Production and characterization of fully selenomethionine-labeled Saccharomyces cerevisiae
Résumé
This paper reports, for the first time, a quantitative replacement of methionine (Met) by selenomethionine (SeMet) at >98% substitution, with up to 4940 μg of SeMet/g of yeast obtained for the entire protein pool of a wild-type yeast grown on a SeMet-containing medium. The incorporation of selenium in yeast proteins, in the form of selenomethionine, and the influence of various organic and inorganic Se and S sources present in the media were monitored during the growth of a wild-type Saccharomyces cerevisiae, which allowed the optimization of the composition of a fully defined synthetic growth medium that ensured maximum SeMet incorporation. Quantitation of SeMet and Met was performed by species-specific isotope dilution GC-MS. The use of ascorbic acid and a minimum concentration of cysteine (5 μg/L) was found to be beneficial to achieve incorporation by limiting the oxidative stress due to the presence of selenium. Except for small amounts of cysteine, no other sources of sulfur were necessary to achieve yeast growth. In a medium containing Se(VI), the maximum replacement of Met with SeMet was 50%, which is considerably higher than that obtained with the current commercial Se yeast formulations. For yeast grown in a Met-free defined medium, which was supplemented with SeMet, nearly total replacement of Met with SeMet could be achieved. The fully Se-labeled yeast could be an important tool for the study of eukaryotic protein structures both by mass spectrometry and by X-ray crystallography through selenomethionine single- and multiple-wavelength anomalous dispersion (SAD and MAD) phasing. In addition, a particular yeast strain, BY4741, that cannot synthesize Met using inorganic sulfur (met15Δ0) was shown to produce SeMet in the presence of inorganic selenium. This might indicate that the incorporation of inorganic selenium salts [Se(VI) and Se(IV)] is obviously not occurring exclusively through the same biological pathways as for sulfur. The reduction of inorganic Se to hydrogen selenide (H2Se), its reactions with organic compounds present in the yeast or in the media, and the possible metabolization through unspecific enzymatic pathways (such as transsulfuration) could also be of considerable importance in the production of selenoamino acids during yeast growth.