FIG. 6.
Effect of RAS activity on Ume3p regulation in response to stress. (A) Cultures expressing Ume3p and either wild-type RAS2 or the constitutively active RAS2Asn126 allele were either harvested directly (glucose), switched to acetate medium or medium lacking any carbon source, or subjected to a 37°C heat shock (see Fig. 2 legend). Protein extracts were prepared, and Ume3p levels were determined by Western blot analysis (see Materials and Methods for details). Tub1p levels served as protein loading controls. (B) Cultures harboring either the wild-type (RAS2) or the activated (RAS2val19) allele of RAS2 were grown to mid-log-phase in glucose medium, harvested, and then resuspended in carbon-depleted medium. Samples were taken at the indicated times, and Ume3p levels were determined. (C) Decay kinetics of Ume3p. The Ume3p signals derived from the experiments in panel B were quantitated by phosphorimaging and plotted with 100% representing the preshift (0-h) values.