Activated Mitofusin 2 Signals Mitochondrial Fusion, Interfereswith Bax Activation, and Reduces Susceptibility toRadical Induced Depolarization*□S

Received for publication, February 10, 2005, and in revised form, March 30, 2005Published, JBC Papers in Press, May 4, 2005, DOI 10.1074/jbc.M501599200

Margaret Neuspiel, Rodolfo Zunino, Sandhya Gangaraju‡, Peter Rippstein, and Heidi McBride§

From the University of Ottawa Heart Institute, Ottawa, Ontario K1Y 4W7, Canada

Mitochondrial fusion in higher eukaryotes requires atleast two essential GTPases, Mitofusin 1 and Mitofusin 2(Mfn2). We have created an activated mutant of Mfn2,which shows increased rates of nucleotide exchangeand decreased rates of hydrolysis relative to wild typeMfn2. Mitochondrial fusion is stimulated dramaticallywithin heterokaryons expressing this mutant, demon-strating that hydrolysis is not requisite for the fusionevent, and supporting a role for Mfn2 as a signalingGTPase. Although steady-state mitochondrial fusion re-quired the conserved intermembrane space tryptophanresidue, this requirement was overcome within the con-text of the hydrolysis-deficient mutant. Furthermore,the punctate localization of Mfn2 is lost in the dominantactive mutants, indicating that these sites are function-ally controlled by changes in the nucleotide state ofMfn2. Upon staurosporine-stimulated cell death, acti-vated Bax is recruited to the Mfn2-containing puncta;however, Bax activation and cytochrome c release areinhibited in the presence of the dominant active mu-tants of Mfn2. The dominant active form of Mfn2 alsoprotected the mitochondria against free radical-induced permeability transition. In contrast to stauro-sporine-induced outer membrane permeability transi-tion, pore opening induced through the introductionof free radicals was dependent upon the conservedintermembrane space residue. This is the firstevidence that Mfn2 is a signaling GTPase regulatingmitochondrial fusion and that the nucleotide-dependent activation of Mfn2 concomitantly protectsthe organelle from permeability transition. The dataprovide new insights into the critical relationshipbetween mitochondrial membrane dynamics andprogrammed cell death.

The mitochondria sit at the crossroad of hundreds of chem-ical reactions that are essential for the life and death of a cell.The dynamic behavior of these organelles has only just begunto be examined, and the implications of steady-state fission,fusion, motility, and cristae remodeling events in the control of

the mitochondrial activity are not yet known. Studies in differ-ent model organisms are addressing this question by investi-gating the molecular mechanisms that govern mitochondrialdynamics to gain insights into the physiological triggers andconsequences of these events. Mitochondrial fusion in mamma-lian cells requires at least two essential outer membraneGTPases, Mitofusin 1 (Mfn1)1 and Mitofusin 2 (Mfn2) (1–6).These proteins span the outer membrane twice, and in additionto their amino-terminal GTPase domain, they have two con-served hydrophobic heptad repeats, HR1 and HR2, which areexposed to the cytosol (2, 7). The second HR2 domain of Mfn1has been shown to facilitate mitochondrial tethering, and thecrystal structure of the purified HR2 domain demonstratesthat it can form a 100 Å antiparallel structure that could bindin trans to bridge mitochondria (7). Another important regionof Mfns is their short, 2–3-amino acid intermembrane spaceloop, which contains a highly conserved tryptophan residue. Inyeast, this region of the protein is required for mitochondrialfusion and has been shown to anchor Fzo1p to sites of mem-brane contact between the inner and outer membrane (8).Although Mfn1 and Mfn2 are 60% identical, recent evidencewith both in vitro mitochondrial docking assays and in rescueexperiments of Mfn1 knock-out cells has shown that Mfn1appears to play a more direct role in mitochondrial docking (7,9) and that it functions in cooperation with the intermembranespace dynamin like GTPase Opa1 (autosomal dominant opticalatrophy 1) (10). The role of Mfn2 in mitochondrial fusion hasremained elusive, although it is clearly required for fusion andcan be found in heterodimeric complexes with Mfn1. Recentstudies have shown that the nucleotide binding and hydrolysisproperties of the two Mfn proteins are distinct (9), consistentwith the idea that the two GTPases regulate different stepsalong the fusion pathway (5). These steps may include theprocesses that drive mitochondrial motility, tethering,assembly of a fusion pore to facilitate lipid bilayer mixing andeventually leading to inner mitochondrial membrane fusion.Given the complexity of these molecular requirements, the Mfnproteins do not act alone, and studies in yeast have identified anumber of additional proteins required for mitochondrial fu-sion, including the outer membrane protein, Ugo1p (11–13),the inner membrane serine protease Rbd1p (14–16), an F-box-

* This work was supported in part by the Canadian Institutes ofHealth Research. The costs of publication of this article were defrayedin part by the payment of page charges. This article must therefore behereby marked “advertisement” in accordance with 18 U.S.C. Section1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org)contains a supplemental figure and video.

‡ Recipient of an Ontario graduate scholarship in science andtechnology.

§ To whom correspondence should be addressed: University of Ot-tawa Heart Institute, 40 Ruskin St., Ottawa, Ontario K1Y 4W7, Can-ada. Tel.: 613-761-4701; Fax: 613-761-5281; E-mail: hmcbride@ottawaheart.ca.

1 The abbreviations used are: Mfn1 and Mfn2, Mitofusin 1 and Mito-fusin 2, respectively; CFP, cyan fluorescent protein; ��, mitochondrialelectrochemical potential; DRP1, dynamin-related protein 1; ECFP,enhanced cyan fluorescent protein; fmk, fluoromethyl ketone; FP, flu-orescent protein; Fzo1p, Fuzzy Onion 1 protein; GFP, green fluorescentprotein; GST, glutathione S-transferase; His6, hexahistidine; HR, hep-tad repeat; IMS, intermembrane space; Mant GMP-PNP, N-methylan-thraniloyl guanosine 5�-(�,�-imino)triphosphate; PBS, phosphate-buff-ered saline; PEG, polyethylene glycol; STS, staurosporine; YFP, yellowfluorescent protein; Z, benzylxoycarbonyl.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 26, Issue of July 1, pp. 25060–25070, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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containing protein Mdm30p (17), along with other candidateslike Mdm35p, Mdm34p, and Mdm39p (18).

One of the outstanding questions in the field of mitochon-drial dynamics remains the physiological importance of mito-chondrial fission and fusion under steady-state conditions.Knock-outs of either Mfn1 or Mfn2 are embryonic lethal (6),demonstrating an essential role of mitochondrial fusion forviability. In addition, mutations within the Mfn2 gene havebeen found in patients suffering from Charcot-Marie-Toothneuropathy type 2A, and six of seven of these mutations werefound within the conserved GTPase region (19, 20). Interest-ingly, evidence that Mfn2 may exhibit intracellular signalingactivity has come from one study that identified the rat Mfn2(called hyperplasia suppressor gene HSG) as an importantantiproliferative protein, which interferes with the Ras path-way and blocks signaling from growth factor receptors at theplasma membrane (21). Although the mechanism for this inhi-bition is unknown, these findings suggest that Mfn2 and/or themorphological state of the mitochondria is highly integratedinto cellular signaling cascades.

