pH homeostasis is critical in managing oxidative stress and iron metabolism

V‐ATPases are highly conserved proton pumps that acidify multiple organelles and contribute to overall cellular pH balance. Mutations in the yeast V‐ATPase ( vma mutants) result in an accumulation of reactive oxygen species and hypersensitivity to exogenous oxidative stress. Our earlier microarray screen revealed TSA2 as the only antioxidant gene to be upregulated in vma2 Δmutant. Multiple AFT1‐regulated genes were also upregulated, consistent with perturbed iron homeostasis in the vma2 Δmutant. This suggested a connection between pH, redox, and iron regulation. We developed GFP biosensors to monitor expression of iron‐dependent AFT1‐regulated genes and the redox regulator TSA2. Inhibition of V‐ATPase activity via the specific inhibitor concanamycin A in wild‐type cells resulted in coordinate upregulation of both AFT1‐ and TSA2‐driven biosensors within 30 minutes. A multi‐copy suppressor screen, to identify genes that suppress sensitivity of vma2Δ mutants to exogenous H 2 O 2 when overexpressed, identified lipid biosynthesis, DNA repair, and mitochondrial genes. These suppressors generally downregulated both biosensors, again suggesting tight coupling of pH, iron and redox control. Overexpression of the nuclear export gene, NUP60, suppressed H 2 O 2 sensitivity. NUP60 likely allows for increased protein levels of TSA2 by facilitating its mRNA export from the nucleus. In all, these suppressors are revealing connections between pH homeostasis, iron metabolism, and resistance to oxidative stress that are likely to be relevant in all eukaryotic cells. NIH R01 GM50322