MARCH-XI, a Novel Transmembrane Ubiquitin Ligase Implicated in Ubiquitin-dependent Protein ...

21 Pages·2007·2.15 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY VOL.282,NO.34,pp.24806–24815,August24,2007
©2007byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
MARCH-XI, a Novel Transmembrane Ubiquitin Ligase
Implicated in Ubiquitin-dependent Protein Sorting in
Developing Spermatids*□S
Receivedforpublication,January16,2007,andinrevisedform,June11,2007 Published,JBCPapersinPress,June29,2007,DOI10.1074/jbc.M700414200
YuriMorokuma‡§,NobuhiroNakamura‡,AkiraKato‡,MichitakaNotoya‡,YokoYamamoto‡,YasuhiroSakai¶,
HidekazuFukuda‡1,ShoheiYamashina¶,YukioHirata§,andShigehisaHirose‡2
Fromthe‡DepartmentofBiologicalSciences,TokyoInstituteofTechnology,Yokohama226-8501,Japan,§DepartmentofClinical
andMolecularEndocrinology,TokyoMedicalandDentalUniversityGraduateSchool,Tokyo113-8519,Japan,andthe
¶DepartmentofAnatomy,KitasatoUniversitySchoolofMedicine,Sagamihara228-8555,Japan
A mechanism by which ubiquitinated cargo proteins are cesses such as neurotransmitter release, hormone secretion,
sorted into multivesicular bodies (MVBs) from plasma and fibroblastmotility,andpathogenengulfment.Italsoplayscer-
trans-Golgi network (TGN) membranes is well established in tainrolesinmammalianspermiogenesis,adifferentiationproc-
yeastandmammaliansomaticcells.However,theubiquitin-de- ess of postmeiotic male germ cells (spermatids) into mature
pendentsortingpathwayhasnotbeenclearlydefinedingerm elongatedspermatozoa,includingacrosomeandflagellumfor-
cells.Inthisstudyweidentifiedanovelmemberofthetrans- mation,nuclearelongationandcondensation,andmitochon-
membraneRING-fingerfamilyofproteins,termedmembrane- drial rearrangement (1, 2). In early developing spermatids,
associatedRING-CH(MARCH)-XI,thatisexpressedpredomi- numerous proteins and membranes necessary for acrosomal
nantly in developing spermatids and weakly in brain and biogenesisaredeliveredtotheimmatureacrosomalvesiclevia
pituitary.MARCH-XIpossessesanE3ubiquitinligaseactivity the Golgi-derived vesicles (1). Some portion of glycoproteins
that targets CD4 for ubiquitination. Immunoelectron micros- has been found to be simultaneously sorted away from this
copyofratroundspermatidsshowedthatMARCH-XIislocal- acrosomepathwaytobetransportedtotheendosomalsystem
izedtoTGN-derivedvesiclesandMVBs.Fluorescencestaining (3–6).Asseeninsomaticcells,theseeventsarethoughttobe
ofratroundspermatidsandimmunoprecipitationofrattestis tightly controlled by the membrane-targeting and fusion
demonstratedthatMARCH-XIformscomplexeswiththeadap- machinery such as Rab GTPase and SNARE3proteins, which
tor protein complex-1 and with fucose-containing glycopro- areassociatedwiththeGolgiandacrosomalmembranes(7–9).
teinsincludingubiquitinatedforms.Furthermore,theC-termi- Recent studies using knock-out mice have indicated that the
nal region of MARCH-XI mediates its interaction with adaptor proteins Hrb and Golgi-associated PDZ- and coiled
(cid:1)1-adaptinandVelithroughatyrosine-basedmotifandaPDZ coilmotif-containingprotein(GOPC)areessentialforfusion
bindingmotif,respectively.OurdatasuggestthatMARCH-XI andtransportoftheproacrosomalvesicles(10,11).However,
actsasaubiquitinligasewitharoleinubiquitin-mediatedpro- majorquestionsremainregardingthemechanismsunderlying
teinsortingintheTGN-MVBtransportpathway,whichmaybe theselectiverecognitionandrecruitmentofcargoproteinsinto
involvedinmammalianspermiogenesis. transportingvesicles.