Another example of how the mitochondrial morphology isintegrated into cellular signaling events is the growing evi-dence for a role of mitochondrial dynamics in the progression ofapoptosis. For example, two of the proteins required for mito-chondrial fission, Fis1p and DRP1, are also essential for pro-grammed cell death (22–25). Fis1p knock-down by small inter-fering RNA blocks recruitment and activation of Bax at thesurface of mitochondria after a death stimulus, indicating anessential role for this small integral membrane protein in apo-ptosis (25). Similarly, although loss of DRP1 does not dramat-ically interfere with Bax activation, cytochrome c release ispartially inhibited, and mitochondrial fission is blocked inthese cells (25, 26). In addition, small interfering RNA knock-down of Opa1, a protein required for mitochondrial fusion,results in fragmented mitochondria, which are highly sensi-tized to the loss of electrochemical potential and cytochrome crelease (25, 27). In contrast, overexpression of the two mito-fusin proteins together provides some protection against differ-ent apoptotic stimuli (28). These recent data highlight the dualroles of mitochondrial GTPases in the regulation of both mito-chondrial dynamics and the mitochondrial contribution to pro-grammed cell death.

Given the increasing evidence that Mfn1 plays a direct rolein mitochondrial tethering (5, 9), we have specifically investi-gated the function of Mfn2 in the process of mitochondrialfusion and examined further how the GTPase activity of Mfn2might contribute to the regulation of programmed cell death.

EXPERIMENTAL PROCEDURES

Construct Preparation and Reagents—The cDNA encoding humanMfn2 (KIAA0214) was graciously provided by Kazusa DNA ResearchInstitute, Japan. Mfn2 cDNA was PCR amplified using standard pro-tocols, for insertion into pECFP-C1 (Clontech) and pcDNA3.1 (Invitro-gen) with BamHI and HindIII restriction sites. Mfn2RasG12V-CFP wasprepared with QuikChange mutagenesis (Stratagene, La Jolla, CA)using pECFP-C1-Mfn2 as the template and a set of oligonucleotidesdesigned to replace amino acids GRTSNGKS with GAVGVGKS. Therestriction site NarI was introduced into the primers for screeningpurposes. The pcDNA3-GST, Mfn2-His6, and Mfn2RasG12V-His6 con-struct vectors for protein purification from transfected cell lysates werealso prepared using subcloning techniques, and all sequences used inthis work were confirmed. Mfn2W631P-CFP was prepared withQuikChange mutagenesis using pECFP-C1-Mfn2 as the template and aset of oligonucleotides designed to replace the tryptophan amino acid atposition 631 with a proline. The ApaI restriction site was introducedinto the primers for screening purposes. Mfn2RVWP-CFP was preparedby isolating the DNA fragment encoding amino acids 1–431 ofMfn2RasG12V-CFP by digestion with HindIII and SalI and subcloningthis fragment containing the RasG12V mutation into the Mfn2W631P-CFP construct cut with the same enzymes, thereby replacing the wild

type GTPase domain of Mfn2W631P-CFP with the Mfn2RasG12V mutation.The cDNA encoding DsRed2 was amplified from pDsRed2-C1 (Clon-tech) using primers designed for digestion with BamHI and XbaI andligation into the pcDNA3-pOCT vector (29). Tom7-GFP was obtainedfrom Mike Ryan, La Trobe University, Melbourne Australia. Mfn2mouse polyclonal antiserum was generated against a mixture contain-ing both recombinantly expressed Mfn2 (710–757)-GST and a syntheticMfn2 NH2-terminal peptide CNSIVTVKKNKRIIM-OH (Dalton Chem-ical Laboratories, Toronto, Canada), conjugated to 5 mg of maleimide-activated keyhole limpet hemocyanin. Antiserum was generated follow-ing a 56-day standard immunization protocol. Polyclonal antibodiesagainst fluorescent proteins (anti-FP) used for immunoelectron micros-copy were purchased from Clontech. Dihydroethidium (MolecularProbes, Eugene, OR) was utilized to determine steady-state radicallevels in transfected cells. Monoclonal 7H8.2C12 anti-cytochrome cantibodies were obtained from BD Biosciences, and rabbit polyclonalanti-Bax antibodies were obtained from Upstate Biotechnology cellsignaling solutions. Alexa Fluor 350 and 594 goat anti-mouse or rabbitsecondary antibodies from Molecular Probes were used for staurospo-rine (STS) experiments. Z-VAD was obtained from Enzyme SystemsProducts (Aurora, Canada), and STS was obtained from Sigma.

Transfection and Imaging of COS-7 Cells—Transfection and imagingmethods were exactly as described in Ref. 29. For electrochemicalpotential determination, cells were incubated with 50 nM MitoFluor Red589 (Molecular Probes) at 37 °C for 20 min. Whole cell images wereacquired for untransfected and transfected cells by exciting at 589 nmwith the CFP/YFP/DsRed triple pass filter (Chroma, Brattleboro, VT).The presence of transfected CFP-tagged protein was confirmed by ex-citing at 434 nm using the same filter set. Areas of interest wereselected for each cell, and total fluorescence arbitrary units weresummed for each cell. The total fluorescence intensity/cell was quanti-fied as the sum of the values of each pixel within the area of interestminus the average background signal obtained/pixel. The number ofcells within each fluorescence distribution range was scored and tabu-lated as a percentage.

For MitoFluor Red 589 (Molecular Probes) flickering experiments, 50nM dye was added to the chamber medium and incubated with the cellsat 37 °C for 20 min. After equilibration of the dye, 400 images werecollected by exciting at 589 nm with the CFP/YFP/DsRed triple passfilter (Chroma) for 1 s followed by a 2.5-s delay. The presence oftransfected CFP-tagged protein was confirmed as indicated above. Thetime series were analyzed, and each frame where all mitochondrial dyewas released from the whole cell was plotted.

To determine steady-state radical loads, cells were incubated with 5�M dihydroethidium at 37 °C for 20 min. Dihydroethidium oxidation bysuperoxide to ethidium was visualized by excitation at 547 nm. Wholecell imaging and fluorescence quantification for untransfected andtransfected cells were performed as indicated above.

