Figure 8.—
The Atg1T226E variant was defective for autophagy. (A) The ALP-based assay was used to assess autophagy activity in an atg1Δ strain expressing the indicated Atg1 proteins. Extracts were prepared from cells that had been treated with 200 ng/ml rapamycin for either 0 (Log) or 4 hr (Rap). The data shown are the average of two independent experiments where the values obtained varied by less than 15% between replicates. The atg1Δ control strain contained a vector plasmid. (B and C) Autophagy activity was assessed with either the GFP–Atg8 (B) or the Ape1 (C) processing assays as described in materials and methods. In each case, the strains were treated with 200 ng/ml rapamycin for the indicated time (hours). B, bottom, shows the relative levels of the different Atg1 proteins examined. (D) A GFP–Atg8 fusion protein was not delivered to the vacuole in cells expressing only Atg1T226E. The subcellular localization of an GFP–Atg8 fusion protein was assessed by fluorescence microscopy in atg1Δ cells carrying the indicated Atg1 proteins. The cells were treated with 200 ng/ml rapamycin for 2 hr prior to examination. Note that the GFP–Atg8 fusion was localized to the PAS in cells containing the Atg1T226E variant.