Understanding phosphatidylinositol-3-phosphate dynamics during autophagosome biogenesis

Autophagosomes, the hallmark of autophagy, are double-membrane vesicles sequestering cytoplasmic components. They are generated at the phagophore assembly site (PAS), the phagophore being the precursor structure of these carriers. According to the current model, autophagosomes result from the elongation and reorganization of membranes at the PAS/phagophore driven by the concerted action of the autophagy-related (Atg) proteins. Once an autophagosome is completed, the Atg proteins that were associated with the expanding phagophore are released in the cytoplasm and reused for the biogenesis of new vesicles. One molecular event required for autophagosome formation is the generation of phosphatidylinositol 3-phosphate (PtdIns3P) at the PAS. Our data indicate that in addition to the synthesis of this lipid, the dephosphorylation of PtdIns3P is also crucial for autophagy progression. In the absence of Ymr1, a specific PtdIns3P phosphatase and the only yeast member of the myotubularin protein family, Atg proteins remain associated with complete autophagosomes, which are thus unable to fuse with the vacuole.     

Quantitative analysis of autophagy-related protein stoichiometry by fluorescence microscopy

In yeast, approximately 31 autophagy-related (Atg) proteins have been identified. Most of them reside at the phagophore assembly site (PAS), although the function of the PAS mostly remains unclear. One reason for the latter is the lack of stoichiometric information regarding the Atg proteins at this site. We report the application of fluorescence microscopy to study the amount of Atg proteins at the PAS. We find that an increase in the amount of Atg11 at the PAS enhances the recruitment of Atg8 and Atg9 to this site and facilitates the formation of more cytoplasm-to-vacuole targeting vesicles. In response to autophagy induction, the amount of most Atg proteins remains unchanged at the PAS, whereas we see an enhanced recruitment of Atg8 and 9 at this site. During autophagy, the amount of Atg8 at the PAS showed a periodic change, indicating the formation of autophagosomes. Application of this method and further analysis will provide more insight into the functions of Atg proteins.     

Double duty of Atg9 self-association in autophagosome biogenesis

The understanding of the membrane flow process during autophagosome formation is essential to illuminate the role of autophagy under various disease-causing conditions. Atg9 is the only identified integral membrane protein required for autophagosome formation, and it is thought to cycle between the membrane sources and the phagophore assembly site (PAS). Thus, Atg9 may play an important role as a membrane carrier. We report the self-interaction of Atg9 and generate an Atg9 mutant that is defective in this interaction. This mutation results in abnormal autophagy, due to altered phagophore formation as well as inefficient membrane delivery to the PAS. Based on our analyses, we discuss a model suggesting dual functions for the Atg9 complex: by reversibly binding to another Atg9 molecule, Atg9 can both promote lipid transport from the membrane origins to the PAS, and also help assemble an intact phagophore membrane.     

Atg9 reservoirs,a new organelle of the yeast endomembrane system?

Despite all the advances in understanding the roles and the regulation of autophagy in health and disease realized during the past decade, the key question about the origin of the initial autophagosomal membranes remains largely unknown. Among the 16 autophagy-related (Atg) proteins composing the conserved machinery required for autophagy, Atg9 is the only integral membrane component and it is one of the first Atg proteins to be recruited to the phagophore assembly site (PAS) emphasizing its relevance in the early stages of autophagosome biogenesis. Because it is

intrinsically associated with lipid bilayers, Atg9 has all the prerequisites to be a major factor in regulating the supply of at least part of the membranes necessary for the formation and expansion of nascent autophagosomes.     

Atg9 reservoirs, a new organelle of the yeast endomembrane system?

Despite all the advances in understanding the roles and the regulation of autophagy in health and disease realized during the past decade, the key question about the origin of the initial autophagosomal membranes remains largely unknown. Among the 16 autophagy-related (Atg) proteins composing the conserved machinery required for autophagy, Atg9 is the only integral membrane component and it is one of the first Atg proteins to be recruited to the phagophore assembly site (PAS) emphasizing its relevance in the early stages of autophagosome biogenesis. Because it is: intrinsically associated with lipid bilayers, Atg9 has all the prerequisites to be a major factor in regulating the supply of at least part of the membranes necessary for the formation and expansion of nascent autophagosomes.     

