Source data for Saravanan and Kane, "Early responses to hyperosmotic stress at the yeast vacuole"

In yeast, early adaptation to hyperosmotic stress involves organelle-based mechanisms, including synthesis of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂) at the vacuole. This low-level signaling lipid drives vacuolar fragmentation and activates the V-ATPase proton pump, which acidifies the vacuole and drives salt sequestration. The vacuole-resident V-ATPase subunit Vph1 interacts with PI(3,5)P₂ via its N-terminal domain (Vph1NT), directly linking lipid signaling to proton pump regulation. Under NaCl stress, PI(3,5)P₂ rapidly accumulates, triggering increased V-ATPase activity and vacuolar remodeling; these responses are impaired by deficient PI(3,5)P₂ synthesis. A Vph1NT-GFP fusion protein with no membrane domain is cytosolic without salt, but upon NaCl addition, rapidly relocalizes to a region adjacent to the vacuole in a PI(3,5)P2-dependent manner. The intensity and duration of this response depend on salt concentration. Vph1NT-GFP returns to the same location upon repeated salt challenge, suggesting that PI(3,5)P2 synthesis occurs at a localized domain/contact site. Disrupting PI(3,5)P₂ signaling, V-ATPase activity, or the high osmolarity glycerol pathway, which coordinates long-term transcriptional changes, compromises cellular adaptation to salt, underscoring the integration of lipid signaling and transcriptional regulation in hyperosmotic stress. These findings suggest activation of the V-ATPase, and possibly other targets, by PI(3,5)P2 synthesis provides immediate protection that primes cells for longer-term survival strategies.