Nucleophagy is promoted by two autophagy receptors and inhibited by chromatin-nuclear envelope tethering in fission yeast

Domain: neuroscience
Published: 2026-03-31
Editorial outcome: major_revision

Abstract
Selective autophagy of the nucleus, known as nucleophagy, targets nuclear components for degradation. The molecular mechanisms underlying nucleophagy remain inadequately understood. In this study, we identify a nucleophagy receptor, Npr1, in the fission yeast Schizosaccharomyces pombe. Npr1 is an Atg8-binding multi-transmembrane protein localized to the outer nuclear membrane. It functions redundantly with another autophagy receptor Epr1 to promote nitrogen starvation-induced nucleophagy. In the absence of both Npr1 and Epr1, starved cells exhibit abnormal nuclear morphology and reduced survival. During nucleophagy, the nuclear envelope (NE) forms outward protrusions where Atg8 co-localizes with Npr1 and/or Epr1. These protrusions subsequently detach from the NE, resulting in the formation of autophagosomes that contain nucleophagy cargo. Notably, artificially enhancing chromatin association with the inner nuclear membrane leads to NE protrusions that fail to detach, thereby aborting nucleophagy. Our findings provide mechanistic insights into nucleophagy and suggest that abortive nucleophagy protects chromatin from degradation.

Introduction
Macroautophagy (hereafter referred to as autophagy) plays a crucial role in maintaining intracellular homeostasis by transporting cytoplasmic components to lysosomes or vacuoles for degradation1-3. During autophagy, various cargos, including organelles, are enclosed by an expanding membrane called the phagophore (also known as the isolation membrane), which eventually forms a double-membrane-bound vesicle known as the autophagosome4, 5. The fusion of the autophagosome with the lysosome or vacuole leads to the degradation of its internal contents2, 6. Autophagy can be classified into non-selective and selective types2, 3. Selective autophagy achieves cargo selectivity by employing proteins known as autophagy receptors7. Autophagy receptors share a common feature: their ability to interact with the Atg8/LC3 family of proteins through a sequence termed the Atg8-interacting motif (AIM) or the LC3-interacting region (LIR). The core sequence of the AIM/LIR is WE/F/YxxL/V/I8-10. By simultaneously associating with their cognate cargos and Atg8/LC3 proteins, autophagy receptors establish a physical link between the cargos and the phagophore, thereby promoting selective cargo encapsulation into the autophagosome.

Selective autophagy targets various organelles for degradation, including mitochondria (mitophagy), the endoplasmic reticulum (ER-phagy), and the nucleus (nucleophagy)11. The first nucleophagy receptor, Atg39, was discovered in Saccharomyces cerevisiae and is found exclusively in budding yeasts12. Atg39 is a transmembrane protein localized at the nuclear envelope (NE) and interacts with Atg8 through an AIM in its N-terminal cytosolic tail12. In cells undergoing nucleophagy, Atg39 forms bright Atg8-positive puncta in a manner dependent on its Atg8-binding ability13. While Atg39 is essential for the selective autophagy of proteins within the outer nuclear membrane (ONM), inner nuclear membrane (INM), nucleolus, and nucleoplasm12, 13. it is dispensable for the autophagic degradation of nuclear pore components14-16. Instead, recent studies have identified the nucleoporin Nup159 as an Atg8-binding protein that promotes the autophagic degradation of nuclear pore components in S. cerevisiae14, 15. The selectivity factors participating in the autophagic degradation of nuclear components in other organisms remain largely undefined.

During nucleophagy in S. cerevisiae, histones and DNA are notably absent from nucleophagy-derived autophagosomes13. Proteomic analyses also revealed a significant depletion of histones in the contents of autophagic bodies in S. cerevisiae17. These findings suggest that nucleophagy in S. cerevisiae selectively avoids chromatin. However, it remains unclear how this selective exclusion of chromatin is achieved.

In the fission yeast Schizosaccharomyces pombe, the soluble autophagy receptor Epr1, which is localized to the ER (including the NE) through its interaction with the integral ER membrane proteins Scs2 and Scs22, mediates ER stress-induced ER-phagy and nucleophagy18, 19. However, Epr1 is dispensable for nitrogen starvation-induced ER-phagy and nucleophagy19. In this study, we identify Npr1, a muti-transmembrane protein localized to the ONM, as a nucleophagy receptor that functions redundantly with Epr1 to facilitate the degradation of nuclear components during nitrogen starvation. Our study also reveals that like the situation in S. cerevisiae, the chromatin is excluded from nucleophagy in S. pombe. Remarkably, we found that artificially tethering the chromatin to the INM inhibits nucleophagy at the step of NE fission, suggesting a mechanism that prevents chromatin degradation by inhibiting nucleophagy.

Results
Nitrogen starvation induces autophagy of specific nuclear components in S. pombe
To investigate nucleophagy in S. pombe, we examined whether nitrogen starvation triggers the autophagic degradation of specific nuclear components. For this purpose, we analyzed the autophagic processing of five fluorescent protein-tagged markers localized to distinct nuclear subcompartments: Pus1-mECitrine, a nucleoplasmic protein; mECitrine-Bqt4, an inner nuclear membrane protein; mECitrine-Nup82, a nucleoporin; Ker1-mECitrine, a nucleolar protein; and H4-mECitrine, a histone protein associated with chromatin. In wild-type cells, but not in the autophagy-defective atg5Δ cells, substantial levels of Pus1-mECitrine (Fig. 1a), mECitrine-Bqt4 (Fig. 1b), mECitrine-Nup82 (Fig. 1c), and Ker1-mECitrine (Fig. 1d) were processed into free mECitrine after 24 hours of nitrogen starvation. In contrast, the chromatin marker H4-mECitrine showed no evident processing in wild-type cells (Fig. 1e). These findings indicate that nitrogen starvation in S. pombe induces the autophagic degradation of the nucleoplasm, nuclear envelope, nuclear pores, and nucleolus, but not chromatin.

Identification of Npr1 as a candidate nucleophagy receptor
To identify autophagy receptors involved in nitrogen starvation-induced selective autophagy in S. pombe, we employed the TurboID-based proximity labeling technique to search for Atg8-binding proteins20. Mass spectrometry analysis of biotin-labeled proteins from nitrogen-starved cells expressing TurboID-tagged Atg8 revealed the enrichment of an uncharacterized protein SPCC70.04c (Fig. 2a and Supplementary Data 1), which we named Npr1 (for nucleophagy receptor). Co-immunoprecipitation and yeast two-hybrid analyses showed that Npr1 indeed interacts with Atg8 (Fig. 2b, c). Co-immunoprecipitation data revealed that Npr1 associates with Atg8 to a similar extent under both nitrogen-replete and nitrogen-starvation conditions (Fig. 2b), indicating that the Npr1–Atg8 interaction is not dependent on nitrogen starvation.

A proteome-wide study had previously shown that Npr1, when overexpressed, localizes to the nuclear envelope21. Consistently, we found that endogenously fluorescent protein-tagged Npr1 exclusively localized to the nuclear envelope in log-phase vegetative cells (Fig. 2d, e). Notably, its distribution on the nuclear envelope was uneven, with a tendency to avoid overlap with nuclear pores (Fig. 2e). The underlying reason for this uneven distribution remains unclear.Membrane protein prediction analysis indicated that Npr1 is an integral membrane protein containing four transmembrane helices, with both its N- and C-terminal tails facing the cytoplasm or nucleoplasm (Supplementary Fig. 2a)22. To experimentally assess the membrane topology, we established a split-GFP assay (Supplementary Fig. 2b)23-25. This assay utilizes two fragments of GFP, GFP1-10 and GFP11, which can assemble into a fluorescent protein if present in the same cellular compartment. By fusing GFP1-10 to proteins of known localization (cytosolic Sum3 or ER luminal Gbs1) and fusing GFP11 to either terminus of Npr1, we found that fluorescence was observed only when the Npr1 fusion constructs were co-expressed with the cytosolic reporter, indicating that both termini of Npr1 face the cytosol (Supplementary Fig. 2b). This orientation suggests that Npr1 localizes to the outer nuclear membrane, a notion supported by electron microscopy (EM) analysis using a genetically encoded EM tag, MTn26, which showed that MTn-tagged Npr1 was distributed along the outer nuclear membrane (Supplementary Fig. 2c).

To further investigate the interaction between Npr1 and Atg8, we utilized AlphaFold2-Multimer, a tool that effectively predicts binding interfaces between Atg8 and its interacting proteins27, 28. The predicted structure of the Atg8-Npr1 complex revealed that Npr1 employs a canonical Atg8-interacting motif (AIM), specifically 22WIDV25, to bind Atg8 (Supplementary Fig. 2d). Mutations of key AIM residues (W22A/V25A) disrupted Npr1's interaction with Atg8, as demonstrated by both co-immunoprecipitation and yeast two-hybrid analyses (Fig. 2b, c). These findings demonstrate that Npr1 interacts with Atg8 through an AIM located in its N-terminal tail facing the cytosol (Fig. 2f, g).

Under nitrogen starvation conditions, wild-type Npr1, but not its AIM-mutated variant, formed bright puncta that colocalized with Atg8 at the nuclear envelope within 2 hours (Fig. 2h). After 6 hours of starvation, wild-type Npr1 showed pronounced vacuolar re-localization, while the AIM-mutated version exhibited significantly reduced vacuolar targeting (Fig. 2i, j).

Taken together, these results suggest that Npr1 is a candidate nucleophagy receptor. It localizes to the outer nuclear membrane, binds Atg8 via an AIM in its cytosolic N-terminal tail, and undergoes nitrogen starvation-induced vacuolar re-localization in an Atg8-binding-dependent manner.

Npr1 and Epr1 are redundantly required for starvation-induced nucleophagy
To investigate the role of Npr1 in nucleophagy, we examined the effects of npr1 deletion on the autophagic processing of various nuclear components during nitrogen starvation. Deletion of npr1 did not substantially affect the processing of the nucleoplasmic protein Pus1-mECitrine, the inner nuclear membrane protein mECitrine-Bqt4, the nucleolar protein Ker1-mECitrine, or the nucleoporin mECitrine-Nup82 (Fig. 3a and Supplementary Fig. 3a). However, overexpression of Npr1 from the P41nmt1 promoter enhanced the autophagic processing of Pus1-mECitrine, particularly at the early time points of nitrogen starvation, suggesting that Npr1 has a nucleophagy-promoting function (Supplementary Fig. 3b).

