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.     

GAL4 protein: purification, association with GAL80 protein, and conserved domain structure.

Expression of the yeast Saccharomyces cerevisiae GAL4 protein under its own (galactose-inducible) control gave 5 to 10 times the level of protein observed when the GAL4 gene was on a high-copy plasmid. Purification of GAL4 by a procedure including affinity chromatography on a GAL4-binding DNA column yielded not only GAL4 but also a second protein, shown to be GAL80 by its reaction with an antipeptide antibody. Sequence comparisons of GAL4 and other members of a family of proteins sharing homologous cysteine finger motifs identified an additional region of homology in the middle of these proteins shown by genetic analysis to be important for GAL4 function. GAL4 could be cleaved proteolytically at the boundary of the conserved region, defining internal and carboxy-terminal folded domains.     

Peptides selected to bind the Gal80 repressor are potent transcriptional activation domains in yeast

The activation domain of the yeast Gal4 protein binds specifically to the Gal80 repressor and is also thought to associate with one or more coactivators in the RNA polymerase II holoenzyme and chromatin remodeling machines. This is a specific example of a common situation in biochemistry where a single protein domain can interact with multiple partners. Are these different interactions related chemically? To probe this point, phage display was employed to isolate peptides from a library based solely on their ability to bind Gal80 protein in vitro. Peptide-Gal80 protein association is shown to be highly specific and of moderate affinity. The Gal80 protein-binding peptides compete with the native activation domain for the repressor, suggesting that they bind to the same site. It was then asked if these peptides could function as activation domains in yeast when tethered to a DNA binding domain. Indeed, this is the case. Furthermore, one of the Gal80-binding peptides binds directly to a domain of the Gal11 protein, a known coactivator. The fact that Gal80-binding peptides are functional activation domains argues that repressor binding and activation/coactivator binding are intimately related properties. This peptide library-based approach should be generally useful for probing the chemical relationship of different binding interactions or functions of a given native domain.