Figure 1

Yeast with mutations in the GCN2-signaling pathway are defective in autophagy. (A) Western blot analysis of lysates from yeast grown in normal media (YEPD) or N deprivation media lacking amino acids and ammonium sulfate (SD-N), with an eIF2α Ser-51-phosphospecific Ab (1:100 dilution; Research Genetics). (B) Quantitation by DIC microscopy of autophagic body formation in yeast grown in normal growth conditions (open bars) or subjected to 4 h of nitrogen deprivation (black bars), treatment with 0.2 μg⋅ml⁻¹ rapamycin (light gray bars), or treatment with 10 μg⋅ml⁻¹ cycloheximide (dark 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. (C) Representative DIC micrographs (left columns of each panel) and electron micrographs (right columns of each panel) of yeast deprived of nitrogen for 4 h. Arrows in light micrographs denote representative cells which would be scored as positive in experiment shown in B. Arrows in electron micrograph denote representative autophagic bodies within the vacuole. (Scale bars, 0.5 μm.)