Characterization of the PI(3,5)P2 production and turnover on endosomes and the vacuole in S. cerevisiae

Abstract

Eucaryotic cells are highly compartmentalized by lipid membranes that define organelles, creating specialized environments for distinct biological processes. Lysosomes in mammalian cells, are central organelles for the degradation and recycling of macromolecules as well as for the storage of ions, metabolites and amino acids. Proteins, lipids, carbohydrates reach the lysosome through cellular trafficking routes, such as endocytosis or autophagy. In the acidic lumen of lysosomes, macromolecules are digested by hydrolases for cellular reuse. Beyond these catabolic functions, lysosomes also play essential roles in growth regulation, by sensing the availability of nutrients, and osmotic adaptation by adjusting their membrane to maintain membrane integrity. In Saccharomyces cerevisiae, the model organism used in this thesis, growth signaling and stress response largely occur at the vacuole, the analogue of the mammalian lysosome. The vacuole harbors the nutrient sensing Target of Rapamycin complex 1 (TORC1) and the phosphatidylinositol 3-5 kinase Fab1, which converts Phosphatidylinositol-3-phosphate (PI(3)P) to Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2). PI(3,5)P2 is a signaling lipid that is essential for membrane remodeling and vacuole fission. Both TORC1 and Fab1 additionally localize to the vacuole-derived signaling endosomes (SEs), which function as secondary signaling platforms, where TORC1 downregulates catabolic processes. TORC1 dependent phosphorylation of Fab1 has been proposed to link vacuole membrane homeostasis to growth control. However, how TORC1 and Fab1 activities are functionally coupled remains unclear. In this study, we employed the newly developed PI(3,5)P2 reporter SnxA together with live-cell microscopy analysis to investigate subcellular dynamics of PI(3,5)P2 during stress and growth conditions. We identified two distinct pools of PI(3,5)P2 that operate in independent pathways. During osmotic stress response, Fab1 primarily acts on the vacuole in a TORC1-independent manner enabling rapid adaptation to hyperosmotic conditions. Under growth-promoting conditions, TORC1 together with the membrane remodeling CROP complex mediate the redistribution of PI(3,5)P2 from the vacuole to SEs. High levels of PI(3,5)P2 promoted the redistribution, indicating a direct role of PI(3,5)P2 in SE biogenesis. PI(3,5)P2 persists on SEs, potentially stabilized through a feedback loop between Fab1 and TORC1. PI(3,5)P2 is then channeled into the endocytic pathway, likely by endosome maturation or fusion with late endosomes, until it is delivered back to the vacuole, where it is degraded by the phosphatase Fig4