Atg18 and Atg21 are homologous S. cerevisiae autophagy proteins. Atg18 is essential for biogenesis of Cvt vesicles and autophagosomes, while Atg21 is only essential for Cvt vesicle formation. We found that mutated Atg18-(FTTGT), which lost almost completely its binding to PtdIns3P and PtdIns(3,5)P 2 , is non-functional during the Cvt pathway but active during autophagy and pexophagy. Since the Cvt pathway does not depend on PtdIns(3,5)P 2 , we conclude that the Cvt pathway requires binding of Atg18 to PtdIns3P. Mutated Atg21-(FTTGT) is inactive during the Cvt pathway but showed only partly reduced binding to PtdIns-phosphates, suggesting further lipid binding domains in Atg21. GFP-Atg18-(FTTGT) and Atg21-(FTTGT)-GFP are released from vacuolar punctae to the cytosol.

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...