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• W2797472953 abstract "Article16 April 2018free access Transparent process The heptad repeat domain 1 of Mitofusin has membrane destabilization function in mitochondrial fusion Frédéric Daste Membrane Traffic in Health & Disease, INSERM ERL U950, Sorbonne Paris Cité, Université Paris Descartes, Paris, France Institut Jacques Monod, CNRS UMR 7592, Sorbonne Paris Cité, Université Paris Diderot, Paris, France Search for more papers by this author Cécile Sauvanet Institut de Biochimie et Génétique Cellulaires, CNRS UMR 5095, Université de Bordeaux, Bordeaux, France Search for more papers by this author Andrej Bavdek Membrane Traffic in Health & Disease, INSERM ERL U950, Sorbonne Paris Cité, Université Paris Descartes, Paris, France Institut Jacques Monod, CNRS UMR 7592, Sorbonne Paris Cité, Université Paris Diderot, Paris, France Search for more papers by this author James Baye Membrane Traffic in Health & Disease, INSERM ERL U950, Sorbonne Paris Cité, Université Paris Descartes, Paris, France Institut Jacques Monod, CNRS UMR 7592, Sorbonne Paris Cité, Université Paris Diderot, Paris, France Search for more papers by this author Fabienne Pierre Membrane Traffic in Health & Disease, INSERM ERL U950, Sorbonne Paris Cité, Université Paris Descartes, Paris, France Institut Jacques Monod, CNRS UMR 7592, Sorbonne Paris Cité, Université Paris Diderot, Paris, France Centre de Psychiatrie et Neurosciences, INSERM UMR 894, Sorbonne Paris Cité, Université Paris Descartes, Paris, France Search for more papers by this author Rémi Le Borgne Institut Jacques Monod, CNRS UMR 7592, Sorbonne Paris Cité, Université Paris Diderot, Paris, France Search for more papers by this author Claudine David Institut de Biochimie et Génétique Cellulaires, CNRS UMR 5095, Université de Bordeaux, Bordeaux, France Search for more papers by this author Manuel Rojo Institut de Biochimie et Génétique Cellulaires, CNRS UMR 5095, Université de Bordeaux, Bordeaux, France Search for more papers by this author Patrick Fuchs Institut Jacques Monod, CNRS UMR 7592, Sorbonne Paris Cité, Université Paris Diderot, Paris, France Search for more papers by this author David Tareste Corresponding Author [email protected] orcid.org/0000-0002-8744-7598 Membrane Traffic in Health & Disease, INSERM ERL U950, Sorbonne Paris Cité, Université Paris Descartes, Paris, France Institut Jacques Monod, CNRS UMR 7592, Sorbonne Paris Cité, Université Paris Diderot, Paris, France Centre de Psychiatrie et Neurosciences, INSERM UMR 894, Sorbonne Paris Cité, Université Paris Descartes, Paris, France Search for more papers by this author Frédéric Daste Membrane Traffic in Health & Disease, INSERM ERL U950, Sorbonne Paris Cité, Université Paris Descartes, Paris, France Institut Jacques Monod, CNRS UMR 7592, Sorbonne Paris Cité, Université Paris Diderot, Paris, France Search for more papers by this author Cécile Sauvanet Institut de Biochimie et Génétique Cellulaires, CNRS UMR 5095, Université de Bordeaux, Bordeaux, France Search for more papers by this author Andrej Bavdek Membrane Traffic in Health & Disease, INSERM ERL U950, Sorbonne Paris Cité, Université Paris Descartes, Paris, France Institut Jacques Monod, CNRS UMR 7592, Sorbonne Paris Cité, Université Paris Diderot, Paris, France Search for more papers by this author James Baye Membrane Traffic in Health & Disease, INSERM ERL U950, Sorbonne Paris Cité, Université Paris Descartes, Paris, France Institut Jacques Monod, CNRS UMR 7592, Sorbonne Paris Cité, Université Paris Diderot, Paris, France Search for more papers by this author Fabienne Pierre Membrane Traffic in Health & Disease, INSERM ERL U950, Sorbonne Paris Cité, Université Paris Descartes, Paris, France Institut Jacques Monod, CNRS UMR 7592, Sorbonne Paris Cité, Université Paris Diderot, Paris, France Centre de Psychiatrie et Neurosciences, INSERM UMR 894, Sorbonne Paris Cité, Université Paris Descartes, Paris, France Search for more papers by this author Rémi Le Borgne Institut Jacques Monod, CNRS UMR 7592, Sorbonne Paris Cité, Université Paris Diderot, Paris, France Search for more papers by this author Claudine David Institut de Biochimie et Génétique Cellulaires, CNRS UMR 5095, Université de Bordeaux, Bordeaux, France