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Preservation of both the integrity and fluidity of biological membranes is a critical cellular homeostatic function. Signaling pathways that govern lipid bilayer fluidity have long been known in bacteria, yet no such pathways have been identified in eukaryotes. Here we identify mutants of the yeast Saccharomyces cerevisiae whose growth is differentially influenced by its two principal unsaturated fatty acids, oleic and palmitoleic acid. Strains deficient in the core components of the cell wall integrity (CWI) pathway, a MAP kinase pathway dependent on both Pkc1 (yeast's sole protein kinase C) and Rho1 (the yeast RhoA-like small GTPase), were among those inhibited by palmitoleate yet stimulated by oleate. A single GEF (Tus1) and a single GAP (Sac7) of Rho1 were also identified, neither of which participate in the CWI pathway. In contrast, key components of the CWI pathway, such as Rom2, Bem2 and Rlm1, failed to influence fatty acid sensitivity. The differential influence of palmitoleate and oleate on growth of key mutants correlated with changes in membrane fluidity measured by fluorescence anisotropy of TMA-DPH, a plasma membrane-bound dye. This work provides the first evidence for the existence of a signaling pathway that enables eukaryotic cells to control membrane fluidity, a requirement for division, differentiation and environmental adaptation. [ABSTRACT FROM AUTHOR]

Protons, the smallest and most ubiquitous of ions, are central to physiological processes. Transmembrane proton gradients drive ATP synthesis, metabolite transport, receptor recycling and vesicle trafficking, while compartmental pH controls enzyme function. Despite this fundamental importance, the mechanisms underlying pH homeostasis are not entirely accounted for in any organelle or organism. We undertook a genome-wide survey of vacuole pH (pHv) in 4,606 single-gene deletion mutants of Saccharomyces cerevisiae under control, acid and alkali stress conditions to reveal the vacuolar pH-stat. Median pHv (5.27±0.13) was resistant to acid stress (5.28±0.14) but shifted significantly in response to alkali stress (5.83±0.13). Of 107 mutants that displayed aberrant pHv under more than one external pH condition, functional categories of transporters, membrane biogenesis and trafficking machinery were significantly enriched. Phospholipid flippases, encoded by the family of P4-type ATPases, emerged as pH regulators, as did the yeast ortholog of Niemann Pick Type C protein, implicated in sterol trafficking. An independent genetic screen revealed that correction of pHv dysregulation in a neo1ts mutant restored viability whereas cholesterol accumulation in human NPC1-/- fibroblasts diminished upon treatment with a proton ionophore. Furthermore, while it is established that lumenal pH affects trafficking, this study revealed a reciprocal link with many mutants defective in anterograde pathways being hyperacidic and retrograde pathway mutants with alkaline vacuoles. In these and other examples, pH perturbations emerge as a hitherto unrecognized phenotype that may contribute to the cellular basis of disease and offer potential therapeutic intervention through pH modulation. [ABSTRACT FROM AUTHOR]