We hypothesized that the absence of a nucleophagy defect in the npr1Δ mutant may be due to redundancy with another autophagy receptor. In S. pombe, the ER-phagy receptor Epr1 has been shown to be essential for ER-phagy and nucleophagy under ER stress but not during nitrogen starvation18, 19. Thus, we reasoned that Npr1 and Epr1 may act redundantly under nitrogen starvation conditions. Indeed, deletion of both npr1 and epr1, but not epr1 alone, nearly completely abolished the nitrogen starvation-induced processing of Pus1-mECitrine, mECitrine-Bqt4, and Ker1-mECitrine (Fig. 3a and Supplementary Fig. 3a). Interestingly, the processing of the nucleoporin mECitrine-Nup82 remained unaffected in the epr1Δ npr1Δ mutant (Fig. 3a and Supplementary Fig. 3a). These results indicate that Npr1 and Epr1 redundantly function as nucleophagy receptors, promoting the autophagic degradation of nucleoplasmic, nuclear envelope, and nucleolar components, but not nuclear pores. This selectivity mirrors the situation in S. cerevisiae, where specialized autophagy receptors mediate nucleoporin degradation14, 15.

Consistent with their redundant roles, re-introducing either Npr1 or Epr1 into the epr1Δ npr1Δ mutant fully restored the autophagic processing of Pus1-mECitrine and mECitrine-Bqt4 (Fig. 3b and Supplementary Fig. 3c). In contrast, introducing AIM-mutated Npr1 (Npr1-W22A/V25A, abbreviated as Npr1*) or AIM-mutated Epr1 (Epr1-F352A/V355A, abbreviated as Epr1*) failed to rescue the nucleophagy defects (Fig. 3b and Supplementary Fig. 3c), demonstrating that the nucleophagy function of Npr1 and Epr1 depends on their abilities to bind Atg8. Using this re-introduction assay, we found that MTn-fused Npr1 and GFP11-fused Npr1 retain nucleophagy function (Supplementary Fig. 3d).

To further investigate the redundant relationship between Epr1 and Npr1, we tested whether the loss of one affects the expression level of the other. We found that the loss of Npr1 did not impact the protein level of Epr1, and conversely, the loss of Epr1 did not affect the protein level of Npr1 after 8 hours of nitrogen starvation in the isp6Δ psp3Δ background, which blocks vacuolar degradation (Supplementary Fig. 3e).

Given that Epr1 localizes to both the cortical ER and nuclear envelope18, while Npr1 is exclusively localized to the nuclear envelope, we hypothesized that only Epr1 contributes to cortical ER-phagy. Consistent with this idea, nitrogen starvation-induced autophagic processing of the cortical ER membrane protein Rtn1-mECitrine was moderately reduced in the epr1Δ mutant but was unaffected in the npr1Δ mutant (Supplementary Fig. 3f, g). Furthermore, the additional deletion of npr1 did not exacerbate the mild phenotype observed in the epr1Δ mutant (Supplementary Fig. 3f, g). These results suggest that other, yet unidentified, ER-phagy receptors may act redundantly with Epr1 during nitrogen starvation-induced cortical ER-phagy.

We previously showed that an artificial AIM (AIMart) fused to the ER membrane protein Erg11 could functionally substitute for Epr1 in ER stress-induced ER-phagy18. To determine whether the nucleophagy functions of Npr1 and Epr1 could similarly be replaced, we employed the same AIMart, composed of three tandem copies of the EEEWEEL sequence29, 30. Substituting the N-terminal 29 amino acids of Npr1 (which contains its AIM) with AIMart generated a chimeric protein, AIMart-Npr1(30-244), which successfully rescued the nucleophagy defect of the epr1Δ npr1Δ mutant (Fig. 3c), indicating that AIMart can functionally substitute for the AIM of Npr1.

We next fused AIMart to various integral membrane proteins present on the NE. Only the ONM protein Kms1 and the ER membrane protein Erg11 with cytosol-facing AIMart fusions rescued the nucleophagy defect of epr1Δ npr1Δ (Fig. 3c and Supplementary Fig. 3h). In contrast, fusion of AIMart to the lumen-facing tails of these proteins, or to the nucleoplasm-facing N-terminus of the INM protein Man1 or the cytosol-facing C-terminus of the cortical ER membrane protein Rtn1, failed to rescue the defect. These findings indicate that the nucleophagy roles of Npr1 and Epr1 can be substituted by a membrane protein localized at the ONM, provided it possesses an AIM that faces the cytosol.

Taken together, these results demonstrate that Npr1 and Epr1 redundantly promote nitrogen starvation-induced nucleophagy by establishing a physical link between the ONM and Atg8. This link is mediated by their AIMs, which are essential for their function.

Spatiotemporal analysis of Atg8 and nucleophagy receptors at the NE
To further investigate the mechanisms of nucleophagy, we used live-cell imaging to study the genetic interdependency, co-localization, and temporal dynamics of Atg8, Npr1, and Epr1. First, we observed that Atg8 puncta still formed at the NE in epr1Δ npr1Δ cells, albeit at slightly lower levels than in wild-type cells (Supplementary Fig. 4a, b). Conversely, NE-localized Npr1 and Epr1 puncta were completely absent in atg8Δ cells, indicating that their formation strictly relies on Atg8 (Supplementary Fig. 4c, d).

Next, we analyzed the co-localization patterns of Atg8, Npr1, and Epr1 at the NE using time-lapse imaging of wild-type cells (Fig. 4). We classified NE-localized Atg8 puncta into four types based on their overlap with Npr1 and Epr1 for at least one time point: Type I (36%), which overlapped with both; Type II (55%), with only Npr1; Type III (8%), with only Epr1; and Type IV (1%), with neither (Fig. 4a, e and Supplementary Fig. 4e, f). Thus, in wild-type cells, nearly all NE-localized Atg8 puncta overlapped with receptor puncta, suggesting that they represent nucleophagy events. The fact that over half of the NE-localized Atg8 puncta overlapped with only one receptor indicates that Npr1 and Epr1 can function independently, consistent with genetic data showing that either receptor is sufficient for nucleophagy.

We analyzed the sequential appearance and disappearance of Npr1, Epr1, and Atg8 at types I-III NE-localized Atg8 puncta (Fig. 4b-d). For type I puncta, Atg8 and Npr1 appeared simultaneously and before Epr1 in 50% of cases, while Atg8 appeared before both receptors in 34%. Regarding disappearance, Epr1 disappeared first in 47% of cases, while puncta of all three proteins disappeared simultaneously in 22%. Among type II puncta, Atg8 and Npr1 appeared simultaneously in 71% of cases, with Atg8 disappearing first in 67%. For type III puncta, Atg8 appeared first in 73% of cases, with Atg8 and Epr1 disappearing simultaneously in 73%. These results indicate that the sequential orders of the appearance and disappearance of Atg8, Npr1, and Epr1 puncta are not fixed.

All analyzed NE-localized Npr1 and Epr1 puncta (72 and 52, respectively) exhibited overlap with Atg8, consistent with the absence of these puncta in atg8Δ cells (Supplementary Fig. 4g). Overall, in 64% of cases, Atg8 appeared simultaneously with the receptors, while in 26%, Atg8 preceded receptor appearance. The rare cases (10%) where Npr1 preceded visible Atg8 puncta suggest that Npr1 puncta formation may require only low levels of Atg8. Notably, Npr1 puncta often persisted after Atg8 disappearance, indicating that their stability does not require continuous high levels of Atg8.

Nucleophagy receptors promote NE protrusion formation
During Atg39-mediated nucleophagy in S. cerevisiae, the NE forms outward protrusions13, 16, 31. To investigate whether NE protrusions also form during nucleophagy in S. pombe, we imaged nitrogen-starved cells expressing Npr1-mCherry and the INM protein mECitrine-Bqt4 (Fig. 4f). Time-lapse analysis revealed that in 52% of cases, Npr1 puncta formation was followed by a short outward projection of the mECitrine-Bqt4 signal at the same position, indicating NE protrusion formation (Fig. 4f, g). Notably, we did not observe any instances where NE protrusions formed before the emergence of Npr1 puncta at the same sites (Fig. 4h). Furthermore, the nucleoplasmic protein Pus1-mTurquoise2 co-localized with Bqt4 projections, indicating the presence of nucleoplasmic components within the NE protrusions (Supplementary Fig. 4h).

Similar observations were made with Epr1, where time-lapse analysis of cells expressing Epr1-ymScarlet2I and mECitrine-Bqt4 showed that in 54% of cases, the formation of NE-localized Epr1 puncta was associated with the subsequent formation of NE protrusions at the same sites (Supplementary Fig. 4i-k). These data suggest that the assembly of Npr1 and Epr1 puncta at the NE frequently precedes and potentially initiates the formation of NE protrusions.

Next, we investigated the relationship between Atg8 puncta and NE protrusions during nucleophagy. Time-lapse imaging of cells expressing ymScarlet2I-Atg8 and mECitrine-Bqt4 revealed that the formation of NE-localized Atg8 puncta was frequently followed by the formation of NE protrusions at the same locations (Supplementary Fig. 4n-p). Electron microscopy analysis confirmed the presence of NE protrusions in nitrogen-starved cells (Fig. 4k and Supplementary Fig. 4s), revealing that these NE protrusions were closely surrounded by membranes that terminated at the necks of the protrusions—likely representing phagophores where lipidated Atg8 resides.

To assess the role of nucleophagy receptors in NE protrusion formation, we compared the frequency of Atg8-positive NE protrusions in wild-type and epr1Δ npr1Δ cells. We found that 40% of NE-localized Atg8 puncta were associated with NE protrusions in wild-type cells, compared to only 4% in epr1Δ npr1Δ cells (Supplementary Fig. 4o). These findings indicate that nucleophagy receptors play a critical role in promoting the formation of NE protrusions during nucleophagy.

Distinct dynamics of nucleophagy components during NE protrusion release
To investigate the fate of NE protrusions formed during nucleophagy, we analyzed time-lapse imaging data, focusing on sites where Atg8 and nucleophagy receptors assembled into puncta (Fig. 4f, i, j and Supplementary Fig. 4i, l-n, q, r). Using the INM protein mECitrine-Bqt4 as an NE marker, we observed that the disappearance of an NE protrusion was often accompanied by the emergence of a nearby Bqt4-labeled punctum in the cytosol (Fig. 4f). This suggests that NE protrusions are released into the cytosol.

