Quantitative analysis of GAL genetic switch of Saccharomyces cerevisiae reveals that nucleocytoplasmic shuttling of Gal80p results in a highly sensitive response to galactose

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 alpha-galactosidase, but not beta-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.     

Analysis of the galactose signal transduction pathway in Saccharomyces cerevisiae: interaction between Gal3p and Gal80p.

The GAL3 gene plays a critical role in galactose induction of the GAL genes that encode galactose- metabolizing enzymes in Saccharomyces cerevisiae. Defects in GAL3 result in a long delay in GAL gene induction, and overproduction of Gal3p causes constitutive expression of GAL. Here we demonstrate that concomitant overproduction of the negative regulator, Gal80p, and Gal3p suppresses this constitutive GAL expression. This interplay between Gal80p and Gal3p is direct, as tagged Gal3p coimmunoprecipitated with Gal80p. The amount of coprecipitated Gal80p increased when GAL80 yeast cells were grown in the presence of galactose. When both GAL80 and GAL3 were overexpressed, the amount of coprecipitated Gal80p was not affected by galactose. Tagged gal3 mutant proteins bound to purified Gal80p, but only poorly in comparison with the wild type, suggesting that formation of the Gal80p-Gal3p complex depends on the normal function of Gal3p. Gal3p appeared larger in Western blots (immunoblots) than predicted by the published nucleic acid sequence. Reexamination of the DNA sequence of GAL3 revealed several mistakes, including an extension at the 3' end of another predicted 97 amino acids.     

Characterization of the Saccharomyces cerevisiae galactose mutarotase/UDP-galactose 4-epimerase protein, Gal10p

Saccharomyces cerevisiae and some related yeasts are unusual in that two of the enzyme activities (galactose mutarotase and UDP-galactose 4-epimerase) required for the Leloir pathway of d-galactose catabolism are contained within a single protein-Gal10p. The recently solved structure of the protein shows that the two domains are separate and have similar folds to the separate enzymes from other species. The biochemical properties of Gal10p have been investigated using recombinant protein expressed in, and purified from, Escherichia coli. Protein-protein crosslinking confirmed that Gal10p is a dimer in solution and this state is unaffected by the presence of substrates. The steady-state kinetic parameters of the epimerase reaction are similar to those of the human enzyme, and are not affected by simultaneous activity at the mutarotase active site. The mutarotase active site has a strong preference for galactose over glucose, and is not affected by simultaneous epimerase activity. This absence of reciprocal kinetic effects between the active sites suggests that they act independently and do not influence or regulate each other.     

Catabolite repression by galactose in overexpressed GAL4 strains of Saccharomyces cerevisiae

Catabolite repression by galactose was investigated in several strains of Saccharomyces cerevisiae grown on different carbon sources. Galactose repressed as much as glucose; raffinose was less effective. Full derepression was achieved with lactate. The functions tested were L-lactate ferricytochrome c oxidoreductase, NAD-glutamate dehydrogenase, and respiration. Galactose repression was observed only in the GAL4 but not in the gal4 strain. The presence of multiple copies of the GAL4 gene enhanced the repression by galactose. Different alleles of the GAL4 gene and the copy number did not affect glucose repression.