Modulators of MAPK pathway activity during filamentous growth in Saccharomyces cerevisiae

Abstract

Mitogen-activated protein kinase (MAPK) pathways control the response to intrinsic and extrinsic stimuli. In the budding yeast Saccharomyces cerevisiae, cells undergo filamentous growth, which is regulated by the fMAPK pathway. To better understand the regulation of the fMAPK pathway, a genetic screen was performed to identify spontaneous mutants with elevated activity of an fMAPK-pathway dependent growth reporter (ste4 FUS1-HIS3). In total, 159 mutants were isolated and analyzed by secondary screens for invasive growth by the plate-washing assay, and filament formation by microscopy. Thirty-two mutants were selected for whole-genome sequencing, which identified new alleles in genes encoding known regulators of the fMAPK pathway. These included gain-of-function alleles in STE11, which encodes the MAPKKK, as well as loss-of-function alleles in KSS1, which encodes the MAP kinase, and RGA1, which encodes a GTPase activating protein (GAP) for CDC42. New alleles in previously identified pathway modulators were also uncovered in ALY1, AIM44, RCK2, IRA2, REG1 and in genes that regulate protein folding (KAR2), glycosylation (MNN4), and turnover (BLM10). C-terminal truncations in the transcription factor Ste12p were also uncovered that resulted in elevated reporter activity, presumably identifying an inhibitory domain in the C-terminus of the protein. We also show that a wide variety of filamentous growth phenotypes result from mutations in different regulators of the response. The alleles identified here expand the connections surrounding MAPK pathway regulation and reveal new features of proteins that function in the signaling cascade. ARTICLE SUMMARYSignaling pathways control the response to stimuli. In yeast, a signaling (MAPK) pathway controls a fungal behavioral response called filamentous growth. A genetic screen was performed to identify spontaneous mutants that show hyperactivity of a MAPK pathway-dependent reporter. Select mutants were analyzed by whole-genome sequencing. New alleles in known regulatory proteins were identified. A potential inhibitory domain in the C-terminus of the transcription factor Ste12p was also uncovered. Our results indicate that filamentous growth is determined by the combinatorial effects of multiple positive and negative regulatory inputs.