Mant GMP-PNP Binding Assay—COS-7 cells were transfected withpOCT-CFP, Mfn2-CFP, Mfn2RasG12V-CFP, and Rab5-CFP fusion con-structs. The day after transfection, the cells were harvested by trypsintreatment, washed with PBS and with nucleotide binding buffer (220mM mannitol, 68 mM sucrose, 200 mM NaCl, 2 mM MgCl2, 0.5 mM EGTA,2.5 mM KH2PO4, 10 mM Hepes, pH 7.4, 1 mg/ml bovine serum albumin)containing protease inhibitors. Cells were then broken in a cell cracker,and the whole lysate was centrifuged at 10,000 rpm to concentrateheavy membrane fractions. Pellets were resuspended in binding buffer,and 50-�l aliquots were mixed with Mant GMP-PNP (1 �M final con-centration, Molecular Probes) and incubated at 37 °C for differenttimes. After incubation the aliquots were scanned for emission fluores-cence of Mant nucleotides in a QuantaMaster 6000SE (Photon Technol-ogy International, London, Canada) (excitation at 360 nm). The peakemission at 448 nm was recorded, and the background fluorescence ofthe nucleotide alone was subtracted. Each value was also normalizedfor total protein concentration in the sample (determined using the DCprotein assay (Bio-Rad)) and for the level of recombinant protein ex-pression by measuring the CFP signal obtained in the fluorometer uponexcitation at 434 nm, emission at 477 nm.

For the purification of GST, Mfn2-GST, and Mfn2RasG12V-GST fromtransfected cell lysates, 10-cm dishes of COS-7 cells were transfectedusing Lipofectamine 2000 (Invitrogen), and after 12 h they were treatedwith trypsin, washed, and lysed with TNE buffer (25 mM Tris-HCl, pH7.4, 150 mM NaCl, 5 mM, EDTA, 1 mM dithiothreitol, 60 �g/ml chymo-trypsin, 1 �M leupeptin, 25 �g/ml antipain, 2 �g/ml aprotinin, 40 �M

4-aminidophenylmethane-sulphonyl fluoride, 1 mM pepstatin A), con-taining 1% Triton X-100 for 2 h at 4 °C. Lysates were adjusted to 40%sucrose and centrifuged at 70,000 rpm for 1 h at 4 °C. Supernatantswere diluted to 10% sucrose with TNE � Triton X-100 buffer and were

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incubated overnight with glutathione-Sepharose beads at 4 °C. Beadswere washed, and GST fusion proteins were eluted with 50 mM reducedglutathione in TNE buffer � 10% sucrose. Eluted aliquots (50-�l elu-tions) were incubated in duplicate with 0.2 �M Mant GMP-PNP for 15min. at 37 °C and assayed for fluorescence (excitation, 360 nm; emis-sion, 448 nm). Normalization was done by assaying the enzymatic activityof GST within each aliquot, with 1-chloro-2,4-dinitrobenzene (Sigma) andglutathione as substrates, with formation of a product with absorbance at340 nm (Amersham Biosciences), being the product formation rate pro-portional to the amount of GST in the aliquots. Known concentrations ofbacterially purified GST were used as a standard in this assay. Bacteriallyexpressed Rab5-GST was purified as described previously (30).

GTP Hydrolysis Assay—COS-7 cells were transfected with Mfn2-His6, Mfn2RasG12V-His6, or LacZ-His6, and the following day cells werewashed with PBS, harvested by scraping, and centrifuged. Cell pelletswere resuspended in a small volume of TNX solubilization buffer (50mM Tris, pH 7.4, 150 mM NaCl, 2% Triton X-100, 1 mM �-mercaptoeth-anol, 2 mM MgCl2, protease inhibitor mixture) and incubated for 1 h at4 C. Lysates were then cleared at 70,000 � g in a TLA 110 rotor for 30min at 4 °C, and supernatants were incubated with nickel-nitrilotriace-tic acid-agarose beads for 1.5 h at 4 °C. Beads were then centrifugedand washed twice with TNX buffer containing 200 �M ATP (to removechaperones and other nonspecific proteins of the lysate) followed by twomore washes with TNX buffer containing 20 mM imidazole. Proteinswere eluted with series of 100 and 200 mM imidazole in TNX buffer, andconcentrations were determined by the Bio-Rad method. 10 �g of thedifferent eluted proteins were preloaded with 1 �l of [�-32P]GTP (2,000Ci/mmol) in exchange buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 1%Triton X-100, 2 mM MgCl2, 1 mM dithiothreitol) by incubating for 30 s at37 °C in a 140-�l volume. The reaction was then diluted 10 times to trapthe nucleotide in place and begin [�-32P]GTP hydrolysis. From this mix,aliquots were taken, in triplicate, at different time points (0, 1, 2, 5, and10 min), applied to nitrocellulose filters in a vacuum manifold, andflushed immediately with 1 ml of PBS (to remove unbound nucleotideand hydrolyzed 32P). Filters were then placed in vials and assayed for32P scintillation counting.

Electron Microscopy—COS-7 cells were transfected with the appro-priate cDNA in 10-cm dishes with Lipofectamine 2000 for 16 h. Follow-ing this, fluorescence was first examined using the light microscope toensure that transfection was at least 70% prior to trypsin treatmentand washing of the cells in PBS. The washed cells were then pelletedand fixed in 1.6% glutaraldehyde in 0.1 M sodium cacodylate bufferprior to osmium tetroxide and uranyl acetate staining, spur resin em-bedding, and final thin sectioning of the blocks, and the grids werestained with lead citrate. For immunoelectron microscopy, the sametransfection procedure was followed. Cells were treated with trypsin,washed in PBS, fixed in 1.6% glutaraldehyde, and centrifuged at3,000 � g for 15 min. Cell pellets were embedded in LR white (Marivac,PQ, Canada), thin sections were cut with a Leica Ultracut E ultramic-rotome, immunolabeled with polyclonal anti-FP antibodies and 10 nmgold-labeled secondary antibodies (Jackson Laboratories, Bar Harbor,ME). Cells were then counterstained with lead citrate and uranylacetate. Digital images were taken using a JEOL 1230 TEM at 60 kVadapted with a 2,000 by 2,000 pixel bottom mount CCD digital camera(Hamamatsu, Japan) and AMT software.

PEG Fusion Assay—The whole cell fusion assay was performed asdescribed previously (4). Given that the broad excitation spectra ofDsRed overlaps with the GFP excitation wavelength, special precau-tions were taken to ensure that the images of fused mitochondria didnot include any bleed-through between the channels. For this, live cellswere imaged on the Olympus IX70 microscope with a 100 � objective UPlan Apochromat, NA 1.35–0.50 objective, exited at 488 nm (GFP) and560 nm (DsRed) with the Polychrome IV monochrometer (TillPhotonics,Grafelfing, Germany) through a fluorescein isothiocyanate/Cy3/Cy5 tri-ple pass filter (Chroma). The emitted light was filtered through anadditional dual view beam splitter (Optical Insights, Santa Fe, NM)equipped with two filters HQ520/20 and D600/40 to separate the GFPand DsRed emission signals, and images were captured as describedabove. The transfected CFP-tagged protein was imaged using the CFP/YFP dual pass filter (TillPhotonics) along with the beam splitterequipped with D465/30 and HQ535/30 filters (data not shown). Theacquired images were saved as .tiff images and overlaid in AdobePhotoshop for image assembly.

