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 loss-of-function alleles in 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, identifying an inhibitory domain of the protein from residues 491 to 688. We also find that a diversity of filamentous growth phenotypes can result from combinatorial effects of multiple mutations and by loss of 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.

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“…Glucose or nitrogen limitation turns on the fMAPK pathway and activates Kss1, a downstream Ser/Thr MAPK ( 83 , 84 ). The TOR pathway, as the name suggests, is inhibited by rapamycin and induced by nitrogen sources ( 85 ).…”

“…Intersections and cross-talk between these pathways and with other signaling networks in the cell are well established. For example, fMAPK is only one of at least three intersecting MAPK pathways, the other two being the pheromone-responsive mating MAPK and the osmotic stress responsive high-osmolarity glycerol 1 (Hog1) pathways ( 84 ). Similarly, Ras2 feeds into the fMAPK as well as the cAMP/PKA pathway ( 88 ).…”