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. 2019 Apr 11;17(4):e3000219. doi: 10.1371/journal.pbio.3000219

Fig 4. The structured regions in the ATG1 and ATG13 ORFs are necessary for the translational regulation by Dhh1 after nitrogen starvation.

Fig 4

(A) The mutations (“mut”) made in the structured regions of ATG1 and ATG13 ORFs are shown as indicated; mutated nucleotides are presented in black boxes. (B) WT Atg1 (XLY316), WT Atg1 dhh1Δ (XLY317), mutant Atg1 (XLY318), and mutant Atg1 dhh1Δ (XLY319) cells were grown in YPD to mid-log phase (-N, 0 h) and then shifted to SD-N for 6 h. Cell lysates were prepared, subjected to SDS-PAGE, and analyzed by western blot. The quantification of Atg1 protein level was conducted as indicated in Fig 2A. NS, not significant. ****p < 0.0001. (C) WT Atg13–PA (ZYY202), WT Atg13–PA dhh1Δ (ZYY203), mutant Atg13–PA (ZYY205), and mutant Atg13–PA dhh1Δ (ZYY206) cells were grown in YPD to mid-log phase (-N, 0 h) and then shifted to SD-N for 6 h. Cell lysates were prepared, subjected to SDS-PAGE, and analyzed by western blot. The quantification of Atg13–PA protein level was conducted as indicated in Fig 2A. The 5′-UTR and 3′-UTR of ATG13 in these strains were not changed. ****p < 0.0001. (D) The WT strain with empty vector (XLY329), the dhh1Δ strain with either empty vector (XLY331), or vectors expressing WT Dhh1–PA (XLY333) or Dhh1D195A,E196A,S226A,T228A–PA (DEAA STAA; XLY342) were grown in YPD to mid-log phase (-N, 0 h) and then shifted to SD-N for 6 h. Cell lysates were prepared, subjected to SDS-PAGE, and analyzed by western blot. The quantification of the Atg1 protein level was conducted as indicated in Fig 2A. **p < 0.01. ***p < 0.001. (E) The strains in (D) were grown in YPD to mid-log phase (-N, 0 h) and then shifted to SD-N for 24 h. Cell lysates were prepared and subjected to SDS-PAGE. The processing of Pgi1–GFP was analyzed by western blot. The quantification of free GFP level was conducted as indicated in Fig 1D. **p < 0.01. ****p < 0.0001. (See also S5 Fig; raw numerical values are shown in S1 Data). Atg, autophagy-related; DEAA STAA, Dhh1D195A,E196A,S226A,T228A; GFP, green fluorescent protein; L.E., long exposure; NS, not significant; ORF, open reading frame; PA, protein A; Pgi1, phosphoglucoisomerase 1; Pgk1, 3-phosphoglycerate kinase 1; SD-N, synthetic minimal medium lacking nitrogen; S.E., short exposure; WT, wild type; YPD, yeast extract–peptone–dextrose.