Figure 5.
Atg1 kinase activity is required for autophagy. (A) Atg1 mutants are defective for autophagy. The alkaline phosphatase activity from Pho8Δ60, a marker for nonspecific autophagy, was monitored to determine the level of autophagy activity. The wild-type (YTS159) and atg1Δ (UNY1) strains harboring plasmids expressing Atg1, Atg1K54A (Atg1K), Atg1K54A,M102A (Atg1KM), or empty vector (pRS415) were grown in SMD lacking auxotrophic amino acids and shifted to SD-N for 4 h. Samples were collected and protein extracts assayed for alkaline phosphatase activity as described in Materials and Methods. The results represent the mean and SD of three experiments. (B) Atg1 mutants are defective for kinase activity. The atg1Δ mutant (WHY001) cells expressing HA-Atg1, HA-Atg1K54A, or HA-Atg1K54A,M102A were treated with 0.2 μg/ml rapamycin for 2 h. The extract of each mutant was immunoprecipitated with anti-HA antibody, and then immunocomplexes were assayed for Atg1 kinase activity as described in Materials and Methods.