Fig. 5.

The putative phosphate sensor of the Atg13 HORMA domain is required for autophagy and PI 3-kinase recruitment. (A) GFP-Atg8 processing assay of atg11Δ atg13Δ cells transformed with ATG13 wild type and mutants designed to disrupt the putative phosphate binding site. GFP-Atg8 processing was monitored by Western blot against GFP. The GFP-Atg8 and GFP bands are labeled. (B) Pho8Δ60 assay to monitor autophagy was performed in SMD (white) and SD-N (gray) using atg11Δatg13Δ cells transformed with the same ATG13 constructs as in A. Samples were normalized to the activity of Atg13 in starved cells. (C) Representative images of atg11Δ atg13Δ ATG14-GFP cells transformed with vector, and ATG13 (as shown also in Fig. 2A) and mutant ATG13 alleles, and treated with rapamycin. (D) Quantification of the Atg14 puncta from C using the procedure described in Fig. 2. A total of three trials with 100 cells counted per trial. Error bars in B and C represent the SD of triplicate experiments.