ADR1 and CAT8 encode carbon source-responsive transcriptional regulators that cooperatively control expression of genes involved in ethanol utilization. These transcription factors are active only after the diauxic transition, when glucose is depleted and energy-generating metabolism has shifted to the aerobic oxidation of non-fermentable carbon sources. The Snf1 protein kinase complex is required for activation of their downstream target genes described previously. Using DNA microarrays, we determined the extent to which these three factors collaborate in regulating the expression of the yeast genome after glucose depletion. The expression of 108 genes is significantly decreased in the absence of ADR1. The importance of ADR1 during the diauxic transition is illustrated by the observation that expression of almost one-half of the 40 most highly glucose-repressed genes is ADR1-dependent. ADR1-dependent genes fall into a variety of functional classes with carbon metabolism containing the largest number of members. Most of the genes in this class are involved in the oxidation of different non-fermentable carbon sources. These microarray data show that ADR1 coordinates the biochemical pathways that generate acetylCoA and NADH from non-fermentable substrates. Only a small number of ADR1-dependent genes are also CAT8-dependent. However, nearly one-half of the ADR1-dependent genes are also dependent on the Snf1 protein kinase for derepression. Many more genes are SNF1-dependent than are either ADR1-or CAT8-dependent suggesting that SNF1 plays a broader role in gene expression than either ADR1 or CAT8. The largest class of SNF1-dependent genes encodes regulatory proteins that could extend SNF1 dependence to additional pathways.During the diauxic shift, when yeast cells deplete the glucose in the medium, the flow of metabolites changes dramatically to adapt to the use of alternative energy and carbon sources, primarily ethanol produced during fermentation. The changes in gene expression that are required for this metabolic reorganization were dramatically revealed by DNA microarray analysis (1). The expression of more than one-quarter of the yeast genome is altered when glucose is exhausted. These alterations allow the cell to utilize carbon sources other than glucose to channel metabolites into the tricarboxylic acid and glyoxylate cycles to generate energy and synthesize intermediates for gluconeogenesis. In addition, these changes allow the cell to alter its growth rate and prepare for growth arrest and stationary phase.Numerous transcription factors and regulatory proteins are responsible for the altered transcriptional program that accompanies the depletion of glucose. Two transcription factors that play important roles during the diauxic transition are Adr1 1 and Cat8.

Glutathione is the most abundant low molecular weight thiol in the eukaryotic cytosol. The compartment-specific ratio and absolute concentrations of reduced and oxidized glutathione (GSH and GSSG, respectively) are, however, not easily determined. Here, we present a glutathione-specific green fluorescent protein–based redox probe termed redox sensitive YFP (rxYFP). Using yeast with genetically manipulated GSSG levels, we find that rxYFP equilibrates with the cytosolic glutathione redox buffer. Furthermore, in vivo and in vitro data show the equilibration to be catalyzed by glutaredoxins and that conditions of high intracellular GSSG confer to these a new role as dithiol oxidases. For the first time a genetically encoded probe is used to determine the redox potential specifically of cytosolic glutathione. We find it to be −289 mV, indicating that the glutathione redox status is highly reducing and corresponds to a cytosolic GSSG level in the low micromolar range. Even under these conditions a significant fraction of rxYFP is oxidized.

In Saccharomyces cerevisiae, glucose depletion causes a profound alteration in metabolism, mediated in part by global transcriptional changes. Many of the transcription factors that regulate these changes act combinatorially. We have analyzed combinatorial regulation by Adr1 and Cat8, two transcription factors that act during glucose depletion, by combining genome-wide expression and genome-wide binding data. We identified 32 genes that are directly activated by Adr1, 28 genes that are directly activated by Cat8, and 14 genes that are directly regulated by both. Our analysis also uncovered promoters that Adr1 binds but does not regulate and promoters that are indirectly regulated by Cat8, stressing the advantage of combining global expression and global localization analysis to find directly regulated targets. At most of the coregulated promoters, the in vivo binding of one factor is independent of the other, but Adr1 is required for optimal Cat8 binding at two promoters with a poor match to the Cat8 binding consensus. In addition, Cat8 is required for Adr1 binding at promoters where Adr1 is not required for transcription. These data provide a comprehensive analysis of the direct, indirect, and combinatorial requirements for these two global transcription factors.Cells respond to stresses such as variations in temperature, pH or osmotic or nutrient conditions by altering the expression of genes that allow the cell to respond to the new environment (10). These transcriptional changes are initiated by signals that converge on a limited number of transcription factors, which then act combinatorially to control many genes in multiple pathways. Microarrays can be used to identify these coordinated, combinatorial responses by detecting both the localization of key transcription factors and the corresponding changes in the transcriptome (1).

Glutathione is present in millimolar concentrations in the cell, but its relative distribution among cellular compartments remains elusive. We have chosen the endoplasmic reticulum (ER) as an example organelle to study compartment-specific glutathione levels. Using a glutaredoxin sensor (sCGrx1pER), which rapidly and specifically equilibrates with the reduced glutathione (GSH)–glutathione disulfide (GSSG) redox couple with known equilibrium constant, we showed that the [GSH]:[GSSG] ratio in the ER of intact HeLa cells is less than 7:1. Taking into consideration the previously determined value for [GSH]2:[GSSG] in the ER of 83 mM, this translates into a total glutathione concentration in the ER ([GStot]=[GSH]+2[GSSG]) of greater than 15 mM. Since the integrated, intracellular [GStot] was measured as ~7 mM, we conclude the existence of a [GStot] gradient across the ER membrane. A possible homeostatic mechanism by which cytosol-derived glutathione is trapped in the ER is discussed. We propose a high [GStot] as a distinguishing feature of the ER environment compared to the extracellular space.