cells
Review
Redox Modifications of Proteins of the Mitochondrial
Fusion and Fission Machinery
Christina Wolf 1,† , Víctor López del Amo 2,† , Sabine Arndt 3 , Diones Bueno 1 ,
Stefan Tenzer 3 , Eva-Maria Hanschmann 4 , Carsten Berndt 4 and Axel Methner 1,*
1 Institute of Molecular Medicine, University Medical Center of the Johannes-Gutenberg University Mainz,
55131 Mainz, Germany; wolf.christina90@gmail.com (C.W.); diones.bueno@gmail.com (D.B.)
2 Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA 92093, USA;
vlopezdelamo@ucsd.edu
3 Institute for Immunology, University Medical Center of the Johannes-Gutenberg University Mainz,
55131 Mainz, Germany; arndt@uni-mainz.de (S.A.); tenzer@uni-mainz.de (S.T.)
4 Department of Neurology, Medical Faculty, Heinrich-Heine University, 40225 Düsseldorf, Germany;
eva-maria.hanschmann@med.uni-duesseldorf.de (E.-M.H.); carsten.berndt@hhu.de (C.B.)
* Correspondence: axel.methner@gmail.com
† These authors contributed equally.




Received: 11 February 2020; Accepted: 24 March 2020; Published: 27 March 2020 

Abstract: Mitochondrial fusion and fission tailors the mitochondrial shape to changes in cellular
homeostasis. Players of this process are the mitofusins, which regulate fusion of the outer
mitochondrial membrane, and the fission protein DRP1. Upon specific stimuli, DRP1 translocates
to the mitochondria, where it interacts with its receptors FIS1, MFF, and MID49/51. Another fission
factor of clinical relevance is GDAP1. Here, we identify and discuss cysteine residues of these proteins
that are conserved in phylogenetically distant organisms and which represent potential sites of
posttranslational redox modifications. We reveal that worms and flies possess only a single mitofusin,
which in vertebrates diverged into MFN1 and MFN2. All mitofusins contain four conserved cysteines
in addition to cysteine 684 in MFN2, a site involved in mitochondrial hyperfusion. DRP1 and FIS1
are also evolutionarily conserved but only DRP1 contains four conserved cysteine residues besides
cysteine 644, a specific site of nitrosylation. MFF and MID49/51 are only present in the vertebrate
lineage. GDAP1 is missing in the nematode genome and contains no conserved cysteine residues. Our
analysis suggests that the function of the evolutionarily oldest proteins of the mitochondrial fusion
and fission machinery, the mitofusins and DRP1 but not FIS1, might be altered by redox modifications.
Keywords: mitochondria; fusion; fission; redox; metabolism; thiol switch
1. Introduction
The function and activity of many proteins is regulated by post-translational modifications (PTMs)
of specific amino acids, including, for instance, phosphorylation of serine and tyrosine residues,
ubiquitylation, and SUMOylation of lysine residues. These modifications are important for adjusting
the cellular physiology to diverse challenges in order to regain homeostasis. Although cysteine is the
least abundant amino acid, with 2.2% in complex eukaryotes [1], 214,000 cysteines are still encoded by
the human genome [2]. Interestingly, the number of cysteines has increased throughout evolution and
seems to correlate with the complexity of the organism [1].
Cysteine is one of the most commonly post-translationally modified amino acids due to the
physical availability and the acid dissociation constant pKa of its thiol (R-SH). Thiols are nucleophilic
and are more reactive in their thiolate state (RS-), which can be stabilized by positively charged or
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Cells 2020, 9, 815 2 of 18
protonatedCelals m202i0n, 9o, 8a15c ids in their proximity [3]. In fact, cysteines can undergo at lea2s otf 1188 different
physiological nonradical post-translational modifications [2] induced by reactive oxygen species (ROS),
reactive nipthryosgioelnogsicpale cnioensra(dRicNalS p)o, sat-ntrdanrselaatciotnivale msoudliffuicratsiopnesc [i2e]s in(RduScSed)  (bFyi greuarcetiv1e) .oxUygnedn esrpepchieys siological
conditions(,R5O–S1)2, %reoacftiavlel pnriotrtoegienn cyspsetceiiens e(rReNsSid), uaend reactive sulfur species (RSS) (Figure 1). Under physiological conditions, 5–12% of all proteins cayrseteoinxei dreiszideudeisn arcee ollxsidainzeddt iins scuelelss a[n4d]. tiTsshueeso [x4i]d. ation and
reduction oTfhec yosxtiediantieosn thanadt fruednuccttiioonn aosf tchyisotelinsews itthcahte fsu,nic.eti.o,nv iaas tthheiorl esvweirtcshibesl,e if.eo.r, mviaa tithoen roefvedrissibullefi de bonds
or via de/gflourtmaathtioionn yofl atdiiosunl,fimdea kbeosndths eomr evsisa endtei/agluftoarthsioignnylaaltitorna, nmsdaukecst iothneman desvseanrtiioalu sfocre lsluiglnaarl processes,
including ctrealnlspdruoctliiofen raatnido nv,adriioffues recenltluialatrio pnr,ogceesnsees,e xinpcrleusdsiniogn c,ealln dprmoliefetraabtioolni,s mdi.ffMereonrtieaotivoenr, , gthenioe l switches
exhibit notexopnrelyssikoeny, asnidg nmiefitacbaonlicsemi. nMvoareroioveurs, tphihoyl sswioitlcohgeisc eaxlhbibuit not only key significance in various physiological but also pathological processes [5,6]. t also pathological processes [5,6].
