Endoplasmic reticulum-associated degradation regulates mitochondrial dynamics in brown adipocytes

Abstract

The endoplasmic reticulum (ER) engages mitochondria at specialized ER domains known as mitochondria-associated membranes (MAMs). Here, we used three-dimensional high-resolution imaging to investigate the formation of pleomorphic “megamitochondria” with altered MAMs in brown adipocytes lacking the Sel1L-Hrd1 protein complex of ER-associated protein degradation (ERAD). Mice with ERAD deficiency in brown adipocytes were cold sensitive and exhibited mitochondrial dysfunction. ERAD deficiency affected ER-mitochondria contacts and mitochondrial dynamics, at least in part, by regulating the turnover of the MAM protein, sigma receptor 1 (SigmaR1). Thus, our study provides molecular insights into ER-mitochondrial crosstalk and expands our understanding of the physiological importance of Sel1L-Hrd1 ERAD.

One Sentence Summary:

This study reveals Sel1L-Hrd1 ERAD as a new regulatory mechanism underlying ER-mitochondrial communication in a physiological setting.

Membrane contact sites mediate interorganellar communication and are key to organelle homeostasis and organismal health (1–3). Endoplasmic reticulum (ER)-derived mitochondria-associated membranes (MAMs) are indispensable for mitochondrial dynamics and function. MAMs, characterized by intimate contact between the ER and mitochondria (~10–25 nm apart) (4), mark sites for mitochondrial DNA synthesis and fission (5–7), and for calcium exchange and lipid biosynthesis (1, 8, 9). During mitochondrial fission, ER tubules envelop and constrict mitochondria, and the activated dynamin-related GTPase protein (Drp1) aggregates to cleave the organelle (5, 10–13). The mechanism underlying interorganellar communication and physiological consequences of miscommunication remain largely unclear.

ER-associated degradation (ERAD) is a conserved quality control mechanism to recruit ER proteins for cytosolic proteasomal degradation (14–19). The Sel1L-Hrd1 protein complex comprises the most conserved form of ERAD from yeast to humans. Sel1L resides on the ER membrane and controls the stability of the E3 ligase Hrd1 (16–18, 20–22). Sel1L-Hrd1 ERAD is indispensable for fundamental physiological processes in vivo such as lipid metabolism, water balance, food intake, and systemic energy homeostasis (15, 19, 23–31).

Formation of megamitochondria in Sel1L^−/− brown adipocytes

Serendipitously, we observed elongated mitochondria in Sel1L^−/− primary brown preadipocytes, resembling those in Drp1^−/− preadipocytes but distinct from those in pro-fusion Opa1^−/− cells (Fig. S1A–B). These findings prompted us to explore a possible role of Sel1L in mitochondria-rich brown adipose tissue (BAT), a potential therapeutic target for obesity (32). Acute cold challenge induces mitochondrial fission in BAT to enhance respiration and maintain body temperature (33), while having no impact on the expression of ERAD genes (Fig. S2A–B).

We generated adipocyte-specific Sel1L-deficient mice (Sel1L^AdipCre) by crossing Sel1L^f/f mice to the adiponectin promoter-driven Cre mice (25). Sel1L was deleted specifically in both white adipose tissue (WAT) and BAT, resulting in lower Hrd1 expression (Fig. S2C). We next performed transmission electron microscopy (TEM) to visualize mitochondria in BAT from mice housed at room temperature (22°C), or 4°C for 6 hr. There was no difference in mitochondrial morphology between Sel1L^f/f and Sel1L^AdipCre BAT at 22°C (Fig. 1A–B). Acute cold challenge led to smaller mitochondria in Sel1L^f/f BAT due to increased fission (Fig. 1A and 1C). By contrast, most mitochondria in Sel1L^AdipCre brown adipocytes were enlarged and pleomorphic, with abnormal cristae architecture (yellow arrows, Fig. 1B–C and S3A–B). Consequently, mitochondrial density per cell was reduced in cold-stimulated Sel1L^AdipCre mice compared to control (Fig. S3C).

Figure 1. Sel1L regulates mitochondrial morphology in BAT during cold exposure.

