The effect of phosphate accumulation on metal ion homeostasis in Saccharomyces cerevisiae

Abstract

Much of what is currently understood about the cell biology of metals involves their interactions with proteins. By comparison, little is known about metal interactions with intracellular inorganic compounds such as phosphate. Here we examined the role of phosphate in metal metabolism in vivo by genetically perturbing the phosphate content of Saccharomyces cerevisiae cells. Yeast pho80 mutants cannot sense phosphate and have lost control of phosphate uptake, storage and metabolism. We report here that pho80 mutants specifically elevate cytosolic and non-vacuolar phosphate and this in turn causes a wide range of metal homeostasis defects. Intracellular levels of the hard metal cations sodium and calcium increase dramatically, and cells become susceptible to toxicity from the transition metals manganese, cobalt, zinc and copper. Disruptions in phosphate control also elicit an iron starvation response, as pho80 mutants were seen to up-regulate iron transport genes. The iron responsive transcription factor Aft1p appears activated in cells with high phosphate in spite of normal intracellular iron levels. The high phosphate of pho80 mutants can be lowered by mutating Pho4p, the transcription factor for phosphate uptake and storage genes. Such lowering of phosphate by pho4 mutations reversed the high calcium and sodium of pho80 mutants and prevented the iron starvation response. However, pho4 mutations only partially reversed toxicity from heavy metals, representing a novel outcome of phosphate dysregulation. Overall these studies underscore the importance of maintaining a charge balance in the cell; a disruption in phosphate metabolism can dramatically impact on metal homeostasis.

Regulation of Manganese Antioxidants by Nutrient Sensing Pathways in Saccharomyces cerevisiae

“…Phosphate signaling through Pho4p is not responsible for the aerobic lethality of sod1D pho80D mutants A loss of phosphate control in S. cerevisiae through disruption of PHO80 or PHO85 renders sod1Δ cells inviable in air and unable to effectively utilize Mn for oxidative stress suppression (Reddi et al 2009;McNaughton et al 2010;Rosenfeld et al 2010). As seen in Figure 1A, Mn supplements to the growth medium only partly restore aerobic growth to a sod1Δ pho80Δ double mutant.…”

“…The limited efficacy of Mn in this regard might be explained by the intolerance of pho80 mutants to Mn toxicity ( Figure 1B). These mutants accumulate very high Mn ( Figure 1C, note log scale) due to uncontrolled uptake of Mn-Pi by the Pho84p transporter (Wykoff and O'shea 2001;Jensen et al 2003;Reddi et al 2009;Rosenfeld et al 2010). To remove effects of Mn toxicity, we disrupted PHO84, which reversed both the high Mn accumulation ( Figure 1C) and Mn sensitivity ( Figure 1B) of the sod1Δ pho80Δ mutant.…”

“…All experiments employed cells freshly obtained from frozen stocks and cultured on YPDE in oxygen-depleted, CO 2 -enriched culture jars (GasPak, BectonDickinson). Growth tests to assay oxygen resistance in SC complete medium were conducted under well-aerated (shaking at 220 rpm) conditions as described (Reddi et al 2009); tests for aerobic lysine auxotrophy and Mn toxicity were conducted under micro-aerobic conditions (not shaking) as described (Reddi et al 2009;Rosenfeld et al 2010).…”

Probing in vivo Mn2+speciation and oxidative stress resistance in yeast cells with electron-nuclear double resonance spectroscopy

“…To alter phosphate levels specifically, we targeted the Vph1p subunit of the vacuolar ATPase, needed for phosphate accumulation (4,25), and the Pho85p kinase, which negatively controls phosphate uptake and storage (25)(26)(27). As shown in Table 1, the resulting vph1 and pho85 strains respectively exhibit a fourfold decrease and an eightfold increase in cellular Pi, polyphosphate (pP), and total phosphates concentrations, with negligible changes in manganese concentrations.…”

Co-precipitation of Phosphate and Iron Limits Mitochondrial Phosphate Availability in Saccharomyces cerevisiae Lacking the Yeast Frataxin Homologue (YFH1)

“…Interestingly, Mir1 is also involved in protein import into mitochondria (24), and Pic2 can reversibly change its transport mode (antiport-uniport) according to the redox conditions (25). More generally, phosphate is involved in the homeostasis of several cations in different cellular compartments (26). Co-precipitation of phosphate with iron in ⌬yfh1 mitochondria is thus expected to have physiological implications.…”