RESULTS

Creation of a Hydrolysis-deficient Mutant of Mfn2—To deter-mine whether nucleotide hydrolysis of Mfn2 was essential for

mitochondrial fusion, we constructed a RasG12V mutant. Ourpurpose in designing the mutant was to maintain nucleotidebinding and exchange properties of Mfn2 and yet reduce theintrinsic rate of nucleotide hydrolysis. Therefore we replacedthe residues within the P-loop with those from the activatedRasG12V (31–33) by replacing amino acids GRTSNGKS withGAVGVGKS (the consensus for GTP binding is GxxxxGKS(34)). We first compared the nucleotide exchange properties ofthis mutation (Mfn2RasG12V) with wild type Mfn2 and controlsby using crude extracts from cells transfected with CFP fusionproteins. COS-7 cells were transfected with matrix-targetedCFP (negative control), wild type Mfn2-CFP, Mfn2RasG12V-CFP, or Rab5-CFP as a positive control. Rab5 is the best char-acterized of the Rab GTPases and is known to regulate earlyendosome fusion (35). Regulatory GTPases of the Ras familyare all characterized by their low intrinsic rates of hydrolysisand high nucleotide affinities, which results in stable nucleo-tide states that require accessory proteins for their activity.Given the evidence that Mfn2 exhibits low rates of nucleotidehydrolysis and high affinity (9), it is relevant to use Rab5 as apositive control in these assays. After transfection with CFP-tagged constructs, the cells were harvested, broken, and themitochondria-enriched heavy membrane fractions were incu-bated with the environment-sensitive nonhydrolyzable GTPanalog Mant GMP-PNP (36, 37). Increased Mant nucleotidefluorescence is a direct measurement of nucleotide exchange.As expected, untransfected cells, or cells transfected with thematrix CFP control plasmid, bound a constant level of MantGMP-PNP because of the endogenous GTPases present in theextracts. Interestingly, overexpression of Mfn2-CFP did notsignificantly alter the basal amount of nucleotide binding inthe heavy membrane fraction (Fig. 1A). Overexpression of Rab5provided a �4-fold increase in Mant GMP-PNP binding withinthe first 10 min after incubation at 37 °C (Fig. 1A). This signalis specific because addition of unlabeled GTP competes for thebinding. Notably, although transfection of Mfn2-CFP had noeffect on basal nucleotide binding in this assay, transfection ofMfn2RasG12V-CFP demonstrated a significant increase in nucle-otide binding (Fig. 1A). We next isolated Mfn2RasG12V-GST orMfn2-GST from transfected mammalian cells using glutathi-one-Sepharose beads. We employed a quantitative GST enzymeassay with 1-chloro-2,4-dinitrobenzene as substrate and deter-mined the total amount of GST fusion proteins purified tonormalize/mol of purified protein (Fig. 1B). GST-tagged proteinwas purified from transfected cell lysates, and, as in total celllysates, the nucleotide binding experiments demonstrate a �2-fold increase in activity of Mfn2RasG12V-GST relative to the wildtype protein (Fig. 1B). In addition, the stimulated exchangeactivity of the Mfn2RasG12V mutant reached levels similar tothose obtained with molar equivalents of recombinant Rab5-GST (Fig. 1B). Although we could detect no significant differ-ences in nucleotide binding to Mfn2-CFP within whole cellextracts (Fig. 1A), the GST-purified Mfn2 did demonstratenucleotide binding/exchange relative to the GST control (Fig.1B). These biochemical data indicate that purified Mfn2 hasextremely low rates of nucleotide exchange, consistent withhigh affinity binding. These data are consistent with publisheddata that have also shown much stronger binding of Mfn2 tonucleotide relative to Mfn1 (9). Our data now show that muta-tions within the P-loop of Mfn2 increase nucleotide exchangerelative to the wild type protein, thereby bringing the ratescloser to the intrinsic rates observed for the Rab GTPases.

We next examined the intrinsic rates of [�-32P]GTP hydrol-ysis using the established filter-based assay quantifying therelease of the labeled tertiary phosphate from the GTP-boundprotein purified from transfected COS-7 cells (Fig. 1D) (38).

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Purified Mfn2-His6 hydrolyzed 7.33 � 10�3 mmol of GTP/mmolof protein/min, which is �4 times faster than the rates obtainedfor molar equivalents of bacterial expressed Rab5 protein(1.55 � 10�3 mmol of GTP/mmol of protein/min) (Fig. 1C).Although part of this signal may be the result of completeloss/exchange of nucleotide rather than nucleotide hydrolysis,we consider this to be negligible because the rates of exchangefor wild type Mfn2 are lower than Rab5 (Fig. 1, A and B). Therates of GTP hydrolysis we have determined for Rab5 aresimilar to previously calculated rates of 2 � 10�3/min (38, 39),further demonstrating the validity of the assay. Although themutant Mfn2 exhibited increased rates of nucleotide exchange,Mfn2RasG12V-His6 demonstrated significantly reduced levels ofhydrolysis (2.08 � 10�3/min) relative to the wild type protein(7.33 � 10�3/min) (Fig. 1C). Although we cannot exclude thecontribution of trace levels of copurifying GTPases, GTP ex-change factors or GTPase-activating proteins in the Mfn2 pu-rification from cell extracts, our data clearly demonstrate thatspecific mutations in Mfn2 alter the nucleotide binding andhydrolysis properties of the purified protein.

Mfn2 Is Localized in Specific Subdomains along Mitochon-drial Tubules in a Nucleotide-dependent Manner—We exam-ined the cellular consequences of the GTPase mutant by trans-fecting COS-7 cells with wild type Mfn2-CFP, GTPase mutantMfn2RasG12V-CFP, or a truncation mutant completely lacking

the GTPase domain Mfn2(430–757)-CFP. Transfection of COS-7cells with the wild type Mfn2 resulted in increased intercon-nectivity among the mitochondrial reticulum (Fig. 2), whichupon high levels of expression appears as a cluster (1, 2, 4). Asreported previously (40), Mfn2-CFP was localized in punctaalong the mitochondrial tubules. Transfection of the GTPasemutant Mfn2RasG12V-CFP resulted in the clustering of mito-chondria, which appeared to be fused even at the lowest levelsof detectable expression (Fig. 2). Importantly, unlike Mfn2-CFP, Mfn2RasG12V-CFP was distributed evenly along the sur-face of these clusters (Fig. 2). In contrast, transfection of theMfn2(430–757)-CFP mutant resulted in mitochondria visiblyfragmented into small, spherical units that cluster together ina stable manner (Fig. 2 and data not shown), similar to thosefound in the Mfn1 GTPase truncation mutant (7). These phe-notypes are specific to Mfn2 because transfection of anothermitochondrial outer membrane protein, Tom7 (24), did notcause significant mitochondrial clustering (Fig. 2). In addition,these phenotypes were not affected by the presence of the CFPtag because transfection of untagged constructs gave similarresults (data not shown). We considered that the assembly ofthe Mfn2 puncta may depend upon the conserved IMS residues.Mammalian Mfn2 contains only 2–3 amino acids separatingthe two transmembrane domains, one of which is a highlyconserved tryptophan residue that we replaced with a proline

