Atg11 directs autophagosome cargoes to the PAS along actin cables

For more than 40 years, autophagy has been almost exclusively studied as a cellular response that allows adaptation to starvation situations. In nutrient-deprived conditions, cytoplasmic components and organelles are randomly sequestered into double-membrane vesicles called autophagosomes, creating the notion that this pathway is a nonselective process (reviewed in Refs 1, 2). Recent results, however, have demonstrated that under certain circumstances, cargoes such as protein complexes, organelles and bacteria can be selectively and exclusively incorporated into double-membrane vesicles.(1) We have recently shown that actin plays an essential role in two selective types of autophagy in the yeast Saccharomyces cerevisiae, the cytoplasm to vacuole targeting (Cvt) pathway and pexophagy, raising the possibility that the structures formed by polymers of this protein helps autophagosomes in recognizing the cargoes that must be delivered to the vacuole.(3) In this addendum, we discuss the possible central role of Atg11 as a molecule connecting cargoes, actin and pre-utophagosomal structure (PAS) elements.     

Atg19 mediates a dual interaction cargo sorting mechanism in selective autophagy

Autophagy is a catabolic membrane-trafficking mechanism conserved in all eukaryotic cells. In addition to the nonselective transport of bulk cytosol, autophagy is responsible for efficient delivery of the vacuolar enzyme Ape1 precursor (prApe1) in the budding yeast Saccharomyces cerevisiae, suggesting the presence of a prApe1 sorting machinery. Sequential interactions between Atg19-Atg11 and Atg19-Atg8 pairs are thought responsible for targeting prApe1 to the vesicle formation site, the preautophagosomal structure (PAS), and loading it into transport vesicles, respectively. However, the different patterns of prApe1 transport defect seen in the atg11Delta and atg19Delta strains seem to be incompatible with this model. Here we report that prApe1 could not be targeted to the PAS and failed to be delivered into the vacuole in atg8Delta atg11Delta double knockout cells regardless of the nutrient conditions. We postulate that Atg19 mediates a dual interaction prApe1-sorting mechanism through independent, instead of sequential, interactions with Atg11 and Atg8. In addition, to efficiently deliver prApe1 to the vacuole, a proper interaction between Atg11 and Atg9 is indispensable. We speculate that Atg11 may elicit a cargo-loading signal and induce Atg9 shuttling to a specific PAS site, where Atg9 relays the signal and recruits other Atg proteins to induce vesicle formation.     

A cycling protein complex required for selective autophagy

Survival of environmental stress conditions requires the maintenance of cellular homeostasis. To preserve this balance, cells utilize a degradative mechanism known as autophagy. During this process, in response to starvation or other stresses, bulk cytoplasm is non-specifically sequestered within double-membrane vesicles and delivered to the lysosome/vacuole for subsequent degradation and recycling. The cytoplasm to vacuole targeting (Cvt) pathway is a type of specific autophagy, which occurs constitutively during growing conditions. Here, we examine three autophagy-related (Atg) proteins, Atg9, Atg23 and Atg27, which exhibit a unique localization pattern, residing both at the pre-autophagosomal structure (PAS) and other peripheral sites. These proteins colocalize, interact with one another in vivo, and form a functional complex. Furthermore, all three proteins cycle between the PAS and the other sites, and depend upon one another for this movement. Our data suggest that Atg9, Atg23 and Atg27 play a role in Atg protein retrieval from the PAS. In addition, Atg9 and Atg27 are the only known integral membrane Atg proteins involved in vesicle formation; a better understanding of their function may offer insight into the mechanism of membrane delivery to the PAS, the site of double-membrane vesicle assembly.     

Lap3 is a selective target of autophagy in yeast, Saccharomyces cerevisiae

Autophagy is a primarily non-selective degradation system of cytoplasmic constituents in lysosomes/vacuoles during starvation. In yeast, autophagy is also involved in the selective transport of Ape1, a vacuolar hydrolase, as a biosynthetic route. Ald6, a soluble cytoplasmic enzyme, is preferentially eliminated from cytoplasm via autophagy. However, little is known about the mechanisms of Ald6 targeting to autophagosomes. Here, we show that Lap3, a soluble cytosolic cysteine protease, is spatially associated with Ape1 and selectively transported to the vacuole during nitrogen starvation. The rate of Lap3 transport is much higher than that of Ald6 and is similar to that of Ape1. Moreover, ATG11 and ATG19, essential factors for Ape1 transport, are important for Lap3 transport. Most Lap3 is degraded within a couple of hours in the vacuole in contrast to Ape1; therefore, we conclude that the machinery required for Ape1 biosynthesis is used for selective degradation of Lap3.     

