate that Atg9 shuttles between this location and mitochondria. These data support a new model where mitochondria may provide at least part of the autophagosomal lipids and suggest a novel cellular function for this well-studied organelle.     

The actin cytoskeleton is required for selective types of autophagy, but not nonspecific autophagy, in the yeast Saccharomyces cerevisiae

Autophagy is a catabolic multitask transport route that takes place in all eukaryotic cells. During starvation, cytoplasmic components are randomly sequestered into huge double-membrane vesicles called autophagosomes and delivered into the lysosome/vacuole where they are destroyed. Cells are able to modulate autophagy in response to their needs, and under certain circumstances, cargoes such as aberrant protein aggregates, organelles and bacteria can be selectively and exclusively incorporated into autophagosomes. In the yeast Saccharomyces cerevisiae, for example, double-membrane vesicles are used to transport the Ape1 protease into the vacuole, or for the elimination of superfluous peroxisomes. In the present study we reveal that in this organism, actin plays a role in these two types of selective autophagy but not in the nonselective, bulk process. In particular, we show that precursor Ape1 is not correctly recruited to the PAS, the putative site of double-membrane vesicle biogenesis, and superfluous peroxisomes are not degraded in a conditional actin mutant. These phenomena correlate with a defect in Atg9 trafficking from the mitochondria to the PAS.     

Exocyst Subcomplex Functions in Autophagosome Biogenesis by Regulating Atg9 Trafficking

During autophagy, double-membrane vesicles called autophagosomes capture and degrade the intracellular cargo. The de novo formation of autophagosomes requires several vesicle transport and membrane fusion events which are not completely understood. We studied the involvement of exocyst, an octameric tethering complex, which has a primary function in tethering post-Golgi secretory vesicles to plasma membrane, in autophagy. Our findings indicate that not all subunits of exocyst are involved in selective and general autophagy. We show that in the absence of autophagy specific subunits, autophagy arrest is accompanied by accumulation of incomplete autophagosome-like structures. In these mutants, impaired Atg9 trafficking leads to decreased delivery of membrane to the site of autophagosome biogenesis thereby impeding the elongation and completion of the autophagosomes. The subunits of exocyst, which are dispensable for autophagic function, do not associate with the autophagy specific subcomplex of exocyst.     

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.     

Atg9 proteins,not so different after all

Macroautophagy (hereafter autophagy) is a catabolic pathway present in all eukaryotic cells. The yeast Saccharomyces cerevisiae has been pivotal in the identification and characterization of the key autophagy-related (Atg) proteins, which play a central role in the generation of autophagosomes. The components of the core Atg/ATG machinery and their functions are highly conserved among species, although mammalian cells also have isoforms and auxiliary factors. Atg9/ATG9 is the only transmembrane protein that is part of the core Atg/ATG machinery, but it appears to have divergent localizations and molecular roles in yeast and mammals. A recent experimental analysis of the yeast endo-lysosomal system by the laboratory of Benjamin Glick, however, suggests a more simple organization of this membrane system. Although this study has not examined yeast Atg9, its findings place this protein in the same compartments as its mammalian counterpart. Here, we will discuss the implications of this conceptual change on the trafficking of yeast Atg9 and its function in autophagy.     

The carboxy terminus of yeast Atg13 binds phospholipid membrane via motifs that overlap with the Vac8-interacting domain

ABSTRACT

Macroautophagy/autophagy is a conserved catabolic recycling pathway involving the sequestration of cytoplasmic components within double-membrane vesicles termed autophagosomes. The autophagy-related (Atg) protein Atg13 is a key member of the autophagy initiation complex. The Atg13 C terminus is an intrinsically disordered region (IDR) harboring a binding site for the vacuolar membrane protein Vac8. Recent reports suggest Atg13 acts as a hub to assemble the initiation complex, and also participates in membrane recognition. Here we show that the Atg13 C terminus directly binds to lipid membranes via electrostatic interactions between positively charged residues in Atg13 and negatively charged phospholipids as well as a hydrophobic insertion of a Phe residue. We identified 2 sets of residues in the Atg13 IDR that affect its phospholipid-binding properties; these residues overlap with the Vac8-binding domain of Atg13. Our data indicate that Atg13 binding to phospholipids and Vac8 is mutually exclusive, and both are required for efficient autophagy.     