 
Figure 1. Representation scheme of reversible oxidative post-translational modifications (Ox-PTMs) 
Figure 1. Rone pcyrestseeinnetsa. tPiorontesincsh ceomnteaionfinrge fvreeer stihbiolels ooxr itdhiaotliavtees p(roedstu-cterda npsrolatetiino)n eaxlhimbiot rdeiafictciavittiyo ntosw(aOrdxs- PTMs) on
cysteines. cPerrotatieni noxsidcaonntst,a siuncihn ags frreeaectitvhei ooxlsygoernt shpieocliaest e(Hs 2(Ore2)d, ruecacetdivpe nroittreoginen)  espxehciibesi t(RrNeaOc)t,i avnidty retaocwtivaer ds certain
oxidants, ssuclfhura spreeciaecs t(iRvSeHo),x tyog feonrms psuelcfieensic( aHci2dO (-2S)O, rHe)a, cSt-igvluetanthitiroongyleantiosnp (eRc-iSeSsG()R, dNisOul)p,haidned-broenadcst ive sulfur
species (R(SRHS-)S,),t So-nfiotrromsylsatuiolfne (n-SiNc Oa)c, iadnd( -SS-pOeHrsu),lfiSd-agtilount a(-tShSHio)n. *y, Rla-StiSoGn an(Rd -RSSS-SG a)l,tedraitsiuonlps rheipdrees-ebnot na ds (RS-S),
S-nitrosyladtiivoenrse(- gSrNouOp )o,fa mnoddSif-icpaetirosnus.l fiAdll athtieosne m(-oSdSifHica).ti*o,nRs -aSffSecGt parnotdeinR Sfu-nSctaioltne raantdi oacntsivriteyp arneds eanret a diverse
reversible via thioredoxins (Trx) or glutaredoxins (Grx). Under oxidative conditions, glutaredoxins 
group of maroed aibfilec atot iiondnusc.eA S-lglltuhtaetsheiomnyoladtiiofinc; oaxtiidoantisona ffvieac Ht 2pOr2o itse minosftu linkecltyi ofancialintadteda cbtyi vpietryoxairneddoaxirnes reversible
via thiored(Porxi).n s (Trx) or glutaredoxins (Grx). Under oxidative conditions, glutaredoxins are able to
induce S-glutathionylation; oxidation via H2O2 is most likely facilitated by peroxiredoxins (Prx).
Hydrogen peroxide (H2O2) belongs to the group of ROS that have been studied for its important 
Hydrfougnecntiopn earso ax sideceon(Hd mOess)enbgeelro inng ssigtnoalt htreangsdrouuctpiono. fWRhOenS Hth2Oa2t rheaacvtse wbiethe na sfrteued pireodteifno rthiitosl, important
sulfenic acid (R-SOH)2 is f2ormed (Figure 1), which is unstable and very reactive [7]. Sulfenic acid can 
function absea fusretchoern odximdizeesds eton gsuelrfininic s(Rig-SnOalHt2 )r aannds dsuulfcotnioicn a.ciWd (hRe-SnOH O3H2). C2yrseteaincet sowxiditahtioan fcraene apltreor tein thiol,
sulfenic acpidro(teRin-S cOonHfo)rmisaftoiornm aend (trFigi guerr eun1f)o,ldwinhgi,c lheaidsinugn tsot ahbiglheear npdrovteeinr yturrenaocvteirv, eto[x7ic] .aSggurlefgeantiiocna, cid can be
further oxiadnidz aeldterteods cuelllfi sniginca(liRn-gS uOp2 tHo c)eall nddeasthu [l8fo]. nic acid (R-SO3H). Cysteine oxidation can alter protein
conformation aRneadcttivrieg sguelfruru snpfeoclidesi n(RgS,Sl)e aanddi nthge troespheicgthiveer thpirool tmeoindiftiucartnioonv Se-rp,etrosuxlifcidaeg (g-SrSeHg)a ctaion na,lsaon d altered
alter cysteines (Figure 1). Since it was first shown that H2S is physiologically produced and RSS has 
cell signalian bgioulopgitcoal cfuenllctdioena itnh m[a8m].mals, there has been a steadily growing interest in this topic [9–12]. Next 
Reacttiov enistruicl fouxridsep (eNcOie)s a(nRdS cSa)rbaonnd mtohneoxreidsep (eCcOti)v, eHt2Sh iios lcmouondteidfi acmatoinognstS t-hpee grassuoltfiradnesm(-itSteSrHs. ) can also
alter cysteHinoewsev(eFri,g huowre H12)S., oSri rnactheeri tdwowansstfirerasmt  sphroodwucnts,t shuacth Has 2sSulfiisdep ahnyiosnio (lHoSg-)i,c paolllysuplfrioddesu, acned and RSS
has a biolosgulifchayldfruyln rcatdiiocanl i(nHSm•-a),m acmt aasl sa, stuhlefurre shigansalb reeemnaianss tlaeragdeliyl yelgursiovwe. iInt gwains tperroepsotseidn tthhaits thtoe pic [9–12].
canonical mitochondrial [11] and heme-dependent sulfide oxidation [13] are both important sources 
Next to nitorfi cRSoSx. iFduert(hNerOmo)raen, ednzcyamrbe-ocnatamlyozendo txriadnesp(eCrsOulf)i,dHati2oSn iiss ccoruucniatle fdora tmheo tnarggsett tshpeecgifiacsitoyt roaf nsmitters.