Next, we investigated the dynamics of Npr1, Epr1, and Atg8 puncta in relation to NE protrusion release. For Npr1 puncta associated with NE protrusions in wild-type cells, 56% exhibited simultaneous release with the NE protrusion into the cytosol(Fig. 4i). Of the remaining 44%, 27% disappeared simultaneously with the NE protrusion, and 17% disappeared after the protrusion had vanished, without subsequent detection of cytosolic puncta. The released Npr1 puncta likely represent autophagosomes that have fully engulfed nuclear cargo and detached from the NE during nucleophagy. To assess whether the disappearance of Npr1 puncta represents entry into vacuoles, we examined the fate of Npr1 puncta and NE protrusions in fsc1Δ background, where autophagosome-vacuole fusion is blocked32. We found that the fates of NE protrusion-associated Npr1 puncta in fsc1Δ cells were similar to those in wild-type cells, with 50% exhibiting simultaneous release with the NE protrusion into the cytosol, 30% disappearing simultaneously with the NE protrusion, and 20% vanishing after the protrusion disappeared (Fig. 4i). These results suggest that the disappearance of Npr1 puncta likely represents premature termination of nucleophagy, rather than entry into vacuoles. Notably, Npr1 puncta not associated with NE protrusions were never observed to be released into the cytosol (Fig. 4j), suggesting that NE protrusion formation is essential for successful nucleophagy-related autophagosome generation.

Epr1 puncta associated with NE protrusions exhibited three patterns: (1) simultaneous release with NE protrusions into the cytosol (30%); (2) disappearance before NE protrusion release (25%); and (3) disappearance of both from the NE without cytosolic puncta detection (45%, including 36% simultaneous disappearance and 9% NE protrusion disappearance first) (Supplementary Fig. 4l). Similar to Npr1, Epr1 puncta without associated NE protrusions were never released into the cytosol (Supplementary Fig. 4m).

The behavior of Atg8 puncta associated with NE protrusions differed notably—none were released simultaneously with NE protrusions into the cytosol. In 53% of instances, Atg8 puncta disappeared from the NE before the release of NE protrusions. In the remaining 47%, both disappeared from the NE, either simultaneously (20%) or with Atg8 puncta disappearing first (27%) (Supplementary Fig. 4q). Atg8 puncta not associated with NE protrusions were never observed to be released into the cytosol (Supplementary Fig. 4r).

Taken together, these findings reveal distinct behaviors among nucleophagy components during NE protrusion release. Atg8 typically disappears from the NE prior to protrusion release, while Npr1 tends to be released concurrently. Epr1 shows an intermediate pattern, being released together with NE protrusions in about half of the protrusion release events. Since NE protrusion release likely follows NE fission and autophagosome closure, these findings suggest that Atg8 is usually recycled before autophagosome closure, likely through Atg4- mediated delipidation.33 It is important to note that the observed “disappearance” of Atg8 does not imply that all Atg8 molecules are recycled, but rather that the level of Atg8 associated with the closed autophagosome falls below the detection limit of microscopy. Conversely, Npr1, being an integral membrane protein, always remains within autophagosomes. As a peripheral membrane protein, Epr1 may escape from autophagosomes before their closure. Importantly, we observed two types of premature termination of nucleophagy in wild-type cells: the presence of nucleophagy receptor puncta without associated NE protrusions and the disappearance of NE protrusions without detectable cytosolic puncta, indicating that nucleophagy can abort naturally at either abort naturally at NE protrusion formation or release.

Npr1- and Epr1-mediated nucleophagy maintains nuclear morphology and survival during nitrogen starvation
It is known that S. cerevisiae mutants defective in nucleophagy exhibit abnormal nuclear morphology during nitrogen starvation12, and that S. pombe autophagy mutants display aberrant nuclear morphology during meiosis, which is induced by nitrogen starvation34. To investigate the role of nucleophagy in maintaining normal nuclear morphology during nitrogen starvation in S. pombe, we examined NE morphology using the INM protein mECitrine-Bqt4 as a marker.

Under nutrient-rich conditions, NE morphology appeared normal in both atg5Δ and epr1Δ npr1Δ mutants (Fig. 5a and Supplementary Fig. 5a, b). However, after 24 hours of nitrogen starvation, these mutants exhibited striking NE aberrations characterized by projections with intensified mECitrine-Bqt4 signals. Other autophagy mutants, including atg1Δ, atg13Δ, atg9Δ, atg14Δ, atg2Δ, atg18aΔ, and atg18bΔ, also exhibited NE aberrations after nitrogen starvation (Supplementary Fig. 5a, b). The abnormal NE structures fell into two categories: extended NE projections, where the projections extend away from the NE, and ring-shaped NE projections, where both ends of a projection are associated with the NE. In atg5Δ, atg13Δ, atg9Δ, atg14Δ, atg2Δ, atg18aΔ, and atg18bΔ cells, extended NE projections were more frequently observed than ring-shaped NE projections, whereas the epr1Δ npr1Δ and atg1Δ mutants predominantly displayed ring-shaped projections. Neither type of aberration was observed in nitrogen-starved wild-type, epr1Δ, or npr1Δ cells (Fig. 5a and Supplementary Fig. 5a, b). This suggests that nucleophagy redundantly promoted by Npr1 and Epr1 is required for maintaining normal nuclear morphology during nitrogen starvation. Supporting this, re-introducing either Npr1 or Epr1, but not their AIM-mutated forms, into the epr1Δ npr1Δ mutant restored normal NE morphology (Fig. 5a and Supplementary Fig. 5a, b). Re-introducing either artificial nuclear envelope-Atg8 tethers, AIMart-Kms1 or Erg11-AIMart, into the epr1Δ npr1Δ mutant also restored normal NE morphology (Supplementary Fig. 5a, b).

To further characterize these NE projections, we examined the localization of the nucleoplasmic protein Pus1-mECitrine after 24 hours of nitrogen starvation (Fig. 5b). Pus1-mECitrine colocalized with mCherry-Bqt4 at the NE projections in both atg5Δ and epr1Δ npr1Δ cells, indicating that these projections are nucleoplasm-containing structures (Fig. 5b). The intensified Bqt4 signals observed at these projections likely result from the presence of two layers of NE sandwiching a thin layer of nucleoplasm.

To visualize the ultrastructures of the NE projections, we employed focused ion beam-scanning electron microscopy (FIB-SEM) (Fig. 5c). Wild-type cells exhibited circular-shaped nuclei in FIB-SEM slices and spherical-shaped nuclei in three-dimensional reconstructions (Fig. 5c and Supplementary Movie 1). In stark contrast, and consistent with our light microscopy observations, epr1Δ npr1Δ and atg5Δ cells exhibited aberrant nuclei with prominent projections in FIB-SEM slices, and these projections contain two closely spaced layers of NE sandwiching nucleoplasm (Fig. 5c). The space within the ring-like profile of the projections is cytoplasm, as evidenced by the presence of cytoplasmic membrane organelles, most prominently vacuoles (Fig. 5c, d). Since an S. pombe cell contains numerous small vacuoles dispersed throughout the cytoplasm, the presence of vacuoles within the ring-like profiles of NE projections may simply result from this; however, we cannot rule out the possibility of a preferential spatial relationship between NE projections and vacuoles. Additionally, three-dimensional reconstruction revealed that the ring-shaped NE projections observed under light microscopy and in FIB-SEM slices correspond to cross-sections of dome-like or semi-dome-like structures extending from the NE surface (Fig. 5c and Supplementary Movies 2, 3). The interiors of these dome-like or semi-dome-like structures often contained a few vacuoles (Supplementary Movies 2, 3).

To further investigate the changes in the NE in epr1Δ npr1Δ and atg5Δ cells compared to wild-type cells, we quantified the volume of the nucleus, as well as the surface area and volume of the NE from FIB-SEM slices. Our analysis revealed that the volume of the nucleus remained unchanged in epr1Δ npr1Δ and atg5Δ cells compared to wild-type cells. However, the surface area and volume of the aberrant NE in these mutants were significantly increased compared to wild-type cells (Supplementary Fig. 5c). These findings suggest that nucleophagy plays a crucial role in maintaining normal NE morphology during nitrogen starvation, and that nuclear deformation results in NE expansion without altering nuclear volume in nucleophagy-deficient cells.

Next, we investigated the contribution of nucleophagy to cell survival during nitrogen starvation using spot assays (Fig. 5e). After 3 days of nitrogen starvation, both epr1Δ npr1Δ and atg5Δ mutants showed reduced survival compared to wild-type cells, while epr1Δ or npr1Δ mutants were unaffected. This survival defect in epr1Δ npr1Δ cells was rescued by re-introducing either Npr1 or Epr1, but not their AIM-mutated forms. Re-introduction of either artificial nuclear envelope-Atg8 tethers, AIMart-Kms1 or Erg11-AIMart, into the epr1Δ npr1Δ mutant also rescued the survival defect (Supplementary Fig. 5d).

Collectively, these results demonstrate that Npr1- and Epr1-mediated nucleophagy plays essential roles in maintaining normal nuclear morphology and promoting cell survival during nitrogen starvation in S. pombe.

Inhibition of nucleophagy by Lem2 overexpression
During our examination of various INM proteins as NE markers, we made a serendipitous discovery that the INM protein Lem2, when expressed exogenously from a medium-strength promoter (P41nmt1), nearly completely blocked the autophagic processing of Npr1-mECitrine and Pus1-mECitrine (Fig. 6a). Notably, this inhibition was specific to cargos of Npr1- and Epr1-mediated nucleophagy, as no effect was observed on the autophagic processing of the nucleoporin mECitrine-Nup82 (Supplementary Fig. 6a) and the cortical ER membrane protein Rtn1-mECitrine (Supplementary Fig. 6b). These findings suggest that elevated Lem2 expression selectively disrupts nucleophagy. We decided to investigate this phenomenon, as it implies the existence of a regulatory constraint on nucleophagy.

To determine the level of Lem2 required to inhibit nucleophagy, we expressed Lem2-mCherry using four different promoters with increasing expression strengths: P81nmt1 < Padf1 < P41nmt1 < Pcyc1 (Supplementary Fig. 6c, d). Interestingly, even a modest level of exogenous Lem2 expression (2-4-fold of the endogenous level) driven by the weakest promoter, P81nmt1, resulted in observable, albeit moderate, inhibition of nucleophagy. Expression from stronger promoters (Padf1 and above), at levels 8-16-fold or higher than the endogenous level, resulted in near-complete inhibition of nucleophagy. These findings indicate that Lem2 inhibits nucleophagy in a dose-dependent manner.

