A proteome-integrated, carbon source dependent genetic regulatory network in Saccharomyces cerevisiae.

Integrated regulatory networks can be powerful tools to examine and test properties of cellular systems, such as modelling environmental effects on the molecular bioeconomy, where protein levels are altered in response to changes in growth conditions. Although extensive regulatory pathways and protein interaction data sets exist which represent such networks, few have formally considered quantitative proteomics data to validate and extend them. We generate and consider such data here using a label-free proteomics strategy to quantify alterations in protein abundance for S. cerevisiae when grown on minimal media using glucose, galactose, maltose and trehalose as sole carbon sources. Using a high quality-controlled subset of proteins observed to be differentially abundant, we constructed a proteome-informed network, comprising 1850 transcription factor interactions and 37 chaperone interactions, which defines the major changes in the cellular proteome when growing under different carbon sources. Analysis of the differentially abundant proteins involved in the regulatory network pointed to their significant roles in specific metabolic pathways and function, including glucose homeostasis, amino acid biosynthesis, and carbohydrate metabolic process. We noted strong statistical enrichment in the differentially abundant proteome of targets of known transcription factors associated with stress responses and altered carbon metabolism. This shows how such integrated analysis can lend further experimental support to annotated regulatory interactions, since the proteomic changes capture both magnitude and direction of gene expression change at the level of the affected proteins. Overall this study highlights the power of quantitative proteomics to help define regulatory systems pertinent to environmental conditions.

Energetic limits to metabolic flexibility: responses of Saccharomyces cerevisiae to glucose-galactose transitions.

Glucose is the favoured carbon source for Saccharomyces cerevisiae, and the Leloir pathway for galactose utilization is only induced in the presence of galactose during glucose-derepressed conditions. The goal of this study was to investigate the dynamics of glucose-galactose transitions. To this end, well-controlled, glucose-limited chemostat cultures were switched to galactose-excess conditions. Surprisingly, galactose was not consumed upon a switch to galactose excess under anaerobic conditions. However, the transcripts of the Leloir pathway were highly increased upon galactose excess under both aerobic and anaerobic conditions. Protein and enzyme-activity assays showed that impaired galactose consumption under anaerobiosis coincided with the absence of the Leloir-pathway proteins. Further results showed that absence of protein synthesis was not caused by glucose-mediated translation inhibition. Analysis of adenosine nucleotide pools revealed a fast decrease of the energy charge after the switch from glucose to galactose under anaerobic conditions. Similar results were obtained when glucose-galactose transitions were analysed under aerobic conditions with a respiratory-deficient strain. It is concluded that under fermentative conditions, the energy charge was too low to allow synthesis of the Leloir proteins. Hence, this study conclusively shows that the intracellular energy status is an important factor in the metabolic flexibility of S. cerevisiae upon changes in its environment.

When transcriptome meets metabolome: fast cellular responses of yeast to sudden relief of glucose limitation.

Within the first 5 min after a sudden relief from glucose limitation, Saccharomyces cerevisiae exhibited fast changes of intracellular metabolite levels and a major transcriptional reprogramming. Integration of transcriptome and metabolome data revealed tight relationships between the changes at these two levels. Transcriptome as well as metabolite changes reflected a major investment in two processes: adaptation from fully respiratory to respiro-fermentative metabolism and preparation for growth acceleration. At the metabolite level, a severe drop of the AXP pools directly after glucose addition was not accompanied by any of the other three NXP. To counterbalance this loss, purine biosynthesis and salvage pathways were transcriptionally upregulated in a concerted manner, reflecting a sudden increase of the purine demand. The short-term dynamics of the transcriptome revealed a remarkably fast decrease in the average half-life of downregulated genes. This acceleration of mRNA decay can be interpreted both as an additional nucleotide salvage pathway and an additional level of glucose-induced regulation of gene expression.