Yeast PI(3,4,5)P3 controls vacuole fusion through Vam7 and membrane microdomain assembly

Membrane trafficking is regulated by the spatiotemporal distribution of phosphoinositides. 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 Saccharomyces cerevisiae produces PI(3,4,5)P3 through Vps34 activity using PI(4,5)P2 as a substrate. In vitro assays showed that dioctanoyl (C8) PI(3,4,5)P3, the PI(3,4,5)P3-binding domain Grp1-PH, and the phosphatase PTEN blocked vacuole fusion. Fluorescence microscopy showed that PI(3,4,5)P3 was present on vacuoles and the plasma membrane and that its presence was eliminated by inhibiting Vps34. Importantly, PI(3,4,5)P3 was required for the enrichment of Ypt7 and the HOPS subunit Vps33 at vertex domains. Blocking PI(3,4,5)P3 also caused the release of the soluble SNARE Vam7, leading to inhibited trans-SNARE pairing. Finally, we show that PI(3,4,5)P3 is needed for vacuole acidification in vivo. These results demonstrate that PI(3,4,5)P3 affects multiple factors in vacuole function.