The ortholog of human REEP1-4 is required for autophagosomal enclosure of ER-phagy/nucleophagy cargos in fission yeast

Selective macroautophagy of the endoplasmic reticulum (ER) and the nucleus, known as ER-phagy and nucleophagy, respectively, are processes whose mechanisms remain inadequately understood. Through an imaging-based screen, we find that in the fission yeast Schizosaccharomyces pombe, Yep1 (also known as Hva22 or Rop1), the ortholog of human REEP1-4, is essential for ER-phagy and nucleophagy but not for bulk autophagy. In the absence of Yep1, the initial phase of ER-phagy and nucleophagy proceeds normally, with the ER-phagy/nucleophagy receptor Epr1 coassembling with Atg8. However, ER-phagy/nucleophagy cargos fail to reach the vacuole. Instead, nucleus- and cortical-ER-derived membrane structures not enclosed within autophagosomes accumulate in the cytoplasm. Intriguingly, the outer membranes of nucleus-derived structures remain continuous with the nuclear envelope-ER network, suggesting a possible outer membrane fission defect during cargo separation from source compartments. We find that the ER-phagy role of Yep1 relies on its abilities to self-interact and shape membranes and requires its C-terminal amphipathic helices. Moreover, we show that human REEP1-4 and budding yeast Atg40 can functionally substitute for Yep1 in ER-phagy, and Atg40 is a divergent ortholog of Yep1 and REEP1-4. Our findings uncover an unexpected mechanism governing the autophagosomal enclosure of ER-phagy/nucleophagy cargos and shed new light on the functions and evolution of REEP family proteins.

cargo separation from source compartments. We find that the ER-phagy role of Yep1 1 4 relies on its abilities to self-interact and shape membranes, and requires its C-terminal 1 5 amphipathic helices. Moreover, we show that human REEP1-4 and budding yeast 1 6 Introduction 1 In eukaryotes, the endoplasmic reticulum (ER) is an intricate membrane 2 organelle composed of interconnected sheet-like structures and tubular networks 1,2 .

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The formation and maintenance of ER morphology involve two conserved families of 4 integral membrane proteins, the reticulons (RTNs) and the REEP family proteins 3,4 .

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The ER plays a crucial role in many cellular processes, such as protein folding, lipid 6 synthesis, ion homeostasis, and communication with other organelles 5 . Disturbances 7 of ER functions have been implicated in a wide range of human diseases 6 .

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Under starvation and ER stress conditions, portions of the ER are turned over 9 through macroautophagy (hereafter autophagy), in a process termed ER-phagy.

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During ER-phagy, ER membrane fragments are sequestered into autophagosomes, 1 1 which are double-membrane vesicles that deliver cargos to the lysosome/vacuole for 1 2 degradation 7,8,9 . In yeasts, the ER mainly consists of the nuclear envelope and the 1 3 cortical ER 10,11 . Both sub-compartments of the ER can be targeted by ER-phagy. The 1 4 autophagic sequestration of the nuclear envelope may result in the engulfment of 1 5 intranuclear components into autophagosomes. Thus, ER-phagy and nucleophagy 1 6 may occur concurrently 12,13 .

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These results demonstrate that Yep1 shares the membrane-shaping ability of Rtn1, 7 Yop1, and Tts1 and contributes to the maintenance of normal ER structure.

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We assessed whether Rtn1, Yop1, and Tts1 function in ER-phagy. DTT and 9 starvation-induced processing of Erg11-GFP was only slightly diminished in the 1 0 rtn1Δ yop1Δ tts1Δ triple deletion mutant ( Figure S4E), suggesting that ER-phagy still 1 1 occurs in the absence of these three proteins. In addition, overexpression of Rtn1,

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It has been recently shown that the formation of curved-shape homo-oligomer of 2 0 ER-shaping proteins is responsible for generating the tubular membrane shape 54 .

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We used AlphaFold-Multimer to predict the structures of Yep1 2 6 homo-oligomers 55 (Figure 3E and S5B). Regardless of the number of Yep1 2 7 1 4 sequences (from two to eight) in the input, AlphaFold-Multimer only predicted one 1 type of oligomeric structure-the structure of the Yep1 dimer, indicating that the 2 dimer is the preferred oligomerization state of Yep1. In the predicted structure of the 3 Yep1 dimer (Figure 3F), within each Yep1 molecule, there are three long α -helices in 4 the N-terminal region. They largely encompass the three transmembrane segments 5 predicted by TOPCONS (Figures 3G and S5C). The C-terminal cytoplasmic region

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In the result of the co-immunoprecipitation assay, both Yep1-GFP and 1 2 Yep1-mCherry appeared as doublets, and the lower band of Yep1-mCherry was 1 3 co-immunoprecipitated with Yep1-GFP ( Figure 3D). Based on the apparent 1 4 molecular weights, the upper band and the low band likely correspond to the 1 5 full-length protein and an N-terminally cleaved protein, respectively. To assess where 1 6 the cleavage occurred, we expressed two N-terminally truncated form, immunoprecipitation conditions and therefore did not affect self-interaction.

