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. 2017 Mar 17:7:44761.
doi: 10.1038/srep44761.

Exposure to selenomethionine causes selenocysteine misincorporation and protein aggregation in Saccharomyces cerevisiae

Pierre Plateau  1 , Cosmin Saveanu  2 , Roxane Lestini  3 , Marc Dauplais  1 , Laurence Decourty  2 , Alain Jacquier  2 , Sylvain Blanquet  1 , Myriam Lazard  1
Affiliations

Exposure to selenomethionine causes selenocysteine misincorporation and protein aggregation in Saccharomyces cerevisiae

Pierre Plateau et al. Sci Rep. .

Abstract

Selenomethionine, a dietary supplement with beneficial health effects, becomes toxic if taken in excess. To gain insight into the mechanisms of action of selenomethionine, we screened a collection of ≈5900 Saccharomyces cerevisiae mutants for sensitivity or resistance to growth-limiting amounts of the compound. Genes involved in protein degradation and synthesis were enriched in the obtained datasets, suggesting that selenomethionine causes a proteotoxic stress. We demonstrate that selenomethionine induces an accumulation of protein aggregates by a mechanism that requires de novo protein synthesis. Reduction of translation rates was accompanied by a decrease of protein aggregation and of selenomethionine toxicity. Protein aggregation was supressed in a ∆cys3 mutant unable to synthetize selenocysteine, suggesting that aggregation results from the metabolization of selenomethionine to selenocysteine followed by translational incorporation in the place of cysteine. In support of this mechanism, we were able to detect random substitutions of cysteinyl residues by selenocysteine in a reporter protein. Our results reveal a novel mechanism of toxicity that may have implications in higher eukaryotes.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. GO term analysis of the SeMet-sensitive and -resistant datasets.
(a) Distribution of SeMet-sensitive mutants according to biological processes affected (upper panel). Hierarchical graph of GO terms enrichment relative to the genome (lower panel). (b) Distribution of SeMet-resistant mutants according to biological processes affected (upper panel). Hierarchical graph of GO terms enrichment relative to the genome (lower panel). The color indicates the p-value of the enrichment according to g:Profiler (yellow: 10−1–10−3, orange: 10−3–10−6, light red: 10−6–10−10, dark red: <10−10). (c) Functional categories significantly enriched (p < 0.001, fold enrichment >2) in the SeMet- or H2Se-sentitive datasets. Only genes for which fitness scores were available in both screens were taken in consideration (137 and 135 genes in the SeMet and H2Se datasets, respectively). The color indicates the fold enrichment for each category. Blank boxes indicates an enrichment <2.
Figure 2
Figure 2. SeMet promotes protein aggregation in vivo.
(a) Induction of Hsp104-GFP by SeMet or heat shock. Exponentially growing BY4741-Hsp104-GFP cells were incubated in SC + 100 μM methionine for 2 h, either at 30 °C in the presence of the indicated concentration of SeMet or at 42 °C in the absence of SeMet. The fluorescence in whole cell extracts was recorded at 508 nm and normalized to the optical density of the extracts at 280 nm. The fluorescence of an extract from untagged BY4741 strain grown in SC + 100 μM methionine was subtracted from the results. The results are the mean ± S. D. of at least 3 experiments. (b) Hsp104-GFP localization was monitored by fluorescence microscopy (GFP) and differential interference contrast (DIC), in living BY4741-Hsp104-GFP cells after 1 h of exposure to various SeMet concentrations in SC + 100 μM methionine. Representative images obtained after maximum intensity z-projection, bar equals 10 μm. (c) Quantification of protein aggregation. The fraction of cells containing at least one Hsp104-GFP focus was determined by visual inspection of 300–600 cells in each condition. The results are the mean and range of at least 2 experiments. (d) Effect of cycloheximide (CHX) on protein aggregation induced by 1 h of exposure to 20 μM SeMet or heat shock at 42 °C. BY4741-Hsp104-GFP cells grown in SC + 100 μM methionine were treated or not with 5 μg/ml cycloheximide for 1 h prior to the SeMet or heat-shock stress. Hsp104-GFP localization was monitored in living cells by fluorescence microscopy. Representative images obtained after maximum intensity z-projection, bar equals 10 μm.
Figure 3
Figure 3. Reduced growth rates affect SeMet toxicity and protein aggregation.
(a) Relationship between SeMet resistance and growth in the absence of SeMet. Dots correspond to the growth fitness score in the absence of SeMet versus the relative fitness score in the presence of 20 μM SeMet, for strains resistant to SeMet (fitness score >1.5). (b) Effect of low cycloheximide concentrations on protein aggregation. Cycloheximide at the indicated concentrations was added to BY4741-Hsp104-GFP cells grown in SC + 100 μM methionine. After 1 h of incubation, 20 μM SeMet was added in the cultures and incubation was continued for 1 h. Hsp104-GFP localization was monitored in living cells by fluorescence microscopy. Representative images obtained after maximum intensity z-projection, bar equals 10 μm. (c) Generation times of BY4741 cells cultured in SC + 100 μM methionine at the indicated concentrations of cycloheximide in the absence (grey boxes) or presence (black boxes) of 20 μM SeMet. The results are the mean and range of at least 2 experiments. (d) Effect of reducing growth rates on SeMet toxicity and protein aggregation. The fraction of cells containing at least one Hsp104-GFP focus (⚫) was determined from the images in panel b, by visual inspection of 150–200 cells in each condition. SeMet growth inhibition (◼) was calculated as the log2(TSeMet/Tcont) value where TSeMet and Tcont are the generation times, calculated for each cycloheximide concentrations in panel c, in the presence and absence of SeMet, respectively. Values were plotted against the generation time in the absence of SeMet (Tcont). The results are the mean and range of at least 2 experiments.
Figure 4
Figure 4. The presence of CYS3 is required for SeMet-induced protein aggregation.
Hsp104-GFP localization in BY4741 and BY4741-∆cys3 cells after 1 h of exposure in SC + 100 μM methionine + 100 μM cysteine to 0, 20 μM and 50 μM SeMet, 100 μM D,L-SeCys in the presence of 1 mM TCEP or after 1 h heat shock at 42 °C was monitored in living cells by fluorescence microscopy. Representative images obtained after maximum intensity z-projection, bar equals 10 μm.
Figure 5
Figure 5. Compounds sharing significantly enriched GO terms with SeMet simultaneously in the sensitive and resistant datasets.
The 160 most sensitive, or 220 most resistant, genes corresponding to SeMet (this study), compounds n°3 (37 °C), 180 (CdCl2), 181 (ZnCl2), 182 (CoCl2), 184 (K2Cr2O7), 374 (Paraquat), 375 (MPP+) in ref. and compounds n°828 (Radicicol), and 4177 (Tunicamycin) in ref. were analyzed for simultaneous functional enrichment with the g:Profiler multiple gene lists tool (http://biit.cs.ut.ee/gprofiler/gcocoa.cgi). Only processes significantly enriched (p-value < 0.001) in the SeMet screen are represented. The number of genes annotated to the GO term in the input lists is indicated with a color code corresponding to the p-value of the enrichment. Abbreviations: BP, Biological process; CC, Cellular component.
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