Nonclassical Protein Sorting to the Yeast Vacuole

Fungal cells spend most of their time in stationary phase (1Werner-Washburne M. Braun E.L. Crawford M.E. Peck V.M. Mol. Microbiol. 1996; 19: 1159-1166Crossref PubMed Scopus (182) Google Scholar, 2Werner-Washburne M. Braun E. Johnston G.C. Singer R.A. Microbiol. Rev. 1993; 57: 383-401Crossref PubMed Google Scholar), yet most research has focused on cells in logarithmic conditions. In the wild, these cells frequently encounter periods of limiting nutrient conditions. Accordingly, recycling, as opposed to simply degradation, of macromolecules is a routine and critical feature of cell biology. It is important to explore the ways in which cells adapt to environmental changes, including starvation, to understand cellular physiology. The basic processes of intracellular transport, as well as many of the specific components, are conserved among yeast and higher eukaryotes, making studies in this experimentally tractable organism relevant to other systems. The vacuole is the most prominent organelle in a yeast cell, and it plays a central role in cellular physiology (3Klionsky D.J. Herman P.K. Emr S.D. Microbiol. Rev. 1990; 54: 266-292Crossref PubMed Google Scholar). The vacuole is involved in cytosolic ion and pH homeostasis and is the main storage site for calcium and other divalent cations. Metabolites such as basic amino acids and polyphosphate, as well as toxic substances, are also stored within this organelle. In addition, the vacuole is important in osmoregulation, sporulation, and regulatory processes that require degradation. The best known role for this organelle is in protein turnover; the vacuole has a large number of membrane-bound and soluble hydrolases (4Van Den Hazel H.B. Kielland-Brandt M.C. Winther J.R. Yeast. 1996; 12: 1-16Crossref PubMed Scopus (145) Google Scholar). With regard to the topic of protein targeting, the vacuole is one of the most complex organelles in a eukaryotic cell. Numerous pathways are used to target both resident hydrolases and substrates destined for degradation to the vacuole (5Klionsky D.J. J. Membr. Biol. 1997; 157: 105-115Crossref PubMed Scopus (43) Google Scholar, 6Scott S.V. Klionsky D.J. Trends Cell Biol. 1997; 7: 225-229Abstract Full Text PDF PubMed Scopus (13) Google Scholar). The best characterized route for delivery of hydrolases to the vacuole is the secretory pathway. Nascent proteins transit from the endoplasmic reticulum to the Golgi complex, and following sorting events in the trans-Golgi network, the proteins are delivered to the vacuole through an endosomal intermediate (7Nothwehr S.F. Stevens T.H. J. Biol. Chem. 1994; 269: 10185-10188Abstract Full Text PDF PubMed Google Scholar, 8Stack J.H. Horazdovsky B. Emr S.D. Annu. Rev. Cell Dev. Biol. 1995; 11: 1-33Crossref PubMed Scopus (172) Google Scholar). A subset of proteins that transit to the vacuole via the secretory pathway, including alkaline phosphatase (ALP) 1The abbreviations used are: ALP, alkaline phosphatase; API, aminopeptidase I; ER, endoplasmic reticulum; AP, autophagosome; AB, autophagic body. and Vam3p, use a partially alternate route, which bypasses the endosome or prevacuolar compartment (9Piper R.C. Bryant N.J. Stevens T.H. J. Cell Biol. 1997; 138: 531-545Crossref PubMed Scopus (134) Google Scholar, 10Cowles C.R. Snyder W.B. Burd C.G. Emr S.D. EMBO J. 1997; 16: 2769-2782Crossref PubMed Scopus (174) Google Scholar, 11Cowles C.R. Odorizzi G. Payne G.S. Emr S.D. Cell. 1997; 91: 109-118Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 12Stepp J.D. Huang K. Lemmon S.K. J. Cell Biol. 1997; 139: 1761-1774Crossref PubMed Scopus (176) Google Scholar). Plasma membrane and cell surface proteins targeted for degradation are delivered to the vacuole by endocytosis (13Riezman H. Munn A. Geli M.I. Hicke L. Experientia. 1996; 52: 1033-1041Crossref PubMed Scopus (82) Google Scholar). Several other degradative pathways also exist. Macroautophagy is used for the turnover of bulk cytosol during starvation (14Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. J. Cell Biol. 1992; 119: 301-311Crossref PubMed Scopus (949) Google Scholar, 15Baba M. Takeshige K. Baba N. Ohsumi Y. J. Cell Biol. 1994; 124: 903-913Crossref PubMed Scopus (402) Google Scholar). The vacuolar import and degradation pathway targets at least the gluconeogenic enzyme fructose 1,6-bisphosphatase into the vacuole under conditions where its enzyme activity is no longer needed (5Klionsky D.J. J. Membr. Biol. 1997; 157: 105-115Crossref PubMed Scopus (43) Google Scholar, 16Hoffman M. Chiang H.