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

Zhang, C.; Calderin, J. D.; Topiwalla, A.; Shah, V.; Karat, J. M.; Knapp, C. T.; Ahmed, R.; Grudzien, D.; Williamson, E. F.; Fratti, R. A.

doi:10.1101/2025.08.01.668199

bioRxiv · 10.1101/2025.08.01.668199

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

Zhang, C.·Calderin, J. D.·Topiwalla, A.·Shah, V.·Karat, J. M.·Knapp, C. T.·Ahmed, R.·Grudzien, D.·Williamson, E. F.·Fratti, R. A.

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Abstract

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 yeast produce PI(3,4,5)P3 through Vps34 activity. 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 at the plasma membrane and vacuoles, and that its detection was blocked by PTEN, C8-PI(3,4,5)P3, the Vps34 inhibitor SAR405 and a VPS34 temperature sensitive mutation. In addition, minimizing PI(4,5)P2 as a substrate for Vps34 with a MSS4 temperature sensitive mutation reduced PI(3,4,5)P3 levels. Importantly, PI(3,4,5)P3 was required for the vertex enrichment of Ypt7 and the HOPS subunit Vps33. Finally, we showed that the soluble SNARE Vam7 was retained in a PI(3,4,5)P3-dependent manner and that its displacement from membranes blocked trans-SNARE pairing. These results demonstrate that vacuolar PI(3,4,5)P3 coordinates vertex assembly and SNARE function. SummarySaccharomyces cerevisiae produces PI(3,4,5)P3 via Vps34 activity on vacuoles to regulate homotypic fusion. PI(3,4,5)P3 functions through controlling vertex microdomain assembly and retaining the soluble SNARE Vam7 on membranes to drive fusion.

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Zhang, C., Calderin, J. D., Topiwalla, A., Shah, V., Karat, J. M., Knapp, C. T., Ahmed, R., Grudzien, D., Williamson, E. F., Fratti, R. A.. 2025-08-02. Vacuolar Phosphatidylinositol 3,4,5-trisphosphate controls fusion through binding Vam7, and membrane microdomain assembly. https://doi.org/10.1101/2025.08.01.668199

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