Figure 2.
Vac8 vacuolar membrane localization is required for correct PAS localization and robust autophagy activity. (A) The vac8Δ strain (YTS178) was transformed with plasmids expressing wild-type VAC8, empty vector, the myristoylation defective vac8 mutant (vac8-2) the palmitoylation defective vac8 mutant (vac8-3) or the myristoylation and palmitoylation double mutant (vac8-4). The strains were grown in SMD and collected at mid-log phase (OD600 = 0.5) and protein extracts were analyzed by western blot using antiserum against Ape1 as described in Materials and Methods. (B) Autophagy activity was measured with the Pho8Δ60 assay in wild-type (WT), Vac8-GFP, Vac8G2A-GFP, Vac8C4,5,7A-GFP and vac8∆ strains under nutrient-rich conditions (+N) and after 3 h of nitrogen starvation (-N 3 h). Error bars indicate the standard deviation of 3 independent experiments. ANOVA, **P < 0.01; ***P < 0.001. ns, no significance. (C) The protein levels of Vac8 tagged with an auxin-inducible degradation (AID) sequence and a MYC peptide were determined by western blot with anti-MYC after 30 min of auxin (IAA) treatment or control DMSO vehicle in strains co-expressing either Vac8-GFP WT, Vac8-GFP Cyt or Vac8-GFP ER. Pgk1 was used as a loading control, and the asterisk represents a nonspecific band. (D) RFP-Ape1 association with the vacuole was determined using fluorescence microscopy after 1 h of nitrogen starvation in strains expressing Vac8-AID-MYC and the Vac8-GFP constructs described in (C). Strains were treated for 30 min with either IAA to induce Vac8-AID-MYC degradation or DMSO. The vacuolar lumen was stained using CellTracker Blue CMAC. (E) Quantification of panel (D). The percentage of cells in which RFP-Ape1 puncta associated with the vacuole is presented. ANOVA, ***P < 0.001. ns, no significance. (F) Autophagy activity was measured with the Pho8Δ60 assay in strains expressing the Vac8-GFP constructs described in (C), or the vac8∆ strain under nutrient-rich conditions (+N) and after 3 h of nitrogen starvation (-N 3 h). Error bars indicate the standard deviation of 3 independent experiments. ANOVA, *P < 0.05; **P < 0.01. ns, no significance