Lrg1p functions as a putative GTPase-activating protein in the Pkc1p-mediated cell integrity pathway in Saccharomyces cerevisiae

In Saccharomyces cerevisiae the ROM2 gene encodes a GDP/GTP exchange factor for the small G-protein Rho1p, a known activator of protein kinase C. In a screen designed to isolate suppressors of a rom2 mutant allele, we identif ied a mutant defective in the gene coding for the putative GTPase-activatin g protein Lrg1p. This protein was previously suggested to be involved in sp orulation and mating. Here we provide evidence for its role in Pkc1p-mediat ed signal transduction based on the following results. (1) Deletion of LRG1 suppresses the growth phenotypes associated with mutations in SLG1 (which codes for a putative sensor of cell wall damage). (2) Using two-hybrid assa ys an interaction between the GAP domain of Lra1p and Rho1p was demonstrate d. (3) The Irg1 mutant shows enhanced activity of the Pkc1p pathway. (4) Ov erexpression of LRG1 leads to a cell lysis defect that can be suppressed by the addition of osmotic stabilizers. Phenotypic comparison of h-gl mutants with mutants defective in other GTPase-activating proteins (Sac7p, Bem2p, Bag7p) presumed to act on Rho1p revealed that deletion of SAC7, but not BEM 2 or BAG7, suppresses the phenotype of rom2 mutants. Pairwise combination o f mutations in all these genes showed that the simultaneous deletion of SAC 7 and LRG1 is synthetically lethal. We therefore suggest that Lrg1p acts as a negative regulator of the Pkc1p pathway in conjunction with its known ho mologue Sac7p.