Aspartyl aminopeptidase is imported from the cytoplasm to the vacuole by selective autophagy in Saccharomyces cerevisiae

Macroautophagy is a catabolic process by which cytosolic components are sequestered by double membrane vesicles called autophagosomes and sorted to the lysosomes/vacuoles to be degraded. Saccharomyces cerevisiae has adapted this mechanism for constitutive transport of the specific vacuolar hydrolases aminopeptidase I (Ape1) and α-mannosidase (Ams1); this process is called the cytoplasm to vacuole targeting (Cvt) pathway. The precursor form of Ape1 self-assembles into an aggregate-like structure in the cytosol that is then recognized by Atg19 in a propeptide-dependent manner. The interaction between Atg19 and autophagosome-forming machineries allows selective packaging of the Ape1-Atg19 complex by the autophagosome-like Cvt vesicle. Ams1 also forms oligomers and utilizes the Ape1 transport system by interacting with Atg19. Although the mechanism of selective transport of the Cvt cargoes has been well studied, it is unclear whether proteins other than Ape1 and Ams1 are transported via the Cvt pathway. We describe here that aspartyl aminopeptidase (Yhr113w/Ape4) is the third Cvt cargo, which is similar in primary structure and subunit organization to Ape1. Ape4 has no propeptide, and it does not self-assemble into aggregates. However, it binds to Atg19 in a site distinct from the Ape1- and Ams1-binding sites, allowing it to "piggyback" on the Ape1 transport system. In growing conditions, a small portion of Ape4 localizes in the vacuole, but its vacuolar transport is accelerated by nutrient starvation, and it stably resides in the vacuole lumen. We propose that the cytosolic Ape4 is redistributed to the vacuole when yeast cells need more active vacuolar degradation.     

Membrane recruitment of Aut7p in the autophagy and cytoplasm to vacuole targeting pathways requires Aut1p, Aut2p, and the autophagy conjugation complex

Autophagy is a degradative pathway by which cells sequester nonessential, bulk cytosol into double-membrane vesicles (autophagosomes) and deliver them to the vacuole for recycling. Using this strategy, eukaryotic cells survive periods of nutritional starvation. Under nutrient-rich conditions, autophagy machinery is required for the delivery of a resident vacuolar hydrolase, aminopeptidase I, by the cytoplasm to vacuole targeting (Cvt) pathway. In both pathways, the vesicle formation process requires the function of the starvation-induced Aut7 protein, which is recruited from the cytosol to the forming Cvt vesicles and autophagosomes. The membrane binding of Aut7p represents an early step in vesicle formation. In this study, we identify several requirements for Aut7p membrane association. After synthesis in the cytosol, Aut7p is proteolytically cleaved in an Aut2p-dependent manner. While this novel processing event is essential for Aut7p membrane binding, Aut7p must undergo additional physical interactions with Aut1p and the autophagy (Apg) conjugation complex before recruitment to the membrane. Lack of these interactions results in a cytosolic distribution of Aut7p rather than localization to forming Cvt vesicles and autophagosomes. This study assigns a functional role for the Apg conjugation system as a mediator of Aut7p membrane recruitment. Further, we demonstrate that Aut1p, which physically interacts with components of the Apg conjugation complex and Aut7p, constitutes an additional factor required for Aut7p membrane recruitment. These findings define a series of steps that results in the modification of Aut7p and its subsequent binding to the sequestering transport vesicles of the autophagy and cytoplasm to vacuole targeting pathways.     

Transport of proteins to the yeast vacuole: autophagy, cytoplasm-to-vacuole targeting, and role of the vacuole in degradation

The vacuole/lysosome performs a central role in degradation. Proteins and organelles are transported to the vacuole by selective and non-selective pathways. Transport to the vacuole by autophagy is the primary mode for degradation of cytoplasmic constituents under starvation conditions. Autophagy overlaps mechanistically and genetically with a biosynthetic pathway termed Cvt (Cytoplasm-to-vacuole targeting) that operates under vegetative conditions to transport the resident vacuolar hydrolase aminopeptidase I (API). API import has been dissected to reveal the action of a novel mechanism that transports cargo within double-membrane vesicles. Recent work has uncovered molecular components involved in autophagy and the Cvt pathway.     

