Quantitative Analysis of GAL Genetic Switch of Saccharomyces cerevisiae Reveals That Nucleocytoplasmic Shuttling of Gal80p Results in a Highly Sensitive Response to Galactose

Abstract

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.

Dynamic analysis of the KlGAL regulatory system in Kluyveromyces lactis: a comparative study with Saccharomyces cerevisiae

“…As seen from the figure the expression levels reached maximum after about 20 h in both species but the maximum level of expression is different. K. lactis requires high galactose concentration (0.44 M) to express at maximum level of 35%, while S. cerevisiae needs only 0.1 M to reach its maximum expression levels of 82% (Verma et al 2003). The expression levels drop once galactose is consumed completely after about 60 h in K. lactis, whereas, in S. cerevisiae the expression levels drop after about 35 h. Since the GAL gene expression is due to the interplay of the regulatory proteins, it is of interest to see how concentration of these protein varies with time for a given steadystate galactose concentration.…”

“…Figure 8a shows the model predictions for the re-engineered system. The distribution coefficient of Gal80p was similar to the original model (K = 0.4), while the distribution coefficient for Gal3p was regressed to match the experimental data (Verma et al 2003). Solid line in Fig.…”

“…However, other than these feedback loops, there exist other regulatory features such as compartmentalization and stoichiometry, which also play a crucial role in the overall performance of the regulatory system. For example, it has been shown that the nucleocytoplasmic shuttling of repressor protein Gal80p 1 in GAL system of S. cerevisiae plays a crucial role in the performance of the switch (Verma et al 2003).…”

“…The GAL system in S. cerevisiae is a well studied regulatory network for which both steady-state and dynamic models have been developed to understand the regulatory structure Verma et al 2003). However, such an in-depth dynamic analysis of K. lactis has not been reported in literature.…”

Feedback control of stochastic noise in the yeast galactose utilization pathway

“…Section 2 explores the role of the feedback loops by comparing two computer models of the galactose pathway. The first is the wild type model of the regulatory and metabolic systems (see also the works of: [14,15] ); the second is an unregulated version, which is exactly the same in every respect, except that the proteins Gal3p and Gal80p are not upregulated during induction, but are maintained at their low, non-induced levels.…”