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

Abstract

The yeast transcriptional activator Gal4 localizes to UAS GAL sites even in the absence of galactose but cannot activate transcription due to an association with the Gal80 protein. By 4 min after galactose addition, Gal4-activated gene transcription ensues. It is well established that this rapid induction arises through a galactose-triggered association between the Gal80 and Gal3 proteins that decreases the association of Gal80 and Gal4. How this happens mechanistically remains unclear. Strikingly different hypotheses prevail concerning the possible roles of nucleocytoplasmic distribution and trafficking of Gal3 and Gal80 and where in the cell the initial Gal3-Gal80 association occurs. Here we tested two conflicting hypotheses by evaluating the subcellular distribution and dynamics of Gal3 and Gal80 with reference to induction kinetics. We determined that the rates of nucleocytoplasmic trafficking for both Gal80 and Gal3 are slow relative to the rate of induction. We find that depletion of the nuclear pool of Gal3 slows the induction kinetics. Thus, nuclear Gal3 is critical for rapid induction. Fluorescence-recovery-after-photobleaching experiments provided data suggesting that the Gal80-Gal4 complex exhibits kinetic stability in the absence of galactose. Finally, we detect Gal3 at the UAS GAL only if Gal80 is covalently linked to the DNA-binding domain. Taken altogether, these new findings lead us to propose that a transient interaction of Gal3 with Gal4-associated Gal80 could explain the rapid response of this system. This notion could also explain earlier observations.

Interplay of a Ligand Sensor and an Enzyme in Controlling Expression of the Saccharomyces cerevisiae GAL Genes

“…Our results are consistent with recently published data suggesting that under inducing conditions, Gal3p transiently associates with a Gal4p-Gal80p complex (5). We go on to show, however, that the dissipation of Gal3p is coincident with the recruitment of the galactokinase (Gal1p) to Gal4p (Fig.…”

“…Chromatin immunoprecipitation (ChIP) experiments (19) and pulldown assays (24) both suggest that the Gal4p-Gal80p complex is somewhat weakened (although perhaps not completely dissociated) when cells are grown in the presence of galactose. Recently published data, however, suggest that nuclear-cytoplasmic shuttling of Gal80p is not required for the expression of the GAL genes and that the induction of GAL gene expression can occur with both Gal80p and Gal3p in the nucleus (5).…”

The Gal3p transducer of the GAL regulon interacts with the Gal80p repressor in its ligand-induced closed conformation

“…It is still unclear, however, whether the three proteins would colocalize-whether Gal3p ever enters the nucleus, as discussed above. A recent study suggested that a tripartite complex might form very transiently in the nucleus (Egriboz et al 2011).…”

“…Other reports claim that Gal3p does enter the nucleus in the presence of galactose, and induction occurs through the association of a tripartite complex formed between Gal4p, Gal80p, and Gal3p (Platt and Reece 1998;Wightman et al 2008;Jiang et al 2009). Recently, Hopper and colleagues (Egriboz et al 2011) suggested that there is sufficient Gal3p in the nucleus prior to galactose induction to account for the rapid induction kinetics.…”

Evolution of gene network activity by tuning the strength of negative-feedback regulation

“…In the GAL network, the galactose signal propagates through a four-stage signalling cascade 4,9,11,12 . The galactose transporter Gal2p imports extracellular galactose into the cell, and intracellular galactose binds to and activates the signal transducer Gal3p.…”