(A-C) Representative TEM images of BAT from Sel1L^f/f (A) and Sel1L^AdipCre mice (B) at 22°C (left) or 4°C (right) for 6 hr, with quantitation of mitochondrial size shown in C (n=870, 601 mitochondria from 3 Sel1L^f/f mice each at 22°C and 4°C; 624, 821 from 3 Sel1L^AdipCre mice each at 22°C and 4°C, one-way ANOVA). Yellow arrows, megamitochondria. M, mitochondrion; LD, lipid droplet. (D-E) Representative TEM images of BAT from Sel1L^Ucp1Cre mice at 4°C for 6 hr, with quantitation of mitochondrial size shown in E (n=676, 618 mitochondria for 3 Sel1L^f/f mice each at 22°C and 4°C; 582, 674 for 3 Sel1L^Ucp1Cre mice each at 22°C and 4°C, one-way ANOVA). (F-H) Representative SBF-SEM images (F) and 3D reconstruction (G) of mitochondria from BAT of Sel1L^f/f and Sel1L^Ucp1Cre mice at 4°C for 6 hr. Mitochondria in pseudo colors were reconstructed from a set of 150 SBF-SEM image stacks (65 nm/slice). (H) Quantitation of 3D volume of mitochondria (n=61 and 63, Sel1L^f/f and Sel1L^Ucp1Cre mice, Student’s t-test). All experiments have been repeated three times. Data are mean ± SEM. *, p< 0.05; **, p< 0.01; ***, p< 0.001; n.s., not significant.

To exclude a possible contribution from WAT, we generated brown adipocyte-specific Sel1L-deficient mice (Sel1L^Ucp1Cre) using Ucp1 promoter-driven Cre mice (Fig. S2D). Consistent with Sel1L^AdipCre mice, mitochondria in Sel1L^Ucp1Cre BAT were dramatically enlarged and pleomorphic upon acute cold exposure (yellow arrows, Fig. 1D–E and S3D). This observation was confirmed using confocal microscopy, with BAT immunolabeled for translocase of outer mitochondrial membrane 20 (Tomm20) and matrix protein pyruvate dehydrogenase (PDH) (Fig. S3E).

To gain further insight into mitochondrial morphology at the three-dimensional (3D) level, we performed serial block-face scanning electron microscopy (SBF-SEM) (Fig. S4A), which allows imaging of a large field (over 10 cells) at 3D planes with nanometer resolution (Fig. S4B–C) (34). In cold-exposed Sel1L^f/f BAT, mitochondria were predominantly spherical or ovoid, with an average volume of ~1.7 μm³ (Fig. S4B and 1F–H). Mitochondrial volume was increased five-fold in Sel1L^−/− BAT (Fig. 1F–H and S4C). Moreover, three-dimensional reconstruction analyses revealed pleomorphic megamitochondria, e.g. branched, or chair- or cup-shaped (Fig. 1G and Movies S1–2). Thus, Sel1L deficiency in brown adipocytes triggers the formation of pleomorphic megamitochondria within hours of cold exposure.

Sel1L controls ER-mitochondria contacts

We next explored whether Sel1L deficiency affected MAMs. In Sel1L^f/f BAT, MAMs were identified as thin sheets aligned with mitochondria, with the distance between the two organelles averaging 10–20 nm (cyan arrows, Fig. 2A, 2C–D and S5A). By contrast, MAMs of Sel1L^−/− BAT formed tubules located closer to mitochondria, with an average distance of 5–10 nm (cyan arrows, Fig. 2B–D and S5B). Moreover, the number of MAMs per mitochondrion was increased in Sel1L^−/− BAT following cold exposure (Fig. 2E). These peri-mitochondrial tubular structures were verified as ER by BiP-specific immunoelectron microscopy (Fig. 2F): pleomorphic megamitochondria appeared to “grow” around ER tubules in cold-stimulated Sel1L^−/− BAT.

Figure 2. Sel1L controls ER-mitochondria contacts in cold-stimulated brown adipocytes.

(A-B) Representative TEM images of BAT from Sel1L^f/f (A) and Sel1L^Ucp1Cre (B) mice at 4°C for 6 hr. M, mitochondrion; Cyan arrows, MAMs; red lines, mitochondrial membranes; the ER, green. (C-E) Representative TEM images of the MAMs in BAT from Sel1L^f/f and Sel1L^Ucp1Cre mice at 4°C for 6 hr, with quantitation of ER-mitochondrion distance (D) and abundance of MAM per mitochondrion (E). n=35 and 40 MAMs (D) and n=450 and 572 mitochondria (E) for Sel1L^f/f and Sel1L^Ucp1Cre. (F) Representative TEM images of BiP-specific immunogold labeling in BAT from Sel1L^AdipCre mice at 4°C for 6 hr. Red dotted line outlines one megamitochondrion; cyan arrows, BiP-positive ER tubule(s). (G) Representative FIB-SEM (left) and the 3D tomography images (300 and 170 slices, 5 nm/slice) of mitochondria in BAT from Sel1L^f/f and Sel1L^Ucp1Cre mice at 4°C for 6 hr. Magenta, mitochondria; green, ER. Cyan arrows, MAMs; orange arrows, MAMs going through the concave surface of a mitochondrion. All experiments have been repeated two to three times. Data are mean ± SEM. ***, p< 0.001 by Student’s t-test.