To explore how this inhibition occurs, we examined the localization of the nucleophagy receptor Npr1 in Lem2-overexpressing cells. After 8 hours of nitrogen starvation, wild-type cells showed Npr1-mECitrine primarily in the vacuole lumen, while both atg5Δ and Lem2-overexpressing cells retained Npr1-mECitrine at the NE (Fig. 6b). Notably, Lem2-overexpressing cells displayed NE-localized Npr1-mECitrine puncta, unlike atg5Δ cells where no such puncta were observed. This indicates that Lem2 overexpression permits the initial phase of nucleophagy—Npr1 puncta formation—but interferes with later stages. Similar to wild-type cells, Npr1-mECitrine puncta in Lem2-overexpressing cells were often associated with NE protrusions (Fig. 6b), which also contained the nucleoplasmic protein Pus1-mECitrine (Supplementary Fig. 6e). Electron microscopy revealed that NE protrusions in Lem2-overexpressing cells were surrounded by phagophores (Fig. 6c and Supplementary Fig. 6f), indicating they are nucleophagy intermediate structures.

Time-lapse imaging showed that in both wild-type and Lem2-overexpressing cells, NE-localized Npr1 puncta were dynamic structures with lifetimes of several minutes (Fig. 6d and Supplementary Fig. 6h). These puncta were often associated with NE protrusions that formed after the puncta appeared (Fig. 6d, e and Supplementary Fig. 6g). However, while more than half of the NE protrusions in wild-type cells eventually detached from the NE and were released into the cytosol (Fig. 4i, 6f), NE protrusions in Lem2-overexpressing cells never detached. Instead, they disappeared from the NE without being released into the cytosol (Fig. 6d, f). In most cases, NE protrusions disappeared simultaneously with Npr1 puncta, although in 23% of instances, NE protrusions disappeared before Npr1 puncta (Fig. 6e). These findings indicate that Lem2 overexpression allows nucleophagy to progress to the stage of NE protrusion formation but blocks the subsequent step of protrusion release.

To further explore this phenomenon, we analyzed the behavior of Atg8 in Lem2-overexpressing cells. While Atg8 and Npr1 co-localized at NE puncta, appearing simultaneously in most cases (Supplementary Fig. 6h-j), Atg8 puncta consistently disappeared from the NE before Npr1 puncta (Supplementary Fig. 6j). Furthermore, Atg8 puncta always disappeared before the NE protrusions (Supplementary Fig. 6k), suggesting that components of the autophagy machinery dissociate before the retraction of NE protrusions during the abortive nucleophagy process.

Chromatin-INM tethering inhibits nucleophagy
To investigate how Lem2 overexpression inhibits nucleophagy, we analyzed which regions of Lem2 are critical for this effect. Lem2 contains an N-terminal LEM domain (amino acids 1-60) that binds DNA35, a Bqt4-binding motif (BBM, amino acids 261-279) that localizes Lem2 to the NE by interacting with the INM protein Bqt436, and two transmembrane helices (Fig. 7a).

Using truncation analysis, we found that an N-terminal soluble fragment of Lem2 (amino acids 1-279), which includes the LEM domain and BBM, was sufficient to inhibit nucleophagy, whereas fragments lacking either domain were ineffective (Supplementary Fig. 7a). Internal deletion analysis confirmed that both domains are necessary for nucleophagy inhibition (Supplementary Fig. 7a). Furthermore, a fusion construct containing just the LEM domain and BBM inhibited nucleophagy, while neither domain alone was sufficient (Fig. 7b). These results establish that the combination of the LEM domain and BBM represents the minimal functional unit required for nucleophagy inhibition.

Because the LEM domain binds DNA and the BBM binds Bqt4, we hypothesized that their combination tethers chromatin to the INM, where Bqt4 resides. To test this hypothesis, we substituted the BBM in the fusion construct with the C-terminal transmembrane helix (TM, amino acids 412-432) of Bqt4, a single-pass INM protein with its N-terminus facing the nucleoplasm37. This LEM-Bqt4(TM) fusion localized to the NE (Supplementary Fig. 7d) and effectively inhibited nucleophagy (Fig. 7c, d), suggesting that the role of the BBM in nucleophagy inhibition is to mediate INM association.

To determine whether the LEM domain’s role is chromatin association, we tested whether it could be replaced by other chromatin-binding domains. We substituted the LEM domain with either the histone-binding bromodomain (BD1) of S. pombe Bdf1 or the non-specific DNA-binding protein Sso7d from Archaea (Fig. 7c, d)38, 39. Fusion proteins containing BD1 or Sso7d, along with the TM of Bqt4, localized to the NE (Supplementary Fig. 7d) and effectively inhibited nucleophagy (Fig. 7d). Notably, when the histone-binding ability of BD1 was disrupted by a point mutation (Y123F, BD1*)40, or the DNA-binding ability of Sso7d was weakened by two mutations (W24A and R43E, Sso7d*)41, these fusion proteins no longer strongly inhibited nucleophagy (Fig. 7d). These results indicate that the main role of the LEM domain in nucleophagy inhibition is to mediate chromatin association.

To further solidify the role of chromatin-INM tethering, we tested Sso7d fused to the multi-transmembrane INM protein Bqt3, a binding partner of Bqt437. This fusion also effectively inhibited nucleophagy (Supplementary Fig. 7b, d). Notably, even though the known physiological functions of Bqt3 and Bqt4 require their interaction with each other37, 42, nucleophagy inhibition by Sso7d-Bqt3 occurred in the absence of bqt4 and nucleophagy inhibition by Sso7d-Bqt4(TM) occurred in the absence of bqt3 (Supplementary Fig. 7b-d), suggesting that nucleophagy inhibition does not depend on Bqt3-Bqt4 interaction. Remarkably, the Sso7d-Bqt3 fusion protein was able to inhibit nucleophagy even when expressed from much weaker promoters, including the P81nmt1 promoter (Supplementary Fig. 7e).

To rule out the possibility that nucleophagy inhibition depends on specific functions of Bqt4(TM) or Bqt3 beyond their INM localization, we employed an artificial INM protein h2NLS-LR2-WALP23, consisting of the NLS of budding yeast Heh2, a random linker sequence LR2, and an artificial transmembrane helix WALP2343. Fusion proteins combining BD1 or Sso7d with h2NLS-LR2-WALP23 inhibited nucleophagy, whereas fusion proteins with the mutated forms BD1* or Sso7d* did not, despite all constructs localizing to the NE (Supplementary Fig. 7 f, g). This definitively demonstrates that simply tethering chromatin to the INM is sufficient to inhibit nucleophagy.

A key feature of nucleophagy inhibition by Lem2 overexpression is the blockage of NE protrusion release into the cytosol (Fig. 6h). We hypothesized that this blockage is caused by excessive chromatin tethering to the INM, leading to the presence of chromatin in the NE protrusions. Analysis of histone H3 localization revealed its presence in 7% of Npr1 puncta-associated NE protrusions in wild-type cells, increasing to 63% in cells overexpressing Lem2 (Fig. 7e). Similarly, H3 was frequently detected in Npr1 puncta-associated NE protrusions in cells expressing Sso7d-h2NLS-LR2-WALP23 but not in cells expressing Sso7d*-h2NLS-LR2-WALP23 (Fig. 7e). Super-resolution microscopy confirmed H3 localization within Npr1-positive structures in Lem2-overexpressing cells (Supplementary Fig. 7h). These findings suggest that chromatin presence in NE protrusions prevents their release, resulting in abortive nucleophagy.

We investigated whether nucleophagy inhibition by chromatin-INM tethering results in NE morphology aberrations and found that cells overexpressing Lem2 and those expressing Sso7d-h2NLS-LR2-WALP23, but not those expressing Sso7d*-h2NLS-LR2-WALP23, displayed abnormal NE morphology (Fig. 7f and Supplementary Fig. 8a). The most striking abnormal NE structures are ring-shaped NE projections, extended NE projections, and NE invaginations (Supplementary Fig. 8a, b). These findings indicate that alterations in NE morphology are a common consequence of all conditions that impede nucleophagy.

Methods
Strain and plasmid construction
The S. pombe strains used in this study are listed in Supplementary Data 2, and the plasmids used in this study are listed in Supplementary Data 3. Unless stated otherwise, the strains were cultured in EMM medium at 30 °C. The compositions of the EMM medium, EMM−N medium, and other media are as decribed47. Standard strain construction methods were used47. Deletion strains were generated through PCR-based gene targeting. The strains with Npr1, Epr1 or Lem2 endogenously tagged at their C-termini were constructed using PCR-based tagging48.

Plasmids expressing proteins fused with TurboID and GFP-Atg8 under the P41nmt1 promoter were constructed using modified pDUAL vectors20, 49. The other protein-expressing plasmids were based on stable integration vectors (SIVs)50, 51. These plasmids allow integration at specific loci, namely ura4, ade6, lys3, or his5. The plasmid expressing histone H3 (Hht2)-yeGFP was derived from the plasmid pDB556852, which was based on the pAde6PmeI SIV plasmid and used a unique PmeI site as the linearization site. Other plasmids, designed to express proteins fused with various N-terminal or C-terminal tags (mCherry, mScarlet2I, ymScarlet2I, mECitrine, or mTurquoise2), were based on modified SIV plasmids with a unique NotI site as the linearization site51.

Plasmids expressing Npr1/Npr1(W22A/V25A)-mECitrine under its endogenous promoter were constructed by introducing the npr1 promoter, together with the coding sequence of Npr1/Npr1(W22A/V25A), into a modified SIV containing the sequence encoding mECitrine. Plasmids expressing AIMart-Npr1(30-244)-mCherry, AIMart-Man1-mCherry, Rtn1-AIMart-mCherry, AIMart-mCherry-Kms1, AIMart-Erg11-mCherry, and Erg11-AIMart-mCherry were constructed by inserting the coding sequences of Npr1(30-244), Man1, Rtn1, Kms1, and Erg11, respectively, into a modified SIV containing the P41nmt1 promoter and the sequence encoding AIMart-mCherry. Plasmids expressing mCherry-Kms1-AIMart were constructed by inserting the coding sequence of Kms1-AIMart into a modified SIV containing the P41nmt1 promoter and the sequence encoding mCherry. AIMart corresponds to 3xEEEWEEL29, 30. The plasmid expressing Lem2(LEM+BBM)-mECitrine was constructed by inserting the coding sequences of amino acids 1-60 and amino acids 261-279 of Lem2 into a modified SIV containing the P41nmt1 promoter and the sequence encoding mECitrine35, 36. Plasmids used to investigate the roles of LEM and BBM, including Lem2(LEM)-mCherry-Bqt4(TM), NLS-BDS1-mCherry-Bqt4(TM), NLS-BD1*-mCherry-Bqt4(TM), NLS-Sso7d-mCherry-Bqt4(TM), NLS-Sso7d*-mCherry-Bqt4(TM), NLS-BD1-mCherry-h2NLS-LR2-WALP23, NLS-BD1*-mCherry-h2NLS-LR2-WALP23, NLS-Sso7d-mCherry-h2NLS-LR2-WALP23, and NLS-Sso7d*-mCherry-h2NLS-LR2-WALP23, were constructed by inserting the coding sequences of Bqt4(412-432) and h2NLS-LR2-WALP23 into modified SIVs, each containing the P41nmt1 promoter and the sequences encoding Lem2(1-60), NLS-BD1, NLS-BD1(Y123F), NLS-Sso7d, or NLS-Sso7d(W24A/R43E), and mCherry37-43. The nucleotide sequence encoding NLS is ATGCCTAAGAAGAAGCGTAAGGTC. Plasmids that encode NLS-Sso7d-mCherry-Bqt3 under the endogenous promoter of Bqt3 were generated by inserting the bqt3 promoter and the coding sequence of Bqt3 into a modified SIV containing the NLS-Sso7d and mCherry sequences. Plasmids expressing NLS-Sso7d-mCherry-Bqt3 under exogenous promoters were constructed by inserting the P81nmt1 promoter, P41nmt1 promoter, or Pcyc1 promoter, along with the coding sequence of Bqt3, into a modified SIV containing the NLS-Sso7d and mCherry sequences.

