Back to article: Balanced CoQ6 biosynthesis is required for lifespan and mitophagy in yeast
FIGURE 5: Mitophagy induction but no autophagy is compromised in Ptc7 yeast.
(A) Yeast strains harboring an expression plasmid expressing the ATG8-GFP gene were grown in YPD 16 hours. Then, yeasts were washed and media was replaced by SDc-N with 2% glucose to activate autophagy. 0.5 OD660 nm of Yeast cells were harvested at indicated time points. Protein extracts were analyzed by SDS-PAGE and Western blot using anti-GFP antibody.
(B) Mitophagy analysis by porin degradation. Yeast strains were grown in YPD for 16 hours. Then, yeasts were washed and media was replaced by SDc-N with 2% glucose to activate mitophagy. Protein extracts were analyzed using anti-porin antibody.
(C) Ptc7 overexpression increases the mitophagy induction. Yeast strains expressing the OM45-GFP gene were grown in YPD for 16 hours. Then, yeasts were washed and media was replaced by YP Lactate. Protein extracts were analyzed using anti-GFP antibody for GFP-Om45, an outer membrane mitochondrial protein, and anti-Kar2 antibody for Kar2, an endoplasmic reticulum marker used as control for non-mitochondrial protein degradation. Original film plates used to prepare the panels are shown in Figure S1. Analysis of densitometry of panels B and C are shown in the Figure S2, S3 and S4. Data are mean ± SD, N ≥ 3 independent assays in all the experiments.