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Updated: Sep 12, 2025

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation
Published on: January 6, 2016
Vacuolar Phosphatidylinositol 3,4,5-trisphosphate controls fusion through binding Vam7, and membrane microdomain
Chi Zhang1,2, Jorge D Calderin1,2, Aliasgar Topiwalla1
1Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL.
Abstract:
Membrane trafficking is regulated by phosphoinositides (PI) and their modification by kinases, phosphatases, and phospholipases. The endolysosomal pathway is primarily 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 from PI(4,5)P2 through class III PI 3-kinase activity. In vitro assays showed that adding 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 also blocked fusion. Fluorescence microscopy showed that PI(3,4,5)P3 was enriched at membrane vertex microdomains, which was blocked by PTEN, C8-PI(3,4,5)P3, and the class III PI 3-kinase inhibitor SAR405. 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 soluble SNARE Vam7 binds PI(3,4,5)P3 and that PTEN abolished trans-SNARE pairing between partner vesicles. Together these data indicate that vacuolar PI(3,4,5)P3 coordinates the assembly of microdomains and SNARE function.
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