Degradation of lipid vesicles in the yeast vacuole requires function of Cvt17, a putative lipase

The vacuole/lysosome serves an essential role in allowing cellular components to be degraded and recycled under starvation conditions. Vacuolar hydrolases are key proteins in this process. In Saccharyomces cerevisiae, some resident vacuolar hydrolases are delivered by the cytoplasm to vacuole targeting (Cvt) pathway, which shares mechanistic features with autophagy. Autophagy is a degradative pathway that is used to degrade and recycle cellular components under starvation conditions. Both the Cvt pathway and autophagy employ double-membrane cytosolic vesicles to deliver cargo to the vacuole. As a result, these pathways share a common terminal step, the degradation of subvacuolar vesicles. We have identified a protein, Cvt17, which is essential for this membrane lytic event. Cvt17 is a membrane glycoprotein that contains a motif conserved in esterases and lipases. The active-site serine of this motif is required for subvacuolar vesicle lysis. This is the first characterization of a putative lipase implicated in vacuolar function in yeast.     

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.     

The Cvt pathway as a model for selective autophagy

Autophagy is a highly conserved, ubiquitous process that is responsible for the degradation of cytosolic components in response to starvation. Autophagy is generally considered to be non-selective; however, there are selective types of autophagy that use receptor and adaptor proteins to specifically isolate a cargo. One type of selective autophagy in yeast is the cytoplasm to vacuole targeting (Cvt) pathway. The Cvt pathway is responsible for the delivery of the hydrolase aminopeptidase I to the vacuole; as such, it is the only known biosynthetic pathway that utilizes the core machinery of autophagy. Nonetheless, it serves as a model for the study of selective autophagy in other organisms.     

Molecular machinery required for autophagy and the cytoplasm to vacuole targeting (Cvt) pathway in S. cerevisiae

Autophagy is a vacuolar trafficking pathway that targets subcellular constituents to the vacuole for degradation and recycling. In nutrient-rich conditions in yeast, a different vacuolar trafficking pathway, the cytoplasm to vacuole targeting (Cvt) pathway, transports the resident hydrolase aminopeptidase I to the vacuole, using many of the same molecular components as autophagy. The Cvt pathway is constitutive, whereas autophagy is induced by starvation. Recent studies have laid important groundwork for understanding the signaling mechanism that induces autophagy. Another key advance has been the identification of two novel conjugation systems that function in vesicle formation in both pathways. Finally, many autophagy- and Cvt-specific gene products, including those involved in lipid modification, vesicle expansion and cargo specificity, have been shown to localize to a novel perivacuolar membrane compartment. Additional analysis of this location will help in further dissecting the early events of vesicle formation and identifying the source of the sequestering membrane.     

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.     

Approaching the molecular mechanism of autophagy

Autophagy is a complex cellular process that involves dynamic membrane rearrangements under a range of physiological conditions. It is a highly regulated process that plays a role in cellular maintenance and development, and has been implicated in a number of genetic diseases. Upon induction of autophagy, cytoplasm is sequestered into vesicles and delivered to a degradative organelle, the vacuole in yeast or the lysosome in mammalian cells. The process is unique in that it converts material that is topologically intracellular into topologically extracellular. Autophagy was first described more than 50 years ago, but it is since the discovery of the pathway in yeast cells that our knowledge about the molecular events taking place during the process has expanded. The generation of autophagy-specific mutants in a variety of yeast cell lines has provided insight into functional roles of more than 15 novel genes, double that number if we include genes whose products function also in other processes. Although we have learned much about autophagy, many questions remain to be answered. This review highlights the most recent advances in the autophagy field in both yeast and mammalian cells.     

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.     

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.     

Autophagy in yeast: a review of the molecular machinery

Autophagy is a membrane trafficking mechanism that delivers cytoplasmic cargo to the vacuole/lysosome for degradation and recycling. In addition to non-specific bulk cytosol, selective cargoes, such as peroxisomes, are sorted for autophagic transport under specific physiological conditions. In a nutrient-rich growth environment, many of the autophagic components are recruited for executing a biosynthetic trafficking process, the cytoplasm to vacuole targeting (Cvt) pathway, that transports the resident hydrolases aminopeptidase I and alpha-mannosidase to the vacuole in Saccharomyces cerevisiae. Recent studies have identified pathway-specific components that are necessary to divert a protein kinase and a lipid kinase complex to regulate the conversion between the Cvt pathway and autophagy. Downstream of these proteins, the general machinery for transport vesicle formation involves two novel conjugation systems and a putative membrane protein complex. Completed vesicles are targeted to, and fuse with, the vacuole under the control of machinery shared with other vacuolar trafficking pathways. Inside the vacuole, a potential lipase and several proteases are responsible for the final steps of vesicle breakdown, precursor enzyme processing and substrate turnover. In this review, we discuss the most recent developments in yeast autophagy and point out the challenges we face in the future.     