Modified SIVs were used in this study to express proteins fused with GFP1-10 and 7×GFP1123-25. The plasmid expressing Sum3-GFP1-10-mCherry under the Padh1 promoter was constructed by inserting the coding sequences of Sum3 and GFP1-10 into a modified SIV containing the Padh1 promoter and mCherry sequences. The plasmid expressing Gbs1-GFP1-10-mCherry under the Padh1 promoter was constructed by inserting the sequences of GFP1-10 and mCherry between codons 496 and 497 of Gbs1 and placing the coding sequence of the fusion protein downstream of the Padh1 promoter in an SIV plasmid. Plasmids expressing 7×GFP11-Erg11, Erg11-7×GFP11, 7×GFP11-Npr1, and Npr1-7×GFP11 under the P41nmt1 promoter were constructed by inserting the coding sequences of Erg11 or Npr1, together with the sequence encoding 7×GFP11, into a modified SIV containing the P41nmt1 promoter.

Plasmids and strains generated in this study are available and can be requested from the corresponding author.

TurboID-based proximity labeling and mass spectrometry analysis
Approximately 1000 OD units of TurboID-mCherry (control) and TurboID-mCherry-Atg8 cells were collected following 4 hours of nitrogen starvation. The collected cells were washed three times with deionized water and subsequently centrifuged to remove the supernatant. The resulting pellet was then reconstituted in 20 mL of deionized water, followed by the addition of 20 mL of 0.7 M sodium hydroxide. The mixture was incubated at room temperature on a rolling wheel for 10 minutes. The alkaline-treated cells were centrifuged to remove the supernatant. Afterward, 1 mL of lysis buffer (2% SDS, 0.06 M Tris-HCl, 5% glycerol, and 4% 2-mercaptoethanol, pH 6.8) was added to resuspend the pellet, which was then incubated at 42°C for 20 minutes. The supernatant was collected after centrifugation at 16,246 × g for 30 minutes. For the purification of biotinylated proteins, approximately 100 µL of Streptavidin Agarose Resin (Thermo Fisher Scientific, Cat#20359) was used. The streptavidin beads were pre-washed twice using washing buffer A (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.4% SDS, 1% NP40, and 1× Roche protease inhibitor cocktail, pH 7.5). After the addition of the streptavidin beads, the supernatant was incubated at room temperature on a rolling wheel for 3 hours to allow the biotinylated proteins to bind to the streptavidin beads. After incubation, the streptavidin beads were pelleted and washed twice with 1 mL of washing buffer A for 5 minutes and then incubated with 1 mL of washing buffer B (50 mM Tris-HCl, 2% SDS, pH 7.5) for 10 minutes. Subsequently, the beads were incubated twice with 1 mL of washing buffer A for 5 minutes each. Following this, biotinylated proteins were eluted from the beads by incubating the beads twice with 200 µL of elution buffer (50 mM Tris-HCl, 2% SDS, 5 mM biotin, pH 8.0) at 60°C for 20 minutes, using a ThermoMixer C (Eppendorf) set at 1,000 × rpm.

To precipitate the proteins, 100 µL of 100% trichloroacetic acid (TCA) was added to approximately 400 µL of eluate. The mixture was then incubated overnight at 4°C. Subsequently, the precipitated proteins were centrifuged at 16,246 × g at 4°C for 30 minutes, and the pellet was washed with acetone three times. The pellet was then resuspended with 30 µL of dissolution buffer (8 M urea, 100 mM Tris-HCl, pH 8.5) and dissolved by sonication using a water bath sonicator at room temperature for 15 minutes. Next, the proteins were reduced using 5 mM tris(2-carboxyethyl)phosphine at room temperature for 20 minutes, followed by alkylation with 10 mM iodoacetamide at room temperature for 15 minutes. The sample was diluted by a factor of 4 and digested into peptide fragments using trypsin at 37°C overnight. To terminate the trypsin digestion, formic acid was added to a final concentration of 5%.

After the completion of the digestion process, LC-MS/MS analysis was conducted using an Easy-nLC II HPLC instrument (Thermo Fisher Scientific), which was coupled to a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific). A total of 8 µL of peptides were loaded onto a pre-column (100 µm ID, 4 cm long, packed with C18 10 µm 120 Å resin from YMC Co., Ltd) and separated on an analytical column (75 µm ID, 10 cm long, packed with Luna C18 1.8 µm 100 Å resin from Welch Materials) using an acetonitrile gradient from 0% to 30% over a duration of 100 minutes. The flow rate during the separation was maintained at 250 nL/min. From each full scan (resolution 70,000), the top 15 most intense precursor ions were selected for higher-energy collisional dissociation tandem mass spectrometry (HCD MS2) analysis, with a normalized collision energy of 27 and a dynamic exclusion time of 30 seconds. The fragment ions obtained from the tandem mass spectrometry were detected using the Orbitrap in normal scan mode. Charge state rejection was enabled, and unassigned charge states as well as charge states 1, 7, 8, and >8 were rejected. Mass spectrometry data were analyzed using pFind software, with a peptide false discovery rate (FDR) cutoff of 1%53. The TurboID-mass spectrometry experiment was independently repeated twice, yielding similar results.

Immunoprecipitation
For immunoprecipitation (IP), approximately 100 OD units of log-phase cells were collected and subjected to three washes with water. The cell pellet was mixed with 100 µL of lysis buffer A (50 mM HEPES, 1 mM EDTA, 150 mM NaCl, 10% glycerol, 3 mM DTT, 3 mM PMSF, Roche 3×Protease inhibitor cocktail, pH 7.5) and 800 µL of glass beads (BioSpec) with a diameter of 0.5 mm for cell lysis. The cells were lysed using the FastPrep-24 instrument at a speed of 6.5 m/sec for 20 seconds. This lysis process was repeated three times. Then, 300 µL of lysis buffer B (50 mM HEPES, 1 mM EDTA, 150 mM NaCl, 10% glycerol, 0.05% NP40, 1 mM DTT, 1 mM PMSF, Roche 1×Protease inhibitor cocktail, pH 7.5) was added to the cell lysate. After centrifugation, 20 µL of the supernatant was retained as input, while the remainder was added to pre-washed GFP-Trap agarose beads at 4°C for 3 hours. The beads had been pre-washed with lysis buffer B. After centrifugation, the agarose beads were washed twice with wash buffer (50 mM HEPES, 1 mM EDTA, 150 mM NaCl, 10% glycerol, 0.05% NP40, 1 mM DTT, pH 7.5) and twice with lysis buffer B. The proteins bound to the GFP-Trap agarose beads were eluted by incubating them in SDS loading buffer (60 mM Tris-HCl, 4% SDS, 4% 2-mercaptoethanol, 5% glycerol, 0.002% bromophenol blue, pH 6.8) at 42°C for 20 minutes for subsequent immunoblotting analysis.

Fluorescence microscopy
We used cells cultured in liquid medium (EMM or EMM−N) for microscopy analysis. Live-cell imaging was performed using a Dragonfly 201-40 high-speed spinning-disk confocal microscope (Andor Technology), equipped with a 100×/1.4 NA objective lens, a Sona sCMOS camera, and two filter sets for mCherry/YFP/CFP and mCherry/GFP, respectively. Super-resolution images were acquired using a High Intelligent and Sensitive Structured Illumination Microscope (HIS-SIM, CSR Biotech Co., Ltd, Guangzhou), equipped with a 100×/1.5 NA objective lens, an sCMOS Flash 4.0 V2 camera, and a filter set for mCherry/GFP54. The microscopy images obtained were analyzed using Fiji55.

For time-lapse imaging, a suspension of nitrogen-starved cells cultured in liquid medium (EMM−N) was applied to an agar pad placed on a microscope glass slide (7.5 cm long)56. Firstly, two double-sided tapes were placed in the middle third of a clean glass slide, about 1.5 cm apart. Then, approximately 50 μL of hot melted agar (in EMM−N) was placed onto the glass slide, and immediately a coverslip (2.2 cm long) was placed on top of the agar drop. After the agar pad solidified in 3-5 minutes, the coverslip was removed, and 1 μL of the cell suspension was placed onto the agar pad. Time-lapse imaging analysis was performed after placing a new coverslip on top of the agar pad and ensuring the coverslip adhered tightly to the glass slide through the double-sided tapes.

Immunoblotting-based protein processing assay
A total of 5 OD units of cells were collected for lysis. The cells were resuspended in 300 µL of 20% TCA, and 700 µL of glass beads with a diameter of 0.5 mm were added. The cells were then lysed using the FastPrep-24 instrument at a speed of 6.5 m/sec for 20 seconds, repeating this process three times. The cell lysates were transferred into new centrifuge tubes through centrifugation. To adjust the TCA concentration to 10%, 300 µL of deionized water was added to the cell lysates. The mixture was vortexed and centrifuged at 865 × g for 30 minutes. The resulting pellet was resuspended in SDS loading buffer (60 mM Tris-HCl, 4% SDS, 4% 2-mercaptoethanol, 5% glycerol, 0.002% bromophenol blue, pH 6.8) and incubated at 42°C for 20 minutes. Subsequently, centrifugation was performed at 16,246 × g for 5 minutes, and the resulting supernatant was separated by 10% SDS-PAGE and subjected to immunoblotting using specific antibodies. The antibodies used for immunoblotting were anti-GFP mouse monoclonal antibody (1:3,000 dilution, Roche, Cat#11814460001) and anti-mCherry rabbit polyclonal antibody (1:3,000 dilution, ThermoFisher, Cat# PA5-34974). Post-immunoblotting staining of the PVDF membrane using Reactive Brown 10 (RB) served as the loading control57. For the statistical analysis of the protein processing data, Welch’s t-test was performed using Excel spreadsheets downloaded from http://www.biostathandbook.com/twosamplettest.html58.

