UC San Diego

Autophagy describes a conserved process that utilizes a double membrane in isolating cytoplasmic cargo for lysosomal degradation. Nutrient-regulation can be used to control the "off/on" state of the process. In yeast and mammals, the PI(3)P binding protein Atg18, has been determined to localize to the omegasome, isolation membranes and autophagosomes, while positively regulating autophagy. Drosophila melanogaster encodes for three Atg18 family homologues : Atg18, CG11975 and CG8678. Here, I explored their role in autophagy, by analyzing their protein sequences and phylogenetic relationships, autophagy associated phenotypes, and regulated cellular localization. I also investigated the possibility of Atg18, CG11975 or CG8678 being autophagy or PI(3)P markers. Here, I show that the D. melanogaster Atg18 family share conserved sequences and motifs. Phylogenetic analysis revealed close ancestry between the D. melanogaster, yeast and mammal Atg18 family. Overexpression and loss of function studies indicated that Atg18 positively regulates autophagy, and partially localizes to both acidified compartments and autophagosomes. In addition, overexpression of Atg18 induced lysosome acidification in larval fat body. In contrast, overexpression of CG11975 inhibited formation of acidified compartments and autophagosomes. CG11975 also showed low localization to acidified compartments and autophagosomes. Furthermore, coexpression of either Atg18 or CG11975 with the class II PI3-kinase Pi3K68D, mutually altered localizations, suggesting that both Atg18 and CG11975 can detect a Pi3K68D-specific PI(3)P pool. I also show that overexpression of CG8678 does not affect starvation- induced autophagy. These studies showing D. melanogaster Atg18s autophagy phenotypes and PI(3)P localization establish inroads for new markers and functional insights into phosphoinositide-dependent functions in autophagy