Vacuolar Phosphatidylinositol 3,4,5-trisphosphate controls fusion through binding Vam7, and membrane microdomain assembly

Membrane trafficking is regulated by phosphoinositides (PI) and their modification. The endolysosomal pathway is controlled by PI3P, PI(4,5)P ₂ and PI(3,5)P ₂ , whereas a role for PI(3,4,5)P ₃ is less clear. We report that yeast vacuoles produce PI(3,4,5)P ₃ through Vps34 activity. In vitro assays showed that dioctanoyl (C8) PI(3,4,5)P ₃ or the PI(3,4,5)P ₃ -binding domain Grp1-PH blocked fusion. Furthermore, modifying endogenous PI(3,4,5)P ₃ with the phosphatase PTEN abolished fusion. Fluorescence microscopy showed that PI(3,4,5)P ₃ was present at the plasma membrane and the vertex microdomains of vacuoles. PI(3,4,5)P ₃ staining was blocked by PTEN, C8-PI(3,4,5)P ₃ , the Vps34 inhibitor SAR405 and a VPS34 temperature sensitive mutation. Importantly, blocking or eliminating PI(3,4,5)P ₃ prevented the vertex enrichment of Ypt7 and the HOPS subunit Vps33. Finally, we show that the SNARE Vam7 binds PI(3,4,5)P ₃ and that both Grp1-PH and PTEN displaced it from membranes to block trans-SNARE pairing. Our results demonstrate that vacuolar PI(3,4,5)P ₃ coordinates vertex assembly and SNARE function.