BARgaining membranes for autophagosome formation: Regulation of autophagy and tumorigenesis by Bif-1/Endophilin B1

Autophagy is an intracellular system for the bulk degradation of cytoplasmic components enclosed within double-membrane structures known as autophagosomes. To date, many autophagy-related (Atg) genes have been identified by independent genetic screens for autophagy-defective mutants in yeast; however, the molecular machinery required for the biogenesis of autophagosomes in mammalian systems has yet to be determined.^1,2 Recently, we have reported that Bif-1 interacts with Beclin 1 through UVRAG and promotes the activation of class III phosphatidylinositol 3-kinase (PI3KC3) and the formation of autophagosomes.³ Moreover, we have found that loss of Bif-1 promotes starvation-induced caspase activation, but prolongs cell survival by suppressing autophagy-dependent cell death, and enhances spontaneous tumorigenesis in mice. Bif-1 is a member of the endophilin family, which possesses membrane binding and liposome tubulation activities.⁴ During nutrient deprivation, Bif-1 accumulates in punctate foci where it co-localizes with LC3, Atg5 and Atg9. Time-lapse microscopy analyses reveal that Bif-1-positive small vesicles expand by recruiting and fusing with Atg9-positive small membranes to form autophagosomes. Taken together, our findings highlight Bif-1 as a potential regulator of autophagosome biogenesis and as a tumor suppressor.

Addendum to: Takahashi Y, Coppola D, Matsushita N, Cualing HD, Sun M, Sato Y, Liang C, Jung JU, Cheng JQ, Mulé JJ, Pledger WJ, Wang H.-G. Bif-1 interacts with Beclin 1 through UVRAG and regulates autophagy and tumorigenesis. Nature Cell Biol 2007; 9:1142-51.     

Autophagosome formation: core machinery and adaptations

Eukaryotic cells employ autophagy to degrade damaged or obsolete organelles and proteins. Central to this process is the formation of autophagosomes, double-membrane vesicles responsible for delivering cytoplasmic material to lysosomes. In the past decade many autophagy-related genes, ATG, have been identified that are required for selective and/or nonselective autophagic functions. In all types of autophagy, a core molecular machinery has a critical role in forming sequestering vesicles, the autophagosome, which is the hallmark morphological feature of this dynamic process. Additional components allow autophagy to adapt to the changing needs of the cell.     

Atg39 links and deforms the outer and inner nuclear membranes in selective autophagy of the nucleus

In selective autophagy of the nucleus (hereafter nucleophagy), nucleus-derived double-membrane vesicles (NDVs) are formed, sequestered within autophagosomes, and delivered to lysosomes or vacuoles for degradation. In Saccharomyces cerevisiae, the nuclear envelope (NE) protein Atg39 acts as a nucleophagy receptor, which interacts with Atg8 to target NDVs to the forming autophagosomal membranes. In this study, we revealed that Atg39 is anchored to the outer nuclear membrane via its transmembrane domain and also associated with the inner nuclear membrane via membrane-binding amphipathic helices (APHs) in its perinuclear space region, thereby linking these membranes. We also revealed that autophagosome formation-coupled Atg39 crowding causes the NE to protrude toward the cytoplasm, and the tips of the protrusions are pinched off to generate NDVs. The APHs of Atg39 are crucial for Atg39 crowding in the NE and subsequent NE protrusion. These findings suggest that the nucleophagy receptor Atg39 plays pivotal roles in NE deformation during the generation of NDVs to be degraded by nucleophagy.     

