Uptake of selenite by Saccharomyces cerevisiae involves the high and low affinity orthophosphate transporters

Abstract

Although the general cytotoxicity of selenite is well established, the mechanism by which this compound crosses cellular membranes is still unknown. Here, we show that in Saccharomyces cerevisiae, the transport system used opportunistically by selenite depends on the phosphate concentration in the growth medium. Both the high and low affinity phosphate transporters are involved in selenite uptake. When cells are grown at low P(i) concentrations, the high affinity phosphate transporter Pho84p is the major contributor to selenite uptake. When phosphate is abundant, selenite is internalized through the low affinity P(i) transporters (Pho87p, Pho90p, and Pho91p). Accordingly, inactivation of the high affinity phosphate transporter Pho84p results in increased resistance to selenite and reduced uptake in low P(i) medium, whereas deletion of SPL2, a negative regulator of low affinity phosphate uptake, results in exacerbated sensitivity to selenite. Measurements of the kinetic parameters for selenite and phosphate uptake demonstrate that there is a competition between phosphate and selenite ions for both P(i) transport systems. In addition, our results indicate that Pho84p is very selective for phosphate as compared with selenite, whereas the low affinity transporters discriminate less efficiently between the two ions. The properties of phosphate and selenite transport enable us to propose an explanation to the paradoxical increase of selenite toxicity when phosphate concentration in the growth medium is raised above 1 mm.

FIGURE 1.

Selenite toxicity is dependent on phosphate concentration. A, cells (strain BY4742) were grown at 30 °C in phosphate-depleted SD medium supplemented with phosphate as follows: 0.1 mm (□, ■), 0.2 mm (△, ▴), 0.4 mm (○, ●), 0.8 mm (⧫, ◊), and 7.3 mm (×, *). The cultures were divided in two, and 5 mm Na₂SeO₃ was added to one sample (filled symbols). Cell growth was monitored by measuring the OD₆₅₀ at various times. B, cells (strain BY4742) were incubated in SD medium supplemented with the indicated phosphate concentration. When the OD₆₅₀ reached 0.1, Na₂SeO₃ was added to the cultures (0 mm, black bars; 5 mm, gray bars; 10 mm, white bars). After 1 h of incubation at 30 °C, the samples were diluted and plated onto YPD-agar. Cell viability was determined after 2 days of growth at 30 °C. The results are expressed as percentages of survival compared with control samples incubated in the absence of selenite. The error bars represent the means and ranges of two independent experiments.

FIGURE 2.

Selenite toxicity in mutants of the phosphate transport pathway. A, various strains, as indicated in the figure, were grown at 30 °C in SD medium supplemented with the indicated phosphate concentration. When the OD₆₅₀ reached 0.1, 5 mm Na₂SeO₃ was added to the cultures. After 1 h of incubation at 30 °C, the samples were diluted and plated onto YPD-agar. Cell viability was determined after 2 days growth at 30 °C. The results are expressed as percentages of survival compared with control samples incubated in the absence of selenite. The values are the means of at least three independent experiments. Standard deviations between these experiments were lower than 15%. B, cells expressing either the control plasmid pRS426 or pPHO84 expressing PHO84 from the ADH1 promoter were grown and treated as in A. The error bars represent the means and ranges of two independent experiments.

FIGURE 3.

Uptake of selenium by BY4742 cells grown in SD medium supplemented with 0.3 mm P_i. A, cells were harvested by centrifugation, washed, and resuspended in SD medium without phosphate. Uptake of 1 mm (□, ■), 2.5 mm (▴, △), 5 mm (○, ●), and 10 mm (⧫, ◊) Na₂SeO₃ was measured as described under “Experimental Procedures,” in the absence (filled symbols) or the presence (open symbols) of 0.5 mm potassium phosphate. The samples were analyzed for their total selenium content. B, rates of selenite uptake in the absence of phosphate (■), determined from A, were fitted to the Michaelis-Menten equation as described under “Experimental Procedures.” In the presence of 0.5 mm phosphate (▴), the rate of selenite uptake increased roughly linearly with the selenite concentration.

FIGURE 4.

Selenite toxicity in high P_i medium. The cells were incubated in SD medium. When the OD₆₅₀ reached 0.1, Na₂SeO₃ was added to the cultures (0 mm, black bars; 5 mm, gray bars; 10 mm, white bars). After 1 h of incubation at 30 °C, the samples were diluted and plated onto YPD-agar. Cell viability was determined after 2 days growth at 30 °C. The results are expressed as percentages of survival compared with control samples incubated in the absence of selenite. The error bars represent the means and ranges of two independent experiments. W.T, wild type.

FIGURE 5.

Sensitivity to selenite toxicity of a Δspl2 strain grown in low P_i medium. Strains BY4742 (■) and Δspl2 (□) were grown at 30 °C in SD medium supplemented with the indicated phosphate concentrations. When the OD₆₅₀ reached 0.1, 5 mm Na₂SeO₃ was added to the cultures. After 1 h of incubation at 30 °C, the samples were diluted and plated onto YPD-agar. Cell viability was determined after 2 days of growth at 30 °C. The results are expressed as percentages of survival compared with control samples incubated in the absence of selenite. The values are the means of at least three independent experiments. Standard deviations between these experiments were lower than 15%.