REEP1-4 subfamily proteins and Atg40 share the same ER-phagy function with Yep1.

(A) Helical wheel representations and the hydrophobic moments (μH) of the 2 C-terminal APHs in Atg40. (B) The alignment of the C-terminal AIM in the proteins whose names are colored red in (C). The AIM core motif is highlighted. (C) Phylogenetic relationships of REEP family proteins and Atg40 proteins in representative Ascomycota species. The sequences of REEP family proteins were retrieved by PSI–BLAST from the NCBI refseq_protein database using the sequences of Yarrowia lipolytica orthologs of S. pombe Yop1 and Yep1 as queries. The sequences of Atg40 proteins were retrieved by PSI–BLAST from the NCBI refseq_protein database using the sequence of S. cerevisiae Atg40 as query. A sequence alignment was generated using MAFFT, and a maximum likelihood tree was constructed using IQ-TREE. The tree was rooted using the REEP5-6 subfamily proteins as outgroup. Branch labels are the SH-aLRT support values (%) and the UFBoot support values (%) calculated by IQ-TREE. The names of proteins containing a C-terminal AIM are colored red. The scale bar indicates 0.8 substitutions per site. (D) S. cerevisiae Atg40 rescued the ER-phagy defect of epr1Δ in a manner dependent on its Atg8-interacting motif (AIM). Atg40^AIMmut harbors the Y242A and M245A mutations. Proteins expressed in yep1Δ were tagged with mCherry, and their expression levels were analyzed by immunoblotting using an antibody against mCherry. (E) S. cerevisiae Atg40 rescued the ER-phagy defect of epr1Δ yep1Δ in a manner dependent on its AIM. Proteins expressed in yep1Δ were tagged with mCherry, and their expression levels were analyzed by immunoblotting using an antibody against mCherry. (F) Schematics of wild-type and truncated Yop1. Yop1 (133–182) appears in Fig 4I. Yop1 [1–52] and Yop1 [35–52] appear in (G). Yop1 (53–182) appears in (H). (G) Adding an extra N-terminal transmembrane helix (TMH) to Yep1 disrupted its ER-phagy function. Yop1 [1–52] includes the N-terminal cytosolic region and the first TMH of Yop1. Yop1 [35–52] includes only the first TMH of Yop1. Proteins expressed in yep1Δ were tagged with mCherry, and their expression levels were analyzed by immunoblotting using an antibody against mCherry. (H) Removing the N-terminal region of Yop1 upstream of its second TMH did not render it capable of substituting for Yep1. Yop1 (53–182) lacks the N-terminal cytosolic region and the first TMH. Proteins expressed in yep1Δ were tagged with mCherry, and their expression levels were analyzed by immunoblotting using an antibody against mCherry. Raw images for panels D, E, G, and H can be found in S1 Raw Images.

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