Sphingolipid synthesis is involved in autophagy in Saccharomyces cerevisiae

In eukaryotes, autophagy is a conserved protein degradation system that degrades cytoplasmic components by encompassing them with double-membrane structures, called autophagosomes, and delivering them to the lytic compartments of vacuoles/lysosomes. Certain Atg proteins are known to be involved in autophagy, yet the identity and function of lipid molecules involved remain largely unknown. We investigated the involvement of sphingolipids in autophagy using Saccharomyces cerevisiae. Inhibiting synthesis of the simplest complex sphingolipid, inositol phosphorylceramide (IPC), resulted in reduced autophagic activities. Similar results were obtained using myriocin, an inhibitor of the first step in sphingolipid synthesis. Our results indicate that sphingolipids, especially IPC, are required for autophagy. Inhibition of sphingolipid synthesis had no effect on formation of Atg12-Atg5 or Atg8-phosphatidylethanolamine conjugates, on maturation of vacuolar proteases, or on formation of the pre-autophagosomal structure (PAS). These results suggest that sphingolipids are not involved in the cellular signaling that leads to formation of the PAS, but may be involved in the process of autophagosome formation.     

A pseudo-receiver domain in Atg32 is required for mitophagy

Mitochondria are targeted for degradation by mitophagy, a selective form of autophagy. In Saccharomyces cerevisiae, mitophagy is dependent on the autophagy receptor, Atg32, an outer mitochondrial membrane protein. Once activated, Atg32 recruits the autophagy machinery to mitochondria, facilitating mitochondrial capture in phagophores, the precursors to autophagosomes. However, the mechanism of Atg32 activation remains poorly understood. To investigate this crucial step in mitophagy regulation, we examined the structure of Atg32. We have identified a structured domain in Atg32 that is essential for the initiation of mitophagy, as it is required for the proteolysis of the C-terminal domain of Atg32 and the subsequent recruitment of Atg11. The solution structure of this domain was determined by NMR spectroscopy, revealing that Atg32 contains a previously undescribed pseudo-receiver (PsR) domain. Our data suggests that the PsR domain of Atg32 regulates Atg32 activation and the initiation of mitophagy.

Abbreviations:AIM: Atg8-interacting motif; GFP: green fluorescent protein; LIR: LC3-interacting region; NMR: nuclear magnetic resonance; NOESY: nuclear Overhauser effect spectroscopy; PDB: protein data bank; PsR: pseudo-receiver; RMSD: root-mean-square deviation     

Autophagy-related Protein 8 (Atg8) Family Interacting Motif in Atg3 Mediates the Atg3-Atg8 Interaction and Is Crucial for the Cytoplasm-to-Vacuole Targeting Pathway

The autophagy-related protein 8 (Atg8) conjugation system is essential for the formation of double-membrane vesicles called autophagosomes during autophagy, a bulk degradation process conserved among most eukaryotes. It is also important in yeast for recognizing target vacuolar enzymes through the receptor protein Atg19 during the cytoplasm-to-vacuole targeting (Cvt) pathway, a selective type of autophagy. Atg3 is an E2-like enzyme that conjugates Atg8 with phosphatidylethanolamine. Here, we show that Atg3 directly interacts with Atg8 through the WEDL sequence, which is distinct from canonical interaction between E2 and ubiquitin-like modifiers. Moreover, NMR experiments suggest that the mode of interaction between Atg8 and Atg3 is quite similar to that between Atg8/LC3 and the Atg8 family interacting motif (AIM) conserved in autophagic receptors, such as Atg19 and p62. Thus, the WEDL sequence in Atg3 is a canonical AIM. In vitro analyses showed that Atg3 AIM is crucial for the transfer of Atg8 from the Atg8∼Atg3 thioester intermediate to phosphatidylethanolamine but not for the formation of the intermediate. Intriguingly, in vivo experiments showed that it is necessary for the Cvt pathway but not for starvation-induced autophagy. Atg3 AIM attenuated the inhibitory effect of Atg19 on Atg8 lipidation in vitro, suggesting that Atg3 AIM may be important for the lipidation of Atg19-bound Atg8 during the Cvt pathway.     

