Trs85 (Gsg1), a component of the TRAPP complexes, is required for the organization of the preautophagosomal structure during selective autophagy via the Cvt pathway.

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-to-Golgi and Golgi trafficking. Growing trs85delta 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 trs85delta 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 trs85delta 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.

Atg21 is required for effective recruitment of Atg8 to the preautophagosomal structure during the Cvt pathway.

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 atg21Delta cells fail to form proaminopeptidase I-containing Cvt vesicles. Quantitative measurement of autophagy in starving atg21Delta 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 atg21Delta ypt7Delta double mutant cells demonstrates the formation of normally sized autophagosomes with an average diameter of 450 nm.

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.

Yeast Mon1p/Aut12p functions in vacuolar fusion of autophagosomes and cvt-vesicles.

Here we identify Mon1p as being essential for the cvt-pathway and autophagy. Thus, mon1Delta cells are impaired in proaminopeptidase I maturation and homozygous diploid mon1Delta cells do not sporulate. Quantitative autophagy measurements suggest a complete autophagy block. The autophagosomal marker protein GFP-Aut7p accumulates in mon1Delta cells at punctate structures outside the vacuole. Furthermore, proaminopeptidase I accumulates in mon1Delta cells in a proteinase-protected form. Our data demonstrate that mon1Delta cells are defective in the fusion of cvt-vesicles and autophagosomes with the vacuole. Consistent with this, GFP-Mon1p localizes to the cytosol and to punctate structures within the cytosol.

Ccz1p/Aut11p/Cvt16p is essential for autophagy and the cvt pathway.

In a reverse genetics screen, we here identify Ccz1p as an essential component of the cvt pathway and autophagy. Ccz1p is identical with the so far unknown Cvt16p. GFP-Aut7p, a specific cargo of autophagosomes, accumulates in ccz1 Delta cells at punctate, vesicular structures in the cytosol, suggesting a block in the autophagic pathway prior to vacuolar fusion of autophagosomes. Proteinase protection experiments using hypotonically lysed ccz1 Delta spheroplasts demonstrate that proaminopeptidase I, another specific cargo of autophagy and the cvt pathway, is trapped inside membrane-enclosed vesicles. Taken together our findings are compatible with a function of Ccz1p in vacuolar fusion of cvt vesicles and autophagosomes.

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.