-L. Genetics. 1996; 143: 1555-1566Crossref PubMed Google Scholar). Similarly, peroxisomes (17Titorenko V.I. Keizer I. Harder W. Veenhuis M. J. Bacteriol. 1995; 177: 357-363Crossref PubMed Scopus (93) Google Scholar, 18Tuttle D.L. Dunn Jr., W.A. J. Cell Sci. 1995; 108: 25-35Crossref PubMed Google Scholar) and mitochondria are selectively imported into the vacuole when environmental signals trigger a cascade leading to their degradation (5Klionsky D.J. J. Membr. Biol. 1997; 157: 105-115Crossref PubMed Scopus (43) Google Scholar). Mammalian cells also utilize both micro- and macroautophagic processes for the delivery of substrates to the lysosome (19Dunn W.A. Trends Cell Biol. 1994; 4: 139-143Abstract Full Text PDF PubMed Scopus (442) Google Scholar, 20Seglen P.O. Bohley P. Experientia. 1992; 48: 158-172Crossref PubMed Scopus (368) Google Scholar, 21Dice J.F. Trends Biochem. Sci. 1990; 15: 305-309Abstract Full Text PDF PubMed Scopus (526) Google Scholar). A specific process of lysosomal uptake has been described by Dice and colleagues (22Cuervo A.M. Dice J.F. Science. 1996; 273: 501-503Crossref PubMed Scopus (700) Google Scholar), who have identified a lysosomal surface receptor that is involved in recognition of proteins bearing a pentapeptide (KFERQ) motif. This import mechanism requires members of the hsp70 family including an intralysosomal hsc73 (23Agarraberes F.A. Terlecky S.R. Dice J.F. J. Cell Biol. 1997; 137: 825-834Crossref PubMed Scopus (249) Google Scholar). These data suggest the presence of protein translocation machinery in the lysosomal membrane, a property not generally ascribed to this organelle. In addition to the pathways mentioned above, other routes are used for protein delivery to the vacuole. One of the recently characterized mechanisms is the cytoplasm to vacuole targeting pathway used to deliver the resident hydrolase aminopeptidase I (API) to the vacuole (24Klionsky D.J. Cueva R. Yaver D.S. J. Cell Biol. 1992; 119: 287-299Crossref PubMed Scopus (305) Google Scholar). An examination of the biosynthesis of API revealed differences from the standard pattern seen with vacuolar proteins that transit through the secretory pathway. All of the characterized vacuolar hydrolases that transit through the ER and Golgi complex undergo proteolytic and/or glycosyl modifications during transit. Following the removal of cleavable signal sequences, these proteins receive a core glycosylation. Subsequent carbohydrate additions, upon delivery to and passage through the Golgi complex, are detected as an increase in molecular mass. Delivery to the vacuole is often accompanied by the removal of a propeptide segment. The half-time for vacuolar delivery through this route is 5–10 min for most of these proteins. In contrast to the secretory pathway proteins, API is not glycosylated even though it has N-linked glycosylation sites (24Klionsky D.J. Cueva R. Yaver D.S. J. Cell Biol. 1992; 119: 287-299Crossref PubMed Scopus (305) Google Scholar). It lacks a signal sequence or consensus signal sequence cleavage site (25Chang Y.-H. Smith J.A. J. Biol. Chem. 1989; 264: 6979-6983Abstract Full Text PDF PubMed Google Scholar,26Cueva R. Garcia-Alvarez N. Suarez-Rendueles P. FEBS Lett. 1989; 259: 125-129Crossref PubMed Scopus (46) Google Scholar). In accordance with the lack of a means to enter the secretory pathway, the precursor form of API is not found within the endoplasmic reticulum or Golgi complex but rather within the cytosol (24Klionsky D.J. Cueva R. Yaver D.S. J. Cell Biol. 1992; 119: 287-299Crossref PubMed Scopus (305) Google Scholar). API contains a propeptide at the N terminus that is removed in the vacuole by a proteinase B-dependent reaction. The half-time for propeptide processing, and presumably vacuolar delivery, is 30–40 min, substantially longer than that for proteins that utilize the secretory pathway. Similarly, the import of API is relatively insensitive tosec mutants, which were isolated based on defects in transit through the secretory pathway. In particular, Sec gene products that are specific to the early steps of the secretory pathway, ER to Golgi complex transport, are not required for API delivery to the vacuole (24Klionsky D.J. Cueva R. Yaver D.S. J. Cell Biol. 1992; 119: 287-299Crossref PubMed Scopus (305) Google Scholar). Many of the vps mutants that were isolated based