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Kane, Patricia
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Fall 2025
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2025-08-22
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V-ATPases are ubiquitous, highly conserved, and tightly regulated ATP-driven proton pumps that acidify organelles, including endosomes, the Golgi apparatus, secretory vesicles, and lysosomes. Especially during hyperosmotic stress in yeast, V-ATPase activation requires the vacuole-enriched signaling lipid PI(3,5)P2. However, the mechanism and dynamics of this stress response are poorly understood.
The yeast V-ATPase vacuolar a-subunit isoform (Vph1) interacts with the PI(3,5)P2 lipid 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 the low-level signaling lipid PI(3,5)P2 independently of other V-ATPase subunits. Under salt stress, PI(3,5)P2 levels transiently increase up to 20-fold within 5 minutes of salt exposure and rapidly return to the basal level. At the same time, Vph1NT-GFP rapidly moves from the cytosol to membranes upon the addition of salt and returns to the cytosol, similar to how the PI(3,5)P2 level increases and returns to the basal level. Using a microfluidic system, we observed that the extent and timing of Vph1NT-GFP recruitment depend on salt concentration. The Vph1NT-GFP recruitment is reduced in vac14Δ mutants, which have very low PI(3,5)P2 levels. The cells also display enlarged vacuoles similar to PI(3,5)P2-deficient cells, independent of salt stress, when Vph1NT is overexpressed, suggesting direct interactions between them. The intensity and duration of Vph1NT recruitment increase proportionally with salt concentration. Surprisingly, Vph1NT recruitment is comparatively higher in the presence of active V-ATPase, and it is also reversible. It repeatedly recruits to an exact subcellular location adjacent to the vacuole.
We propose that recruiting Vph1NT-GFP to membranes reflects stabilizing conformational changes when Vph1 binds to 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 longer-term transcriptional mechanisms are activated. Vph1NT-GFP recruitment is sustained longer in hog1Δ cells, which lack a parallel osmotic stress response pathway in yeast. These findings underscore the importance of PI(3,5)P2 interaction with V-ATPase in protecting cells during salt stress, especially when the transcriptional response is impaired.
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