Ubiquitinisknowntoserveasasortingsignaloftransmem-
braneproteinsforincorporationintothemultivesicularbody
Membranetraffickingisessentialforeukaryoticcellstopro- (MVB),apartoftheendosomalsystem,fromtheplasmamem-
liferate, differentiate, and perform various physiological pro- braneandtrans-Golginetwork(TGN)(12,13).Ubiquitination
iscatalyzedbythesequentialactionofthreeenzymes:aubiq-
uitin-activatingenzyme(E1),aubiquitin-conjugatingenzyme
*Thisworkwassupportedbythe21stCenturyCenterofExcellenceProgram (E2),andaubiquitinligase(E3).Ubiquitinligasesconfersub-
andtheGrant-in-AidforScientificResearch14104002,forYoungScientists
stratespecificityanddirectthetransferoftheactivatedubiq-
16710145 and 16770144, and for Scientific Research on Priority Areas
“Transportsome”18059010fromtheMinistryofEducation,Culture,Sports, uitinmoietytosubstrateproteins.Theyaregroupedintotwo
Science,andTechnologyofJapan.Thecostsofpublicationofthisarticle major families based on the presence of the E3 catalytic core
weredefrayedinpartbythepaymentofpagecharges.Thisarticlemust
domain; that is, the RING-finger and HECT families. Several
thereforebeherebymarked“advertisement”inaccordancewith18U.S.C.
Section1734solelytoindicatethisfact. ubiquitin ligases have been shown to ubiquitinate plasma
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains membraneproteins,includingreceptors,transporters,andion
supplementalmaterialincludingFigs.S1–S6.
Thenucleotidesequence(s)reportedinthispaperhasbeensubmittedtothe
GenBankTM/EBI Data Bank with accession number(s) AB048841 and
AB048842. 3Theabbreviationsusedare:SNARE,solubleN-ethylmaleimidefactorattach-
1Presentaddress:DepartmentofPhysiology,KitasatoUniversitySchoolof mentproteinreceptor;AAL,Aleuriaaurantialectin;AP,adaptorproteincom-
Medicine,Sagamihara228-8555,Japan. plex;GOPC,Golgi-associatedPDZ-andcoiledcoilmotif-containingprotein;
2Towhomcorrespondenceshouldbeaddressed:Dept.ofBiologicalSci- GST,glutathioneS-transferase;MARCH,membrane-associatedRING-CH;MVB,
ences,TokyoInstituteofTechnology,4259-B-19Nagatsuta-cho,Midori-ku, multivesicularbody;PBS,phosphate-bufferedsaline;PVDF,polyvinylidene
Yokohama 226-8501, Japan. Tel.: [phone]; Fax: [phone]; fluoride;TGN,trans-Golginetwork;E1,ubiquitin-activatingenzyme;E2,aubiq-
E-mail:[email protected]. uitin-conjugatingenzyme;E3,ubiquitinligase;HA,hemagglutinin.
24806 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER34•AUGUST24,2007
This is an Open Access article under the CC BY license.