The molecular mechanism of autophagy

Autophagy is a conserved trafficking pathway that is highly regulated by environmental conditions. During autophagy, portions of cytoplasm are sequestered into a double-membrane autophagosome and delivered to a degradative organelle, the vacuole in yeast and the lysosome in mammalian cells, for breakdown and recycling. Autophagy is induced under starvation conditions and in mammalian cells is also invoked in response to specific hormones. In yeast, under nutrient-rich conditions, a constitutive biosynthetic pathway, termed the cytoplasm to vacuole targeting (Cvt) pathway, utilizes most of the same molecular machinery and topologically similar vesicles for the delivery of the resident hydrolase aminopeptidase I to the vacuole. Both autophagy and the Cvt pathway have been extensively studied and comprehensively reviewed in the past few years. In this review, we focus on the yeast system, which has provided most of the insight into the molecular mechanism of autophagy and the Cvt pathway, and highlight the most recent additions to our current knowledge of both pathways.     

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.     

Convergence of multiple autophagy and cytoplasm to vacuole targeting components to a perivacuolar membrane compartment prior to de novo vesicle formation.

Under starvation conditions, the majority of intracellular degradation occurs at the lysosome or vacuole by the autophagy pathway. The cytoplasmic substrates destined for degradation are packaged inside unique double-membrane transport vesicles called autophagosomes and are targeted to the lysosome/vacuole for subsequent breakdown and recycling. Genetic analyses of yeast autophagy mutants, apg and aut, have begun to identify the molecular machinery as well as indicate a substantial overlap with the biosynthetic cytoplasm to vacuole targeting (Cvt) pathway. Transport vesicle formation is a key regulatory step of both pathways. In this study, we characterize the putative compartment from which both autophagosomes and the analogous Cvt vesicles may originate. Microscopy analyses identified a perivacuolar membrane as the resident compartment for both the Apg1-Cvt9 signaling complex, which mediates the switching between autophagic and Cvt transport, and the autophagy/Cvt-specific phosphatidylinositol 3-kinase complex. Furthermore, the perivacuolar compartment designates the initial site of membrane binding by the Apg/Cvt vesicle component Aut7, the Cvt cargo receptor Cvt19, and the Apg conjugation machinery, which functions in the de novo formation of vesicles. Biochemical isolation of the vesicle component Aut7 and density gradient analyses recapitulate the microscopy findings although also supporting the paradigm that components required for vesicle formation and packaging concentrate at subdomains within the donor membrane compartment.     

Selective Transport of α-Mannosidase by Autophagic Pathways: IDENTIFICATION OF A NOVEL RECEPTOR,Atg34p*

In Saccharomyces cerevisiae, aminopeptidase I (Ape1p) and α-mannosidase (Ams1p) are known cargoes of selective autophagy. Atg19p has been identified as an Ape1p receptor and targets Ape1p to the preautophagosomal structure (PAS). Under nutrient-rich conditions, transport of Ams1p to the vacuole largely depends on Atg19p. Here, we show that Atg34p (Yol083wp), a homolog of Atg19p, is a receptor for Ams1p transport during autophagy. Atg34p interacted with Ams1p, Atg11p, and Atg8p using distinct domains. Homo-oligomerized Ams1p bound to the Ams1-binding domain of Atg34p; this binding was important for the formation of a higher order complex named the Ams1 complex. In the absence of the interaction of Atg34p with Atg8p, the Ams1 complex was targeted to the preautophagosomal structure but failed to transit to the vacuole, indicating that the interaction of Atg34p with Atg8p is crucial for the Ams1 complex to be enclosed by autophagosomes. Atg34p and Atg19p have similar domain structures and are important for Ams1p transport during autophagy.     

