CLCC1 governs ER bilayer equilibration to maintain lipid homeostasis

Nature News · Feb 25, 2026 · Collected from RSS

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MainThe cross-bilayer transit of amphipathic phospholipids synthesized asymmetrically on the cytosolic leaflet of the ER bilayer has remained an unaddressed problem until the recent identification of biogenic lipid scramblases, particularly TMEM41B9,11,12. Notably, deficient phospholipid scrambling due to hepatic TMEM41B inactivation not only blocks neutral lipid loading and the biogenesis of very-low-density lipoproteins (VLDLs), but also triggers massive lipid overproduction and substantially accelerates metabolic-dysfunction-associated liver steatohepatitis (MASH)11. Transmission electron microscopy (TEM) revealed that the innumerable lipid droplets in TMEM41B-deficient hepatocytes were all tightly surrounded by membranes (Fig. 1a and Extended Data Fig. 1a), probably originating from the ER. To further understand these unusual lipid storages, we used cryo-electron tomography (cryo-ET) coupled with high-pressure cryo-fixation (HPF) of hepatic tissues17,18. As expected19,20, lipid droplets in wild-type (WT) hepatocytes were observed to be situated in the cytosol (Fig. 1b (left)), often juxtaposed to the ER with an average lumen width of around 50 nm (Fig. 1b (arrowhead)). By contrast, lipid droplets in TMEM41B-deficient hepatocytes were enclosed by the ER bilayer and exhibited an average diameter of around 1 μm (Fig. 1b (right)). Subsequent 3D reconstruction and segmentation further highlighted the enclosure by extensively curved membranes of the giant lipid droplets, occupying much larger surface areas than those of ER tubules (Fig. 1b and Extended Data Fig. 1b,c). Correlative light and electron microscopy (CLEM) experiments further confirmed that ER membranes enclosed the unique lipid droplets in TMEM41B-deficient Huh7 hepatoma cells (Extended Data Fig. 1d). Further cryo-electron tomography (cryo-ET) analysis revealed that these giant lipid droplets were tightly wrapped within the ER lumen by a single ER bilayer characterized by the smooth, ribosome-free appearance, while covering the spheric surface of giant ER-enclosed lipid droplets (geLDs) and therefore adapting curved morphology (Fig. 1c and Extended Data Fig. 1e). Collectively, the ultrastructural analysis revealed an unusual lipid storage, featured by the emergence of geLDs within the ER lumen, accompanied by curved ER bilayers with imbalanced leaflets that appear to contain different amount of phospholipids (Extended Data Fig. 1f).Fig. 1: CLCC1 or TMEM41B loss triggers geLDs.a, TEM analysis of livers from wild-type (WT) or Tmem41b liver-specific knockout (LKO) mice. The arrowheads show the ER. LD, lipid droplet. b, Cryo-ET reconstruction of HPF WT or Tmem41b-deficient livers. The arrowhead shows the ER lumen. The arrow indicates the ER lumen filled with a LD. Right, 3D rendering of a geLD. Mito, mitochondria. c, Cryo-ET reconstruction of the ER bilayer encircling the geLD in HPF Tmem41b-deficient liver. Bottom, relative electron intensity (left) along the orange line crossing the ER bilayer (right). d, Silver staining of LDs isolated from WT and Tmem41b-deficient livers, with equal load. e, Quantitative proteomics of LDs from WT and Tmem41b-deficient livers. Red and blue indicates substantially altered ER-associated or cytosolic-LD-associated proteins. f,g, Differential labelling of cLDs and geLDs by the cLD marker PLIN2 (f) and the ER marker CB5 (g). Control and TMEM41B-KO Huh7 cells were infected with adeno-associated viruses (AAVs) expressing GFP–PLIN2 or GFP–CB5 before analysis using confocal microscopy. h, Immunoblot analysis of cLDs from WT liver, geLDs from Tmem41b-deficient liver and LDLs from plasma, with an equal load. i, Model of a geLD accompanied by curved ER bilayers in TMEM41B-deficient hepatocytes. j, The geLD-enriched proteome, TMEM41B interactome and SURF4 interactome were analysed using MS. k, Schematic of the targeted CRISPR–Cas9 screen for geLDs. The diagram was created using BioRender; Wang, X. https://BioRender.com/xtx5els (2025). l, CLCC1 deficiency induces geLDs. CRISPR–Cas9-mediated CLCC1-KO Huh7 cells were infected with AAVs expressing GFP–PLIN2 or GFP–CB5 before microscopy analysis. In a, representative images from five mice for each group are shown. In b and c, representative views from two control mice and three Tmem41b-LKO mice were reconstructed. For d–h and l, representative results of at least three biologically independent replicates are shown. Scale bars, 200 nm (a), 100 nm (b,c) and 5 μm (f,g,l). Gel source data are provided in Supplementary Fig. 1.Source dataFull size imageBiochemical isolation and silver staining revealed distinctive protein compositions in conventional cytosolic lipid droplets (cLDs) purified from wild-type liver and geLDs from TMEM41B-deficient liver (Fig. 1d and Extended Data Fig. 2a,b). Subsequent quantitative mass spectrometry (MS) revealed that marker proteins associated with cLD, notably perilipin 2 (PLIN2)21,22, were depleted from geLD (Fig. 1e (blue dots)). Instead, these unusual lipid droplets exhibited enrichment of selective ER membrane proteins, such as torsins, sigma-1 receptor, cytochrome b5 and UGT1A9 (Fig. 1e (red dots)). Consistent with these MS data, confocal microscopy confirmed that PLIN2 delineated the surface of cLDs in wild-type cells, but lacking from geLDs in TMEM41B-deficient cells (Fig. 1f). Conversely, the geLDs were encircled by an ER membrane marker generated from the cytochrome b5 protein (GFP–CB5), which was absent from cLDs in wild-type cells (Fig. 1g). Immunoblotting further confirmed the absence of cLD markers including PLIN2, HSD17B13 or the lipolytic enzyme ATGL from geLDs and the enrichment of a subset of ER membrane proteins (Fig. 1h (lane 1 versus 2)). FIT2, which is known to regulate lipid partition in yeast yet has a less-pronounced role in mammals23,24,25, was not detected. Notably, geLDs also lacked apolipoprotein B (APOB), the principal structural protein of VLDLs (Fig. 1h (lane 2 versus 3)). By contrast, geLDs contained the exchangeable apolipoprotein APOE26.The combination of cryo-ET, microscopy and biochemical characterizations collectively revealed the emergence of the unique geLD storage, accompanied by curved ER bilayers with imbalance between the cytosolic versus the lumenal leaflets (Fig. 1i), due to defective phospholipid scrambling11. Consistent with this notion, mathematical modelling revealed that geLD expansion could accommodate a greater absolute number of imbalanced phospholipids within the surrounding ER bilayer, while reducing the percentage of imbalance (Extended Data Fig. 2c,d), together providing a potential adaptive response to cope with sustained deficiency in lipid scrambling. Consistent with the modelling, analysis of bilayer phospholipids and characterization of the enriched proteins both confirmed a large induction of curved bilayers enclosing geLDs in TMEM41B-deficient liver, accounting for a substantial portion of the ER membranes (Extended Data Fig. 2e,f).The massive induction of geLDs, accompanied by the pronounced alterations ER bilayers, prompted us to further investigate a potential shortfall of phospholipids on the lumenal leaflet and the geLD surface (Extended Data Fig. 2g (top right)). Using cLDs of comparable sizes as a reference, we reasoned that fully matched ER bilayers and the geLD surface—comprising a total of three phospholipid monolayers surrounding geLDs as observed by cryo-ET (Fig. 1c)—would contain around three times phospholipids (around 3N) relative to cLDs (1N). Notably, thin-layer chromatography (TLC) quantification of biochemically isolated geLDs and cLDs revealed that geLDs contained only around 1.5N phospholipids, despite having a similar size distribution to reference cLDs and largely retained the enwrapping ER bilayers (Extended Data Fig. 2h–k). The data therefore suggested that there is a phospholipid shortfall on the lumenal/geLD monolayers, supporting an asymmetric phospholipid distribution between ER leaflets caused by scrambling defects. Further fractionation of geLDs into membrane, interspaced and droplet fractions confirmed the enrichment of lumenal amphipathic proteins such as APOE and lumen-facing proteins such as torsins, indicating a selective recruitment of a unique set of functional proteins to the lumenal sites with a shortfall of phospholipids, thereby stabilizing or compensating for the imbalanced phospholipid distribution (Extended Data Fig. 2l–n).This massive emergence of structurally altered leaflets prompted us to hypothesize that geLDs may in turn functionally enrich selective factors promoting ER bilayer equilibration, coincided with efficient phospholipid scrambling and also lipoprotein secretion. To test this hypothesis, we compiled the geLD enriched proteome with the interactomes associated with the TMEM41B scramblase11 and the SURF4 cargo receptor27 (Fig. 1j), resulting in a short list of proteins led by the poorly understood ER membrane protein CLCC1, which was highly enriched in membrane fractions surrounding geLDs (Extended Data Fig. 2m). Subsequently, we initiated a targeted CRISPR-mediated screen focusing on the hits identified in Fig. 1j, guided by the characteristics of geLD (Fig. 1k). Notably, loss of CLCC1 led to induction of geLDs that lacked PLIN2 decoration and instead were encircled by GFP–CB5 (Fig. 1l), recapitulating those resulting from TMEM41B deficiency. Notably, the rough ER marker GFP–Sec61β showed little enrichment around geLDs in TMEM41B- or CLCC1-deficient cells and the ER lumen marker GFP–KEDL exhibited moderate enrichment (Extended Data Fig. 3a,b), whereas APOE–GFP displayed marked enrichment to delineate the surface of geLDs (Extended Data Fig. 3c). Moreover, the cytosolic-targeted APOE mutant did not localize to the geLDs (Extended Data Fig. 3d), and markers of ER tubules or ER sheets did not decorate the geLDs (Extended Data Fig. 3e–h), further implicating the functional importance of these unique structures emerged from defective lipid scr