The intrinsically disordered regions of organellophagy receptors are interchangeable and control organelle fragmentation, ER-phagy and mitophagy flux

Rudinskiy, Mikhail; Galli, Carmela; Raimondi, Andrea; Molinari, Maurizio

doi:10.1038/s41556-025-01728-4

Download PDF

Article
Open access
Published: 04 August 2025

The intrinsically disordered regions of organellophagy receptors are interchangeable and control organelle fragmentation, ER-phagy and mitophagy flux

Nature Cell Biology volume 27, pages 1431–1447 (2025)Cite this article

10k Accesses
40 Altmetric
Metrics details

Subjects

Abstract

Organellophagy receptors control the generation and delivery of portions of their homing organelle to acidic degradative compartments to recycle nutrients, remove toxic or aged macromolecules and remodel the organelle upon physiologic or pathologic cues. How they operate is not understood. Here we show that organellophagy receptors are composed of a membrane-tethering module that controls organellar and suborganellar distribution and by a cytoplasmic intrinsically disordered region (IDR) with net cumulative negative charge that controls organelle fragmentation and displays an LC3-interacting region (LIR). The LIR is required for lysosomal delivery but is dispensable for organelle fragmentation. Endoplasmic reticulum (ER)-phagy receptors’ IDRs trigger DRP1-assisted mitochondrial fragmentation and mitophagy when transplanted at the outer mitochondrial membrane. Mitophagy receptors’ IDRs trigger ER fragmentation and ER-phagy when transplanted at the ER membrane. This offers an interesting example of function conservation on sequence divergency. Our results imply the possibility to control the integrity and activity of intracellular organelles by surface expression of organelle-targeted chimeras composed of an organelle-targeting module and an IDR module with net cumulative negative charge that, if it contains a LIR, eventually tags the organelle portions for lysosomal clearance.

Functional multi-organelle units control inflammatory lipid metabolism of macrophages

Article Open access 05 July 2024

Determinants that enable disordered protein assembly into discrete condensed phases

Article 05 February 2024

Replication licensing regulated by a short linear motif within an intrinsically disordered region of origin recognition complex

Article Open access 13 September 2024

Main

Nutrient restriction, pathologic and physiologic cues including accumulation of aged or toxic material, of misfolded proteins, pathogen attack and cell maturation or differentiation may activate lysosomal clearance of parts of intracellular organelles such as the endoplasmic reticulum (ER), the mitochondria and the Golgi complex^1,2,3,4,5,6. These parts are physically separated from the bulk of the organelle, which must be preserved, and are delivered within degradative acidic organelles. ER-phagy, mitophagy and Golgiphagy (here collectively mentioned as organellophagy) are driven upon activation of membrane-bound organellophagy receptors (for example, the ER-phagy receptors FAM134B⁷, SEC62 (refs. ^8,9), TEX264 (refs. ^10,11), CCPG1 (ref. ¹²) and others (Fig. 1a); the mitophagy receptors FUNDC1 (refs. ^13,14), BNIP3, BNIP3L/NIX^15,16 and others (Fig. 1b); and the Golgiphagy receptors YIPF3 and YIPF4 (refs. ^17,18) (Fig. 1c)). All membrane-bound organellophagy receptors display a cytoplasmic intrinsically disordered region (IDR) module (Fig. 1a–c, green) that engages lipidated LC3/GABARAP proteins via short consensus LC3-interacting region (LIR) motifs (Fig. 1a–c, grey boxes). The activation of organellophagy receptors has two major consequences: (1) the fragmentation of the homing organelle to separate portions to be degraded and (2) the delivery of the organelle fragments within acidic degradative organelles via macro-autophagy, micro-autophagy or LC3-dependent transport pathways^{1,2,6,19,20,21,22,23}. While it has been clearly established that delivery of organelle portions within degradative compartments requires the engagement of LC3 proteins via cytoplasmic organellophagy receptors’ LIRs, how organelles are portioned is unclear. For ER-phagy, a crucial role has been ascribed to the membrane remodelling function of the reticulon homology domains (RHD) that tether members of the FAM134 family at the ER membrane^{7,23,24,25,26,27,28}. However, membrane remodelling is not a conserved trait of membrane-tethering modules of organellophagy receptors. In fact, other ER-phagy, mitophagy and Golgiphagy receptors^17,18 are anchored via conventional multi- or single-spanning transmembrane domains (Fig. 1a–c) that are not expected to promote membrane remodelling per se. Thus, a model assuming that the membrane-tethering modules of organellophagy receptors determine or give essential contribution to organelle fragmentation²³ cannot be generalized. To clarify these issues, we investigated the consequences on ER and mitochondrial integrity of the expression at their limiting membranes of full-length, membrane-tethering or IDR modules of the ER-phagy receptors FAM134B, SEC62 and TEX264 and the mitophagy receptor FUNDC1.

**Fig. 1: Membrane-bound organellophagy receptors.**

Our study shows that the membrane-anchoring domains of organellophagy receptors can be replaced by exogenous membrane anchors with no impact on organelle fragmentation and lysosomal delivery. This implies, for example, that the membrane remodelling activity of FAM134B is dispensable for execution of ER-phagy. We report that ER and mitochondrial fragmentation is controlled by the cytoplasmic IDR modules of the organellophagy receptors. Consistent with the concept of functional conservation on sequence divergency, the IDR modules of organellophagy receptors have different sequences but maintain conserved traits. Among them, we identify a net cumulative negative charge at physiologic pH, a length above the 47 residues and a minimal distance of the LIR from the membrane of 24 residues. Conservation of these and possibly other features that remain to be characterized make them interchangeable. Thus, IDR modules of ER-phagy receptors expressed at the outer mitochondrial membrane (OMM) trigger mitochondrial fragmentation and mitophagy; IDR modules of mitophagy receptors expressed at the ER membrane trigger ER fragmentation and ER-phagy.

Results

Membrane-anchoring and cytosolic IDR modules

The ER, the mitochondria and the Golgi complex display a large array of autophagy receptors at their limiting membrane¹^,⁴^,²¹^,²⁹ (Fig. 1a–c). Organellophagy receptors are composed of a membrane-tethering module that spans the organelle membrane once or multiple times (Fig. 1a–c, blue boxes) and a cytoplasmic IDR module (Fig. 1a–c, green lines) that contains the LIR domain engaging LC3/GABARAP proteins (Fig. 1a–c, grey boxes). Mammalian FAM134 family members and RTN3L are anchored at the ER membrane with RHDs that only partially span the lipid bilayer, with intrinsic membrane remodelling activity (shown in curved regions of the ER membrane in Fig. 1a). All other organellophagy receptors are anchored at membranes via conventional transmembrane domains (Fig. 1a–c).

We notice that the IDR modules of mammals and yeast ER-phagy, mitophagy and Golgiphagy receptors have a length between 47 and 250 residues (Fig. 1d) and include a single LIR (Fig. 1a–c, grey boxes). The LIR of the mitophagy receptor FUNDC1 is placed at 24 residue distance from the last residue of the transmembrane anchor. All other organellophagy receptors’ LIRs are characterized by longer distances from the membrane of the homing organelles. The IDR module of RTN3L, a protein involved in endosome maturation³⁰, spans over 800 residues and contains multiple LIRs^{1,4,5,20,21,31,32,33}.

The IDR modules of organellophagy receptors have diverging sequences but are all characterized by net cumulative negative charge at physiologic pH (for example, −19.95 for SEC62_IDR, −33.94 for FAM134B_IDR, −13.96 for TEX264_IDR, −3.93 for FUNDC_IDR and −8 and −11.11 for YIPF3_IDR and YIPF4_IDR, respectively; Fig. 1e and https://www.biosynth.com/peptide-calculator). IDRs are often present in the proteome and a survey of the literature reveals that their functions include the processing of regulatory cues, molecular communication³⁴ and, notably, sensing and driving membrane curvature^35,36,37. For their role in organellophagy, nothing is known beyond a putative role in ER-phagy as spacers to bridge the distance between the ER membrane in rough ER subdomains covered by ribosomes and LC3 lipidated at the membrane of phagophores (for macro-autophagic pathways) or of endolysosomes (EL, for micro-autophagy or LC3-mediated delivery pathways)¹¹.

Expression of ER-phagy receptors triggers ER-phagy

ER-phagy is activated by pleiotropic (for example, nutrient restriction) and ER-centric cues (for example, luminal accumulation of misfolded proteins, ER stress and ribosome stalling)^1,20. These cues induce expression of individual or multiple ER-phagy receptors and/or increase their local concentration and activity upon derepression, post-translational modifications and/or formation of hetero- or homo-meric clusters^{8,12,24,25,27,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55}. The ectopic expression of ER-phagy receptors induces ER-phagy bypassing the need for activating exogenous or intrinsic cues and has been used to characterize their functions, their interactors and their regulation (for example, FAM134B^7,53,56, SEC62 (refs. ^8,9,57) and TEX264 (refs. ^10,11,54)). Here, we first express Halo-tagged^58,59,60 versions of FAM134B, SEC62 and TEX264 (Fig. 2, left) to compare their capacity to induce ER-phagy. Please note that in all figures, the 4 nm × 3 nm GFP (Fig. 3, green circles) or HaloTag barrels (Fig. 2, green circles)⁶¹ are drawn in approximate scale with the IDRs, where each amino acid contributes to the length of the unstructured IDR for an average of 0.38 nm (ref. ⁶²). ER-phagy is monitored by confocal laser scanning microscopy (CLSM) in mouse embryonic fibroblasts (MEF) treated with Bafilomycin A1 (BafA1) to inhibit lysosomal hydrolases and preserve material (in this case, calnexin (CNX)-positive ER portions) delivered in the lumen of degradative LAMP1-positive EL⁶³. Expression of FAM134B, SEC62 or TEX264 increases delivery of ER portions within LAMP1-positive EL (Fig. 2c,g,k) above the constitutive levels observed in mock-transfected cells (Fig. 2a). The variations in ER-phagy activity (that is, the accumulation of CNX-positive ER portions within LAMP1-positive EL upon inactivation of lysosomal hydrolases) are quantified with the deep learning tool LysoQuant developed in our lab⁶⁴ (Fig. 2e,i,m). The expression of the membrane-anchoring modules of the ER-phagy receptors (FAM134B_RHD (Fig. 2b,e), SEC62_TMD (Fig. 2f,i) or TEX264_TMD (Fig. 2j,m)) does not trigger ER delivery within EL. Likewise, the expression of versions of the full-length ER-phagy receptors carrying mutations that prevent engagement of LC3 proteins (-₄₅₃DDFELLD₄₅₈- to -AAAAAAA- amino acid substitution for FAM134B_LIR (ref. ⁷) (Fig. 2d,e), -₃₆₃FEMI₃₆₆- to -AAAA- for SEC62_LIR (ref. ⁸) (Fig. 2h,i) and -₂₇₃FEEL₂₇₆- to -AAAA- for TEX264_LIR (refs. ^10,11) (Fig. 2l,m)) does not trigger ER delivery within EL. To confirm the activation of an autophagic flux also in cells, where the lysosomal activity has not been inhibited (that is, in absence of BafA1 exposure), we made use of the HaloTag assay, which has been developed in our lab^{59,60,65,66,67}. Briefly, MEF expressing the ER-phagy reporter HT₁₇, a chimeric polypeptide of 37 kDa composed of a HaloTag tethered to the ER membrane with a 17-residue transmembrane domain⁶⁸ (Fig. 2n, second panel from the top, lanes 1–11) were mock-transfected (Fig. 2n, lane 1) or were transfected with the same modules analysed in Fig. 2b–m tagged with a monomeric superfolder (sf)GFP moiety⁶⁹. Lysosomal delivery of ER portions and the subsequent hydrolytic processing of the HT₁₇ reporter generates a protease-resistant Halo fragment of about 33 kDa, whose fluorescence can be directly monitored in gel (Fig. 2n, third panel from the top)^{59,60,65,66,67}. The HaloTag assay confirms the imaging data (Fig. 2a–m). Thus, expression of sfGFP-tagged FAM134B, SEC62 and TEX264 (Fig. 2n, top panel) triggers ER-phagy as shown by the generation of the Halo fragment (Fig. 2n, third panel from the top, lanes 3, 6 and 9), which requires hydrolytic lysosomal activity (that is, the cells exposure to BafA1 inhibits the generation of the Halo fragment, lane 11). The expression of FAM134B_RHD (Fig. 2n, lane 2), SEC62_TMD (lane 5), TEX264_TMD (lane 8) or the expression of FAM134B_LIR (Fig. 2n, lane 4), SEC62_LIR (lane 7) and TEX264_LIR (lane 10)) does not trigger ER-phagy as testified by the absence of the fluorescent Halo fragment.

**Fig. 2: CLSM analyses of ER-phagy induction.**

Notably, as already shown by others in silico²⁶ and double checked here in cellula by room temperature-transmission electron microscopy (RT-TEM) (Extended Data Fig. 1a–e), the membrane-anchoring RHD module of FAM134B has the intrinsic property of remodelling the ER membrane. However, the compartment remains connected by thin tubules (Fig. 4f,g, fluorescence recovery after photobleaching (FRAP) experiments, and Extended Data Fig. 1c, red arrowheads) as it has been observed upon overexpression of members of the conserved family of ER remodelling proteins that generate high ER membrane curvature such as reticulons or REEP proteins^{30,70,71,72,73,74,75,76,77,78,79}. The membrane-anchoring modules of SEC62 and TEX264 do not remodel the ER membrane as shown by the ER ultrastructure in mock-transfected cells (Extended Data Fig. 1b), which remains unchanged in cells expressing SEC62_TMD and TEX264_TMD (Extended Data Fig. 1d,e). Altogether, these data confirm that the expression of ER-phagy receptors competent for LC3 engagement at the ER membrane induces ER-phagy bypassing the needs of external cues. Importantly, the induction of ER-phagy occurs independent of the capacity of the membrane-anchoring modules of the receptors to remodel the ER membrane.

