Cytotoxic mechanism of selenomethionine in yeast

Abstract

Although selenium is an essential element, its excessive uptake is detrimental to living organisms. The significance of selenium for living organisms has been exploited for various purposes. However, the molecular basis of selenium toxicity is not completely understood. Here, we applied a capillary electrophoresis time-of-flight mass spectrometry-based metabolomics approach to analysis of yeast cells treated with selenomethionine. The data indicated that intracellular thiol compounds are significantly decreased, and diselenide and selenosulfide compounds are increased in selenomethionine-treated cells. The growth defect induced by selenomethionine was recovered by extracellular addition of cysteine and by genetic modification of yeast cells that have an additional de novo synthetic pathway for cysteine. Because cysteine is an intermediate of thiol compounds, these results suggested that the loss of a reduced form of thiol compounds due to selenomethionine causes a growth defect of yeast cells.

FIGURE 1.

Metabolic profile of SeMet-treated yeast. A, S. cerevisiae W303-1A strain was grown in SC-Met liquid medium at 30 °C until mid-log phase, and then SeMet was added (final concentration, 0.25 mm). The A₆₀₀ values of the cultures with (solid line) or without (dashed line) SeMet were plotted. B, metabolites extracted from SeMet-treated cells were analyzed using CE-TOFMS. Time-dependent changes in metabolite pools are mapped on the sulfur metabolic pathway. Metabolites whose levels changed significantly (p < 0.05) are indicated in bold frames. C, selenium compounds assigned based on their m/z values and isotopic patters are shown. These graphs are representative results of the assigned peaks (see also supplemental Table S3). The mean values of the relative intensity obtained from three independent experiments were plotted, and the vertical bars indicate S.E. (B and C).

FIGURE 2.

SeMet toxicity is suppressed by cysteine. The growth of wild-type cells at 30 °C in SC-Met medium containing both SeMet and cysteine (0 mm, circles; 0.05 mm, triangles; 0.2 mm, squares; 0.8 mm, diamonds) was monitored. The means of percentage growth normalized to growth without SeMet were plotted. Vertical bars indicate S.E. from triplicate experiments.

FIGURE 3.

Alternative pathway for de novo synthesis of cysteine. A, metabolic pathway of sulfur compounds in S. cerevisiae is shown (black arrows). Endogenous proteins involved in methionine synthesis (Met2p, Met17p, Met6p), the methyl cycle (Sam1p, Sam2p, Sah1p), and the transsulfuration pathway (Cys4p, Cys3p, Str2p, Str3p), are also shown. The de novo synthetic pathway of cysteine that was introduced in this study is indicated (gray arrows). CysE and CysK are E. coli enzymes that display serine acetyltransferase and O-acetylserine sulfhydrylase activities, respectively. B, cysteine auxotrophic mutant (cys3Δ::HIS3MX) was co-transformed with various combinations of multi-copy plasmids that express E. coli cysE (YEp351GAPII-cysE), cysK (YEp352GAPII-cysK), and parental plasmids (empty vectors, YEp351GAPII, and YEp352GAPII). Transformants were grown on an SC-Met/Leu/Ura plate with or without 1 mm cysteine at 30 °C for 2 days. C, yeast strains were grown in SC-Met/Leu/Ura at 30 °C until they reached mid-log phase, and metabolites were then extracted from 20 A₆₀₀ units of cells. Extracted cysteine, homocysteine, GSH, and CoA were labeled with a fluorogenic reagent (SBD-F) and were quantified. Data represent means ± S.E. (vertical lines) for three experiments.

FIGURE 4.

Effect of metabolic engineering of the cysteine synthetic pathway on SeMet sensitivity. TKY3030 (open circles), TKY3035 (open squares), and TKY4175 (closed squares) were grown at 30 °C for 24 h in SC-Met/Leu/Ura containing various concentrations of SeMet, and cell density was then monitored as in Fig. 2. A, comparison of TKY3030 with TKY3035 at concentrations of SeMet ranging from 0 to 4 μm. B, comparison of TKY3035 with TKY4175 at concentrations of SeMet ranging from 0 to 64 μm. C, intracellular levels of thiol compounds extracted from SeMet-treated cells. SeMet was added to the resting cultures of Fig. 3C to give a final concentration of 0.25 mm followed by incubation for 30 min. Extraction and quantification of thiol compounds were performed using the same procedures described in Fig. 3C. Data represent means ± S.E. (vertical lines) for three experiments.