Two nuclear localization signals present in the basic-helix 1 domains of MyoD promote its active nuclear translocation and can function independently

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Two nuclear localization signals present in the basic-helix 1 domains of MyoD promote its active nuclear translocation and can function independently

M. Vandromme, J.C. Cavadore, Anne Bonnieu, A. Froeschlé, N. Lamb, A.

Fernandez

To cite this version:

M. Vandromme, J.C. Cavadore, Anne Bonnieu, A. Froeschlé, N. Lamb, et al.. Two nuclear localization signals present in the basic-helix 1 domains of MyoD promote its active nuclear translocation and can function independently. Proceedings of the National Academy of Sciences of the United States of America , National Academy of Sciences, 1995, 92, pp.4646-4650. �hal-02711865�

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Proc. Natl. Acad. Sci. USA Vol. 92,pp.4646-4650,May1995 CellBiology

Two nuclear localization

signals present

in the basic-helix 1 domains of

MyoD promote

its active nuclear translocation and can function

independently

MARIEVANDROMME*, JEAN-CLAUDECAVADORE*, ANNEBONNIEUt, ANNEFROESCHLEt, NEDLAMB*,

ANDANNEFERNANDEZ*

*CellBiologyUnit,Centre deRecherches de BiochimieMacromoleculaire,Centre National delaRechercheScientifique,Institut Nationalde laSanteetdela RechercheM6dicale,BP5051,34033MontpellierCedex, France;andtLaboratoirede Differentiation Cellulaire etCroissance,United'EndocrinologieCellulaire, InstitutNational de la RechercheAgronomique,34060MontpellierCedex,France

CommunicatedbyHaroldWeintraub, Fred HutchinsonCancerResearchCenter, Seattle, WA, January3, 1995 ABSTRACT MyoD,a member ofthefamilyofhelix-loop-

helix myogenic factors that plays a crucial role in skeletal muscle differentiation, is a nuclear phosphoprotein. Using microinjectionofpurifiedMyoDproteininto ratfibroblasts,

weshow that the nuclearimportofMyoDisarapidand active

process,beingATP andtemperature dependent.Two nuclear localization signals (NLSs), one present in the basic region

and the other in the helix 1 domain ofMyoD protein, are demonstrated tobefunctional inpromotingtheactive nuclear

transport ofMyoD. Synthetic peptides spanning these two NLSs and biochemically coupled to IgGs ^can promote the nuclearimportofmicroinjectedIgG conjugatesin muscle and nonmuscle cells. Deletionanalysisreveals that eachsequence

can function independently within the MyoD protein since concomittant deletion of bothsequences is required toalter the nuclearimport of this myogenic factor. Inaddition, the

complete'cytoplasmic retention of a P-galactosidase-MyoD

fusion mutant protein, double deleted at these two NLSs, arguesagainsttheexistenceof another functional NLS motif inMyoD.

MyoD belongs to the family of muscle-specific helix-loop-

helix(HLH) proteins that includesMyf5, myogenin, MRF4/

Myf6/herculin. Its expressionis restricted to skeletal muscle cellswherein it acts as atranscriptional activator of muscle-

specificgeneexpression duringskeletalmyogenesis (reviewed

inrefs. 1and2). Dimerization betweenmyogenicfactors and

ubiquitousHLH proteinssuchas theproducts ofE2Agene,

E12andE47, is essential forsequence-specific DNAbinding

and subsequent transcriptional activation (3). Immunofluo- rescence analysis has shown that MyoDhas a nuclear localization in different cell lines(C2, azamyoblasts) (4),aswellas in MyoDtransfected cells (5),which is inagreementwith its

transcriptional activity.

