Wsc1 and Mid2 are cell surface sensors for cell wall integrity signaling that act through Rom2, a guanine nucleotide exchange factor for Rho1

Wsc1 and Mid2 are highly O-glycosylated cell surface proteins that reside i n the plasma membrane of Saccharomyces cerevisiae. They have been proposed to function as mechanosensors of cell wall stress induced by wall remodelin g during vegetative growth and pheromone-induced morphogenesis. These prote ins are required for activation of the cell wall integrity signaling pathwa y that consists of the small G-protein Rho1, protein kinase C (Pkc1), and a mitogen-activated protein kinase cascade. We show here by two-hybrid exper iments that the C-terminal cytoplasmic domains of Wsc1 and Mid2 interact wi th Rom2, a guanine nucleotide exchange factor (GEF) for Rho1. At least with regard to Wsc1, this interaction is mediated by the Rom2 N-terminal domain . This do main is distinct from the Rho1-interacting domain, suggesting tha t the GEF can interact simultaneously with a sensor and with Rho1. We also demonstrate that extracts from wsc1 and mid2 mutants are deficient in the a bility to catalyze GTP loading of Rho1 in vitro, providing evidence that th e function of the sensor-Rom2 interaction is to stimulate nucleotide exchan ge toward this G-protein. In a related line of investigation, we identified the PMT2 gene in a genetic screen for mutations that confer an additive ce ll lysis defect with a wsc1 null allele. Pmt2 is a member of a six-protein family in yeast that catalyzes the first step in O mannosylation of target proteins. We demonstrate that Mid2 is not mannosylated in a pmt2 mutant and that this modification is important for signaling by Mid2.