We next performed focused ion-beam SEM (FIB-SEM) to reconstruct mitochondria with MAMs at the 3D level. In comparison to SBF-SEM, FIB-SEM offers higher resolution at the z plane (35) with a resolution of 5 nm isotropic voxels (Fig. S6). Three-dimensional reconstruction revealed more intimate interactions between the ER (green) and mitochondria (purple) in Sel1L^Ucp1Cre BAT vs. a Sel1L^f/f mitochondria (pink) (Fig. 2G). In many U-shaped mitochondria, several ER tubules were found along the entire concavity of the organelle (orange arrows, Fig. 2G and Movie S3).

Formation of mitochondria-perforating ER tubules in the absence of Sel1L

In many U- or dumbbell-shaped megamitochondria, the outer membrane folded back on itself, with less than 10 nm between membranes (green arrows, Fig. 3A and S7A), which we speculated may fuse to embed ER tubules within the mitochondria. Indeed, there were many tubular structures embedded within mitochondrial profiles in Sel1L^−/− BAT (red arrows, Fig. 3B–C, S7B–C). In some, double- or triple- membrane structures were noted. Moreover, these tubular structures served as a “hub” from which cristae folds radiated (Fig. 3B–C and S7B–D). To determine whether these peculiar structures inside mitochondria were ER, we performed BiP-specific immunoelectron microscopy. Clusters of BiP-positive signal were detected both perimitochondrially (cyan arrows) and within mitochondrial profiles (red arrows, Fig. 3D and S7E). We then performed SBF- and FIB-SEM to reconstruct whole mitochondria with perforating ER tubules. For example, we present 12 consecutive images of one megamitochondrion with two parallel penetrating ER tubules (Fig. S8 and 3E–F). Two ER tubules (arrows) perforated the mitochondrial profiles in parallel, with a distance of 1–1.3 μm apart, with radiating and interconnecting cristae folds, resembling wagon wheel spokes. Similarly, independent FIB-SEM analyses showed two parallel ER tubules (0.7–1 μm apart) penetrating another megamitochondrion in Sel1L^Ucp1Cre BAT upon cold (Fig. S9 and Movie S4). Thus, Sel1L deficiency leads to the formation of pleomorphic megamitochondria with perforating ER tubule(s) in brown adipocytes upon cold challenge.

Figure 3. Sel1L deficiency leads to the formation of megamitochondria with perforating ER tubules.

(A) Representative TEM images of BAT in Sel1L^Ucp1Cre mice housed at 4°C for 6 hr, showing a megamitochondrion wrapping around the tubular structures (cyan arrows). Green arrows, two opposite sides of a mitochondrion. (B-C) Representative TEM images of BAT from Sel1L^Ucp1Cre (B) and Sel1L^AdipCre (C) mice at 4°C for 6 hr, showing megamitochondria with tubular structures (red arrows). (D) Representative BiP-immunogold TEM images of BAT in Sel1L^AdipCre mice at 4°C for 6 hr. Red and cyan arrows, mitochondria-perforating ER tubules and peri-mitochondria ER tubules. (E-F) Representative SBF-SEM images (E) and 3D reconstruction (F) in BAT of Sel1L^Ucp1Cre mice at 4°C for 6 hr, showing four different slices of a megamitochondrion with two parallel perforating ER tubules (red and magenta arrows). All 12 slices (65 nm/slice) are shown in Fig. S8. All experiments have been repeated two to three times.

