A new class of mutants deficient in dodecamerization of aminopeptidase 1 and vacuolar transport

Vacuolar aminopeptidase 1 is transported to the vacuole by cytoplasmic double-membrane vesicles, the nonclassic Cvt pathway. The cytosolic protein dodecamerizes and is enclosed in a double-membrane vesicle, which is transported to and fuses with the vacuole releasing a single-membrane autophagic body into the vacuolar lumen. This is degraded and the precursor sequence of aminopeptidase 1 is removed. This pathway resembles autophagy, and most proteins identified to function in the Cvt pathway are also required for autophagy and vice versa. The cytosolic precursor protein and the matured vacuolar protein form a homododecameric complex, and only this complex has enzymatic activity. We developed a new genetic screen to isolate mutants in the biogenesis of vacuolar aminopeptidase 1 based on its enzymatic activity. The sensitivity of this assay made it possible for us to search for mutants under conditions where autophagy is down-regulated, and we describe two new mutants defective in the biogenesis pathway of vacuolar aminopeptidase 1. Mutants are defective in dodecamerization of pApe1p and in Cvt vesicle formation. Complex assembly and transport vesicle formation appear to be linked processes. This mechanism can control the potentially harmful cytoplasmic proteolytic activity and could be the driving force for this nonclassic mechanism of vacuolar enzyme transport.     

Vps51 is part of the yeast Vps fifty-three tethering complex essential for retrograde traffic from the early endosome and Cvt vesicle completion

Autophagy, pexophagy, and the Cvt pathway are processes that deliver hydrolytic enzymes and substrates to the yeast vacuole/lysosome via double-membrane cytosolic vesicles. Whereas these pathways operate under different nutritional conditions, they all employ common machinery with only a few specific factors assisting in the choice of the delivery program and the membrane source for the sequestering vesicle. We found that the YKR020w gene product is essential for Cvt vesicle formation but not for pexophagy or induction of autophagy. Autophagosomes in the ykr020wdelta mutant, however, have a reduced size. We demonstrate that Ykr020 is a subunit of the Vps fifty-three tethering complex, composed of Vps52, Vps53, and Vps54, which is required for retrograde traffic from the early endosome back to the late Golgi, and for this reason we named it Vps51. This complex participates in a fusion event together with Tlg1 and Tlg2, two SNAREs also shown to be necessary for Cvt vesicle assembly. In particular, those factors are essential to correctly target the prApe1-Cvt19-Cvt9 complex to the preautophagosomal structure, the site of Cvt vesicle formation.     

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.     

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.     

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.     

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.     

Atg27 is a second transmembrane cycling protein

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

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.     

Molecular mechanisms of autophagic peroxisome degradation in yeasts

Autophagy, Cvt pathway and pexophagy belong to membrane transport routes, which are able to enwrap into double-membrane vesicles and deliver to the vacuole various cytosolic material, including organelles. Pexophagy is a selective pathway of vacuolar degradation of redundant peroxisomes and can be induced by certain changes of carbon sources in yeasts. Here we review the most general molecular mechanisms of autophagic transport routes with a special emphasis on their features and functions in the yeast peroxisome degradation. Special attention has been also paid to differences in functioning of the basic autophagic machinery during micro- and macroautophagic peroxisome degradation in methylotrophic yeasts. The requirements of autophagic pathways for the sources of membrane for transport vesicle formation are also analyzed. Finally, we point to the gaps in our understanding of peroxisome degradation, which should be filled for complete integration of pexophagy into the network of autophagic transport routes to the vacuole in yeast.     

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.     

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.     