ANovelMARCHProteininSpermiogenesis
channels, leading to their endocytic sorting to the MVB and GAGCAGGGT-3(cid:1)and5(cid:1)-CTCGAGTCAAGACGTCACTCT-
subsequent lysosomal degradation (14). By contrast, little is CATCACTAC-3(cid:1), digested with BglII and XhoI, and then
knownaboutE3moleculesresponsibleforubiquitin-mediated inserted into the same sites of pBS-Kdg5 , yielding pBS-
#9(cid:2)4
sortingattheTGN,withtheexceptionoftwoyeastproteins, Kdg5(cid:3). Then, the SacII-XhoI fragment of pBS-Kdg5(cid:3) was
Rsp5(15)andTul1(16).Inratspermatids,ubiquitinatedpro- insertedintothesamesitesofpcDNA3-MARCH-XI,yielding
teins have been shown to be abundant on membranes of the pcDNA3-MARCH-XI(cid:3).TogenerateaglutathioneS-transfer-
TGNandMVBs(17),suggestingthepresenceofaubiquitin- ase(GST)-His -Xpressexpressionvector(pGex-His -Xpress),
6 6
dependentproteinsortingmechanismintheTGN-MVBpath- acDNAfragmentencodingaHis -Xpresstagwasamplifiedby
6
way.AsyettheE3moleculesresponsibleformediatingthese PCRfrompTrcHis(Invitrogen)withtheprimers5(cid:1)-GGCCA-
ubiquitinationhavenotbeenidentified. GATCTATGGGGGGTTCTCATCAT-3(cid:1)and5(cid:1)-GGCCGGA-
Recently,anovelfamilyofRING-fingerubiquitinligaseshas TCCTTTATCGTCATCGTC-3(cid:1). The PCR product was
been identified in mammals, termed membrane-associated digested with BglII and BamHI and then inserted into the
RING-CH(MARCH)orc-MIR(cellularmodulatorofimmune BamHI site of pGex4T (GE Healthcare). GST-His -Mar11
6 N
regulation)(18,19).TenMARCHgeneproducts(MARCH-Ito was constructed by cloning the fragment encoding residues
-X)havebeenrecognized(20),andthemajoritysharesasimilar 1–146 of MARCH-XI into the BamHI-EcoRI sites of pGex-
structure,anN-terminalC4HC3-typeRINGfinger(RING-CH His -Xpress.His-Mar11 andHis-Mar11 (cid:3)wereconstructed
6 C C
finger)andtwoormoreC-terminaltransmembranespans(18). bycloningthefragmentsencodingresidues323–398and323–
Amongthem,MARCH-IV,-VIII,and-IXhavebeenshownto 397ofMARCH-XI,respectively,intotheXhoI-EcoRIsitesof
ubiquitinateanddown-regulatetransmembraneglycoproteins, pRSET (Invitrogen). GST-RING was constructed by cloning
suchasmajorhistocompatibilitycomplexclassIandII,B7-2, the fragment encoding residues 147–227 of MARCH-XI into
CD166,andICAM-1(18,19,21–23).Particularly,ubiquitina- the BamHI-EcoRI sites of pGex4T. GST-GOPC was con-
tion of major histocompatibility complex class I leads to its structed by cloning of the coding region of rat GOPC into
endocytosis and subsequent sorting to the MVB pathway for pGex4T. FLAG-CD4 was constructed by cloning of the frag-
lysosomaldegradation(18).Previouslywehavedemonstrated ment encoding residues 29–457 of rat CD4 into the EcoRI-
thatMARCH-IIand-IIIareendosomalproteinsboundtothe XbaIsitesoftheFLAG-furinexpressionvector(25).3(cid:4)FLAG-
SNAREproteinsyntaxin6andareinvolvedintheregulationof tagged (cid:2)1-adaptin was constructed by cloning of the coding
endosomaltrafficking(24,25).Hence,itisconceivablethatthe regionofrat(cid:2)1A-adaptinintotheHindIII-XhoIsitesofp3(cid:4)
MARCH family could function in protein sorting and trans- FLAG-CMV10(Sigma).Myc-taggedVeli-3wasconstructedby
port.InthisstudyweidentifiedanovelmemberoftheMARCH cloningofthecodingregionofratVeli-3intotheEcoRI-XhoI
family,termedMARCH-XI,thatishighlyexpressedindevel- sitesofpCMV-Myc(Clontech).GST-Veliwasdescribedprevi-
opingspermatidsofrats.ToaddresstheroleofMARCH-XIin ously(25).Allpointmutationswereintroducedbysite-direct
spermiogenesis,wedetermineditssubcellularlocalizationand mutagenesisusingPCR.
identified associated proteins. Our results suggest that PreparationofRecombinantProteins—TheGST-fusionand
MARCH-XIislikelyaubiquitinligasemediatingproteinsort- His -tagged proteins were produced in the Escherichia coli
6
ingintheTGN-MVBpathway. strainBL21(DE3)pLysS(Novagen)andpurifiedwithglutathi-
one-Sepharose 4B beads (GE Healthcare) and Talon metal
EXPERIMENTALPROCEDURES
affinity resins (Clontech), respectively. Purified recombinant
Cloning of MARCH-XI—A partial cDNA fragment of proteins were dialyzed against saline, with the exception that
MARCH-XIwasobtainedfromratbrainbyPCRamplification His-Mar11 and His-Mar11 (cid:3) were done against 2% Triton
C C
with primers 5(cid:1)-TGTCTAGACACTTGGTCCACCCAAC-3(cid:1) X-100 solution. For detail information, see supplemental
and 5(cid:1)-CCTCTAGAAGTGCTATGGTTTTGCCATTC-3(cid:1). “ExperimentalProcedures.”