However,rhevoewrsibHle2 PST, Mors. rSaotmhe rredsuoltws snusgtgresat mthapt -rSoSdHu isc tnso,t sfourcmheda sthsrouulfighd ae raeancitoion (oHf fSre-e) ,thpiollsy wsiuthlfi des, and
sulfhydrylRSrSa dbuict aralt(hHer Sfr•o-m), a ncut calesopahsiluicl afuttarcksi fgronmal HrSe-m waitihn aslrleardgye olxyideizlueds itvhieo.ls,I stuwcha ass pdirsoulpfiodsees,d that the
canonical mgluittoatchhioonnydlartieadl o[r1 n1i]traonsydlahteedm cyes-tdeienpees,n adnden stulsfuenlific daceido x[1i4d].a Otinoen h[y1p3o]thaerseisb iso tthhati min pthoirs twanayt, sources of
the reaction with excess ROS to sulfinic acid and/or RNS is hindered and the protein is hence 
RSS. Furthperromteoctreed, efrnozmy mpeerm-caanteanlty dzaemdatgrea nbysp ReOrsSu alsfi pdeartsiuolfnidiess ccraunc biael rfeodrutchede. tMarogreeotvsepr,e creicfiecnitt ydaotaf reversible
PTMs. Somsuegrgesstu tlhtasts puegrsguelfsidt athteadt c-ySsSteHineis rensoidtufeosr cmane dbet ohxrioduizgedh tao r–eSaScOtHio, n-SoSOf f2rHe,e anthdi –oSlSsOw3Hit,h wRhSicSh but rather
from a nuc leophilic attack from HS- with already oxidized thiols, such as disulfides, glutathionylated
or nitrosylated cysteines, and sulfenic acid [14]. One hypothesis is that in this way, the reaction with
excess ROS to sulfinic acid and/or RNS is hindered and the protein is hence protected from permanent
damage by ROS as persulfides can be reduced. Moreover, recent data suggest that persulfidated
cysteine residues can be oxidized to -SSOH, -SSO2H, and -SSO3H, which can be reduced because
of the disulfide bridge [15]. H2S was shown to have roles in vasodilation [10], ER stress [16], and
anti-apoptotic pathways [17]. For example, H2S regulates vasodilation through an increase of cyclic
Cells 2020, 9, 815 3 of 18
guanosine monophosphate (cGMP), potassium (K+) increase via opening of ATP-sensitive potassium
channels (KATP), and activation of endothelial nitric oxide synthase (eNOS), which also elevates cGMP
levels. The cGMP degradation by phosphodiesterase 5 (PDE5) is inhibited by H2S, which leads to
prolonged elevated cGMP levels [18–20]. In addition, H2S relaxes vascular smooth muscle cells [21].
Reactive nitrogen species (RNS) include nitric oxide (•NO), nitroxyl (HNO), and peroxynitrite
(ONOO-). They are formed from the reaction of NO with ROS in case of ONOO- or H2S for
HNO, respectively. •NO itself is produced endogenously by eNOS, inducible nitric oxide synthase
(iNOS), and neuronal NOS (nNOS) from L-arginine and oxygen. The cysteine PTM caused by NO is
called S-nitrosylation (R-SNO) (Figure 1). The most abundant endogenously present S-nitrosothiol
is S-nitrosoglutathione (GSNO), which is the major •NO donor for proteins. It is formed by the
transfer of a nitrosyl group (R-NO) from the heme of cytochrome c onto glutathione (GSH) [22].
This reaction takes place within mitochondria from which GSNO diffuses to the rest of the cell,
where the –NO can be transferred to other proteins, such as the nuclear factor NF-kappa-B (NFκB),
hypoxia-inducible factor 1 (HIF-1) [23], and glycerinaldehyd-3-phosphat-dehydrogenase (GAPDH) [24].
Within mitochondria, all complexes of the electron transport chain, ATP synthase, enzymes of the Krebs
cycle [25,26], and β-oxidation enzymes [27] are S-nitrosylation targets. Most modified mitochondrial
proteins seem to be inhibited by S-nitrosylation to restrict ROS production. However, at higher
concentrations and in reaction with the superoxide radical O •-2 , •NO forms the more reactive ONOO-,
leading to tyrosine nitration [28] and cellular damage [29]. GSH not only transfers NO to other
proteins, it can also modify proteins itself by S-glutathionylation (R-SSG). Cysteines can spontaneously
react with the glutathione disulfide (GSSG) through a nucleophilic attack of glutathione on ionized
protein thiol or thiolate (Pr-S-) [30], sulfenic acid, or protein thionyl radical [31,32]. Even though
S-glutathionylation has been shown to regulate protein function, for instance, through modification
of transcription factors, such as the nuclear factor erythroid 2–related factor 2 (Nrf2) and NFκB [33]
and mitochondrial membrane proteins [34], protein glutathionylation during oxidative distress is
rather considered a protective mechanism, which is also enzymatically facilitated by glutaredoxins
(see below). In general, oxidative distress describes the pathological oxidative damage of biomolecules
that has been linked to various pathologies, whereas oxidative eustress describes physiological
redox alterations important for the regulation of several signaling pathways and the activity of
a variety of proteins [35]. For this physiological signaling function, most of the abovementioned
post-translational modifications are reversible and enzymatically controlled via oxidoreductases of the
thioredoxin (Trx) family (Figure 1) [36]. Oxidizing as well as reducing molecules potentially inducing
post-translational modifications on their own, e.g., H2O2 or GSH, show very low reactivity with thiols.
Therefore, enzymes/oxidoreductases are needed for (i) the formation and removal of these modifications
and (ii) to ensure the specificity for proper signaling events [37]. Whereas Trxs are able to reduce
disulfides, S-nitrosylated [38], S-persulfidated [39], and further oxidized S-persulfidated cysteine
residues [15], glutaredoxins (Grxs) reduce disulfides, S-glutathionylated [40], and S-persulfidated
cysteine residues [39]. Sulfenic acid cannot be reduced directly; it needs to be converted into a
disulfide or a S-glutathionylation. Sulfinic and sulfonic acids are generally considered irreversible [7],
although there are indications that sulfinic acid can be reversed in an ATP-dependent reaction by
sulfiredoxin (Srx) [41,42]. In fact, recent data increase the number of Srx targets and indicate regulatory
functions [43]. Peroxiredoxins (Prx) constitute cellular peroxidases that can reduce peroxides and
transfer oxidizing equivalents to target proteins [44,45]. In addition, protein disulfide isomerases also
belong to the Trx family. These enzymes are known to mediate thiol/disulfide exchange reactions,
function as chaperones, and regulate protein activity [46–48].
