Real-time detection of PRT1-mediated ubiquitination via ... fileMot et al. Fluorescent N-end rule...

Motetal. FluorescentN-endrulesubstrates

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Real-timedetectionofPRT1-mediatedubiquitinationviafluo-1

rescentlylabeledsubstrateprobes2

3

AugustinC.Mot,1,2ErikPrell,3MariaKlecker,1,2ChristinNaumann,1,2Frederik4

Faden,1,2BernhardWestermann3&NicoDissmeyer1,2,*5

6

71 Independent JuniorResearchGroup onProteinRecognition andDegradation, Leibniz Institute of PlantBiochemistry8(IPB),Weinberg3,D-06120Halle(Saale),Germany92ScienceCampusHalle–Plant-basedBioeconomy,Betty-Heimann-Str.3,D-06120Halle(Saale),Germany103DepartmentofBioorganicChemistry,LeibnizInstituteofPlantBiochemistry(IPB),Weinberg3,D-06120Halle(Saale),11Germany1213* Correspondence should be addressed to N.D. (phone: +49 176 2355 8710; [email protected], Twitter:14@NDissmeyer).15

16

RUNNINGTITLE17

LiveassayswithfluorescentN-endrulesubstrates18

19

Totalwordcount20

mainbody(Introduction:1639,MaterialsandMethods:2081,Results:1174,Discussion:21

2053,Acknowledgements:204)22

23

Numberoffigures/tables/supportinginformation24

3figures(allcolor),notables,1SupportingFigure,2SupportingTables,1Supporting25

Methods 26

.CC-BY-NC 4.0 International licensepeer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not. http://dx.doi.org/10.1101/062067doi: bioRxiv preprint first posted online Sep. 3, 2016;

http://dx.doi.org/10.1101/062067

http://creativecommons.org/licenses/by-nc/4.0/


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SUMMARY27

28

• TheN-endrulepathwayhasemergedasamajorsystemforregulatingprotein29

functionsbycontrollingtheirturn-overinmedical,animalandplantsciences30

aswellasagriculture.Althoughnovel functionsandenzymesofthepathway31

werediscovered,ubiquitinationmechanismandsubstratespecificityofN-end32

rulepathwayE3Ubiquitin ligases remained elusive. Taking the first discov-33

eredbona fideplantN-endruleE3 ligasePROTEOLYSIS1(PRT1)asamodel,34

weuseanoveltooltomolecularlycharacterizepolyubiquitinationlive,inre-35

al-time.36

• Wegainedmechanistic insights inPRT1substratepreferenceandactivation37

bymonitoringliveubiquitinationbyusingafluorescentchemicalprobecou-38

pled to artificial substrate reporters. Ubiquitinationwasmeasuredby rapid39

in-gel fluorescencescanningaswellas inrealtimebyfluorescencepolariza-40

tion.41

• Enzymatic activity, substrate specificity,mechanisms and reaction optimiza-42

tionofPRT1-mediatedubiquitinationwereinvestigatedadhoc inshorttime43

andwithsignificantlyreducedreagentconsumption.44

• Wedemonstrated for the first time that PRT1 is indeed an E3 ligase,which45

washypothesizedforovertwodecades.TheseresultsdemonstratethatPRT146

has the potential to be involved in polyubiquitination of various substrates47

andthereforepavethewaytounderstandingrecentlydiscoveredphenotypes48

ofprt1mutants.49

50

51

52

53

KEYWORDS54

ubiquitination,proteolysis,E3ligases,activityprofiling,fluorescentdyes,labelingchemis-55

try,proteinlabeling,N-endrulepathway56


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INTRODUCTION57

The ON/OFF status of protein function within the cells’ proteome, their general58

abundance and specific distribution throughout the compartments and therefore their59

functions and activities are precisely controlled by protein quality control (PQC)mecha-60

nismstoensureproperlifeofanyorganism.Therefore,thebiochemicalanalysisoftheun-61

derlyingmechanisms safeguardingproteostatic control ispivotal. It ranges from themo-62

lecular characterization of enzymes involved in PQC and their catalyzed reactions to en-63

zyme-substrate and non-substrate protein-protein interactions. The so-called Ubiquitin64

(Ub)26Sproteasomesystem(UPS) isamastercomponentofPQCwiththekeyelements65

beingnon-catalyticUbligases(E3),theUb-conjugatingenzymes(E2),andtheUb-activating66

enzymes(E1).67

Toinvestigateanelementconferringsubstratespecificity,wechosePROTEOLYSIS168

(PRT1)fromArabidopsisthalianaasamodelE3ligase,whichisabonafidesingle-subunit69

E3withunknownsubstrateportfolio(Bachmairetal.,1993;Potuschaketal.,1998;Staryet70

al.,2003).Itsbiologicalfunctionremainselusivebutitpresumablyrepresentsahighlyspe-71

cificenzymewithE3ligasefunctionoftheN-endrulepathwayoftargetedproteindegrada-72

tion,which is apartof theUPS.TheN-endrule relates thehalf-lifeof aprotein to itsN-73

terminalaminoacid(Bachmairetal.,1986)andcausesrapidproteolysisofproteinsbear-74

ingso-calledN-degrons,N-terminalsequencesthatleadtothedegradationoftheprotein.75

N-degrons are created by endoproteolytic cleavage of protein precursors (pro-proteins)76

andrepresenttheresultingneo-N-terminioftheremainingC-terminalproteinmoiety,al-77

beitnotallfreshlyformedN-terminiautomaticallypresentdestabilizingresidues(Fig.1a).78

TheN-endrulepathwayisanemergingvibrantareaofresearchandhasamultitude79

of functions in all kingdoms (Dougan et al., 2010; Varshavsky, 2011; Tasaki et al., 2012;80

Gibbs et al., 2014a; Gibbs, 2015). Identified substrates are mainly important regulatory81

proteinsandplaykeyrolesinanimalandhumanhealth(Zenkeretal.,2005;Piatkovetal.,82

2012;Brower etal.,2013;Shemorry etal.,2013;Kim etal.,2014),plantstressresponse83

and agriculture (Gibbs et al., 2011; Licausi et al., 2011; Gibbs et al., 2014a; Gibbs et al.,84

2014b;Weitsetal.,2014;deMarchietal.,2016;Mendiondoetal.,2016).85

86


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Inplants,functionsofN-endruleenzymesareassociatedwithcentraldevelopmen-87

talprocessesincludingseedripeningandlipidbreakdown,hormonalsignalingofabscisic88

acid(ABA),gibberellinandethylene,seeddormancyandgermination(Holmanetal.,2009;89

Abbasetal.,2015;Gibbsetal.,2015),leafandshootmorphogenesis,flowerinduction,and90

apicaldominance(Gracietetal.,2009),andthecontrolof leafsenescence(Yoshidaetal.,91

2002).Then,thepathwaywasshowntobeasensorformolecularoxygenandreactiveox-92

ygenspecies(ROS)bymediatingnitricoxide(NO)signalingandregulatingstressresponse93

afterhypoxia,e.g.after floodingandplantsubmergence(Gibbs etal.,2011;Licausi etal.,94

2011;Gibbsetal.,2014b).Anovelplant-specificclassofenzymeswasassociatedwiththe95

pathway, i.e.plantcysteineoxidases(PCOs),highlightingplant-specificmolecularcircuits,96

enzymeclassesandmechanisms(Weitsetal.,2014).InthemossPhyscomitrellapatens,N-97

endrulemutantsaredefectiveingametophyticdevelopment(Schuesseleetal.,2016)and98

protein targets of N-end rule-mediated posttranslational modifications were discovered99

(Hoernstein etal.,2016).Also inbarley, thepathway isconnectedwithdevelopmentand100

stress responses (Mendiondo etal., 2016).Onlyvery recently, a linkbetweenN-endrule101

function and plant-pathogen response and innate immunitywas found (deMarchi et al.,102

2016),sheddinglightonnovelfunctionsoftheyetunderexploredbranchoftargetedpro-103

teolysis.However,todate,theidentityofplantN-endruletargetsstillremainsobscureand104

clear evidences frombiochemical data of in vitro and in vivo studies such asN-terminal105

sub-proteomicsorenzymaticassaysarestilllacking.106

Anovelinvivoproteinstabilizationtoolforgeneticstudiesindevelopmentalbiology107

andbiotechnologicalapplications, the 'lt-degron',works inplantsandanimalsbydirectly108

switchingthelevelsoffunctionalproteinsinvivo(Fadenetal.,2016).Themethodisbased109

on conditional and specific PRT1-mediated protein degradation, the process studied in110

depthwiththehere-generatedfluorescentsubstratereporters.111

N-degronsarebydefinitionrecognizedandthecorrespondingproteinubiquitinated112

byspecializedN-endruleE3ligases,so-calledN-recognins(Srirametal.,2011;Varshavsky,113

