In the yeast Saccharomyces cerevisiae, the interplay between Gal3p, Gal80p and Gal4p determines the transcriptional status of the genes needed for galactose utilization. The interaction between Gal80p and Gal4p has been studied in great detail; however, our understanding of the mechanism of Gal3p in transducing the signal from galactose to Gal4p has only begun to emerge recently. Historically, Gal3p was believed to be an enzyme (catalytic model) that converts galactose to an inducer or co‐inducer, which was thought to interact with GAL80p, the repressor of the system. However, recent genetic analyses indicate an alternative ‘protein–protein interaction model’. According to this model, Gal3p is activated by galactose, which leads to its interaction with Gal80p. Biochemical and genetic experiments that support this model provided new insights into how Gal3p interacts with the Gal80p–Gal4p complex, alleviates the repression of Gal80p and thus allows Gal4p to activate transcription. Recently, a galactose‐independent signal was suggested to co‐ordinate the induction of GAL genes with the energy status of the cell.

The transcriptional activation function of the Saccharomyces cerevisiae GAL4 protein is modulated by the GAL8O and GAI3 proteins. In the absence of galactose, GAL8O inhibits the function of GAL4, presumably by direct binding to the GALA protein. The presence of galactose triggers the relief of the GAL8O block. The key to this relief is the GA13 protein. How GAL3 and galactose activate GALA is not understood, but the long-standing notion has been that a galactose derivative formed by catalytic activity of GAL3 is the inducer that interacts with GAL8O or the GAL8O-GAL4 complex. Here we report that overproduction of the GALO protein causes constitutive expression of GAL/MEL genes in the absence of exogenous galactose. Overproduction of the GALl protein (galactokinase) also causes constitutivity, consistent with the observations that GALl is strikingly similar in amino acid sequence to GAL3 and has GAL3-like induction activity. Cells lacking the GAL10-encoded UDP-galactose-UDP-glucose epimerase retained the constitutivity response to overproduction of GAL3, making it unlikely that constitutivity is due to endogenously produced galactose. A galactoseindependent mechanism of constitutivity is further indicated by the inducing properties of two newly created galactokinaseless alleles of GALI. On the basis of these data, we propose a new model for galactose-induced activation of the GALA protein. This model invokes galactose-activation of the GAL3 and GALl proteins which in turn elicit an alteration of the GAL8O-GAL4 complex to activate GALA. This model is consistent with all the known features of the system and has important implications for manipulating GALA-dependent transcriptional activation in vitro.The GAL4 protein of the yeast Saccharomyces cerevisiae is a DNA-binding transcriptional activator of galactose pathway genes. It is part of a complex molecular switch that responds to carbon sources (16). In the absence of galactose, the transcription activation function of the GAL4 protein is blocked by its association with the GAL80 protein (8,18,21,22,26). The presence of galactose triggers a rapid relief of the GAL80 block, resulting in GAL4-mediated galactose pathway gene activation (16). The key to this rapid activation of GAL4 activity is the GAL3 protein (31, 33). Cells lacking the GAL3 protein and its counterpart activity in the GALl protein cannot activate GALA-mediated gene transcription in response to galactose (2,3,7,24,31). The long-standing model of GAL3 function holds that GAL3 catalyzes the conversion of galactose to an inducer molecule (2,3,7,16,24,31,32). On the basis of a variety of data, the likely target for the presumed inducer is thought to be the GAL8O protein (21,25,27,35).Recent efforts have been directed toward identifying the presumed galactose-derived inducer molecule, with the aim of elucidating the mechanism of the transcriptional switch. However, attempts by several investigators to identify a galactose-derived or galactose-dependent low-molecularweight inducer have been unsuccessful (8...

