Nitrate and Phosphate Transporters Rescue Fluoride Toxicity in Yeast

Abstract

Organisms are exposed to fluoride in the air, water, and soil. Yeast and other microbes utilize fluoride channels as a method to prevent intracellular fluoride accumulation and mediate fluoride toxicity. Consequently, deletion of fluoride exporter genes (FEX) in S. cerevisiae resulted in over 1000-fold increased fluoride sensitivity. We used this FEX knockout strain to identify genes, that when overexpressed, are able to partially relieve the toxicity of fluoride exposure. Overexpression of five genes, SSU1, YHB1, IPP1, PHO87, and PHO90, increase fluoride tolerance by 2- to 10-fold. Overexpression of these genes did not provide improved fluoride resistance in wild-type yeast, suggesting that the mechanism is specific to low fluoride toxicity in yeast. Ssu1p and Yhb1p both function in nitrosative stress response, which is induced upon fluoride exposure along with metal influx. Ipp1p, Pho87p, and Pho90p increase intracellular orthophosphate. Consistent with this observation, fluoride toxicity is also partially mitigated by the addition of high levels of phosphate to the growth media. Fluoride inhibits phosphate import upon stress induction and causes nutrient starvation and organelle disruption, as supported by gene induction monitored through RNA-Seq. The combination of observations suggests that transmembrane nutrient transporters are among the most sensitized proteins during fluoride-instigated stress.

Genome-Wide Identification of Genes Involved in General Acid Stress and Fluoride Toxicity in Saccharomyces cerevisiae

“…One particularly important stored ion is phosphate, which is used by the vacuole to sequester metals (Nguyen et al, 2019). High concentrations of stored phosphate have been demonstrated to resist oxidative stress in yeast (Lev et al, 2017;Johnston and Strobel, 2019). We assessed phosphate storage using 31 P NMR, and as expected, no vacuolar polyphosphate could be detected in V-ATPase mutants, and the overall concentration of intracellular phosphate was lower than wild type ( Figure 6C).…”

“…Each of these were not part of a conserved pathway of genes related to fluoride resistance, but most probably function in general stress resistance. Fluoride is well established to cause metal imbalance, DNA damage, oxidative stress, and metabolic arrest; expression of these genes would counteract these effects (He and Chen, 2006;Fina et al, 2014;Johnston and Strobel, 2019).…”

“…In vivo NMR was conducted with yeast grown to log phase, as outlined in Johnston and Strobel, 2019 .…”

“…Strongly acidic environments feature charged ions that cannot readily cross biological membranes without protein transporters. Consequently, exposure of fungi to highly acidic extracellular environments disrupts the electrochemical potential and function of the fungal plasma membrane (Carmelo et al, 1996;Russell and Gould, 2003;Johnston and Strobel, 2019). In contrast, exposure of fungi to weak acids can result in intracellular acidification (Mira et al, 2010b).…”

“…Our lab recently discovered that Saccharomyces cerevisiae lacking fluoride exporters undergoes cell cycle arrest at 50 μM NaF, well below the concentration required for any known metalloprotein inhibition ( Li et al, 2013 ). We consequently found that fluoride induced cytoplasmic acidosis in yeast, resulting in a disruption of membrane potential and nutrient uptake ( Johnston and Strobel, 2019 ). From this, the question becomes: What aspects of fluoride toxicity are the direct consequence of acid stress?…”