Search for more papers by this author Manuel Rojo Institut de Biochimie et Génétique Cellulaires, CNRS UMR 5095, Université de Bordeaux, Bordeaux, France Search for more papers by this author Patrick Fuchs Institut Jacques Monod, CNRS UMR 7592, Sorbonne Paris Cité, Université Paris Diderot, Paris, France Search for more papers by this author David Tareste Corresponding Author [email protected] orcid.org/0000-0002-8744-7598 Membrane Traffic in Health & Disease, INSERM ERL U950, Sorbonne Paris Cité, Université Paris Descartes, Paris, France Institut Jacques Monod, CNRS UMR 7592, Sorbonne Paris Cité, Université Paris Diderot, Paris, France Centre de Psychiatrie et Neurosciences, INSERM UMR 894, Sorbonne Paris Cité, Université Paris Descartes, Paris, France Search for more papers by this author Author Information Frédéric Daste1,2,‡, Cécile Sauvanet3,†,‡, Andrej Bavdek1,2, James Baye1,2, Fabienne Pierre1,2,4, Rémi Le Borgne2, Claudine David3, Manuel Rojo3, Patrick Fuchs2,† and David Tareste *,1,2,4 1Membrane Traffic in Health & Disease, INSERM ERL U950, Sorbonne Paris Cité, Université Paris Descartes, Paris, France 2Institut Jacques Monod, CNRS UMR 7592, Sorbonne Paris Cité, Université Paris Diderot, Paris, France 3Institut de Biochimie et Génétique Cellulaires, CNRS UMR 5095, Université de Bordeaux, Bordeaux, France 4Centre de Psychiatrie et Neurosciences, INSERM UMR 894, Sorbonne Paris Cité, Université Paris Descartes, Paris, France †Present address: Department of Molecular Biology and Genetics, Weill Institute for Molecular and Cell Biology, Cornell University, Ithaca, NY, USA †Present address: École Normale Supérieure, Laboratoire des Biomolécules, CNRS UMR 7203, Sorbonne Universités, Paris Sciences et Lettres Université, Université Pierre et Marie-Curie, Paris, France ‡These authors contributed equally to this work *Corresponding author. Tel: +33 1 40 78 92 49; E-mail: [email protected] EMBO Rep (2018)19:e43637https://doi.org/10.15252/embr.201643637 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mitochondria are double-membrane-bound organelles that constantly change shape through membrane fusion and fission. Outer mitochondrial membrane fusion is controlled by Mitofusin, whose molecular architecture consists of an N-terminal GTPase domain, a first heptad repeat domain (HR1), two transmembrane domains, and a second heptad repeat domain (HR2). The mode of action of Mitofusin and the specific roles played by each of these functional domains in mitochondrial fusion are not fully understood. Here, using a combination of in situ and in vitro fusion assays, we show that HR1 induces membrane fusion and possesses a conserved amphipathic helix that folds upon interaction with the lipid bilayer surface. Our results strongly suggest that HR1 facilitates membrane fusion by destabilizing the lipid bilayer structure, notably in membrane regions presenting lipid packing defects. This mechanism for fusion is thus distinct from that described for the heptad repeat domains of SNARE and viral proteins, which assemble as membrane-bridging complexes, triggering close membrane apposition and fusion, and is more closely related to that of the C-terminal amphipathic tail of the Atlastin protein. Synopsis The GTPase Mitofusin mediates mitochondrial fusion, but the underlying mechanism of membrane fusion remains unclear. This study shows that the amphipathic nature of the heptad repeat domain 1 (HR1) of Mitofusin perturbs the lipid bilayer structure to drive mitochondrial fusion. HR1 is required for Mitofusin-mediated mitochondrial fusion in cultured cells, and induces liposome fusion in vitro. The membrane fusion activity of HR1 is associated with its capacity to insert into lipid bilayers, notably in regions presenting lipid packing defects. This property is conferred by a conserved amphipathic helix located at the C-terminus of the HR1 sequence. Introduction Membrane fusion allows communication between two compartments delimited by lipid bilayer structures. Fusion occurs during many fundamental physiological processes, including viral entry into cells, inter-organellar, and inter-cellular communications 1. In order to fuse, membranes must first be brought into very close proximity and then remodeled/destabilized to allow the merging of their lipid bilayer. The sequence of events leading to lipid bilayer fusion often includes the passage through a hemifusion intermediate structure, in which the outer lipid monolayers are mixed while the inner monolayers remain separated 2. Biological membrane fusion uses specialized proteins that lower the successive energy barriers of the intermediate states on the fusion pathway. