Atg21 Is Required for Effective Recruitment of Atg8 to the Preautophagosomal Structure during the Cvt Pathway

Abstract

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...

Autophagy Competes for a Common Phosphatidylethanolamine Pool with Major Cellular PE-Consuming Pathways in Saccharomyces cerevisiae

“…This suppression also was observed by deleting ATG7 ( Figure 1B) or ATG14 ( Figure 1C), and as expected, the effect was lost after complementation of the deletions with plasmids expressing WT Mcd4, Atg7, and Atg14, respectively. In contrast, deletion of ATG21, a gene required for the selective cytoplasmto-vacuole targeting (Cvt) pathway (Meiling-Wesse et al 2004), was unable to rescue the lethality of mcd4-174 at the restrictive temperature ( Figure 1D). To examine whether the observed phenotype was a general feature of cells with perturbed Mcd4 function, we analyzed whether deletion of ATG7 was able to restore growth of mcd4D or cells harboring the mcd4-P301L allele (Storey et al…”

WIPI proteins: essential PtdIns3P effectors at the nascent autophagosome

“…However, both Atg18 and Atg21 enable the recruitment of Atg8 (LC3) to the nascent autophagosome (Meiling-Wesse et al, 2004;Nair et al, 2010).…”

Atg18 function in autophagy is regulated by specific sites within its β-propeller

“…Although Atg21 has a function in Atg8 association with the PAS rather than recruiting Atg2 to the same location (Meiling-Wesse et al, 2004;Strømhaug et al, 2004), we examined whether the Atg2-GFP localization observed in our atg18D background was due to Atg21 presence. As shown in Fig.…”