As a separate note, individual ER-phagy receptors are located in and control lysosomal clearance of distinct ER subdomains (FAM134B for ER tubuli, SEC62 and TEX264 for ER sheets and outer nuclear membrane^{1,8,9,10,11,57,80,81}). Notably, the distinct suborganellar localization is also observed upon cellular expression of the membrane-anchoring domains of the ER-phagy receptors lacking the cytoplasmic IDR modules. For example, the endogenous interactome of FAM134B_RHD reveals its distribution in subdomains hosting proteins of the reticulon and REEP families (as reported for the full-length receptor⁵⁶) (Extended Data Fig. 1f,g). The interactions of the SEC62_TMD and of the TEX264_TMD reveal their localization in ER sheets also containing subunits of the translocation machinery including the oligosaccharyltransferase complex and chaperones assisting protein folding (as reported for the full-length receptors⁸) (Extended Data Fig. 1f,g).

Expression of ER-phagy receptors IDR modules elicits ER-phagy

The finding that ER-phagy receptors expression induces ER-phagy (that is, ER fragmentation and delivery of ER portions within degradative LAMP1-positive organelles), independent of the capacity of their membrane-tethering modules to remodel the ER membrane, led us to verify the dispensability of the FAM134B, SEC62 and TEX264 membrane anchors to launch the catabolic programme. To verify this, we expressed in MEF the cytoplasmic IDR modules FAM134B_IDR, SEC62_IDR or TEX264_IDR anchored at the ER membrane with a single-spanning transmembrane domain of 17 residues (T₁₇)⁶⁸. The chimeras were tagged with a luminal sfGFP (Fig. 3, schematics, and Extended Data Fig. 2). Induction of ER-phagy was assessed, as above, both with the imaging and with the HaloTag assays. Analyses by CLSM (Fig. 3a–g and Extended Data Fig. 2a–g for entire cells) and LysoQuant quantifications (Fig. 3h) show the enhanced lysosomal delivery of CNX-positive ER portions in cells expressing the three IDR modules (Fig. 3b,h for GT₁₇-FAM134B_IDR, Fig. 3c,h for GT₁₇-SEC62_IDR and Fig. 3d,h for GT₁₇-TEX264_IDR and Extended Data Fig. 2b–d) compared with cells that express the ER membrane targeted sfGFP that does not display a cytoplasmic IDR (GT₁₇) (Fig. 3a,h and Extended Data Fig. 2a). The inactivation of the LC3-binding function of the three IDR modules abolishes delivery of ER portions within the LAMP1-positive EL (Fig. 3e–h and Extended Data Fig. 2e–g).

**Fig. 3: Membrane-associated IDRs trigger ER-phagy.**

The dispensability of the ER-phagy receptors membrane-tethering modules for ER-phagy induction was confirmed with the HaloTag assay mentioned above that reports on the generation of the Halo fragment (Fig. 3i, third from the top) in cells with functional EL that express the membrane-bound, sfGFP-tagged IDR modules of SEC62 (Fig. 3i, lane 4), FAM134 (Fig. 3i, lane 6) and TEX264 (Fig. 3i, lane 8). The fragment is not produced in cells that express a membrane-tethered GT₁₇ control protein lacking cytoplasmic IDR (Fig. 3i, third from the top, lane 2) or the IDR modules that cannot engage LC3 (lanes 5, 7 and 9, respectively).

Finally, the competence of membrane-tethered IDR modules to fragment the ER and to deliver ER portions within EL was also monitored with anti-GFP immunoelectron microscopy (IEM) in MEF treated with BafA1. The gold particles reveal the localization of the GFP moiety of the membrane-tethered GT₁₇-SEC62_IDR and of the GT₁₇-SEC62_IDRLIR chimeras in the lumen of the ER and of ER portions in the cytoplasm (Fig. 3j,k, red arrowheads). Notably, the cells that express the IDR module that can engage LC3 show GFP-positive ER portions also within the lumen of EL (Fig. 3j, blue arrowheads), whereas the cells that express the IDR module that cannot engage LC3 do not show GFP-positive ER portions within the lumen of EL (Fig. 3k). This confirms that LC3 engagement is required for lysosomal delivery.

Monitoring IDR modules-induced ER fragmentation

The detection of ER portions within LAMP1-positive EL in cells expressing full-length ER-phagy receptors (Fig. 2) or their IDR modules anchored at the ER membrane with a dissimilar transmembrane domain (Fig. 3 and Extended Data Fig. 2) indirectly reports on ER fragmentation, which is a prerequisite for ER portions being delivered within the degradative compartments. Direct monitoring of ER fragmentation (that is, the visualization of ER portions in the cytoplasm) is challenging due to the ultrastructure of the compartment and to the fact that the portions are delivered within EL for clearance. To overcome this hurdle and directly monitor the ER fragmentation function of ER-phagy receptor’s IDR modules, the experiments shown in Fig. 3 were repeated in MEF lacking ATG7 (Fig. 4a), where the absence of LC3 lipidation⁸² inhibits the delivery of ER portions within the LAMP1-positive degradative organelles^7,8,9,10,11, thus preserving them in the cytoplasm.

**Fig. 4: ER fragmentation in ATG7-KO MEF expressing the IDR module of SEC62.**

Analyses by RT-TEM of the ER ultrastructure in cells mock transfected (Fig. 4b), expressing membrane-bound sfGFP (GT₁₇, Fig. 4c) or membrane-bound sfGFP displaying the IDR modules of SEC62 that can (GT₁₇-SEC62_IDR, Fig. 4d) or cannot engage LC3 proteins (GT₁₇-SEC62_IDRLIR, Fig. 4e), reveal that only the expression of GT₁₇-SEC62_IDR and of GT₁₇-SEC62_IDRLIR drives the formation of ER-derived vesicles visible in the cytoplasm (Fig. 4d,e, red arrowheads).

A FRAP assay of ER lumen connectivity performed with the HaloTagged variants of the same constructs (HT₁₇, HT₁₇-SEC62_IDR and HT₁₇-SEC62_IDRLIR) confirms a substantial inhibition of fluorescence recovery in cells expressing the IDR modules SEC62_IDR and SEC62_IDRLIR (Fig. 4f,g, green curves), as compared with mock-transfected cells (Fig. 4f,g, black curve) and with cells expressing HT₁₇ (Fig. 4f,g, violet curve). This is consistent with the RT-TEM images showing that expression of the IDR modules results in physical separation of ER portions from the bulk ER and shows that the LIR function of the IDR module is dispensable for organelle fragmentation. Notably, FRAP experiments also confirm that the expression of the RHD of FAM134B that induces ER remodelling and formation of ER constrictions (Extended Data Fig. 1c, red arrowheads) does not inhibit the recovery of the fluorescent signal, consistent with the incompetence of the RHD of FAM134B expressed alone in cellula to fragment the ER (Fig. 4f,g, blue curve).

ER-phagy receptors IDRs-driven mitophagy

Motivated by these findings and by the fact that also autophagy receptors at the limiting membrane of other organelles display cytoplasmic IDR modules with similar features (Fig. 1), we verified whether the IDR modules of ER-phagy receptors encode a signal driving lysosomal delivery of mitochondrial portions, when exposed at the limiting membrane of mitochondria. To this end, FAM134B_IDR, SEC62_IDR and TEX264_IDR were expressed at the OMM. Briefly, MEF were mock-transfected (Fig. 5a) or were transiently transfected with a plasmid for expression of a sfGFP moiety targeted at the OMM by the transmembrane domain of the mitochondrial protein TOMM20 (Mito-GFP)⁸³ (Fig. 5b). Alternatively, the cells were transfected with plasmids for expression of the same membrane-tethered sfGFP moiety displaying at the C-terminus SEC62_IDR, FAM134B_IDR or TEX264_IDR modules (Fig. 5c,e,g), or the same IDR modules carrying a mutation in the LIR domain that prevents LC3 engagement (Fig. 5d,f,h and Extended Data Fig. 3a–j, for the same experiment replicated in HEK293 cells and for the control of LC3 binding). The cells were exposed to BafA1 to preserve the endogenous TOMM20-positive mitochondrial fragments delivered within endolysosomal degradative compartments⁶³. In mock-transfected cells and in cells expressing the Mito-GFP at the OMM (Fig. 5a,b), a low percentage of the LAMP1-positive EL accumulates the endogenous mitochondrial TOMM20 protein marker (quantifications with LysoQuant in Fig. 5j), reporting on the constitutive lysosomal turnover of mitochondrial portions. Mitochondrial transplantation of the SEC62_IDR, FAM134B_IDR or TEX264_IDR modules, enhances the delivery of the mitochondrial fragments within degradative LAMP1-positive EL (Fig. 5c,e,g,j). Mutations of the IDR’s LIR motifs that inhibit engagement of LC3 proteins (Fig. 5d,f,h,j) or transplantation of the ER-phagy receptors IDR modules at the OMM in MEF lacking ATG7, which are defective in LC3 lipidation (Extended Data Fig. 4) hamper the delivery of TOMM20-positive mitochondrial portions within the degradative LAMP1 compartment.

**Fig. 5: Monitoring lysosomal delivery of fragmented mitochondria in wild-type MEF expressing the ER-phagy receptors IDR modules.**

Next, the HaloTag assay in cells expressing the mitophagy reporter HMAVS_TM (HaloTag tethered to the mitochondrial OMM with the 31-residue-long transmembrane domain of the mitochondrial antiviral-signalling protein (MAVS)) reports on the absence of Halo fragment in mock-transfected cells and in cells expressing Mito-GFP at the OMM (Fig. 5i, lanes 1 and 2), indicating low detectable constitutive mitophagy. Expression of ER-phagy IDR modules tethered to the OMM (Fig. 5c,e,g) induces the formation of Halo fragments (Fig. 5i, lanes 4, 6 and 8), which requires hydrolytic lysosomal activity (that is, exposure of cells to BafA1 inhibits the generation of the Halo fragment, lane 3), indicating the delivery of mitochondrial fragments to the hydrolytically active degradative compartments. Importantly, this delivery is abrogated in cells expressing LC3-binding-deficient IDR modules (Fig. 5i, lanes 5, 7 and 9), once again indicating that mitophagy requires LC3 engagement by the ER-phagy receptors’ IDR modules that have been transplanted at the limiting membrane of mitochondria.

In MEF, we also monitored the lysosomal delivery of the mitochondrial fragments generated by the expression of the Mito-GSEC62_IDR polypeptide by IEM (Fig. 5k). The Mito-GSEC62_IDR polypeptide immunolabelled with gold particles is seen at the OMM of mitochondria in the process of being engulfed by an EL (Fig. 5k, left) or of mitochondria within the lumen of the EL upon inactivation of hydrolytic enzymes with BafA1 (Fig. 5k, right), thus confirming the results of the CLSM analyses and of the HaloTag assay (Fig. 5c,i,j). The IEM also reveals that the mitochondrial portions captured by the LAMP1-positive EL maintain the characteristic morphology characterized by a double membrane and luminal cristae. The IEM analyses of cells that display at the OMM the Mito-GSEC62_IDRLIR polypeptide (Fig. 5d) that fragments mitochondria (‘ER-phagy receptors IDRs-driven mitochondrial fragmentation’ section) but cannot engage LC3 (Extended Data Fig. 3j) reveal that these mitochondrial portions fail to be delivered within the EL and remain in the cytoplasm (Fig. 5l).

ER-phagy receptors IDRs-driven mitochondrial fragmentation

The accumulation of mitochondrial portions within LAMP1-positive EL implies that expression of IDR modules at the OMM triggers mitochondrial fragmentation. As previously shown for the ER fragmentation induced by the expression at the ER membrane of the SEC62_IDR IDR module, to directly monitor the mitochondrial fragmentation triggered upon transplantation of the same IDR modules at the OMM, the experiments shown in Fig. 5b–h were repeated in MEF lacking ATG7 (Fig. 6a–g), where the absence of LC3 lipidation⁸² inhibits the delivery of mitochondria portions within the LAMP1-positive degradative organelles (Extended Data Fig. 4), thus preserving them in the cytoplasm. The morphology of mitochondria stained with a TOMM20-reactive antibody that decorates the OMM was first compared by CLSM in cells visible in the same coverslips that do express (Fig. 6a–g, green cells) or do not express (Fig. 6a–g, red cells) the GFP chimeras. The expression at the OMM of Mito-GFP has minor consequences on the network of mitochondria (Fig. 6a, insets 1 and 2, comparing the mitochondria in non-transfected cells with the mitochondria in the cell expressing the Mito-GFP). In cells expressing the cytoplasmic ER-phagy receptors’ IDR modules attached to Mito-GFP, the mitochondria appear smaller (Fig. 6b–g, inset 2) compared with non-transfected cells in the same coverslips (inset 1) and to cells expressing Mito-GFP. Importantly, as previously shown for the IDR modules exposed at the ER membrane, the analyses of the light microscopy (Fig. 6b–g) and of the IEM micrographs (Fig. 6h–k and quantification in Fig. 6l) confirm that the LC3-binding function of the IDR modules is dispensable for organelle fragmentation. These results were also replicated in HEK293 cells, in which mitochondria labelled with IDR modules at the OMM (Extended Data Fig. 5b–g, inset 2, and quantification in Extended Data Fig. 5h) are smaller compared with the mitochondria in cells expressing Mito-GFP (Extended Data Fig. 5a, inset 2) or non-transfected cells (Extended Data Fig. 5a–g, inset 1, and quantification in Extended Data Fig. 5h), indicating the induction of mitochondrial fragmentation by ER-phagy IDR modules transplanted at the OMM.