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and fusion proteins from enveloped viruses, notably the hemagglutinin (HA) protein from the influenza virus, constitute the best described fusion machineries, which both use the energy of protein folding to mediate membrane fusion. SNARE proteins are membrane-anchored, coiled-coil forming proteins, involved in intracellular vesicle traffic and exocytosis 3. SNARE-mediated fusion occurs when the heptad repeat domains of the vesicular (v-) and target membrane (t-) SNARE proteins assemble in a zipper-like fashion (from the membrane-distal parts to the membrane-proximal parts of the proteins) to form a highly stable membrane-bridging coiled-coil complex that pulls the membranes close together and triggers their fusion 4-8. Viral fusion proteins are transmembrane proteins residing in the viral envelope membrane, and displaying an initially closed and inactive conformation, in which a fusion peptide is sequestered. During viral infection, the fusion protein opens up and releases the fusion peptide, which anchors the viral envelope to the target membrane. The viral protein then folds back on itself, which brings the viral and target membranes in close proximity. This membrane apposition effect combined with lipid bilayer perturbation by the fusion peptide leads to membrane fusion 9. In addition to these heterotypic fusion events, homotypic fusion events also occur within cells such as those that control the dynamics and morphology of endoplasmic reticulum (ER) and mitochondria. The homotypic fusion of ER tubules is mediated by Atlastin, a large membrane-anchored GTPase protein from the dynamin superfamily. Recent structural and biochemical data suggest that Atlastin mediates ER membrane docking through GTP-dependent trans-dimerization of its GTPase domain, and ER membrane fusion through lipid bilayer destabilization by a C-terminal amphipathic helix 10. Mitochondria also form a network of highly dynamic organelles, which undergo frequent cycles of fusion and fission within cells. The balance between membrane fusion and fission defines mitochondrial morphology and is important for normal mitochondrial and cellular function 11. Dysfunctions of mitochondrial dynamics are associated with several major neurodegenerative disorders, including Parkinson, Alzheimer, and Huntington diseases 12. Mitochondrial fusion involves four membranes (an inner and an outer membrane for each mitochondrion) that must fuse in a coordinated manner. The key molecular players of mitochondrial fusion have been identified but the underlying molecular mechanisms of the fusion event remain largely unknown. Like ER fusion, mitochondrial fusion is controlled by membrane-anchored dynamin-related proteins: optic atrophy 1 (OPA1) in the inner membrane and Mitofusins in the outer membrane 11, 13. Mammalian cells possess two Mitofusin proteins (Mfn1 and Mfn2) that display highly similar primary structures, and are both involved in mitochondrial fusion 14-16. Mitofusins are transmembrane proteins, containing an N-terminal GTPase domain, and whose U-shaped bipartite transmembrane (TM) region spans the outer mitochondrial membrane twice 15. As a result, both the N- and the C-terminal portions of Mitofusins face the cytosol. The two TM domains of Mitofusins are flanked by two heptad repeat domains (HR1 and HR2) with the potential to form coiled-coil structures (Fig 1A and Appendix Fig S1). Mutations in any of these functional domains impair Mitofusin function, but their exact role in mitochondrial fusion remains unknown 15, 17-20. Structural and in situ studies showed that the HR2 domain of Mfn1 can form an antiparallel coiled-coil dimer, suggesting a role in mitochondrial docking 18. A recent study questioned the cytosolic orientation of the HR2 domain and proposed that it resides instead in the