2. The Mitochondrial Fusion and Fission Machinery
Most of the cellular reactive species are produced by mitochondria [49,50], cellular organelles
with an outer (OMM) and inner (IMM) membrane that convert most of the cell’s energy to ATP by
generating a proton (∆pHm) and electrical (∆ψm) gradient across the inner membrane through the
Cells 2020, 9, 815 4 of 18
respiratory chain, which drives the ATP synthase. Complex I of the respiratory chain can produce
large amounts of ROS under conditions, such as a high NADH/NAD+ ratio caused by inhibition
or damage to complex I [51] or reverse electron transport due to a backlog of electrons during low
ATP demand [52,53], cytochrome c (Cyt c) loss, or inhibition of oxygen consumption. This leads to a
relatively reduced coenzyme Q (CoQ) pool and a high proton motive force ∆p [54], which then results
in oxygen reduction at complex I and hence ROS production [55]. Apparently, complex I activity can
also be impaired via the thiol switch of cysteine 39 of the ND3 subunit by nitric oxide, which shows
a cardioprotective effect in conditions of ischemia [56]. Additionally, though still under debate and
shown by in vitro conditions only, complex III of the respiratory chain has been proven as a second
important ROS-producing source primarily affecting the proteins of the OMM and intermembrane
space via oxidative modification, whereas the matrix proteins are rather targeted by ROS produced
by complex I, indicating two separate redox-signaling pathways [57–59]. However, one mechanism
of how ROS produced by the ETS can also enter the cytosol is via the voltage-dependent anion
channel (VDAC), which resides in the outer mitochondrial membrane, thus allowing the diffusion of
inner-mitochondrial superoxides into the cytosol [60]. Mitochondria also contain various members of
the Trx family [36]. Trx2, and Grx1 (intermembrane space) and Grx2 (matrix) indicate functioning redox
signaling [36,61,62]. In addition, mitochondria also contain two peroxiredoxins, i.e., peroxiredoxins 3
and 5 [36]
Mitochondria can form a long tubular network, which continually undergoes fusion and fission
events in a regulated process termed mitochondrial dynamics necessary to adapt to environmental
changes. The overall shape of the mitochondrial tubular network is determined by the balance between
fusion and fission. Mitochondrial fission events also regulate and enable mitochondrial transport
throughout the cells, especially throughout the neurons’ axons, while the fusion process, in contrast,
can spare mitochondria from mitophagy by allowing an exchange of biolipids and mitochondrial DNA.
These mitochondrial dynamics are thus vital for cellular wellbeing (reviewed in [63]).
The fusion of adjacent mitochondria can be induced by homo- or heterodimers of the GTPases
mitofusin 1 and 2 (MFN1/MFN2) located at the OMM. Fusion of the IMM, in contrast, is mediated by
the protein optical atrophy 1 (OPA1). Proteolytic processing of OPA1 is triggered by a loss of membrane
potential and results in the induction of IMM fusion [64]. This pro-fusion mechanism seems to be
controlled by cellular metabolism, as OPA1-induced fusion events are supposedly strongly linked to
OXPHOS activity [64]. Fragmentation of mitochondria is primarily mediated by the cytosolic GTPase
dynamin-related protein 1 (DRP1), which, after activation, translocates to the outer mitochondrial
membrane, oligomerizes, and encircles the mitochondrion, thereby inducing fragmentation. Crucial
for DRP1′s binding to mitochondria are other OMM proteins, including the fission 1 protein (FIS1),
mitochondrial dynamics proteins of 49 and 51 kDa (MID49/51) [65], and the mitochondrial fission
factor (MFF) [66]. Overexpression of the OMM protein ganglioside-induced differentiation-associated
protein 1 (GDAP1), which shares homology with some glutathione-S-transferases, is closely related to
mitochondrial fission, although the precise mechanism is still unclear [67–69].
We here hypothesize that the cysteine modification of proteins involved in mitochondrial dynamics
enables cellular adaptation to altered physiological conditions that involve reactive cellular species,
and review the relevant literature on this topic.
3. Evolutionary Conservation of Proteins Involved in Mitochondrial Dynamics
Important redox modification sites have been shown to be conserved over different taxa. Hence,
to identify potential sites of the aforementioned proteins, we decided to first investigate the presence
of these proteins in phylogenetically distant organisms and detect evolutionarily conserved cysteines
assuming that such sites are more likely to represent sites of physiologically relevant redox modifications.
Wherever possible, we obtained the protein sequence of these proteins from Homo sapiens, the mouse
Mus musculus, the chicken Gallus gallus, the zebrafish Danio rerio, the fruit fly Drosophila melanogaster,
and the nematode Caenorhabditis elegans using the Uniprot database (www.uniprot.org) and conducted
Cells 2020, 9, 815 5 of 18 
assuming that such sites are more likely to represent sites of physiologically relevant redox 
modifications. Wherever possible, we obtained the protein sequence of these proteins from Homo 
sapiens, the mouse Mus musculus, the chicken Gallus gallus, the zebrafish Danio rerio, the fruit fly 
Cells 2020, 9, 815 5 of 18
Drosophila melanogaster, and the nematode Caenorhabditis elegans using the Uniprot database 
(www.uniprot.org) and conducted multiple sequence alignments with Clustal Omega 
m(wuwltiwp.lcelusesqtaule.onrcge).a lOigunrm aenatslywsiist hdCemluostnasltOramteedg ath(awt wCw. .eclleugsatnasl. oarngd). DO.u rmaenlaanlyogsiasstdeer mponssetrsast eodnltyh aat 
Csi.neglelgea mnsitaonfdusDin.  m(FelZaOno1g asntedr pMoAssReFss1,o nrelyspaescitnivgelelym), iwtohfuicshin i(nF ZvOer1teabnrdatMesA dRivFe1r,gredsp ienctoiv MelyF)N, w1 hainchd 
iMnFvNer2t e(bFrigauterse d2iAve).r gDeRdPi1n taondM FINS 1 arned aMlsoF Nev2ol(uFtigiounraeri2lAy )c.oDnRsePr1veadn dinF aIlSl 1thaerseea olsrogaenviosmlusti o(Fniagruilrye 
c2oBn) swerhvieled GinDaAllPth1e isse aolrsgoa pnrisemsesn(tF iing utrhee 2fBru) iwt hfliyle bGuDt AnoPt1 iins tahlseo npermesaetnotdien gtheenofrmuiet (flFyigbuurten 2oCt )i.n Tthe 
nDeRmPa1t ordeecegpetnoorms Me (FFig uanred2 MC)I.DT4h9e/5D1R aPr1e,r eicne pctoonrtsraMstF,F eavnodluMtioIDna4r9i/l5y1 naerew,  inancdo notrnalsyt ,perveosleunttio inna rtihlye 
nvewrteabnrdatoe nlilnyepargees e(Fnitgiunrteh 2eDv)e. rtebrate lineage (Figure 2D).