Impaired mitochondrial function and thermogenic response in Sel1L^−/− mice

Next, we asked how Sel1L deficiency affected mitochondrial function and thermogenesis. Purified mitochondria from cold-exposed Sel1L^AdipCre mice had reduced oxygen consumption rate (OCR) from oxidation of pyruvate and malate compared to those from Sel1L^f/f mice (Fig. 4A). In addition, differentiated Sel1L^−/− brown adipocytes showed defective respiration in response to adrenergic receptor agonist norepinephrine (NE) stimulation (Fig. 4B). Mitochondrial functional defects were further confirmed using a targeted metabolomics analysis of 119 intracellular metabolites in NE-treated differentiated Sel1L^f/f vs. Sel1L^−/− brown adipocytes (Fig. S10). Pathway analysis of the 30 significantly altered metabolites revealed three main pathways altered in Sel1L^−/− adipocytes: TCA cycle, pyrimidine and purine metabolism (Fig. 4C), all of which are associated with mitochondrial function.

Figure 4. Sel1L in brown adipocytes regulates mitochondrial function and cold-induced thermogenesis.

(A-B) Oxygen consumption rate (OCR) of purified mitochondria from BAT of cold-exposed mice (A) and differentiated brown adipocytes (B) with the addition of various stimuli as indicated. NE, norepinephrine; Oligo, oligomycin; Pyr/Mal, pyruvate/malate; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; Anti, antimycin; Rot, Rotenone. (C) Metaboanalyst pathway enrichment analysis for the metabolites in Sel1L^f/f and Sel1L^−/− brown adipocytes treated with 1 μM NE for 1 hr (n=3/group). (D) Rectal temperature of 8- to 10- week-old mice housed at 22°C or 4°C for 6 hr (n=7–10 mice each, one-way ANOVA). (E-F) Representative H&E (E) and Perilipin1 (F) staining of BAT from Sel1L^f/f and Sel1L^AdipCre mice housed at 22°C or 4°C for 6 hr. All experiments have been repeated three times except panel C (3 samples per group). Data are mean ± SEM. ***, p< 0.001.

Both Sel1L^AdipCre and Sel1L^Ucp1Cre mice appeared normal and grew comparably to their Sel1L^f/f littermates up to 22 weeks on low-fat diet at room temperature (Fig. S11A). While core body temperatures (~37°C) were similar between cohorts when housed at room temperature, Sel1L^AdipCre mice were cold sensitive and dropped core body temperature to 26°C within 6 hr of cold exposure (Fig. 4D, vs. 33°C in Sel1L^f/f). Histological examination of BAT revealed that, while largely indistinguishable at room temperature, Sel1L^AdipCre BAT exhibited larger lipid droplets upon cold exposure compared to Sel1L^f/f mice (Fig. 4E), pointing to defects in lipid mobilization. This was confirmed using immunostaining of lipid droplet binding protein Perilipin 1 (Fig. 4F). Similar observations were made with Sel1L^Ucp1Cre mice (Fig. 4D and S11B-C). Phosphorylation of hormone-sensitive lipase (HSL) was not affected in response to β3-adrenergic receptor agonists (Fig. S11D–E), thereby uncoupling β-adrenergic signaling from the thermogenic defect of Sel1L^−/− mice.

Sel1L effect on mitochondria is mediated through Hrd1 ERAD

Sel1L may have an Hrd1-independent function, and vice versa (25, 36, 37). To determine whether Sel1L and Hrd1 act similarly to regulate mitochondria in BAT, we generated brown adipocyte-specific Hrd1-deficient (Hrd1^Ucp1Cre) mice (Fig. S12A). Deletion of Hrd1 stabilized Sel1L protein (Fig. S12A), in line with the notion that Sel1L is a substrate of Hrd1. Hrd1^Ucp1Cre mice grew normally (Fig. S12B), but were cold sensitive (Fig. 5A), with more lipid droplets in cold-challenged BAT compared to Hrd1^f/f mice (Fig. S12C). Moreover, Hrd1^Ucp1Cre BAT demonstrated enlarged mitochondrial, some with penetrating ER tubule(s), upon cold stimulation (Fig. 5B–D, S12D).

Figure 5. Hrd1, but not Ire1α of UPR, controls mitochondrial morphology and thermogenesis in brown adipocytes.

