Novel Gal3 Proteins Showing Altered Gal80p Binding Cause Constitutive Transcription of Gal4p-Activated Genes in Saccharomyces cerevisiae

Abstract

Gal4p-mediated activation of galactose gene expression in Saccharomyces cerevisiae normally requires both galactose and the activity of Gal3p. Recent evidence suggests that in cells exposed to galactose, Gal3p binds to and inhibits Gal80p, an inhibitor of the transcriptional activator Gal4p. Here, we report on the isolation and characterization of novel mutant forms of Gal3p that can induce Gal4p activity independently of galactose. Five mutant GAL3 c alleles were isolated by using a selection demanding constitutive expression of a GAL1 promoter-driven HIS3 gene. This constitutive effect is not due to overproduction of Gal3p. The level of constitutive GAL gene expression in cells bearing different GAL3 c alleles varies over more than a fourfold range and increases in response to galactose. Utilizing glutathione S-transferase-Gal3p fusions, we determined that the mutant Gal3p proteins show altered Gal80p-binding characteristics. The Gal3p mutant proteins differ in their requirements for galactose and ATP for their Gal80p-binding ability. The behavior of the novel Gal3p proteins provides strong support for a model wherein galactose causes an alteration in Gal3p that increases either its ability to bind to Gal80p or its access to Gal80p. With the Gal3p-Gal80p interaction being a critical step in the induction process, the Gal3p proteins constitute an important new reagent for studying the induction mechanism through both in vivo and in vitro methods.

Gene activation by interaction of an inhibitor with a cytoplasmic signaling protein

“…Gal3p is a paralog of Gal1p (4), the galactokinase and the first enzyme of the galactose utilization pathway, but Gal3p does not have galactokinase activity (5,6). Any one of several mutations dispersed in the Gal3p sequence (GAL3 constitutive mutant) causes the activation of GAL genes in the absence of galactose (7). Biochemical studies have shown that Gal3p directly interacts with Gal80p in a galactose-and ATPdependent manner (3,8), and that the Gal3p constitutive mutants interact with Gal80p in the absence of galactose (7).…”

“…Any one of several mutations dispersed in the Gal3p sequence (GAL3 constitutive mutant) causes the activation of GAL genes in the absence of galactose (7). Biochemical studies have shown that Gal3p directly interacts with Gal80p in a galactose-and ATPdependent manner (3,8), and that the Gal3p constitutive mutants interact with Gal80p in the absence of galactose (7). How the formation of the Gal3p-Gal80p complex transmits the galactose signal to the Gal80p-Gal4p complex in the nucleus to activate Gal4p has been unclear.…”

Genetic Evidence for Sites of Interaction Between the Gal3 and Gal80 Proteins of the Saccharomyces cerevisiae GAL Gene Switch

“…ScPD2 was derived from ScVP2-103 (Pilauri et al 2005) by disruption of the GAL1 gene. Sc750 and Sc751 were derived from Sc724 (Blank et al 1997) by disruption of the GAL80 gene and integration of the GAL80 S-0 and GAL80 S-1 alleles, respectively. The GAL80 S-2 mutation was not integrated into the genome, but instead was carried on a CEN plasmid (pMPW87).…”

“…Gal3 C proteins bind to Gal80 in the absence of galactose and cause constitutive expression of the GAL genes (Blank et al 1997). A suppressor (GAL3 SOC ) of GAL3 C mutations and four suppressible alleles (GAL3 C -F237Y, -V396A, and -S509P/L) colocalize to a region in the Gal3 model that contains a putative ligand-regulated hinge region previously identified in a KlGal1 homology model (Menezes et al 2003).…”

“…Recessive gal3 mutations impair induction, whereas dominant GAL3 C mutations encode proteins that bind to Gal80 independently of galactose and confer constitutive GAL gene expression (Blank et al 1997). Gal3 is a paralogue of the Gal1 galactokinase (Bajwa et al 1988;Wolfe and Shields 1997;Platt et al 2000).…”