Fig. 1.
Elevated levels of Ras/PKA signaling activity inhibited autophagy. A, RAS2val19 mutants exhibited very low levels of autophagy following nitrogen starvation. The indicated strains were grown to mid-log phase at 30 °C and transferred to the nitrogen starvation medium, SD-N, for 0 or 15 h. Autophagy levels were measured with the alkaline phosphatase-based assay as described under “Experimental Procedures.” The levels of autophagy induction are indicated by the relative increase in alkaline phosphatase activity that is induced by the nitrogen starvation. The strains used were wild type (TN125), atg1Δ (YYK126), atg13Δ (YYK130), and RAS2val19 (PHY3513). All strains except PHY3513 carried the control vector, pRS413. B, increased levels of PKA activity inhibited autophagy activity. Alkaline phosphatase assays were performed as described above in A. The strains used were wild-type (TN125 with the control vector, pRS426), HC-TPK1 (wild-type carrying the high-copy number TPK1 plasmid, pPHY2056) and atg1Δ (YYK126 with pRS426). C, elevated levels of Ras/PKA signaling activity inhibited the rapamycin-mediated induction of autophagy. The indicated strains were grown to mid-log phase in YM-glucose medium and then treated with 0.2 μg/ml rapamycin for 0, 1, or 3 h. Autophagy levels were then assessed with the alkaline phosphatase-based assay system. The strains analyzed were those used in A.