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Yep1 lacking residues 98-113 can no longer support ER-phagy ( Figure 3J). Together, 9 these results suggest that Yep1 self-interaction is important for its membrane-shaping 1 0 ability and imply that the membrane-shaping ability is important for its role in 1 1 ER-phagy. can self-interact and can rescue rtn1Δ tts1Δ yep1Δ to the same extent as full-length 2 3 Yep1 (Figures S6B-S6E).

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HeliQuest analyses of the C-terminal helices and visual inspection of the 2 5 AlphaFold-predicted structure indicated that these two long helices are amphipathic 2 6 helices (APHs), whereas the two upstream short helices do not exhibit obvious 2 7 1 6 amphipathicity 57 (Figures S6F-H). To examine whether the amphipathic nature of 1 these two APHs is functionally important, we substituted three hydrophobic amino 2 acids with aspartates in each APH to disrupt their hydrophobic face 58,59 ( Figure S6I).

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Mutating the first APH substantially weakened, but did not abolish, the ER-phagy 4 function of Yep1 ( Figure 3L). Mutating the second APH slightly weakened the 5 ER-phagy function, while mutating both APHs rendered Yep1 nonfunctional in 6 ER-phagy ( Figure 3L). Together, these results demonstrate that these APHs are 7 redundantly essential for the ER-phagy function of Yep1. TMHs. They all contain APHs in the C-terminal cytoplasmic region. REEP5-6 1 8 subfamily proteins also possess APHs in the N-terminal cytoplasmic region.

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heterologous expression of Atg40 in S. pombe can also rescue the ER-phagy defect of 2 6 epr1Δ, and this rescue requires its AIM ( Figure S7D). Moreover, Atg40 can even 2 7 1 8 rescue the ER-phagy defect of the yep1Δ epr1Δ double mutant (Figure S7E), 1 suggesting that Atg40 fulfills the combined roles of Yep1 and Epr1 in ER-phagy.

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We examined the role of the APHs in Atg40 by truncation analysis.

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To address the question why REEP1-4 subfamily proteins and Atg40 but not ER-phagy function of Yep1 (Figures S7F and S7G)  re-associating with the ER network through homotypic fusion. The first hypothesis 2 2 more readily explains the autophagosomal enclosure defect.

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The best understood ER-phagy factors are ER-phagy receptors, which mediate nucleophagy and ER-phagy, suggesting that these two processes share a common set 2 4 of factors.

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The exact role of Yep1 in ER-phagy/nucleophagy remains unclear. Here, we 2 6 discuss two possibilities based on the requirement of its membrane-shaping ability: Yep1 may remodel cargo membranes, or it may help shaping autophagic membranes.

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These two possibilities are not mutually exclusive.

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In the first possibility, as an integral ER membrane protein, Yep1 may exert its immunoprecipitation coupled with mass spectrometry analysis using Yep1-GFP as 1 0 bait, we found that a large number of ER membrane proteins were 1 1 co-immunoprecipitated with Yep1 (Table S1). Among them are Scs2 and Scs22, two

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In the second possibility, some Yep1 molecules may re-localize from the ER to 2 0 the isolation membrane and play a role in shaping the isolation membrane. We 2 1 speculate that this re-localization may take the route of COPII vesicles, which have 2 2 been shown to transport an integral ER membrane protein to the autophagic 2 3 membranes 65 . If Yep1 functions on the isolation membrane, it begs the question: why 2 4 is Yep1 essential for ER-phagy/nucleophagy but dispensable for bulk autophagy? We 2 5 speculate that one possible explanation is that different types of autophagy may utilize 2 6 different membrane sources for the isolation membrane. As a result, the isolation 2 7 membrane for ER-phagy/nucleophagy may have a protein composition different from 2 8 2 2 the isolation membrane for bulk autophagy, and Yep1 is not important for bulk 1 autophagy because there are other factors playing a similar function on the isolation 2 membrane for bulk autophagy. Another possibility is that the size and shape of 3 ER-phagy/nucleophagy cargos impose a special requirement for the shape of the 4 isolation membrane, and Yep1 is needed to meet this requirement.

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During the preparation and submission of this manuscript, Wang et al. and

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reported the cargo structure accumulation phenotype that we observed in yep1Δ cells, 1 9 likely because they did not examine the localization of nuclear proteins, which 2 0 provides the clearest evidence of this phenotype.

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To investigate the Atg8-interacting ability, Yep1 and Epr1 were fused to GFP as the 2 3 prey, and Atg8 was fused to Pil1-mCherry as the bait 45 . Log-phase cells co-expressing 2 4