Self-interaction is critical for Atg9 transport and function at the phagophore assembly site during autophagy

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.     

Mammalian Atg18 (WIPI2) localizes to omegasome-anchored phagophores and positively regulates LC3 lipidation

Autophagosome formation is a complex process that begins with the nucleation of a pre-autophagosomal structure (PAS) that expands into a phagophore or isolation membrane, the precursor of the autophagosome. A key event in the formation of the phagophore is the production of PtdIns3P by the phosphatidylinsitol kinase Vps34. In yeast the two closely related proteins, Atg18 and Atg21, are the only known effectors of PtdIns3P that act in the autophagy pathway. The recruitment of Atg18 or Atg21 to the PAS is an essential step in the formation of the phagophore. Our bioinformatic analysis of the Atg18 and Atg21 orthologues in all eukaryotes shows that WIPI1 and WIPI2 are both mammalian orthologues of Atg18. We show that WIPI2 is a mammalian effector of PtdIns3P and is ubiquitously expressed in a variety of cell lines. WIPI2 is recruited to early autophagosomal structures along with Atg16L and ULK1 and is required for the formation of LC3-positive autophagosomes. Furthermore, when WIPI2 is depleted, we observe a remarkable accumulation of omegasomes, ER-localized PtdIns3P-containing structures labeled by DFCP1 (double FYVE domain-containing protein 1), which are thought to act as platforms for autophagosome formation. In view of our data we propose a role for WIPI2 in the progression of omegasomes into autophagosomes.     

The Atg17-Atg31-Atg29 complex and Atg11 regulate autophagosome-vacuole fusion

The macroautophagy (hereafter autophagy) process involves de novo formation of double-membrane autophagosomes; after sequestering cytoplasm these transient organelles fuse with the vacuole/lysosome. Genetic studies in yeasts have characterized more than 40 autophagy-related (Atg) proteins required for autophagy, and the majority of these proteins play roles in autophagosome formation. The fusion of autophagosomes with the vacuole is mediated by the Rab GTPase Ypt7, its guanine nucleotide exchange factor Mon1-Ccz1, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. However, these factors are not autophagosome-vacuole fusion specific. We recently showed that 2 autophagy scaffold proteins, the Atg17-Atg31-Atg29 complex and Atg11, regulate autophagosome-vacuole fusion by recruiting the vacuolar SNARE Vam7 to the phagophore assembly site (PAS), where an autophagosome forms in yeast.     

Atg4 recycles inappropriately lipidated Atg8 to promote autophagosome biogenesis

Atg8 is a ubiquitin-like protein required for autophagy in the budding yeast Saccharomyces cerevisiae. A ubiquitin-like system mediates the conjugation of the C terminus of Atg8 to the lipid phosphatidylethanolamine (PE), and this conjugate (Atg8-PE) plays a crucial role in autophagosome formation at the phagophore assembly site/pre-autophagosomal structure (PAS). The cysteine protease Atg4 processes the C terminus of newly synthesized Atg8 and also delipidates Atg8 to release the protein from membranes. While the former is a prerequisite for lipidation of Atg8, the significance of the latter in autophagy has remained unclear. Here, we show that autophagosome formation is significantly retarded in cells deficient for Atg4-mediated delipidation of Atg8. We find that Atg8-PE accumulates on various organelle membranes including the vacuole, the endosome and the ER in these cells, which depletes unlipidated Atg8 and thereby attenuates its localization to the PAS. Our results suggest that the Atg8-PE that accumulates on organelle membranes is erroneously produced by lipidation system components independently of the normal autophagic process. It is also suggested that delipidation of Atg8 by Atg4 on different organelle membranes promotes autophagosome formation. Considered together with other results, we propose that Atg4 acts to compensate for the intrinsic defect in the lipidation system; it recycles Atg8-PE generated on inappropriate membranes to maintain a reservoir of unlipidated Atg8 that is required for autophagosome formation at the PAS.     

