TT foci are cleared by autophagy. (A) Strains were transformed with pRP1657 and examined in mid-log or early stationary phase. White arrows indicate examples of Pab1 autophagic bodies within vacuoles; red arrows indicate empty vacuoles. Data representative of observations in three biological replicates (separate culture and transformant). Scale bar = 2.5 µM. (B) Quantitation of data in panel A; three biological replicates (separate cultures and transformants) with mean ± SD shown. (C) Pab1-GFP Western blot in mid-log cells; GFP fragment (GFP) indicative of autophagic turnover of Pab1-GFP. PGK1 serves as a loading control. Data representative of observations in three biological replicates (separate culture and transformant) (D) Quantitation of data in C; ratio of GFP fragment levels to full length were calculated for each strain and normalized to the WT ratio. Mean ± SD shown. (E) Loss of Atg15 impairs Pab1-GFP turnover both in THO-TREX-2 mutants and during nonselective autophagy (4h–N media). (F) GFP-Atg8 colocalizes with TT foci in mid-log hpr1Δ cells. Blue arrowhead indicates colocalization (see Supplemental Table S5 for quantitation).