ThePCRproductwasinsertedintotheXbaIsiteofpBluescript Antibodies—The following primary antibodies were pur-
IISK(cid:2)(Stratagene),yieldingpBS-Kdg5 .Arattestis(cid:1)cDNA chased. Anti-ubiquitin (Ubi-1) was from Zymed Laboratories
Xba
library(Stratagene)wasscreenedbythestandardmethodwith Inc., anti-(cid:3)-adaptin (clone 88) and anti-TGN38 (clone 2)
theXbaIfragmentofpBS-Kdg5 asaprobe.Positiveclones were from BD Transduction Laboratories, anti-ubiquitin
Xba
were plaque-purified, and the cDNA inserts were subcloned (FK1) was from Biomol, anti-FLAG (M2), anti-His , and
6
into the EcoRI site of pBluescript II SK(cid:2), yielding pBS- anti-GST were from Sigma, and anti-Myc (9E10) and
Kdg5 . The nucleotide sequence for rat MARCH-XI has anti-HA (3F10) were from Roche Applied Science. Rabbit
#9(cid:2)4
been deposited in the DDBJ/GenBankTM/EMBL databases andratpolyclonalanti-Mar11antiserawereraisedagainsta
withaccessionnumberAB048841. GST-His -Mar11 protein. Rabbit polyclonal anti-Veli and
6 N
Plasmids—AmammalianexpressionvectorforMARCH-XI anti-GOPC antisera were raised against GST-Veli and GST-
(pcDNA3-MARCH-XI)wasconstructedbycloningthe1.4-ki- GOPCproteins,respectively.Theimmunizationprotocolsand
lobase PstI-EcoRI fragment of pBS-Kdg5 into pcDNA3 the specificity of rabbit and rat anti-Mar11 antisera are
#9(cid:2)4
(Invitrogen).Toconstructamammalianexpressionvectorfor described in the supplemental data. The anti-Veli and anti-
the deletion mutant lacking the last amino acid residue of GOPC immunoaffinity beads were prepared as follows. The
MARCH-XI (pcDNA3-MARCH-XI(cid:3)), the cDNA fragment appropriateantigen(2mg)wascovalentlycoupledto1mlofthe
encoding residues 167–397 of MARCH-XI was amplified by HiTrapNHS-activatedHPcolumn(GEHealthcare)accordingto
PCRwiththeprimers5(cid:1)-AAGATCTGCTTCCAGGGCGCG- themanufacture’sprotocol.Theantiserum(5ml)wasincubated
AUGUST24,2007•VOLUME282•NUMBER34 JOURNALOFBIOLOGICALCHEMISTRY 24807
ANovelMARCHProteininSpermiogenesis
withtheaffinitycolumnfor30minatroomtemperature.Then, transferredtoanImmobilon-Ppolyvinylidenefluoride(PVDF)
afterwashingwith15mlof75mMTris-HCl,pH8.0,boundanti- membrane (Millipore). The filter was blocked with T-TBS
bodieswereelutedwith0.5MNaCland0.1Mglycine-HCl,pH2.7 (0.05%Tween20,150mMNaCl,and10mMTris-HCl,pH7.6)
andsubsequentlydialyzedagainst0.1MNa PO ,pH7.0.Theaffin- containing 5% bovine serum albumin (BSA) for 2 h atroom
2 4
ity-purifiedanti-Veliandanti-GOPCantibodies(500(cid:2)gofIgG temperature and then incubated with biotin-labeled Aleuria
each)werecoupledto500(cid:2)loftheCarbolinkgel(Pierce)accord- aurantia lectin (AAL) (10 (cid:2)g/ml in T-TBS; Vector Laborato-
ingtothemanufacturer’sprotocol. ries)overnightat4°C.Thefilterwasincubatedwithgoatanti-
Northern Blot Analysis—Northern blot analysis was per- biotin antibody (2.5 (cid:2)g/ml; Vector Laboratories) followed by
formed with 32P-labeled XbaI fragment of pBS-Kdg5 as peroxidase-conjugatedanti-goatIgGantibody(1:100,000;Jack-
Xba
describedpreviously(24). sonImmunoResearchLaboratories).