space between outer and inner mitochondrial membranes, where it can form cis-complexes driving Mitofusin oligomerization 21. Expression of Mitofusin mutants lacking GTP binding activity abolished mitochondrial fusion in situ 16, and in vitro fusion between isolated mitochondria required GTP hydrolysis 22, 23, showing that a functional GTPase domain is essential for mitochondrial fusion. Because dynamin-related proteins are known for their membrane tubulation and constriction properties depending on their GTPase domain 24, it was suggested that the N-terminal GTPase domain of Mitofusins could be involved in mitochondrial membrane remodeling events 1, 22, 25. Two recent X-ray structural studies of the GTPase domain of Mfn1 linked to the HR2 domain via an artificial flexible linker revealed a closed conformation closely resembling that of the membrane-distal region of the bacterial dynamin-like protein (BDLP) in its GDP-bound state 26, 27. In addition, this GTPase-HR2 fragment was shown to dimerize in the presence of GTP. By analogy with BDLP, which transits from an open to a closed conformation upon GTP hydrolysis, it was therefore proposed that Mfn1 could bring membranes in close apposition through GTP-dependent conformational changes of trans-Mfn1 dimers. Despite these recent insights into how Mitofusin mediates mitochondrial membrane docking, the molecular trigger of the fusion event remains to be identified. Notably, the exact function of the HR1 domain of Mitofusins in outer mitochondrial membrane fusion is still unknown. Recent works proposed that the HR1 domain of Mfn2 could allow the HR2 domain to adopt an active extended conformation 28, 29. Figure 1. The HR1 and HR2 domains are required for Mitofusin function in mitochondrial fusion Domain architecture of Mitofusin proteins. Mitofusins have an N-terminal GTPase domain (in purple) and two heptad repeat domains (HR1 in red and HR2 in blue); these three domains face the cell cytosol and are anchored to the outer mitochondrial membrane via a bipartite transmembrane (TM) region (in green). Scheme of Mfn1 variants used for transfection of Mfn1 KO MEFs. Mfn1 KO MEFs were transfected with a plasmid expressing mtEGFP alone (control) or in combination with a plasmid expressing Mfn1-Myc full length (Mfn1 WT), Mfn1-Myc lacking the HR1 domain (Mfn1-∆HR1), or Mfn1-Myc lacking the HR2 domain (Mfn1-∆HR2). Mfn1 variants were stained with an anti-Myc antibody and actin was stained with Phalloidin. Co-transfected cells were identified as those expressing both Mfn1 variants and mtEGFP on mitochondria. The right panels show magnified views of the boxed areas in the left panels. Mfn1 KO MEFs expressing Mfn1 WT display normal filamentous mitochondrial morphology, whereas expression of Mfn1-∆HR1 or Mfn1-∆HR2 cannot rescue mitochondrial morphology. The scale bar is 10 μm for the left panels and 2 μm for the right panels. The morphology of the mitochondrial network was quantitatively analyzed using the MiNA Image J macro tool (˜ 30 cells for each Mfn1 variant; n = 3–4 independent experiments). Mitochondrial morphologies were classified as either individuals (structures with no junction, which can be puncta or rods) or networks (structures with at least one junction and three branches). The program also calculated the mean length of rods and network branches, and the mean number of branches per network. Box plots represent the 25th and 75th percentiles around the median, and the whiskers represent the maximum and minimum values. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 by Mann–Whitney U-test. Download figure Download PowerPoint In vitro approaches using the reconstitution of fusion proteins into artificial membrane systems have proven very successful in elucidating the molecular mechanisms of various cellular fusion machineries, including SNARE, Atlastin, and viral proteins 6, 10, 30-35. In this work, we sought to determine the role of the two heptad repeat domains (HR1 and HR2) of Mitofusin in membrane docking and fusion using a combination of in situ cell assays and in vitro reconstitution assays with liposomes mimicking the outer mitochondrial membrane. Expression of Mitofusin variants lacking specific protein fragments was used to reveal the importance of