**Fig. 6: Monitoring mitochondria fragmentation in AT7G7-KO MEF expressing the mitochondria-targeted ER-phagy receptors IDR modules.**

IDRs-induced mitochondrial fragmentation is driven by DRP1

The fission of mitochondria that precedes delivery of the fragmented organelles to endolysosomal degradative compartments via mitophagy pathways as induced by various cues including nutrient deprivation⁸⁴, hypoxia⁸⁵ or oxidative stress⁸⁶ is controlled by the DRP1 GTPase^87,88, although DRP1-independent mitophagy has also been observed⁸⁹. To assess whether DRP1 controls the mitochondrial fission as induced by the transplantation of ER-phagy receptor’s IDR modules at the OMM, Mito-GFP, Mito-GSEC62_IDR, and Mito-GSEC62_IDRLIR polypeptides were expressed at the OMM of wild-type MEF (Fig. 7b–d) and in MEF lacking DRP1 (DRO1 knockout (KO))⁹⁰ (Fig. 7a,e–g). DRP1-KO cells have enlarged mitochondria (Fig. 7e–g, inset 1, DRP1-KO, and Fig. 7b–d, inset 1, wild-type MEF) confirming the functional KO of DRP1 leading to a general dysfunction of mitochondrial fission⁹⁰. Expression of the Mito-GFP polypeptide does not affect the mitochondrial size and morphology in either wild-type or DRP1-KO MEF (Fig. 7b,e, a comparison of inset 2 with the corresponding inset 1). The expression of Mito-GSEC62_IDR and Mito-GSEC62_IDRLIR polypeptides induces mitochondrial fragmentation in wild-type MEF (Fig. 7c,d, respectively) as shown above for MEF (Fig. 6) and HEK293 cells (Extended Data Fig. 5) but not in DRP1-KO cells (Fig. 7f,g, a comparison of inset 2 with the corresponding inset 1). Thus, DRP1 is necessary for the mitochondrial fragmentation induced by transplantation of ER-phagy receptors IDR modules at the OMM.

**Fig. 7: IDR modules fail to induce mitochondrial fragmentation in DRP1-KO cells.**

Next, we confirmed the involvement of DRP1 in the mitophagy induced by the transplantation of the FAM134B_IDR, SEC62_IDR or TEX264_IDR IDR modules at the OMM by monitoring the lysosomal accumulation of TOMM20-positive mitochondrial portions in cells treated with BafA1. As shown in Fig. 5, in mock-transfected wild-type MEF (Extended Data Fig. 6a) and in MEF expressing the Mito-GFP chimera (Extended Data Fig. 6b) mitochondria are virtually absent from the lumen of LAMP1-positive EL, reporting on low levels of constitutive mitophagy. Transplantation of the LC3-binding-competent chimeras at the OMM of wild-type MEF enhances mitophagy (Extended Data Fig. 6c,e,g and LysoQuant quantification in Extended Data Fig. 6q, black dots), which is substantially inhibited upon mutations of the LIR that prevent LC3 engagement (Extended Data Fig. 6d,f,h and LysoQuant quantification in Extended Data Fig. 6q). In the DRP1-KO MEF, the delivery of TOMM20-positive mitochondrial portions within LAMP1-positive EL is substantially inhibited (Extended Data Fig. 6a–h versus Extended Data Fig. 6i–p and Extended Data Fig. 6q, quantifications, and Extended Data Fig. 7a versus Extended Data Fig. 7b and Extended Data Fig. 7l, quantification). This shows that DRP1 contributes to the fission of mitochondria that display the ER-phagy receptors IDR modules at their OMM, before the delivery of formed mitochondrial fragments within degradative EL.

IDRs-induced mitophagy requires the GTPase activity of DRP1

Finally, to confirm that the GTPase activity of DRP1 controls the mitophagy programme elicited by exposure of ER-phagy receptors IDR modules at the OMM, the active (mCherry–DRP1), or the inactive forms of the GTPase (mCherry–DRP1_K38A)⁹¹ were back-transfected in the DRP1-KO MEF. The analyses show that the back-transfection of the active form of DRP1 partially restores the capacity of the SEC62, FAM134B and TEX264 IDR modules competent for LC3 engagement to trigger mitophagy (Extended Data Fig. 7c,f,i,l, respectively) but the back-transfection of the inactive GTPase DRP1_K38A does not (Extended Data Fig. 7d,g,j,l). As expected, reconstitution of the DRP1 activity does not activate mitophagy, if the IDR modules cannot engage LC3 (Extended Data Fig. 7e,h,k,l). Notably, the fissionase DRP1 is not involved in ER-phagy programmes induced by ER-phagy IDR modules (Extended Data Fig. 8).

Expression of the FUNDC1 IDR at the OMM triggers mitophagy

The conservation amongst organellophagy receptors of membrane-tethered IDR modules with net cumulative negative charge led us to postulate a functional conservation that would allow heterologous transplantation to trigger autophagic regulation of a given organelle on demand. So far, we demonstrated that the IDR modules of ER-phagy receptors trigger mitophagy when transplanted at the OMM. Are IDR modules of mitophagy receptors (for example, of FUNDC1 (refs. ^13,14)) competent for ER-phagy induction when transplanted at the ER membrane?

First, we assessed dispensability of the FUNDC1 tri-spanning membrane-tethering part in mitophagy induction. As shown above for the ER-phagy receptors, also for FUNDC1 the substitution of the original membrane anchor with a dissimilar transmembrane domain (the membrane anchor of the mitochondrial protein MAVS) does not affect the capacity of the polypeptide to control generation and delivery of TOMM20-positive mitochondrial portions within LAMP1-positive EL (Fig. 8a,b,d). The inactivation of the LC3-engaging function of the LIR embedded in the FUNDC1_IDR-GMAVS module upon mutation of the -YEVL- tetrapeptide to -AAAA-, substantially inhibits mitophagy induction (FUNDC1_IDRLIR-GMAVS) (Fig. 8c,d).

**Fig. 8: Transplantation of the IDR module of the mitophagy receptor FUNDC1 at the mitochondrial membrane triggers mitophagy and at the ER membrane triggers ER-phagy.**

Expression of the FUNDC1 IDR at the ER triggers ER-phagy

Next, the IDR module of the mitophagy receptor FUNDC1 was targeted and anchored at the ER membrane with a conventional 17-residue membrane anchor (the T17 used above) (Fig. 8f). The ER-targeted FUNDC1_IDR-GT₁₇ engages lipidated LC3B-II as shown by co-immunoprecipitation (Fig. 8i, lane 6, compared with the LC3-II engagement by the membrane-anchored IDR module of the ER-phagy receptor SEC62, lane 3). LC3 engagement is abolished upon mutation of the -YEVL- tetrapeptide of FUNDC1 to -AAAA- (Fig. 8i, lane 7). The expression of FUNDC1_IDR-GT₁₇ substantially enhances the delivery of CNX-positive ER fragments within the LAMP1-positive EL (GT₁₇) (Fig. 8f,h) compared with the expression of ER-targeted GFP (GT₁₇) (Fig. 8e,h). The inactivation of the LC3-engaging function of the FUNDC1 IDR module transplanted at the ER membrane substantially inhibits the delivery of ER portions within LAMP1-positive EL (Fig. 8g,h).

Conservation of features, net cumulative negative charge

To close the loop and go back to the functional conservation of cytoplasmic IDR modules among organellophagy receptors (Fig. 1), we analysed the capacity of the IDR of REEP1 (refs. ^92,93) to fragment mitochondria, when targeted at the OMM. REEP1 is member of a conserved family of ER remodelling proteins that generate high ER membrane curvature. It displays a C-terminal IDR module of 51 residues, a length that is in the range of sizes observed for the IDR modules of organellophagy receptors (Fig. 1d). However, the IDR module of REEP1 is characterized by a net cumulative positive charge of 4.03 (Fig. 1e). In contrast to the IDR modules of ER-phagy and mitophagy receptors tested in this work, the transplantation of REEP1_IDR at the OMM does not trigger mitochondria fragmentation (Extended Data Fig. 9a,b), even though this module is expressed at much higher level (Extended Data Fig. 9c, lane 11) than the corresponding modules derived from the ER-phagy receptors (Extended Data Fig. 9c, lane 8) that induce mitochondria fragmentation. Notably, the addition in the REEP1 IDR module of a nonapeptide containing the extended LIR sequence of SEC62 (-GNDFEMITK-) results in poor engagement of LC3, both when the REEP1 IDR module is expressed at the ER membrane (GT₁₇-REEP1_IDRFEMI) (Extended Data Fig. 9c, lane 5) or at the mitochondrial membrane (Mito-GREEP1_IDRFEMI, lane 10). Not surprisingly, therefore, the expression of the IDR module of REEP1 at the OMM or at the ER membrane does not induce mitochondrial (Extended Data Fig. 9d–h) nor ER delivery (Extended Data Fig. 9f–i) to the degradative LAMP1 compartment.

Conservation of features, LIR distance from the membrane

As shown above, the engagement of LC3 proteins is dispensable for the organelle fragmentation function of the IDR modules of organellophagy receptors. However, it is required to execute the delivery of organelle portions within the degradative compartments. To monitor how the distance from the membrane of the LIR impacts on organellophagy, we made use of the ER-exposed IDR module of SEC62. The full-length IDR module (GT₁₇-GSEC62_IDR148 in Extended Data Fig. 10a) has a total length of 148 residues (including a 4-residue-long linker between the IDR and the T₁₇ transmembrane domain) and has 111 residues between the end of the membrane anchor and the LIR. This distance was progressively reduced to 55 (GT₁₇-GSEC62_IDR92), 28 (GT₁₇-SEC62_IDR65) and 10 residues (GT₁₇-SEC62IDR₄₇) (Extended Data Fig. 10b–d). Analyses by CLSM show that the induction of ER-phagy is maintained by the GT₁₇-GSEC62_IDR92 and the GT₁₇-SEC62_IDR65 modules (Extended Data Fig. 10b,c,f, quantification of ER delivery to the degradative LAMP1-positive EL). The delivery of CNX-positive ER portions within EL drops substantially on ER expression of the GT₁₇-SEC62_IDR47 module that fails to engage LC3 proteins (Extended Data Fig. 10g), possibly because the short distance of the LIR from the lipid bilayer, 10 residues, impedes the access of LC3 proteins.

Conservation of features, length of the IDR modules

All the IDR modules examined in these experiments conserve the net negative charge (−19.95 for GT₁₇-GSEC62_IDR148 and for GT₁₇-GSEC62_IDR92, −16.00 for GT₁₇-SEC62_IDR65 and −14.06 for GT₁₇-SEC62_IDR47) and would have a length consistent with the length of cytoplasmic IDR modules displayed by organellophagy receptors (Fig. 1d, the shortest IDR module tested in our experiments is the GT₁₇-SEC62_IDR47, with a total length of 47 amino acids that, despite the incapacity of engaging LC3 proteins, could be competent to induce organelle fragmentation).

To assess how the length of the IDR modules affects their capacity to induce organelle fragmentation, we progressively reduced the length of the SEC62 IDR modules exposed on a sfGFP moiety at the limiting membrane of mitochondria (Extended Data Fig. 10h–m). Our tests show that only the Mito-GFP23 IDR has lost the capacity to fragment the organelle, when exposed at the OMM (Extended Data Fig. 10h,m). This IDR maintains the net cumulative negative charge at physiological pH (−1.03). However, it is shorter—about half the length—of the shortest organellophagy receptors known so far (SAMM50 and FUNDC1) (Fig. 1d).

Discussion

Delivery of organelle portions to degradative compartments requires the engagement of LC3 proteins by membrane-bound organellophagy receptors. An open question in the field is how the organelle portions to be removed from the cell are physically separated from the bulk of the organelle that must be preserved. Seminal studies on ER-phagy receptors of the FAM134 family (FAM134A, FAM134B and FAM134C) established that their transcriptional induction, phosphorylation, ubiquitylation and homo-/hetero-oligomerization control ER fragmentation and lysosomal clearance^{7,24,25,27,38,53,55,94,95}. FAM134 proteins are anchored at the ER membrane via a RHD, a membrane-tethering module found in ER shaping proteins of the reticulon and REEP families that generates high curvature and constrictions of the ER membrane^{30,70,71,72,73,74,75,76,77,78,79}. The capacity of the FAM134B-RHD to remodel the ER membrane has been highlighted by molecular dynamics simulations and in vitro liposome remodelling and has been proposed to promote the ER fragmentation required for lysosomal clearance of select ER portions^{7,23,24,25,26,27,28}. During revision of our manuscript, the same group reported that molecular dynamics simulations revealed that the IDR module of FAM134B might enhance the capacity of the RHD of FAM134B to fragment the ER⁹⁶. Notably, the vast majority of organellophagy receptors are anchored at the membrane of their homing organelle (the ER, the mitochondria or the Golgi complex), via conventional multi- or single-spanning transmembrane domains that do not share the membrane remodelling function of RHD modules (Fig. 1). Thus, a model assuming that the membrane-tethering modules of organellophagy receptors determine or give essential contribution to organelle fragmentation cannot be generalized. As shown in Fig. 1, the cytoplasmic IDR modules with net cumulative negative charge are a shared trait of autophagy receptors at the surface of organelles that rely on fragmentation for homoeostatic control. Our work highlights the functional conservation of the cytoplasmic IDR modules of membrane-tethered organellophagy receptors in promoting the organelle fragmentation, which is a conditio sine qua non for the execution of the autophagic programmes that regulate size, activity and homoeostasis of the organelles in our cells. We report that (1) mammalian ER-phagy and mitophagy receptors expose at the cytoplasmic face of their homing organelle a functionally conserved IDR module with diverse primary sequence, a length between 47 and 250 residues, a net cumulative negative charge at physiologic pH and a LIR separated by at least 24 residues from the membrane of the organelle (mammalian Golgiphagy receptors and yeast ER-phagy and mitophagy receptors, whose function has not been analysed here, also expose these modules); (2) the expression of full-length ER-phagy receptors recapitulates ER fragmentation and lysosomal delivery independent of the ER remodelling capacity of their membrane-anchoring modules and bypassing the requirement for external triggering signals; (3) the membrane-anchoring modules of the three ER-phagy receptors analysed in this study (FAM134B, SEC62 and TEX264) determine the suborganelle distribution of the receptors but are dispensable for ER fragmentation and execution of the ER-phagy programme. Their substitution with an unrelated transmembrane domain has no impact on the induction of ER fragmentation and ER-phagy; (4) likewise, the membrane-anchoring module of the mitophagy receptor FUNDC1 can be replaced by an unrelated sequence that targets the IDR module at the OMM, with no consequences on activation of mitochondrial fragmentation and mitophagy; (5) tethering the IDR modules of ER-phagy receptors at the OMM induces mitochondrial fragmentation driven by the DRP1 fissionase and mitophagy; (6) tethering the IDR module of the mitophagy receptor FUNDC1 at the ER membrane triggers ER fragmentation and ER-phagy; (7) the engagement and lipidation of LC3 proteins is dispensable for organelle fragmentation but required for delivery of the organelle portions within LAMP1-positive degradative organelles; (8) finally, the non-compliance with features conserved amongst organellophagy receptors (that is, change from net cumulative negative to positive charge, reduction in size or in the distance of the LIR from the membrane) negates the capacity of the cytoplasmically exposed IDR modules to fragment the organelles and to deliver organelle portions to degradative compartments.

