ER-phagy and nucleophagy cargos accumulated in the cytoplasm of yep1Δ cells.

(A) Yep1 is not required for the colocalization of Epr1 and Atg8 at punctate structures shortly after ER-phagy induction. Wild-type and yep1Δ cells coexpressing Epr1-mCherry and mTurquoise2-Atg8 were examined by microscopy after 2.5-hour DTT or 1.5-hour starvation treatment. Bar, 5 μm. (B) Quantification of the colocalization between Bqt4 puncta and Pus1 puncta in the analysis shown in Fig 2C (more than 250 puncta were examined for each sample). (C, D) The vast majority of cytoplasmic Pus1 puncta (C) and Bqt4 puncta (D) in yep1Δ cells did not colocalize with Atg8 puncta. Bar, 5 μm. Over 200 Pus1 or Bqt4 puncta were examined per sample. (E) Electron microscopy analysis of nitrogen-starved and DTT-treated wild-type and yep1Δ atg5Δ cells. N, nucleus; V, vacuole; M, mitochondrion; A, autophagosome. Bar, 1 μm. (F) Quantification of the number of cytoplasmic ring-shaped structures in the analysis shown in Figs 2D and S2E (more than 30 cells were examined for each sample). Autophagosomes, which are ring-shaped structures juxtaposed to vacuoles, were excluded from this quantification. (G) Quantification of the diameters of the ring-shaped structures in yep1Δ cells in the analysis shown in Fig 2D and the inner rings in the double-ring structures in fsc1Δ cells in the analysis shown in S1F Fig. P values were calculated using Welch’s t test. Numerical data underlying panels B-D, F, and G can be found in S1 Data.

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