(A) Rectal temperature of 8- to 10- week-old Hrd1^f/f and Hrd1^Ucp1Cre mice housed at 22°C or 4°C for 6 hr, n=4/group (one-way ANOVA). (B-D) Representative TEM images of BAT from Hrd1^f/f and Hrd1^Ucp1Cre mice housed at 22°C or 4°C for 6 hr with quantitation shown in C. n= 543, 581 mitochondria for 22°C and 4°C, Student’s t-test. Yellow arrows, megamitochondria; red arrows, ER tubules within mitochondrial profiles. (E) RT-PCR analysis of Xbp1 mRNA splicing in BAT at 22°C or 4°C for 6 hr with quantitation of the ratio of spliced Xbp1 (s) to total Xbp1 (spliced + unspliced (u)) shown below the gel (n = 6 mice per group). BAT from wildtype mice injected with vehicle or tunicamycin (Tuni, 1 mg/kg) included as controls. (F-G) Representative TEM pictures of BAT from Ire1a^AdipCre mice at 4°C for 6 hr with quantitation shown in G (n=500, 665 from 2 mice each, Student’s t-test). All experiments have been repeated two to three times. Data are mean ± SEM. **, p< 0.01; ***, p< 0.001.

Because ER stress has been linked to mild mitochondrial elongation (38, 39), we next asked whether the effect of ERAD deficiency on mitochondria was mediated through ER stress. Basal Xbp1 mRNA splicing was slightly higher in Sel1L^AdipCre BAT compared to Sel1L^f/f BAT at room temperature; however, cold exposure reduced Xbp1 mRNA splicing in Sel1L^AdipCre BAT (Fig. 5E). There was no detectable cell death in Sel1L^AdipCre BAT (Fig. S13A). To study a possible impact of ER stress signaling on mitochondria in vivo, we generated mice lacking Ire1α, a key sensor of UPR, specifically in adipocytes (Ire1a^AdipCre). These knockout mice grew normally and were not cold-sensitive (Fig. S13B–D). Mitochondria from Ire1a^AdipCre exhibited normal morphology, with reduced size at 4°C (Fig. 5F–G). Thus, the effect of Sel1L on mitochondria is mediated by ERAD, independent of ER stress or cell death.

Sel1L-Hrd1 ERAD regulates ER-mitochondria contacts and dynamics via SigmaR1

We next explored how mitochondrial dynamics were affected by ERAD dysfunction. While total protein levels of mitochondrial respiratory proteins and Ucp1 were largely comparable between the cohorts (Fig. S14A), cold-induced phosphorylation of Drp1 at Ser616, a key activating event in mitochondrial division (11, 40) was reduced in Sel1L^AdipCre BAT compared to Sel1L^f/f BAT (Fig. 6A). Opa1 processing to its shorter form upon cold exposure did not occur in Sel1L^AdipCre BAT (Fig. 6A). Protein levels of outer mitochondrial membrane Drp1 receptors Fis1 and Mff were unchanged (Fig. S14A). Similar observations were obtained in Hrd1^−/− BAT (Fig. S14B), but not in Ire1α^−/− BAT (Fig. S13E). Furthermore, in Sel1L^−/− BAT, cold exposure increased oligomerization of key fusion factor Mfn2 into high molecular weight (HMW) complexes as revealed by blue-native gels and sucrose gradient fractionation (Fig. S14C–D). Indeed, the Mfn2 HMW complex promotes mitochondrial fusion (41–43). We then tested the protein levels of several known MAM proteins including Mfn2, Vapb, Bap31 and SigmaR1. Only SigmaR1 levels were increased in Sel1L- and Hrd1-deficient BAT (Fig. 6B and S15A–B), but not in Ire1α^−/− BAT (Fig. S13E). However, SigmaR1 mRNA level was comparable in Sel1L^−/− vs. WT BAT in response to cold (Fig. S15C), pointing to a post-transcriptional regulation of SigmaR1 protein.

Figure 6. Sel1L-Hrd1 ERAD regulates MAM and mitochondrial dynamics via SigmaR1.

