Fig. 6. Rom2p is required for the lethal effects of PtdIns(3)P accumulation in ymr1 sjl2 sjl3 cells. (A) ymr1 sjl2^ts sjl3 rom2 cells were transformed with a pRS415-derived plasmid carrying either wild-type ROM2, YMR1 or no insert. Transformants were then streaked to 5-FOA plates lacking the amino acid leucine to select for maintenance of the pRS415 vector. Growth was scored after 5 days at 26°C. Results are representative of two independent transformants. (B) Cellular PtdIns(3)P levels are not significantly reduced in ymr1 sjl2^ts sjl3 rom2 cells. Cells were grown to early log phase under appropriate selection, then pre-incubated at either 26°C or 38°C for 20 minutes, and deacylated [³H]-glycero-phosphoinositols were analyzed by HPLC as in Fig. 1. Data represent the mean±s.d. of two experiments performed in duplicate. (C) Deletion of ROM2 attenuates hyperactivation of the Rho1p/Pkc1p-mediated Slt2p MAPK pathway. Samples were resolved together by SDS-PAGE and the nitrocellulose filter was probed as in Fig. 4. Results represent a 20-second exposure under the same conditions described for Fig. 4A. Note that the basal level of active Slt2p in ymr1 sjl2^ts sjl3 cells is greater than the maximal heat-stress-induced signal in wild-type cells, indicating constitutive Rho1p/Pkc1p signaling in PI 3-phosphatase-deficient cells.