Autophagosomes are formed at a distinct cellular structure

Autophagy is characterized by the formation of double-membrane vesicles called autophagosomes, which deliver bulk cytoplasmic material to the lytic compartment of the cell for degradation. Autophagosome formation is initiated by assembly and recruitment of the core autophagy machinery to distinct cellular sites, referred to as phagophore assembly sites (PAS) in yeast or autophagosome formation sites in other organisms. A large number of autophagy proteins involved in the formation of autophagosomes has been identified; however, how the individual components of the PAS are assembled and how they function to generate autophagosomes remains a fundamental question. Here, we highlight recent studies that provide molecular insights into PAS organization and the role of the endoplasmic reticulum and the vacuole in autophagosome formation.     

A Cycling Protein Complex Required for Selective Autophagy

Survival of environmental stress conditions requires the maintenance of cellular homeostasis. To preserve this balance, cells utilize a degradative mechanism known as autophagy. During this process, in response to starvation or other stresses, bulk cytoplasm is non-specifically sequestered within double-membrane vesicles and delivered to the lysosome/vacuole for subsequent degradation and recycling. The cytoplasm to vacuole targeting (Cvt) pathway is a type of specific autophagy, which occurs constitutively during growing conditions. Here, we examine three autophagy-related (Atg) proteins, Atg9, Atg23 and Atg27, which exhibit a unique localization pattern, residing both at the pre-autophagosomal structure (PAS) and other peripheral sites. These proteins colocalize, interact with one another in vivo, and form a functional complex. Furthermore, all three proteins cycle between the PAS and the other sites, and depend upon one another for this movement. Our data suggest that Atg9, Atg23 and Atg27 play a role in Atg protein retrieval from the PAS. In addition, Atg9 and Atg27 are the only known integral membrane Atg proteins involved in vesicle formation; a better understanding of their function may offer insight into the mechanism of membrane delivery to the PAS, the site of double-membrane vesicle assembly.     

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.     

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.     

Apg9p/Cvt7p is an integral membrane protein required for transport vesicle formation in the Cvt and autophagy pathways

In nutrient-rich, vegetative conditions, the yeast Saccharomyces cerevisiae transports a resident protease, aminopeptidase I (API), to the vacuole by the cytoplasm to vacuole targeting (Cvt) pathway, thus contributing to the degradative capacity of this organelle. When cells subsequently encounter starvation conditions, the machinery that recruited precursor API (prAPI) also sequesters bulk cytosol for delivery, breakdown, and recycling in the vacuole by the autophagy pathway. Each of these overlapping alternative transport pathways is specifically mobilized depending on environmental cues. The basic mechanism of cargo packaging and delivery involves the formation of a double-membrane transport vesicle around prAPI and/or bulk cytosol. Upon completion, these Cvt and autophagic vesicles are targeted to the vacuole to allow delivery of their lumenal contents. Key questions remain regarding the origin and formation of the transport vesicle. In this study, we have cloned the APG9/CVT7 gene and characterized the gene product. Apg9p/Cvt7p is the first characterized integral membrane protein required for Cvt and autophagy transport. Biochemical and morphological analyses indicate that Apg9p/Cvt7p is localized to large perivacuolar punctate structures, but does not colocalize with typical endomembrane marker proteins. Finally, we have isolated a temperature conditional allele of APG9/CVT7 and demonstrate the direct role of Apg9p/Cvt7p in the formation of the Cvt and autophagic vesicles. From these results, we propose that Apg9p/Cvt7p may serve as a marker for a specialized compartment essential for these vesicle-mediated alternative targeting pathways.     

Atg11 links cargo to the vesicle-forming machinery in the cytoplasm to vacuole targeting pathway

Proteins are selectively packaged into vesicles at specific sites and then delivered correctly to the various organelles where they function, which is critical to the proper physiology of each organelle. The precursor form of the vacuolar hydrolase aminopeptidase I is a selective cargo molecule of the cytoplasm to vacuole targeting (Cvt) pathway and autophagy. Precursor Ape1 along with its receptor Atg19 forms the Cvt complex, which is transported to the pre-autophagosomal structure (PAS), the putative site of Cvt vesicle formation, in a process dependent on Atg11. Here, we show that this interaction occurs through the Atg11 C terminus; subsequent recruitment of the Cvt complex to the PAS depends on central regions within Atg11. Atg11 was shown to physically link several proteins, although the timing of these interactions and their importance are unknown. Our mapping shows that the Atg11 coiled-coil domains are involved in self-assembly and the interaction with other proteins, including two previously unidentified partners, Atg17 and Atg20. Atg11 mutants defective in the transport of the Cvt complex to the PAS affect the localization of other Atg components, supporting the idea that the cargo facilitates the organization of the PAS in selective autophagy. These findings suggest that Atg11 plays an integral role in connecting cargo molecules with components of the vesicle-forming machinery.     