Various studies on nuclear localization have led to the

concept that transport across the nuclear envelope (which separates the cytoplasm from the nucleoplasm) is an active

processmediatedbyoneorseveral nuclear localization signal sequences(NLSs)presentwithin either theproteinitselfor an associated cofactor (reviewed ⁱⁿ ^refs. ⁶ ^and 7). Specialized transporterproteinsknownasNLS-binding proteinsallow the

transport of NLS-containing proteins to the nucleus. Initial evidence for the existence of NLSsarosefromanalysisofyeast

MATa2 protein (8),Xenopus nucleoplasmin (9),^{and simian}

virus 40largetumorantigen(SV40Tantigen) (10).Such NLSs have since been characterized inagrowingnumber of nuclear

proteins by ^two criteria: an NLS is sufficient to promote

nuclear accumulation of an otherwise cytoplasmic protein

when fusedto itgeneticallyorbiochemically; incounterpart,

Thepublicationcostsof this articleweredefrayedinpartbypagecharge payment.This article must therefore beherebymarked"advertisement"in accordance with 18 U.S.C. §1734solelytoindicatethis fact.

deletion of an NLS(s) from a nuclear protein leads to its

cytoplasmicretention.Althoughastrict consensusdoesnotyet exist, ^it appears that most NLSs are short linear sequences [from 5 to 12amino acids (aa)] generallycontaining several basicresidues (arginineor/andlysine).

With a recent report suggesting that the localization of

MyoDcanbedevelopmentally regulatedinXenopus(11),and our data showing the requirement for A kinase in MyoD

nuclearimport(12), the identification ofMyoDNLSsrepre-

sents a necessary and important step in understanding the

regulationofMyoDlocalization.Here,wereportthatMyoD

containstwo independent functional NLSs, one localized in the basicregionandthe other in the helix1 domain.Each of them is sufficientto translocate rabbitIgGto the nucleus of nonmuscleormuscle cells.Additionally, bydeletionanalysis,

we showed that either sequence can function independently

withintheMyoDprotein,since both NLSs mustbedeletedto alter the nuclearimportofeitherMyoD^{or a}P-galactosidase- MyoDfusionprotein.

MATERIALS AND METHODS

Peptide SynthesisandConjugation.Solid-phase synthesisof NLSpeptideswasperformedasdescribed (13).^When^necessary, a terminal cysteine was added to the MyoD peptide sequenceinordertoprovideadisulfide bond with the carrier

protein.Conjugationofpeptides/protein^wasperformedusing

the heterobifunctionalcrosslinking reagentsulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane 1-carboxylate (Pierce,In-

terchim, Montlucon, France) following the manufacturer's instructions. Peptide-IgG conjugates were resuspended at 2

mg/mlin100 mMHepes(pH 7.2)forsubsequent microinjec-

tion experiments. Theapproximate numberofpeptidescou-

pledpermol ofIgGwasevaluated from theresultingshift in molecular mass of the IgG heavy chains analyzed by SDS/

PAGE.

Mutagenesis. The MyoD ^cDNA ^was mutagenized in the

Moloneysarcomaviruslongterminalrepeatexpressionvector

pEMSV-scribe (agiftfrom H.Weintraub, Seattle, WA) (14) using thePromegain vitro mutagenesis kit (Coger, Paris) ^as

described(12).Mutationswereconfirmedbysequencingusing

theUnited States Biochemical 2.0sequenasekit(Amersham).

PurifiedMyoD ProteinandAntibody. Production andpu-

rification of thefull-length murine MyoDaswell as affinity- purified anti-MyoDantibodies have been describedelsewhere

(12).

Microinjection Experiments. Rat embryonic fibroblasts REF52 were grown on glass coverslips as described (13).

Peptide-IgG conjugates (1 mg/ml) were microinjected to-

gether with inert mouse IgG antibodies (1 mg/ml) as an Abbreviations:NLS,nuclear localizationsignal;SV40,simian virus40;

Tantigen, largetumorantigen;HLH,helix-loop-helix.

4646

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Proc. Natl.Acad. Sci. USA 92 (1995) 4647

injection marker into the cytoplasm of REF52 cells. At different times after injection, cells were fixed with 3.7%

formalin before extraction with-20°C acetoneasdescribed. In the caseofwild-type MyoDanddeletedmutants,cellsgrowing

on plastic dishes were microinjected into the nucleus with

pEMSV-MyoD(0.5mg/ml)orwiththepurified protein(0.5-1 mg/ml)togetherwith mouse inert IgGantibodies (1 mg/ml)

to serve subsequentlyinidentifying injectedcells. Vizualiza- tion of MyoD localization and injected cells was done by

immunofluorescence asdescribed (12).

