Localization and Interaction of the Proteins Constituting the GAL Genetic Switch in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, the GAL genes encode the enzymes required for galactose metabolism. Regulation of these genes has served as the paradigm for eukaryotic transcriptional control over the last 50 years. The switch between inert and active gene expression is dependent upon three proteins-the transcriptional activator Gal4p, the inhibitor Gal80p, and the ligand sensor Gal3p. Here, we present a detailed spatial analysis of the three GAL regulatory proteins produced from their native genomic loci. Using a novel application of photobleaching, we demonstrate, for the first time, that the Gal3p ligand sensor enters the nucleus of yeast cells in the presence of galactose. Additionally, using Förster resonance energy transfer, we show that the interaction between Gal3p and Gal80p occurs throughout the yeast cell. Taken together, these data challenge existing models for the cellular localization of the regulatory proteins during the induction of GAL gene expression by galactose and suggest a mechanism for the induction of the GAL genes in which galactose-bound Gal3p moves from the cytoplasm to the nucleus to interact with the transcriptional inhibitor Gal80p.

Transcriptional Regulation inSaccharomyces cerevisiae: Transcription Factor Regulation and Function, Mechanisms of Initiation, and Roles of Activators and Coactivators

“…Although this tripartite complex has been observed in vitro in the presence of galactose and ATP (Platt and Reece 1998), it has eluded detection in vivo. Recent fluorescence resonance energy transfer (FRET) experiments showed that a Gal80-Gal3 complex is present in both the cytoplasm and the nucleus after galactose induction; however, association of Gal80-Gal3 with Gal4 at a promoter has not yet been observed (Wightman et al 2008). Other conflicting evidence supports a dissociation The dissociation model Hopper 2000, 2002) suggests that Gal3 bound to galactose and ATP in the cytoplasm sequesters Gal80 from the nucleus and thus frees Gal4 AD for interaction with coactivators.…”

“…One focus of current research is the nature of promoter-bound but transcriptionally inactive Gal4 (Wightman et al 2008;). In the absence of galactose, the C-terminal AD cannot recruit coactivators because it is occluded by Gal80 (Johnston et al 1987;Ma and Ptashne 1987a).…”

Rapid GAL Gene Switch of Saccharomyces cerevisiae Depends on Nuclear Gal3, Not Nucleocytoplasmic Trafficking of Gal3 and Gal80

“…One suggests that Gal3 interacts with Gal80 exclusively in the cytoplasm and sequesters it away from nuclear Gal4 (Peng and Hopper 2002). A strikingly different hypothesis specifies that cytoplasmic Gal3 binds galactose and then moves into the nucleus to bind to Gal80 (Wightman et al 2008). These two hypotheses specify not only different subcellular compartments in which the initial interaction of Gal3 and Gal80 occurs, but also different nucleocytoplasmic trafficking dynamics for these proteins.…”

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

“…Recent experimental studies on S. cerevisiae GAL system have indicated that the inducer protein Gal3p can also shuttle into the nucleus and interact with the repressor protein Gal80p to relieve inhibition on activator protein Gal4p (Wightman et al 2008). To mimic the experimental findings, we re-engineered the GAL system in S. cerevisiae to include the nucelocytoplasmic shuttling of both repressor (Gal80p) and inducer (Gal3p) proteins.…”

“…Recent studies indicate that in addition to the nucleocytoplasmic shuttling of Gal80p, the inducer Gal3p also shuttles to the nucleus (Wightman et al 2008). To study the effect of nucleocytoplasmic transport of Gal3p on the response of GAL switch, the S. cerevisiae model was re-engineered to include the transport of Gal3p.…”