2011;Tasakietal.,2012;Gibbs,2015).Inplants,onlytwoofthese,namelyPRT1andPRT6,114

areassociatedwiththeN-endruleandassumedtofunctionasN-recognins(Fig.1b).This115

isincontrasttothehighnumberofproteolyticallyprocessedproteinswhichcarryintheir116

matureformN-terminalaminoacidsthatcouldpotentiallyentertheenzymaticN-endrule117


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pathwaycascade(Venneetal.,2015). Inthelightofmorethan800putativeproteasesin118

themodelplantArabidopsisthaliana, it is likelythattheN-endrulepathwayplaysanim-119

portantroleforproteinhalf-lives inaproteome-widemanner.Examplesarefoundinthe120

METACASPASE9degradome,i.e.thatpartoftheproteomewhichisassociatedwithdegra-121

dation(Tsiatsianietal.,2013),ortheN-degradomeofE.coli(Humbardetal.,2013)witha122

possiblyanalogousoverlapwithendosymbioticplantorganelles(Apeletal.,2010).123

PRT1,comparedtotheSaccharomycescerevisiaeN-recogninUbr1(225kDa),isa124

relativelysmallprotein(46kDa)andtotallyunrelatedtoanyknowneukaryoticN-125

recogninsbutwithfunctionalsimilaritiestoprokaryotichomologs(Fig.1b).Itistherefore126

perceivedasaplantpioneerE3ligasewithbothdiversifiedmechanisticsandfunction.Arti-127

ficialsubstratereportersbasedonmousedihydrofolatereductase(DHFR)comprisingan128

N-terminalphenylalaninegeneratedviatheubiquitin-fusion(UFT)techniqueleadtothe129

isolationofaprt1mutantinaforwardmutagenesisscreen(Bachmairetal.,1993).Inthe130

mutantcellsandafterMG132treatment,theF-DHFRreporterconstructwasshowntobe131

stabilizedwhereasitwasinstableintheuntreatedwildtype(Potuschaketal.,1998;Stary132

etal.,2003).PRT1wasabletoheterologouslycomplementaSaccharomycescerevisiae133

ubr1ΔmutantstrainwherePhe-,Tyr-,andTrp-initiatedβ-galactosidasetestproteinswere134

stabilized.Thesereporterswererapidlydegradedinubr1ΔtransformedwithPRT1(Stary135

etal.,2003).AnewstudyrevealedthatcleavageoftheE3ligaseBIGBROTHERbyprotease136

DA1formsaC-terminal,Tyr-initiatedfragment.ItsstabilitydependsontheN-terminal137

aminoacidTyrandthefunctionofPRT1E3ligase(Dongetal.,2016).However,untiltoday,138

therearenomoreinvivotargetsordirectfunctionsassociatedwithPRT1,butrecently,a139

potentialroleofPRT1inplantinnateimmunitywasflagged(deMarchietal.,2016).140

The spectrumofN-termini possibly recognizedbyplantN-end ruleE3 ligases in-141

cludingPRT1isnotsufficientlyexplored.OnlyPhe-startingtestsubstrateswerefoundto142

bestabilized inaprt1mutantwhereas initiationbyArgandLeustill causeddegradation143

(Potuschaketal.,1998;Staryetal.,2003;Garzónetal.,2007).Inthelightofsubstrateiden-144

tification, it is cardinal to determine PRT1 mechanistics in more detail because several145

posttranslationallyprocessedproteinsbearingPhe,TrpandTyrattheneo-N-terminiwere146

found(Tsiatsianietal.,2013;Venneetal.,2015)andhencerepresentputativePRT1tar-147


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getsaltogether.ElucidatingthesubstratespecificityofPRT1willbeanimportantstepfor-148

wardtowardssubstrate identificationandassociationofPRT1andtheN-endrulewitha149

biologicalcontext.150

Weestablished a technique that allows real timemeasurements of ubiquitination151

using fluorescencescanningofSDS-PAGEgelsand fluorescencepolarization.Wepropose152

itsuseasagenerictoolformechanisticandenzymologicalcharacterizationofE3ligasesas153

mastercomponentsoftheUPSdirectingsubstratespecificity.Withaseriesofartificialtest154

substratescomprisingvariousbonafidedestabilizingN-endruleN-termini,substratespec-155

ificitywasanalyzedandrevealedPRT1preferenceforPheasarepresentativeofthebulky156

hydrophobicclassofaminoacids.Themethodscommonlyusedtoassayinvitroubiquitina-157

tionarebasedonend-timemethodswhere the reaction is stoppedat agiven timepoint158

and analyzed by SDS-PAGE followed by immunostaining with anti-Ub versus anti-target159

specific antibodies. This detection via western blot often gives rise to the characteristic160

hallmarkof polyubiquitinatedproteins, a "ubiquitination smear" or amoredistinct "lad-161

dering"oftheposttranslationallyUb-modifiedtargetproteins.Alltheinformationofwhat162

occurredduringthetimeofreactionisunknownunlesstheassayisrunatseveraldifferent163

timepointswhichdrasticallyincreasesbothexperimentaltimeandreagentconsumption.164

Besidesthemostcommonmethodsusedforubiquitinationassessmentthatinvolveimmu-165

nodetectionwithanti-Ubandanti-targetantibodies,therearefewotherapproachesmak-166

inguseofdifferentreagents.Comparablemethods,theiradvantagesanddisadvantagesare167

listedinSupportingInformationTableS1.Thenoveltyofferedbythepresentstudyisthe168

development of a fluorescence-based assay that allows real-timemeasurement ofUb in-169

corporationinbulkysolutioneliminatingshortcomingsoftheexistingmethodsandthusa170

morerealmechanisticinvestigation.Ourmethodmonitorstheubiquitinationprocesslive,171

inreal time,using fluorescently labeledsubstrateproteinsand fluorescence-baseddetec-172

tionassays,namelyfluorescencepolarization(FP).Inaddition,theprotocolwascoupledto173

fastandconvenientscanningfluorescencein-geldetection.Thistypeofassaycanbeeasily174

adapted for high-throughput measurements of ubiquitination activity and probably also175

similarproteinmodificationprocessesinvolvingchangesinsubstratemoleculeproperties176

overtime invitro.Ratherthanmerelyanalyzingenzyme–substrateorprotein–proteinin-177

teractions,theheredescribedmethodforthefirsttimeemploysFPmeasurementsforthe178


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characterizationofenzymeactivityandparametersaffectingtheperformanceoftheubiq-179

uitinationreaction(Xiaetal.,2008;Kumaretal.,2011;Smithetal.,2013).180

Here,wereportanoveladvancedapproachtomolecularlycharacterizeE3ligases,181

tomeasureandtrackpolyubiquitinationliveandinatime-resolvedmanner.Ithasthepo-182

tentialtogiverisetoprofoundimplicationsonourunderstandingoftheinteractionsofE3183

ligaseswith substrates and cofactors (non-substrates) and can impact ubiquitination re-184

searchingeneralasourworksuggeststobetransferabletootherE3ligasesandenzyme-185

substratepairs.Themethodreliesonrapid,easyandcheapprotocolswhicharecurrently186

lacking for in-depth biochemical analysis of E3 ligases and is at the same time non-187

radioactive, sterically not interfering, andworkswith entire proteins in form of directly188

labeledsubstrates.189

Sofar,onlythreereportsmentionworkonPRT1atall,i.e.thetwofirstbriefdescriptions190

(Potuschaketal.,1998;Staryetal.,2003)andonehighlightingtheroleoftheN-endrule191

pathway,inparticularanovelfunctionforPRT1,inplantimmunity(deMarchietal.,2016).192

