Abstract
Metabolomics is considered as an emerging new tool for functional proteomics in the identification of new protein function or in projects aiming at modeling whole cell metabolism. When combined with proteome studies, metabolite-profiling analyses revealed unanticipated insights into the yeast sulfur pathway. In response to cadmium, the observed overproduction of glutathione, essential for the detoxification of the metal, can be entirely accounted for by a marked drop in sulfur-containing protein synthesis and a redirection of sulfur metabolite fluxes to the glutathione pathway. A kinetic analysis showed sequential and dramatic changes in intermediate sulfur metabolite pools within the first hours of the treatment. Strikingly, whereas proteome and metabolic data were positively correlated under cadmium conditions, proteome and metabolic data were negatively correlated during other growth conditions, i.e. methionine supplementation or sulfate starvation. These differences can be explained by alternative mechanisms in the regulation of Met4, the activator of the sulfur pathway. Whereas Met4 activity is controlled by the cellular cysteine content in response to sulfur source and availability, the present study suggests that Met4 activation under cadmium conditions is cysteine-independent. The results clearly indicate that the metabolic state of a cell cannot be safely predicted based solely on proteomic and/or gene expression data. Combined metabolome and proteome studies are necessary to draw a comprehensive and integrated view of cell metabolism.
Highlights
Metabolomics is considered as an emerging new tool for functional proteomics in the identification of new protein function or in projects aiming at modeling whole cell metabolism
Pools of Sulfur Metabolites under Standard Growth Conditions—Due to their high polarity, sulfur metabolites were analyzed on a liquid chromatographic system based on “hydrophilic interaction liquid chromatography,” which is suitable for electrospray mass spectrometric detection (17, 18)
The intracellular concentrations of most sulfur metabolites were in the range 30 – 80 M except for homocysteine (Ͻ4 M), methionine, and GSH (ϳ2 mM)
Summary
Reagents—Homocysteine, methionine, cadmium chloride, and sulfide were from Sigma. Acetonitrile was from SDS (Peypin, France). [15N]ammonium sulfate was from Eurisotop (Gif-sur-Yvette, France). Strains and Growth Conditions—S. cerevisiae strain S288c (MAT␣ SUC2 mal mel gal CUP1) was grown at 30 °C in a minimum medium corresponding to yeast nitrogen base medium modified for sulfate concentration (1 mM) and for ammonium concentration (20 mM) In these growth conditions, the generation time is 2 h. Concerning cadmium treatments, cells were treated with 50 M Cd2ϩ and collected at the indicated times (0 h; 0 h, 30 min; 1 h, 10 min; 2 h; 3 h, 10 min; 4 h, 20 min; 6 h; 16 h; and 24 h) for extraction of metabolites. Cells were grown for more than 10 generations with 50 M sulfate and labeled for 1 h with 100 Ci [35S]methionine. The 35S-labeled metabolites, GSH, cystathionine, and ␥-Glu-Cys were quantified by PhosphorImager (Amersham Biosciences) as described previously (7)
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