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. 2022 Apr 10;19(2):457–473. doi: 10.1080/15548627.2022.2059744

Figure 3.

Figure 3.

Multiple ATG5 uORFs mediate Atg5 translation and autophagy in yeast. (A) Schematic representation of partial ATG5 mRNA transcript with 4 potential functional uORFs identified by ribosome profiling (represented by yellow, blue, green, and purple boxes). Each ATG5 uORF has a start codon (AUG) in the 5’ UTR, and a stop codon preceding the ATG5 CDS. (B) The mutations made in the start codon of ATG5 uORFs in the plasmid constructs are shown as indicated (represented as a change from a red triangle to a green triangle). (C) ATG5-PA mRNA levels were determined in WT and ATG5 uORF mutant cells under growing and 1-h nitrogen-starvation conditions by RT-qPCR. Mean±SD of n = 3 independent experiments are shown as indicated. ANOVA; ns: not significant. (D) Atg5-PA levels were measured by western blot in WT and ATG5 uORF mutant cells under growing conditions and after 2 h of nitrogen starvation. Pgk1 was used as a loading control. (E) The ratio of total Atg5-PA to Pgk1 was quantified. Mean±SD of n = 3 independent experiments are shown as indicated. ANOVA; ns: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.005. (F) The ratio of Atg12–Atg5-PA to Pgk1 was quantified. Mean±SD of n = 3 independent experiments are shown as indicated. ANOVA; ns: not significant, *: p < 0.05, **: p < 0.01, ***: p < 0.005. (G) Autophagy activity was measured using the Pho8Δ60 assay in WT, and ATG5 uORF mutant cells under growing conditions and after 3 h of nitrogen starvation. Mean±SD of n = 3 independent experiments are shown as indicated. ANOVA; *: p < 0.05, ns: not significant. (H) Autophagy was measured with the GFP-Atg8 processing assay in WT, and ATG5 uORF mutant strains under growing conditions and after 1.5 h of nitrogen starvation; a representative image is shown. (I) The ratio of free GFP to GFP-Atg8 after 1.5 h of nitrogen starvation was quantified. Mean±SD of n = 3 independent experiments are shown as indicated. ANOVA; *: p < 0.05. SD-N, synthetic minimal medium lacking nitrogen; +N (YPD), yeast extract-peptone-dextrose; WT, wild type; PA, protein A.