However, the community lacksproofsdemonstrating thatPRT1andotherE3candidates193

are indeed involved in substrateproteinubiquitination. Todate, ubiquitinationactivities194

ofE3ligasecandidatesfromtheplantN-endrulepathwaywereonlyspeculated.195

196

197

MATERIALSANDMETHODS198

Cloningandexpressionofrecombinantproteins199

ArtificialN-endrulesubstrates200

Escherichia coli flavodoxin (Flv,uniprot ID J7QH18) coding sequencewas cloneddirectly201

from E. coli DNA BL21(DE3) and flanked by an N-terminal triple hemagglutinin (HAT)202

epitope sequence using the primers Flv_rvs (5‘-TTATTTGAGTAAATTAATCCACGATCC-3‘)203

and Flv_eK_HAT(oh)_fwd (5‘-CTGGTGCTGCAGATATCACTCTTATCAGCGG-3‘). The X-eK se-204

quencescomprisingcodonsforvariousN-terminalaminoacidsexposedafterTEVcleavage205

oftheexpressedX-eK-FlvfusionproteinwereclonedfromaneK:HATtemplateusingthe206

primers eK(X)_TEV(oh)_fwd (5’-GAGAATCTTTATTTTCAGxxx CACGGATCTGGAGCTTG-3’207

with xxx=GTT (for Phe), GGG (for Gly), GAG (for Arg), and GTT (for Leu)) and208


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eK_HAT_flav(oh)_rvs (5’-CCGCTGATAAGAGTGATATCTGCAGCACCAG-3’). This sequence209

containsaTEVproteaserecognitionsequence(ENLYFQ|XwithXbeingtheneo-N-terminal210

aftercleavage,i.e.TEVP1'residue)attheN-terminaloftheexpressedX-eK-Flvfusionpro-211

tein.InordertoattachGatewayattBsitesandfusethePCRproducts,aPCRwasperformed212

using Flv_attB2(oh)_rvs (5’-GGGACCACTTTGTACAAGAAAGCTGGGTA TCATTATTTGAG-213

TAAATTAATCCACGATCC-3’) and adapter_tev_fwd (5’-GGGGACAAGTTTG TACAAAAAA-214

GCAGGCAGGCTTAGAAAACCTGTATTTTCAGGGAATG-3’).Allprimersequencesarelistedin215

SupportingInformationTableS2.AnLRreactionintopVP16(Thaoetal.,2004)(kindgift216

fromRussellL.Wrobel,UniversityofWisconsin-Madison) lead to the final construct that217

consists of an N-terminal 8xHis:MBP double affinity tag. The expression vector218

pVP16::8xHis:MBP:tev:eK:3xHA:FlvwastransformedintoE.coliBL21(DE3)andthefusion219

proteinwasexpressedby0.2mMIPTGinductioninLBmediumfor16hat26°C.Cellswere220

harvestedviacentrifugation(3,500g,4°C,20min),resuspendedinNi-buffer(50mMsodi-221

umphosphatepH8.0,300mMNaCl),treatedwith1mg/mLlysozyme(Sigma)inthepres-222

enceofPMSF(SantaCruzBiotechnology,sc-3597)addedtoafinalconcentrationof1mM223

followed by sonication (4 min 40%, 6 min 60% intensity). The lysate was centrifuged224

(12,500g,30min),thesupernatantloadedontoaNi-NTAagarosecolumn(Qiagen)equili-225

bratedwithNi-buffer,followedbyNi-bufferwashing,thentheproteinwaselutedwithNi-226

buffercontaining200mMimidaziole(Merck)andloadedontoamyloseresin(NEB).After227

washingwithamylose-buffer(25mMsodiumphosphatepH7.8,150mMNaCl),theprotein228

waselutedwithamylose-buffercontaining10mMmaltose.ForTEVdigest,thefusionpro-229

teinwas incubated overnight at 4°Cwith 0.27 µg/µL self-madeTEVprotease, expressed230

frompRK793(Addgene,plasmid8827),in50mMphosphatepH8.0,0.5mMEDTA,1mM231

DTTandloadedontoaNi-agarosecolumn(Qiagen)equilibratedwithNi-buffer.Theflow-232

throughcontainingthetag-freeX-eK-FlvsubstratewasconcentratedwithanAmiconUltra-233

15(MerckMillipore).234

235

PRT1cloning,expressionandpurification236

The coding sequence of Arabidopsis PRT1 was cloned according to gene annotations at237

TAIR(www.arabidopsis.org)fromcDNA.TheSequencewasflankedbyanN-terminalTEV238

recognitionsequenceforfacilitateddownstreampurificationusingtheprimersss_prt1_tev239


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(5’-GCTTAGAGAATCTTTATTTTCAGGGGATGGCCGAAACTATGAAAGATATTAC-3’) and240

as_prt1_gw (5’-GGGTATCATTCTGTGCTTGATGACTCATTAG-3’). A second PCR using the241

primers adapter (5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAGAGAATCTTTATTTTCAG242

GGG-3’) and prt1_pos2_as (5’-GGGGACCACTTTGTACAAGAAAGCTGGGTATCATTCTGTGCTT243

GATGA-3’)wasperformedtoamplifytheconstructtouseitinaBPreactionforcloninginto244

pDONR201 (Invitrogen) followed by an LR reaction into the vector pVP16 (Thao et al.,245

2004).RecombinationintothisGatewaydestinationvectorcontaininga8xHis:MBPcoding246

sequence5’oftheGatewaycassetteleadstoanN-terminal8xHis:MBPdoubleaffinitytag.247

The8xHis:MBP:PRT1isolation,cleavageandpurificationwasdoneasdescribedabovefor248

theX-eK-FlvbuttheNi-buffercontained10%glyceroland0.1%Tween20.249

250

Chemicallabeling251

10µMofpurifiedX-eK-Flvwasincubatedfor1hatroomtemperaturewith100µMofthe252

synthesized thiol reactive fluorogenic labelingdye in20mMTris-ClpH8.3, 1mMEDTA253

and 1 mM tris(2-carboxy-ethyl)phosphine (TCEP, Thermo Scientific). The reaction was254

stoppedwith1mMcysteinehydrochloride,theunreactivedyeremovedusing10kDacut-255

off Amicon filters (MerckMillipore) by three successivewashing steps, and the labeling256

efficiencyevaluatedbyfluorescenceintensityofthelabeleddye(TecanM1000)andtotal257

proteinconcentrationusinginfra-redspectroscopy(DirectDetect,MerckMillipore).258

259

Chemicalsynthesis260

The detailed synthesis protocols of the labeling probe NBD-NH-PEG2-NH-haloacetamide261

aredescribedinSupportingInformationMethods.Inbrief,thefollowingsynthesissteps262

were accomplished: 1) tert-butyl {2-[2-(2-aminoethoxy)ethoxy)ethyl}carbamate (NH2-263

PEG2-NHBoc);2)NBD-NH-PEG2-NHBoc;3)NBD-NH-PEG2-NH2hydrochloride;4)NBD-NH-264

PEG2-NH-iodo-acetamide; 5) NBD-NH-PEG2-NH-iodoacetamide; 6) NBD-NH-PEG2-NH-265

chloroacetamide.266

267

tert-butyl{2-[2-(2-aminoethoxy)ethoxy)ethyl}carbamate(NH2-PEG2-NHBoc)268

Toasolutionof2,2'-(ethylenedioxy)-bis(ethylamine)(50.00mL,33.83mmol;495.6269

%)indrydioxane(190mL),di-tert-butyldicarbonate(14.90g,68.27mmol,100%)indry270


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dioxane(60mL)wasaddedslowlyandtheresultingmixturewasstirredat25°Cfor12h.271

Thereactionmixturewas filtered, thesolventwasremovedunderreducedpressureand272

theremainingresiduewasdissolvedindistilledwater(300mL).Theaqueousphasewas273

extractedwithdichloromethane(3x250mL).Finally,thecombinedorganicphaseswere274

dried(Na2SO4)andthesolventwasremovedunderreducedpressuretoyieldtert-butyl{2-275

[2-(2-aminoethoxy)ethoxy)ethyl}carbamate(NH2-PEG2-NHBoc)aslightyellowoil(16.09g,276

64.8mmol,94.9%).1HNMR(400MHz;CDCl3)δ:1.42(br.s.,2H),1.42−1.46(m,9H),2.87277

−2.90(m,2H),3.32(m,2H),3.52(m,,2H),3.55(m,,2H),3.61–3.64(m,4H),5.13(br.s.,278

1H)ppm;13CNMR(100MHz,CDCl3)δ:28.4,40.3,41.8,67.1,70.2,73.5,79.2,156.0ppm;279

ESI-MS m/z: 248.7 [M + H]+, 497.4 [2M + Na+]+; HRMS (ESI) calculated for C11H25N2O4280

249.1809,found249.1809.281

282

NBD-NH-PEG2-NHBoc283

Toasuspensionoftert-butyl{2-[2-(2-aminoethoxy)ethoxy)ethyl}carbamate(1.50g,284

6.04mmol,100%)andsodiumbicarbonate(1.01g,12.08mmol;200%)inacetonitrile(30285

mL), 4-chloro-7-nitrobenzofurazan (NBD) (1.80 g, 9.06mmol, 150%) in acetonitrile (30286

mL)wasaddedslowlyoveraperiodof2handtheresultingmixturewasstirredat25°C287

for12h.Thereactionmixturewasfiltered,thesolventwasremovedunderreducedpres-288

sure,and theremainingresiduewassubjected tochromatography(silicagel,methanol/289

ethyl acetate,5 : 95) toyieldNBD-NH-PEG2-NHBocas abrownsolid (1.89g,4.58mmol,290

75.9%).M.p.:85–86°C;RF=0.56(methanol/ethylacetate,5:95);1HNMR(400MHz;291

CDCl3)δ[ppm]:1.42–1.45(m,9H),3.31–3.37(m,2H),3.54–3.56(m,2H),3.58–3.60(m,292

2H),3.61–3.71(m,4H),3.87(m,2H),5.02(m,1H),6.20(d,J=8.6Hz,1H),6.88(m,1H),293

8.49(d,J=8.6Hz,1H);13CNMR(100MHz;CDCl3)δ[ppm]:28.4,43.6,68.1,70.2,70.2,70.4,294

70.5,77.2,98.7,136.3,143.9,144.0,144.0,144.3,155.9;ESI-MSm/z:410.5[M−H]+,434.2295

