Selective disintegration of membrane-enclosed autophagic bodies is a feature of eukaryotic cells not studied in detail. Using a Saccharomyces cerevisiae mutant defective in autophagic-body breakdown, we identified and characterized Aut5p, a glycosylated integral membrane protein. Site-directed mutagenesis demonstrated the relevance of its putative lipase active-site motif for autophagic-body breakdown. aut5⌬ cells show reduced protein turnover during starvation and are defective in maturation of proaminopeptidase I. Most recently, by means of the latter phenotype, Aut5p was independently identified as Cvt17p. In this study we additionally checked for effects on vacuolar acidification and detected mature vacuolar proteases, both of which are prerequisites for autophagic-body lysis. Furthermore, biologically active hemagglutinin-tagged Aut5p (Aut5-Ha) localizes to the endoplasmic reticulum (nuclear envelope) and is targeted to the vacuolar lumen independent of autophagy. In pep4⌬ cells immunogold electron microscopy located Aut5-Ha at ϳ50-nm-diameter intravacuolar vesicles. Characteristic missorting in vps class E and fab1⌬ cells, which affects the multivesicular body (MVB) pathway, suggests vacuolar targeting of Aut5-Ha similar to that of the MVB pathway. In agreement with localization of Aut5-Ha at intravacuolar vesicles in pep4⌬ cells and the lack of vacuolar Aut5-Ha in wild-type cells, our pulse-chase experiments clearly indicated that Aut5-Ha degradation with 50 to 70 min of half-life is dependent on vacuolar proteinase A.

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. aut10v v 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 aut10v v diploids do not sporulate. Vacuolar acidification indicated by accumulation of quinacrine is normal in aut10v v 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. ß

Atg21 and Atg18 are homologue yeast proteins. Whereas Atg18 is essential for the Cvt pathway and autophagy, a lack of Atg21 only blocks the Cvt pathway. Our proteinase protection experiments now demonstrate that growing atg21⌬ cells fail to form proaminopeptidase I-containing Cvt vesicles. Quantitative measurement of autophagy in starving atg21⌬ cells showed only 35% of the wild-type rate. This suggests that Atg21 plays a nonessential role in improving the fidelity of autophagy. The intracellular localization of Atg21 is unique among the Atg proteins. In cells containing multiple vacuoles, Atg21-yellow fluorescent protein clearly localizes to the vertices of the vacuole junctions. Cells with a single vacuole show most of the protein at few perivacuolar punctae. This distribution pattern is reminiscent to the Vps class C(HOPS) (homotypic fusion and vacuolar protein sorting) protein complex. In growing cells, Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure. Consistently, the covalent linkage of Atg8 to the lipid phosphatidylethanolamine is significantly retarded. Lipidated Atg8 is supposed to act during the elongation of autophagosome precursors. However, despite the reduced autophagic rate and the retardation of Atg8 lipidation, electron microscopy of starved atg21⌬ ypt7⌬ double mutant cells demonstrates the formation of normally sized autophagosomes with an average diameter of 450 nm.Autophagy is a starvation-induced bulk flow transport pathway delivering cytosolic material, including whole organelles for breakdown to the lysosome (vacuole) (1-4). Autophagy recycles amino acids and provides the cells with energy, thus helping them to survive when external nutrient supplies are limited. The autophagic pathway is well conserved between mammals, plants, and yeasts. Over the last decade, pace-setting work in Saccharomyces cerevisiae led to the identification of more than 20 autophagy (ATG) genes (5). Based on the knowledge of the yeast ATG genes, not only mammalian homologues were identified (6), but also the importance of autophagy in the development of severe diseases such as cancer (7-9), cardiomyopathy (10), and Huntington's and Parkinson's disease (11-14) has been discovered. Increasing evidence also points to a relationship between autophagy and aging (15,16).Autophagy starts at the so-called preautophagosomal structure (PAS), 1 where numerous autophagy proteins colocalize (17, 18). Out of this structure, double membrane-layered autophagosomes filled with parts of the cytosol are formed (1,19,20). The outer membrane of the autophagosomes then fuses with the vacuole, and still membrane-bound autophagic bodies are released into the vacuolar lumen, where they are broken down.In S. cerevisiae, proaminopeptidase I (pAPI), a resident vacuolar proteinase, has been shown to be selectively targeted to the vacuole via autophagy using the receptor-like Atg19 protein (21,22). Once in the vacuole pAPI is proteolytically matured. Under nutrient-rich conditions, when autophagy is not in...

Autophagosomes and Cvt vesicles are limited by two membrane layers. The biogenesis of these unconventional vesicles and the origin of their membranes are hardly understood. Here we identify in Saccharomyces cerevisiae Trs85, a nonessential component of the TRAPP complexes, to be required for the biogenesis of Cvt vesicles. The TRAPP complexes function in endoplasmic reticulum-toGolgi and Golgi trafficking. Growing trs85⌬ cells show a defect in the organization of the preautophagosomal structure. Although proaminopeptidase I is normally recruited to the preautophagosomal structure, the recruitment of green fluorescent protein-Atg8 depends on Trs85. Autophagy proceeds in the absence of Trs85, albeit at a reduced rate. Our electron microscopic analysis demonstrated that the reduced autophagic rate of trs85⌬ cells does not result from a reduced size of the autophagosomes. Growing and starved cells lacking Trs85 did not show defects in vacuolar biogenesis; mature vacuolar proteinase B and carboxypeptidase Y were present. Also vacuolar acidification was normal in these cells. It is known that mutations impairing the integrity of the ER or Golgi block both autophagy and the Cvt pathway. But the phenotypes of trs85⌬ cells show striking differences to those seen in mutants with defects in the early secretory pathway. This suggests that Trs85 might play a direct role in the Cvt pathway and autophagy.Starvation-induced autophagy is an unselective, degradative pathway that delivers cytosolic material to the lysosome (vacuole) (1-3). It is well conserved between eukaryotes such as fungi, plants, and mammals. During the last decade work initially using the model eukaryote Saccharomyces cerevisiae led to the identification of a set of more than 20 ATG genes essential for the autophagic process (4 -7). Studies on the mammalian counterparts of the yeast ATG genes uncovered the importance of autophagy in the development of severe diseases such as cancer, cardiomyopathy, Huntington and Parkinson disease (8). Autophagy also plays an important role in the removal of intracellular pathogens (9), and increasing evidence points to a relationship between autophagy and aging (10,11).Autophagy starts at the preautophagosomal structure (PAS), 2 a perivacuolar organelle where numerous Atg proteins colocalize (12, 13). Out of the PAS double membrane-layered transport vesicles, the autophagosomes are formed (14, 15). The outer membrane of the autophagosome then fuses with the vacuolar membrane, and the inner part of the vesicle is released as a still membrane-limited autophagic body into the vacuole. Within the vacuole the autophagic bodies are lysed dependent on the putative lipase Atg15 (16), and the cytosolic material is degraded by the various vacuolar hydrolases. The use of transport vesicles limited by two membrane layers distinguishes autophagy from other transport pathways. Consistently, the molecular mechanisms used for the biogenesis of these vesicles are also unconventional. For example, the homotypic membrane fusion event during t...

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 mai1v v cells mature vacuolar proteinases are detectable and vacuolar acidification is normal. In mai1v v 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 mai1v v 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 membraneassociated. In indirect immunofluorescence it localizes to the vacuolar membrane or structures nearby. In some cells Hatagged Mai1p appears concentrated at regions adjacent to the nucleus. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.