FIG. 1. Creation of a hydrolysis-deficient mutant of Mfn2. A, Mant GMP-PNP binding of cell extracts either untransfected (Utf), ortransfected with pOCT-CFP (negative control), Mfn2-CFP, Mfn2RasG12V-CFP, or Rab5-CFP (positive control). Incubations performed at 37 °C for1 and 10 min were analyzed in the fluorometer (see “Experimental Procedures”). Competition with unlabeled GTP was performed for the 10 mintime point only, as indicated. All values obtained were normalized for total protein concentration and for expression levels of CFP-tagged proteinsand are expressed as fluorescence arbitrary units (A.U.). These results are representative of four independent experiments. p values (unpaired ttest) show insignificant differences when comparing untransfected, pOCT-CFP, and Mfn2-CFP. p values demonstrate a significant increasebetween Mfn2-CFP and Mfn2RasG12V-CFP (p � 0.013), but the values between Mfn2RasG12V-CFP and Rab5-CFP are insignificant (p � 0.347). B,Mant GMP-PNP binding to GST-tagged proteins purified from COS-7 cells transfected with GST, Mfn2-GST, or Mfn2RasG12V-GST. Incubationsperformed at 37 °C for 15 min were analyzed in the fluorometer, and the values are expressed as arbitrary units of fluorescence/�g of GST protein.Competition in the presence of cold GTP is shown in black. The data presented are representative of three independent experiments. p values areless than 0.05 when comparing GST with Mfn2-GST (p � 0.019) and also when comparing Mfn2-GST and Mfn2RasG12V-GST (p � 0.016),demonstrating that they represent statistically significant differences. There was no statistical significance to the differences observed betweenMfn2RasG12V-GST and Rab5-GST (p � 0.404). C, purified Mfn2-His6, Mfn2RasG12V-His6, and Rab5-GST were loaded with [�-32P]GTP, and theamount of radiolabeled phosphate remaining bound/nmol of protein over time was quantified. Each point was collected in triplicate, and the dataare representative of four individual experiments. D, Western blot analysis of the purification of proteins used in the hydrolysis experimentsindicates the initial levels of expression relative to the endogenous Mfn2 (arrows). Control, LacZ-His6-transfected cell extracts are shown in lane1, and transfections of Mfn2-His6 (lane 2) and Mfn2RasG12V-His6 (lane 3) reveal at least a 10-fold increased expression over endogenous protein.Eluates of tagged, purified proteins are shown in lane 4 (LacZ control), lane 5 (Mfn2-His6), and lane 6 (Mfn2RasG12V-His6).

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residue (Mfn2W631P). This proline residue is naturally occur-ring within the IMS loop in Drosophila melanogaster Fzo, in-dicating that this substitution should not alter the topology ofMfn2. Mfn2W631P-CFP remains sharply localized in distinctfoci; however, the tubular morphology of the mitochondria isreduced, and the organelles eventually fragment (Fig. 1). Cre-ation of a double mutant where both the Mfn2RasG12V andMfn2W631P mutations are present (Mfn2RVWP-CFP) also re-sulted in a loss of Mfn2 puncta, indicating that the punctatelocalization does not depend upon the conserved IMS residues,rather it is determined by the nucleotide state (Fig. 2). Videoanalysis showed that the Mfn2-CFP puncta remained highlyimmotile and were not observed at sites of mitochondrial fu-sion, nor did they significantly colocalize with the cytoskeleton(actin filaments or microtubules), and they did not accumulateat sites of endoplasmic reticulum/mitochondrial contact (datanot shown).

To ensure that the clustered mitochondria within cells trans-fected with Mfn2 and mutants maintained their electrochemi-

cal potential, we quantified the total fluorescence intensitywithin cells loaded with a ��-dependent dye and scored theresults in a distribution profile as shown in Table I. In untrans-fected cells, the fluorescence intensity was between 1 and 3 �106 arbitrary units in 90% of the cells examined. Transfectionof Mfn2W631P showed some loss in total dye uptake, with 61% ofthe cells examined exhibiting fluorescent arbitrary units offewer than 1 � 106. Transfection of the other constructs did notshow significant loss in potential, but showed a broader distri-bution profile than untransfected cells, with many transfectedcells exhibiting fluorescence units higher than normal. Forexample, transfection of Mfn2, Mfn2RasG12V, or Mfn2RVWP

showed 15–20% of cells having greater than 4 � 106 fluorescentarbitrary units. These data demonstrate that electrochemicalpotential is maintained, with a slight reduction in cells trans-fected with Mfn2W631P.

PEG-induced Cell Fusion Demonstrates That Mfn2RasG12V Isa Dominant Active Mutant—To assess directly the fusion com-petence of mitochondria in cells expressing these proteins, weemployed a well characterized assay that induces fusion be-tween whole cells transfected with different matrix markerproteins (4–6). Mitochondria from heterokaryons transfectedwith marker proteins alone fused completely within 8–12 h(Fig. 3, top left panels) (4–6). Mitochondria from donor cellsexpressing Mfn2-CFP also fused with acceptor mitochondriawithin a similar time course (Fig. 3, middle left, n � 29);however, �20% of the heterokaryons expressing Mfn2-CFPscored complete content mixing within 2 h. Strikingly, fusionbetween mitochondria expressing Mfn2RasG12V-CFP with ac-ceptor GFP organelles occurred within 30 min after the addi-tion of PEG in 90% of the observed cell fusions (Fig. 3, bottomleft, n � 20). Video analysis of the PEG fusion assay shows thatthe Mfn2RasG12V-CFP cluster remains immobile, whereas theGFP-containing acceptor mitochondria are motile within theheterokaryon (see supplemental video). This efficient fusionbetween mitochondria was highly significant because the mi-gration of mitochondria from one cell into another is a slowprocess in mammalian cells. In control experiments very fewmitochondria within the heterokaryon had migrated across thecell boundary in the 1st h (Fig. 3). In contrast, the rapid and/ordirected migration of the GFP-expressing mitochondria towardthe Mfn2RasG12V-CFP-expressing DsRed mitochondria allowedcomplete fusion of all mitochondria to occur within 30 min. Thisindicates that the presence of Mfn2RasG12V within one popula-tion of mitochondria initiates a cascade of events that lead tohighly efficient mitochondrial fusion. Therefore, we considerthe Mfn2RasG12V to be the first characterized dominant active,GTPase-deficient mutant that stimulates mitochondrial fusion.