on defects in vacuolar targeting of carboxypeptidase Y show essentially normal processing kinetics for API. Finally, unlike vacuolar proteins that traverse the secretory pathway, overexpression of API does not lead to secretion from the cell. These results indicate that API does not enter the vacuole through the secretory pathway but rather uses an alternate mechanism to attain its correct subcellular localization. Many vacuolar hydrolases are synthesized as zymogens containing a propeptide segment that keeps the enzyme inactive. The maintenance of a latent state may serve to protect the cell from the hydrolytic activity prior to the arrival of the enzyme in the vacuole. The propeptides of vacuolar hydrolases may also be involved in folding and/or targeting of the hydrolase (4Van Den Hazel H.B. Kielland-Brandt M.C. Winther J.R. Yeast. 1996; 12: 1-16Crossref PubMed Scopus (145) Google Scholar). The propeptides of proteinase A and carboxypeptidase Y have been shown to contain vacuolar targeting determinants; however, no consensus sequences have been defined for yeast vacuolar proteins. The API propeptide is composed of two α-helices (27Martinez E. Jimenez M.A. Segui-Real B. Vandekerckhove J. Sandoval I.V. J. Mol. Biol. 1997; 267: 1124-1138Crossref PubMed Scopus (17) Google Scholar). The first of these forms an amphipathic structure; unlike mitochondrial targeting signals, the charged half is composed of both basic and acidic residues. Site-specific mutagenesis was used to analyze the location of targeting information in the API propeptide (28Oda M.N. Scott S.V. Hefner-Gravink A. Caffarelli A.D. Klionsky D.J. J. Cell Biol. 1996; 132: 999-1010Crossref PubMed Scopus (77) Google Scholar, 29Segui-Real B. Martinez M. Sandoval I.V. EMBO J. 1995; 14: 5476-5484Crossref PubMed Scopus (31) Google Scholar). Small deletions anywhere within the first helix of the propeptide resulted in a complete block in targeting. Similar deletions within the second helix had no affect on targeting; in this case, the altered proteins were delivered to the vacuole with wild type kinetics. Deletion of the entire second helix, however, blocks API targeting suggesting some role for this part of the precursor protein in the import process. A series of random mutants was also generated within the API propeptide (28Oda M.N. Scott S.V. Hefner-Gravink A. Caffarelli A.D. Klionsky D.J. J. Cell Biol. 1996; 132: 999-1010Crossref PubMed Scopus (77) Google Scholar). All mutations that caused precursor accumulation mapped within the first helix. The precursor API was shown to reside in the cytosol in a protease-sensitive form. This result confirmed that the propeptide mutations caused a defect in targeting and not simply processing of API. Hence, the first helix of the API propeptide contains information that is necessary for delivery to the vacuole. The targeting information in the first and second helices is also sufficient for vacuolar localization; this segment of API can target a passenger protein to the vacuole. Hybrid proteins containing the N-terminal helices of API fused to the green fluorescent protein, are localized to the vacuole. 2I. Sandoval, personal communication. The API propeptide presumably interacts with subcellular sorting components such as a binding protein and/or receptor. To identify these sorting components, an in vitro system was established to reconstitute API import (30Scott S.V. Klionsky D.J. J. Cell Biol. 1995; 131: 1727-1735Crossref PubMed Scopus (30) Google Scholar). The import reaction is temperature-dependent, having an optimum at approximately 30 °C. In addition, import is inhibited both in vitro andin vivo at 14 °C. Inhibition at 14 °C suggests that import does not occur through a proteinaceous channel as in the ER, mitochondria, or chloroplasts; translocation in these organelles can occur at temperatures as low as 0 °C, as long as protein unfolding is not limiting. A block in transport at 14 °C suggests a vesicle-mediated event. The in vitro import reaction is also energy-dependent (30Scott S.V. Klionsky D.J. J. Cell Biol. 1995; 131: 1727-1735Crossref PubMed Scopus (30) Google Scholar). When import was carried out in the presence of non-hydrolyzable ATP or GTP analogs, or when ATP was depleted, processing of the API precursor was blocked. Inhibition of vacuolar ATPase activity with specific inhibitors or usingvma mutants resulted in reduced processing of API. In these cases, however, the block may have