[M + Na]+, 845.4 [2M + Na]+; HRMS (ESI) calculated for C17H25N5O7Na 434.1646, found296

434.1647.297

298

NBD-NH-PEG2-NH2hydrochloride299

ToasolutionofNBD-NH-PEG2-NHBoc(2.08g,5.06mmol,100%)indrymethanol300

(20mL),trimethylsilylchloride(2.70mL,21.27mmol,500%)wasaddedviasyringeand301


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the resultingmixturewas stirred at 25 °C for 12 h. The solventwas removed under re-302

ducedpressure. The remaining residuewas suspended indiethyl ether (15mL), filtered303

andthesolidwaswashedwithseveralportionsofdiethylether,and theremainingsolid304

wasdriedunder reducedpressure toyieldNBD-NH-PEG2-NH2hydrochlorideasabrown305

solid(1.56g,5.01mmol,98.9%).Thecrudeproductwasusedwithoutfurtherpurification.306

M.p.:192–193°C;1HNMR(400MHz;CD3OD)δ[ppm]:3.09–3.11(m,2H),3.64–3.76(m,307

8H),3.87–3.90(m,2H),6.19(d, J=8.4Hz,1H),8.45(d, J=8.7Hz,1H); 13CNMR(100308

MHz; CD3OD) δ [ppm]: 41.5, 41.7, 70.1, 70.3, 70.8, 73.2, 98.8, 123.0, 136.5, 144.1, 144.4,309

144.8; ESI-MSm/z: 310.5 [M − 2H]+, 312.3 [M]+; HRMS (ESI) calculated for C12H18N5O5310

312.1303,found312.1303.311

312

NBD-NH-PEG2-NH-iodoacetamide313

Toasolutionof NBD-NH-PEG2-NH2hydrochloride(202.3mg,0.65mmol;100%)314

andN,N'-diisopropylethylamine(134.3µL,0.77mmol,120%)indryacetonitril(4.0mL),315

iodoacetic anhydride (401.0mg,1.13mmol;174%)wasadded slowlyand the resulting316

mixturewasstirredat25°C for12h.Thesolventwasremovedunderreducedpressure317

andtheremainingresiduewassubjected tochromatography(silicagel,methanol/ethyl318

acetate,10:90)toyieldNBD-NH-PEG2-NH-iodoacetamideasabrownsolid(151.1mg,0.32319

mmol,48.5%).RF=0.45(methanol/ethylacetate,10:90);1HNMR(400MHz;CDCl3)δ320

[ppm]:3.50–3.54(m,2H),3.62–3.65(m,2H),3.69–3.71(m,8H),3.73–3.76(m,2H),321

6.21(d,J=8.7Hz,1H),6.55(br.s.,1H),6.95(br.s.,1H),8.48(d,J=8.6Hz,1H);13CNMR322

(100MHz;CDCl3)δ[ppm]:0.56,40.1,43.6,68.1,69.4,70.3,70.5,136.4,143.9,144.3,167.1;323

ESI-MSm/z:478.3[M–H]+,502.1[M+Na]++981.3[2M+Na]+;HRMS(ESI(negativemo-324

dus))calculatedforC14H17N5O6I478.0229,found478.0222.325

326

NBD-NH-PEG2-NH-chloroacetamide327

Toasolutionof NBD-NH-PEG2-NH2hydrochloride(202.5mg,0.65mmol;100%)328

andN,N'-diisopropylethylamine(134.3µL,0.77mmol,120%)indryacetonitril(4.0mL),329

chloroaceticanhydride(221.7mg,1.30mmol;200%)wasaddedslowlyandtheresulting330

mixturewasstirredat25°C for12h.Thesolventwasremovedunderreducedpressure331

andtheremainingresiduewassubjected tochromatography(silicagel,methanol/ethyl332


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acetate, 10 : 90) to yield NBD-NH-PEG2-NH-chloroacetamid as a brown solid (150.5mg,333

0.39mmol,59.7%).RF=0.46(methanol/ethylacetate,10:90);1HNMR(400MHz;CDCl3)334

δ[ppm]:3.54–3.58(m,2H),3.64–3.75(m,8H),3.87–3.90(m,2H),4.06(m,2H),6.20(d,335

J=8.7Hz,1H),6.90 (br.s.,1H),6.98 (br.s.,1H),8.48(d, J=8.6Hz,1H); 13CNMR(100336

MHz;CDCl3) δ [ppm]: 30.51, 42.7, 43.6, 68.1, 69.5, 70.3, 70.5, 136.3, 143.9, 144.3, 166.0;337

ESI-MSm/z:386.1[M–H]+,410.1[M+Na]+;HRMS(ESI(negativemodus))calculatedfor338

C14H17N5O6Cl386.0873,found386.0863.339

340

Ubiquitinationassayandin-gelfluorescencedetection341

3.4µM(totalproteinconcentration,bothlabelandunlabeled)oftheX-eK-Flvfluorescently342

labeledsubstrate(X-eK-Flv-NBD)weresolvedin25mMTris-ClpH7.4,50mMKCl,5mM343

MgCl2,0.7mMDTTcontaining16µMUbfrombovineerythrocytes(Sigma-Aldrich,U6253).344

For ubiquitination, 2mM of ATP (New England Biolabs), 40 nM of E115, 0.31 µM of E2345

(UBC8)15,and5nMofE3(8xHis:MBP-taggedoruntaggedPRT1)wereaddedtotheprevi-346

ousmixinafinalvolumeof30µLandincubatedat30°Cfor1h.Thereactionwasstopped347

byadding5X reductive SDS-PAGE loadingbuffer and incubating for10min at 96 °C fol-348

lowedbySDS-PAGE.ThegelswerescannedusingfluorescencedetectiononaTyphoonFLA349

9500biomolecularimager(GEHealthcare)withablueexcitationlaser(473nm)LDandan350

LBP emission filter (510LP), then blotted onto a cellulosemembrane and detectedwith351

eithermousemonoclonalanti-Ubantibody(Ub(P4D1),sc-8017,SantaCruzBiotechnology,352

1:5,000 dilution in blocking solution [150mMNaCl, 10mMTris-Cl pH 8, 3% skimmilk353

powder, 0.1% Tween 20]) or mouse monoclonal anti-HA epitope tag antibody (HA.11,354

clone16B12:MMS-101R,Covance;1:1,000to1:5,000,inblockingsolution)andgoatanti-355

mouse IgG-HRP (1858415, Pierce; 1:2,500 to 1:5,000 dilution in blocking solution). The356

acquiredimagesofthegels(priorblotting)wereanalyzedusingtheGelAnalyserdensito-357

metricsoft(Gel.Analyser.com).Thus,onemayusethesamegelforbothin-gelfluorescence358

detectionfollowedbyblottingandimmunodetection.359

Thesamegelsthatweredetectedviafluorescencescanningwereblottedanddetectedwith360

ECLwithoutfurtherprocessingsuchasstripping.Thus,fluorescentdetectioncanbecom-361


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binedwithECLinonesimpleworkflow.ForevaluationofpHdependence,50mMTris-Cl362

wasusedasabufferingagentatpH6.75,7.0,7.5,8.0,8.5and9.0.363

364

Real-timeubiquitinationassayusingfluorescencepolarization365

Forfluorescencepolarization(FP),thereactionmixture(24µL)containingallthecompo-366

nents except theATPwas incubated in a384wellmicroplate (Corning,Cat.No.3712or367

3764)at30°C inaM1000 infiniteplate reader (Tecan)until the temperaturewas stable368