The Mfn2(430–757)-CFP construct completely inhibited thefusion, confirming the requirement for the GTPase domain(Fig. 3, top right). In addition to a role in contact site formation,previous studies with yeast Fzo1p have demonstrated the re-quirement for the IMS loop of Fzo1p for mitochondrial fusion(8). Consistent with this, fusion was inhibited between mito-chondria containing Mfn2W631P-CFP and acceptor GFP-con-taining mitochondria. Surprisingly, the inhibition of mitochon-drial fusion conferred by the Mfn2W631P mutation (Fig. 3,middle right) was rescued by the Mfn2RasG12V mutation withinthe double mutant (Fig. 3, bottom right), indicating that theconserved IMS domain is not directly required for the fusionevent, but likely plays a more regulatory role in the activationof Mfn2.

Fused Mitochondria Are Interconnected by Novel MembraneElements—Ultrastructural analysis revealed that the mito-chondria do not fuse into a single organelle, but instead theyare interconnected through unusual membranous networks

FIG. 2. Mitochondrial morphology and distribution of Mfn2mutants. COS-7 cells were cotransfected with pOCT-YFP (left panels)and the CFP-tagged constructs indicated (middle panels). As a controlTom7-GFP was cotransfected with pOCT-DsRed2 (top panels). Imageswere taken from living cells 16 h after transfection. In the overlay, thematrix marker is shown in red and FP fusion proteins in green. Note thepunctate appearance of Mfn2-CFP and Mfn2W631P-CFP (arrows) com-pared with the smooth distribution of Mfn2RasG12V-CFP and Mfn2RVWP-CFP. Scale bars are 1 �m.

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(Fig. 4A). The fused clusters in Mfn2RasG12V-transfected cellswere qualitatively similar to the wild type (Fig. 4A), indicatingthat the end point of the fused mitochondrial reticulum issimilar, even though the PEG fusion assay demonstrated thatkinetics of the fusion event are different (Fig. 3). Analysis ofMfn2W631P-transfected cells did not reveal any interconnectingmembranes between the clustered organelles. Notably, some ofthe mitochondria in the Mfn2W631P-transfected cells appearedto “unravel,” with membranous material emanating beyond theclear boundaries of the outer membrane (Fig. 4A). This may becaused by the loss of contact site formation (8), leading toaberrant membrane architecture. Mitochondria in cells ex-pressing Mfn2RVWP-CFP contained stacks of parallel mem-branes and extensive membrane whorls that were intercon-nected throughout the cluster. Immunolabeling of the fusedmitochondrial clusters indicates that the Mfn2 protein is foundwithin the interconnecting membranes, demonstrating thatthese membranes are derived, at least in part, from the outermitochondrial membrane (Fig. 4B). Regardless of this massivealteration in membrane architecture, the cristae and electrondense matrix compartments appeared normal, consistent withtheir ability to maintain electrochemical potential (Table I) andstimulate mitochondrial fusion (Fig. 3). Mitochondria withincells expressing Mfn2(430–757)-CFP were docked togetherwithin a cluster, consistent with the proposed tethering func-

tion of the HR2 domain (7). However, there was no fusedmembrane material between the mitochondria (Fig. 4A), dem-onstrating that the fused membranes do not arise simply be-cause of nonspecific mitochondrial clustering.

Activated Mfn2 Represses Bax Activation, Cytochrome c Re-lease, and Permeability Transition—Given the increasing in-volvement of dynamic changes in mitochondrial morphologyduring the progression of apoptosis, we next examined thespecific consequence of Mfn2 activation on the mitochondrialresponse to two different types of stimuli. Stimulation of pro-grammed death by STS treatment resulted in the efficientrecruitment and activation of Bax to the mitochondria, as re-vealed by an antibody that specifically recognizes the confor-mationally active form of Bax (41). As expected (42, 43), theamount of Bax activation correlated with the amount of cyto-chrome c release (�50% by 3 h, Fig. 5, A and B, n � 379). As anegative control, we transfected cells with DRP1(K38E) (44–46), which has been shown to block mitochondrial fission andinhibit cytochrome c release from mitochondria (26, 47). Asexpected, only 3% of these cells showed cytochrome c releaseafter 3 h of STS treatment, with �10% of cells showing Baxactivation (n � 66). By 5 h of treatment, this level of Baxactivation increased without a release of cytochrome c (data notshown), as has been reported previously within cells trans-fected with DRP1(K38A) (40). Transfection of Mfn2-CFP

TABLE IQuantification of MitoFluor Red 589 uptake into mitochondria of transfected cells

FluorescenceaDistribution profile of intensities of ��-dependent dye uptakeb

UTFc Mfn2-CFP Mfn2RasG12V-CFP Mfn2W631P-CFP Mfn2RVWP-CFP Mfn2(430–757)-CFP DRP1(K38E)-CFP

A.U. � 106 % % % % % % %

1 4.8 19 9 61.1 14.3 16.7 6.31–2 57.1 28.6 25 33.3 38.1 33.3 18.82–3 33.3 28.6 25 5.6 9.5 38.9 37.53–4 4.8 9.5 19 0 23.8 11.1 37.54 0 15 22 0 15 0 0

a A.U., arbitrary units.b Percent of total cells.c UTF, untransfected.

FIG. 3. Mfn2RasG12V stimulates mitochondrial fusion in PEG cell fusion assays. COS-7 cells transfected with pOCT-GFP or pOCT-DsRed2and Mfn2-CFP constructs (as indicated) were seeded together for 12 h, and 50% PEG 5000 was added for 60 s after a 2-h preincubation withcycloheximide. Images of both fluorophores were taken from live cells after 1 h and 8 h. The overlay of the pOCT-GFP (green) and pOCT-DsRed(red) is shown for each time point. Mitochondrial fusion is indicated by GFP and DsRed matrix content mixing, which appears yellow in the image.Scale bars are 1 �m.

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showed a reduction of STS-induced Bax activation and cyto-chrome c release (Fig. 5A), with only �35% of cells showingsusceptibility to STS treatment (Fig. 5B, n � 92). Interestingly,in the 35% of STS-sensitive cells, many of the Mfn2-CFP-containing puncta colocalized with activated Bax (insets, Fig.5A). Mfn2RasG12V-CFP and Mfn2RVWP-CFP both repressed Baxactivation and cytochrome c release, where only �20% of cellswere susceptible to STS treatment (Fig. 5B, n � 69 and 72,respectively). In contrast, the fusion-incompetent Mfn2W631P-CFP did not provide protection against Bax activation or cyto-chrome c release (Fig. 5A, n � 73). As with Mfn2-CFP, acti-vated Bax often colocalized with Mfn2W631P-CFP-containingpuncta (see insets, Fig. 5A). Cells transfected with the domi-nant negative mutant Mfn2(460–757)-CFP were highly sensitiveto STS treatment, with 80% of cells showing complete cyto-chrome c release by 3 h (Fig. 5A, n � 95). Oddly, the amount ofcytochrome c release in this condition (�80%) did not mirrorthe amount of Bax activation (�40%), which could be explainedbecause cytochrome c was released in Mfn2(430–757)-CFP-trans-fected cells in a Bax-independent manner, without any deathstimuli (Fig. 5A). As expected, transfection of the other Mfn2constructs showed normal, mitochondrial cytochrome c stain-ing in the absence of any death stimuli (data not shown). Takentogether, the data show that activated Mfn2 is a repressor ofBax activation and outer membrane pore formation.