Negatively charged IDRs are common in the proteomes of nucleated cells, where they are mainly found in long aspartic and glutamic acid repeats⁹⁷. Organellophagy receptors IDR modules seem to be characterized by more uniform negative charge distribution along their sequence, which can be further amplified by post-translational modifications such as phosphorylation that has been reported as an activation signal for Metazoan’s ER-phagy receptors^{53,54,55,95,98,99} and receptors controlling autophagic turnover of other organelles^{100,101,102,103} (reviewed in ref. ¹⁰⁴ for Fungi). Another peculiarity of IDR modules of organellophagy receptors is obviously the presence of LIR motifs that, upon activation, engage components of the autophagy machinery. It should not be considered a coincidence that the LIR located closer to a lipid bilayer is the one of FUNDC1, placed 24 residues apart. In our tests, the progressive shortening of the distance of the LIR from the membrane clearly generates spatial constraints for the engagement of LC3 proteins. Calculating an elongated structure and an estimated average length for a single amino acid of 0.38 nm (ref. ⁶²), the GT₁₇-SEC62_IDR65 module places the LIR at a distance of 28 residues from the membrane (that is, an estimated 10.64 nm that still allows LC3 engagement). By contrast, for the GT₁₇-SEC62_IDR47 module the LIR is placed at 10 residues (that is, 3.8 nm) from the lipid bilayer, a distance too short to accommodate an LC3 molecule bound to the IDR. This was confirmed in cellula, where the shortened IDR modules progressively lost the capacity to engage LC3 proteins and to deliver ER portions within degradative compartments. Clearly, the shortening of the IDR module also eliminates domains outside the LIR that contribute to the stable association with lipidated LC3.

For IDR-driven mitochondrial fragmentation our data reveal the intervention of the fissionase and GTPase DRP1, whose involvement in the fragmentation of mitochondria that precedes mitophagy has been previously reported^{13,84,85,86,87,88,90}. The identity and/or the involvement of a GTPase that contributes with the ER-phagy receptors in the fragmentation of the ER, if any, remains to be established. The combination of a membrane-tethering module that specifies and pre-remodels the organelle and the suborganellar portion to be removed from cells^8,9,10,11,81 and of a functionally conserved IDR module that facilitates organelle fragmentation and engages cytosolic autophagy gene products to deliver organelle portions to the degradative compartments builds an elegant and modulable sensor to adapt organelle size and function to cellular needs.

The capacity to control the integrity of organelles and the lysosomal clearance of select portions of organelles may find applications in the treatment of diseases associated with dysfunctional regulation of organelle size and activity. For example, the enhanced expression of ER-phagy receptors and the resulting hyperactivation of constitutive ER-phagy render tumour cells more resistant to cellular stresses such as those induced upon therapeutic interventions^{105,106,107,108,109}. Giant and megamitochondria have been described by pathophysiologists as early hallmarks of human disorders including liver diseases^110,111, cardiomyopathies¹¹² and alcohol-induced heart disease^113,114 as well as in pathophysiological conditions such as ageing^115,116,117, which leads to organelle dysfunction. Other organelles may also be affected by gigantism: pathological ER expansion due to chronic ER stress is a hallmark of metabolic and of ER storage disorders^118,119, while giant peroxisomes are present in hepatic tissues of patients affected with rhizomelic chondrodysplasia punctata and acyl-CoA oxidase deficiency¹²⁰.

Our work leads the way to develop organelle-targeting chimeras (ORGATACs), where an organelle-targeting signal is fused to a short IDR sequence that conserves the functional features of organellophagy IDR modules. Organelle-targeting chimeras may favour organelle fragmentation (in the absence of engagement of the autophagic machinery) or organelle fragmentation and turnover (upon engagement of LC3 proteins). This is expected to alleviate the symptoms of diseases characterized by organelle dysfunction also related to ageing, intraorganelle accumulation of harmful macromolecules including misfolded proteins, and to promote de novo biogenesis of functional replacements. All in all, our study offers a method to control integrity and activity of intracellular organelles by surface activation of IDR modules with net negative charges. It highlights a remarkable example of functional conservation on sequence divergency that would support a model, where IDRs characterized by different protein primary structure may retain biophysical features that are important for their function¹²¹ and paves the way to study the evolution of IDR modules to control organellar homoeostasis across species.

Methods

Cell culture, transient transfection and inhibitors

MEF and human embryonic kidney 293 (HEK293) cells were cultured in DMEM supplemented with 10% foetal calf serum (FCS, Dubco) at 37 °C in a 5% CO₂ atmosphere. Transient transfections were carried out using jetPRIME transfection reagent (PolyPlus) according to the manufacturer’s instructions. Wild-type and ATG7-KO MEF are a kind gift from M. Komatsu. Wild-type and DRP1-KO MEF are a kind gift from Mike Ryan and Susanna Manley. BafA1 (Calbiochem) was used at 50 nM for 15 h if not otherwise specified. HEK293 were purchased from ATCC (CRL-1573). ATG7-KO were checked for absence of ATG7 and for lack of LC3 lipidation (for example, refs. ^8,82,122). DRP1-KO MEFs were checked for absence of DRP1 and enlarged mitochondria. Wild-type MEF cells resulted positive for DRP1 and ATG7, displayed classical fibroblast morphology and were positive for LAMP1 staining with mouse-specific LAMP1 antibody. HEK293 were obtained from ATCC, and their identity was confirmed by morphology.

Expression plasmids and antibodies

ER-targeted IDR constructs of ER-phagy receptors were subcloned in a pcDNA3.1(+) backbone with an N-terminal sfGFP or HALO tag, an inactive bacterial hydrolase that covalently binds cell permeable ligands coupled to fluorescent probes⁵⁸. The ER targeting of ER-phagy receptors’ IDR portions was achieved by incorporating an N-terminal prolactin signal sequence, followed by sfGFP, -AGG-TPSETLITTVESNSSW- linker derived from the mouse Cyb5a protein and a -T₁₇ transmembrane anchor (-WTNWVIPAISALVVALM-) linker with the -YRGS- cytosolic flanking sequence. Constructs containing transmembrane domain modules were fused with a C-terminal cytosolic GFP or HALO moiety preceded by -AAAGT- or -AAASGAGS- linkers, respectively. ER-targeting of FUNDC1 IDR–GFP and GFP control constructs was achieved by adding the T₁₇ sequence at the C-terminal (-WTNWVIPAISALVVALM-) preceded by the sfGFP-GGS-PSETLITTVESNSSW- linker. Mitochondrial targeting of ER-phagy IDRs was achieved by fusing the first 33 amino acids of the human TOMM20 protein to the N-terminus, followed by a -GPVAT- linker, sfGFP, and the IDRs of ER-phagy receptors. Mitochondrial targeting of the FUNDC1 IDR–GFP and GFP control constructs was achieved by adding the transmembrane sequence of MAVS (-RPSPGALWLQVAVTGVLVVTLLVVLYRRRLH-) at the C-terminus, preceded by AAAA-GA-sfGFP and -GGS-PSETLITTVESNSSW- linker. The extended LIR sequence of SEC62 (-GNDFEMITK-) was generated by replacing the QP residues in the REEP1 IDR sequence -GQPK- with -NDFEMIT- to create REEP1_IDRFEMI. The sequences of IDRs and the corresponding linkers, cumulative charges and lengths are summarized in Supplementary Table 1. The antibodies used in this study, along with their respective dilutions, are listed in Supplementary Table 2.

Cell lysis and western blot

Following the respective treatments, MEF were washed with ice-cold phosphate-buffered saline (PBS) containing 20 mM N-ethylmaleimide (NEM). The cells were then lysed using either 2% CHAPS (in HEPES-buffered saline (HBS), pH 6.8) or RIPA buffer (1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate in HBS, pH 7.4), both supplemented with 20 mM NEM and protease inhibitors (200 mM phenylmethylsulfonyl fluoride, 16.5 mM chymostatin, 23.4 mM leupeptin, 16.6 mM antipain, 14.6 mM pepstatin) for 20 min on ice. The postnuclear supernatant (PNS) was obtained by centrifugation at 10,600gfor 10 min at 4 °C. The PNS was denatured by adding 100 mM dithiothreitol (DTT) and heated for 5 min at 95 °C before being subjected to SDS–polyacrylamide gel electrophoresis (PAGE). For in gel GFP-fluorescent assays, the PNS supplemented with DTT was kept for 5 min at room temperature (RT). GFP-fluorescent SDS–PAGE gels were scanned with on the Amersham Typhoon scanner (Cytiva) with 488 nm laser and 525BP20 filter. HaloTag(TMR)-fluorescent SDS–PAGE gels were scanned with the same instrument with 532 nm laser and 570BP20 filter. The protein bands were then stained with Coomassie or transferred onto polyvinylidene difluoride membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). The membranes were blocked with 8% (w/v) non-fat dry milk (Bio-Rad) in Tris-buffered saline containing 1% Tween 20 (TBS-T, Sigma-Aldrich) and incubated overnight with primary antibodies diluted in TBS-T. Afterward, horseradish peroxidase (HRP)-conjugated protein A diluted in TBS-T was applied for 45 min. The protein bands were visualized using the WesternBright Quantum detection system (Advansta), and the signals were captured with the FusionFX chemiluminescence imaging system (VILBER/Witec). The quantification of western blot bands was performed using Fusion FX Edge 18.12 and ImageJ 2.16.0/1.54p software¹²³.

Protein cross‐linking with DSP

After treatment, the cells were washed with PBS and incubated with 1 mM dithiobis-succinimidyl propionate (DSP) (Thermo Fisher Scientific) (prepared from a 100× stock in dimethylsulfoxide) in PBS for 30 min at RT. The reaction was quenched by adding 1 M Tris (pH 7.8) to a final concentration of 20 mM, followed by a 15-min incubation at RT. The cells were then washed with PBS, treated with 20 mM NEM and lysed using RIPA buffer (1% Triton X-100, 0.1% SDS, 20 mM NEM, 0.5% sodium deoxycholate in HBS, pH 7.4) for 20 min on ice. The PNS were collected by centrifugation at 10,600gfor 10 min and used for immunoprecipitation of IDR chimera–LC3 complexes.

Affinity purification-liquid chromatography/mass spectrometry

The HEK293 cells were transfected with GFP-tagged TMD/RHD, SEC62/SEC62LIR or GT₁₇-SEC62_IDR/IDRLIR chimeras. Fifteen-hour post-transfection, cells were treated with 100 nM BafA1 for 6 h, crosslinked with DSP as described above, and the supernatant was collected. The protein complexes were isolated with 100 µl GFP–TRAP (Chromotek) following manufacturer’s protocol. The samples were digested on beads following a modified version of the iST method (named miST method). A total of 25 µl of miST lysis buffer (1% sodium deoxycholate, 100 mM Tris pH 8.6, 10 mM DTT), were added to the beads. After mixing and dilution 1:1 (v:v) with H₂O, the samples were heated 5 min at 75 °C. The reduced disulfides were alkylated by adding 13 µl of 160 mM chloroacetamide (33 mM final) and incubating for 45 min at 25 °C in the dark. After digestion with 0.5 µg of trypsin/LysC mix (Promega #V5073) for 2 h at 25 °C, the sample supernatants were transferred in new tubes. To remove sodium deoxycholate, two sample volumes of isopropanol containing 1% TFA were added to the digests, and the samples were desalted on a strong cation exchange plate (Oasis MCX; Waters) by centrifugation. After washing with isopropanol/1% TFA, peptides were eluted in 200 µl of 40% MeCN, 59% water and 1% (v/v) ammonia and dried by centrifugal evaporation.

Tryptic peptide mixtures were injected on a Vanquish Neo nanoHPLC system interfaced via a nanospray Flex source to a high resolution Orbitrap Exploris 480 mass spectrometer (Thermo Fisher). The peptides were loaded onto a trapping microcolumn PepMap100 C18 (5 mm × 1.0 mm ID, 5 μm, Thermo Fisher) before separation on a C18 custom packed column (75 μm ID × 45 cm, 1.8 μm particles, Reprosil Pur, Dr. Maisch), using a gradient from 2% to 80% acetonitrile in 0.1% formic acid for peptide separation at a flow rate of 250 nl min⁻¹(total time 130 min). Full MS survey scans were performed at 120,000 resolution. A data-dependent acquisition method controlled by Xcalibur software (Tune 2.9, Thermo Fisher Scientific) was used that optimized the number of precursors selected (‘top speed’) of charge 2+ to 5+ while maintaining a fixed scan cycle of 2 s. The peptides were fragmented by higher energy collision dissociation with a normalized energy of 30% at 15,000 resolution. The window for precursor isolation was of 1.6 m/zunits around the precursor and selected fragments were excluded for 60 s from further analysis. The intensity values were obtained following MaxQuant (V. 2.1.4.0)¹²⁴ analysis. Only the proteins with at least two detected peptides were considered for analyses. The results were normalized by median abundance, the missing values were imputed from a normal distribution of 1.8 standard deviation down shift and with a width of 0.3 of each sample, and unpaired two-tailed tests were used to calculate the Pvalue. Data analyses and graph plotting were done with the Perseus platform (version 2.0.5)¹²⁵ and MATLAB (The MathWorks, R2023b Update 5 23.2.0.24591. The raw data are deposited in the Pride database (project accession PXD060519).