HeterodimerizationExperiments.Twohundredto500ng^of bacterially produced glutathione-S-transferase-E12 proteins

bound to glutathione-Sepharose 4B (Pharmacia) was incu- bated with either in vitro translated MyoD labeled with

[35S]methionine (5 uld),bacterially produced MyoD (1 ,ug),^or IgG-NLS conjugates(1 ,tg)inbindingbuffer[10mMHepes, pH 7.9/50 mM KC1/2.5 mM MgCl2/10% glycerol/1 mM

dithiothreitol/1 mMphenylmethylsulfonyl fluoride (PMSF)]

overnightat4°C. El2proteinswerecollectedbycentrifugation

and washedtwicewith RIPA buffer(10^mMTris,pH7.5/150

mM NaCl/1 mM EDTA/0.2%Nonidet P-40/1 mMPMSF).

Proteins bound to E12 were separated by SDS/PAGE and

analyzed byWesternblotting^todetect rabbitIgGin the case ofNLS-IgG conjugates andbyautoradiographyof the gelin thecase ofthe in vitro translatedMyoD.

Fusion of MyoD to P-Galactosidase Using the pCHO10

Vector and Transient Transfection Experiments. Wild-type

and mutant forms ofMyoD^were genetically fused to P-ga-

lactosidase cDNAusing thepCH110 expression^vector (Pro- mega).cDNAswereamplified byPCRusingTaqpolymerase.

The sequenceofthe5' primer, 5'-CACGCCCAAGCTTAT-

GGAGCTTCTATCGCCGCCA-3',spannedthepCH110vec- torsequencenearthe ATG andincludedaHindIIIrestriction site for subsequent cloning. The 3' primer sequence, 5'- CGGCGGGTACCGCAAGCACCTGATTAAATC- GCATT-3',spannedtheendofthecodingsequenceofMyoD

and included a Kpn I site for subsequent cloning. After

digestion with Hindll and Kpn I, the purified restriction

fragmentwasligatedintothepCH110 expressionvectorafter

cuttingwith thesamerestrictionenzymes. Theresultingplas-

mid was used to transfect REF52 cells using N-[1-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sul- fatereagent(Boehringer) asdescribedbythesupplier. Forty- eight hours after transfection, localization of the expressed

fusion protein was assessed by immunofluorescence using anti-3-galactosidaseantibodies(Promega)^andanti-MyoDan- tibodies.

RESULTS

Nuclear Import of MyoD is an Active Process. Nuclear

importofMyoD ^wasinvestigated through cytoplasmic injec-

tion ofpurified bacterially produced MyoD into rat embryo

fibroblasts (REF52). The use of nonmuscle cells allows the

study of nuclear localization ofectopic MyoDwithout inter- ference from endogenous MyoD. Additionally, MyoD has been showntoactivatemyogenesiswhenexpressed ectopically

in nonmuscle cells (14, 15), showing that it can function

independently of othermuscle-specific factors. The purified MyoD protein was injected into the cytoplasm of REF52 fibroblasts and cellswerefixed 15-60 min thereafter. As shown in Fig. 1,MyoD acquiresanuclear localization within 15min ofcytoplasmic injection(Fig.1AandB)andispredominantly

nuclear after 30min (Fig. 1 C andD).After 45 min, MyoD

could be detectedin only50-60% ofinjectedcells(Fig. 1 E andF)and by 1 hafterinjection, the proteinwas nolonger

detectable ininjectedcells(Fig.1 GandH),showingthat it is

rapidly degraded, as expected from the short half-life of the

protein describedby Thayer^et^al. (16).

