Atg18 Regulates Organelle Morphology and Fab1 Kinase Activity Independent of Its Membrane Recruitment by Phosphatidylinositol 3,5-Bisphosphate

摘要

The lipid kinase Fab1 governs yeast vacuole homeostasis by generating PtdIns(3,5) P ₂ on the vacuolar membrane. Recruitment of effector proteins by the phospholipid ensures precise regulation of vacuole morphology and function. Cells lacking the effector Atg18p have enlarged vacuoles and high PtdIns(3,5) P ₂ levels. Although Atg18 colocalizes with Fab1p, it likely does not directly interact with Fab1p, as deletion of either kinase activator— VAC7 or VAC14 —is epistatic to atg18Δ: atg18 Δ vac7 Δ cells have no detectable PtdIns(3,5) P ₂. Moreover, a 2xAtg18 (tandem fusion) construct localizes to the vacuole membrane in the absence of PtdIns(3,5) P ₂, but requires Vac7p for recruitment. Like the endosomal PtdIns(3) P effector EEA1, Atg18 membrane binding may require a protein component. When the lipid requirement is bypassed by fusing Atg18 to ALP, a vacuolar transmembrane protein, vac14 Δ vacuoles regain normal morphology. Rescue is independent of PtdIns(3,5) P ₂, as mutation of the phospholipid-binding site in Atg18 does not prevent vacuole fission and properly regulates Fab1p activity. Finally, the vacuole-specific type-V myosin adapter Vac17p interacts with Atg18p, perhaps mediating cytoskeletal attachment during retrograde transport. Atg18p is likely a PtdIns(3,5) P ₂“sensor,” acting as an effector to remodel membranes as well as regulating its synthesis via feedback that might involve Vac7p.