Electron microscopy
For conventional transmission electron microscopy (TEM) analysis, a total of 50 OD units of cells were collected after either 2 or 6 hours of nitrogen starvation. The cells were fixed with glutaraldehyde and KMnO4 59. Following fixation, the cells underwent 13 rounds of water washing to remove any brownish particles. They were then dehydrated by passing through a series of graded ethanol solutions. Ultimately, the dehydrated samples were embedded in Spurr's resin59. Thin sections of 90 nm were examined using an FEI Tecnai G2 Spirit electron microscope equipped with a Gatan 895 4k × 4k CCD camera. For electron microscopy analysis utilizing the genetically encoded EM tag MTn, samples containing 20 OD units of vegetative cells expressing Npr1-mECitrine-MTn were processed18, 26.

For focused ion beam-scanning electron microscopy (FIB-SEM), a total of 50 OD units of cells were collected after 24 hours of nitrogen starvation. Cell samples were processed using the same method as that used for preparing conventional TEM samples. The resin-embedded samples were mounted on aluminum stubs. FIB-SEM datasets were acquired using a Zeiss Crossbeam 550 microscope equipped with ATLAS 3D software (ZEISS). During the milling process with the focused gallium-ion beam, a milling current of 700 pA at 30 kV was used from the gallium emitter. The resin-embedded samples were milled in 10 nm layers. Scanning EM images were captured using an SE2 detector set at 2 kV and 1 nA. The image resolution in the xy plane was 10 nm/pixel. The alignment of image stacks, visualization, 3D reconstructions, movie creation, and quantification of nuclear volume, as well as the surface area and volume of the NE, were carried out using the Dragonfly Pro software (version 2022.2). For the statistical analysis, Student’s two-sample t-test was performed using Excel spreadsheets downloaded from http://www.biostathandbook.com/twosamplettest.html.

Yeast two-hybrid (Y2H) assay
The yeast two-hybrid (Y2H) analysis was conducted using the Matchmaker system 3 (Clontech) to express two fusion proteins, namely the bait and prey. Bait plasmids were constructed by inserting the coding sequence of Atg8 into the pGBKT7 vector. Prey plasmids were constructed by inserting the coding sequence of Npr1/Npr1(W22A/V25A) into the pGADT7 vector. The AH109 yeast strain was co-transformed with the bait and prey plasmids and then selected on double dropout medium (SD/−Leu/−Trp). The activation of the HIS3 and ADE2 reporter genes was evaluated using quadruple dropout medium (SD/−Leu/−Trp/−His/−Ade). Photographs were taken after incubating the transformants on double dropout medium and quadruple dropout medium at 30°C for 3 to 4 days.

Growth phenotype assay (spot assay)
The S. pombe strains for the spot assay were inoculated into EMM medium supplemented with histidine, leucine, and uracil. The cells were cultured at 30°C until they reached the logarithmic phase. A portion of the cells was collected, while the remaining cells were subjected to nitrogen starvation for 3 days and 7 days, respectively. Subsequently, the harvested cells were subjected to a five-fold dilution and spotted onto YES plates. The plates were then incubated at 30°C and photographed after 3 days.

Prediction of transmembrane topology and protein complex structure
The transmembrane topology was predicted using the CCTOP web server (https://cctop.ttk.hu)22. The structure of the Atg8-Npr1 complex was predicted using AlphaFold2-Multimer (version 2.3.1) with default parameters27, 28. The structure exhibiting the highest confidence score among the predicted outputs was chosen for subsequent analysis. The visual representation of the predicted structure was generated using the Mol* Viewer (version 4.2.0)60.

Statistics and reproducibility
No statistical method was used to predetermine sample size. No data were excluded. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. All experiments were independently repeated at least twice with similar results, indicating reproducibility. For the statistical analysis of the protein processing data, Welch’s t-test was performed. For the statistical analysis of the FIB-SEM data, Student’s two-sample t-test was performed.