IniectedCells MvoD

15min

30min

45min

60min

FIG. 1. Purified MyoDproteinsisolatedfromabacterialexpres-

sionsystem^weremicroinjectedintoratembryofibroblasts(REF52)

to assess its intracellular localization and half-life. Injected REF52 cells were stained for the distribution of aninert antibodyusedto

identifyinjectedcells(A,C,E,andG)orforinjected MyoD proteins

withmonospecific anti-MyoD antisera(B,D, F, andH). ^Cells^were

fixed15(AandB),30(CandD),45(EandF),or60(GandH)min after injection. Arrowheads in B, D, F, andH indicate the MyoD stainingin theinjectedcells.(Bar⁼ 10,tm.)

MyoD (318 aa: apparent molecular mass of 45-48 kDa) spontaneouslyformshomodimers andmayform heterodimers withvariouspartnerssuchasE12andE47proteinsinside the cell. Thus,its native size ispredicted tobe in the order ofat least80-90kDa,makingfree diffusionthroughnuclearpores unlikely(17).TotestwhethernuclearimportofMyoD^{was an}

active process,weassayedthe effect of lowtemperatureand

depletion of ATPon nuclearimportofMyoD.When MyoD

wasinjectedinto cellskeptat4°Corintocellsdepletedof ATP

by^treatmentwithamixture of 6 mMdeoxyglucoseand 10 mM sodium azide(18),weobserved thatnuclearimportofMyoD

wasabolished(datanotshown).Takentogether,theseresults showed that the transport of MyoD, being energy and tem-

peraturedependent, is anactiveprocess.

TwoMyoD Sequencesinthe Basic-Helix1 Domains Func- tionasNLSs. WefurtherquestionedwhetherMyoD^contains

functional NLSs thatmaydirect its nuclearlocale.Byhomol-

ogy withareported NLS consensus sequence (19),we identified the presence of sixputative and partially overlapping

NLSs inMyoDprotein (Fig.2Upper).All of thesesequences

arepresentinthe basic-helix 1 regionofMyoD(aa102-136).

TotesttheactivityoftheseputativeNLSs,threepeptidesthat mimic the MyoD NLS were synthesised. As shown in Fig. 2

Upper,the firstpeptide(CKRKTTNADRRKA; NLS234)cov- ersthethreeputativeamino-terminal NLSs(NLS2,NLS3,^and

NLS4)^andonlylacks thefirstaminoacidofNLS1,because of the extensiveoverlappingofthese foursequences.We choseto omit the first aminoacidof NLS1tohaveanaturalcysteineat the amino-terminalpositionforsubsequent coupling.Thetwo other peptides (NLS5, CATMRERRRLSK; and NLS6

VNEAFETLKRC) contain the fifth and the sixth putative NLSs,respectively. Each peptidewas biochemically coupled CellBiolo·gy: Vandrommeetal.

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4648 CellBiology: Vandromme etaL

100ACKIiNADRRKAAMR VN 135

ACKRK RRLSK TLKRC

RKaTTR

NAt^K 1,1CVNEAFEITXRC

NLAS2 CKRKTINADRI K

N1S^CATMlER4MlM

injecteacells INL-lg' Injectedcells NL-lgG FIG. 2. (Upper) Shown from top to bottom is the amino acid sequence for murine MyoD spanning aa 100-135, the six putative

NLSs with the amino acids that match the consensus sequence

(RKTA)-K-K-(RQNTSG)-K(19)inboldfacetype,and thesequence ofthe threepeptides synthesized spanningthe NLSsdetected in the

MyoDsequence:NLS234, NLS5,andNLS6.(Lower)The threeMyoD peptidesaswellastheSV40-NLSpeptide(CPKKRKV)^wereconju- gated to rabbit IgGs. The NLS-IgG conjugates ^weremicroinjected togetherwithunconjugatedmouseIgGsinto thecytoplasmofREF52 cells. Cellswerefixedatdifferent times thereafter beforestainingfor localization of theNLS-conjugatedrabbitIgGs(B,D, F, H, J, L, N,and