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.     

Passing membranes to autophagy: Unconventional membrane tethering by Atg17

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.     

Atg4 recycles inappropriately lipidated Atg8 to promote autophagosome biogenesis

Atg8 is a ubiquitin-like protein required for autophagy in the budding yeast Saccharomyces cerevisiae. A ubiquitin-like system mediates the conjugation of the C terminus of Atg8 to the lipid phosphatidylethanolamine (PE), and this conjugate (Atg8–PE) plays a crucial role in autophagosome formation at the phagophore assembly site/pre-autophagosomal structure (PAS). The cysteine protease Atg4 processes the C terminus of newly synthesized Atg8 and also delipidates Atg8 to release the protein from membranes. While the former is a prerequisite for lipidation of Atg8, the significance of the latter in autophagy has remained unclear. Here, we show that autophagosome formation is significantly retarded in cells deficient for Atg4-mediated delipidation of Atg8. We find that Atg8–PE accumulates on various organelle membranes including the vacuole, the endosome and the ER in these cells, which depletes unlipidated Atg8 and thereby attenuates its localization to the PAS. Our results suggest that the Atg8–PE that accumulates on organelle membranes is erroneously produced by lipidation system components independently of the normal autophagic process. It is also suggested that delipidation of Atg8 by Atg4 on different organelle membranes promotes autophagosome formation. Considered together with other results, we propose that Atg4 acts to compensate for the intrinsic defect in the lipidation system; it recycles Atg8–PE generated on inappropriate membranes to maintain a reservoir of unlipidated Atg8 that is required for autophagosome formation at the PAS.     

The Atg1 kinase complex is involved in the regulation of protein recruitment to initiate sequestering vesicle formation for nonspecific autophagy in Saccharomyces cerevisiae

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.     

Dual role of Atg1 in regulation of autophagy-specific PAS assembly in Saccharomyces cerevisiae

Most autophagy-related (Atg) proteins are assembled at the phagophore assembly site or pre-autophagosomal structure (PAS), which is a potential site for vesicle formation during vegetative or starvation conditions. To understand the initial step of vesicle formation, it is important to know how Atg proteins are recruited to the PAS. Atg11 facilitates PAS assembly for the cytoplasm to vacuole targeting (Cvt) pathway in vegetative conditions. To examine autophagy-specific PAS formation, an ATG11 deletion mutant was used to eliminate the PAS formation that occurs in vegetative conditions. We found that Atg1, Atg13 and Atg17 play a similar role for PAS formation under autophagy-inducing conditions as seen for Atg11 during vegetative growth. In particular, Atg1 is proposed to have dual roles for autophagy-specific PAS recruitment. Atg1 plays a structural role for efficient recruitment of Atg proteins to the PAS, which is mediated by interaction with Atg13 and Atg17. In contrast, Atg1 kinase activity is needed for dissociation of Atg proteins from the PAS during autophagy inducing conditions, a function which is also critical for autophagy activity.     

Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation

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.     

Atg11

Selective macroautophagy uses double-membrane vesicles, termed autophagosomes, to transport cytoplasmic pathogens, organelles and protein complexes to the vacuole for degradation. Autophagosomes are formed de novo by membrane fusion events at the phagophore assembly site (PAS). Therefore, precursor membrane material must be targeted and transported to the PAS. While some autophagy-related (Atg) proteins, such as Atg9 and Atg11, are known to be involved in this process, most of the mechanistic details are not understood. Previous work has also implicated the small Rab-family GTPase Ypt1 in the process, identifying Trs85 as a unique subunit of the TRAPPIII targeting complex and showing that it plays a macroautophagy-specific role; however, the relationship between Ypt1, Atg9 and Atg11 was not clear. Now, a recent report shows that Atg11 is a Trs85-specific effector of the Rab Ypt1, and may act as a classic coiled-coil membrane tether that targets Atg9-containing membranes to the PAS. Here, we review this finding in the context of what is known about Atg11, other Rab-dependent coiled-coil tethers, and other tethering complexes involved in autophagosome formation.     

