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

• Published in: The FEBS journal Vol. 287; no. 22; pp. 4881 - 4901
• Main Authors: Jiménez‐Gutiérrez, Elena, Alegría‐Carrasco, Estíbaliz, Alonso‐Rodríguez, Esmeralda, Fernández‐Acero, Teresa, Molina, María, Martín, Humberto
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01-11-2020
• Subjects: Cell activation; Cell surface; cell wall integrity; Cell walls; Control theory; Exploitation; Feedback; Feedback circuits; Feedback loops; Glycerol; Hog1 protein; Integrity; Kinases; MAP kinase; MAPK; Osmolarity; Osmoregulation; Phenotypes; phosphorylation; Positive feedback; positive feedback loop; Protein kinase; Protein kinase C; Proteins; Rewiring; Signal transduction; Signaling; yeast; Yeasts; cell wall integrity; MAPK; phosphorylation; positive feedback loop; yeast
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.15288

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 Integrity Pathway Activation Circuit (IPAC), 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 SDS. Among these proteins, the MAPK kinase kinase Ssk2 of the osmoregulatory high‐osmolarity glycerol (HOG) pathway, but not its paralogue 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 MAP kinase kinases 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.
We constructed a synthetic feedback loop that rewires the cell wall integrity (CWI) mitogen‐activated protein kinase (MAPK) pathway of Saccharomyces cerevisiae by placing a hyperactive MKK1 under the control of a CWI‐induced promoter. This genetic circuit promotes hypersensitivity to cell wall damage, providing a useful tool to find components of this pathway. By this means, we identified a novel signalling axis to the CWI MAPK Slt2 involving the high‐osmolarity glycerol pathway MAP kinase kinase kinase Ssk2.