Splice variants of mitofusin 2 shape the endoplasmic reticulum and tether it to mitochondria

In eukaryotic cells, different organelles interact at membrane contact sites stabilized by tethers. Mitochondrial mitofusin 2 (MFN2) acts as a membrane tether that interacts with an unknown partner on the endoplasmic reticulum (ER). In this work, we identified the MFN2 splice variant ERMIT2 as the ER tethering partner of MFN2. Splicing of MFN2 produced ERMIT2 and ERMIN2, two ER-specific variants. ERMIN2 regulated ER morphology, whereas ERMIT2 localized at the ER-mitochondria interface and interacted with mitochondrial mitofusins to tether ER and mitochondria. This tethering allowed efficient mitochondrial calcium ion uptake and phospholipid transfer. Expression of ERMIT2 ameliorated the ER stress, inflammation, and fibrosis typical of liver-specific Mfn2 knockout mice. Thus, ER-specific MFN2 variants display entirely extramitochondrial MFN2 functions involved in interorganellar tethering and liver metabolic activities. Description Editor’s summary Inside eukaryotic cells, different types of organelles interact at membrane contact sites that arestabilized by protein bridges called tethers. The mitochondrial fusion protein mitofusin 2 (MFN2) links mitochondria to an unknown partner on the endoplasmic reticulum (ER). Naón et al. found two splice variants of mitofusin 2, ERMIT2 and ERMIN2. ERMIN2 affected ER morphology, and ERMIT2 was localized to the ER and enriched at the contact sites with mitochondria. Treatment with adenovirus encoding for ERMIT2 allowed for the transfer of calcium and lipids between the two organelles and reduced liver damage in models of reduced MFN2 expression. —Stella M. Hurtley Endoplasmic reticulum–specific variants explain MFN2 function beyond mitochondria and its role in the pathogenesis of nonalcoholic steatohepatitis. INTRODUCTION In the cytosol of eukaryotic cells, organelles are closely juxtaposed at membrane contact sites. Membrane contact sites provide hotspots for the metabolic and signaling cascades shared between the interacting organelles. Protein bridges called tethers stabilize membrane contact sites to generate the microenvironment essential for the exchange of metabolites and second messengers. The interaction between the endoplasmic reticulum (ER) and mitochondria at membrane contact sites allows ER-to-mitochondria Ca2+ and phosphatidylserine (PS) transfer and serves as a site of autophagy and mitochondrial fission initiation. In mammalian cells, the mitochondrial fusion protein mitofusin 2 (MFN2) also functions as a structural ER-mitochondria tether, but its partner on the ER membrane is unknown. RATIONALE Interactions between mitofusins on separate mitochondria drive organelle fusion. MFN2 can also support heterotypic organelle interaction, as indicated by the ability of an artificial ER-restricted MFN2 mutant to physically interact with mitofusins on mitochondria. Because of this MFN2 propensity for homotypic interactions, and because mitochondrial dynamics genes are alternatively spliced in variants with divergent subcellular localizations and functions, we hypothesized that the partner of MFN2 on the ER could be a splice variant of the MFN2 gene. We studied MFN2 splice variants dubbed ERMIN2 (ER mitofusin 2) and ERMIT2 (ER mitofusin 2 tether) that we found in human skeletal muscle and mouse fibroblasts. RESULTS ERMIN2 and ERMIT2 were expressed in multiple human tissues and were induced upon stresses that drive ER-mitochondria proximity. We combined imaging and biochemical assays of subcellular localization and found that the MFN2 variants were exclusively at the ER. ERMIT2 and MoV-MFN2 were enriched at the ER-mitochondria interface with their N and C termini exposed to the cytosol. Neither variant was able to correct the disrupted morphology of Mfn2−/− mitochondria. However, ERMIN2 was essential to shape the ER, and ERMIT2 and MoV-MFN2 were essential to tether it to mitochondria. The coiled coil (CC) domain of ER-located ERMIT2 interacted with the CC regions of the mitochondrial mitofusins, forming a complex that was energetically stable enough to tether the two organelles at least in molecular modeling simulations. Functionally, organelle tethering mediated by ERMIT2 allowed mitochondrial uptake of ER-released calcium in cells. Furthermore, organelle tethering by ERMIT2 allowed for mitochondrial uptake of ER-derived phospholipids in the liver of genetic and diet-induced models of reduced ER-mitochondria juxtaposition. This tethering ultimately helped to alleviate hepatic ER stress and inflammation. CONCLUSION In this work, we identified alternatively spliced, ER-restricted variants of the single MFN2 gene. These variants acted as the molecular mediators of MFN2 extramitochondrial functions in ER morphology and tethering to mitochondria. We also identified the role of the tethering function of MFN2 in liver pathophysiology. In particular, in the context of nonalcoholic steatohepatitis, ERMIT2 could correct the altered phospholipid transfer from ER to mitochondria and the ensuing hepatic distress. Thus, alternative splicing can extend the function of the mitochondria-shaping machinery beyond the mitochondria, and altered ER-mitochondria communication plays a key role in the pathobiology of metabolic liver diseases. Alternatively spliced, ER-specific MFN2 variants ERMIN2 and ERMIT2 expand MFN2 function beyond the mitochondria. (Top) Alternative splicing of the human MFN2 gene results in two additional transcripts coding for two proteins restricted at the ER. (Bottom left) ERMIT2 is enriched at the ER-mitochondria interface and is the ER partner of mitochondrial mitofusins in tethering, allowing efficient Ca2+ and phospholipid transfer between the organelles. (Bottom right) ERMIN2 is not enriched in mitochondria-associated membranes (MAMs), and it regulates ER morphology. IMM, inner mitochondrial membrane; OMM, outer mitochondrial membrane; PE, phosphatidylethanolamine.