Dual role of Atg1 in regulation of autophagy-specific PAS assembly in Saccharomyces cerevisiae

Most autophagy-related (Atg) proteins are assembled at the phagophore assembly site or pre-autophagosomal structure (PAS), which is a potential site for vesicle formation during vegetative or starvation conditions. To understand the initial step of vesicle formation, it is important to know how Atg proteins are recruited to the PAS. Atg11 facilitates PAS assembly for the cytoplasm to vacuole targeting (Cvt) pathway in vegetative conditions. To examine autophagy-specific PAS formation, an ATG11 deletion mutant was used to eliminate the PAS formation that occurs in vegetative conditions. We found that Atg1, Atg13 and Atg17 play a similar role for PAS formation under autophagy-inducing conditions as seen for Atg11 during vegetative growth. In particular, Atg1 is proposed to have dual roles for autophagy-specific PAS recruitment. Atg1 plays a structural role for efficient recruitment of Atg proteins to the PAS, which is mediated by interaction with Atg13 and Atg17. In contrast, Atg1 kinase activity is needed for dissociation of Atg proteins from the PAS during autophagy inducing conditions, a function which is also critical for autophagy activity.

Addendum to: Cheong H, Nair U, Geng J Klionsky DK. The Atg1 kinase complex Is involved in the regulation of protein recruitment to initiate sequestering vesicle formation for nonspecific autophagy in Saccharomyces cerevisiae. Mol Biol Cell 2008; 19:668-81.     

Phosphorylation of Atg31 is required for autophagy

Autophagy is an evolutionarily conserved cellular process which degrades intracellular contents. The Atg17- Atg31-Atg29 complex plays a key role in autophagy induction by various stimuli. In yeast, autophagy occurs with autophagosome formation at a special site near the vacuole named the pre-autophagosomal structure (PAS). The Atg17-Atg31-Atg29 complex forms a scaffold for PAS organization, and recruits other autophagy-related (Atg) proteins to the PAS. Here, we show that Atg31 is a phosphorylated protein. The phosphorylation sites on Atg31 were identified by mass spectrometry. Analysis of mutants in which the phosphorylated amino acids were replaced by alanine, either individually or in various combinations, identified S174 as the functional phosphorylation site. An S174A mutant showed a similar degree of autophagy impairment as an Atg31 deletion mutant. S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment. Mass spectrometry analysis showed that S174 is phosphorylated constitutively, and expression of a phosphorylation-mimic mutant (S174D) in the Atg31 deletion strain restores autophagy. In the S174A mutant, Atg9-positive vesicles accumulate at the PAS. Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS. Our data demonstrate the role of phosphorylation of Atg31 in autophagy.     

Bif-1 regulates Atg9 trafficking by mediating the fission of Golgi membranes during autophagy

Atg9 is a transmembrane protein essential for autophagy which cycles between the Golgi network, late endosomes and LC3-positive autophagosomes in mammalian cells during starvation through a mechanism that is dependent on ULK1 and requires the activity of the class III phosphatidylinositol-3-kinase (PI3KC3). In this study, we demonstrate that the N-BAR-containing protein, Bif-1, is required for Atg9 trafficking and the fission of Golgi membranes during the induction of autophagy. Upon starvation, Atg9-positive membranes undergo continuous tubulation and fragmentation to produce cytoplasmic punctate structures that are positive for Rab5, Atg16L and LC3. Loss of Bif-1 or inhibition of the PI3KC3 complex II suppresses starvation-induced fission of Golgi membranes and peripheral cytoplasmic redistribution of Atg9. Moreover, Bif-1 mutants, which lack the functional regions of the N-BAR domain that are responsible for membrane binding and/or bending activity, fail to restore the fission of Golgi membranes as well as the formation of Atg9 foci and autophagosomes in Bif-1-deficient cells starved of nutrients. Taken together, these findings suggest that Bif-1 acts as a critical regulator of Atg9 puncta formation presumably by mediating Golgi fission for autophagosome biogenesis during starvation.     

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.