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.

In hair cells of the inner ear, robust /Hϩ exchange, which we propose is performed by NHE9 in hair bundles, exploits the high-K ϩ endolymph, responds only to pH imbalance across the bundle membrane, is unaffected by the ϩ80 mV endocochlear potential, and uses mechanisms already present in the ear for K ϩ recycling. This mechanism allows the hair cell to remove H ϩ generated by Ca 2ϩ pumping without ATP hydrolysis in the cell.

Background: Nhx1/Vps44 is proposed to be a Class E gene involved in formation of the multivesicular body (MVB). However, this hypothesis has not been tested. Results: Nhx1 is not required for cargo sorting or MVB formation and shows synthetic phenotypes with select ESCRT mutants. Conclusion: Nhx1 functions independently of the ESCRT pathway. Significance: Nhx1 may have a post-ESCRT role in endosomal membrane fusion.