P)andforcoinjectedmouseIgGstoidentifytheinjectedcells(A,C, E,G, I,KM,and0). (AandB) InjectionofSV40-NLS-IgG conjugate

into cells fixed 1 h afterward. (CandD, E andF, and G andH) Injection of NLS234-conjugated IgGs and fixation 1, 2, and 3 h afterward, respectively. (Iand J and K andL) Injectionof NLS5-

conjugated IgGsand fixation2and3hafterward,respectively.(Mand NandOandP) InjectionofNLS6-conjugatedIgGsandfixation1and 2hafterward,respectively. (Bar = 10jum.)

with rabbitIgG ^asdetailed inMaterialsandMethods. Rabbit

IgGswere chosenbecause of their inertactivity incells and

large size, which prohibits passive diffusion through nuclear pores. As a positive control, ^we also coupled ^a peptide corresponding to the SV40 T antigen NLS (CPKKRKV) ^to

rabbit IgG. ^Ineach case,thecouplingratio, estimatedbythe

resulting shift in molecular mass of the IgG heavy chains observedbySDS/PAGE,wasevaluatedto 5-10peptidesper mol of IgG (data not shown). Each peptide-conjugate was

microinjectedintothecytoplasmof REF52 cellstogetherwith

an inert mouse antibody for subsequent identification of

injected cells. The localization ofconjugates^wasassessedby

immunofluorescence 1, 2,and 3 h afterinjection.Asshownin

Fig. 2Lower, SV40 NLS-coupled IgGs were already totally

nuclear 1 h after their cytoplasmic injection (Fig. 2 Lower A andB).NLS234-IgGswerealsotranslocatedtothenucleus but

ataslowerratesincethey^weredetectable in the nucleusonly

2 hafterinjectionat alevel of nuclearstainingsimilartothe

cytoplasmic staining(Fig.2 Lower EandF),and 3 hfollowing

theirinjection, theNLS234-IgGs^were mostlynuclear (Fig. 2 LowerG andH).NLS5 wasineffective inaddressing IgGsto the nucleus since theyremainedtotallycytoplasmic2 h (Fig.

2 Lower IandJ)and 3 hafter theirinjection (Fig.2 Lower K andL). Incontrast, ^asshown in Fig. 2Lower, NLS6allowed efficient translocation of the IgGs into the nucleus; they

became nuclear 1 h after theirinjection(Fig. 2Lower M and

N)andweretotallynuclearafter2h(Fig. 2 Lower0 andP).

Similar experimentsinmouse C2musclecells gaveidentical results (datanotshown).

Toensurethatthe activityofNLS6, located in helix1,was notduetoitsassociation with another HLHproteininside the cell that wouldserve as acotransporter^todriveNLS6-IgGto thenucleus,^wetestedwhetherNLS6-IgGcouldheterodimer- izewith E12. Inagreementwithpreviousdata from Daviset al. (20) showing that the integrity of the HLH region is

necessary^for heterodimerization, we found that NLS6-IgGs

were unable to heterodimerize with E12 (data not shown).

Taken together, these results show that among the three

peptides containing putative NLSs in MyoD, two of them,

NLS234 and NLS6, canfunction as true localization signals

when coupled to a nonnuclear carrier protein, here rabbit

IgGs.

Deletionof NLS234 and NLS6 Is RequiredToImpairthe

Ability^ofMyoDToTranslocate to the Nucleus.Thefunction-

ality of the two NLSs identified in MyoD was probed by deletingthem from thewild-type MyoD andlooking for the effect of such mutationson MyoDnuclear localization. Mu- tantsofMyoDdeletedateither NLS234 (aa100-112),^NLS6 (aa 130-135),^or^both^wereprepared by mutagenizing MyoD

cDNAin thepEMSVeukaryote expressionvector(Fig. 3),as described in Materials and Methods. Wild-type and deleted

MyoD plasmids were injected directly into the nucleus of

growing REF52 cells. Injected plasmids were allowed to

express for 10-15 h before fixation of cells and analysis of

MyoDexpressionandlocalizationbyimmunofluorescence.As

before,cellswerecoinjectedwith mouseIgGsforsubsequent

identification of injected cells. As shown in Fig. 3, in cells

injectedwith theplasmid codingforwild-type MyoD, staining

forexpressed MyoDwasfoundexclusivelyin the nucleus(Fig.