CLSM

MEFs were seeded onto alcian blue-coated (Sigma) glass coverslips (VWR) and transiently transfected using jetPRIME reagent following the manufacturer’s protocol. For LysoQuant analysis, 8 h post transfection, the cells were treated with 50 nM BafA1 for 15 h. Moreover, the cells transfected with HaloTag-fusion proteins were incubated with 100 nM TMR HaloTag ligand (Promega). A total of 24 h post transfection, the cells were fixed at RT for 20 min using 3.7% formaldehyde in PBS. To permeabilize the membranes, coverslips were incubated for 20 min in a permeabilization solution containing 10% goat serum, 10 mM HEPES, 15 mM glycine and 0.05% saponin. After permeabilization, the cells were incubated with primary antibodies diluted in permeabilization solution (as specified in Supplementary Table 2) for 120 min. Following three washes in permeabilization solution, the cells were then incubated with Alexa Fluor-conjugated secondary antibodies diluted 1:300 in permeabilization solution for 45 min. The cells were rinsed three times with permeabilization solution and water, then mounted onto glass microscope slides (Epredia) using a drop of Mount Liquid anti-fade (Abberior). Confocal images were captured using Leica TCS SP5, STELLARIS 5 and STELLARIS 8 microscopes equipped with a Leica HCX PL APO lambda blue 63×/1.40 oil objective and a pinhole set at 1 a.u. Image acquisition was performed with Leica LAS X software, utilizing diode at 405 nm or laser beams at 489, 499, 552, 561, 587 and 653 nm wavelengths for excitation. Fluorescence emissions were collected at the following ranges: 430–490 nm (Alexa Fluor 405), 494–557 nm (GFP), 504–587 nm (Alexa Fluor 488), 557–663 nm (TMR), 592–644 nm (Alexa Fluor 568) and 658–750 nm (Alexa Fluor 646). Image analysis and quantification were performed using LysoQuant and ImageJ 2.16.0/1.54p software^64,123. Image post-processing was performed with Adobe Photoshop (v26.2.0).

RT-TEM and electron tomography

MEFs were plated on gridded glass-bottom dishes (MatTek Corporation) and transiently transfected using jetPRIME reagent following the manufacturer’s protocol. A total of 24 h after transfection, the cells were prefixed with a 4% formaldehyde EM-grade solution supplemented with 0.1% glutaraldehyde. For morphological ER analyses, the coordinates of the cells on the finder grid were determined using wide‐field microscopy on the Leica STELLARIS 8 microscope with Leica LAS X 4.5.0.025531 software with Leica PL APO 10× 0.40 air objective with pinhole 2 a.u., and the transfected and non-transfected cells were identified based on GFP fluorescence (493–556 nm range). The cells were then fixed with a solution containing 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). After several washes in sodium cacodylate buffer, the cells were post-fixed in 1% osmium tetroxide (OsO4), 1.5% potassium ferricyanide (K4(Fe(CN)6)) in 0.1 M sodium cacodylate buffer for 1 h on ice, washed with distilled water and stained en bloc with 0.5% uranyl acetate in distilled water overnight at 4 °C in the dark. The samples were then rinsed in distilled water, dehydrated with increasing concentrations of ethanol and embedded in Epoxy resin (Sigma-Aldrich). After curing for 48 h at 60 °C, ultrathin sections (70–90 nm thickness) were collected using an ultramicrotome (UC7, Leica microsystem), stained with uranyl acetate and Sato’s lead solutions and observed under a Transmission Electron Microscope Talos L120C (FEI, Thermo Fisher Scientific) operating at 120 kV. The images of transfected and non-transfected cells were acquired with a Ceta CCD camera (FEI, Thermo Fisher Scientific). For three-dimensional serial section electron tomography, serial sections (130–150 nm thickness) were collected on formvar-carbon coated slot grids, tilted images series (+60°) were acquired with a Talos L120C TEM using Tomography 5 software (FEI, Thermo Fisher Scientific). The tilted images were aligned, and tomograms reconstructed using IMOD software (v. 4.11.24) (organelle segmentation was performed in Microscopy Image Browser (MIB, v. 2.84 (ref. ¹²⁶)) and visualized in IMOD v4.11.24 (ref. ¹²⁷)).

Immunogold electron microscopy

MEFs were seeded onto glass-bottom dishes (MatTek Corporation) and transiently transfected with GFP-tagged chimeras using jetPRIME reagent, following the manufacturer’s instructions. A total of 8 h after transfection, the cells were either treated with 50 nM BafA1 for 15 h and then fixed for 2 h in periodate-lysine-paraformaldehyde¹²⁸ (FUJIFILM Wako) at RT or fixed 25 h post-transfection without treatment. After the fixative was removed by washing with 1× PBS, the cells were incubated with 50 mM glycine and blocked for 30 min in a blocking solution (0.2% bovine serum albumin, 5% goat serum, 50 mM NH₄Cl, 0.1% saponin, 20 mM phosphate buffer, 150 mM NaCl) at RT. The cells were stained with primary rabbit anti-GFP antibody (Abcam) and gold-labelled secondary antibodies (Nanoprobes) in the blocking buffer at RT. Afterwards, the cells were refixed for 30 min in 1% glutaraldehyde, and the nanogold particles were enhanced using a gold enhancement solution (Nanoprobes) as per the manufacturer’s protocol. The cells were post-fixed with osmium tetroxide (OsO₄) and processed for electron microscopy. The samples were then rinsed with distilled water, dehydrated through a graded ethanol series and embedded in epoxy resin (Sigma-Aldrich). After curing for 48 h at 60 °C, ultrathin sections (70–90 nm) were cut using an ultramicrotome (UC7, Leica Microsystems), stained with uranyl acetate and Sato’s lead solutions. The images were captured using a Ceta CCD camera (FEI, Thermo Fisher Scientific) and Velox 3.6.0 software (FEI, Thermo Fisher Scientific) on a Talos L120C TEM (FEI, Thermo Fisher Scientific) operating at 120 kV. For live FRAP analyses, ATG7-KO MEF expressing GFP–KDEL and HaloTag fusion proteins were imaged for 50 frames before bleach, then the GFP signal was bleached by 20 pulses of 405 diode at 100% intensity and imaged post-bleaching for 500 frames with the frame rate of 0.261 s. The bleaching and intensity measurements were done over a peripheral ER area of 29 µm². The intensity values were then normalized to prebleach measurements, and the maximum normalized postbleach value was used to assess ER fragmentation.

Statistical analyses, data acquisition and blinding

The statistical comparisons and graphical plots were performed using GraphPad Prism 10 (GraphPad Software, version 10.1.2 for Windows). A data analysis for proteomics data was performed in Perseus v2.0.5. Tests employed for statistical analysis are reported in figure legends. Data distributions (individual points) are always shown. Whenever data distribution was not assumed to be normal, an appropriate statistical test was used to take it into account. An adjusted P value <0.05 (for one-way and two-way analysis of variance (ANOVA) with multiple comparison tests) was considered statistically significant. The ANOVA test results were corrected for multiple comparisons. All t-tests performed are two-sided. All experimental replicates represent independent biological replicates. Figure legends specify the number of cells, EL or mitochondria analysed (n) and the number of independent biological replicates of the experiment (N). No statistical methods were used to predetermine sample sizes, but our sample sizes are similar to those reported in previous publications^{57,64,65,129,130}. No randomization was performed as this is not common for western blot or microscopy analysis. The covariates were controlled with the corresponding negative controls.

Unbiased imaging data collection/analyses were performed by at least two different scientists, one of which was blind to the identity of the sample. Morphological analyses of mitochondrial size were performed by the scientists blinded to the identity of the sample. The fluorescent image quantifications of ER and mitochondria delivery to ELs were performed using LysoQuant, an unbiased and automated deep learning tool. Acquisition of MS data was done by scientist blinded to the nature of the sample and the scope of the experiment. For western blot and immunoprecipitation analyses, conclusions were drawn based on qualitative presence/absence of band signal; therefore, blinding is not relevant. For electron microscopy, acquisition of samples was performed by a scientist blinded to the nature of the sample. No data were excluded from the analyses.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The MS proteomics data supporting the data in Extended Data Fig. 1f,g can be accessed from the ProteomeXchange Consortium via the PRIDE partner repository under accession code PXD060519. Uncropped blots and gels are included in image source data. No restrictions on data availability apply. All materials used in the analysis are available, without restriction upon reasonable request to reproduce or extend the analyses. Source data are provided with this paper.

Code availability

No custom code was developed or used in this study.