(typically4-5min)andthereactiontriggeredbyadding6uLof10mMATPpreheatedto369

30°C.FPwasmonitoredevery2minat562nmwhiletheexcitationwavelengthwassetto370

470nm.TheM1000fluorescencepolarizationmodulewascalibratedusing10nMfluores-371

ceinin10mMNaOHatP=20mP.372

373

Structuremodelingoftheartificialsubstrate374

TheaminoacidsequenceoftheartificialF-eK-FlvsubstratewassubmittedtotheProtein375

Homology/AnalogyRecognitionEngineV2.0(Kelleyetal.,2015)(Phyre2,StructuralBioin-376

formaticsGroup,ImperialCollege,London)inbothnormalandintensivemodes.Thebests377

selectedtemplateswerefoundtobePBDID:3EDCfortheeKregionand2M6RfortheFlv378

part)andthemodelwasvisualizedusingViewerLite(AccelrysInc.).379

380

381

RESULTS382

383

PRT1isanE3ubiquitinligaseandprefersbulkyN-termini384

FortheanalysisofPRT1E3ligasefunction,i.e.recognitionofN-endrulesubstrates,385

weusedrecombinantPRT1togetherwithgenericsubstratereagentswithunprecedented386

detectionfeaturescombiningchemicallysynthesizedfluorophoresandrecombinantubiq-387

uitinationacceptorswhichwereusedasliveproteinmodificationdetectors.TodescribeN-388

terminalaminoacidspecificityofPRT1,theN-terminallyvariableproteinpartsofthere-389

porters were engineered as N-terminal His8:MBP fusions comprising a recognition se-390

quenceoftobaccoetchvirus(TEV)proteaseatthejunctiontothesubsequentgenericsub-391

strateproteinmoiety(Fig.2a,SupportingInformationFig.S1a).CleavagebyTEVgave392


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risetosmallC-terminal fragmentsoftheHis8:MBP-substratefusionsofwhichtheneo-N-393

terminal, i.e. theP1' residueof theTEV cleavage site, canbe altered to all proteinogenic394

aminoacidsexceptproline(Kapustetal.,2002;Phanetal.,2002;Naumannetal.,2016).395

For a novel fluorescence-based approach, we covalently coupled a synthetic fluorescent396

probe(Fig.2b)totheartificialsubstrateprotein.Theresultingreagentservedasfluores-397

centproteinUbacceptor inN-endruleubiquitinationassays.Thearchitectureoftherea-398

gentisasfollows:afterthecleavableHis8:MBPtag,eK,apartofE.colilacZ(Bachmairetal.,399

1986)followedbyatriplehemagglutininepitopetag(3HA)forimmunodetectionandanE.400

coliflavodoxin(Flv)werecombined.Flvwaschosenasahighlysolubleandstableprotein401

andincludesflavinmononucleotideasacofactor.Itssemiquinoneisfluoresentbutnotsta-402

bleenoughtobeusedasfluorophorefordetectioninitsplainform.Therefore,wedecided403

toadditionally label theFlvprotein.The junctionsbetweenHis8:MBPandeKencode for404

theN-terminiglycin (Gly,G),phenylalanin (Phe,F), arginine (Arg,R), and leucin (Leu,L)405

thatgetN-terminallyexposedafterTEVcleavage.TheG/F/L/R-eK-Flvconstructscontaina406

singlecysteine(Cys101ofFlv)thatallowedthelabelingofthepurifiedrecombinantfusion407

proteinwith a novel thiol-reactive probe that comprises an iodoacetamide-polyethylene408

glycol(PEG)linkerandthefluorogenicsubunitof4-nitro-2,1,3-benzoxadiazole(NBD;Fig.409

2b).Wechosethelatterduetoitssmallsizecomparedtootherlabelingreagentssuchas410

largefluoresceinmoietiesandbecauseitcanbedetectedveryspecificallybybothUVab-411

sorptionandUVfluorescencewithlowbackgroundinterferences.412

Inaninvitroubiquitinationassay,weusedrecombinantUBC8asapromiscuousE2413

conjugatingenzymeandUBA1asE1activatingenzyme(Stegmannetal.,2012)andshow414

here for the first timeE3 ligaseactivityofPRT1dependingonE1,E2andATP (Fig.2c).415

PRT1discriminatedasubstratebyitsN-terminal,aidingthetransferofUbtothesubstrate416

andleadingtopolyubiquitination.Afterimmunostainingwithanti-Ubantibodies,usually,a417

typicalsmearofhighermolecularweightcomparedtothetargetprotein'ssizeisobserved418

or afterprobingwith target-specific antibodies, amoreor lessdistinct laddering, alsoof419

highmolecularweight,becomesevident.Thesearethecommonsignsforpolyubiquitina-420

tion and a clear laddering was also visualized by fluorescent scanning in our novel ap-421

proach.Weidentifieddistinctsubspeciesviain-geldetection(Fig.2c).Aclassicalend-time422

pointassaywherethereactionwasstoppedatdifferentreactiontimepoints followedby423


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SDS-PAGEandin-gelfluorescencedetectionrevealedthekineticsofPRT1activityusingF-424

eK-Flvassubstrate(Fig.2d).425

However,areal-timemonitoringof thekineticprofileof theenzymaticreaction is426

onlypossibleviaFPinlivedetectionmeasurements.Thekineticprofileisbest-fittedwith427

anS-shapedcurveandagrowthcurvemodelof logistic type (Richards’ equation) rather428

thanexponentiallyasexpectedforsimplekinetics(Fig.2e).429

ItwaspreviouslysuggestedthatPRT1bindstoN-degronscarryingbulkydestabiliz-430

ing residues (Stary et al., 2003) but biochemical evidence for that was still lacking. By431

changing theN-terminal residueof theX-eK-Flv-NBDsubstrate, itwaspossible to reveal432

thatPRT1indeeddiscriminatesthesubstratesaccordingtotheN-terminalresidue,asex-433

pected(Fig.2f,SupportingInformationFig.S1b,c).WhilethesubstratescarryingG-,R-,434

L-initiatedN-terminishowedpoorubiquitination,F-eK-Flv-NBDwasheavilyubiquitinated.435

WhiletheeK-basedsubstrateshowedthekineticcurvediscussedabove,thecontrolF-eΔK-436

Flvsubstratewithmutatedlysines(expectedsiteofubiquitination,Lys15andLys17,both437

replacedbyArg)presenteda faster initial rateofubiquitinationbut levelsofonlyhalfof438

thefinalFPvalue(Fig.2f).Thisisingoodagreementwiththein-gelfluorescencedetection439

wherelowerdegreesofubiquitinationofF-eΔK-Flv,reducedmono-anddi-ubiquitination-440

butstillclearpolyubiquitination-wereobserved(SupportingInformationFig.S1c).441

Anotherremarkableobservationoftheubiquitinationpatterninthein-gelfluores-442

cenceimage(usingthreedifferentindependentsubstrateproteinpurificationsofF-eK-Flv-443

NBD)wasthatthetri-ubiquitinatedformpresentsthreedistinctsubspecieswhicheventu-444

allyleadtoamultitudeofotherspeciesathigherlevel(SupportingInformationFig.S1b).445

There was only one species of tri-ubiquitinated F-eΔK-Flv-NBD generated, where two446

ubiquitination acceptors siteswithin eK (Lys15 and Lys17)were replaced by Arg (Sup-447

portingInformationFig.S1b).448

449

FluorescentlylabeledsubstrateproteinsunravelmechanismofPRT1-mediated450

ubiquitination451

Thecombinationoftheproposedtwofluorescence-basedmethodsallowedfastand452

efficientinvitroinvestigationoftheubiquitinationprocessviatheE3ligasePRT1andthe453


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optimizationofthereactionconditions.Asafirstapproachutilizingthereal-timeassayin454

thecontextofsubstrateubiquitination,westudiedtheroleofchangesinpHontheubiqui-455

tination processmediated by PRT1. A classical end-time approach revealed the reaction456

optimum to be clearly above pH 7 but below pH 9 as indicated by the occurrence of457

polyubiquitinatedspeciesofthefluorescentsubstrateprobeF-eK-Flv-NBD(Fig.3a).How-458

ever, using our real-time FP protocol,we additionally acquired the kinetic profile of the459

PRT1-mediated ubiquitination process (Fig. 3b) and themaximum reached polarization460

valuesofthisreaction(Fig.3c).Thesecorrelatedwiththeamountofpolyubiqutinatedspe-461

cies detected in the SDS-PAGE gel-based end-time experiment (Fig. 3a) and the highest462

initialrate(Fig.3c)whereasthelatterappearstobedifferentfromthereactionoptimum463

accordingtothedetectedmax.FP.Wealsohadpreviouslyobserved,thatF-eΔK-Flvubiqui-464

tinationpresentedafaster initialratebutonlyhalfofthefinalFP(Fig.2f)andlowerde-465

greesoffinalubiquitination(SupportingInformationFig.S1c).Bothbell-shapedformsof466

the pH dependence for the highest initial reaction rate (pH 8.0) and themaximum sub-467

stratepolyubiquitinationrate(pH7.5)indicatedtwocompetingprocessesthatgeneratea468

localmaximum(Fig.3c).469

A strong decrease of the ubiquitination rate mediated by PRT1 was observed at470

higherconcentrationsoftheE2-conjugatingenzymeUBC8(>2µM)bothviain-gelfluores-471

cence (Fig. 3d) and FP (Fig. 3e-g). Based on the FPmeasurements using up to 2 µM of472