(A) Immunoblot analysis of mitochondrial dynamic proteins in BAT from Sel1L^f/f and Sel1L^AdipCre mice at 22°C or 4°C for 6 hr with quantitation of phos-/total Drp1 and large (L)-/small (S)-Opa1 shown below the gel (n = 7 per group). (B) Immunoblot analysis of MAM proteins in BAT of Sel1L^f/f and Sel1L^AdipCre mice with quantitation of SigmaR1/Hsp90 shown below the gel (n = 7 per group). (C) Immunoblot analysis of endogenous SigmaR1 in WT and Hrd1-deficient (Hrd1^−/−) HEK293T cells with or without CHX and MG132 treatment. (D) Immunoblot analysis of GFP or endogenous SigmaR1 immunoprecipitates in HEK293T (with or without MG132 treatment), showing Hrd1-mediated SigmaR1 ubiquitination. (E) Immunoblot analysis of Flag immunoprecipitates in HEK293T cells transfected with a combination of plasmids, showing a dose-dependent interaction between SigmaR1-Flag and Mfn2-EGFP. (F) Immunoblot analysis of Mfn2 oligomers or -containing high molecular weight (HMW) complexes in brown adipocytes treated with or without NE (1 μM, 1 hr) in blue native (BN)-PAGE and regular SDS-PAGE. (G-H) Representative images of mito-DsRed- and mEmerald-Sec61b- expressing pre-adipocytes. DAPI (blue) with quantitation of ER-mitochondrial colocalization using Manders’ overlap coefficient (50 cells each) shown in H, one-way ANOVA. (I-J) Confocal images showing the spread and decay of mitochondria-targeted photoactivated GFP (mito-PAGFP) in brown adipocytes 60 min following NE stimulation and photoactivation. Active mitochondrial were stained with TMRE (red). Quantitation of changes of GFP signal intensity over time shown in J. (n=13, 15, and 10 for Sel1L^f/f, Sel1L^−/− and Sel1L^−/−;SigmaR1^−/−, one-way ANOVA). All were repeated two to three times. Data are mean ± SEM. **, p< 0.01; ***, p< 0.001.

SigmaR1 is a single-span ER-resident protein residing at the MAMs where it may regulate ER-mitochondria contacts and mitochondrial dynamics (44, 45), although a detailed mechanism remains vague. SigmaR1 protein was degraded by the proteasome with a half-life of approximately 6 hr (Fig. 6C), interacted with the E3 ligase Hrd1 (Fig. S15D–E) and was ubiquitinated in a largely Hrd1-dependent manner (Fig. 6D). In the absence of Sel1L or Hrd1, SigmaR1 protein was stabilized (Fig. 6C) and accumulated in the ER (Fig. S15F). SigmaR1 interacted with Mfn2 as well as other MAM proteins Bap31 and Vapb, but not Ip3r (Fig. 6E and S15G), and were localized at very close proximity to mitochondria in Sel1L^−/− cells (Fig. S16H). Genetic deletion of SigmaR1 reduced cold-induced Mfn2 HMW complexes in Sel1L^−/− brown adipocytes (Fig. 6F and S16A). Deletion of SigmaR1 reversed mitochondrial elongation (Fig. S16B–C) and reduced ER-mitochondrial contacts in Sel1L^−/− cells (Fig. 6G–H). In a mitochondrial fusion assay (33) using mitochondria-targeted photoactivated GFP (mito-PAGFP), deletion of SigmaR1 attenuated the spread and decay of GFP intensity seen in Sel1L^−/− brown adipocytes upon NE treatment (Fig. 6I–J). Thus, SigmaR1 mediates mitochondrial hyperfusion in Sel1L-deficient cells, thereby linking ERAD to ER-mitochondrial contacts and mitochondrial dynamics in brown adipocytes.

Discussion

Here, we found a critical role for Sel1L-Hrd1 ERAD in regulating ER-mitochondria contacts and mitochondrial function. We observed profound changes in mitochondrial morphology, beyond mere elongation or swelling that is commonly associated with mitochondrial dysfunction or ER stress. Using 3D imaging techniques, we reconstructed these pleomorphic mitochondria and their associated MAMs. We speculate that the ER tubules embedded within profiles of pleomorphic megamitochondria represent halted fission and/or accelerated fusion intermediates. Our mechanistic studies identified MAM protein SigmaR1 as an ERAD substrate, which, when deleted, reduced ER-mitochondria contacts and rescued abnormal mitochondrial dynamics and morphology in Sel1L^−/− cells. SigmaR1 may regulate mitochondrial dynamics by interacting with Mfn2 as well as other MAM proteins and promoting Mfn2 oligomerization in an unknown manner.

MAMs are disturbed in many diseases such as obesity (46), cardiac disease (47), and neurodegeneration (48); however, to date, the nature of this disruption in disease pathogenesis remains vague. Our data suggest that Sel1L-Hrd1 ERAD may regulate the dynamics of ER-mitochondria interactions via modulation of MAMs. Indeed, an inverse correlation between Sel1L and SigmaR1 expression levels has been reported in patients with neurodegeneration (49–51). Considering the recent identification of Sel1L mutants in progressive early-onset cerebellar ataxia in canines (52) and various cancers (53, 54), this study may have implications not only for interorganelle communication, but also for the pathophysiological role of Sel1L-Hrd1 ERAD.