Figure 5.
The YPT6 ARL1 conditional double knockout strain is defective for autophagy at 30°C. The YSA021 strain, containing the ARL1AID allele, or the ypt6Δ strain was transformed with the pRS316-GFP-Atg8 plasmid. Cells were cultured until OD600 = 0.6 and divided into 2 portions. One portion was cultured with 1 mM NAA for 30 min, another one was with 0.17% ethanol alone. Autophagy was induced as described. For the YSA021 strain, after 3 h in SD-N medium, an aliquot of the + NAA culture was washed 3 times by SD-N medium and cells were cultured in SD-N medium without NAA for an additional 3 h. All the samples were collected and subjected to either Western blot with the anti-GFP antibody and anti-FLAG antibody, or live-cell fluorescence microscopy. (A) GFP-Atg8 assay shows the degradation of Arl1 caused the autophagy defect in the ypt6Δ strain background (YSA021) at 30°C. This defect can be reversed by washing out NAA (“washed”). (B) GFP-Atg8 assay shows that adding NAA to the ypt6Δ strain did not affect autophagy. (C) Fluorescence microcopy shows the punctate phenotype of mislocalized GFP-Atg8 after Arl1 was degraded. The diffuse green phenotype of normal autophagy reappeared after NAA was removed. Scale bar: 3 µm.