Atg27 is a Second Transmembrane Cycling Protein

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

Addendum to:

Atg27 is Required for Autophagy-Dependent Cycling of Atg9

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

Mol Biol Cell 2006; In press     

Membrane Delivery to the Yeast Autophagosome from the Golgi�CEndosomal System

While many of the proteins required for autophagy have been identified, the source of the membrane of the autophagosome is still unresolved with the endoplasmic reticulum (ER), endosomes, and mitochondria all having been evoked. The integral membrane protein Atg9 is delivered to the autophagosome during starvation and in the related cytoplasm-to-vacuole (Cvt) pathway that occurs constitutively in yeast. We have examined the requirements for delivery of Atg9-containing membrane to the yeast autophagosome. Atg9 does not appear to originate from mitochondria, and Atg9 cannot reach the forming autophagosome directly from the ER or early Golgi. Components of traffic between Golgi and endosomes are known to be required for the Cvt pathway but do not appear required for autophagy in starved cells. However, we find that pairwise combinations of mutations in Golgi-endosomal traffic components apparently only required for the Cvt pathway can cause profound defects in Atg9 delivery and autophagy in starved cells. Thus it appears that membrane that contains Atg9 is delivered to the autophagosome from the Golgi-endosomal system rather than from the ER or mitochondria. This is underestimated by examination of single mutants, providing a possible explanation for discrepancies between yeast and mammalian studies on Atg9 localization and autophagosome formation.     

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.     

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.     

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 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.     

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.     

Structure of yeast Ape1 and its role in autophagic vesicle formation

In Saccharomyces cerevisiae, a constitutive biosynthetic transport pathway, termed the cytoplasm-to-vacuole targeting (Cvt) pathway, sequesters precursor aminopeptidase I (prApe1) dodecamers in the form of a large complex into a Cvt vesicle using autophagic machinery, targeting it into the vacuole (the yeast lysosome) where it is proteolytically processed into its mature form, Ape1, by removal of an amino-terminal 45-amino acid propeptide. prApe1 is thought to serve as a scaffolding cargo critical for the assembly of the Cvt vesicle by presenting the propeptide to mediate higher-ordered complex formation and autophagic receptor recognition. Here we report the X-ray crystal structure of Ape1 at 2.5 Å resolution and reveal its dodecameric architecture consisting of dimeric and trimeric units, which associate to form a large tetrahedron. The propeptide of prApe1 exhibits concentration-dependent oligomerization and forms a stable tetramer. Structure-based mutagenesis demonstrates that disruption of the inter-subunit interface prevents dodecameric assembly and vacuolar targeting in vivo despite the presence of the propeptide. Furthermore, by examining the vacuolar import of propeptide-fused exogenous protein assemblies with different quaternary structures, we found that 3-dimensional spatial distribution of propeptides presented by a scaffolding cargo is essential for the assembly of the Cvt vesicle for vacuolar delivery. This study describes a molecular framework for understanding the mechanism of Cvt or autophagosomal biogenesis in selective macroautophagy.     

Proteinase protection of prApe1 as a tool to monitor Cvt vesicle/autophagosome biogenesis

Due in part to the increasing number of links between autophagy malfunction and human diseases, this field has gained tremendous attention over the past decade. Our increased understanding of the molecular machinery involved in macroautophagy (hereafter autophagy) seems to indicate that the most complex step, or at least the stage of the process where the majority of the autophagy-related (Atg) proteins participate, is in the formation of the double-membrane sequestering vesicle. Thus, it is important to establish reliable approaches to monitor this specific process. One of the most commonly used methods is morphological analysis by electron microscopy of the cytosolic vesicles used in the cytoplasm-to-vacuole targeting (Cvt) pathway and autophagy, or the single-membrane intralumenal products, termed Cvt or autophagic bodies, that are formed after the fusion of these vesicles with the yeast vacuole. This method, however, can be costly and time consuming, and reliable analysis requires expert input. Furthermore, it is extremely difficult to detect an incomplete autophagosome by electron microscopy because of the difficulty of obtaining a section that randomly cuts through the open portion of the phagophore. The primary Cvt pathway cargo, precursor amminopeptidase I (prApe1), is enwrapped within either a Cvt vesicle or autophagosome depending on the nutritional conditions. The proteolytic sensitivity of the prApe1 propeptide can therefore serve as a useful tool to determine the completion status of double-membrane Cvt vesicles/autophagosomes in the presence of exogenously added proteinase. Here, we describe an assay that examines the proteinase protection of prApe1 for determining the completion of Cvt vesicles/autophagosomes.