A synthetic analysis of the Saccharomyces cerevisiae stress sensor Mid2p, and identification of a Mid2p-interacting protein, Zeo1p, that modulates the PKC1-MPK1 cell integrity pathway

• Published in: Microbiology (Society for General Microbiology) 2003-09, Vol.149 (9), p.2487-2499
• Main Authors: Green, Robin, Lesage, Guillaume, Sdicu, Anne-Marie, Menard, Patrice, Bussey, Howard
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing; Biological and medical sciences; Calcium-Binding Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Growth, nutrition, metabolism, transports, enzymes. Molecular biology; Intracellular Signaling Peptides and Proteins; Membrane Glycoproteins; Membrane Proteins - metabolism; Microbiology; MID2 gene; Mitogen-Activated Protein Kinases; Mycology; ROM2 gene; Rom2 protein; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - metabolism; Signal Transduction; WSC1 gene; Zeo1 protein
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/mic.0.26471-0

Department of Biology, McGill University, Montreal, Quebec, Canada H3A 1B1 Correspondence Howard Bussey howard.bussey{at}mcgill.ca Mid2p is a plasma membrane protein that functions in Saccharomyces cerevisiae as a sensor of cell wall stress, activating the PKC1–MPK1 cell integrity pathway via the small GTPase Rho1p during exposure to mating pheromone, calcofluor white, and heat. To examine Mid2p signalling, a global synthetic interaction analysis of a mid2 mutant was performed; this identified 11 interacting genes. These include WSC1 and ROM2 , upstream elements in cell integrity pathway signalling, and FKS1 and SMI1 , required for 1,3- -glucan synthesis. These synthetic interactions indicate that the Wsc1p sensor acts through Rom2p to activate the Fks1p glucan synthase in a Mid2p-independent way. To further explore Mid2p signalling a two-hybrid screen was done using the cytoplasmic tail of Mid2p; this identified ZEO1 ( YOL109w ), encoding a 12 kDa peripheral membrane protein that localizes to the plasma membrane. Disruption of ZEO1 leads to resistance to calcofluor white and to a Mid2p-dependent constitutive phosphorylation of Mpk1p, supporting a role for Zeo1p in the cell integrity pathway. Consistent with this, zeo1 -deficient cells suppress the growth defect of mutants in the Rho1p GDP–GTP exchange factor Rom2p, while exacerbating the growth defect of sac7 mutants at 37 °C. In contrast, mid2 mutants have opposing effects to zeo1 mutants, being synthetically lethal with rom2 , and suppressing an 18 °C growth defect of sac7 , while overexpression of MID2 rescues a rom2 37 °C growth defect. Thus, MID2 and ZEO1 appear to play reciprocal roles in the modulation of the yeast PKC1–MPK1 cell integrity pathway. Abbreviations: HA, haemagglutinin