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 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     

Atg9 vesicles are an important membrane source during early steps of autophagosome formation

During the process of autophagy, cytoplasmic materials are sequestered by double-membrane structures, the autophagosomes, and then transported to a lytic compartment to be degraded. One of the most fundamental questions about autophagy involves the origin of the autophagosomal membranes. In this study, we focus on the intracellular dynamics of Atg9, a multispanning membrane protein essential for autophagosome formation in yeast. We found that the vast majority of Atg9 existed on cytoplasmic mobile vesicles (designated Atg9 vesicles) that were derived from the Golgi apparatus in a process involving Atg23 and Atg27. We also found that only a few Atg9 vesicles were required for a single round of autophagosome formation. During starvation, several Atg9 vesicles assembled individually into the preautophagosomal structure, and eventually, they are incorporated into the autophagosomal outer membrane. Our findings provide conclusive linkage between the cytoplasmic Atg9 vesicles and autophagosomal membranes and offer new insight into the requirement for Atg9 vesicles at the early step of autophagosome formation.     

Harpooning the Cvt complex to the phagophore assembly site

Autophagy is a catabolic process employed by eukaryotes to degrade and recycle intracellular components. When this pathway is induced by starvation conditions, part of the cytoplasm and organelles are sequestered into double-membrane vesicles called autophagosomes, and delivered into the lysosome/vacuole for degradation. In addition to the random bulk elimination of cytoplasmic contents, the selective removal of specific cargo molecules has also been described. These selective types of autophagy are characterized by the recruitment of the cargo destined for degradation in close proximity to the forming double-membrane vesicle that results in an exclusive incorporation (that is, without bulk cytoplasm). A number of factors required for selective types of autophagy have been identified. In particular, we have recently shown that actin and the actin-binding Arp2/3 protein complex are involved in the cytoplasm to vacuole targeting (Cvt) pathway, a yeast selective type of autophagy. The contribution at a molecular level of these factors, however, remains unknown. In this addendum, we present mechanistic models that take into account possible roles of actin and the Arp2/3 complex in the Cvt pathway.

Addendum to: Monastyrska I, He C, Geng J, Hoppe D, Li Z, Klionsky DJ.Arp2 links autophagic machinery with the actin cytoskeleton. Mol Biol Cell 2008; 19:1962-75.     

Pex3 confines pexophagy receptor activity of Atg36 to peroxisomes by regulating Hrr25-mediated phosphorylation and proteasomal degradation

In macroautophagy (hereafter autophagy), cytoplasmic molecules and organelles are randomly or selectively sequestered within double-membrane vesicles called autophagosomes and delivered to lysosomes or vacuoles for degradation. In selective autophagy, the specificity of degradation targets is determined by autophagy receptors. In the budding yeast Saccharomyces cerevisiae, autophagy receptors interact with specific targets and Atg11, resulting in the recruitment of a protein complex that initiates autophagosome formation. Previous studies have revealed that autophagy receptors are regulated by posttranslational modifications. In selective autophagy of peroxisomes (pexophagy), the receptor Atg36 localizes to peroxisomes by binding to the peroxisomal membrane protein Pex3. We previously reported that Atg36 is phosphorylated by Hrr25 (casein kinase 1δ), increasing the Atg36–Atg11 interaction and thereby stimulating pexophagy initiation. However, the regulatory mechanisms underlying Atg36 phosphorylation are unknown. Here, we show that Atg36 phosphorylation is abolished in cells lacking Pex3 or expressing a Pex3 mutant defective in the interaction with Atg36, suggesting that the interaction with Pex3 is essential for the Hrr25-mediated phosphorylation of Atg36. Using recombinant proteins, we further demonstrated that Pex3 directly promotes Atg36 phosphorylation by Hrr25. A co-immunoprecipitation analysis revealed that the interaction of Atg36 with Hrr25 depends on Pex3. These results suggest that Pex3 increases the Atg36–Hrr25 interaction and thereby stimulates Atg36 phosphorylation on the peroxisomal membrane. In addition, we found that Pex3 binding protects Atg36 from proteasomal degradation. Thus, Pex3 confines Atg36 activity to the peroxisome by enhancing its phosphorylation and stability on this organelle.