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

Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210, USA.
Genetics (Impact Factor: 5.96). 09/2011; 189(3):825-36. DOI: 10.1534/genetics.111.131839
Source: PubMed

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.

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    • "Signaling pathways activated by other sugars have been reported to sense the nonphosphorylated form that is imported into the cell [33]. For example, galactose taken up by S. cerevisiae cells is bound by Gal3, which then promotes activation of the Gal4 transcription factor [34]. Also, it is common for metabolites to be direct mediators of cell signaling [35]. "
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    12/2012; 2012(16). DOI:10.6064/2012/489208
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    • "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). Gene regulation is ultimately and always dictated by molecular interactions between regulatory molecules. "
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    ABSTRACT: A wealth of genetic information and some biochemical analysis have made the GAL regulon of the yeast Saccharomyces cerevisiae a classic model system for studying transcriptional activation in eukaryotes. Galactose induces this transcriptional switch, which is regulated by three proteins: the transcriptional activator Gal4p, bound to DNA; the repressor Gal80p; and the transducer Gal3p. We showed previously that NADP appears to act as a trigger to kick the repressor off the activator. Sustained activation involves a complex of the transducer Gal3p and Gal80p mediated by galactose and ATP. We solved the crystal structure of the complex of Gal3p-Gal80p with α-D-galactose and ATP to 2.1 Å resolution. The interaction between the proteins occurs only when Gal3p is in a "closed" state induced by ligand binding. The structure of the complex provides a rationale for the phenotypes of several well-known Gal80p and Gal3p mutants as well as the lack of galactokinase activity of Gal3p.
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    ABSTRACT: The regulation of the Saccharomyces cerevisiae GAL genes in response to galactose as a source of carbon has served as a paradigm for eukaryotic transcriptional control over the last 50 years. Three proteins--a transcriptional activator (Gal4p), an inhibitor (Gal80p), and a ligand sensor (Gal3p)--control the switch between inert and active gene expression. The molecular mechanism by which the recognition of galactose within the cell is converted into a transcriptional response has been the subject of considerable debate. In this study, using a novel and powerful method of localizing active transcription factors within the nuclei of cells, we show that a short-lived complex between Gal4p, Gal80p, and Gal3p occurs soon after the addition of galactose to cells to activate GAL gene expression. Gal3p is subsequently replaced in this complex by Gal1p, and a Gal4p-Gal80p-Gal1p complex is responsible for the continued expression of the GAL genes. The transient role of the ligand sensor indicates that current models for the induction and continued expression of the yeast GAL genes need to be reevaluated.
    Eukaryotic Cell 12/2011; 11(3):334-42. DOI:10.1128/EC.05294-11 · 3.18 Impact Factor
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