UBC8,theKMofsubstrateubiquitinationbyPRT1atdifferentE2concentrationswasfound473

tobeinthesubmicromolarrange,0.08±0.01µM,indicatingaverytightbindingoftheE2to474

PRT1comparedtootherRINGE3ligases(Ye&Rape,2009)(Fig.3e).Moreover,thedistri-475

butionpatternoftheubiquitinatedsubstratespeciesattheendofthereaction(Fig.3f)and476

thekineticprofilesofubiquitination(Fig.3g)aredifferent,dependingontheusedE2con-477

centration.478

479

480

DISCUSSION481

TheN-endrulepathway isanemergingvibrantareaof research inplant sciences482

and agriculture (Gibbs et al., 2011; Licausi et al., 2011; Gibbs et al., 2014b;Weits et al.,483

2014;deMarchietal.,2016;Mendiondoetal.,2016)andreviewedin(Gibbsetal.,2014a;484


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Gibbs, 2015;Gibbs et al., 2016). Taking the firstbona fide plantN-end ruleE3Ub ligase485

PRT1asthemodel,wedescribeanoveltooltomolecularlycharacterizepolyubiquitination486

live,inreal-time,anduseittogainthefirstmechanisticinsightsinPRT1substrateprefer-487

ence,activationandfunctionalpairingwithanE2-conjugatingenzyme.Todate,activityand488

functionofenzymaticN-endrulepathwaycomponentswasonlyspeculatedandthe field489

was lacking investigations onmolecular level. Here, we showed the first molecular evi-490

denceforubiquitinationactivityofanE3ligasecandidatefromtheentireplantN-endrule491

pathway.492

Here,wedemonstratedPRT1E3Ubligaseactivityandsubstratepreferencebyus-493

ingrecombinantPRT1togetherwithartificialproteinsubstratesinaninvitrofluorescence-494

basedlifeubiquitinationassay.Wefoundthatfirstofall, thereporterconstructbasedon495

bacterialFlvchemicallycoupledtoNBD(Fig.2b)worksasubiquitinationacceptor.Second,496

this reaction reflects substrate specificity and cannot be considered an in vitro artifact,497

sinceN-terminal amino acids other thanPhe rendered the substrate aweaker target for498

PRT1(Fig.2f,SupportingInformationFig.S1b,c).Third,ourtestsystemallowedtode-499

scribeE3ligasefunctionandtargetspecificitybyusingvariantsoflabeledsubstrates.500

Similar experiments are usually evaluated based on immunochemical and colori-501

metricdetection,incorporationofradioisotopessuchas125Ior32P,orfluorescentlylabeled502

nativeorrecombinantUb(Ronchi&Haas,2012;Melvinetal.,2013;Luetal.,2015a;Luet503

al.,2015b)(SupportingInformationTab.S1).However,problemsofsterichindranceby504

modifyingUbanddifficultiestodiscriminatebetweenauto-andsubstrateubiquitinationif505

usinglabeledUbmayoccur.Alsoartificialexperimentalsetupssuchassingle-moleculeap-506

proaches or extreme buffer conditionsmight not represent or support formation of the507

requiredcomplexubiquitinationmachinery(SupportingInformationTable1).Ourassay508

allowedbothdirectassessmentintheflowoftheactualFPexperimentandgel-basedeval-509

uationaftercompletingSDS-PAGE.Thisrendersproteintransferviawesternblottingplus510

thesubsequenttime-consumingstepsofblocking, immuno-andchemicaldetectionobso-511

lete.Theprotocoldescribed is rapid,non-radioactive, usesonly a small fluorophoreas a512

covalentdye,workswithfullsubstrateproteinsinsteadofonlypeptides,andcanberead513

out live in real-time.Moreover, the FP approach conveys superimposable kinetic curves514

withdata fromclassical end-timepoint assays, but faster,withhigher resolution in time515


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andusing fewer reagents. The advantage of a combination of the described two fluores-516

cence-based approaches, that is, gel-based and FP, is the possibility to gainmechanistic517

insightswhichwasnotpossiblebyapplyingonlyoneofthesingleprotocols.Anexampleis518

the determination of KM and kcat of the interaction of the E3 ligase PRT1 with E2-519

conjugatingenzymes.ThisincludedtheinfluenceoftheE2concentrationonboththeubiq-520

uitinatedsubstratespeciesandthekineticprofileoftheubiquitinationreaction.521

UsingFPcoupledtoimmunoblotanalysis,wewereabletoconfirmthatPRT1isan522

active E3 ligase acting in concertwith E2-conjugating enzyme UBC8. In a buffer system523

closetophysiologicalconditions,itcouldbeshownthatPRT1notonlymonoubiquitinates524

N-degron containing substrates, but alsomediates polyubiquitinationwithout the aid of525

furthercofactors.Therefore,itwasruledoutthatPRT1onlymonoubiquitinateswhichwas526

speculatedpreviously(Staryetal.,2003).Moreover, theactionofa typeII-N-recogninas527

smallasPRT1(46kDa) ismost likelysufficient forsubsequenttargetdegradationbythe528

proteasome. Since PRT1 lacks the conserved ClpS domain that confers affinity to type II529

substrates inotherN-recognins, thebindingmechanismofPRT1to itssubstrateremains530

anintriguingopenquestion.531

ByFP-facilitated real-timemonitoring of the kinetic profile of the PRT1-mediated532

ubiquitination,weobservedtheS-shapedcurveofthereaction(Fig.2e).Oneexplanation533

forthiskineticsandthepresenceofaninitiallagphaseisanincreaseoftheaffinityofPRT1534

forthemonoubiquitinatedsubstratescomparedtothenon-ubiquitinatedpopulation.Pref-535

erencesofE2sandE3s formono-orpolyubiquitinatedsubstratesand their influenceon536

ubiquitinationvelocitybutalsothatinitialubiquitinationgreatlyenhancesthebindingaf-537

finityofE3stothesubstrate insubsequentreactionswasshownpreviously(Sadowski&538

Sarcevic,2010;Luetal.,2015b).Thechainelongation(Ub-Ubisopeptidebondformation)539

canbefasterthanthechain initiationwhichmightrepresenttherate limiting-stepofthe540

reaction, rather than an E1-E2-controlled limiting-step. Thus, the chain elongation and541

chaininitiationstepsappeartobedistinctprocessesthathavedistinctmolecularrequisites542

inagreementwithpreviousfindingsforotherE3s(Petroski&Deshaies,2005;Deshaies&543

Joazeiro,2009).Thelagphaseisreducediftherateisincreasedbyhigherconcentrationof544

PRT1(Fig.2e).545


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TheFP-basedassay revealed that thekineticprofileof theubiquitinationwasde-546

pendentonthepositionandavailabilityoflysinesasUbacceptorsitesassuggestedtobe547

characteristicofN-degrons(Bachmair&Varshavsky,1989).Byloweringtheoverallnum-548

berofavailablelysinesintheF-eΔK-Flv-NBDsubstrate(twoLyslessthaninX-eK-Flvcon-549

structswith11Lysintotal)theoverallubiquitinationwasdetectablyreduced.Differences550

inthekineticcurvesofF-eK-FlvversusF-eΔK-Flvindicatedthatareductionoftheavailable551

numberofLysresidues leadtoa faster initial rateofubiquitinationwhereas the finalFP552

valuesreachedonlyhalfofthelevelscomparedtotheassayapplyingthesubstratewiththe553

full setofLys residues (Fig.2f,Supporting InformationFig. S1c).However, the simple554

gel-basedend-pointassaycouldnotunravelifthiswasduetoalteredvelocityofchainini-555

tiationversuschainelongation.TheinitiationperLysresiduewasexpectedtobesimilarin556

F-eK-versusF-eΔK-FlvsubstratesbutchainelongationcouldapparentlystartfasterinF-557

eΔK-Flv.ThisdemonstratedthatthepresenceofE2togetherwiththeparticularsubstrate558

playsakeyroleintheformationofthemolecularassemblyfacilitatingtheubiquitination559

process. Already the intermolecular distance between the E3 ligase and theUb acceptor560

lysinesofthesubstrateaswellastheaminoacidresiduesproximaltotheacceptorlysines561

determinetheprogressofthereactionandubiquitinationspecificity(Sadowski&Sarcevic,562

