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.

Figure 1:

Fluoride toxicity in the fluoride sensitive FEX double knockout yeast. (A) Ratio of intracellular fluoride to extracellular fluoride measured by a fluoride electrode. (B) Cell viability over time, as measured by flow cytometry. Viability was determined as the percent of cells in the lower left gate after staining with propidium iodide and annexin V. (C) Measurement of intracellular ATP using cell titer glo and (D) mitochondrial activity as the incorporation of FUN-1 dye at 6 hours. (E) Fold change in the concentration of cytoplasmic calcium over time, assed using Indo-1. (F) RT-qPCR of oxidative stress response genes at 6 hours of growth. The change is compared with gene expression at 1-hour growth in YPD without fluoride and using actin for normalization.

Figure 2:

Overexpression of proteins conferring fluoride resistance. Serial dilutions of (A) FEX DKO or (B) wild type cells +/− pRS426 plasmids, tested for growth on YPD-agar supplemented with the designated concentrations of fluoride. On the right of the serial dilutions is the fold-change in fluoride resistance based on the change in IC₅₀’s. Liquid growth assay of (C) FEX DKO and (D) wild type cells +/− pRS426 plasmids over a 24-hour period in increasing fluoride. SSU1 and YHB1 plasmids also contain 1 kb of the corresponding gene’s native promoter.

Figure 3:

Nitrosative stress in fluoride-treated FEX DKO yeast. (A) Liquid growth assay of the combined effect on growth of yeast with fluoride and the IC₂₅ of nitrate (850 mM) or sulfite (20 mM). (B) Serial dilution of FEX DKO cells +/− pRS426 plasmids over a 24-hour period. For H. polymorpha genes SSU2 and NAR1, both plasmids contain the promoter region upstream of the S. cerevisiae SSU1 gene. (C) RT-qPCR of FZF1, YHB1, and SSU1 gene expression over time, compared with one-hour growth in YPD and using actin as a housekeeping gene. (D) Efflux of extracellular nitrate in cells over time. (E) Fold change in intracellular ions of FEX DKO + 50 μM NaF after 24 hours using ICP-MS. SO and PO are oxidized sulfur and phosphorous, respectively.

Figure 4:

Intracellular phosphate in FEX DKO with high copy plasmids. (A) ³¹P NMR spectra of yeast after 12 hours growth. The peaks are (from bottom to top) buffer alone, FEX DKO, and FEX DKO with a pRS426GPD promoter expressing PHO87, PHO90, or IPP1. (B) ³²PO₄ influx assay measuring the rate of phosphate import over 60 minutes. (C) Liquid growth assay of cells grown in synthetic minimal media over 24 hours in increasing fluoride, with the noted final concentration of phosphate present in the media. For all assays, buffer is kept at pH 6.5. (D) Fold change in the concentration of cytoplasmic calcium over time, assed using Indo-1.

Figure 5:

RNA-Seq analysis of FEX DKO treated with 50 μM NaF for four hours. (A) Functional classification graph of genes with more than 1.5-fold difference in expression compared with genes in FEX DKO grown in YPD for four hours. (B) Pie chart of the protein class and function, as mapped using the Saccharomyces Genome Database gene ontology slim mapper. (C) Fold-change in expression of genes from the list of hits linked to stress response and DNA repair, (D) metal homeostasis, (E) mitochondrial and endoplasmic reticulum stress, and (F) glucose starvation.

Figure 6:

³²P influx assay as a measurement of phosphate uptake over 60 minutes in 10 mM PO₄ buffer at pH 6.5. FEX DKO yeast were grown three hours in (A) YPD or (B) NaF before transferring to ³²PO₄ and increasing NaF.

Figure 7:

Assessment of phosphate influx as a function of pH. (A) Intracellular pH of FEX DKO cells after three hours growth in YPD or 50 μM NaF, as determined using 5(6)-CFDA. (B) Measurement of electrochemical potential of cells in either 3 hours exposure to fluoride (dashed line), or immediate exposure to NaF, CCCP (an agent that causes a rapid drop in pH_intra), or HCl (an agent that causes a rapid drop in pH_extra),. The (◆) indicates the point in which compounds were added for immediate exposure. (C) FEX DKO yeast grown for three hours in YPD, before transferring to buffer with pH from 5.5-7.5, using HCl and NaOH. (D) Cells grown in acetic acid for 30 minutes to pH_intra of 3.5-6.5, then transferred to buffer at pH 6.5 for measurement of ³²PO₄ uptake. For further information as to the protocol, see Experimental Procedures.