been indirect and reflect decreased access to the precursor protein (see below). The vacuolar localization of API was also analyzed through a classical genetic approach (31Harding T.M. Morano K.A. Scott S.V. Klionsky D.J. J. Cell Biol. 1995; 131: 591-602Crossref PubMed Scopus (397) Google Scholar, 32Harding T.M. Hefner-Gravink A. Thumm M. Klionsky D.J. J. Biol. Chem. 1996; 271: 17621-17624Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Because the mature form of API is stable in the vacuole lumen, wild type cells accumulate this form of the protein. To identify mutants in the import pathway, mutagenized yeast cells were screened for precursor accumulation. Mutants were obtained that are termed cvt, for cytoplasm to vacuole targeting defective. These mutants accumulate the precursor form of API but (for most) do not affect vacuolar delivery of proteins that transit through the secretory pathway. In all but two cvt mutants, precursor API is located in the cytosol, indicating that these mutants are blocked in targeting and do not deliver the precursor protein to the vacuole. To determine if these mutants are unique, complementation studies were carried out between cvt mutants and other mutant strains known to affect vacuolar protein delivery. The complementation studies revealed that some of the cvt mutants are probably allelic to certain vps mutants. This is expected because some of thevps mutants display major defects in vacuole morphology, in some cases lacking a detectable vacuole; these mutants do not present a proper target for API delivery. In addition, some of the components needed for API delivery may reach the vacuole through the secretory pathway. A more substantial overlap is detected between the cvtmutants and two groups of mutants that were isolated based on defects in macroautophagy, the apg and aut mutants (32Harding T.M. Hefner-Gravink A. Thumm M. Klionsky D.J. J. Biol. Chem. 1996; 271: 17621-17624Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 33Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1390) Google Scholar, 34Thumm M. Egner R. Koch M. Schlumpberger M. Straub M. Veenhuis M. Wolf D.H. FEBS Lett. 1994; 349: 275-280Crossref PubMed Scopus (479) Google Scholar, 35Scott S.V. Hefner-Gravink A. Morano K.A. Noda T. Ohsumi Y. Klionsky D.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12304-12308Crossref PubMed Scopus (213) Google Scholar). This overlap is surprising because there are substantial differences between the Cvt pathway and macroautophagy. For example, API import is kinetically slower than secretory pathway transit but is much faster than macroautophagy. Along these lines, macroautophagy also shows a lower yield of protein uptake. The Cvt pathway is biosynthetic, delivering a resident hydrolase to the vacuole. Accordingly, API import occurs during vegetative growth. In contrast, macroautophagy is a degradative pathway and, while occurring at a basal level in rich media, is induced under starvation conditions. Finally, in agreement with their respective roles in cellular physiology, Cvt import is specific for API and perhaps other hydrolases, whereas macroautophagy is nonspecific and is used to deliver bulk cytosol to the vacuole. To understand the molecular basis for the overlap between the Cvt and macroautophagic pathways, the native state of API was examined during the import process. The mature hydrolase was reported to be a dodecamer in the vacuole (36Metz G. Rohm K.-H. Biochim. Biophys. Acta. 1976; 429: 933-949Crossref PubMed Scopus (59) Google Scholar, 37Metz G. Marx R. Röhm K.H. Z. Naturforsch. Sect. C Biosci. 1977; 32: 929-937Crossref PubMed Scopus (29) Google Scholar, 38Löffler H.G. Röhm K.H. Z. Naturforsch. Sect. C Biosci. 1979; 34C: 381-386Crossref PubMed Scopus (9) Google Scholar). To determine when oligomerization occurs, and specifically if the precursor is a dodecamer, a kinetic analysis was carried out (39Kim J. Scott S.V. Oda M. Klionsky D.J. J. Cell Biol. 1997; 137: 609-618Crossref PubMed Scopus (116) Google Scholar). The results indicated that oligomerization into a dodecamer occurred rapidly, with a half-time of approximately 3 min. This is much shorter than the half-time of processing and indicates that oligomerization is not rate-limiting in the import process and occurs prior to vacuolar delivery. Oligomerization studies combined with subcellular fractionation showed that the oligomer forms in the cytosol; monomeric API can only be