these fragments for mitochondrial fusion in situ. Membrane docking and fusion events were monitored in vitro through a combination of spectroscopy and electron microscopy assays. Conformational changes and biophysical/biochemical properties of HR1 and HR2, as they pertain to their capacity to mediate membrane fusion, were revealed using circular dichroism experiments, partitioning assays and in silico bioinformatics analysis of their sequence. Using these different approaches, we show that both the HR1 and the HR2 domains of Mitofusin have the capacity to mediate membrane docking. We also show that the HR1 domain possesses a conserved amphipathic helix that induces fusion by interacting with the lipid bilayer structure. Results The HR1 and HR2 domains are essential for Mitofusin-mediated mitochondrial fusion To investigate the importance of the heptad repeat domains of Mitofusin in mitochondrial fusion, we examined the properties of wild-type and mutant Mfn1 proteins (Fig 1B) in mouse embryo fibroblasts devoid of endogenous Mfn1 (Mfn1 KO MEFs). In agreement with previous work 16, Mfn1 KO MEFs displayed largely fragmented mitochondria, and expression of wild-type Mfn1 led to the appearance of elongated branched mitochondria (Fig 1C). In contrast, expression of Mfn1 molecules lacking the HR2 domain (Mfn1-ΔHR2) did not restore the tubular morphology of the mitochondrial network (Fig 1C), confirming that the HR2 domain is essential for Mitofusin function 18. Remarkably, Mfn1 molecules lacking the HR1 domain (Mfn1-ΔHR1) also failed to restore mitochondrial fusion and tubular mitochondrial morphology (Fig 1C). Western blot analysis of cell lysates revealed that all constructs were expressed at similar levels in Mfn1 KO MEFs (Appendix Fig S2A), and immunofluorescence microscopy showed that they were all strongly co-localizing with an EGFP expressed in the mitochondrial matrix (Appendix Fig S2B), indicating that the observed phenotypes were not due to a default in expression level and/or mitochondrial targeting of Mfn1 mutants. To confirm the effect of wild-type and mutant Mfn1 by unambiguous quantitative procedures, we used a recently developed method for quantitative analysis of mitochondrial morphology—the mitochondrial network analysis (MiNA) toolset—that allows one to (i) categorize mitochondrial morphologies into two different structures, either individuals (for isolated puncta or short filaments) or networks (for interconnected filaments), and (ii) evaluate the extent of branching and the length of the filaments 36. MiNA revealed that expression of wild-type Mfn1 was associated with a decrease in the number of individuals and networks that was paralleled by an increase of mitochondrial length and of the number of branches per mitochondrial network (Fig 1D). In contrast, neither Mfn1-ΔHR1 nor Mfn1-ΔHR2 expression led to a significant alteration of mitochondrial morphology in Mfn1 KO MEFs (Fig 1D). These results confirm that Mfn1 expression in Mfn1 KO cells restores mitochondrial fusion and mitochondrial network morphology, and that the HR1 and HR2 domains are essential for Mfn1-mediated mitochondrial fusion. The requirement of HR2 may be related to its proposed role in mitochondrial docking 18, 29 or in the formation of a helix-bundle important for the integrity of Mitofusin GTPase domain 26, 27. The HR1 domain has been shown to interact with HR2 15, 19, 28, and this interaction may modulate HR2 activity 29, but it cannot be excluded that the HR1 domain also plays a direct role in the fusion process. In order to unveil the exact role of HR1 and HR2 in mitochondrial membrane fusion, we next used in vitro biochemical/biophysical assays with defined membrane systems. The HR1 domain of Mitofusin induces liposome fusion To elucidate the function of the heptad repeat domains of Mitofusin in membrane fusion, we reconstituted HR1 or HR2 fragments into liposomes and monitored the fusion between these liposomes using a fluorescence resonance energy transfer (FRET)-based lipid-mixing assay 6. Because the heptad repeat domains of Mitofusin can engage in homotypic interactions 18, possibly occurring during liposome reconstitution and storage, we chose to reconstitute them at the very beginning of the fusion assay using