Quantitative regulation of vesicle formation in yeast nonspecific autophagy

In eukaryotic cells, autophagy is a degradative pathway necessary for the turnover of bulk cytoplasm. In yeast, this pathway also mediates the specific transport of a vacuolar hydrolase zymogen, precursor aminopeptidase (prApe1), from the cytoplasm to the vacuole. Autophagy is under precise regulation, not only qualitatively but also quantitatively, especially in the steps involved in the vesicle formation process. We have recently used a fluorescence microscopy-based method to study the stoichiometry of autophagy-related (Atg) proteins during different conditions. This analysis shows that increased expression of Atg11 in the cytoplasm to vacuole targeting (Cvt) pathway increases the amount of this protein localized at the phagophore assembly site (PAS). In turn, under nutrient-rich conditions, the increased level of Atg11 causes the recruitment of higher than normal levels of Atg8 and Atg9 to the PAS, resulting in the formation of more Cvt vesicles, whereas the vesicle size is not affected. Combined with results from previous studies in starvation conditions, in this addendum we discuss the possible role of Atg8 and Atg9 in quantitatively regulating the vesicle formation process.     

Cargo proteins facilitate the formation of transport vesicles in the cytoplasm to vacuole targeting pathway

Selective incorporation of cargo proteins into the forming vesicle is an important aspect of protein targeting via vesicular trafficking. Based on the current paradigm of cargo selection in vesicular transport, proteins to be sorted to other organelles are condensed at the vesicle budding site in the donor organelle, a process that is mediated by the interaction between cargo and coat proteins, which constitute part of the vesicle forming machinery. The cytoplasm to vacuole targeting (Cvt) pathway is an unconventional vesicular trafficking pathway in yeast, which is topologically and mechanistically related to autophagy. Aminopeptidase I (Ape1) is the major cargo protein of the Cvt pathway. Unlike the situation in conventional vesicular transport, precursor Ape1, along with its receptor Atg19/Cvt19, is packed into a huge complex, termed a Cvt complex, independent of the vesicle formation machinery. The Cvt complex is subsequently incorporated into the forming Cvt vesicle. The deletion of APE1 or ATG19 compromised the organization of the pre-autophagosomal structure (PAS), a site that is thought to play a critical role in Cvt vesicle/autophagosome formation. The proper organization of the PAS also required Atg11/Cvt9, a protein that localizes the cargo complex at the PAS. Accordingly, the deletion of APE1, ATG19, or ATG11 affected the formation of Cvt vesicles. These observations suggest a unique concept; in the case of the Cvt pathway, the cargo proteins facilitate receptor recruitment and vesicle formation rather than the situation with most vesicular transport, in which the forming vesicle concentrates the cargo proteins.     

Recruitment of Atg9 to the preautophagosomal structure by Atg11 is essential for selective autophagy in budding yeast

Autophagy is a conserved degradative pathway that is induced in response to various stress and developmental conditions in eukaryotic cells. It allows the elimination of cytosolic proteins and organelles in the lysosome/vacuole. In the yeast Saccharomyces cerevisiae, the integral membrane protein Atg9 (autophagy-related protein 9) cycles between mitochondria and the preautophagosomal structure (PAS), the nucleating site for formation of the sequestering vesicle, suggesting a role in supplying membrane for vesicle formation and/or expansion during autophagy. To better understand the mechanisms involved in Atg9 cycling, we performed a yeast two-hybrid-based screen and identified a peripheral membrane protein, Atg11, that interacts with Atg9. We show that Atg11 governs Atg9 cycling through the PAS during specific autophagy. We also demonstrate that the integrity of the actin cytoskeleton is essential for correct targeting of Atg11 to the PAS. We propose that a pool of Atg11 mediates the anterograde transport of Atg9 to the PAS that is dependent on the actin cytoskeleton during yeast vegetative growth.