 
FFiigguurree 22.. PPhhyylologgeenneetitcict rtereeseos botabitnaeindebdy bmyu mltiupllteipallieg namligennmt oefnatm oifn aomacinidos eaqciude nsceeqsufernocmesH formomo s aHpioemnso, 
Msapuisenmsu, sMcuulus sm, Gusaclululus sg,a Gllualsl,uDs agnailolurse,r iDo,aanniod rDerrioos, oapnhdil aDmroeslaonpohgilaas tmerelwanhoegnasptoers swibhlee.n Tphoessmibolset. aTbhuen mdaonstt 
iasboufonrdmasntw iseorefoursmeds wfoerrteh uesaeldig fnomr ethnet. a(lAig)nMmFeNnt2. i(sAp) rMesFeNnt2i nis apllrecsoemnpt ainre adlls cpoemciepsarwedhi lsepMecFieNs 1wwhialse 
nMoFt Nfo1u nwdaisn nDorto sfoopuhnilda ainn dDCroesloepgahnilsa. a(Bn)dD CR Pel1egaannds.F (IBS1) pDrResPe1n taenddo rFtIhSo1l opgrseisnenatleldco omrtphaorleodgssp ienc iaelsl. 
(C) GDAP1 is present in all compared species except in C elegans. (D) MFF and MID49/51 proteins were
compared species. (C) GDAP1 is present in all compared species except in C elegans. (D) MFF and 
found in Homo sapiens, Mus musculus, and Danio rerio.
MID49/51 proteins were found in Homo sapiens, Mus musculus, and Danio rerio. 
4. Redox Modifications of Proteins Involved in Mitochondrial Dynamics
4. Redox Modifications of Proteins Involved in Mitochondrial Dynamics 
4.1. Mitofusins
4.1. MMitFoNfu1sianns d MFN2 are OMM proteins that mediate mitochondrial fusion by forming homo- or
heterModFiNm1e rasn. dT hMeyFNar2e aerxep OreMssMed pmrotsetinabs uthnadta mntelydiiantet hme intoecrhvonuds rsiyasl tfeumsi,oans bwye lfloarms iingsk hino,mhoe-a ortr, 
ahnedtermoduismclertsi.s Tsuhey[7 a0r,e7 1e]x. pIrtehsasesdb emeonsst uagbguensdteadnttlhy aitnM thFeN n2ermvoedusia styestoermg,a anse lwleetlel tahse irnin sgkibne, thweeaernt, 
manidto cmhuonscdleri atiisnsutrea [n7s0o,7r1b].e tIwt heeans bmeietonc shuogngdersiateadn tdhtaht eMEFRNw2h mil edMiaFtNes1 ofargcialnitealtlees ttehtehefurisniogn bpertwoceesns 
tmogiteotchheornwdirtiha OinP tAra1nast othr ebeintwnerenm memitborcahnoen[d7r2ia,7 a3n].dA tfhte rEGRT wPhhilyed MroFlyNz1a tfiaocnil,iMtatFeNs 1thaep fpuasrieont plyrolocseess 
ittosgaeffithneirt ywtiothi tOs cPoAu1n atetr tphaer itnbnuetrh masemgrberaatenre G[7T2P,7t3u]r. nAofvter GacTtPiv hitydwrohliylezaMtiFoNn,2 MpFroNte1i nasppstailrlernetmlya lionsaes 
ditism aeffrisn, imtya tkoi nitgs McoFuNnt2erapmarotr beuetff heactsi vgeretaetehre GrinTgP mtuorlneocvueler [a7c2t]iv. ity while MFN2 proteins still remain 
as diMmeFrNs,1 mcaokninsigs tMs FoNf 27 4a1 maonrde eMffFecNti2veo tfet7h5e7rianmg imnoleacuidles ;[7b2o].t h proteins share 82% structural
similMariFtNie1s [c7o4n]saisntds coof n7t4a1in aannd NM-tFeNrm2 inoaf l 7G5T7 Pa-mdoinmoa iancifdosl;l obwoethd bpyroatecinosil esdh-acroei l8h2e%p tastdr-urcetpueratl 
dsiommilaairnit(iHesD [17)4a] nadndtw cootnrtaaninsm aenm Nbr-atenremdionmal aGinTsP(-TdMomDsa)inse fpoalrloawtededb ybyo nae caomiliendo-caocild h(Fepigtuadre-r3eAp,eBa)t. 
O nly MFN2 harbors a proline-rich domain between the TMD and the HD1 domain, which is most
likely involved in protein–protein interactions [75,76]. The C-terminus of both mitofusins contains a
second HR2 domain, which seems to be located in the intermembrane space (IMS) [77] (Figure 3A).
Cells 2020, 9, 815 6 of 18 
domain (HD1) and two transmembrane domains (TMDs) separated by one amino acid (Figure 3A,B). 
Only MFN2 harbors a proline-rich domain between the TMD and the HD1 domain, which is most 
Celllsi2k0e2l0y, 9in, 8v1o5lved in protein–protein interactions [75,76]. The C-terminus of both mitofusins contain6so af 18
second HR2 domain, which seems to be located in the intermembrane space (IMS) [77] (Figure 3A). 
 
FigFuirgeu3re.  S3.c hScehmeme iel liullsutsrtartaintigngt htheer erelalatitviveep poossiittiioonn ooff ccoonnsseerrvveedd ccyysstteeinineess wwitihthinin rerceocgonginziazbalbe lperporteoitne in
domdoaminasinosf otfh ethien dinicdaicteadtedp rportoetienisn.s. CCoolloorrss aarree uusseedd ttoo hhiigghhlliigghhtt pprrootteeinin ddoommaainins,s ,eveovloultuiotinoanrialryi ly
concsoenrsveerdvecdy sctyesitneiense,sa, nadndc ycsytsetieninesesw witihtha ad deemmoonnssttrraatteedd ffuunnccttiioonn.. (A(A) )MMFFNN2 2hahsa sfivfiev ecocnosnesrevrevde d
cysctyesinteeisneosu otuotf owf hwichhichth teheC 6C86484h ahsasa ap prervevioiouusslylyd deemmoonnssttrraatteedd rreeddooxx--iinndduuccibiblel efufunnctcitoino.n (.B()B M) MFNF1N 1
conctoanintasinfos uforucro cnosnesrevrevdedcy csytsetieniensesb buut ty yeettw witithhoouutt aannyy oobbsseerrvveedd rreeddooxx ffuunncctitoionn. .(C(C) )DDRRP1P 1popsossessesesss es
 four conserved cysteines, whereas a fifth not fully conserved cysteine (C644) can be redox modified.
