 Yeast 15, 1719–1731 (1999) RGD1 Genetically Interacts with MID2 and SLG1, Encoding Two Putative Sensors for Cell Integrity Signalling in Saccharomyces cerevisiae GEOFFROY DE BETTIGNIES 1 , CHRISTOPHE BARTHE 1 , CARINE MOREL 1 , MARIE FRANCE PEYPOUQUET 1 , FRANC q OIS DOIGNON 1 AND MARC CROUZET 1 * 1 Laboratoire de Biologie Mole ´culaire et de Se ´quenc ¸age, UPR CNRS 9026, BP 64, 146 rue Le ´o Saignat, 33076 Bordeaux cedex, France The RGD1 gene was identified during systematic genome sequencing of Saccharomyces cerevisiae. To further understand Rgd1p function, we set up a synthetic lethal screen for genes interacting with RGD1. Study of one lethal mutant made it possible to identify the SLG1 and MID2 genes. The gene SLG1/HCS77/WSC1 was mutated in the original synthetic lethal strain, whereas MID2/SMS1 acted as a monocopy suppressor. The SLG1 gene has been described to be an upstream component in the yeast PKC pathway and encodes a putative cell surface sensor for the activation of cell integrity signalling. First identified by viability loss of shmoos after pheromone exposure, and since found in different genetic screens, MID2 was recently reported as also encoding an upstream activator of the PKC pathway. The RGD1 gene showed genetic interactions with both sensors of cell integrity pathway. The rgd1 slg1 synthetic lethality was rescued by osmotic stabilization, as expected for mutants altered in cell wall integrity. The slight viability defect of rgd1 in minimal medium, which was exacerbated by mid2, was not osmoremediated. As for mutants altered in PKC pathway, the accumulation of small-budded dead cells in slg1, rgd1 and mid2 after heat shock was prevented by 1  sorbitol. In addition, the rgd1 strain also displayed dead shmoos after pheromone treatment, like mid2. Taken together, the present results indicate close functional links between RGD1, MID2 and SLG1 and suggest that RGD1 and MID2 interact in a cell integrity signalling functionally linked to the PKC pathway. Copyright  1999 John Wiley & Sons, Ltd.   — Saccharomyces cerevisiae; RGD1; MID2; SLG1; PKC pathway; synthetic lethality INTRODUCTION We have already identified the gene RGD1 (R e- lated G AP D omain), also referred to as YBR260c, during the systematic sequencing of a region of chromosome II of Saccharomyces cerevisiae (Doignon et al., 1993). This gene is expressed in different growth conditions and in particular in the stationary phase (Barthe et al., 1998). The main characteristic of the 666 amino acid RGD1 product is the existence of a putative carboxy-terminal domain which resembles human and yeast Rho/ Rac GTPase activating proteins (GAP). Compari- son with data banks has revealed a significant similarity between the C-terminal part of Rgd1p and those of the human GAPs Bcr and Abr (Barthe et al., 1998) and of the Saccharomyces cerevisiae Bem2p and Bem3p. These latter pro- teins, which are involved in the control of bud emergence, possess at the C-terminal extremity a GAP activity on Rho1p and Cdc42p (Wang et al., 1995; Zheng et al., 1994). This carboxy-terminal similarity suggests that Rgd1p might also possess a GAP activity. Beyond the demonstration of the GAP activity, the main objective is now to determine the RGD1 cellular function, as for the thousands of ORFs discovered during systematic genome sequencing (Dujon, 1996). This is the aim of functional analy- sis, which turns classic notions of genetic research upside down (Oliver, 1996). The RGD1 gene was *Correspondence to: M. Crouzet, Laboratoire de Biologie Mole ´culaire et de Se ´quenc ¸age, UPR CNRS 9026, BP 64, 146 rue Le ´o Saignat, 33076 Bordeaux cedex, France. Tel.: 05 57 57 10 92; fax: 05 56 51 85 61; e-mail: crouzet@lbms.u-bordeaux2.fr Contract/grant sponsor: University of Bordeaux II, France. Contract/grant sponsor: CNRS, France. Contract/grant sponsor: MENRT Fellowship, France CCC 0749–503X/99/161719–13$17.50 Copyright  1999 John Wiley & Sons, Ltd. Received 17 May 1999 Accepted 16 August 1999