Targeting of aminopeptidase I to the yeast vacuole is mediated by Ssa1p, a cytosolic member of the 70-kDa stress protein family

The two cytosolic members of the highly conserved 70-kDa stress protein family, Ssa1p and Ssa2p, were specifically retained by the prepro-NH(2) extension of the vacuolar aminopeptidase I precursor (pAPI) conjugated to agarose (Sulfolink). A temperature-sensitive mutant strain a1(ts)a234 (ssa1(ts) ssa2 ssa3 ssa4), when incubated at the restrictive temperature, was able to assemble the API precursor into dodecamers, but failed to pack pAPI into vesicles and to convert it into mature API (mAPI), a process that occurs in the vacuole. Altogether these results indicate that Ssa1p mediates the targeting of pAPI to the vacuole.     

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 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.     

Phosphatidylethanolamine, a limiting factor of autophagy in yeast strains bearing a defect in the carboxypeptidase Y pathway of vacuolar targeting

Vps4p and Vps36p of Saccharomyces cerevisiae are involved in the transport of proteins to the vacuole via the carboxypeptidase Y pathway. We found that deletion of VPS4 and VPS36 caused impaired maturation of the vacuolar proaminopeptidase I (pAPI) via autophagy or the cytosol to vacuole targeting pathway. Supplementation with ethanolamine rescued this defect, leading to an increase of the cellular amount of phosphatidylethanolamine (PtdEtn), an enhanced level of the PtdEtn-binding autophagy protein Atg8p and a balanced rate of autophagy. We also discovered that maturation of pAPI was generally affected by PtdEtn depletion in a psd1Delta psd2Delta mutant due to reduced recruitment of Atg8p to the preautophagosomal structure. Ethanolamine supplementation provided the necessary amounts of PtdEtn for complete maturation of pAPI. Since the expression level of Atg8p was not compromised in the psd1Delta psd2Delta strain, we concluded that the amount of available PtdEtn was limiting. Thus, PtdEtn appears to be a limiting factor for the balance of the carboxypeptidase Y pathway and autophagy/the cytosol to vacuole targeting pathway in the yeast.     

Yeast aminopeptidase I is post-translationally sorted from the cytosol to the vacuole by a mechanism mediated by its bipartite N-terminal extension.

Transport of aminopeptidase I (API) to the vacuole appears to be insensitive to blockage of the secretory pathway. Here we show that the N-terminal extension of the 61 kDa precursor of API (pAPI) is proteolytically processed in two sequential steps. The first step involves proteinase A (PrA) and produces a 55 kDa unstable intermediate (iAPI). The second step involves proteinase B (PrB) and converts iAPI into the 50 kDa stable, mature enzyme (mAPI). Reversion of the cup1 growth phenotype by a pAPI-CUP1 chimera indicates that pAPI is transported to the vacuole by a post-translational mechanism. Deletion of the first 16 amino acids results in accumulation of the truncated protein in the cytosol, indicating that pAPI is actively transported to the vacuole. The chimera pAPI-myc, constructed by fusing a myc tag to the C-terminus of pAPI, was exploited to dissect the mechanism of pAPI transport. Cell fractionation studies show the presence of iAPI-myc and mAPI in a fraction of vacuoles purified by density centrifugation. This and the sequential conversion of pAPI-myc into iAPI-myc and mAPI lacking the myc tag is consistent with insertion of pAPI into the vacuolar membrane through its N-terminal extension. The specific mechanism of API sorting demonstrates a new pathway of protein transport in vacuolar biogenesis.     

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.     

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.     

Autophagy and the cytoplasm to vacuole targeting pathway both require Aut10p.

We here report the identification of AUT10 as a novel gene required for both the cytoplasm to vacuole targeting of proaminopeptidase I and starvation-induced autophagy. aut10Delta cells are impaired in maturation of proaminopeptidase I under starvation and non-starvation conditions. A lack of Aut10p causes a defect in autophagy prior to vacuolar uptake of autophagosomes. Homozygous aut10Delta diploids do not sporulate. Vacuolar acidification indicated by accumulation of quinacrine is normal in aut10Delta cells and mature vacuolar proteinases are present. A biologically active Ha-tagged Aut10p, chromosomally expressed from its endogenous promoter, localizes in indirect immunofluorescence microscopy in the cytosol and on granulated structures, which appear clustered around the vacuolar membrane. This localization differs from known autophagy proteins.     

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.     

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.     

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.     

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.     

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.     

ATG23, a novel gene required for maturation of proaminopeptidase I, but not for autophagy

In rich media proaminopeptidase I is targeted to the vacuole via the Cvt pathway and during starvation via autophagy. We here identify Atg23 (Ylr431c), a protein of so far unknown function, as a novel component essential for proaminopeptidase I maturation under non-starvation conditions. Maturation of proaminopeptidase I takes place in starved atg23Delta cells. Selective vacuolar targeting of the autophagosomal marker GFP-Aut7 and the accumulation of autophagic bodies during starvation in the presence of phenylmethylsulfonyl fluoride suggest that autophagy occurs in atg23Delta cells but at a reduced rate. In atg23Delta cells mature vacuolar carboxypeptidase Y is present and accumulation of quinacrine suggests no significant defect in vacuolar acidification. Furthermore, growth of atg23Delta cells on nitrocellulose detects no significant secretion of carboxypeptidase Y.     

