Self-Association of the Gal4 Inhibitor Protein Gal80 Is Impaired by Gal3: Evidence for a New Mechanism in the GAL Gene Switch

Abstract

The DNA-binding transcriptional activator Gal4 and its regulators Gal80 and Gal3 constitute a galactose-responsive switch for the GAL genes of Saccharomyces cerevisiae. Gal4 binds to GAL gene UASGAL (upstream activation sequence in GAL gene promoter) sites as a dimer via its N-terminal domain and activates transcription via a C-terminal transcription activation domain (AD). In the absence of galactose, a Gal80 dimer binds to a dimer of Gal4, masking the Gal4AD. Galactose triggers Gal3-Gal80 interaction to rapidly initiate Gal4-mediated transcription activation. Just how Gal3 alters Gal80 to relieve Gal80 inhibition of Gal4 has been unknown, but previous analyses of Gal80 mutants suggested a possible competition between Gal3-Gal80 and Gal80 self-association interactions. Here we assayed Gal80-Gal80 interactions and tested for effects of Gal3. Immunoprecipitation, cross-linking, and denaturing and native PAGE analyses of Gal80 in vitro and fluorescence imaging of Gal80 in live cells show that Gal3-Gal80 interaction occurs concomitantly with a decrease in Gal80 multimers. Consistent with this, we find that newly discovered nuclear clusters of Gal80 dissipate in response to galactose-triggered Gal3-Gal80 interaction. We discuss the effect of Gal3 on the quaternary structure of Gal80 in light of the evidence pointing to multimeric Gal80 as the form required to inhibit Gal4.

Analyzing negative feedback using a synthetic gene network expressed in the Drosophila melanogaster embryo

“…Both mathematical modeling, as well as expression of the Gal80-binder Gal3, validate our findings [35]. This work demonstrates how in spatial systems, gene networks can produce very different outputs depending on the relative spatial domains of inputs.…”

The yeast galactose network as a quantitative model for cellular memory

“…When galactose enters the cell, either via a passive diffusion process or facilitated by the transporter Gal2p [15], it binds and activates the inducer Gal3p in the cytoplasm. Activated Gal3p frees Gal4p from the inhibition of Gal80p [16], perhaps by reducing Gal80p dimerization [17]. Gal4p then instigates transcription of the target genes, increasing their expression by several-fold (regulatory genes GAL3,80) or up to 1000-fold (structural genes GAL1,2,7,10) over the course of a few hours [3].…”

Polymorphisms in the yeast galactose sensor underlie a natural continuum of nutrient-decision phenotypes

“…This includes nonconservative amino-acid substitutions in the Gal3p dimerization interface and the Gal3-Gal80p interface [48]. Gal3p and Gal80p are both homodimers and the Gal3p-Gal80p interaction, which is crucial to the mechanism of GAL pathway activation, is thought to depend on this homodimerization [49,50]. We are less able to predict the impact of promoter variation, but we found 13 SNPs in the promoter (500 bp upstream of the start codon), none of which were in known transcription factor binding sites (S3 Table).…”