Snf1 Protein Kinase and the Repressors Nrg1 and Nrg2 Regulate FLO11, Haploid Invasive Growth, and Diploid Pseudohyphal Differentiation

The budding yeast Saccharomyces cerevisiae responds to a variety of different environmental signals by altering its gene expression, metabolism, and morphology. Diploid pseudohyphal differentiation and haploid invasive growth are related but distinct cell type-specific developmental processes that occur in response to distinct nutrient limitation signals (6, 16, 22). Pseudohyphal differentiation is a response of diploid cells to nitrogen limitation (8). Cells assume an elongated morphology, change their budding pattern, and generate chains of filamentous-form cells projecting from the main colony of yeast-form cells. At the molecular level, one of the key events is transcriptional activation of the FLO11 (MUC1) gene, which encodes a cell-surface glycoprotein with roles in cell-cell adhesion and adherence to surfaces (15, 17, 18). FLO11 has a large and complex promoter, and its transcription is regulated by at least two pathways: the cyclic AMP-dependent protein kinase A (PKA) pathway, which acts through the transcriptional activator Flo8 and the repressor Sfl1, and the Kss1 mitogen-activated protein kinase (MAPK) cascade, which regulates the activator Ste12-Tec1 (17, 21, 26, 31, 33). Haploid invasive growth is a related process that similarly requires the Flo11 flocculin (15, 17, 30). Haploid cells elongate, alter their budding pattern, and invade the agar during growth on rich medium. Agar invasion does not initiate immediately but rather occurs after several days, suggesting that limitation for a nutrient triggers this process. Haploid invasive growth requires the same regulatory pathways as pseudohyphal differentiation, namely the PKA and MAPK pathways (30, 31); however, invasive growth occurs in response to limitation for glucose, not nitrogen (3). As is the case with many glucose limitation-induced processes, invasive growth also depends on the Snf1 protein kinase (3). Finally, a distinct type of pseudohyphal and invasive growth occurs in haploids and diploids that lack the forkhead transcription factors Fkh1 and Fkh2, which control the CLB2 cluster of cell cycle-regulated genes (10, 39). This pseudohyphal growth is constitutive, rather than induced by nutrient starvation, and does not require FLO11 or STE12. The Snf1 kinase is conserved in fungi, plants, and animals (AMP-activated kinase in mammals) and has broad roles in transcriptional and metabolic responses to cellular stress (9, 13). In S. cerevisiae, the Snf1 kinase is primarily required for adaptation to glucose limitation but has also been implicated in other stress responses (2, 7). The dependence of haploid invasive growth on both the Snf1 kinase and the FLO11 gene suggested that Snf1 regulates FLO11 in response to glucose limitation. In support of this idea, FLO11 transcription increases during the postdiauxic phase (33) and during growth on poor carbon sources (5), when the Snf1 kinase is active. In addition, the FLO11 promoter is nearly identical over 3.9 kb to that of a Snf1-dependent gene, the glucoamylase gene STA2 (5, 14). The similarity of FLO11 to STA2 further suggested a role for the zinc finger repressor proteins Nrg1 and Nrg2, which interact physically with Snf1 (37). Nrg1 and Nrg2 contribute to glucose repression of several Snf1-dependent genes, including SUC2, GAL, and DOG2 (37, 38). Most importantly, Nrg1 plays a major role in glucose repression of STA genes (27) and the Nrg1 binding site is conserved in FLO11. Here, we have explored the role of the Snf1 kinase pathway in regulating invasive growth. We present evidence that Snf1 regulates transcription of the FLO11 gene in response to glucose depletion. We further show that Nrg1 and Nrg2 negatively regulate FLO11 and invasive growth. Finally, we present genetic evidence that Snf1, Nrg1, and Nrg2 also affect two other Flo11-dependent processes, the initiation of biofilm formation and diploid pseudohyphal differentiation. The genetic and physical relationships between Snf1 and Nrg proteins lead us to propose a model in which Snf1 positively regulates Flo11-dependent developmental processes by antagonism of Nrg-mediated repression of FLO11.