(D) FIS1 and (E) MFF do not have any conserved cysteines along their structure. (F) MID51 has six
fully conserved cysteines, including C452, regulating the protein’s dimerization. (G) MID49 has two
conserved cysteines (H) GDAP1 has no fully conserved cysteines.
Cells 2020, 9, 815 7 of 18
As mitofusins are crucial for the mitochondrial fusion process, mitofusin turnover is a highly
regulated process. Mitofusins are ubiquitinated by parkin (PARK2) [78,79] during mitochondrial
quality control, where PARK2 translocates to the mitochondria to orchestrate their selective elimination
through mitophagy [80]. Ubiquitination of mitofusins by E3 ligases like MGRN1 and Gp78 not
only controls mitochondrial turnover via mitophagy but also affects mitochondrial morphology and
mitochondria-ER-contact sites (MERCS), which constitute important intracellular signaling hubs [81–83].
By treating primary neurons with the NO-donor A-nitrosocysteine (SNOC), Barsoum et al. observed
that concomitant MFN1 expression prevented NO-induced mitochondrial fragmentation and even
NO-induced cell death [84]. This may emphasize the importance of mitochondrial fusion’s ability
due to mitofusin expression in order to cope with nitrosative stress. The process of mitochondrial
hyperfusion, a cellular stress response induced by changes in the intracellular redox state, involves
both mitofusins [85]. In line, oxidized glutathione (GSSG) addition, the core cellular stress indicator, to
mitochondrial preparations stimulated mitochondrial fusion by inducing disulphide bond-mediated
oligomer formation of MFN1 and cysteine 684 of MFN2 [85]. This points towards a regulation of
redox-regulated mitochondrial hyperfusion into the IMS. Here, cysteines trigger dimerization of the
mitofusins through disulfide bonds to drive mitochondrial fusion [77]. Accordingly, work from our
laboratory demonstrated that this cysteine is involved in sensing the redox environment in the IMS [86].
In addition to the abovementioned conserved cysteine 684, both MFN1 and MFN2 contain four
and five highly conserved cysteines, respectively. Three are located in the GTPase domain while the
fourth one is located next to the HR1 domain in both mitofusins (Figure 3A,B and complete alignment
in Supplementary Materials). The role of these evolutionarily conserved cysteines located in the
cytosol remains unclear with regard to their function as potential PTMs in response to changes in the
redox milieu.
4.2. DRP1
DRP1 is the major player in the mitochondrial fission process. The protein consists of an N-terminal
GTPase and a C-terminal GTPase effector domain (GED) separated by a helical segment of amino
acids [87,88]. In humans, six DRP1 isoforms are generated by alternative splicing. Isoform 1 consists of
736 amino acids and is expressed exclusively in the brain (Figure 3C). The isoforms 2 (710 amino acids)
and 3 (699 amino acids) are predominantly expressed in the testis and skeletal muscle, respectively.
DRP1 isoform 4 (725 amino acids) is weakly expressed in the brain, heart, and kidney. The isoform 5
(710 amino acids) occurs predominantly in the liver, heart, and kidney, while isoform 6 (749 amino
acids) is expressed in neurons [89–93].
DRP1 exists in the cytoplasm in an equilibrium between dimeric and tetrameric forms [94]. Upon
stimulation, DRP1 is recruited to mitochondria and promotes fission via interaction with its receptors
FIS1, MFF, and MID49/51 located at the OMM, which allows DRP1 to form ring-like oligomers that
constrict and divide the mitochondria, a process accompanied by GTP hydrolysis [95–97]. The ability
of DRP1 to promote mitochondrial fission can be regulated by phosphorylation, ubiquitination, and
SUMOylation [98].
Most importantly in the redox context, DRP1 can be regulated by S-nitrosylation of cysteine
644 (C644; position in the human protein), which is conserved from flies to humans (Figure 3C).
Cho et al. showed that an NO increase provoked by amyloid-β peptide induces DRP1 S-nitrosylation
at C644, leading to subsequent DRP1 dimerization and increased GTPase activity, and resulting in more
fragmented mitochondria [99]. Furthermore, postmortem brain samples from Alzheimer’s disease
(AD) patients presented higher levels of S-nitrosylated DRP1 when compared to control samples or
samples from patients suffering from Parkinson’s disease (PD) [99]. In contrast, Bossy et al. suggested
that DRP1 S-nitrosylation does not increase its GTPase activity, and that the levels of S-nitrosylated
DRP1 are not changed when comparing postmortem brain samples from control, AD, and PD
patients. Instead, they suggested that NO production stimulates DRP1 activity via serine 616 (S616)
phosphorylation [100]. Nakamura and Lipton counterargued that Bossy et al. used recombinant DRP1
Cells 2020, 9, 815 8 of 18
for their experiments, which was already oxidized, preventing its S-nitrosylation [101]. Additionally,
it was shown that DRP1 can be trans-S-nitrosylated by cyclin-dependent kinase 5 (CDK5) in AD
models [102], and that an increase in S-nitrosylated DRP1 induced by decreased S-nitrosoglutathione
reductase (GSNOR) expression can occur in primary cells undergoing senescence [103]. A recent study
demonstrated that the protein disulfide isomerase A1 (PDIA1) acts as a thiol reductase for DRP1. In
neurodegenerative and cardiovascular diseases, PDIA1 deficiency results in increased S-nitrosylated
DRP1, mitochondrial fragmentation, and endothelial senescence [104]. DRP1 phosphorylation
was also associated with changes in the redox milieu. Zhou et al. showed that c-Abl-induced
DRP1 phosphorylation triggers mitochondrial fragmentation upon H2O2 treatment in primary
neurons [105], while Lee and Kim suggested that PDI-mediated DRP1 S-nitrosylation facilitates
DRP1 S616 phosphorylation in hippocampal neurons [106]. Additionally, Tsushima et al. showed
increased DRP1 S616 phosphorylation in response to lipid overload-induced oxidative stress in cardiac
myocytes [107].
