Jen1p: a high affinity selenite transporter in yeast

Abstract

Selenium is a micronutrient in most eukaryotes, including humans, which is well known for having an extremely thin border between beneficial and toxic concentrations. Soluble tetravalent selenite is the predominant environmental form and also the form that is applied in the treatment of human diseases. To acquire this nutrient from low environmental concentrations as well as to avoid toxicity, a well-controlled transport system is required. Here we report that Jen1p, a proton-coupled monocarboxylate transporter in S. cerevisiae, catalyzes high-affinity uptake of selenite. Disruption of JEN1 resulted in selenite resistance, and overexpression resulted in selenite hypersensitivity. Transport assay showed that overexpression of Jen1p enables selenite accumulation in yeast compared with a JEN1 knock out strain, indicating the Jen1p transporter facilitates selenite accumulation inside cells. Selenite uptake by Jen1p had a Km of 0.91 mM, which is comparable to the Km for lactate. Jen1p transported selenite in a proton-dependent manner which resembles the transport mechanism for lactate. In addition, selenite and lactate can inhibit the transport of each other competitively. Therefore, we postulate selenite is a molecular mimic of monocarboxylates which allows selenite to be transported by Jen1p.

Figure 1.

JEN1 expression leads to sensitive phenotype to selenite. Four yeast strains [BY4741 (WT), QZ1 (JEN1Δ), QZ1pDR196, and QZ1pJEN1] were grown in minimal SD medium containing either 2% glucose or galactose and supplied with the appropriate amino acids, and 10-fold serial dilutions were spotted onto 1.5% agar SD plates. Selenite was added to medium at final concentration of 50 or 200 μM, respectively. The plates were scanned and aligned for comparison.

Figure 2.

Detection of JEN1 expression in yeast membrane by immunofluorescence. Immunofluorescence staining of the four yeast strains from Figure 1 was performed using cells grown in SD medium containing either 2% glucose or galactose, as indicated. Images of bright field and immunofluorescence fields are shown side by side.

Figure 3.

Jen1p facilitates selenite uptake. Yeast strains BY4741 (●), QZ1 (○), QZ1pDR196 (▾), and QZ1pJEN1 (▵) were grown in minimal SD medium supplied with either 2% galactose or glucose to exponential phase (OD₆₀₀ ∼1.0). The cells were harvested and suspended in transport buffer. Sodium selenite was added to the yeast suspensions at a final concentration of 1 mM, and at the indicated times portions (0.1 ml) were passed though the membrane filters. The filters were digested, and total selenium was quantified by ICP-MS. Three repeats of each were used to derive the means and SEs.

Figure 4.

Jen1p catalyzes high-affinity uptake of both selenite and lactate. Uptake of selenite (A) or lactate (B) by strain QZ1pJEN1 was determined after 30 min, as described under Materials and Methods. The values from the deletion strain QZ1 were subtracted at each time point to correct for nonspecific binding. The K_m and V_max values were determined using a hypobolic fit of the data with SigmaPlot.

Figure 5.

(A) Selenite uptake as a function of pH. Selenite transport in QZ1pJEN1was assayed at the indicated pHs at a final concentration of 1 mM. (B) FCCP and nigericin inhibit selenite uptake. Cells of QZ1pJEN1 (black bar) were incubated with FCCP (100 μM) or nigericin (300 μM) for 5 min before addition of selenite at a final concentration of 1 mM, compared with QZ1 (white bar). Selenium accumulation was measured after 30-min incubation.

Figure 6.

Inhibition of selenite and lactate uptake by monocarboxylic acids. Uptake of 0.5 mM of either selenite (A) or lactate (B) by strain QZ1pJEN1 was determined after 30 min in the presence 5 mM of the indicated monocarboxylates, as described under Materials and Methods. The values from the deletion strain QZ1 were subtracted at each time point to correct for nonspecific binding.

Figure 7.

Inhibition kinetics between selenite and lactate. Uptake of indicated concentration of either selenite (A) or lactate (B) by strain QZ1pJEN1 was determined after 5 and 2 min in the presence of indicated concentration of lactate (A) or selenite (B), which is used as inhibitor. Each point represents three to four replications. Mean values and SEs were calculated from Sigma Plot 10.0. Each plot was linearized using Sigma Plot 10.0.

Figure 8.

Mercury and arsenite inhibit selenite and lactate transport via Jen1p. Uptake of 1 mM of either selenite (A) or lactate (B) by strain QZ1pJEN1 (black bar) was determined after 30 min in the presence of either 0.2 mM mercuric chloride (Hg²⁺) or 5 mM sodium arsenite final concentrations. The cells were preincubated with mercury or arsenite for 5 min before initiation of the assay. Strain QZ1 was used as a control (white bar).

Figure 9.

Selenite is an inorganic molecular mimic of monocarboxylates. Shown are the structures of (A) formate (PubChem CID: 284), (B) acetate (PubChem CID: 176), (C) pyruvate (PubChem CID: 450648), (D) lactate (PubChem CID: 612), and (E) selenite (Larsen and Søtofte Inger, 1971). The bond angles used to create the pdb files were derived from published data (PubChem data base) and the structures rendered with JMOL.