Insights into Lysosomal PI(3,5)P2 Homeostasis from a Structural-Biochemical Analysis of the PIKfyve Lipid Kinase Complex

The phosphoinositide PI(3,5)P-2, generated exclusively by the PIKfyve lipid kinase complex, is key for lysosomal biology. Here, we explore how PI(3,5)P-2 levels within cells are regulated. We find the PIKfyve complex comprises five copies of the scaffolding protein Vac14 and one copy each of the lipid kinase PIKfyve, generating PI(3,5)P-2 from PI3P and the lipid phosphatase Fig4, reversing the reaction. Fig4 is active as a lipid phosphatase in the ternary complex, whereas PIKfyve within the complex cannot access membrane-incorporated phosphoinositides due to steric constraints. We find further that the phosphoinositide-directed activities of both PIKfyve and Fig4 are regulated by protein-directed activities within the complex. PIKfyve autophosphorylation represses its lipid kinase activity and stimulates Fig4 lipid phosphatase activity. Further, Fig4 is also a protein phosphatase acting on PIKfyve to stimulate its lipid kinase activity, explaining why catalytically active Fig4 is required for maximal PI(3,5)P-2 production by PIKfyve in vivo.