a maleimide lipid-anchorage strategy (Fig 2A). Maleimide anchorage has been previously used to successfully reconstitute SNARE proteins into liposomes and recapitulate SNARE-mediated fusion in vitro 7. Efficient fusion required that SNAREs are anchored to maleimide lipids with long hydrophobic chains (with at least 45 carbons) which can span both leaflets of lipid bilayers. In our work, we used either a short (18 carbons hydrophobic chain) or a long (45 carbons hydrophobic chain) maleimide lipid, called C18 and C45 maleimide lipids, respectively. The heptad repeat domains of Mitofusins were modified to contain only a single terminal cysteine residue, at the C-terminus in the case of HR1 and at the N-terminus in the case of HR2, therefore allowing their coupling to maleimide-containing liposomes with the same orientation as on mitochondrial membranes (Fig 1A). We chose to work with liposomes exclusively made of phosphatidylcholine lipids (besides the reactive C18 or C45 maleimide lipid) to allow direct comparison with liposome fusion mediated by reconstituted synaptic SNARE proteins. The main lipids found in synaptic vesicles are phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) 37, whereas the main lipids of the outer mitochondrial membrane are PC, PE, phosphatidylinositol (PI), and cardiolipins (CL) 38. PE lipids could not be used here because they were reacting with the maleimide groups during liposome reconstitution 39, therefore preventing chemical linkage of heptad repeat domains to maleimide lipids after liposome formation. Figure 2. The HR1 domain of Mitofusin mediates lipid mixing between liposomes Experimental system used to monitor the docking and fusion properties of the heptad repeat domains of Mitofusin. The HR1 and HR2 domains were reconstituted into liposomes by chemical linkage to maleimide lipids having either short (18 carbon atoms, C18) or long (45 carbon atoms, C45) hydrophobic chains. FRET-based lipid-mixing assay between POPC:C45:DOPE-NBD:DOPE-Rho(92:5:1.5:1.5) and POPC:C45(95:5) liposomes (mol:mol ratios) to monitor homotypic fusion events in the absence or presence of the heptad repeat domains of Mfn1 or SNARE proteins added at t = 0 (500 μM of lipids and 12.5 μM of proteins, leading to an actual lipid-to-protein ratio of ˜ 130 in the liposome membrane; see Appendix Fig S5A). The HR1 domain of Mfn1 induced robust lipid mixing when it was anchored to either short or long maleimide lipids. No lipid mixing was measured under the same conditions with the HR2 domain of Mfn1, or with the heptad repeat domains of the v-SNARE protein VAMP2 (V2) or the t-SNARE protein Syn1A/SNAP25 (T). The left panel shows one representative set of kinetics experiments, and the right panel the average extent of lipid mixing after 90 min (n = 8–11 independent experiments; error bars are standard deviations). Download figure Download PowerPoint Liposome fusion experiments were first performed with the HR1 and HR2 domains of Mfn1. Fluorescence dequenching (signature of lipid mixing between the two distinct populations of liposome) occurred 10 min after addition of the HR1 domain to the liposomes (Fig 2B). This delay corresponds to the time required for the HR1-maleimide coupling reaction to proceed (Appendix Fig S3). Interestingly, similar extent of lipid mixing was observed after 90 min of reaction whether HR1 was coupled to C18 or C45 maleimide lipids. In addition, no liposome fusion was measured when HR1 was added to liposomes lacking maleimide lipids or in which maleimide lipids had been inactivated (Fig 2B and Appendix Fig S4A), showing that only membrane-anchored HR1 has the capacity to induce lipid mixing. Under the same experimental conditions, HR2 was unable to induce lipid mixing and so were the heptad repeat domains of the synaptic t- or v-SNARE proteins in an homotypic configuration (i.e., with the same SNARE protein in both liposome populations; Fig 2B). In the heterotypic configuration (i.e., with the t-SNARE in one liposome population and its cognate v-SNARE in the other one), the heptad repeat domains of SNAREs mediated lipid mixing only when they were anchored to long C45 maleimide lipids (Appendix Fig S4B), in agreement with previous work 7. SNARE- and HR1-mediated liposome fusions