Figure 2.

Mammalian pkr promotes autophagy in GCN2-disrupted yeast. (A) Western blot analysis of lysates from N-starved Δgcn2 yeast by using an eIF2α Ser-51-phosphospecific Ab. (B) Quantitative effects of GCN2 and pkr transformation on autophagic body formation in Δgcn2 yeast grown in normal growth conditions (open bars), or subjected to 4 h of nitrogen deprivation (black bars) or treatment with 0.2 μg⋅ml⁻¹ rapamycin (light gray bars). Cells with one or more autophagic bodies within the vacuole were scored as positive. A minimum of 100 cells was counted for each sample for each yeast strain. The results shown represent the mean ± SEM percentage of cells with autophagic bodies within the vacuole for triplicate samples. Similar results were obtained in five independent experiments. Similar results were also observed for five independent clones of pkr-transformed Δgcn2 yeast that displayed normal growth phenotypes in nutrient rich media. (C) Representative light micrographs (Left column) and electron micrographs (Right column) of GCN2- and pkr-transformed Δgcn2 yeast deprived of nitrogen for 4 h. Arrows in light micrographs denote representative cells that would be scored as positive in experiment shown in B. Arrows in electron micrographs denote representative autophagic bodies within the vacuole. (Scale bars, 0.5 μm.)