Abstract
Vacuolar H(+)-ATPases (V-ATPases) acidify intracellular organelles and help to regulate overall cellular pH. Yeast vma mutants lack V-ATPase activity and allow exploration of connections between cellular pH, iron, and redox homeostasis common to all eukaryotes. A previous microarray study in a vma mutant demonstrated up-regulation of multiple iron uptake genes under control of Aft1p (the iron regulon) and only one antioxidant gene, the peroxiredoxin TSA2 (Milgrom, E., Diab, H., Middleton, F., and Kane, P. M. (2007) Loss of vacuolar proton-translocating ATPase activity in yeast results in chronic oxidative stress. J. Biol. Chem. 282, 7125-7136). Fluorescent biosensors placing GFP under transcriptional control of either an Aft1-dependent promoter (P(FIT2)-GFP) or the TSA2 promoter (P(TSA2)-GFP) were constructed to monitor transcriptional signaling. Both biosensors were up-regulated in the vma2Δ mutant, and acute V-ATPase inhibition with concanamycin A induced coordinate up-regulation from both promoters. PTSA2-GFP induction was Yap1p-dependent, indicating an oxidative stress signal. Total cell iron measurements indicate that the vma2Δ mutant is iron-replete, despite up-regulation of the iron regulon. Acetic acid up-regulated P(FIT2)-GFP expression in wild-type cells, suggesting that loss of pH control contributes to an iron deficiency signal in the mutant. Iron supplementation significantly decreased P(FIT2)-GFP expression and, surprisingly, restored P(TSA2)-GFP to wild-type levels. A tsa2Δ mutation induced both nuclear localization of Aft1p and P(FIT2)-GFP expression. The data suggest a novel function for Tsa2p as a negative regulator of Aft1p-driven transcription, which is induced in V-ATPase mutants to limit transcription of the iron regulon. This represents a new mechanism bridging the antioxidant and iron-regulatory pathways that is intimately linked to pH homeostasis.
Highlights
Loss of V-ATPase activity triggers oxidative stress and altered iron homeostasis
Tsa2p is not known to have a role in iron regulation, we show that its transcription responds to iron levels and that Tsa2p helps to limit Aft1p-driven up-regulation of the iron regulon
To assess if protein levels reflect the preferential transcriptional up-regulation of TSA2 in the vma mutant, a triple hemagglutinin tag was integrated at the C terminus of each peroxiredoxin
Summary
Loss of V-ATPase activity triggers oxidative stress and altered iron homeostasis. Results: The peroxiredoxin Tsa2p is induced when V-ATPase activity is lost and limits up-regulation of iron intake genes. The data suggest a novel function for Tsa2p as a negative regulator of Aft1p-driven transcription, which is induced in V-ATPase mutants to limit transcription of the iron regulon. This represents a new mechanism bridging the antioxidant and iron-regulatory pathways that is intimately linked to pH homeostasis. In an attempt to understand which of the defects in the vma mutant trigger the distinctive induction of TSA2 and the iron regulon, we generated GFP biosensors under control of the TSA2 promoter and Aft1p-dependent FIT2 promoter. Our data suggest that TSA2 overexpression in the vma mutant may be a unique, protective mechanism that bridges the antioxidant and iron homeostasis pathways
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