The nucleocytoplasmic shuttling of the repressor Gal80p is known to play a pivotal role in the signal transduction process of GAL genetic switch of Saccharomyces cerevisiae (Peng, G., and Hopper, J. E. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 8548 -8553). We have developed a comprehensive model of this GAL switch to quantify the expression from the GAL promoter containing one or two Gal4p-binding sites and to understand the biological significance of the shuttling process. Our experiments show that the expression of proteins from the GAL promoter containing one and two binding sites for Gal4p is ultrasensitive (a steep response to a given input). Furthermore, the model revealed that the shuttling of Gal80p is the key step in imparting ultrasensitive response to the inducer. During induction, free Gal80p concentration is altered by sequestration, without any change in the distribution coefficient across the nuclear membrane. Furthermore, the estimated concentrations of Gal80p and Gal3p allow basal expression of ␣-galactosidase, but not ␤-galactosidase, from the GAL promoter containing one and two binding sites for Gal4p, respectively. Conversely, the expression from genes with two binding sites is more sensitive to inducer concentration as compared with one binding site. We show that autoregulation of Gal80p is coincidental to the autoregulation of Gal3p, and it does not impart ultrasensitivity. We conclude from our analysis that the ultrasensitivity of the GAL genetic switch is solely because of the shuttling phenomena of the repressor Gal80p across the nuclear membrane.

The budding yeast, Saccharomyces cerevisiae, responds to various environmental cues by invoking specific adaptive mechanisms for their survival. Under nitrogen limitation, S. cerevisiae undergoes a dimorphic filamentous transition called pseudohyphae, which helps the cell to forage for nutrients and reach an environment conducive for growth. This transition is governed by a complex network of signaling pathways, namely cAMP-PKA, MAPK and TOR, which controls the transcriptional activation of FLO11, a flocculin gene that encodes a cell wall protein. However, little is known about how these pathways co-ordinate to govern the conversion of nutritional availability into gene expression. Here, we have analyzed an integrative network comprised of cAMP-PKA, MAPK and TOR pathways with respect to the availability of nitrogen source using experimental and steady state modeling approach. Our experiments demonstrate that the steady state expression of FLO11 was bistable over a range of inducing ammonium sulphate concentration based on the preculturing condition. We also show that yeast switched from FLO11 expression to accumulation of trehalose, a STRE response controlled by a transcriptional activator Msn2/4, with decrease in the inducing concentration to complete starvation. Steady state analysis of the integrative network revealed the relationship between the environment, signaling cascades and the expression of FLO11. We demonstrate that the double negative feedback loop in TOR pathway can elicit a bistable response, to differentiate between vegetative growth, filamentous growth and STRE response. Negative feedback on TOR pathway function to restrict the expression of FLO11 under nitrogen starved condition and also with re-addition of nitrogen to starved cells. In general, we show that these global signaling pathways respond with specific sensitivity to regulate the expression of FLO11 under nitrogen limitation. The holistic steady state modeling approach of the integrative network revealed how the global signaling pathways could differentiate between multiple phenotypes.

A suppressor of galactose toxicity in a gal7 yeast strain (lacking galactose 1-phosphate uridyl transferase) has been isolated from a HeLa cell cDNA library. Analysis of the plasmid clone indicated that the insert has an ORF identical to that of hIMPase (human myo-inositol monophosphatase). The ability of hIMPase to suppress galactose toxicity is sensitive to the presence of Li(+) in the medium. A gal7 yeast strain harboring a plasmid containing cloned hIMPase grows on galactose as a sole carbon source. hIMPase mediated galactose metabolism is dependent on the functionality of GAL1 as well as GAL10 encoded galactokinase and epimerase respectively. These results predicted that the UDP-glucose/galactose pyrophosphorylase mediated pathway may be responsible for the relief of galactose toxicity. Experiments conducted to test this prediction revealed that expression of UGP1 encoded UDP-glucose pyrophosphorylase can indeed overcome the relief of galactose toxicity. Moreover, expression of UGP1 allows a gal7 strain to grow on galactose as a sole carbon source. Unlike the hIMPase mediated relief of galactose toxicity, UGP1 mediated relief of galactose toxicity is lithium insensitive. Based on our results and on the basis of available information on galactose toxicity, we suggest an alternative explanation for the molecular mechanism of galactose toxicity.