Mechanism of cargo selection in the cytoplasm to vacuole targeting pathway

The proper functioning of eukaryotic organelles is largely dependent on the specific packaging of cargo proteins within transient delivery vesicles. The cytoplasm to vacuole targeting (Cvt) pathway is an autophagy-related trafficking pathway whose cargo proteins, aminopeptidase I and alpha-mannosidase, are selectively transported from the cytoplasm to the lysosome-like vacuole in yeast. This study elucidates a molecular mechanism for cargo specificity in this pathway involving four discrete steps. The Cvt19 receptor plays a central role in this process: distinct domains in Cvt19 recognize oligomerized cargo proteins and link them to the vesicle formation machinery via interaction with Cvt9 and Aut7. Because autophagy is the primary mechanism for organellar turnover, these results offer insights into physiological processes that are critical in cellular homeostasis, including specific packaging of damaged or superfluous organelles for lysosomal delivery and breakdown.     

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.     

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.     

Apg2 is a novel protein required for the cytoplasm to vacuole targeting, autophagy, and pexophagy pathways

To survive starvation conditions, eukaryotes have developed an evolutionarily conserved process, termed autophagy, by which the vacuole/lysosome mediates the turnover and recycling of non-essential intracellular material for re-use in critical biosynthetic reactions. Morphological and biochemical studies in Saccharomyces cerevisiae have elucidated the basic steps and mechanisms of the autophagy pathway. Although it is a degradative process, autophagy shows substantial overlap with the biosynthetic cytoplasm to vacuole targeting (Cvt) pathway that delivers resident hydrolases to the vacuole. Recent molecular genetics analyses of mutants defective in autophagy and the Cvt pathway, apg, aut, and cvt, have begun to identify the protein machinery and provide a molecular resolution of the sequestration and import mechanism that are characteristic of these pathways. In this study, we have identified a novel protein, termed Apg2, required for both the Cvt and autophagy pathways as well as the specific degradation of peroxisomes. Apg2 is required for the formation and/or completion of cytosolic sequestering vesicles that are needed for vacuolar import through both the Cvt pathway and autophagy. Biochemical studies revealed that Apg2 is a peripheral membrane protein. Apg2 localizes to the previously identified perivacuolar compartment that contains Apg9, the only characterized integral membrane protein that is required for autophagosome/Cvt vesicle formation.     

Vacuolar localization of oligomeric alpha-mannosidase requires the cytoplasm to vacuole targeting and autophagy pathway components in Saccharomyces cerevisiae

One challenge facing eukaryotic cells is the post-translational import of proteins into organelles. This problem is exacerbated when the proteins assemble into large complexes. Aminopeptidase I (API) is a resident hydrolase of the vacuole/lysosome in the yeast Saccharomyces cerevisiae. The precursor form of API assembles into a dodecamer in the cytosol and maintains this oligomeric form during the import process. Vacuolar delivery of the precursor form of API requires a vesicular mechanism termed the cytoplasm to vacuole targeting (Cvt) pathway. Many components of the Cvt pathway are also used in the degradative autophagy pathway. alpha-Mannosidase (Ams1) is another resident hydrolase that enters the vacuole independent of the secretory pathway; however, its mechanism of vacuolar delivery has not been established. We show vacuolar localization of Ams1 is blocked in mutants that are defective in the Cvt and autophagy pathways. We have found that Ams1 forms an oligomer in the cytoplasm. The oligomeric form of Ams1 is also detected in subvacuolar vesicles in strains that are blocked in vesicle breakdown, indicating that it retains its oligomeric form during the import process. These results identify Ams1 as a second biosynthetic cargo protein of the Cvt and autophagy pathways.     

Studies of cargo delivery to the vacuole mediated by autophagosomes in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, aminopeptidase I (API), a vacuolar hydrolase, is selectively transported to the vacuole via the autophagosome. API forms a cytosol to vacuole targeting (Cvt) complex in the cytoplasm. The complex is engulfed by the autophagosome under starvation conditions. In this study, the Cvt complex is visualized as a dot in the cytoplasm by fluorescence microscopy with API-GFP. The Cvt complex associates with the preautophagosomal structure (PAS), which plays a central role in autophagosome formation. In a Deltacvt19 mutant, which is specifically defective in API transport, but not in autophagy, the Cvt complex forms normally but never associates with the PAS. This indicates that Cvt19p mediates association between the Cvt complex and the PAS.     