Data availability
The authors declare that all data supporting the findings of this study are available within the paper and its supplementary information files. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, the authors investigated the molecular mechanisms of nucleophagy, selective autophagic degradation of nuclear components, in Schizosaccharomyces pombe. They identified a novel nucleophagy receptor, Npr1, an outer nuclear membrane protein that binds Atg8 and functions redundantly with Epr1, which was previously identified by the same group as a receptor for both the ER and the nucleus.\n\nA particularly significant finding of this study is the discovery that chromatin tethering to the inner nuclear membrane (INM) inhibits nucleophagy. Based on the result that artificial chromatin–INM tethering impaired the release of nucleus-derived vesicles from NE protrusions, the authors propose that chromatin–INM tethering may serve as a safeguard mechanism that preserves genome integrity during nucleophagy. The authors also demonstrated that the epr1-delta npr1-delta double mutant, which is completely defective in nucleophagy, exhibits pronounced defects in nuclear morphology and reduced cell viability under starvation conditions, emphasizing the physiological importance of nucleophagy in this organism.\n\nThus, this work provides new insights into the mechanisms underlying nucleophagy. Overall, the study is well-designed and the data are compelling; however, the following issues should be addressed prior to publication.\n1. On pages 12 and 13, the authors state that Atg8 “disappears” from NE protrusions and that Atg8 is deconjugated from the isolation membrane prior to its closure. However, this explanation appears to contradict their model shown in Fig. 7f, in which Atg8 is retained within autophagosomes. It seems that the Atg8 signal just decreases under levels undetectable by fluorescence microscopy at the late stages of nucleophagosome formation, so the authors should reconsider their wording (e.g., replacing “disappears” with “decreases”).\n2. In Fig. 3a, degradation of nuclear proteins appears to be partially defective in epr1-delta cells and npr1-delta cells. To more precisely evaluate the effects of single deletion of npr1 and epr1, the authors should quantify the results and perform statistical analysis.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The manuscript by Zhu-Hui Ma investigates the process of nucleophagy in fission yeast and reports that nucleophagy is promoted by two autophagy receptors and inhibited by chromatin–nuclear envelope (NE) tethering. The authors identify Npr1 as an outer nuclear membrane (ONM) transmembrane protein that functions as a nucleophagy receptor by interacting with Atg8 via its N-terminal AIM. Npr1 functions redundantly with Epr1, and both receptors independently bind Atg8 on the ONM, consistent with genetic data showing that either receptor is sufficient to mediate nucleophagy.\n\nThe formation of Npr1 and Epr1 puncta with Atg8 at the NE appears to precede the development of NE protrusions, suggesting this may represent an early—possibly initiating—step in the process. While Atg8 eventually dissociates, Npr1 and Epr1 are captured by the autophagosome and delivered into vacuoles.\n\nNpr1- and Epr1-mediated nucleophagy is required to maintain normal nuclear morphology, which, in turn, appears to contribute to cell survival during starvation. This part of the manuscript is compelling and the results are interesting and important. I would recommend concluding the manuscript at this point.\n\nThe authors also present evidence that chromatin tethering to the inner nuclear membrane interferes with or prevents the completion of nucleophagy. This second part of the study, which explores this chromatin tethering, is disconnected from the core findings. I suggest to either link it to mechanistically to Npr1, Epr1, or to remove this section. Currently the results in Figures 6 and 7 neither enhance nor diminishes the impact of the primary discoveries.\n\nMajor Points:\n1. Genetic data clearly support redundant functions of Npr1 and Epr1 in nucleophagy. Mechanistically, however, this redundancy is less well explained and somewhat puzzling. Does loss of Npr1 result in upregulation of Epr1 (or vice versa)? What could be other compensatory mechanism?\n2. The electron microscopy (EM) images of NE protrusions are compelling, but it would strengthen the manuscript if the authors could demonstrate the presence of Atg8, Npr1, or Epr1 on these structures.\n3. The morphology of the NE protrusions observed by FIB-SEM should be described more clearly. In the images vacuolar structures appear to be captures by the NE protrusions. What is the role of vacuoles in the context of these protrusions?\n4. In my view, the experiments involving overexpression of Lem2 are not essential, as they are not directly linked to Npr1 or Epr1. I recommend removing Figures 6 and 7 unless a mechanistic connection to Npr1 and Epr1 can be established.", "reviewer_4": "The work of Ma et al investigated mechanisms of nucleophagy in fission yeast. They identified a novel protein, Npr1, from Atg8 affinity purification, and established that it functions as a receptor for nucleophagy under starvation. They showed that Npr1 was present on the outer nuclear membrane, away from nuclear pores, with both termini facing the cytosol. They confirmed that Npr1 interacted with Atg8 via an AIM motif, and demonstrated via an elegant set of experiments that the main role of Npr1 is to bridge Atg8 with the nuclear envelope.\n\nIn live cell imaging, the authors found that Npr1 formed concentrated dots at a pace that was similar to Atg8. Their imaging data suggest that the concentrated Npr1 dots were pieces of nuclear envelope that eventually separated from the rest of nuclear envelope, possibly at the same time as they were sequestered into autophagosomes. The related timing, and the dependence of Npr1 dot formation on its interaction with Atg8, implies that the concentration of Npr1 occurs during the formation of autophagosomes. Under electron microscopy, a protruding piece of nuclear envelope could be seen wrapped with another piece of membrane, most likely autophagosome membrane.\n\nIn cells lacking nucleophagy receptors, nuclear envelope displayed irregular shapes, instead of spheres as in wild type cells. These mutants were also unable to survive starvation.\n\nThey further showed that when chromatin was artificially tethered to nuclear envelope, nucleophagy was blocked at a stage with protruding structures positive for Npr1, but unable to complete separation from the rest of nuclear envelope. Such a block could be achieved by over expressing a native protein with such tethering capacity, Lem2, or with artificially designed proteins.\n\nOverall, the work of Ma et al reported a new autophagy receptor, and dissected how this protein functions. These novel discoveries will be of interest to autophagy and membrane biology field. However, there are several areas where the manuscript needs to be improved.\n\nMain issues:\n(1) How is nucleophagy triggered?\nThe authors’ data analyzing the structural elements of Npr1 suggests that the sole function of a nucleophagy receptor is to link the substrate, the nuclear envelope, with the autophagosome membrane. In this case, nucleophagy is essentially a byproduct of general autophagy. But I am not convinced this is the whole story, and suggest the authors test the following:\nObserve Npr1 dependent nucleophagy under other autophagy inducing conditions. The above model predicts that in the presence of receptors, it will always occur whenever general autophagy occurs.\nTarget AIMs to other organelles will lead to their AIM dependent turnover under starvation. Try Golgi, peroxisomes, or even the plasma membrane?\nExamine whether the interaction between Npr1 and Atg8 is constitutive or starvation induced.\n\n(2) The physiological significance of nucleophagy.\nThe authors found that nuclear envelope displayed abnormal shapes in receptor mutants, and in atg5D mutant. To show that the phenotype is indeed due to nucleophagy, instead of some other functions of Npr1 or Atg8, the authors need to test if their artificial nuclear envelope – Atg8 tethers can recover the morphology and survival phenotypes, and other classes of atg mutants beyond the Atg8 system also have similar phenotypes.\nThe authors did not mention similar morphological abnormalities with AIM mutations, or with artificial chromatin tethering. Please confirm whether nuclear envelope morphology change is a shared consequence of all conditions blocking nucleophagy.\nBesides shape, can the authors provide quantifications of the volumes and surface areas of nuclear envelopes with vs without nucleophagy? While detailed mechanistic investigation of nuclear envelope morphorlogy change may be beyond the scope of this work, such simple quantifications may provide some clues as for the physiological function of nucleophagy.\n\nMinor issues:\n(1) The role of chromatin.\nIt is a very interesting observation that forcing chromatin into autophagosomes prevented their proper formation. However, the conditions the authors tested were all artificial. Is there a strong decrease in the degree of chromatin attachment to nuclear envelope from growing condition to starvation condition? In other words, what is the physiological relevance?\n(2) The authors used several constructs when analyzing the membrane topology of Npr1. The functionality of these constructs need to be verified.\n(3) In time lapse imaging, the authors observed about ¼ of the cases where no inner membrane protein dots were released into cytosol. The authors interpreted these events as premature termination of nucleophagy. My two alternative hypotheses are that the disappearance of Npr1 dots represents entry into vacuoles, and that the cases where no cytosolic inner membrane protein dots were observed represent cases with dots being dim to begin with. In particular for Npr1, their dots stayed at stable signal level before a complete disappearance at the next time point, unlike the gradual decline of Atg8, which implies Npr1 disappearance represents vacuole entry (similar to Ape1 in budding yeast). Can the authors provide more definitive evidence supporting their premature termination claim?\n(4) In time lapse imaging, I do not see Npr1 signal on nuclear envelope, only dots. Is it because the nuclear envelope signal was too weak?\n(5) When labeling lanes in immunoblots, the authors used a format that results in very long text lines. This makes it difficult to read and catch the most important information. The authors should change the labeling format."}, "author_response": "Response: We thank the reviewer for the positive evaluation of our manuscript and for raising the concern regarding the interpretation of the “disappearance” of Atg8 puncta. We agree with the reviewer that not all Atg8 molecules are recycled; some must remain within the closed autophagosomes. We have added the following sentence to the main text of the revised manuscript: “It is important to note that the observed ‘disappearance’ of Atg8 does not imply that all Atg8 molecules are recycled, but rather that the level of Atg8 associated with the closed autophagosome falls below the detection limit of microscopy.”\n2. In Fig. 3a, degradation of nuclear proteins appears to be partially defective in epr1-delta cells and npr1-delta cells. To more precisely evaluate the effects of single deletion of npr1 and epr1, the authors should quantify the results and perform statistical analysis.\nResponse: We have conducted the quantification and statistical analysis of the processing of Pus1-mECitrine, mECitrine-Bqt4, mECitrine-Nup82, and Ker1-mECitrine, as suggested by the reviewer. The results are presented in Supplementary Fig. 3a of the revised manuscript. As noted by the reviewer, the levels of processing were slightly reduced in the absence of Epr1 or Npr1, but the P values are mostly greater than 0.05.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nResponse: We thank the reviewer for contributing to the evaluation of our manuscript and for providing constructive suggestions.\nReviewer #3 (Remarks to the Author):\nThe manuscript by Zhu-Hui Ma investigates the process of nucleophagy in fission yeast and reports that nucleophagy is promoted by two autophagy receptors and inhibited by chromatin–nuclear envelope (NE) tethering. The authors identify Npr1 as an outer nuclear membrane (ONM) transmembrane protein that functions as a nucleophagy receptor by interacting with Atg8 via its N-terminal AIM. Npr1 functions redundantly with Epr1, and both receptors independently bind Atg8 on the ONM, consistent with genetic data showing that either receptor is sufficient to mediate nucleophagy.\n\nThe formation of Npr1 and Epr1 puncta with Atg8 at the NE appears to precede the development of NE protrusions, suggesting this may represent an early—possibly initiating—step in the process. While Atg8 eventually dissociates, Npr1 and Epr1 are captured by the autophagosome and delivered into vacuoles.\n\nNpr1- and Epr1-mediated nucleophagy is required to maintain normal nuclear morphology, which, in turn, appears to contribute to cell survival during starvation. This part of the manuscript is compelling and the results are interesting and important. I would recommend concluding the manuscript at this point.\n\nThe authors also present evidence that chromatin tethering to the inner nuclear membrane interferes with or prevents the completion of nucleophagy. This second part of the study, which explores this chromatin tethering, is disconnected from the core findings. I suggest to either link it to mechanistically to Npr1, Epr1, or to remove this section. Currently the results in Figures 6 and 7 neither enhance nor diminishes the impact of the primary discoveries.\nResponse: We sincerely thank the reviewer for the thoughtful comments and for recognizing the significance of our findings on Npr1- and Epr1-mediated nucleophagy. We appreciate the suggestion to streamline the manuscript and agree that the chromatin tethering section of the manuscript (Figures 6 and 7) does not have a strong connection to the earlier parts.\nHowever, we believe this section addresses a fundamental and unresolved question in the field: how do cells selectively degrade nuclear material via nucleophagy without risking genomic DNA loss? Our observation that artificial chromatin tethering to the inner nuclear membrane blocks nucleophagy provides the first experimental evidence that a mechanism may exist to safeguard against inadvertent chromatin degradation. To better integrate this section with the main narrative, we have revised the text in the Results and Discussion to emphasize chromatin-nuclear envelope association as a regulatory constraint on nucleophagy, rather than an independent phenomenon. We believe these revisions strengthen the conceptual link.\nMajor Points:\n1. Genetic data clearly support redundant functions of Npr1 and Epr1 in nucleophagy. Mechanistically, however, this redundancy is less well explained and somewhat puzzling. Does loss of Npr1 result in upregulation of Epr1 (or vice versa)? What could be other compensatory mechanism?\nResponse: To address the reviewer’s question, we have added Supplementary Fig. 3e in the revised manuscript, which shows that the loss of Npr1 did not affect the protein level of Epr1, and the loss of Epr1 did not affect the protein level of Npr1. We agree with the reviewer that the redundancy between Npr1 and Epr1 is intriguing and raises the question of why such redundancy arose during evolution. We speculate that under circumstances other than nitrogen starvation, these two proteins may not function redundantly and thus provide more opportunities for regulatory controls. Further investigations are needed to address this possibility.\n2. The electron microscopy (EM) images of NE protrusions are compelling, but it would strengthen the manuscript if the authors could demonstrate the presence of Atg8, Npr1, or Epr1 on these structures.