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.     

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.     

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.     

Cvt9/Gsa9 functions in sequestering selective cytosolic cargo destined for the vacuole

Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation. Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling. Most of the import machinery characterized thus far is required for all three modes of transport. However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection.We have identified Cvt9 and its Pichia pastoris counterpart Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy. These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.     

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.     

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.     

Yol082p, a novel CVT protein involved in the selective targeting of aminopeptidase I to the yeast vacuole

The yeast vacuolar enzyme aminopeptidase I (API) is synthesized in the cytoplasm as a precursor (pAPI). Upon its assembly into dodecamers, pAPI is wrapped by double-membrane saccular structures for its further transport within vesicles that fuse with the vacuolar membrane and release their content in the vacuolar lumen. Targeting of API to the vacuole occurs by two alternative transport routes, the cvt and the autophagy pathways, which although mechanistically similar specifically operate under vegetative growth or nitrogen starvation conditions, respectively. We have studied the role of Yol082p, a protein identified by its ability to interact with API, in the transport of its precursor to the vacuole. We show that Yol082p interacts with mature API, an interaction that is strengthened by the amino extension of the API protein. Yol082p is required for targeting of pAPI to the vacuole, both under growing and short term nitrogen starvation conditions. Absence of Yol082p does not impede the assembly of pAPI into dodecamers, but precludes the enclosure of pAPI within transport vesicles. Microscopy studies show that during vegetative growth Yol082p is distributed between a cytoplasmic pool and a variable number of 0.13--0.27-microm round, mobile structures, which are no longer observed under conditions of nitrogen starvation, and become larger in cells expressing the inactive Yol082 Delta C32p, or lacking Apg12p. In contrast to the autophagy mutants involved in API transport, a Delta yol082 strain does not lose viability under nitrogen starvation conditions, indicating normal function of the autophagy pathway. The data are consistent with a role of Yol082p in an early step of the API transport, after its assembly into dodecamers. Because Yol082p fulfills the functional requisites that define the CVT proteins, we propose to name it Cvt19.     

Apg13p and Vac8p are part of a complex of phosphoproteins that are required for cytoplasm to vacuole targeting

We have been studying protein components that function in the cytoplasm to vacuole targeting (Cvt) pathway and the overlapping process of macroautophagy. The Vac8 and Apg13 proteins are required for the import of aminopeptidase I (API) through the Cvt pathway. We have identified a protein-protein interaction between Vac8p and Apg13p by both two-hybrid and co-immunoprecipitation analysis. Subcellular fractionation of API indicates that Vac8p and Apg13p are involved in the vesicle formation step of the Cvt pathway. Kinetic analysis of the Cvt pathway and autophagy indicates that, although Vac8p is essential for Cvt transport, it is less important for autophagy. In vivo phosphorylation experiments demonstrate that both Vac8p and Apg13p are phosphorylated proteins, and Apg13p phosphorylation is regulated by changing nutrient conditions. Although Apg13p interacts with the serine/threonine kinase Apg1p, this protein is not required for phosphorylation of either Vac8p or Apg13p. Subcellular fractionation experiments indicate that Apg13p and a fraction of Apg1p are membrane-associated. Vac8p and Apg13p may be part of a larger protein complex that includes Apg1p and additional interacting proteins. Together, these components may form a protein complex that regulates the conversion between Cvt transport and autophagy in response to changing nutrient conditions.     

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.     

Cytoplasm to vacuole trafficking of aminopeptidase I requires a t-SNARE-Sec1p complex composed of Tlg2p and Vps45p.

Aminopeptidase I (API) is imported into the yeast vacuole/lysosome by a constitutive non-classical vesicular transport mechanism, the cytoplasm to vacuole targeting (Cvt) pathway. Newly synthesized precursor API is sequestered in double-membrane cytoplasmic Cvt vesicles. The Cvt vesicles fuse with the vacuole, releasing single-membrane Cvt bodies containing proAPI into the vacuolar lumen, and maturation of API occurs when the Cvt body is degraded, releasing mature API. Under starvation conditions, API is transported to the vacuole by macroautophagy, an inducible, non-selective mechanism that shares many similarities with the Cvt pathway. Here we show that Tlg2p, a member of the syntaxin family of t-SNARE proteins, and Vps45p, a Sec1p homologue, are required in the constitutive Cvt pathway, but not in inducible macroautophagy. Fractionation and protease protection experiments indicate that Tlg2p is required prior to or at the step of API segregation into the Cvt vesicle. Thus, the early Vps45-Tlg2p-dependent step of the Cvt pathway appears to be mechanistically distinct from the comparable stage in macroautophagy. Vps45p associates with both the Tlg2p and Pep12p t-SNAREs, but API maturation is not blocked in a pep12(ts) mutant, indicating that Vps45p independently regulates the function of multiple t-SNARES at distinct trafficking steps.     

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.     

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.     

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 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.     

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.     

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.     

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.     

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.     

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.     

Atg23 is essential for the cytoplasm to vacuole targeting pathway and efficient autophagy but not pexophagy

Cells must regulate both biosynthesis and degradation to ensure proper homeostasis of cellular organelles and proteins. This balance is demonstrated in a unique way in the yeast Saccharomyces cerevisiae, which possesses two distinct, yet mechanistically related trafficking routes mediating the delivery of proteins from the cytoplasm to the vacuole: the biosynthetic cytoplasm to vacuole targeting (Cvt) and the degradative autophagy pathways. Several components employed by these two transport routes have been identified, but their mechanistic interactions remain largely unknown. Here we report a novel gene involved in these pathways, which we have named ATG23. Atg23 localizes to the pre-auto-phagosomal structure but also to other cytosolic punctate compartments. Our characterization of the Atg23 protein indicates that it is required for the Cvt pathway and efficient autophagy but not pexophagy. In the absence of Atg23, cargo molecules such as prApe1 are correctly recruited to a pre-autophagosomal structure that is unable to give rise to Cvt vesicles. We also demonstrate that Atg23 is a peripheral membrane protein that requires the presence of Atg9/Apg9 to be specifically targeted to lipid bilayers. Atg9 transiently interacts with Atg23 suggesting that it participates in the recruitment of this protein.