Besides C644, DRP1 contains four additional evolutionarily conserved cysteine residues with
yet unknown functions, one at the end of the GTPase domain and three located in the middle region
that connects the GTPase domain with the effector domain (Figure 3C and complete alignment in
Supplementary Materials).
4.3. FIS1
FIS1 was the first protein shown to interact with DRP1 and to allow mitochondrial fission in
the budding yeast Saccharomyces cerevisiae [108]. In contrast, FIS1 does not seem to be essential for
mitochondrial fission in mammalian cells [109,110] and shows low tissue specificity [111]. FIS1 is
a 17-kDa protein with a C-terminal OMM anchor and two cytosolic tetratricopeptide repeat (TPR)
motifs (Figure 3D) [112]. A concave surface formed by the residues in the TPR motifs is believed to
be the interacting region for DRP1 [113] while the N-terminal arm controls DRP1 access to the TPR
motifs [114]. However, an understanding of how the position of the N-terminal arm is regulated is
still lacking.
Most of the reported FIS1 regulation is associated with the regulation of its protein abundance
in response to cellular stress. Glutamate-challenged HT22 cells have an upregulated FIS1
(and phosphorylated DRP1) and have a more fragmented mitochondrial shape [115]. Zhang et al.
reported increased FIS1 protein levels in mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP; a precursor of the mitochondrial complex I inhibitor 1-methyl-4-phenylpyridinium (MPP+), a
mouse model of PD, while overexpression of DJ-1 (a protein mutated in familiar PD) repressed FIS1
upregulation via RING-finger protein-5-mediated FIS1 ubiquitination and degradation [116]. In line
with this, 6-hydroxydopamine (6-OHDA; another PD model) increased FIS1 (and DRP1) protein levels
in PC12 cells [117], while H2O2 induced an increase in FIS1 protein levels in SH-SY5Y cells [118]. In
summary, although FIS1 does not seem to be directly regulated via redox mechanisms, it seems to
respond to the redox milieu via upregulation or decreased degradation. In line with this, FIS1 contains
only a single cysteine, which is also not evolutionarily conserved (Figure 3D and complete alignment
in Supplementary Materials). We therefore believe that a direct redox-mediated regulation of FIS1 by
cysteine modification is unlikely.
4.4. MFF
The protein mitochondrial fission factor (MFF) was first described in a Drosophila cell line screen
for proteins triggering mitochondrial morphology alterations. MFF knockdown caused elongated
mitochondria similar to what was observed for DRP1 and FIS1 knockdown [119]. MFF contains 342
amino acids and is one of three DRP1 receptors located in the OMM facing the cytosol [119]. This
protein contains two consecutive cytosolic motifs that are necessary for DRP1 recruitment [109] and a
third motif of unknown function. MFF dimerization depends on a well-preserved coiled-coil domain,
Cells 2020, 9, 815 9 of 18
which precedes the C-terminal transmembrane (TM) domain (Figure 3E) [119]. MFF is broadly present
in different human tissues, such as the heart, kidney, liver, brain, muscle, and stomach [119].
A PTM predictive model identified multiple serine and threonine phosphorylation sites that
are potential targets for post-translational modification [120]. Mutating MFF serine 155 (S155)
and S172 residues precludes mitochondrial fission promoted by AMP-activated protein kinase
(AMPK) [121], a well-known intracellular energy sensor that phosphorylates multiple downstream
effectors participating in cellular homeostasis [122]. These serine residues are located between the
DRP1-interacting region at the N-terminus and the C-terminal mitochondrial TM domain. Additionally,
S129 and S146 have been also identified as AMPK targets in hepatocytes using proteomics [123].
Interestingly, similar to MFN2, MFF has been identified as a PARK2 target for ubiquitination. Four
glycine residues (G28, G88, G251, and G264) were identified within four different ubiquitin-interacting
motifs [124]. Eliminating PARK2 was not sufficient to preclude MFF ubiquitination, suggesting that
other proteins may play a role in MFF proteostasis [125].
MFF only contains one cysteine residue (human C209), which is located after the DRP1-interacting
region but before the TM domain (Figure 3E). This residue is not conserved as it is absent in G. gallus
(complete alignment in Supplementary Materials). In line with this, an unbiased proteomic approach,
where cysteine reactivity within mitochondrial proteins was monitored by labelling cysteine-reactive
residues with chemical probes followed by mass spectrometry, also failed to identify functional cysteine
residues in MFF [126]. These data imply that the single and not fully conserved cysteine residue in
MFF probably has no relevant function.
4.5. MID49/51
The MID49/51 proteins were first described in a large-scale analysis as mitochondrial proteins [127],
and later redefined as novel elements of the mitochondrial fission protein group. The overexpression
of MID49/51 proteins sequestered DRP1 from mitochondria and triggered an elongated mitochondrial
network whereas abolishing their expression caused a fragmented mitochondrial pattern [65]. MID51
contains 463 amino acids while MID49 is slightly smaller with 454 amino acids (Figure 3F,G). MID49/51
are encoded by the mitochondrial elongation factor 1 (MIEF1) and MIEF2 genes, respectively [128].
Both proteins are located in the OMM, with most of the protein present in the cytosol to regulate
DRP1-mediated mitochondrial fission. The highly conserved residues within the TMD, amino acids
26-47 of MID49, and 24-46 of MID51 are necessary for mitochondrial anchoring but are not required
for interaction with DRP1 [129]. Following the TMD, an N-terminal disordered region with an
absent secondary structure has been described in the crystal structure of these proteins [130]. Both
proteins contain nucleotidyl transferase (NT) domains without enzymatic activity but a still preserved
ADP-binding capacity for MID51 (Figure 3F,G) [130,131]. Although DRP1 recruitment by MID51
is ADP independent, ADP binding to MID51 facilitates its dimerization, which in turn stimulates
DRP1 oligomerization [131]. MID49/51 also share similar α-helices and β-strands at the N-terminal
position, which precedes a linker before the C-terminal containing five α helices [130]. Although both
proteins share α-helices and β-sheets from the N- to C-terminus, their oligomerization domains are
different [131]. While MID51 appears to be dimeric, MID49 adopts a monomeric structure. It has been
proposed that MID49 weakly dimerizes and that this was not apparent in the crystal structure due to
technical issues [130].