found in a soluble fraction whereas a low speed pellet fraction contains exclusively the dodecameric form. The transition from a soluble to sedimentable form may be indicative of binding to a target membrane and/or the formation of a pelletable complex. A temperature-sensitive API propeptide mutant was utilized to follow the import of API into the vacuole following the initial oligomerization event. An alteration of to at of the results in a temperature-sensitive targeting (28Oda M.N. Scott S.V. Hefner-Gravink A. Caffarelli A.D. Klionsky D.J. J. Cell Biol. 1996; 132: 999-1010Crossref PubMed Scopus (77) Google at the precursor API in the pelletable form. Because the is it can be used to follow the of import to the initial oligomerization reaction and binding event. of the mutant revealed that precursor API to and into the vacuole in the dodecameric form (39Kim J. Scott S.V. Oda M. Klionsky D.J. J. Cell Biol. 1997; 137: 609-618Crossref PubMed Scopus (116) Google Scholar). This translocation through a proteinaceous channel because the precursor is approximately in mass. This with the in vitro and genetic suggests that precursor API the vacuole through a vesicle-mediated process that is to macroautophagy. The process of macroautophagy had been characterized in yeast through studies and more recently by molecular and classical genetic (14Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. J. Cell Biol. 1992; 119: 301-311Crossref PubMed Scopus (949) Google Scholar, 15Baba M. Takeshige K. Baba N. Ohsumi Y. J. Cell Biol. 1994; 124: 903-913Crossref PubMed Scopus (402) Google Scholar, 33Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1390) Google Scholar). Macroautophagy with the formation of an membrane that this membrane forms a membrane that is termed an The of the membrane is not The that to be in suggests that may from a organelle such as a of the it is not that the necessary targeting components, needed for delivery to the reside in the membrane of this organelle. In addition, by (14Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. J. Cell Biol. 1992; 119: 301-311Crossref PubMed Scopus (949) Google Scholar) suggests that at least some of macroautophagy require protein Following targeting to the the membrane of the with the vacuole The machinery needed for targeting, and have not been well characterized but are to be to involved in processes in other of the cell J. Cell Biol. 1997; 139: PubMed Scopus Google Scholar, Z. A. E. W. J. Cell Biol. 1997; PubMed Scopus Google Scholar, A. W. EMBO J. 1996; 15: PubMed Scopus Google Scholar, A. T. W. EMBO J. 1995; 14: PubMed Scopus Google Scholar). For example, the vacuolar Vam3p, which is required for vacuole A. 1997; PubMed Scopus Google Scholar), is also needed for API delivery to the vacuole T. Emr S.D. J. Cell Biol. 1997; 138: PubMed Scopus Google Scholar). Along these lines, indicates a role for the protein and the in API Scott and J. of the with the vacuole the of the membrane into the vacuole This termed an autophagic is by vacuolar hydrolases, access to the strains in vacuolar hydrolase activity accumulate within the vacuole (14Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. J. Cell Biol. 1992; 119: 301-311Crossref PubMed Scopus (949) Google Scholar). of the is N. A. Y. Ohsumi Y. J. Biochem. 1997; PubMed Scopus Google Scholar). The vacuolar pH in mutants This may the block in API processing in the data on macroautophagy to API import specific the Cvt pathway. In particular, if API is imported by a process to macroautophagy, precursor API reside in the cytosol in a membrane to an Similarly, the precursor protein accumulate within membrane to in the vacuole in mutants that are in This for API import was mutants in of the localization process. The protein is part of a complex that is required for transport or the vacuole membrane Emr S.D. Mol. Biol. Cell. 1997; PubMed Scopus Google Scholar). A mutant precursor API S.V. Baba M. Ohsumi Y. Klionsky D.J. J. Cell Biol. 1997; 138: PubMed Scopus Google Scholar). studies showed that precursor API in the mutant in rich from a protease-sensitive to a with its within a membrane-bound fractionation studies that the precursor was not located within the with its in cytosolic The mutant precursor API within the vacuole. indicates that the mutant is in vacuolar API with in the of the vacuole membrane but not revealed that the precursor API be from vacuolar proteins S.V. Baba M. Ohsumi Y. Klionsky D.J. J. Cell