Rewiring the yeast Cell Wall Integrity (CWI) pathway through a synthetic positive feedback circuit unveils a novel role for the MAPKKK Ssk2 in CWI pathway activation.

The Cell Wall Integrity (CWI) pathway mediates the response of Saccharomyces cerevisiae to cell wall alterations. Stress at the cell surface is detected by mechanosensors, which transduce the signal to a protein kinase cascade that involves Pkc1, Bck1, Mkk1/Mkk2, the mitogen-activated protein kinase (MAPK) Slt2 and the transcription factor Rlm1. We incorporated a positive feedback loop into this pathway by placing a hyperactive MKK1 allele under the control of the Rlm1-regulated MLP1 promoter. This circuit operates as a signal amplifier and leads to a highly increased Slt2 activation under stimulating conditions. Triggering the CWI pathway in cells engineered with this circuit, which we have named the IPAC (Integrity Pathway Activation Circuit), results in strong growth inhibition. Exploitation of this hypersensitive phenotype allowed the identification of novel proteins that contribute in signalling to Rlm1 in response to cell-surface stressing agents such as Congo red, zymolyase and sodium dodecyl sulfate (SDS). Among these proteins, the MAPK kinase kinase Ssk2 of the osmoregulatory High-Osmolarity Glycerol (HOG) pathway, but not its paralog Ssk22, proved to be necessary for the SDS-induced IPAC-mediated growth inhibition. We found the existence of an Ssk1-independent Ssk2-Pbs2-Hog1-CWI pathway signalling axis that contributes to Slt2 activation in response to cell-surface stress. We also demonstrated that the MAPKKs Mkk1 and Pbs2 and the MAPKs Slt2 and Hog1 of the HOG and CWI pathways interact physically, forming a complex. Our results show how a simple synthetic circuit can be used as a powerful tool for a better understanding of signalling pathways.