3a). A similar nuclear localization was observed in cells

injectedwith eithersingledeletionmutant,MyoDANLS234^or MyoDANLS6,althoughin both cases.somecytoplasmicstain-

ing could also beobserved, perhaps reflecting a diminished

efficiency in nuclear import (Fig. 3 b and c). However, as revealed by the bright nuclear staining, each single mutant could efficiently translocate to the nucleus showing that NLS234 and NLS6mayactaloneandindependentlyindriving

nuclear import. In contrast, anti-MyoD staining of cells in-

jectedwith the MyoD double mutant (deletedfor NLS234 and NLS6) showed a bright immunoreactivity in the cytoplasm (Fig. 3d). The nuclear staining also observed was

MyoD1 102 162 318

BASIC I HEID 1 LOOPI E11 X

AqKiTTNADRLAATMRER1LSIVNEAFETLKRCTSSNPNQ0LPfVELRNAIRYIEGLQAI

Localization

a t N

b A1MU2 (ACKRKTTNADRRK) N

C ail3-13 (TLKRC) N

d-a.lI23.i ACDmRmK)r (TLRC) C> N

FIG. 3. Shown is the amino acid sequence of MyoD with the localization of the basic, helix 1, loop, and helix2 domains in the molecule. Representedbelow are the single deletionperformed ^at

eitherNLS234(b)^orNLS6(c)andthedouble deletion of NLS234 and NLS6(c). Photomicrographsshow theimmunofluorescence staining

forexpressed MyoD proteins15hafterinjectionof eitherwild-type MyoDcoding plasmid(a)orthedifferentdeleted mutantsrepresented

onthe left(b-d).Solid arrowheadsindicate the distributionofstaining

in the nucleus;open arrowheads indicate the cytoplasmic compart-

ment.(Bar = 10,Lm.)

Proc. Natl.Acad. Sci. USA 92 (1995)

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Proc. NatlAcad. Sci. USA 92(1995) ⁴⁶⁴⁹

never detectably higher than the cytoplasmic staining and could reflect the passive diffusion of the double mutant

MyoDprotein.Indeed,one canexpectthatdeletion of NLS6

presentin the helix1regionwillinterfere withdimerization,

thus generating MyoD monomers, which could undergo passivenuclear diffusion (17). We confirmed that, incontrast to MyoDwt and MyoDANLS234, MyoDANLS6 and

MyoDANLS234+ANLS6wereunable todimerize with glu-

tathione-S-transferase-E12 bacterially produced protein (datanot shown).

To ensurethat the partialcytoplasmic/nuclear stainingof the double mutantproteinwas notduetothepresenceofan NLS other than the twowehadmutatedinMyoD,wild-type

and deletionmutantMyoD^weregenetically coupledto 1-ga-

lactosidase. After transfection of REF52cells,the subcellular localization of the expressed P-galactosidase fusion protein

wasexaminedbydouble-immunofluorescenceusingmonoclo- nalanti-P-galactosidaseandpolyclonalanti-MyoDantibodies.

As a control, REF52 cellswere transfected either with the

parental pCH110 or the SV40 NLS-pCH110vectors,which

express ^a cytoplasmic (Fig. 4C) or a nuclear localized j-ga-

lactosidase(Fig. 4A), respectively.^As^{shown in}Fig.4 EandF, MyoDwt-j3galactosidasefusionproteinwasfoundexclusively

inthenucleus,furtherdemonstratingthecapacityofMyoDto be actively translocated into the nucleus. Single deletion of either NLS234(Fig. 4G andH) ^or^NLS6(Fig. ^{4 I}^andJ)^led

toahybrid proteinthatwasstill activelytransportedinto the

nucleus,asrevealedbythebrightnuclearstaining, confirming

thateachNLScan actindependently.However,inbothcases,

acytoplasmic stainingcouldclearlybeobserved,showingthat thecapacityofMyoDtodrive 3-galactosidaseinto the nucleus wasdiminishedinthe absence ofoneofthetwoNLSs.Fusion of 3-galactosidase to MyoD deleted for NLS234 and NLS6

gave rise to a protein that could not be imported into the nucleus. Only a cytoplasmic staining was observed in the double mutanttransfected cells(Fig. 4 KandL). Thisresult shows that the 1-galactosidase fused to MyoD ^deleted at NLS234 and NLS6 fails to be actively transported into the nucleus and demonstrates that thenuclearstainingobserved in thecase ofthe nonfused MyoD ^double ^mutant ^described

below(seeFig.3d)wastheconsequenceofpassivediffusion.

