Roles and Regulation of the Lipid Kinase, Vps34
Steinfeld, Noah
2021
Abstract
Lipids serve as structural components of biological membranes. Additionally, a small subset of lipids serve as signaling molecules. Phosphoinositide (PPI) lipids are a class of lipids that are low in abundance, but nonetheless control a variety of signal transduction pathways and play crucial roles in cellular homeostasis. PPI species are generated by phosphorylation of phosphatidylinositol (PI) on the 3, 4, or 5 position of its inositol ring head group in every combination to yield a total of seven PPI species. The levels of these lipids are dynamically regulated by PPI lipid kinases and phosphatases that interconvert PPI lipids in response to stimuli. The generation of a specific PPI species on a membrane leads to the recruitment of distinct effector proteins that can then perform downstream functions. The dynamic regulation of PPI lipids suggests that in addition to being required for specific pathways, changes in PPI lipids may drive downstream processes. Most of the downstream functions of PPI lipids have been identified in studies that use knockout or knockdown of PPI kinases to deplete a specific PPI lipid and thereby test the necessity for that PPI species in a process. However, little is known about the impact of elevating phosphoinositides. Understanding the impact of elevating phosphoinositides is critical as the levels of PPI lipids can be dynamically elevated in response to stimuli. To test this hypothesis, we elevated phosphatidylinositol-3-phosphate (PI3P) levels by generating hyperactive alleles of the yeast phosphatidylinositol 3-kinase, Vps34. We find that hyperactive Vps34 has complex effects on cellular function. Hyperactive Vps34 drives phosphatidylinositol 3,5 bisphosphate (PI(3,5)P2) synthesis during hyperosmotic shock and retrograde transport from the vacuole. This demonstrates that elevating PI3P can accelerate some pathways and is rate limiting in those pathways. We also show that hyperactive Vps34 does not affect ESCRT (endosomal sorting complexes required for transport) function at endosomes or on the vacuole. Thus, elevating PI3P does not always increase the overall rate of a complex pathway. We also show that elevating PI3P can delay a pathway. Hyperactive Vps34 does not affect the induction of autophagy, but inhibits late steps in autophagy, in part via a delay in disassembly of the autophagy machinery from the surface of mature autophagosomes and also a delay in fusion of autophagosomes with the vacuole. This latter defect is likely due to a more general defect in vacuole fusion, as evidenced by an increase in the number of vacuole lobes per cell, which is consistent with a defect in homotypic vacuole fusion. Overall, these studies suggest that stimulus-induced elevation of PI3P levels regulates some, but not all, PI3P-dependent membrane trafficking pathways and that phosphoinositide lipids are commonly rate-limiting in pathways where they are required. Because the hyperactive Vps34 mutations we have identified are conserved in the mammalian Vps34 homolog PIK3C3, we think it is likely that these mutations in PIK3C3 will elevate PI3P. Thus, in the future, this work provides a roadmap to test the downstream effects of elevating PI3P in mammalian systems.Deep Blue DOI
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phosphoinositide signaling lipids PI3P Vps34 hyperactive mutant screen intracellular membrane trafficking autophagy
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