FIG. 3.
Wsc1 and Mid2 regulate guanine nucleotide exchange activity toward Rho1HA. (A) The ability of crude extracts from wild-type (WT [1788]), mid2Δ (DL2394), rom2Δ (DL2069), and wsc1Δ (DL1987) strains to catalyze loading of [35S]GTPγS onto immunoprecipitated Rho1HA was measured with a filter binding assay (see Materials and Methods). Rho1HA was immunoprecipitated from wild-type (1788) cells overexpressing pRS424[RHO1HA] by using the 12CA5 antibody. The rate of [35S]GTPγS loading onto Rho1HA by the mutant extracts was expressed as a percentage of the rate catalyzed by the wild-type extract. All strains were grown in YEPD. (B) The ability of crude extracts from wild type (WT; 1783), mid2Δwsc1Δ (DL2282), and pkc1Δ (DL376) strains to catalyze loading of [35S]GTPγS onto immunoprecipitated Rho1HA was measured. All strains were grown in YEPD supplemented with 10% sorbitol for osmotic support. (C) The effect of in vivo-generated cell wall stress on in vitro-catalyzed guanine nucleotide exchange on immunoprecipitated Rho1HA was measured. Wall stress was induced in wild-type (1783) cells grown in YEPD at 23°C by treatment for 90 min with 40 μg of calcofluor white per ml.