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)P2 and PI(3,5)P2, whereas a role for PI(3,4,5)P3 is less clear. We report that yeast vacuoles produce PI(3,4,5)P3 through Vps34 activity. In vitro assays showed that dioctanoyl (C8) PI(3,4,5)P3 or the PI(3,4,5)P3-binding domain Grp1-PH blocked fusion. Furthermore, modifying endogenous PI(3,4,5)P3 with the phosphatase PTEN abolished fusion. Fluorescence microscopy showed that PI(3,4,5)P3 was present at the plasma membrane and the vertex microdomains of vacuoles. PI(3,4,5)P3 staining was blocked by PTEN, C8-PI(3,4,5)P3, the Vps34 inhibitor SAR405 and a VPS34 temperature sensitive mutation. Importantly, blocking or eliminating PI(3,4,5)P3 prevented the vertex enrichment of Ypt7 and the HOPS subunit Vps33. Finally, we show that the SNARE Vam7 binds PI(3,4,5)P3 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)P3 coordinates vertex assembly and SNARE function.