Biol. 1997; 138: PubMed Scopus Google Scholar). studies confirmed that the precursor API is within a of proteins that these termed Cvt were from vacuole membrane These results that precursor API within both cytosolic and vacuolar The only for a to be from the of a cytosolic with the vacuole is for the cytosolic to be The of API during import and the of the transit were examined S.V. Baba M. Ohsumi Y. Klionsky D.J. J. Cell Biol. 1997; 138: PubMed Scopus Google Scholar, M. M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: PubMed Scopus Google Scholar). API is from the of the of a random it into are to be initial are of membrane or at least of a membrane The of these termed Cvt is not The Cvt are by membrane, in the formation of membrane Cvt API was also detected in in the vacuole in the These data of the and genetic data suggesting that API import occurs through a vesicle-mediated process. the data indicated a substantial overlap between the Cvt and macroautophagic pathways, there are To the two pathways a macroautophagic was is a resident vacuolar membrane protein that to the vacuole through a of the secretory pathway. is a type membrane protein with a propeptide that the vacuole and that is removed upon vacuolar delivery D.J. Emr S.D. EMBO J. 1989; PubMed Scopus Google Scholar). The N terminus of contains a that as an signal sequence that is needed to into the of a of the N terminus of including the generated a that is no longer to enter the secretory pathway T. A. Y. Ohsumi Y. Biochem. Biophys. 1995; PubMed Scopus Google Scholar). The only for to be delivered to the vacuole is by macroautophagy. delivery can be by following removal of the was used as a for macroautophagy and API as a for the Cvt pathway. vacuolar uptake of these two proteins under vegetative and starvation conditions revealed differences between the two pathways S.V. Hefner-Gravink A. Morano K.A. Noda T. Ohsumi Y. Klionsky D.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12304-12308Crossref PubMed Scopus (213) Google Scholar). API import is and complete under both conditions. The kinetics of import are essentially in rich or In contrast, uptake of does not occur in rich is into the vacuole upon of macroautophagy by The kinetics of however, are much slower than seen for API, and the yield of the import process is uptake of at approximately the conditions of the and the the Cvt pathway and macroautophagy are API import occurs under both vegetative and starvation conditions. import for API import under vegetative conditions. To the of the import process under starvation a analysis was M. M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: PubMed Scopus Google Scholar). API import was during a from vegetative to starvation conditions. vegetative precursor API in the form of Cvt was detected in Cvt as described These are approximately in and contain an core that to be from starvation, the Cvt are seen The of Cvt to decreased as starvation conditions The are substantially than Cvt having a of to In addition, the contain cytosol with the Cvt complex. These data suggest that API is imported into the vacuole by two processes that substantially overlap and that many components vegetative import occurs through the Cvt pathway. signals the for the turnover of cytosolic proteins. some Cvt are no longer synthesized or are to The mechanism is not known but may such as T. Ohsumi Y. J. Biol. Chem. 273: Full Text Full Text PDF PubMed Scopus Google Scholar). Because API uptake and complete under starvation specific machinery to uptake during It is to that API increase substantially during S.V. Hefner-Gravink A. Morano K.A. Noda T. Ohsumi Y. Klionsky D.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12304-12308Crossref PubMed Scopus (213) Google Scholar). The increase in API is by an increase in the import via the use of API is a critical hydrolase during starvation, the cell an of this enzyme by its biosynthesis to macroautophagic uptake. The and degradation of organelles and macromolecules are in part by the of such as or mitochondrial Similarly, peroxisomes in the presence of or These organelles are selectively delivered to the vacuole for degradation if or for or When yeast cells conditions of or starvation following in rich macroautophagic uptake of bulk cytosol is The turnover of macromolecules and organelles within the vacuole the cell with critical of membrane and are involved in these and degradation yet are not well