Given that activated Mfn2 can provide protection againstBax activation and cytochrome c release triggered by external

apoptotic stimuli, we next wanted to investigate whether Mfn2could also protect the mitochondria from damage induced byinternal metabolic stress. We therefore adapted an assay thatartificially produces free radicals within the matrix of the mi-tochondria, thereby triggering permeability transition and per-meability transition pore opening in the absence of Bax activa-tion (48–50). This approach allowed us to damage themitochondria from the matrix side, simulating physiologicalsystems where mitochondrial radical loads are increased be-cause of excessive respiratory activity or other stresses. The��-sensitive dye tetramethylrhodamine ester and its deriva-tives, such as MitoFluor Red 589, become photoactivated uponexposure to light and subsequently produce free radicals withinthe matrix of the mitochondria. With the increasing accumu-lation of free radicals, the permeability transition pore opens,and protons become equilibrated across the inner membrane(49, 50). Concomitant with the loss in ��, the potential-sensi-tive dye is redistributed within the cell, a process observedusing time lapse video microscopy. The mitochondria then re-gain ��, which allows the reuptake of the potential-sensitivedye, and in time lapse they appear to “flicker” until they be-come terminally depolarized. 400 images were collected over 25min by exciting MitoFluor Red 589 for 1,000 ms followed by a2,500-ms delay. Fig. 6 shows that 84% of the untransfectedcells incubated with 50 nM MitoFluor Red 589 reached terminaldepolarization within the first frame category (frames 1–250,n � 56). Mfn2-CFP-expressing cells revealed a moderate, but

FIG. 4. Ultrastructural analysis re-veals that clustered mitochondriaare interconnected by novel Mfn2-containing membrane elements. A,representative images taken at high mag-nification are shown from cells expressingthe constructs indicated. Mitochondriafrom untransfected cells are also shownfor comparison (upper left panel). B, im-munoelectron micrographs using anti-FPantibodies to label mitochondrial clustersexpressing the activated Mfn2RasG12V-CFP and Mfn2RVWP-CFP, as indicated.

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significant protection from free radical-induced damage, with avaried distribution of rates of dye loss across the four framecategories (p 0.003 Fisher’s exact test, n � 19). Similar to theprotection granted against STS treatment, 62% of the cellscontaining a fused mitochondrial reticulum induced by trans-fection of Mfn2RasG12V-CFP were significantly protectedagainst free radical-induced damage until the fourth framecategory (350–400 frames, p 0.001 from Fisher’s exact test,n � 21). In contrast, the mitochondria within cells expressingMfn2W631P-CFP rapidly reached their damage threshold with92% of cells losing their dye (n � 13) within the first framecategory, consistent with their susceptibility to STS. Unexpect-edly, although the morphological phenotypes were similar be-tween Mfn2RVWP-CFP and Mfn2RasG12V, and they both signif-icantly protected mitochondria against STS treatment, the

double mutant was not protected from the radical-induceddamage, with 78% (n � 18) of mitochondria terminally depo-larized in the first frame category. These data suggest thatboth the activation of Mfn2 GTPase domain and the IMS tryp-tophan residue are essential for permeability transition. Frag-mented mitochondria expressing Mfn2(430–757)-CFP but did notaffect the loss of dye in these experiments because 82% (n � 22)of the mitochondria were terminally depolarized within thefirst frame category. These effects are not caused by differencesin either MitoFluor dye loading or absolute free radical produc-tion because fluorescent quantification of initial MitoFluor dyeuptake and the radical dye dihydroethidium shows no differ-ences between the transfected versus untransfected controls(Tables I and II). The specificity of this assay was furtherverified by creating a fused reticulum after transfection with

FIG. 5. Activation of Mfn2 protectsagainst STS-induced cell death. A,transfected COS-7 cells were incubatedwith 2 �M STS in the presence of 10 �M

Z-VAD-fmk and immunolabeled with an-ti-cytochrome c antibodies (blue) and anti-Bax antibodies (red). Representative im-ages of cells are shown. Cytochrome crelease of cells transfected withMfn2(430–757)-CFP in the absence of deathstimuli is shown in lower right panel. In-sets show higher magnifications of the in-dividual channels, as indicated. Note thecolocalization of Bax with Mfn2-CFP pro-teins upon activation. B, the percentage ofcells with cytochrome c release and Baxactivation were scored at 3 h after STStreatment, and the data were plotted as apercentage distribution of total cells.

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the dominant interfering DRP1 mutant, DRP1(K38E). In con-trast with the protection granted by DRP1(K38E) in terms ofSTS-induced cytochrome c release, the fused mitochondrialreticulum within cells expressing DRP1(K38E)-CFP were notprotected from the free radical load (Fig. 6, n � 26), demon-strating that a fused mitochondrial morphology alone does notinhibit permeability transition.

DISCUSSION

We have shown for the first time that Mfn2 exhibits proper-ties of a signaling GTPase capable of regulating not only mito-chondrial fusion, but that its nucleotide state also plays acritical role in regulating the mitochondrial response to apo-ptotic and free radical-induced damage. Through the creationof mutants, we have dissected the functional role of the GTPaseactivity and the intermembrane space domain within Mfn2.Biochemical evidence indicates that Mfn2 exhibits nucleotideproperties similar to Rab5, with low intrinsic rates of hydroly-sis and nucleotide exchange. These data are consistent with therecently published work showing that Mfn2 has a higher affin-ity to nucleotide and dramatically slower rates of hydrolysisrelative to Mfn1 (9). Most importantly, we have characterizedthe nucleotide binding and hydrolysis properties of a mutantform of Mfn2, Mfn2RasG12V, and showed that this mutant hasslower hydrolysis and increased nucleotide exchange whencompared directly with the wild type protein (Fig. 1). Thismutant allowed us to examine the functional consequences of aGTP hydrolysis-deficient, dominant active form of Mfn2. Ourmutants have demonstrated a number of important findings.First, the hydrolysis-deficient mutant of Mfn2 results in adramatic stimulation of mitochondrial fusion, and the ultra-structural analysis of the fused mitochondrial clusters indi-

cates a striking proliferation of interconnected membranes,shedding new light on the plasticity of the mitochondrial mem-branes. Furthermore, we have determined that the conservedtryptophan residue within the intermembrane space region ofMfn2 is not essential to form a fusion pore but is required toactivate fusion within the context of the wild type GTPase. Themutational analysis also allowed us to determine that thepunctate localization of Mfn2 (40) is regulated by the nucleo-tide state. The hydrolysis-deficient mutants of Mfn2 that stim-ulate mitochondrial fusion do not readily form puncta, whereasthe mutants that inhibit mitochondrial fusion are found inthese foci. Finally, the activated Mfn2 constructs significantlyrepresses Bax activation, cytochrome c release, and free radi-cal-induced permeability transition. Taken together, thesedata reveal a role for Mfn2 as a regulator of mitochondrialfusion and as a nucleotide-dependent modulator of the apo-ptotic response.