References

Reggiori, F. & Molinari, M. ER-phagy: mechanisms, regulation and diseases connected to the lysosomal clearance of the endoplasmic reticulum. Physiol. Rev. 102, 1393–1448 (2022).
Article CAS PubMed PubMed Central Google Scholar
Adriaenssens, E., Ferrari, L. & Martens, S. Orchestration of selective autophagy by cargo receptors. Curr. Biol. 32, R1357–R1371 (2022).
Article CAS PubMed Google Scholar
Conway, O., Akpinar, H. A., Rogov, V. V. & Kirkin, V. Selective autophagy receptors in neuronal health and disease. J. Mol. Biol. 432, 2483–2509 (2020).
Article CAS PubMed Google Scholar
Popelka, H. & Uversky, V. N. Theater in the self-cleaning cell: intrinsically disordered proteins or protein regions acting with membranes in autophagy. Membranes 12, 457 (2022).
Article CAS PubMed PubMed Central Google Scholar
Chino, H. & Mizushima, N. ER-phagy: quality and quantity control of the endoplasmic reticulum by autophagy. Cold Spring Harb. Perspect. Biol 15, a041256 (2023).
Article CAS PubMed PubMed Central Google Scholar
Lamark, T. & Johansen, T. Mechanisms of selective autophagy. Annu. Rev. Cell Dev. Biol. 37, 143–169 (2021).
Article CAS PubMed Google Scholar
Khaminets, A. et al. Regulation of endoplasmic reticulum turnover by selective autophagy. Nature 522, 354–358 (2015).
Article CAS PubMed Google Scholar
Fumagalli, F. et al. Translocon component Sec62 acts in endoplasmic reticulum turnover during stress recovery. Nat. Cell Biol. 18, 1173–1184 (2016).
Article CAS PubMed Google Scholar
Loi, M., Raimondi, A., Morone, D. & Molinari, M. ESCRT-III-driven piecemeal micro-ER-phagy remodels the ER during recovery from ER stress. Nat. Commun. 10, 5058 (2019).
Article PubMed PubMed Central Google Scholar
An, H. et al. Tex264 is an endoplasmic reticulum-resident ATG8-interacting protein critical for ER remodeling during nutrient stress. Mol. Cell 74, 891–908 (2019).
Article CAS PubMed PubMed Central Google Scholar
Chino, H., Hatta, T., Natsume, T. & Mizushima, N. Intrinsically disordered protein TEX264 mediates ER-phagy. Mol. Cell 74, 909–921 (2019).
Article CAS PubMed Google Scholar
Smith, M. D. et al. CCPG1 is a non-canonical autophagy cargo receptor essential for ER-phagy and pancreatic ER proteostasis. Dev. Cell 44, 217–232.e11 (2018).
Article CAS PubMed PubMed Central Google Scholar
Chen, M. et al. Mitophagy receptor FUNDC1 regulates mitochondrial dynamics and mitophagy. Autophagy 12, 689–702 (2016).
Article CAS PubMed PubMed Central Google Scholar
Liu, L. et al. Mitochondrial outer-membrane protein FUNDC1 mediates hypoxia-induced mitophagy in mammalian cells. Nat. Cell Biol. 14, 177–185 (2012).
Article PubMed Google Scholar
Sandoval, H. et al. Essential role for Nix in autophagic maturation of erythroid cells. Nature 454, 232–235 (2008).
Article CAS PubMed PubMed Central Google Scholar
Schweers, R. L. et al. NIX is required for programmed mitochondrial clearance during reticulocyte maturation. Proc. Natl Acad. Sci. USA 104, 19500–19505 (2007).
Article CAS PubMed PubMed Central Google Scholar
Hickey, K. L. et al. Proteome census upon nutrient stress reveals Golgiphagy membrane receptors. Nature 623, 167–174 (2023).
Article CAS PubMed PubMed Central Google Scholar
Kitta, S. et al. YIPF3 and YIPF4 regulate autophagic turnover of the Golgi apparatus. EMBO J. 43, 2954–2978 (2024).
Article CAS PubMed PubMed Central Google Scholar
Mizushima, N. Organelle degradation. Mol. Cell 82, 1604–1605 (2022).
Article PubMed Google Scholar
Molinari, M. ER-phagy responses in yeast, plants, and mammalian cells and their crosstalk with UPR and ERAD. Dev. Cell 56, 949–966 (2021).
Article CAS PubMed Google Scholar
Johansen, T. & Lamark, T. Selective autophagy: ATG8 family proteins, LIR motifs and cargo receptors. J. Mol. Biol. 432, 80–103 (2020).
Article CAS PubMed Google Scholar
Gubas, A. & Dikic, I. A guide to the regulation of selective autophagy receptors. FEBS J. https://doi.org/10.1111/febs.15824 (2021).
Article PubMed Google Scholar
Gubas, A. & Dikic, I. ER remodeling via ER-phagy. Mol. Cell 82, 1492–1500 (2022).
Article CAS PubMed PubMed Central Google Scholar
Gonzalez, A. et al. Ubiquitination regulates ER-phagy and remodelling of endoplasmic reticulum. Nature https://doi.org/10.1038/s41586-023-06089-2 (2023).
Foronda, H. et al. Heteromeric clusters of ubiquitinated ER-shaping proteins drive ER-phagy. Nature 618, 402–410 (2023).
Article CAS PubMed PubMed Central Google Scholar
Bhaskara, R. M. et al. Curvature induction and membrane remodeling by FAM134B reticulon homology domain assist selective ER-phagy. Nat. Commun. 10, 2370 (2019).
Article PubMed PubMed Central Google Scholar
Jiang, X. et al. FAM134B oligomerization drives endoplasmic reticulum membrane scission for ER-phagy. EMBO J. 39, e102608 (2020).
Article CAS PubMed PubMed Central Google Scholar
Hoyer, M. J. & Harper, J. W. ER membrane curvature and ubiquitin as drivers of ER phagy. Dev. Cell 58, 1219–1220 (2023).
Article CAS PubMed Google Scholar
Gonzalez, A. & Dikic, I. Decoding Golgiphagy: selective recycling under stress. Cell Res. 34, 277–278 (2024).
Article PubMed Google Scholar
Wu, H. & Voeltz, G. K. Reticulon-3 promotes endosome maturation at ER membrane contact sites. Dev. Cell 56, 52–66 (2021).
Article CAS PubMed PubMed Central Google Scholar
Delorme-Axford, E., Popelka, H. & Klionsky, D. J. TEX264 is a major receptor for mammalian reticulophagy. Autophagy 15, 1677–1681 (2019).
Article CAS PubMed PubMed Central Google Scholar
van der Lee, R. et al. Classification of intrinsically disordered regions and proteins. Chem. Rev. 114, 6589–6631 (2014).
Article PubMed PubMed Central Google Scholar
Lemke, E. A. et al. Intrinsic disorder: a term to define the specific physicochemical characteristic of protein conformational heterogeneity. Mol. Cell 84, 1188–1190 (2024).
Article CAS PubMed Google Scholar
Holehouse, A. S. & Kragelund, B. B. The molecular basis for cellular function of intrinsically disordered protein regions. Nat. Rev. Mol. Cell Biol. https://doi.org/10.1038/s41580-023-00673-0 (2023).
Zeno, W. F. et al. Molecular mechanisms of membrane curvature sensing by a disordered protein. J. Am. Chem. Soc. 141, 10361–10371 (2019).
Article CAS PubMed PubMed Central Google Scholar
Busch, D. J. et al. Intrinsically disordered proteins drive membrane curvature. Nat. Commun. 6, 7875 (2015).
Article CAS PubMed Google Scholar
Zeno, W. F. et al. Synergy between intrinsically disordered domains and structured proteins amplifies membrane curvature sensing. Nat. Commun. 9, 4152 (2018).
Article PubMed PubMed Central Google Scholar
De Leonibus, C. Sestrin2 drives ER-phagy in response to protein misfolding. Dev. Cell 59, 2035–2052.e10 (2024).
Article CAS PubMed PubMed Central Google Scholar
Liao, Y. J., Duan, B., Zhang, Y. F., Zhang, X. M. & Xia, B. Excessive ER-phagy mediated by the autophagy receptor FAM134B results in ER stress, the unfolded protein response, and cell death in HeLa cells. J. Biol. Chem. 294, 20009–20023 (2019).
Article CAS PubMed PubMed Central Google Scholar
Zielke, S. et al. ATF4 links ER stress with reticulophagy in glioblastoma cells. Autophagy https://doi.org/10.1080/15548627.2020.1827780 (2020).
Koenig, U. et al. Cell death induced autophagy contributes to terminal differentiation of skin and skin appendages. Autophagy 16, 932–945 (2020).
Article CAS PubMed Google Scholar
Cinque, L. et al. MiT/TFE factors control ER-phagy via transcriptional regulation of FAM134B. EMBO J. 39, e105696 (2020).
Article CAS PubMed PubMed Central Google Scholar
Kohno, S., Shiozaki, Y., Keenan, A. L., Miyazaki-Anzai, S. & Miyazaki, M. An N-terminal-truncated isoform of FAM134B (FAM134B-2) regulates starvation-induced hepatic selective ER-phagy. Life Sci. Alliance 2, e201900340 (2019).
Article PubMed PubMed Central Google Scholar
Shiozaki, Y., Miyazaki-Anzai, S., Keenan, A. L. & Miyazaki, M. MEF2D-NR4A1-FAM134B2-mediated reticulophagy contributes to amino acid homeostasis. Autophagy https://doi.org/10.1080/15548627.2021.1968228 (2021).
Tian, X. et al. RAB26 and RAB3D are direct transcriptional targets of MIST1 that regulate exocrine granule maturation. Mol. Cell. Biol. 30, 1269–1284 (2010).
Article CAS PubMed Google Scholar
Shi, W. et al. Transcriptional profiling of mouse B cell terminal differentiation defines a signature for antibody-secreting plasma cells. Nat. Immunol. 16, 663–673 (2015).
Article CAS PubMed Google Scholar
Reimold, A. M. et al. Plasma cell differentiation requires the transcription factor XBP-1. Nature 412, 300–307 (2001).
Article CAS PubMed Google Scholar
Pengo, N. et al. Plasma cells require autophagy for sustainable immunoglobulin production. Nat. Immunol. 14, 298–305 (2013).
Article CAS PubMed Google Scholar
Cenci, S. et al. Progressively impaired proteasomal capacity during terminal plasma cell differentiation. EMBO J. 25, 1104–1113 (2006).
Article CAS PubMed PubMed Central Google Scholar
Mizuno, T., Muroi, K. & Irie, K. Snf1 AMPK positively regulates ER-phagy via expression control of Atg39 autophagy receptor in yeast ER stress response. PLoS Genet. 16, e1009053 (2020).
Article CAS PubMed PubMed Central Google Scholar
Mizuno, T. & Irie, K. Msn2/4 transcription factors positively regulate expression of Atg39 ER-phagy receptor. Sci. Rep. 11, 11919 (2021).
Article CAS PubMed PubMed Central Google Scholar
Cui, Y. et al. A COPII subunit acts with an autophagy receptor to target endoplasmic reticulum for degradation. Science 365, 53–60 (2019).
Article CAS PubMed PubMed Central Google Scholar
Di Lorenzo, G. et al. Phosphorylation of FAM134C by CK2 controls starvation-induced ER-phagy. Sci. Adv. 8, eabo1215 (2022).
Article PubMed PubMed Central Google Scholar
Chino, H. et al. Phosphorylation by casein kinase 2 enhances the interaction between ER-phagy receptor TEX264 and ATG8 proteins. EMBO Rep. 23, e54801 (2022).
Article CAS PubMed PubMed Central Google Scholar
Berkane, R. et al. The function of ER-phagy receptors is regulated through phosphorylation-dependent ubiquitination pathways. Nat. Commun. 14, 8364 (2023).
Article CAS PubMed PubMed Central Google Scholar
Grumati, P. et al. Full length RTN3 regulates turnover of tubular endoplasmic reticulum via selective autophagy. eLife 6, e25555 (2017).
Article PubMed PubMed Central Google Scholar
Kucinska, M. K. et al. TMX4-driven LINC complex disassembly and asymmetric autophagy of the nuclear envelope upon acute ER stress. Nat. Commun. 14, 3497 (2023).
Article CAS PubMed PubMed Central Google Scholar
England, C. G., Luo, H. M. & Cai, W. B. HaloTag technology: a versatile platform for biomedical applications. Bioconjugate Chem. 26, 975–986 (2015).
Article CAS Google Scholar
Mizushima, N. Autophagic flux measurement: cargo degradation versus generation of degradation products. Curr. Opin. Cell Biol. 93, 102463 (2025).
Article CAS PubMed Google Scholar
Rudinskiy, M., Morone, D. & Molinari, M. Fluorescent reporters, imaging, and artificial intelligence toolkits to monitor and quantify autophagy, heterophagy, and lysosomal trafficking fluxes. Traffic 25, e12957 (2024).
Article CAS PubMed Google Scholar
Yang, F., Moss, L. G. & Phillips, G. N. Jr The molecular structure of green fluorescent protein. Nat. Biotechnol. 14, 1246–1251 (1996).
Article CAS PubMed Google Scholar
Zhou, H. X. Polymer models of protein stability, folding, and interactions. Biochemistry 43, 2141–2154 (2004).
Article CAS PubMed Google Scholar
Klionsky, D. J., Elazar, Z., Seglen, P. O. & Rubinsztein, D. C. Does bafilomycin A(1) block the fusion of autophagosomes with lysosomes? Autophagy 4, 849–850 (2008).
Article CAS PubMed Google Scholar
Morone, D., Marazza, A., Bergmann, T. J. & Molinari, M. Deep learning approach for quantification of organelles and misfolded polypeptide delivery within degradative compartments. Mol. Biol. Cell 31, 1512–1524 (2020).
Article CAS PubMed PubMed Central Google Scholar
Rudinskiy, M., Bergmann, T. J. & Molinari, M. Quantitative and time-resolved monitoring of organelle and protein delivery to the lysosome with a tandem fluorescent Halo-GFP reporter. Mol. Biol. Cell 33, ar57 (2022).
Article CAS PubMed PubMed Central Google Scholar
Rudinskiy, M. & Molinari, M. ER-to-lysosome-associated degradation in a nutshell: mammalian, yeast, and plant ER-phagy as induced by misfolded proteins. FEBS Lett. https://doi.org/10.1002/1873-3468.14674 (2023).
Yim, W. W., Yamamoto, H. & Mizushima, N. A pulse-chasable reporter processing assay for mammalian autophagic flux with HaloTag. eLife 11, e78923 (2022).
Article CAS PubMed PubMed Central Google Scholar
Pedrazzini, E., Villa, A. & Borgese, N. A mutant cytochrome b5 with a lengthened membrane anchor escapes from the endoplasmic reticulum and reaches the plasma membrane. Proc. Natl Acad. Sci. USA 93, 4207–4212 (1996).
Article CAS PubMed PubMed Central Google Scholar
Pedelacq, J. D., Cabantous, S., Tran, T., Terwilliger, T. C. & Waldo, G. S. Engineering and characterization of a superfolder green fluorescent protein. Nat. Biotechnol. 24, 79–88 (2006).
Article CAS PubMed Google Scholar
Hu, J. et al. Membrane proteins of the endoplasmic reticulum induce high-curvature tubules. Science 319, 1247–1250 (2008).
Article CAS PubMed Google Scholar
Shibata, Y. et al. The reticulon and DP1/Yop1p proteins form immobile oligomers in the tubular endoplasmic reticulum. J. Biol. Chem. 283, 18892–18904 (2008).
Article CAS PubMed PubMed Central Google Scholar
Voeltz, G. K., Prinz, W. A., Shibata, Y., Rist, J. M. & Rapoport, T. A. A class of membrane proteins shaping the tubular endoplasmic reticulum. Cell 124, 573–586 (2006).
Article CAS PubMed Google Scholar
Shibata, Y. et al. Mechanisms determining the morphology of the peripheral ER. Cell 143, 774–788 (2010).
Article CAS PubMed PubMed Central Google Scholar
Hu, J. et al. A class of dynamin-like GTPases involved in the generation of the tubular ER network. Cell 138, 549–561 (2009).
Article CAS PubMed PubMed Central Google Scholar
Terasaki, M. et al. Stacked endoplasmic reticulum sheets are connected by helicoidal membrane motifs. Cell 154, 285–296 (2013).
Article CAS PubMed PubMed Central Google Scholar
De Craene, J. O. et al. Rtn1p is involved in structuring the cortical endoplasmic reticulum. Mol. Biol. Cell 17, 3009–3020 (2006).
Article PubMed PubMed Central Google Scholar
Wang, S., Romano, F. B., Field, C. M., Mitchison, T. J. & Rapoport, T. A. Multiple mechanisms determine ER network morphology during the cell cycle in Xenopus egg extracts. J. Cell Biol. 203, 801–814 (2013).
Article CAS PubMed PubMed Central Google Scholar
Kiseleva, E., Morozova, K. N., Voeltz, G. K., Allen, T. D. & Goldberg, M. W. Reticulon 4a/NogoA locates to regions of high membrane curvature and may have a role in nuclear envelope growth. J. Struct. Biol. 160, 224–235 (2007).
Article CAS PubMed PubMed Central Google Scholar
Tolley, N. et al. Overexpression of a plant reticulon remodels the lumen of the cortical endoplasmic reticulum but does not perturb protein transport. Traffic 9, 94–102 (2008).
Article CAS PubMed Google Scholar
Loi, M., Fregno, I., Guerra, C. & Molinari, M. Eat it right: ER-phagy and recovER-phagy. Biochem. Soc. Trans. 46, 699–706 (2018).
Article CAS PubMed PubMed Central Google Scholar
Loi, M. & Molinari, M. Mechanistic insights in recov-ER-phagy: micro-ER-phagy to recover from stress. Autophagy 16, 385–386 (2020).
Article PubMed PubMed Central Google Scholar
Komatsu, M. et al. Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. J. Cell Biol. 169, 425–434 (2005).
Article CAS PubMed PubMed Central Google Scholar
Kanaji, S., Iwahashi, J., Kida, Y., Sakaguchi, M. & Mihara, K. Characterization of the signal that directs Tom20 to the mitochondrial outer membrane. J. Cell Biol. 151, 277–288 (2000).
Article CAS PubMed PubMed Central Google Scholar
Webster, B. R., Scott, I., Traba, J., Han, K. & Sack, M. N. Regulation of autophagy and mitophagy by nutrient availability and acetylation. Biochim. Biophys. Acta 525, 534 (2014).
Google Scholar
Chen, C. W. et al. NME3 is a gatekeeper for DRP1-dependent mitophagy in hypoxia. Nat. Commun. 15, 2264 (2024).
Article CAS PubMed PubMed Central Google Scholar
Frank, M. et al. Mitophagy is triggered by mild oxidative stress in a mitochondrial fission dependent manner. Biochim. Biophys. Acta 1823, 2297–2310 (2012).
Article CAS PubMed Google Scholar
Smirnova, E., Griparic, L., Shurland, D. L. & van der Bliek, A. M. Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells. Mol. Biol. Cell 12, 2245–2256 (2001).
Article CAS PubMed PubMed Central Google Scholar
Fonseca, T. B., Sanchez-Guerrero, A., Milosevic, I. & Raimundo, N. Mitochondrial fission requires DRP1 but not dynamins. Nature 570, 34–42 (2019).
Article Google Scholar
Graef, M. A dividing matter: Drp1/Dnm1-independent mitophagy. J. Cell Biol. 215, 599–601 (2016).
Article CAS PubMed PubMed Central Google Scholar
Osellame, L. D. et al. Cooperative and independent roles of the Drp1 adaptors Mff, MiD49 and MiD51 in mitochondrial fission. J. Cell Sci. 129, 2170–2181 (2016).
Article CAS PubMed PubMed Central Google Scholar
Smirnova, E., Shurland, D. L., Ryazantsev, S. N. & van der Bliek, A. M. A human dynamin-related protein controls the distribution of mitochondria. J. Cell Biol. 143, 351–358 (1998).
Article CAS PubMed PubMed Central Google Scholar
Wang, N., Shibata, Y., Paulo, J. A., Gygi, S. P. & Rapoport, T. A. A conserved membrane curvature-generating protein is crucial for autophagosome formation in fission yeast. Nat. Commun. 14, 4765 (2023).
Article CAS PubMed PubMed Central Google Scholar
Shibata, Y. et al. The membrane curvature-inducing REEP1-4 proteins generate an ER-derived vesicular compartment. Nat. Commun. 15, 8655 (2024).
Article CAS PubMed PubMed Central Google Scholar
Reggio, A. et al. Role of FAM134 paralogues in endoplasmic reticulum remodeling, ER-phagy, and collagen quality control. EMBO Rep. 22, e52289 (2021).
Article CAS PubMed PubMed Central Google Scholar
Iavarone, F., Di Lorenzo, G. & Settembre, C. Regulatory events controlling ER-phagy. Curr. Opin. Cell Biol. 76, 102084 (2022).
Article CAS PubMed Google Scholar
Poveda-Cuevas, S. A. et al. Intrinsically disordered region amplifies membrane remodeling to augment selective ER-phagy. Proc. Natl Acad. Sci. USA 121, e2408071121 (2024).
Article CAS PubMed PubMed Central Google Scholar
Bigman, L. S., Iwahara, J. & Levy, Y. Negatively charged disordered regions are prevalent and functionally important across proteomes. J. Mol. Biol. 434, 167660 (2022).
Article CAS PubMed Google Scholar
Mochida, K. et al. Super-assembly of ER-phagy receptor Atg40 induces local ER remodeling at contacts with forming autophagosomal membranes. Nat. Commun. 11, 3306 (2020).
Article CAS PubMed PubMed Central Google Scholar
De Leonibus, C., Cinque, L. & Settembre, C. Beating the ER: novel insights into FAM134B function and regulation. EMBO J. https://doi.org/10.15252/embj.2020104546 (2020).
Rogov, V. V. et al. Phosphorylation of the mitochondrial autophagy receptor Nix enhances its interaction with LC3 proteins. Sci. Rep. 7, 1131 (2017).
Article PubMed PubMed Central Google Scholar
Farre, J. C., Burkenroad, A., Burnett, S. F. & Subramani, S. Phosphorylation of mitophagy and pexophagy receptors coordinates their interaction with Atg8 and Atg11. EMBO Rep. 14, 441–449 (2013).
Article CAS PubMed PubMed Central Google Scholar
Heo, J. M., Ordureau, A., Paulo, J. A., Rinehart, J. & Harper, J. W. The PINK1-PARKIN mitochondrial ubiquitylation pathway drives a program of OPTN/NDP52 recruitment and TBK1 activation to promote mitophagy. Mol. Cell 60, 7–20 (2015).
Article CAS PubMed PubMed Central Google Scholar
Richter, B. et al. Phosphorylation of OPTN by TBK1 enhances its binding to Ub chains and promotes selective autophagy of damaged mitochondria. Proc. Natl Acad. Sci. USA 113, 4039–4044 (2016).
Article CAS PubMed PubMed Central Google Scholar
Mochida, K. & Nakatogawa, H. ER-phagy: selective autophagy of the endoplasmic reticulum. EMBO Rep. 23, e55192 (2022).
Article CAS PubMed PubMed Central Google Scholar
Zimmermann, J. S. M., Linxweiler, J., Radosa, J. C., Linxweiler, M. & Zimmermann, R. The endoplasmic reticulum membrane protein Sec62 as potential therapeutic target in overexpressing tumors. Front. Physiol. 13, 1014271 (2022).
Article PubMed PubMed Central Google Scholar
Bergmann, T. J., Fumagalli, F., Loi, M. & Molinari, M. Role of SEC62 in ER maintenance: a link with ER stress tolerance in SEC62-overexpressing tumors?. Mol. Cell. Oncol. 4, e1264351 (2017).
Article PubMed PubMed Central Google Scholar
Tang, W. K. et al. Oncogenic properties of a novel gene JK-1 located in chromosome 5p and its overexpression in human esophageal squamous cell carcinoma. Int. J. Mol. Med. 19, 915–923 (2007).
CAS PubMed Google Scholar
Chen, W., Mao, H., Chen, L. & Li, L. The pivotal role of FAM134B in selective ER-phagy and diseases. Biochim. Biophys. Acta 1869, 119277 (2022).
Article CAS Google Scholar
Humpton, T. J. et al. Oncogenic KRAS induces NIX-mediated mitophagy to promote pancreatic cancer. Cancer Discov. 9, 1268–1287 (2019).
Article CAS PubMed PubMed Central Google Scholar
Shami, G. J. et al. Giant mitochondria in human liver disease. Liver Int. https://doi.org/10.1111/liv.15711 (2023).
Feldmann, G. et al. Hepatocyte giant mitochondria: an almost constant lesion in systemic scleroderma. Virchows Arch. A 374, 215–227 (1977).
Article CAS Google Scholar
Kraus, B. & Cain, H. Giant mitochondria in the human myocardium—morphogenesis and fate. Virchows Arch. B 33, 77–89 (1980).
Article CAS PubMed Google Scholar
Hibbs, R. G., Ferrans, V. J., Black, W. C., Weilbaecher, D. G. & Burch, G. E. Alcoholic cardiomyopathy; an electron microscopic study. Am. Heart J. 69, 766–779 (1965).
Article CAS PubMed Google Scholar
Li, A. M. Y. et al. Giant mitochondria in cardiomyocytes: cellular architecture in health and disease. Basic Res. Cardiol. 118, 39 (2023).
Article PubMed PubMed Central Google Scholar
Coleman, R., Silbermann, M., Gershon, D. & Reznick, A. Z. Giant mitochondria in the myocardium of aging and endurance-trained mice. Gerontology 33, 34–39 (1987).
Article CAS PubMed Google Scholar
Yoon, Y. S. et al. Formation of elongated giant mitochondria in DFO-induced cellular senescence: Involvement of enhanced fusion process through modulation of Fis1. J. Cell. Physiol. 209, 468–480 (2006).
Article CAS PubMed Google Scholar
Miwa, S., Kashyap, S., Chini, E. & von Zglinicki, T. Mitochondrial dysfunction in cell senescence and aging. J. Clin. Invest. 132, e158447 (2022).
Article CAS PubMed PubMed Central Google Scholar
Ozcan, L. & Tabas, I. Role of endoplasmic reticulum stress in metabolic disease and other disorders. Annu. Rev. Med. 63, 317–328 (2012).
Article CAS PubMed PubMed Central Google Scholar
Wang, M. & Kaufman, R. J. Protein misfolding in the endoplasmic reticulum as a conduit to human disease. Nature 529, 326–335 (2016).
Article CAS PubMed Google Scholar
De Craemer, D. et al. Very large peroxisomes in distinct peroxisomal disorders (rhizomelic chondrodysplasia punctata and acyl-coa oxidase deficiency)—novel data. Virchows Arch. A 419, 523–525 (1991).
Article Google Scholar
Gonzalez-Foutel, N. S. et al. Conformational buffering underlies functional selection in intrinsically disordered protein regions. Nat. Struct. Mol. Biol. 29, 781–790 (2022).
Article CAS PubMed PubMed Central Google Scholar
Fasana, E., Fregno, I., Galli, C., Soldà, T. & Molinari, M. ER-to-lysosome-associated degradation acts as failsafe mechanism upon ERAD dysfunction. EMBO Rep. 25, 2773–2785 (2024).
Article CAS PubMed PubMed Central Google Scholar
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Article CAS PubMed Google Scholar
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).
Article CAS PubMed Google Scholar
Tyanova, S. et al. The Perseus computational platform for comprehensive analysis of (prote)omics data. Nat. Methods 13, 731–740 (2016).
Article CAS PubMed Google Scholar
Belevich, I., Joensuu, M., Kumar, D., Vihinen, H. & Jokitalo, E. Microscopy image browser: a platform for segmentation and analysis of multidimensional datasets. PLoS Biol. 14, e1002340 (2016).
Article PubMed PubMed Central Google Scholar
Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).
Article CAS PubMed Google Scholar
McLean, I. W. & Nakane, P. K. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J. Histochem. Cytochem. 22, 1077–1083 (1974).
Article CAS PubMed Google Scholar
Fregno, I., Fasana, E., Solda, T., Galli, C. & Molinari, M. N-glycan processing selects ERAD-resistant misfolded proteins for ER-to-lysosome-associated degradation. EMBO J. 40, e107240 (2021).
Article CAS PubMed PubMed Central Google Scholar
Fregno, I. et al. ER-to-lysosome-associated degradation of proteasome-resistant ATZ polymers occurs via receptor-mediated vesicular transport. EMBO J. 37, e99259 (2018).
Article PubMed PubMed Central Google Scholar