Together,these results show that NLS234 and NLS6haveto bedeletedtoimpairthe nucleartransportofMyoDandimply

that each oftheseNLSs is efficientautonomously.Moreover,

the failure of MyoD ^deleted ^at NLS234 and NLS6 to drive

,3-galactosidaseto the nucleusarguesagainstthe presenceof additionalfunctional NLS motifsinMyoD.

DISCUSSION

We report here that the nuclear import of the myogenic

factor MyoD is mediated by ^two NLS sequences present

in the basic-helix 1 region of MyoD. Although the two

peptides spanning the signals CKRKTTNADRRKA and VNEAFETLKRCwereefficient in translocatingbiochemi-

cally coupled rabbit IgG to the nucleus, they functioned

weaklywhencomparedtotheefficiencyof the NLS of SV40 Tantigenandrequiredahighcouplingratio ofpeptide/IgG

to drive the coupled protein into the nucleus (ca. ^5-10 peptides perIgG). Nevertheless, we found that deletion of onesingle MyoDNLSdidnotsignificantly impairtheability

of MyoD or MyoD fused to P3-galactosidase to enter the

nucleus,showingthat eachsequencecanfunctionefficiently

inthecontextoftheMyoDprotein,evenwhenpresentina

singlecopy.Thisobservation suggeststhattheefficiencyof the NLS innucleartransport maybehighlysensitive tothe

protein environment inwhich the NLS is located. Indeed,

conformational structureof the NLSmaybecritical for the interactionwithNLSbindingproteinsthatparticipateinthe nuclear import process (21). Such a structure may be par-

P-Galactosidase

T^A^HA

MyoD

C)

.qo

0.

CL

--4S:L

D._c, z

z^rqI

FIG. 4. MyoDwild-type and deletion mutants were genetically coupled^to 3-galactosidaseusingthepCH110 expression^vector.The

resultingplasmidscalledpCH110-MyoDwt, pCH110-MyoDANLS234, pCH110-MyoDANLS6,andpCH110-MyoDANLS234+ANLS6andas

control theparental pCH110andpCH110-SV40^weretransfected into REF52 cells. Localization of expressed proteins ^was assessed by

immunofluorescenceusinganti-3-galactosidase(A,C,E,G,I,andK)

andanti-MyoD(B,D, F, H, J,andL)antibodies. Shownarephotomi- crographsofpCH110-SV40 (AandB),pCH110 (CandD),pCH110- MyoDwt (E andF), pCH110-MyoDANLS234(G andH), pCH110- MyoDANLS6(IandJ),andpCH110-MyoDANLS234+ANLS6(Kand

L)transfected REF52cells. (x800.)

tiallylost inpeptides,ahypothesisreinforced inlightof the

recently described crystal structure ofMyoD showing the basic-helix1regionpresentsahelical conformation(22).In contrast to the lack of effect ofone singleNLS deletionin

MyoD,we found that the deletion ofboth NLS sequences resulted in a great loss of MyoD nuclear import, further

demonstratingthat the twoNLSswehavecharacterizedare

individually sufficient to promote nuclear translocation of the MyoD protein. Moreover, since deletion of both NLSs abolished theabilityofMyoDtodrive fused 3-galactosidase

tothe nucleus, it showsthat nootherregionin MyoDthan thetwo NLSs we identified has NLS capability in the cell systemswe have used.

The existenceofmultiplekaryophilicsignals is a frequent phenomenon among nuclear proteins. Like MyoD, several nuclear proteins have been shown to contain two mutually independentNLSsthat havetobedeletedtogethertoinhibit

CellBiology: Vandrommeetall