Abstract 1385 Crosstalk between V-ATPase and PI(3,5)P2 lipid in Osmotic Stress. How, When, and Where? Kalaivani Saravanan and Patricia Kane Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, United States

V-ATPases are ubiquitous, conserved, and highly regulated, ATP-driven proton pumps that acidify organelles such as endosomes, Golgi, and lysosomes. V-ATPases in the lysosome-like vacuole participate in the response to many stress signals, and V-ATPase activation in response to osmotic stress requires the vacuole-enriched signaling lipid PI(3,5)P2. The mechanism and dynamics of this stress response are poorly understood. The yeast V-ATPase vacuolar a-subunit isoform (Vph1) interacts with PI(3,5)P2 via its cytosolic N-terminal domain (Vph1NT), and this interaction stabilizes and activates the V-ATPase. To better understand the dynamics of the V-ATPase-PI(3,5)P2 interaction, we utilized a Vph1NT-GFP fusion that interacts with PI(3,5)P2 lipid independent of other V-ATPase subunits1. Under salt stress, PI(3,5)P2 levels transiently increase up to 20-fold within 5 minutes of salt exposure and rapidly return to basal level2. At the same time, Vph1NT-GFP rapidly moves from the cytosol to membranes upon the addition of salt. Using a microfluidic system, we observe that the extent and timing of Vph1NT-GFP recruitment is dependent on salt concentration. Importantly, recruitment is compromised in vph1 mutants that do not bind PI(3,5)P2, and when Vph1NT is overexpressed, cells display morphological phenotypes (I.e. enlarged vacuoles) indicative of PI(3,5)P2 depletion. This suggests the direct interactions of Vph1NT directly interacts with PI(3,5)P2 and this interaction is independent of saltthe yeast cell vacuole exhibited PI(3,5)P2 deficient morphology. Interestingly, Vph1NT-GFP is consistently recruited to an area immediately adjacent to the vacuole. Recruitment is reversible, but upon washout and re-addition of salt, Vph1NT-GFP returns to the same location. We propose that the recruitment of Vph1NT-GFP to membranes reflects the stabilizing conformational changes that occur in response to Vph1 binding PI(3,5)P2 in the intact V-ATPase. These data suggest that osmotic stress may induce a short-term, localized activation of V-ATPase activity that could protect cells by driving vacuolar salt sequestration until transcriptional protection mechanisms can be activated. To support this data, when we expressed Vph1NT-GFP in the pho85D cells, in which signals upstream of PI(3,5)P2 synthesis are disrupted3. WWwe noted that Vph1NT-GFP recruitment with salt was much lower than in due to the low level of PI(3,5)P2 compared to the WT cells. In contrast, Vph1NT-GFP recruitment was sustained longer in the hog1D cells, which lack the parallel transcriptional pathway of the osmotic stress response in the yeast. These data highlight the importance of PI(3,5)P2-dependent V-ATPase activation in protecting the cells during osmotic stresswhen the downstream is compromised. In addition, the Vph1NT-GFP can provide new insights into the localization and duration of PI(3,5)P2 signals in vivo. 1. Li SC, Diakov TT, Xu T, Tarsio M, Zhu W, Couoh-Cardel S, Weisman LS, Kane PM. The signaling lipid PI(3,5)P₂ stabilizes V₁-V(o) sector interactions and activates the V-ATPase. Mol Biol Cell. 2014 Apr;25(8):1251-62. https://doi.org/10.1091/mbc. E13-10-0563. Epub 2014 Feb 12. PMID: 24523285; PMCID: PMC3982991. 2. Duex JE, Tang F, Weisman LS. The Vac14p-Fig4p complex acts independently of Vac7p and couples PI3,5P2 synthesis and turnover. J Cell Biol. 2006 Feb 27;172(5):693-704. https://doi.org/10.1083/jcb.200512105. Epub 2006 Feb 21. PMID: 16492811; PMCID: PMC2063702. 3. Jin N, Jin Y, Weisman LS. Early protection to stress mediated by CDK-dependent PI3,5P2 signaling from the vacuole/lysosome. J Cell Biol. 2017 Jul 3;216(7):2075-2090. https://doi.org/10.1083/jcb.201611144. Epub 2017 Jun 21. PMID: 28637746; PMCID: PMC5496620. Supported by NIH R35GM145256 to PMK.