Cell Culture—Maintenance of cultured cells and transient Fluorescence Microscopy—Wister rats (8 weeks old) were
transfectionweredescribedpreviously(24).Forubiquitination perfusedwithPBSfollowedbyice-coldfixative(2%paraform-
experiments, at 16–20 h post-transfection, cells were incu- aldehyde and 100 mM NaH PO , pH 7.4). Testes were
2 4
bated for 4 h at 37°C inculture medium containing 50 n M extracted,bisect,immersedwiththesamefixativefor2h,and
concanamycin A (Wako), a specific inhibitor of vacuolar thenimmersedwithPBScontaining15%sucrosefollowedby
H(cid:5)-ATPaseusedtopreventthelysosomaldegradationofubiq- PBScontaining30%sucroseovernightat4°C.Preparationand
uitinatedCD4(26). staining of sections and smear preparations of rat testis were
InVitroUbiquitinationAssay—PurifiedGST-RINGorGST- describedpreviously(28)withtheexceptionthatprimaryanti-
RINGmut (1 (cid:2)g) was incubated with a mixture (20 (cid:2)l) com- bodieswereincubatedfor2daysat4°C.Forstainingoffucose
posedof0.1(cid:2)gofrabbitE1(BostonBiochem),1(cid:2)gofpurified glycoproteins,spermatidswereincubatedwith10(cid:2)g/mlbio-
recombinant His -tagged E2 (24), 1 (cid:2)g of ubiquitin (Sigma), tin-labeledALLinPBSatovernightat4°CfollowedbyAlexa
6
and1mMcreatinephosphokinase(Sigma)inanATP-regener- Fluor488-conjugatedmouseanti-biotinmonoclonalantibody
ation buffer (25 mM Tris-HCl, pH 7.5, 120 mM NaCl, 1 mM (2F5;5(cid:2)g/ml;MolecularProbes)for30minatroomtempera-
MgCl , 0.3 mM dithiothreitol, 2 mM ATP, and 1 mM creatine ture. Dilutions of primary and secondary antibodies were as
2
phosphate) for 4 h at 30°C. Thesamples were subjected to follows:anti-ubiquitinFK1,1:200;anti-(cid:3)-adaptin,1:500;anti-
Western blot analysis with a tank-blotting apparatus (Atto) as TGN38,1:250;anti-Mar11,anti-Veli,andanti-GOPC,1:1000;
described previously (27). Ubiquitin conjugates were detected Alexa Fluor 488 or 546-conjugated anti-rabbit or mouse IgG
withanti-ubiquitin(P4D1)antibody(SantaCruzBiotechnology). antibody(MolecularProbes),1:2000;Cy3-conjugatedanti-rat
Immunoprecipitation—All operations were carried out at IgGantibody(JacksonImmunoResearchLaboratories),1:2000.
4°C, and all solutions and buffers were added with protease AfterthenucleiwerestainedwithTO-PRO3(2(cid:2)M;Molecular
inhibitors(10mMleupeptin,1mMpepstatin,5mg/mlaproti- Probes) or Hoechst 33342 (5 (cid:2)g/ml; Molecular Probes), the
nin,and1mMphenylmethylsulfonylfluoride).Forimmunopre- sampleswereobservedwithaconfocalmicroscope(LSM510;
cipitation of FLAG-CD4, the membrane fractions were lysed CarlZeiss).
