FIGURE 1:

snx4Δpsd1Δ cells display a synthetic autophagy defect. (A) Representative immunoblot analysis of GFP-Atg8 processing in cells incubated with rapamycin (RAP) for 4 h to induce autophagy. Anti-GFP was used to detect GFP-Atg8 and the released GFP proteolytic fragment. Note the large increase in the proportion of full-length GFP-Atg8 to free GFP in snx4Δpsd1Δ cells. Loading control is anti-PGK immunoblot. (B) Quantitation of GFP-Atg8 processing. “Proportion released GFP” is measured as the ratio of free GFP/(free GFP + GFP-Atg8 signal within the same lane). The results from three experiments were averaged and standard error of the mean indicated. The proportion of processed GFP-Atg8 is reduced in snx4Δpsd1Δ cells compared with wild-type or to single mutations. **p < 0.01; ***p < 0.001. (C) Immunoblot analysis of native Atg8. The positions of nonlipidated (Atg8) and lipidated Atg8 (Atg8-PE) are indicated. (D) Quantification of three independent experiments with standard error of the mean is shown in the graph. Anti-PGK immunoblot was used to normalize loads. ***p < 0.001.