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.     

Mai1p is essential for maturation of proaminopeptidase I but not for autophagy

We here identify Mai1p, a homologue of the autophagy protein Aut10p, as a novel component essential for proaminopeptidase I (proAPI) maturation under non-starvation conditions. In mai1Delta cells mature vacuolar proteinases are detectable and vacuolar acidification is normal. In mai1Delta cells autophagy occurs, though at a somewhat reduced level. This is indicated by proAPI maturation during starvation and accumulation of autophagic bodies during starvation with phenylmethylsulfonyl fluoride. Homozygous diploid mai1Delta cells sporulate, but with a slightly reduced frequency. Biologically active Ha-tagged Mai1p, chromosomally expressed under its native promoter, is at least in part peripherally membrane-associated. In indirect immunofluorescence it localizes to the vacuolar membrane or structures nearby. In some cells Ha-tagged Mai1p appears concentrated at regions adjacent to the nucleus.     

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.     

Cytosolic Hsp70s are involved in the transport of aminopeptidase 1 from the cytoplasm into the vacuole

Eukaryotic 70 kDa heat shock proteins (Hsp70s) are localized in various cellular compartments and exhibit functions such as protein translocation across membranes, protein folding and assembly. Here we demonstrate that the constitutively expressed members of the yeast cytoplasmic Ssa subfamily, Ssa1/2p, are involved in the transport of the vacuolar hydrolase aminopeptidase 1 from the cytoplasm into the vacuole. The Ssap family members displayed overlapping functions in the transport of aminopeptidase 1. In SSAI and SSAII deletion mutants the precursor of aminopeptidase 1 accumulated in a dodecameric complex that is packaged in prevacuolar transport vesicles. Ssa1/2p was prominently localized to the vacuolar membrane, consistent with the role we propose for Ssa proteins in the fusion of transport vesicles with the vacuolar membrane.     

The Ccz1-Mon1 protein complex is required for the late step of multiple vacuole delivery pathways

Mon1 and Ccz1 were identified from a gene deletion library as mutants defective in the vacuolar import of aminopeptidase I (Ape1) via the cytoplasm to vacuole targeting (Cvt) pathway. The mon1Delta and ccz1Delta strains also displayed defects in autophagy and pexophagy, degradative pathways that share protein machinery and mechanistic features with the biosynthetic Cvt pathway. Further analyses indicated that Mon1, like Ccz1, was required in nearly all membrane-trafficking pathways where the vacuole represented the terminal acceptor compartment. Accordingly, both deletion strains had kinetic defects in the biosynthetic delivery of resident vacuolar hydrolases through the CPY, ALP, and MVB pathways. Biochemical and microscopy studies suggested that Mon1 and Ccz1 functioned after transport vesicle formation but before (or at) the fusion step with the vacuole. Thus, ccz1Delta and mon1Delta are the first mutants identified in screens for the Cvt and Apg pathways that accumulate precursor Ape1 within completed cytosolic vesicles. Subcellular fractionation and co-immunoprecipitation experiments confirm that Mon1 and Ccz1 physically interact as a stable protein complex termed the Ccz1-Mon1 complex. Microscopy of Ccz1 and Mon1 tagged with a fluorescent marker indicated that the Ccz1-Mon1 complex peripherally associated with a perivacuolar compartment and may attach to the vacuole membrane in agreement with their proposed function in fusion.     

The prepropeptide of vacuolar aminopeptidase I is necessary and sufficient to target the fluorescent reporter protein GFP to the vacuole of yeast by the Cvt pathway

We have studied the capacity of the prepro amino extension of vacuolar protease leucine aminopeptidase I (API) to target the fluorescent reporter protein GFP to the vacuole of yeast. The preproGFP chimera constructed by extending the amino end of GFP with the prepro-part of API is rapidly degraded in both wild-type WCG cells and WCG 11/21a cells deficient in the proteasome. In contrast, the chimera expressed in WCG-PP cells deficient in both proteasome activity and vacuolar proteinase A accumulates in the vacuole, where it remains stable. Replacement of Gly by Ile-7, a substitution that prevents folding of the pre-part into an amphipathic helix and inhibits the targeting of the API precursor to the vacuole, inhibits the targeting of preproGFP to the vacuole. The separated pre- and pro-parts of the API precursor do not target GFP to the vacuole. Targeting of preproGFP to the vacuole is independent of its levels of expression, as the fluorescent protein localizes to the vacuole in cells expressing the protein under the control of both the GAL 1/10 or the API promoter. The preproGFP expressed under both promoters is recovered as monomers from cytosolic cell extracts. PreproGFP expressed under the API promoter is packed into cytoplasmic bodies that penetrate into the vacuolar lumen to release the protein. Altogether our results show that the prepro-part of the API precursor is necessary and sufficient to target the green fluorescent reporter protein to the vacuole.