UC Berkeley

Multiple mitogen-activated protein kinases (MAPKs) enable eukaryotic cells to evoke an appropriate response when presented with a particular stimulus. In the yeast Saccharomyces cerevisiae, MAPK Hog1 is activated by osmosensors in the high-osmolarity glycerol (HOG) pathway during hyperosmotic stress, MAPK Fus3 is activated by pheromone-binding receptors in the mating pathway, and MAPK Kss1 is activated by mucins in the filamentous growth (FG) pathway during nutrient limitation. These pathways provide an excellent model for studying mechanisms and principles of signal transduction in a genetically and biochemically tractable organism because these conserved pathways have served countless species in their struggle to adapt to change throughout evolution.

Upon hyperosmotic shock, yeast cells accumulate intracellular glycerol to balance the osmotic gradient. It had been accepted that Hog1 elevates glycerol production by inducing the transcription of enzymes necessary for glycerol synthesis. Using global microarray analysis, I found that Hog1-dependent transcription is not necessary for hyperosmotic shock survival. Instead, Hog1 increases glycerol production by directly regulating metabolism and work presented in this thesis describes progress made towards understanding how this control is exerted.

The HOG, mating and FG pathways share common upstream activators, including Ste50 (adapter protein), Ste20 [p21-activated protein kinase (PAK)], Ste11 [MAPK kinase kinase (MAPKKK)] and Cdc42 [guanosine tri-phosphatase (GTPase)]. Activation of Ste11 within the HOG pathway does not result in Ste11-mediated activation of the mating or FG pathways. Tellingly, if Hog1 function is absent, hyperosmotic stress does result in Ste11-mediated activation of these other MAPK pathways, a situation called crosstalk. Therefore, a mechanism of Hog1-enforced crosstalk prevention exists. Using single-cell analysis of both HOG and mating pathway activation, I found that crosstalk is prevented by insulation of the HOG pathway from other MAPK pathways, over-turning a previously established erroneous model of cross-inhibition. Through a genetic selection, I found that Rga1 [a Cdc42 GTPase-activating protein (GAP)] is required for HOG pathway insulation, that Rga1 is a substrate of Hog1, that it contributes to negative feedback regulation of the HOG pathway, and that Rga1 presumably helps prevent crosstalk by limiting the extent and duration of Cdc42 activation.