Transport of a Large Oligomeric Protein by the Cytoplasm to Vacuole Protein Targeting Pathway

Aminopeptidase I (API) is transported into the yeast vacuole by the cytoplasm to vacuole targeting (Cvt) pathway. Genetic evidence suggests that autophagy, a major degradative pathway in eukaryotes, and the Cvt pathway share largely the same cellular machinery. To understand the mechanism of the Cvt import process, we examined the native state of API. Dodecameric assembly of precursor API in the cytoplasm and membrane binding were rapid events, whereas subsequent vacuolar import appeared to be rate limiting. A unique temperature-sensitive API-targeting mutant allowed us to kinetically monitor its oligomeric state during translocation. Our findings indicate that API is maintained as a dodecamer throughout its import and will be useful to study the posttranslational movement of folded proteins across biological membranes.     

Characterization of a novel autophagy-specific gene, ATG29

Autophagy is a process whereby cytoplasmic proteins and organelles are sequestered for bulk degradation in the vacuole/lysosome. At present, 16 ATG genes have been found that are essential for autophagosome formation in the yeast Saccharomyces cerevisiae. Most of these genes are also involved in the cytoplasm to vacuole transport pathway, which shares machinery with autophagy. Most Atg proteins are colocalized at the pre-autophagosomal structure (PAS), from which the autophagosome is thought to originate, but the precise mechanism of autophagy remains poorly understood. During a genetic screen aimed to obtain novel gene(s) required for autophagy, we identified a novel ORF, ATG29/YPL166w. atg29Delta cells were sensitive to starvation and induction of autophagy was severely retarded. However, the Cvt pathway operated normally. Therefore, ATG29 is an ATG gene specifically required for autophagy. Additionally, an Atg29-GFP fusion protein was observed to localize to the PAS. From these results, we propose that Atg29 functions in autophagosome formation at the PAS in collaboration with other Atg proteins.     

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.     

The itinerary of a vesicle component, Aut7p/Cvt5p, terminates in the yeast vacuole via the autophagy/Cvt pathways

Aminopeptidase I (API) is delivered to the yeast vacuole by one of two alternative pathways, cytoplasm to vacuole targeting (Cvt) or autophagy, depending on nutrient conditions. Genetic, morphological, and biochemical studies indicate that the two pathways share many of the same molecular components. The Cvt pathway functions during vegetative growth, while autophagy is induced during starvation. Both pathways involve the formation of cytosolic vesicles that fuse with the vacuole. In either case, the mechanism of vesicle formation is not known. Autophagic uptake displays a greater capacity for cytosolic protein sequestration. This suggests the involvement of an inducible protein(s) that allows the vesicle-forming machinery to adapt to the increased degradative needs of the cell. We have analyzed the biosynthesis of Aut7p, a protein required for both pathways. We find Aut7p expression is induced by nitrogen starvation. Aut7p is degraded by a process dependent on both proteinase A and Cvt/autophagy components. Protease accessibility assays demonstrate that Aut7p is located within vesicles in strains defective in vesicle delivery or breakdown. Finally, the aut7/cvt5 mutant accumulates precursor API at a stage prior to vesicle completion. These data suggest that Aut7p is induced during autophagy and delivered to the vacuole together with precursor API by Cvt/autophagic vesicles.     

Cooperative binding of the cytoplasm to vacuole targeting pathway proteins,Cvt13 and Cvt20, to phosphatidylinositol 3-phosphate at the pre-autophagosomal structure is required for selective autophagy

Autophagy is a catabolic membrane-trafficking mechanism involved in cell maintenance and development. Most components of autophagy also function in the cytoplasm to vacuole targeting (Cvt) pathway, a constitutive biosynthetic pathway required for the transport of aminopeptidase I (Ape1). The protein components of autophagy and the Cvt pathway include a putative complex composed of Apg1 kinase and several interacting proteins that are specific for either the Cvt pathway or autophagy. A second required complex includes a phosphatidylinositol (PtdIns) 3-kinase and associated proteins that are involved in its activation and localization. The majority of proteins required for the Cvt and autophagy pathways localize to a perivacuolar pre-autophagosomal structure. We show that the Cvt13 and Cvt20 proteins are required for transport of precursor Ape1 through the Cvt pathway. Both proteins contain phox homology domains that bind PtdIns(3)P and are necessary for membrane localization to the pre-autophagosomal structure. Functional phox homology domains are required for Cvt pathway function. Cvt13 and Cvt20 interact with each other and with an autophagy-specific protein, Apg17, that interacts with Apg1 kinase. These results provide the first functional connection between the Apg1 and PtdIns 3-kinase complexes. The data suggest a role for PtdIns(3)P in the Cvt pathway and demonstrate that this lipid is required at the pre-autophagosomal structure.