Combined Proteome and Metabolite-profiling Analyses Reveal Surprising Insights into Yeast Sulfur Metabolism

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.Assimilable sulfur is essential for all living organisms. The cell requirement for sulfur can be fulfilled by the uptake of sulfur-containing amino acids or by assimilation of inorganic sulfur into organic compounds such as cysteine or homocysteine (1, 2). In yeast, homocysteine is the precursor of methionine through the methyl cycle and of cysteine through the transsulfuration pathway ( Fig. 1) (3). Cysteine is the sensor of the metabolic state in the sulfur amino acid pathway (4) and is required for the synthesis of GSH, an essential antioxidant molecule also important for detoxification.The yeast sulfur pathway has been extensively investigated at the genetic, enzymatic, and regulatory levels (3). The pools of most metabolites of the pathway have been analyzed (5, 6), and the K m values of many enzymes have been measured (3). However, some metabolic data such as the metabolite fluxes in the pathway and the concentration of the metabolites of the transsulfuration pathway (homocysteine and cystathionine) are lacking. Moreover, the levels of some sulfur metabolites are presumed to be modified in different mutants and under different physiological conditions (i.e. sulfur starvation, the presence of a sulfur metabolite, or a toxic metal in the medium), but the few quantitative data that are available are restricted to a small part of the pathway (5). Thus, it has been shown that cadmium (Cd 2ϩ ) strongly increases GSH synthesis (7), which is consistent with the primary impor...

Single yeast cells vary in transcription activity not in delay time after a metabolic shift

“…10B) decreases in a manner very similar to the nutrient downshift experiment (although the medium still contains high concentrations of methionine). A similar decrease has been observed previously by Lafaye et al 39 and is likely explained by a shift in the pools of sulphurcontaining metabolites after cadmium treatment to increase the GSH concentration.…”

“…This reduction is not enough to sustain measurable growth as the sulphur requirement of yeast is E90 nmol per mg dry weight 37 , indicating rapid sulphur limitation. Even if GSH reserves, with GSH concentrations about 10-fold higher than methionine concentrations 38,39 , would be used as a sulphur source this would not lead to significant growth. So, cells need to activate the sulphate assimilation pathway to synthesize methionine before they can continue growing.…”

The Yeast Genes ROX1, IXR1, SKY1 and Their Effect upon Enzymatic Activities Related to Oxidative Stress

“…In spite of the fact that metal detoxification requires GSH consumption, it is not probable that always metal treatment causes GSH depletion. The intracellular GSH concentration is in the millimolar range in yeast, whereas Cd (II) is toxic in the micromolar range (Lafaye et al, 2005). However, As (V) is toxic in the millimolar range and therefore it cannot be excluded that some metals could decrease the GSH pool to an extent where GSH-dependent enzyme activities, such as glutathione peroxidases, glutathione S-transferases and glutaredoxins, might be affected.…”

Dug1p Is a Cys-Gly Peptidase of the γ-Glutamyl Cycle of Saccharomyces cerevisiae and Represents a Novel Family of Cys-Gly Peptidases

“…Three independent replicates were made for each sample. The extraction of metabolites was adapted from a procedure described previously (27). Briefly, cells (4 -5 ϫ 10 8 cells) were collected by centrifugation (4000 rpm for 3 min at 4°C), washed once with cold water (4°C), and centrifuged again (4000 rpm for 3 min at 4°C).…”