2010).TakingtheslowerinitiationofpolyubiquitinationofF-eK-Flvintoaccount,theavail-563

abilityof lysinesat theN-terminusmight interferewith themonoubiquitinationofother,564

moredistallysinesandtheE3couldremainassociatedwithsubstratesthataremonoubiq-565

uitinatedattheN-terminal.566

Whensubjecting theF-eK-Flv-NBDsubstrate fusionprotein to invitroubiquitina-567

tion assays, three distinct subspecies of the tri-ubiquitinated formwere detected versus568

onlyoneform,ifF-eΔK-Flv-NBDwasused(SupportingInformationFig.S1c).Thiscould569

beexplainedbyaformationofvariousubiquitinatedisoformsofthesubstratebyutilizing570

different lysine side chainsasubiquitinationacceptor sites.These couldbeeitherwithin571

the sequence of eK (e.g. Lys15 and Lys17) orwithin Flv (e.g. Lys100 and Lys222which572

seem structurally more favored according to the structural model, Supporting Infor-573

mationFig.S1a).Thiswasfurthersupportedbythefactthatthereisonlyonespeciesof574

tri-ubiquitinatedF-eΔK-Flv-NBD,wheretwoubiquitinationacceptorsiteswithineK(Lys15575

andLys17)werereplacedbyArg(SupportingInformationFig.S1b).576


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WhenanalyzingtheinfluenceofthepHonPRT1functionasE3Ubligase,wedocu-577

mentedbell-shaped formsof thepHdependence for thehighest initial reaction rate (pH578

8.0)anddeterminedthemaximumsubstratepolyubiquitinationrate(pH7.5).Theseindi-579

catedtwocompetingprocessesthatgeneratealocalmaximum(Fig.3c).Inthelightofre-580

cently discussedmechanisms of E3 ligase action (Berndsen&Wolberger, 2014) and the581

predictionof twoRINGdomains inPRT1 (Stary et al., 2003), higherubiquitination rates582

withincreasedpHcouldbeduetodeprotonationoftheattackinglysinesidechainoftheE2583

activesite.ThiswouldfacilitatethioestercleavagebetweenE2andUbandtherebymediate584

Ubtransfertothesubstratelysines.Asimilareffectwasobservedregardingtheinfluence585

oftheacidicresiduesinclosevicinityoftheE2activesite,whichalsocausedeprotonation586

ofthelysinesidechainoftheincomingsubstrate(Plechanovovaetal.,2012).Thispossibly587

explainsthedrastic increaseintheinitialrateofPRT1substrateubiquitinationinthepH588

6.8topH8range(Fig.3c).Thecompetingprocessesleadingtothedecreaseinubiquitina-589

tion atpH>8 couldbedestabilizationof ionic andhydrogenbonds at alkalinepH simply590

interferingwithprotein-proteininteractionorATPhydrolysisaffectingtheUbchargingof591

theE2bytheE1.Thiscouldalsoexplaintheprematurelevelingofthekineticcurvesinthe592

FPmeasurementsatpH>8(Fig.3b)whileinalongerreactiontimescale,themaximumFP593

valueswouldbeexpectedtobethesamefrompH6.8topH7.5.594

The apparent catalytic rate constant (kcat) of the Ub transfer, more precisely the595

transferof the firstUbmolecule, i.e. therate limitingstep,was foundtobe1.30±0.07s-1.596

ThissuggestedthatontheonehandPRT1hadahighturnovernumberduetoahighlyac-597

tivecatalyticcenterandontheotherhandthattheE2concentrationdoesnotonly influ-598

ence the rate of theUb transfer to the substrate but also themechanism itself. Possible599

causesarethetwoseparateandpotentiallydistinctlyfavoredchaininitiationandelonga-600

tionprocessesmentionedabove.These could result in lowering the rateof the initiation601

stepathigherE2concentrationssinceboththekineticprofileandtheformationofubiqui-602

tinatedspeciesareaffectedandalsotheattackinglysinesmightbestructurallydifferently603

favored.This isespecially suggestedby thevariableoccurrenceof thedistinctpatternof604

triubiquitinated substrate species (Fig. 3d,f) asmentioned above anddiscussed in other605

systemsaswell(Ye&Rape,2009).606

607


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Byusingfluorescentlylabeledsubstrateproteinsinthetwodescribedapproaches,608

thatis,gel-basedfluorescencescanningafterSDS-PAGEandFP,wewereabletoinvestigate609

themechanismofPRT1-mediatedubiquitinationandoptimizethereactionconditions.The610

presentedworkservesasamodelforthedemonstrationofdifferentialmechanismsofsub-611

straterecognitionandtightinteractor-bindingintheN-endrulepathway.612

PRT1isaplantpioneerenzymelackinghomologsintheotherkingdomsalbeitsmall613

andeasytoproduceinanactiveformasrecombinantproteinrenderingitanexcitingcan-614

didateforfurtherfunctionalandstructuralstudiesofkeyfunctionsofonebranchoftheN-615

endrulepathway.Sofar,onlythreeresearcharticlesmentionworkonPRT1, i.e. thetwo616

first brief descriptions (Potuschak et al., 1998; Stary et al., 2003) and one recently pub-617

lishedstudyhighlightingtheroleoftheN-endrulepathway-andinparticularanovelfunc-618

tionforPRT1-inplantimmunity(deMarchietal.,2016).However,todate,thecommunity619

lacksproofsdemonstratingthatPRT1andotherE3candidatesareindeedinvolvedinsub-620

strateproteinubiquitination.621

TheheredescribedtoolcanbeadoptedbylaboratoriesinvestigatingN-endrulere-622

lated posttranslational modifications such as deformylation, methionine excision, oxida-623

tion, deamidation, arginylation, ubiquitination and degradation. Moreover, we are con-624

vincedthatitmayalsobeextendedtoassaysforotherposttranslationalmodificationssuch625

asphosphorylationandtootherE3Ubligasesaslongasatleastonenativeorartificialsub-626

strateproteinforthemodificationof interest isknown.Becauseitmakesuseofchemical627

labelingof substrateproteins rather than labelingproteinmodifiers suchasUborphos-628

phate themselves,onecommonreagentcanbeused forvariousmodificationassays.The629

approachallowstomeasureandtrackposttranslationalproteinmodificationliveandina630

time-resolvedmannerandhasprofoundimplicationsforourunderstandingoftheinterac-631

tionsofE3 ligaseswith substratesandnon-substrates.Concerning the fieldof theN-end632

rulepathway,thismightapplytoothercandidatesofE3UbligasessuchasPROTEOLYSIS6633

(PRT6) and BIG (AT3G02260) or potential N-end rule adapter proteins like PRT7634

(AT4G23860)(Tasakietal.,2005;Garzón,2008;Talloji,2011).Theseexperimentswillbe635

ofpremier interest in the futurebecausephenotypesofbiological importanceandgenet-636

ically determined causalitieswere described and need to be substantiated onmolecular637

level.Therefore,weseepotential forabroaderimpactforubiquitinationresearchas it is638


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conceivablethatthemethodistransferabletootherE3ligasesandenzyme-substratepairs.639

Inthecourseofourstudies,wefeltthatrapid,easyandcheapprotocolswerelackingfor640

in-depthbiochemicalanalysisofE3ligasekinetics,thesameholdstruefornon-radioactive641

and sterically not interfering protocols and thosewhere entire proteins and directly la-642

beledsubstratescanbeapplied.643

In terms of further applications, the kinetic approach allowed collecting data that644

canassisttosetuphigh-throughputassays,e.g.forscreensofinhibitorsandtheinfluence645

ofsmallmoleculespotentiallyfacilitatingorenhancingubiquitination.Inourexample,this646

includedtestingoftheenzymaticparametersofE2-E3interactionsandsubstratespecifici-647

tiesforPRT1.Similarapproacheshaveusedlabelingwithradionuclidesorfluorescentdyes648

coupledtoUb(Ronchi&Haas,2012;Melvinetal.,2013;Luetal.,2015a;Luetal.,2015b).649

ThelattercovalentmodificationofUbwithfluorescentmoietiesisoftenimpracticalsince650

thesegroupscanstericallyhindertheE1-catalyzedactivationandE2-dependenttransthio-651

lationreactions(Ronchi&Haas,2012).Thisinturncanaltertherate-limitingstep.Theuse652

of radioactive isotopes requiresat least runninganSDS-PAGEandgel-dryingorwestern653

blottingfollowedbyautoradiographyforhourstodays(SupportingInformationTab.1).654

Besides thedescribed invitromethods, severalprotocolsand toolsweresuccessfullyap-655

pliedinvivo,mainlybasedontranslationalfusionsoffluorescentproteinstodegronsofthe656

Ubfusiondegradation(UFD)pathway(Hameretal.,2010;Matilainenetal.,2016),theN-657

endrulepathway(Speeseetal.,2003;Fadenetal.,2016)orboth(Dantumaetal.,2000).658

OthermethodsmakeuseofUb-bindingsystemstoachievevariousread-outs(Marblestone659

etal.,2012;Matilainenetal.,2013)(SupportingInformationTab.1).660

Inconclusion,wedescribeasystemforreal-timemeasurementsofubiquitinationin661

bulky solutionwith combined fluorescence scanning of SDS-PAGE gels and fluorescence662

polarization.This setupwasused toestablishanartificial substrateprotein-baseddetec-663

tionreagentthatrevealsimportantmechanisticinsightsofE2-PRT1-substrateinteraction.664