thus display different membrane anchor length requirements, suggesting that they might proceed through a different molecular mechanism. SNARE-mediated liposome fusion requires that both v- and t-SNARE proteins have surface densities of at least 1 protein for 300 lipids 32. To determine the minimal protein surface density allowing liposome fusion by HR1, we have performed lipid-mixing experiments with various concentrations of HR1 added at t = 0 of the assay and quantified the actual lipid-to-protein ratio of each liposome preparation in a separate liposome co-floatation assay (Appendix Fig S5A). These titration experiments revealed that significant lipid mixing (larger than 5% after 90 min of reaction, i.e., twofold higher than the fusion background) required an HR1 surface density of at least 1 protein for 470 lipids (Appendix Fig S5B). This value is consistent with the physiological concentration of Mitofusins that we estimated to be around 1 protein for 450 lipids using mitochondria purified from wild-type MEFs (Appendix Supplementary Text and Appendix Fig S5C). Lipid mixing between liposomes can occur through hemifusion, that is, the mixing of only the outer leaflets of the liposome bilayers, or full fusion (Fig 3A). To determine the contribution of fluorescence dequenching arising from outer or inner leaflets mixing, we specifically eliminated the NBD fluorescence signal of the outer leaflets by pre-incubating fluorescent liposomes with sodium dithionite 40. Thus, any fluorescence increase observed in the fusion assay after HR1 addition would result from mixing of the inner leaflets. Significant fluorescence dequenching was still measured after dithionite treatment (Fig 3B). By comparing the extent of liposome fluorescence dequenching measured with or without prior dithionite treatment, we could estimate the percentage of liposomes that underwent hemifusion or had completed full fusion. After 90 min of incubation with HR1, ~ 60% of the liposomes had hemifused and ~ 40% had undergone full fusion (Fig 3B), showing that the HR1 domain of Mfn1 can induce fusion of both the outer and inner leaflets of liposome membranes. Figure 3. The HR1 domain of Mitofusin induces both hemifusion and full fusion of liposome membranes Scheme of the dithionite assay to quantify the percentage of liposomes that undergo full fusion. When fluorescent liposomes are pre-treated with sodium dithionite to eliminate the fluorescence of their outer leaflet, only full fusion events lead to fluorescence dequenching in the FRET-based lipid-mixing assay. Liposomes were prepared as in Fig 2 except that DOPE-NBD was replaced by DOPS-NBD to prevent flip-flop of the fluorescent lipids. Fluorescent liposomes indicated with an asterisk were treated with sodium dithionite prior to start the lipid-mixing assay in order to quench NBD fluorescence from the outer leaflets. Comparison of the fluorescence dequenching signals obtained with or without prior dithionite treatment allows the estimation of the percentage of liposomes that have undergone hemifusion or full fusion (see Materials and Methods). The left panel shows one representative set of kinetics experiments (performed with the same lipid and protein concentrations as in Fig 2), and the right panel the average percentage of liposomes that have fully fused after 90 min (n = 4 independent experiments; error bars indicate standard deviations). Download figure Download PowerPoint Qualitatively, similar results were obtained with the HR1 and HR2 domains of Mfn2 (Appendix Fig S4C). However, the preparations of Mfn2-HR1 peptides displayed variable fusion activities and a strong tendency to aggregate, as observed by SDS–PAGE following high speed centrifugation (Appendix Fig S6). Therefore, we pursued the functional characterization of the heptad repeat domains of Mitofusins in fusion using exclusively Mfn1 fragments. The HR1 and" @default.
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• W2797472953 title "The heptad repeat domain 1 of Mitofusin has membrane destabilization function in mitochondrial fusion" @default.
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• W2797472953 doi "https://doi.org/10.15252/embr.201643637" @default.
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