The identification and initial characterization of Yep1 as a factor required for ER-phagy and nucleophagy.

(A) Bulk segregant analysis identifying a mutation in SPBC30D10.09c (yep1) as a candidate phenotype-causing mutation in an ER-phagy defective mutant. The scatter plot depicts the reference allele frequencies at SNP sites in the pool of the ER-phagy defective segregants derived from a cross between the mutant strain and a wild-type strain. The T17M mutation in SPBC30D10.09c (yep1) is highlighted in red. (B) Subcellular localization of Yep1-mCherry expressed from the P41nmt1 promoter. Log-phase yep1Δ cells coexpressing Yep1-mCherry and the ER marker Erg11-GFP were examined by fluorescence microscopy. Bar, 5 μm. (C) Yep1-mECitrine formed puncta colocalizing with Epr1 and Atg8 double positive puncta after ER-phagy induction by nitrogen starvation and DTT treatment. Red arrows denote puncta where Yep1-mECitrine, Epr1-mCherry, and mTurquoise2-Atg8 colocalize. Bar, 5 μm. (D) Quantification of the percentage of Epr1 and Atg8 double positive (Atg8+/Epr1+) puncta that are also positive for Yep1 in the analysis shown in (C) (more than 100 Atg8+/Epr1+ puncta were examined for each sample). (E) Autophagic processing of the bulk autophagy marker Tdh1-CFP was largely normal in yep1Δ cells. (F) Electron microscopy analysis of starved and DTT-treated fsc1Δ and fsc1Δ yep1Δ cells. N, nucleus; V, vacuole; A, autophagosome. Double-ring structures are denoted by pink arrows. Bar, 1 μm. (G) Quantification of the number of double-ring structures per cell in the analysis shown in (E) (more than 50 cells with autophagosomes were examined for each sample). (H) Yep1 did not interact with Atg8 in a Pil1 co-tethering assay. Log-phase cells coexpressing the bait (Pil1-mCherry or Pil1-mCherry-Atg8) and the prey Yep1-GFP were examined by fluorescence microscopy. Cells coexpressing Pil1-mCherry-Atg8 and Epr1-GFP served as a positive control. Peripheral planes of the cells were imaged. Bar, 5 μm. (I) Yep1 is not required for the DTT-induced increase of the protein level of Epr1. isp6Δ psp3Δ background, which lacks vacuolar protease activities, was used to prevent the degradation of Epr1. Endogenously tagged Epr1-mCherry was analyzed by immunoblotting. (J) Yep1 did not interact with Epr1 in a Y2H assay. Crb2 served as a specificity control, and the self-interaction of Crb2 and the interaction between Epr1 and Atg8 served as positive controls. (K) Ectopic expression of Erg11-AIM^art but not Yep1 from the P41nmt1 promoter suppressed the ER-phagy defect of epr1Δ. (L) Ectopic expression of Erg11-AIM^art or Epr1 did not rescue the ER-phagy defect of yep1Δ. Numerical data underlying panels A, D, and G can be found in S1 Data, and raw images for panels E, I, K, and L can be found in S1 Raw Images.

(TIF)