Figure 5.

Hypersensitivity to caffeine and rapamycin is due to decrease in TORC2 activity. (A) Protein extracts were prepared from log phase cultures of wild-type, erg2, scs7, and erg2 scs7 mutant strains. The extent of phosphorylation of the hydrophobic motifs in Ypk1 and Sch9 (indicative of TORC2 and TORC1 activity, respectively) were determined by SDS-PAGE and Western blotting. Rapamycin treatment was for 20 min with 200 ng/ml. B) The same yeast strains, transformed with empty vector (Yep352) or an activated allele of Ypk2p (Yep352::Ypk2D239A-HA were grown to stationary phase, diluted, and 10-fold serial dilutions were plated onto plates containing 2 mM caffeine or 20 ng/ml rapamycin.