Wedemonstrate for the first time that PRT1 is indeed involved in polyubiquitination of665

substrateproteinsdependingonitsN-terminalaminoacidandthereforeapproachedPRT1666

asanplayeroftheN-endrulepathwayforthefirsttimeonamolecularlevel.667

668

669


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ACKNOWLEDGEMENTS670

WethankMarcoTrujilloforexpressionclonesofHis-taggedUBC8andUBA1,discussions671

and constant support inubiquitination-related issues andAngela Schaks for synthesis of672

thechemicalprobe.Thisworkwassupportedbyagrantforsettingupthejuniorresearch673

group of the ScienceCampus Halle – Plant-based Bioeconomy to N.D., by the grant WE674

1467/13-1oftheGermanResearchFoundation(DeutscheForschungsgemeinschaft,DFG)675

toB.W.fundingE.P.,agrantoftheLeibniz-DAADResearchFellowshipProgrammebythe676

LeibnizAssociationandtheGermanAcademicExchangeService(DAAD)toA.C.M.andN.D.,677

andPh.D. fellowshipsoftheLandesgraduiertenförderungSachsen-AnhaltawardedtoC.N.678

andF.F.Financial supportcame fromtheLeibnizAssociation, thestateofSaxonyAnhalt,679

theDeutscheForschungsgemeinschaft(DFG)GraduateTrainingCenterGRK1026“Confor-680

mationalTransitions inMacromolecular Interactions”atHalle,andtheLeibniz Instituteof681

PlantBiochemistry(IPB)atHalle,Germany.Tocompleteworkonthisproject,aShortTerm682

ScientificMission(STSM)of theEuropeanCooperation inScienceandTechnology(COST,683

www.cost.eu)wasgrantedtoA.C.M.andN.D.bytheCOSTActionBM1307–“Europeannet-684

worktointegrateresearchonintracellularproteolysispathwaysinhealthanddisease(PRO-685

TEOSTASIS)”.ThisworkwaspartiallyfundedbythegrantDI1794/3-1oftheGermanRe-686

searchFoundationtoN.D.687

688

AUTHORCONTRIBUTION689

A.C.M.performedtheubiquitinationreactionsandrelatedanalysis.E.P.andB.W.designed690

andsynthesizedthefluorescentprobe,B.W.supervisedthechemicalsynthesis,M.K.estab-691

lishedPRT1ubiquitinationreactions,C.N.clonedandpurifiedPRT1,F.F.clonedtheX-eK-692

HAT fragment and performed site-directed mutagenesis. N.D. and A.C.M. designed the693

study,wrote themanuscriptunderconsultationwithall co-authorsanddesignedthe fig-694

ures.Allauthorsreadandapprovedthefinalversionofthismanuscript. 695


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857

858


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FIGURES859

860

861

862Figure1.GenerationofN-endrulesubstratesbyproteolyticprocessingandpredictedfeaturesof thetwobona863fideplantN-recognins.a)SubstratescontainingN-degronscanbegeneratedfrom(pre-)pro-proteinsasprecursorse-864quencesafterproteolyticcleavage(indicatedbythescissors).TheN-degronshownherecomprisesaPheresidueaspri-865marydestabilizing residueat theprotein-C’ and internal lysines forpolyubiquitination.TheseN-degronscanbe recog-866nizedbyN-endruleE3Ubligases(N-recognins)whichinturnassociatewithUb-conjugatingenzymes(E2)carryingUb867whichwaspreviouslyactivatedbyE1enzymes.Onepossibleresultofubiquitinationisproteindegradationandtodate,in868thecontextoftheN-endrule,ubiquitinationisassumedto leadtodegradationinmostofthecases.b)Thetwoknown869ArabidopsisN-recogninswereidentifiedbytheirfunction(PRT1,46kDa)andbyhomologytotheUBR-boxfromS.cere-870visiaeUBR1p(PRT6,224kDa).UBR:boxbindingtypeIsubstrates;RING*:compositedomaincontainingRINGandCCCH-871typeZnfingers;ZZ:ZincbindingdomainsimilartoRING;RING:protein-proteininteractiondomainforE2–E3interaction;872AI:predictedautoinhibitorydomain (intramolecular interaction);P:phosphorylationsite (PhosPhAt4.0;phosphat.uni-873hohenheim.de).bismodifiedfromTasakietal.,2012.874

875


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876

877

Figure2.Fluorescentproteinconjugatesformonitoringinvitrosubstrateubiquitinationinrealtime.a)Designof878recombinantfusionproteinsusedasN-endrulesubstrates.AfterTEVcleavageandremovaloftheHis8:MBPaffinitytag,879theartificialsubstratebasedonE.coliflavodoxin(Flv)isinitiatedwithaneo-N-terminal,herePhe(F),Gly(G),Leu(L)or880Arg(R).b)Skeletalformulaofthesynthesizedthiol-reactivefluorescentcompound.Thesubstratewascovalentlytagged881withthereagentcomposedof iodoacetamide,polyethyleneglycol(PEG) linkerand4-nitro-2,1,3-benzoxadiazole(NBD).882Thereactiveiodine-containinggroupontheleftcouplestothethiolgroupofinternalCysresiduesofFlv.NBDservesasa883fluorophorewithexcitationat470nmandemissionat520nm.c)Detectionviafluorescenceandimmunoblottingofthe884F-eK-Flv-NBDafter invitroubiquitination.Thelabeledproteinanditsubiquitinatedvariantsweredetectedviafluores-885cencescanningdirectlyfromtheSDS-PAGEgelfollowedbywesternblottingandimmunodetectionwithanti-HAandanti-886Ub antibodies. Lane 6 showsubiquitinatedE2 like in all lanes and autoubiquitination of PRT1 as very highmolecular887weight ‘smear’.CleavedPRT1aswell asHis8:MBP-taggedPRT1wereused togetherwithHis:UBA1 (E1)andHis:UBC8888(E2)(Stegmannetal.,2012).dande)KineticprofilesofPRT1-mediatedubiquitination.F-eK-Flv-NBDubiquitinationwas889monitored by FP and in-gel fluorescence scanning. The S-shaped kinetic curve is observed in both in-gel fluorescence890scanningdetection and fluorescencepolarization. f)N-terminal specificity evaluatedby real-timeubiquitinationdetec-891


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tion.Fluorescently labelledR-eK-Flv,L-eK-Flv,G-eK-Flv,F-eΔK-FlvandF-eK-Flvwerecomparativelyevaluatedfortheir892degreeofubiquitinationbyPRT1. 893


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894

895

Figure3.Applicationsof fluorescentproteinconjugates formonitoringpHdependentubiquitinationandenzy-896maticparametersofPRT1E3ligase.a-c)pHdependentubiquitinationoftheF-eK-Flvsubstrate.a)In-geldetectionof897F-eK-Flvubiquitinatedspeciesafter1hreactionatseveralpHvaluesdemonstratingdifferentpatternsofpolyubiquitina-898tionpreferencesdependingonthepH.b)Kineticprofiles,c)initialratesandmaximumend-timeFPvaluesformingabell-899shapeddistributiondependingonthepH.d-g)PRT1-mediatedubiquitinationofF-ek-Flvdependentontheconcentration900ofE2-conjugatingenzyme(UBC8).d)TimedependenceofubiquitinationatseveralE2concentrationsforthefirst60min901at5nMPRT1,timescale:5-60min.e)Michaelis-MentencurveplottedusingtheinitialratefromFPdatasuggestanE2-902driveninhibitioneffect.f)Thequalitativeevaluationofubiquitinationwasdoneusingin-gelscanningfluorescenceandg)903kineticprofileswereobtainedusingFPmeasurements,similarconditionsasind)butwithtentimeshigherconcentration904ofPRT1,i.e.50nM.905

906


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SUPPORTINGINFORMATION907

908

Additionalsupportinginformationmaybefoundintheonlineversionofthisarticle.909

910

SUPPORTINGFIGURES911

Supporting Information Figure 1.Modeled structure of the F-eK-Flv substrate and912

PRT1N-terminalspecificity.913

914

SUPPORTINGTABLES915

SupportingInformationTable1.State-of-the-artubiquitinationdetectionmethods.916

SupportingInformationTable2.Oligonucleotidesusedinthisstudy.917

918

SUPPORTINGMETHODS919

SynthesisofthechemicalprobeNBD-NH-PEG2-NH-haloacetamide.920

921

SUPPORTINGREFERENCES922


http://dx.doi.org/10.1101/062067


Date post:	23-Jul-2019
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Real-time detection of PRT1-mediated ubiquitination via ... fileMot et al. Fluorescent N-end rule...

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