MID49/51 proteins contain DRP1 recruitment sites [132,133] and their interaction with DRP1
is independent of MFF or FIS1 [134]. The interaction of DRP1 with MID51 is GTP dependent and
MID51 dimerization is necessary for mitochondrial morphology control [135]. Indeed, these differences
between both MID49/51 proteins could explain different roles in mitochondrial fission.
MID49/51 proteins were not found in a proteomic screen searching for conserved functional
cysteines in pure mitochondrial fractions [126]. However, MID51 can dimerize under non-reducing
conditions [136]. This dimerization can be abolished by the mutation of C452 [136]. Five additional
conserved cysteines distributed through the NT domain, DRP1 recruitment site, and the C-terminus
Cells 2020, 9, 815 10 of 18
were identified within MID51 (Figure 3F and complete alignment in Supplementary Materials), but
no function has been defined for these until now. MID49 contains two conserved cysteine residues
located within the DRP1 recruitment domain and another one close to the C-terminus (Figure 3G),
which also do not have any demonstrated function so far.
4.6. GDAP1
GDAP1 [137] is a 41.5 kDa protein, which causes the hereditary polyneuropathy Charcot–Marie–
Tooth disease 4A when mutated on both alleles [138,139]. It is anchored in the outer mitochondrial
and peroxisomal membranes and mainly expressed in neurons and less in Schwann cells [140,141].
Overexpression of GDAP1 causes a more fragmented mitochondrial shape while its knockdown
results in more elongated mitochondria [140]. This fission-like activity can be counterbalanced by
fusion-promoting factors like mitofusins or dominant-negative DRP1 [140]. GDAP1 is anchored in the
OMM with a C-terminal transmembrane domain (TMD) and faces the cytosol (Figure 3H). It has two
cytosolic domains that share similarities to glutathione-S-transferases (GSTs; Figure 3H) [140]. The
C-terminal hydrophobic domain (HD) of GDAP1 presumably resides in the cytosol. Truncated GDAP1
lacking the TMD has no GST-like activity [141,142], but recently, a theta-class-like GST activity was
described for GDAP1. Huber et al. proposed a model where the HD domain of GDAP1 is able to
regulate the GST activity of the protein. In an inactive state, the HD domain blocks the GST activity
while in the active state, the amphipathic pattern of HD would trigger membrane curvature and
mitochondrial fission [68]. In this model, dominant GDAP1 mutations, which cause a hereditary
polyneuropathy, would adopt a super-active conformation resulting in toxic hyper-fission activity
while recessive GDAP1 mutations with reduced fission activity would adopt the inactive state [68].
Furthermore, Noack et al. observed an increase of GSH in response to overexpression of wildtype but
not mutant GDAP1 [143]. In line with this, Drosophila melanogaster studies demonstrated that not only
overexpression of GDAP1 wildtype but also knockdown results in both a higher GSH concentration
and higher GSH to GSSG ratio in fly thoraxes [144]. Additional studies are required to confirm the
Drosophila observations since muscle tissue is different from neurons, where GDAP1 seems to be
more important. Structurally, Drosophila GDAP1 shares similarities with the human GDAP1 protein,
especially in respect to the GST domains. In fact, human GDAP1 overexpression was able to rescue the
alterations caused by Gdap1 interference in flies [144]. This reinforces the functional similarity between
the species. Niemann et al. described a disturbed GSH/GSSG balance towards increased oxidized
glutathione in a mouse model with a deletion of exon 5 in the Gdap1 gene, leading to a truncated protein
that lacks the TMD anchoring in the OMM [145]. These mice developed a mild hypomyelinating
peripheral neuropathy, and most importantly, the lack of phenotype in the central nervous system was
attributed to a compensatory function mediated by the paralogue GDAP1L1 [145], which shares a 70%
sequence similarity with GDAP1 [142], including the GST domains and the C-terminal transmembrane
domain [146]. GDAP1L1 expression is restricted to the brain and testes (www.proteinatlas.org) and, in
contrast to GDAP1, resides in the cytosol under basal conditions but translocates to mitochondria upon
increased levels of oxidized glutathione, where it is also able to induce mitochondrial fission [145].
Although all four cysteines in the human GDAP1 protein (respectively five in GDAP1L1) are
only conserved in vertebrates but not in Drosophila melanogaster (Figure 3H and complete alignment
in Supplementary Materials), potential cysteine modifications and their impact on GDAP1 function
warrant further investigation. No pathogenic mutations are associated with GDAP1L1. However,
its ability to translocate from the cytosol to mitochondria under altered redox conditions suggests a
possible role in cellular homeostasis through redox-mediated modifications.
5. Conclusions
Our analysis suggests that the evolutionarily oldest proteins of the mitochondrial fusion and
fission machinery, the mitofusins and DRP1 but not FIS1, are subject to redox modifications functionally
relevant for mitochondrial dynamics and potentially also for mitochondrial quality control. The lack of
Cells 2020, 9, 815 11 of 18
evolutionary conservation rendered the analysis of other proteins relevant for mitochondrial dynamics
more difficult. Overall, however, conserved cysteine residues are present in almost all the proteins of
the mitochondrial fission/fusion machinery, offering new avenues for future investigation.
Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4409/9/4/815/s1,
Protein alignments performed with CLUSTAL O (1.2.4).
Author Contributions: Conceptualization, A.M.; methodology, D.B.; writing—original draft preparation, C.W.,
V.L.d.A., S.A., D.B., S.T., E.-M.H., C.B.; writing—review and editing, A.M.; visualization, D.B., V.L.d.A.; supervision,
A.M. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Deutsche Forschungsgemeinschaft grant numbers TE599/5-1,
HA8334/2-2, BE3259/5-2, and ME1922/16-1 within the priority program SPP 1710 “Dynamics of thiol-based
redox switches in cellular physiology”.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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