{"doi":"10.1101/2025.08.11.669746","title":"Early responses to hyperosmotic stress at the yeast vacuole","abstract":"ABSTRACT 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. Significance Statement --Adaptation to high salt involves early responses at organelle membranes and slower transcriptional responses. The vacuolar/lysosomal signaling lipid, PI(3,5)P2 is critical for the early response, but the timing, localization, and targets of salt-induced PI(3,5)P2 synthesis are not fully understood. --Experiments using Vph1NT-GFP as a low-affinity PI(3,5)P₂ biosensor suggest lipid synthesis occurs at a specific domain of the vacuolar membrane, with the level and duration of synthesis dependent on salt concentration and V-ATPase activity. A hog1Δ mutation ablates the slower response but elevates and extends PI(3,5)P2 activation. --Controlled PI(3,5)P2 synthesis at the vacuole supports V-ATPase-driven salt sequestration; long-term adaptation requires both V-ATPases and the HOG pathway.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":558764,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":1,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9441,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":291110,"name":"Patricia M. Kane","orcid":"0000-0003-4210-1187","position":1,"is_corresponding":false},{"id":1342566,"name":"Kalaivani Saravanan","orcid":null,"position":0,"is_corresponding":true}],"reference_count":71,"raw_metadata":null,"created_at":"2026-07-19T02:55:30.312295Z","pmid":"40832326","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}