The PEG fusion assay demonstrates that Mfn2 is a regula-tory GTPase, which, when in the activated form, is capable ofsignaling to neighboring mitochondria to fuse in an acceleratedmanner. Because Mfn2RasG12V-CFP is anchored within the mi-tochondrial outer membrane of the DsRed-containing mito-chondria in Fig. 3, it follows that Mfn2RasG12V initiated cytoso-lic events resulting in the efficient recruitment and fusion ofGFP-containing mitochondria with the Mfn2RasG12V-CFP/DsRed reticulum. Clearly there are a number of molecularevents that contribute to this dramatic increase in fusion, in-cluding increased motility events, as well as the activation ofthe fusion machinery. Our data therefore indicate that Mfn2may play a dual role, both as a direct constituent of the teth-ering/fusion machinery (minimally through the coiled coil do-

FIG. 6. Activation of Mfn2 protectsagainst permeability transition.COS-7 cells, as indicated, were incubatedwith 50 nM MitoFluor Red 589, exposed tolight (see “Experimental Procedures”),and scored for terminal depolarization.The results were converted to a percentvalue and plotted as a frequency distribu-tion for each transfection condition, as in-dicated in the graph.

TABLE IIQuantification of hET oxidation within transfected cells

FluorescenceaDistribution profile of steady-state radical loadb

UTFc Mfn2-CFP Mfn2RasG12V-CFP Mfn2W631P-CFP Mfn2RVWP-CFP Mfn2(430–757)-CFP DRP1(K38E)-CFP

A.U. � 106 % % % % % % %

1 41.7 40 26.7 61.5 12.5 12 101–2 25 50 40 38.5 62.5 59 34.52–3 25 10 20 0 18.8 29 24.13 8.3 0 13.3 0 6.3 0 31

a A.U., arbitrary units.b Percent of total cells.c UTF, untransfected.

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mains of Mfn430–757) and as a GTPase capable of initiatingsecondary cellular events. These secondary cellular eventslikely include the cooperation and/or activation of Mfn1, whichhas been shown to function directly with Opa1 in driving mi-tochondrial tethering and fusion (7, 9, 10). Evidence supportinga primary role for Mfn2 as a signaling GTPase has come fromprevious work demonstrating that mouse embryonic fibroblastcells lacking Mfn2 show a loss of long range motility events (6),consistent with a role for Mfn2 in regulating mitochondrialmovement. Our data are also consistent with the recently iden-tified role for Mfn2 as a regulator of the Ras signaling pathway(21). Given that Ras signaling occurs at the plasma membrane,the ability of Mfn2 to invoke a signaling cascade would providea mechanism for it to act upstream of events at a separateintracellular location.

What are the physiological triggers that might activateMfn2? Because Mfn2W631P-CFP results in fusion inhibition, weconsider that this IMS residue is required for the stimulation ofGTP nucleotide exchange in Mfn2. In this case, the GTPasedomain within the Mfn2W631P protein would remain in theinactive, GDP-bound state locked within the puncta wherefusion could not be initiated, as observed in Figs. 2 and 3.Because the IMS tryptophan residue is so critical in providingprotection against free radical-induced permeability transition(Fig. 6), we speculate that internal mitochondrial signals wouldcommunicate with Mfn2 through this residue to initiate mito-chondrial fusion in response to local free radical production andother forms of metabolic damage. Mitochondrial fusion maythen buffer and rescue local radical damage by sharing scav-engers and other metabolites between healthy and damagedorganelles. Under physiological conditions, this novel link be-tween mitochondrial fusion and permeability transition poreinhibition suggests a robust system to protect against accumu-lated cellular damage that could potentially lead to inoppor-tune cell death. Consistent with this, mouse embryonic fibro-blast cells lacking Mfn2 showed a loss in membrane potential(6), and antisense experiments to reduce Mfn2 levels in cul-tured myotubes demonstrated a loss in glucose oxidation, mem-brane potential, and oxygen consumption (51).

In addition to providing protection against free radical-in-duced permeability transition, the data demonstrate that acti-vated Mfn2 represses STS-induced Bax activation and cyto-chrome c release. This places a primary component of themitochondrial fusion machinery as a regulator of the apoptoticresponse. Given that the activation of Mfn2 concomitantlyblocks permeability transition and activates mitochondrial fu-sion, the data suggest that these two pathways are mutuallyexclusive. It has been shown previously that mitochondrialfusion events are inhibited during an apoptotic stimulus (52),consistent with our data indicating that Mfn2 must be in theinactive, GDP-bound form to allow for activation of Bax. Al-though the mechanism by which Mfn2 might regulate Baxactivation is unknown, it is possible that the punctate Mfn2sites represent preassembled channels competent for activatedBax recruitment and outer membrane permeability transition,and upon activation of Mfn2, critical components within thesemicrodomains may disassemble. Alternatively, the activationof Mfn2 for fusion may directly inhibit the fission machinery sothat the two events remain exclusive. Because fission precedescytochrome c release and appears requisite for apoptosis (53,54), any interference of activated Mfn2 with components of thefission machinery would effectively block Bax activation andcytochrome c release. Obvious candidates would be Fis1 andDRP1 because they function together in promoting mitochon-drial fission and eliciting an efficient apoptotic response (22,23, 25, 26, 47). It will be important to characterize the Mfn2-

interacting proteins, those both cytosolic and mitochondrial, todefine better the molecular events that link the GTPase cycle ofMfn2 with mitochondrial fusion and the control of permeabilitytransition.

Acknowledgments—We are extremely thankful to the anonymousreviewers, who have played an essential role in the development of thiswork. We are grateful to Marino Zerial (MPI-CBG Dresden, Germany)for the use of Rab5-CFP and Rab5-GST, Mike Ryan (La Trobe Univer-sity, Australia) for Tom7-GFP, and to Kazuhisa Nakayama (Universityof Tsukuba, Japan) for the DRP1(K38A) constructs used in this study.We thank members of the laboratory, Gordon Shore, andMarta Miaczynska for critical comments on the manuscript.

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Activated Mfn2 Signals Mitochondrial Fusion25070

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McBrideMargaret Neuspiel, Rodolfo Zunino, Sandhya Gangaraju, Peter Rippstein and Heidi

and Reduces Susceptibility to Radical Induced DepolarizationActivated Mitofusin 2 Signals Mitochondrial Fusion, Interferes with Bax Activation,

doi: 10.1074/jbc.M501599200 originally published online May 4, 20052005, 280:25060-25070.J. Biol. Chem. 

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