\nResponse: We thank the reviewer for the suggestion. Unfortunately, despite numerous attempts, we have not been able to identify EM sample preparation conditions that effectively preserve the structures of NE protrusions while also allowing for clear visualization of the MTn-tagged Atg8, Npr1, or Epr1. We hope to overcome these challenges in future experiments.\n3. The morphology of the NE protrusions observed by FIB-SEM should be described more clearly. In the images vacuolar structures appear to be captures by the NE protrusions. What is the role of vacuoles in the context of these protrusions?\nResponse: We have revised the paragraph describing the morphology of the NE projections observed by FIB-SEM and have added a schematic diagram illustrating these structures in Fig. 5d of the revised manuscript. We also included the following sentence regarding their relationship with vacuoles: “Since an S. pombe cell contains numerous small vacuoles dispersed throughout the cytoplasm, the presence of vacuoles within the ring-like profiles of NE projections may simply result from this; however, we cannot rule out the possibility of a preferential spatial relationship between NE projections and vacuoles.”\n4. In my view, the experiments involving overexpression of Lem2 are not essential, as they are not directly linked to Npr1 or Epr1. I recommend removing Figures 6 and 7 unless a mechanistic connection to Npr1 and Epr1 can be established.\nResponse: We appreciate this kind suggestion. Although there is not a strong connection between the nucleophagy receptor section of the manuscript and the section on the inhibition of nucleophagy by chromatin-nuclear envelope tethering, we believe both sections contribute to a comprehensive understanding of nucleophagy. We have revised the text in the Results and Discussion to emphasize chromatin-nuclear envelope association as a regulatory constraint on nucleophagy, rather than an independent phenomenon. We believe these revisions strengthen the conceptual link.\nReviewer #4 (Remarks to the Author):\nThe work of Ma et al investigated mechanisms of nucleophagy in fission yeast. They identified a novel protein, Npr1, from Atg8 affinity purification, and established that it functions as a receptor for nucleophagy under starvation. They showed that Npr1 was present on the outer nuclear membrane, away from nuclear pores, with both termini facing the cytosol. They confirmed that Npr1 interacted with Atg8 via an AIM motif, and demonstrated via an elegant set of experiments that the main role of Npr1 is to bridge Atg8 with the nuclear envelope.\nIn live cell imaging, the authors found that Npr1 formed concentrated dots at a pace that was similar to Atg8. Their imaging data suggest that the concentrated Npr1 dots were pieces of nuclear envelope that eventually separated from the rest of nuclear envelope, possibly at the same time as they were sequestered into autophagosomes. The related timing, and the dependence of Npr1 dot formation on its interaction with Atg8, implies that the concentration of Npr1 occurs during the formation of autophagosomes. Under electron microscopy, a protruding piece of nuclear envelope could be seen wrapped with another piece of membrane, most likely autophagosome membrane.\nIn cells lacking nucleophagy receptors, nuclear envelope displayed irregular shapes, instead of spheres as in wild type cells. These mutants were also unable to survive starvation.\nThey further showed that when chromatin was artificially tethered to nuclear envelope, nucleophagy was blocked at a stage with protruding structures positive for Npr1, but unable to complete separation from the rest of nuclear envelope. Such a block could be achieved by over expressing a native protein with such tethering capacity, Lem2, or with artificially designed proteins.\nOverall, the work of Ma et al reported a new autophagy receptor, and dissected how this protein functions. These novel discoveries will be of interest to autophagy and membrane biology field. However, there are several areas where the manuscript needs to be improved.\nMain issues:\n(1) How is nucleophagy triggered?\nThe authors’ data analyzing the structural elements of Npr1 suggests that the sole function of a nucleophagy receptor is to link the substrate, the nuclear envelope, with the autophagosome membrane. In this case, nucleophagy is essentially a byproduct of general autophagy. But I am not convinced this is the whole story, and suggest the authors test the following:\nObserve Npr1 dependent nucleophagy under other autophagy inducing conditions. The above model predicts that in the presence of receptors, it will always occur whenever general autophagy occurs.\nTarget AIMs to other organelles will lead to their AIM dependent turnover under starvation. Try Golgi, peroxisomes, or even the plasma membrane?\nExamine whether the interaction between Npr1 and Atg8 is constitutive or starvation induced.\nResponse: We agree with the reviewer that nucleophagy is not simply a byproduct of general autophagy. Nucleophagy receptors are only one type of nucleophagy-specific factor that is important for nucleophagy but not general autophagy. Even though the nucleophagy role of the two fission yeast nucleophagy receptors, Npr1 and Epr1, can be largely substituted by an artificial tether connecting Atg8 to the nuclear envelope, other nucleophagy-specific factors are still indispensable. For example, we have previously shown that the fission yeast protein Yep1 (also known as Hva22 or Rop1), an ortholog of budding yeast Atg39 and human REEP1-4 proteins, is essential for nucleophagy but not for general autophagy (Zou et al. 2023, PMID:37939137). Budding yeast Atg39 appears to serve a combined role of fission yeast nucleophagy receptors and Yep1, thus fulfilling functions beyond merely linking Atg8 to the nuclear envelope. Given the complexity of the nucleophagy process, we anticipate that many additional nucleophagy-specific factors remain to be discovered.\nRegarding autophagy-inducing conditions, we have identified only two that can robustly induce general autophagy in fission yeast: nitrogen starvation and DTT treatment, both of which also stimulate nucleophagy (Zou et al. 2023, PMID:37939137). It remains possible that there are undiscovered conditions that induce nucleophagy without inducing general autophagy in fission yeast.\nThe suggestion of artificially tethering Atg8 to other organelles is intriguing but lies beyond the scope of this study. We will pursue this line of investigation in the future.\nThe interaction between Npr1 and Atg8 is constitutive, as supported by co-immunoprecipitation analysis conducted prior to nitrogen starvation, as shown in Fig. 2b.\n(2) The physiological significance of nucleophagy.\nThe authors found that nuclear envelope displayed abnormal shapes in receptor mutants, and in atg5D mutant. To show that the phenotype is indeed due to nucleophagy, instead of some other functions of Npr1 or Atg8, the authors need to test if their artificial nuclear envelope – Atg8 tethers can recover the morphology and survival phenotypes, and other classes of atg mutants beyond the Atg8 system also have similar phenotypes.\nThe authors did not mention similar morphological abnormalities with AIM mutations, or with artificial chromatin tethering. Please confirm whether nuclear envelope morphology change is a shared consequence of all conditions blocking nucleophagy.\nBesides shape, can the authors provide quantifications of the volumes and surface areas of nuclear envelopes with vs without nucleophagy? While detailed mechanistic investigation of nuclear envelope morphorlogy change may be beyond the scope of this work, such simple quantifications may provide some clues as for the physiological function of nucleophagy.\nResponse: We thank the reviewer for suggesting these additional experiments to strengthen our conclusion. We have added new experimental data in Supplementary Fig. 5a-b showing that reintroducing either nuclear envelope–Atg8 tethers, AIMart-Kms1 or Erg11-AIMart, into the epr1Δ npr1Δ mutant largely restores NE morphology. Moreover, we have included data showing that either tether also restores the survival phenotype of the epr1Δ npr1Δ mutant (Supplementary Fig. 5d). AIM-mutated forms of Npr1 or Epr1 cannot rescue the NE morphology defect of the epr1Δ npr1Δ mutant (Fig. 5a and Supplementary Fig. 5a).\nAdditionally, we have added new data in Supplementary Fig. 5a, b showing that other autophagy mutants, including atg1Δ, atg13Δ, atg9Δ, atg14Δ, atg2Δ, atg18aΔ, and atg18bΔ, also exhibit NE morphology aberrations. In Fig. 7f and Supplementary Fig. 8, we show that either overexpression of Lem2 or expression of the artificial chromatin tether Sso7d-h2NLS-LR2-WALP23 leads to abnormal NE morphology.\nFinally, we have added Supplementary Fig. 5c, which shows that the surface area and volume of the NE are increased in epr1Δ npr1Δ and atg5Δ cells compared to wild-type cells, whereas the volume of the nucleus remains largely unchanged. Thus, nuclear deformation allows for NE expansion without altering nuclear volume in nucleophagy-deficient cells.\nMinor issues:\n(1) The role of chromatin.\nIt is a very interesting observation that forcing chromatin into autophagosomes prevented their proper formation. However, the conditions the authors tested were all artificial. Is there a strong decrease in the degree of chromatin attachment to nuclear envelope from growing condition to starvation condition? In other words, what is the physiological relevance?\nResponse: In Discussion, we propose that the presence of chromatin in an NE protrusion being enclosed by an autophagic membrane results in abortive nucleophagy, and this serves as a safeguarding mechanism preventing the inadvertent degradation of genomic DNA during nucleophagy. Based on this model, we do not expect that the level of chromatin-NE association necessarily decreases under starvation conditions, because nucleophagy should be able to proceed as long as there are NE regions free of chromatin attachment. It would be interesting to determine whether the extent of chromatin-NE attachment changes when cells are transitioned from growth conditions to starvation conditions. However, this cannot be easily accomplished, and therefore we chose not to pursue it during the revision.\n(2) The authors used several constructs when analyzing the membrane topology of Npr1. The functionality of these constructs need to be verified.\nResponse: To address the reviewer’s question, we added experimental data in Supplementary Fig. 3d examining whether various Npr1 fusion proteins retain the nucleophagy function. Reintroducing MTn-tagged Npr1 into the epr1Δ npr1Δ mutant fully restores the autophagic processing of Pus1-mCherry, whereas reintroducing Npr1 fused with GFP at either the N-terminus or C-terminus into the epr1Δ npr1Δ mutant partially restores the autophagic processing of Pus1-mCherry. Thus, these fusions do not abrogate the function of Npr1.\n(3) In time lapse imaging, the authors observed about ¼ of the cases where no inner membrane protein dots were released into cytosol. The authors interpreted these events as premature termination of nucleophagy. My two alternative hypotheses are that the disappearance of Npr1 dots represents entry into vacuoles, and that the cases where no cytosolic inner membrane protein dots were observed represent cases with dots being dim to begin with. In particular for Npr1, their dots stayed at stable signal level before a complete disappearance at the next time point, unlike the gradual decline of Atg8, which implies Npr1 disappearance represents vacuole entry (similar to Ape1 in budding yeast). Can the authors provide more definitive evidence supporting their premature termination claim?\nResponse: To address the reviewer’s question, we imaged nitrogen-starved cells expressing Npr1-mCherry and the INM protein mECitrine-Bqt4 in a fsc1Δ background, where autophagosome-vacuole fusion is blocked. If the disappearance of Npr1 puncta indeed represents entry into vacuoles, we would expect to see a reduction in this type of Npr1 puncta and an increase in Npr1 puncta released into the cytosol in fsc1Δ cells compared to wild-type cells. What we observed is that in fsc1Δ cells, 50% of Npr1 puncta associated with NE protrusions exhibited simultaneous release into the cytosol, compared to 56% in wild-type cells. Additionally, 30% of puncta in fsc1Δ cells (27% in wild-type cells) disappeared simultaneously with the NE protrusion, while 20% in fsc1Δ cells (17% in wild-type cells) disappeared after the protrusion had vanished, with no subsequent detection of cytosolic puncta (Fig. 4i in the revised manuscript). These results suggest that the disappearance of NE protrusion-associated Npr1 puncta cannot be attributed to vacuole entry.\nIt is unclear why the disappearance of Npr1 appears abrupt rather than exhibiting a gradual decline in signal like Atg8. We speculate that this difference may be due to the distinct disassembly kinetics of the local assemblies where these two proteins concentrate.\nFurthermore, in our time-lapse imaging results, the signals of NE protrusions released into the cytosol are not initially stronger than those that vanished without ever being detected in the cytosol.\n(4) In time lapse imaging, I do not see Npr1 signal on nuclear envelope, only dots. Is it because the nuclear envelope signal was too weak?\nResponse: The signal of Npr1 puncta in cells undergoing nucleophagy is much stronger than the signal of Npr1 on the nuclear envelope. In time-lapse imaging, to avoid bleaching, we used low laser power and short exposure times. Under such imaging conditions, the Npr1 puncta are clearly visible, but the signal of Npr1 on the nuclear envelope is no longer readily visible.\n(5) When labeling lanes in immunoblots, the authors used a format that results in very long text lines. This makes it difficult to read and catch the most important information. The authors should change the labeling format.\nResponse: We thank the reviewer for the suggestion. We have made adjustments to enhance readability in our revised manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all the concerns we raised for the original manuscript in a satisfactory manner.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The authors have addressed most of my original concerns.", "reviewer_4": "The revised manuscript is substantially improved. The authors provided reasonable responses to most of my questions, and provided new data clarifying existing issues.\n\nThere is one comment that I feel the authors did not address properly. In my major point 1, I asked if nucleophagy was a byproduct of general autophagy given that the reported role of the receptor was solely to link the substrate membrane with Atg8, and one specific request was to test if the interaction between the receptor and Atg8 was induced by starvation. The authors responded by referring to a pre-existing CoIP data done under normal growing condition. While the data indeed demonstrates the existence of interaction under growing condition, it is fundamentally unrelated to whether the interaction is regulated by starvation or not."}, "author_response": "Response: We thank the reviewer for the positive evaluation.\nResponse: We thank the reviewer for the positive evaluation.\nResponse: We thank the reviewer for the positive evaluation.\nResponse: We thank the reviewer for highlighting this important point and acknowledge that our initial response did not adequately address whether the Npr1–Atg8 interaction is regulated by nitrogen starvation. To directly test this, we have now performed co-immunoprecipitation (CoIP) experiments comparing Npr1–Atg8 interaction levels under normal growth conditions and after nitrogen starvation. As shown in the new Fig. 2b, Npr1 interacts with Atg8 to a comparable extent in both conditions, indicating that this interaction is constitutive and not induced or enhanced by starvation."}, {"round": 3, "reviewer_comments": {"reviewer_4": "The authors have addressed all my major concerns. I have no further comments."}, "author_response": ""}]

Decision letter