Download references

Acknowledgements

We thank the members of Molinari’s laboratory for discussions and critical reading of the manuscript and T. Calì for suggestions on mitochondrial targeting. We thank S. Manley, M. Ryan, T. Saitoh and M. Komatsu for gifts of cell lines. Funding was provided by the Swiss National Science Foundation (grant nos 310030_214903 and 320030-227541 to M.M.).

Author information

Authors and Affiliations

Institute for Research in Biomedicine, Università della Svizzera italiana, Bellinzona, Switzerland
Mikhail Rudinskiy, Carmela Galli, Andrea Raimondi & Maurizio Molinari
Department of Biology, Swiss Federal Institute of Technology, Zurich, Switzerland
Mikhail Rudinskiy
School of Life Sciences, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
Maurizio Molinari

Authors

Mikhail Rudinskiy
View author publications
Search author on:PubMed Google Scholar
Carmela Galli
View author publications
Search author on:PubMed Google Scholar
Andrea Raimondi
View author publications
Search author on:PubMed Google Scholar
Maurizio Molinari
View author publications
Search author on:PubMed Google Scholar

Contributions

Conceptualization: M.M., M.R. and C.G. Methodology: M.R., C.G., A.R. and M.M. Investigation: M.R. and C.G. Visualization: M.R., C.G., A.R. and M.M. Funding acquisition: M.M. Project administration: M.M. Supervision: M.M. Writing—original draft: M.M. Writing—review and editing: M.M., M.R. and C.G.

Corresponding author

Correspondence to Maurizio Molinari.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Cell Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Membrane remodeling and sub-compartmental localization functions of membrane-tethering modules.

(a) ER-phagy receptors FAM134B, SEC62, and TEX264. TMD: transmembrane domain; RHD: reticulon homology domain; LIR: LC3-interacting region; IDR: intrinsically disordered region. (b) RT-TEM showing ER ultrastructure in mock-transfected MEF (also in Supplementary Video 1). Scale bar: 1 μm. N = 1 biological replicate. (c) Same as (b) showing constricted ER tubuli in MEF expressing FAM134B_RHD (Supplementary Video 2). (d) Same as (c) for SEC62_TMD-transfected cell, showing intact ER morphology (Supplementary Video 3). (e) Same as (d) for TEX264_TMD (Supplementary Video 4). (f) Vulcano plot, endogenous ER proteins among FAM134B_RHD or SEC62_TMD interactors in HEK293 cells, N = 2 biological replicates. Two-tailed t-test, with permutation-based FDR not considered. -Log10(Adjusted p-value) is plotted on the y axis. (g) Same as (f) for FAM134B_RHD and TEX264_TMD.

Source data

Extended Data Fig. 2 Membrane-associated IDRs trigger ER-phagy.

(a-g) Schematics and full cell CLSM images of insets shown in Fig. 3a–g. Scale bar 1 μm.

Extended Data Fig. 3 Monitoring lysosomal delivery of mitochondrial fragments in HEK293 cells expressing the mitochondria-targeted ER-phagy receptors IDR modules.

(a) CLSM images of mock-transfected cells exposed to BafA1 to stabilize the cargo delivered within degradative endolysosomes and chemically fixed for immunostaining. Mitochondria are labeled with anti-TOMM20, endolysosomes with anti-LAMP1. Scale bar: 10 µm, inset magnification: 4x. (b) Same as (a) in cells transfected with Mito-GFP chimera. (c-h) Same as (b) in cells exposed to BafA1 to stabilize the cargo delivered within LAMP1-positive degradative endolysosomes and expressing mitochondria-targeted IDR chimeras. (i) LysoQuant quantification of the percentage of cellular endolysosomes that contain TOMM20-positive fragmented mitochondria in panels a-h (N = 3 biological replicates for a-d, N = 2 for e-h, n = 15, 28, 38, 41, 36, 20, 21, and 24 cells, for a-h, respectively). Ordinary one-way ANOVA with Turkey’s multiple comparisons test, F = 149.6. Mean bar is shown. Adjusted P value: ****p < 0.0001, ns (P = 0.9960), not significant. (j) Mitochondria-targeted ER-phagy receptors IDR modules interact with LC3B-II through their LC3-interacting motif. Co‐immunoprecipitation of GFP-tagged IDR modules (bait, upper panel) with LC3B (lower panel) from WT MEF cells treated with 50 nM BafA1 for 6 hours. LC3B-II interacts with IDR modules containing active LIR domains (lanes 2, 4, and 6). Lane 9: detergent-soluble total cell extracts (TCE) is shown. N = 3 biological replicates for lanes 1-3 and 9, N = 1 for lanes 4-8.

Source data

Extended Data Fig. 4 Monitoring mitophagy in ATG7 KO MEF expressing the ER-phagy receptors IDR modules.

ATG7KO MEF were exposed to BafA1 to preserve mitochondria (labeled with TOMM20, red) possibly transported within LAMP1-positive degradative endolysosomes (cyan) and imaged in CLSM. The absence of mitochondria in the lumen of the endolysosomes certifies absence of detectable mitophagy. Scale bar: 10 µm, inset magnification: 4x. (b) Same as the CLSM image in (a) for cells expressing Mito-GFP at the OMM. (c) same as (b) for cells expressing Mito-GSEC62_IDR. (d) Same as (c) for cells expressing Mito-GSEC62_IDRLIR. (e) same as (c) for Mito-GFAM134B_IDR. (f) Same as (d) for Mito-GFAM134B_IDRLIR. (g-h) Same as (c-d) for Mito-GTEX264_IDR and Mito-GTEX264_IDRLIR. (i) LysoQuant quantification of the percentage of cellular endolysosomes that are degrading TOMM20-positive mitochondria fragments in panels A-H (N = 3 biological replicates for a-d, N = 1 for e-h, n = 31, 19, 21, 29, 37, 26, 28, and 29 cells for a-h, respectively). Ordinary one-way ANOVA with Turkey’s multiple comparisons test, F = 1.4. Mean bar is shown. Adjusted P value: ns (P = 0.2070), not significant.

Extended Data Fig. 5 Transplant of ER-phagy receptors IDR modules onto OMM triggers mitochondria fragmentation in HEK293 cells.

(a) The representative CLSM micrograph shows one cell expressing sfGFP at the OMM (Mito-GFP, green in the panel on the left), and non-transfected cells, whose mitochondria are labeled with TOMM20 (red). Inset 1: mitochondrial structure in a non-transfected cell; inset 2: mitochondrial structure in a cell expressing Mito-GFP. Scale bar: 10 µm, inset magnification: 10x. (b) same as (a) for a representative cell that expresses Mito-GSEC62_IDR. (c) Same as (b) for a representative cell that expresses Mito-GSEC62_IDRLIR, which does not engage LC3. (d) same as (b) for Mito-GFAM134B_IDR. (e) Same as (c) for Mito-GFAM134B_IDRLIR. (f-g) Same as (b-c) for Mito-GTEX264_IDR and Mito-GTEX264_IDRLIR. (h) Quantification of mitochondrial cross-section area of HEK293 cells (n = 708, 787, 766, 1007, 911, 1128, 924, and 994 mitochondria for non-transfected cells and transfected cells in a-g, respectively), measured in CLSM. Kruskal-Wallis test with Dunn’s multiple comparisons test, Kruskal-Wallis statistic=1152. Median bar (red) is shown, y axis is Log2. N = 3 biological replicates. Adjusted P value: ****P < 0.0001.

Extended Data Fig. 6 Monitoring mitophagy in DRP1 KO MEF expressing the ER-phagy receptors IDR modules.

(a) WT MEF were exposed to BafA1 to preserve mitochondria (labeled with TOMM20, red) possibly transported within LAMP1-positive degradative endolysosomes (cyan). The absence of mitochondria in the lumen of the endolysosomes certifies absence of detectable mitophagy. Scale bar: 10 µm, inset magnification: 4x. (b) Same as (a) for cells expressing Mito-GFP at the OMM. (c) same as (b) for cells expressing Mito-GSEC62_IDR. (d) Same as (c) for cells expressing Mito-GSEC62_IDRLIR. (e) same as (c) for Mito-GFAM134B_IDR. (f) Same as (d) for Mito-GFAM134B_IDRLIR. (g) Same as (c) for Mito-GTEX264_IDR. (h) Same as (d) for Mito-GTEX264_IDRLIR. (i-p) Same as (a-h) for DRP1 KO MEF cells, showing reduced delivery of TOMM20 to endolysosomes. (q) LysoQuant quantification of the percentage of cellular endolysosomes that are degrading TOMM20-positive mitochondria fragments in panels a-h (black dots) and i-p (red dots). (N = 2 biological replicates, n = 15, 11, 10, 11, 11, 12, 10, and 11 cells for panels (a-h), n = 18, 12, 19, 16, 23, 21, 19, and 13 cells for (i-p), respectively). Two-way ANOVA with Turkey’s multiple comparisons test. Mean bar is shown. Adjusted P value: **** p < 0.0001.

Extended Data Fig. 7 Reconstitution of DRP1 KO cells with GTPase-active WT DRP1 rescues IDR-induced mitophagy.

(a) WT MEF were exposed to BafA1 to preserve mitochondria (labeled with TOMM20, red) possibly transported within LAMP1-positive degradative endolysosomes (cyan). Mitochondria were delivered into the lumen of the endolysosomes in the absence of ectopically expressed DRP1. Scale bar: 10 µm, inset magnification: 4x. (b) DRP1 KO MEF cells transfected with Mito-GSEC62_IDR show reduced delivery of mitochondria into the lumen of LAMP1-positive endolysosomes, demonstrating reduced mitophagy. (c) Co-transfection of mCherry-DRP1 construct rescues mitophagy induced by Mito-GSEC62_IDR as shown by the increased prevalence of TOMM20-positive mitochondria inside LAMP1-positive endolysosomes. (d) GTPase-inactive mutant DRP1 K38A fails to rescue mitophagy inhibition in DRP1 KO MEF cells. (e) Same as (c) for Mito-GSEC62_IDRLIR, showing no induction of mitophagy. (f-h) Same as (c-e) for Mito-GFAM134B_IDR chimeras. (i-k) Same as (c-e) for Mito-GTEX264_IDR chimeras. (l) LysoQuant quantification of the percentage of cellular endolysosomes that are degrading TOMM20-positive mitochondria fragments in panels (a-k) (N = 3 biological replicates for panels c-e, N = 2 for a-b and f-k, n = 9, 11, 40, 37, 28, 9, 5, 11, 12, 15, and 10 cells for panels a-k, respectively). Ordinary one-way ANOVA with Turkey’s multiple comparisons test. F = 22.48. Mean bar is shown. Adjusted P value: **** p < 0.0001, ***p = 0.0003, **p = 0082.

Extended Data Fig. 8 Knock outs of DRP1 has no effect on the ER-phagy induced by the IDR chimeras.

(a-b) WT and (c-d) DRP1 KO MEF were exposed to BafA1 to preserve ER portions (labeled with CNX, red) possibly transported within LAMP1-positive degradative endolysosomes (cyan). Scale bar: 10 µm, inset magnification: 4x. (a) WT MEF cells transfected with GT₁₇-SEC62_IDR show efficient delivery of ER portions into the lumen of LAMP1-positive endolysosomes (b) Same for cells expressing GT₁₇-SEC62_IDRLIR, demonstrating no induction of ER-phagy. (c-d) Same as (b-c) for DRP1 KO MEFs expressing SEC62 IDR chimeras. ER portions were efficiently delivered into the lumen of the endolysosomes in the absence of endogenous DRP1. (e) Lysoquant quantification of the percentage of cellular endolysosomes that are degrading CNX-positive ER fragments in panels (a-d) (N = 2 biological replicates, n = 10, 10, 10, and 9 cells). Two-way ANOVA with Turkey’s multiple comparisons test. Adjusted ****P < 0.0001, ns (P = 0.9733), not significant. Mean bar is shown.

Extended Data Fig. 9 Non-autophagy IDR does not induce organelle fragmentation nor organellophagy.

(a) Schematic of Mito-GREEP1_IDR construct targeting the OMM with the transmembrane domain of the mitochondrial protein TOMM20 (green in the panel on the left). The representative CLSM micrograph shows a HEK293 cell expressing Mito-GREEP1_IDR at the OMM (green) and non-transfected cells, whose mitochondria are labeled with TOMM20 (red). Inset 1: mitochondrial structure in a non-transfected cell; inset 2: mitochondrial structure in a cell expressing Mito-GREEP1_IDR. Scale bar: 10 µm, inset magnification: 10x. (b) Quantification of mitochondrial cross-section area in (a) (n = 787 mitochondria), compared with Mito-GFP-expressing cells (Extended Data Fig. 5a, Extended Data Fig. 5h, n = 1120 mitochondria) showing that the Mito-GREEP1_IDR construct does not induce mitochondrial fragmentation. (c) IP-WB showing bait level (upper panel) and engagement of LC3B (lower panel) by ER-targeted (lanes 3-6) or mitochondria-targeted (lanes 8-11) IDRs of SEC62 and REEP1 expressed in wild type MEF. N = 3 biological replicates for lanes 1-4 and 7-9, N = 1 for lanes 5-6 and 10–11. (d) MEF cells expressing the Mito-GREEP1_IDR were exposed to BafA1 to preserve mitochondria fragments (labeled with TOMM20, red) possibly transported within LAMP1-positive degradative endolysosomes (cyan). The representative CLSM micrograph shows a cell expressing Mito-GREEP1_IDR at the OMM (green). Scale bar: 10 µm, inset magnification: 4x. The absence of mitochondrial portions within LAMP1-positive ELs indicates the absence of mitophagy. (e) Same as (d) for cells expressing Mito-GREEP1_IDRFEMI construct equipped with the LIR of SEC62. (h) LysoQuant quantification of (d-e) (N = 1 biological replicate, n = 11 and 16 cells for panels d and e, respectively). Unpaired two-tailed t test. Mean bar is shown. P-value = 0.3691, ns, not significant (p = 0.3691). (f-i) Same as (d-h) for GT₁₇-REEP1_IDR and GT₁₇-REEP1_IDRFEMI, showing no delivery of the ER marker CNX (red). N = 1 biological replicate, n = 16 and 19 cells for panels f and g, respectively. Unpaired two-tailed t test. Mean bar is shown. P-value = 0.0863, ns, not significant.

Source data

Extended Data Fig. 10 ER-phagy and Mitochondrial fragmentation induced by IDR chimeras of different lengths.

(a) WT MEF were exposed to BafA1 to preserve ER portions (labeled with CNX, red) possibly transported within LAMP1-positive degradative endolysosomes (cyan). Scale bar: 10 µm, inset magnification: 4x. (a) WT MEF cells transfected with GT₁₇-SEC62_IDR148 (GFP, green) show efficient delivery of ER portions into the lumen of LAMP1-positive endolysosomes (b-d) Same for cells expressing truncated GT₁₇-SEC62_IDR chimeras. (e) Same as (a) for cells expressing GT₁₇-SEC62_IDR148LIR, demonstrating no induction of ER-phagy. (f) Lysoquant quantification of the area of LAMP1-positive EL occupied by CNX in MEF from (a-e) (each dot represents a single EL, n = 811, 831, 739, 629, and 615 endolysosomes analyzed in cells from (a-e) respectively, N = 2 biological replicates). Kruskal-Wallis ANOVA test with Dunn’s multiple comparison test, Kruskal-Wallis statistic=521.3. Adjusted ****P < 0.0001, ns: not significant (P = 0.8932). Median bar is shown. (g) Association of endogenous LC3B-II with GT₁₇-SEC62_IDR chimeras in MEF treated with 50 nM BafA1. Immunocomplexes were revealed with anti-GFP (upper panel) or anti-LC3B antibodies (lower panel). N = 3 biological replicates. (h) The representative CLSM micrographs showing one ATG7 KO MEF cell expressing Mito-GFP construct exposing a 23 residues long linker at the OMM (Mito-GFP₂₃, green in the panel on the left) that does not induce mitochondrial fragmentation, and non-transfected cells, whose mitochondria are labeled with TOMM20 (red). Inset 1: mitochondrial structure in a non-transfected cell; Inset 2: mitochondrial structure in a cell expressing Mito-GFP₂₃. Scale bar: 10 µm, inset magnification: 6x. (i-l) Same as (a) for truncated Mito-GSEC62_IDR chimeras that show mitochondria fragments in transfected cells. (m) Quantification of mitochondrial cross-section area of ATG7 KO cells (n = 505, 478, 453, 505, and 623 mitochondria for cells in g-k, respectively), measured in CLSM. Kruskal-Wallis test with Dunn’s multiple comparisons test, Kruskal-Wallis statistic=502.4. Median bar (red) is shown, y axis is Log2. N = 1 biological replicate. Adjusted P value: ****P < 0.0001.

Source data

Supplementary information

Reporting Summary

Peer Review File

Supplementary Video 1

Electron tomography and three-dimensional reconstruction of ER morphology in a mock-transfected cell.

Supplementary Video 2

Electron tomography and three-dimensional reconstruction of ER morphology in a cell expressing FAM134B-RHD.

Supplementary Video 3

Electron tomography and three-dimensional reconstruction of ER morphology in a cell expressing SEC62-TMR.

Supplementary Video 4

Electron tomography and three-dimensional reconstruction of ER morphology in a cell expressing TEX264-TMR.

Supplementary Table 1

Characteristics and sequences of ER- and mitochondria-transplanted IDRs modules and the corresponding linkers used in the study.

Supplementary Table 2

Details of antibodies and fluorescent ligands used. IB, immunoblotting.