withTNEbuffer(50mMTris,pH7.4,150mMNaCl,1%Nonidet ImmunoelectronMicroscopy—Pre-embeddingimmunoelec-
P-40, 10% glycerol, and 1 mM EDTA). The lysates were sub- tron microscopy was performed by the previous method (29)
jected to immunoprecipitation with anti-FLAG M2 beads withmodifications.Wisterratswereperfusedwithice-coldfix-
(Sigma)followedbyWesternblottingasdescribedpreviously ative(4%paraformaldehydeinPBS).Testeswereextracted,cut
(27).ForimmunoprecipitationofMARCH-XI,rattestes(3g) intosmallpieces((cid:6)20(cid:4)20(cid:4)20mm),andfixedinthesame
werehomogenizedin30mlofSPHbuffer(0.25Msucrose,0.2M fixativefor2h.Thepiecesoftesteswerefurthercut((cid:6)3(cid:4)3(cid:4)
KCl,and10mMHEPES-KOH,pH7.0).Thehomogenatewas 3mm),fixedinthesamefixativefor8h,immersedwithPBS
layeredontoa1.5Msucrosecushion(12ml)inanSW28tube containing 7% sucrose at 4°C, and then frozen in optimum
(BeckmanCoulter)andcentrifugedfor2hat25,000rpmonan cutting temperature compound. Cryosections (15-(cid:2)m thick-
SW28rotor(BeckmanCoulter).Themembranefraction(0.25/1.5 ness)wereblockedwithPBScontaining1%bovineserumalbu-
M sucrose interface) was diluted in phosphate-buffered saline min for 1 h atroom temperature and incubated with rabbit
(PBS)andcentrifugedfor30minat40,000rpmonanSW41rotor. anti-Mar11(1:300inPBS)overnightat4°C.Thesampleswere
Theresultingmembranepelletwaslysedwith6mlofPBScontain- treatedwithbiotinylatedanti-rabbitIgGantibody(Dako)fol-
ing 0.5% Triton X-100. The lysate was incubated with 20 (cid:2)l of lowed by peroxidase-conjugated streptavidin (Dako). Signals
protein G-Sepharose beads (GE Healthcare) and 2 (cid:2)l of rabbit were developed with diaminobenzidine tetrahydrochloride.
anti-Mar11antiserumovernight.ForimmunoprecipitationofVeli Thesampleswerepost-fixedwith1%OsO ,dehydratedwith
4
orGOPC,thelysatewasincubatedfor4hwith500(cid:2)loftheanti- ethanol, and embedded in Epon. Ultrathin sections (80-nm
Velioranti-GOPCimmunoaffinitybeads.Afterwashing7times thickness)werecontrastedwithuranylacetatefor10minfol-
with PBS containing 0.5% Triton X-100, the beads were eluted lowedbyleadcitratefor5minandobservedwithanelectron
with0.5%TritonX-100and0.1Mglycine-HCl,pH2.5. microscope(H-600,Hitachi).
LectinBlotting—Therattesticularmembraneswerelysedin YeastTwo-hybridAssay—TheMATCHMAKERtwo-hybrid
6mlofTNEbuffercontainingproteaseinhibitors.Thelysate system3waspurchasedfromClontech.ThecDNAfragments
(800(cid:2)gofprotein)wassubjectedtoimmunoprecipitationwith encoding rat (cid:2)1A-adaptin and (cid:2)2-adaptin were inserted into
rabbit anti-Mar11 or preimmune serum. The eluted proteins theClaI-XhoIsitesofpGADT7.Thefragmentsencodingresi-
were separated on a 10% SDS-polyacrylamide gel and then dues 323–398 of MARCH-XI and its Y367A mutant were
24808 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER34•AUGUST24,2007
ANovelMARCHProteininSpermiogenesis
FIGURE1.CharacterizationofMARCH-XI.A,deducedaminoacidsequenceofratMARCH-XIisshown.Dots,proline-richsequences;underline,aRING-CH
fingerdomain;boxes,putativetransmembraneregions;doubleunderline,atyrosine-basedmotif;wavyline,aPDZbindingmotif.B,phylogeneticanalysisofrat
(r)andhuman(h)MARCHproteins.Thetreewasconstructedbytheneighbor-joiningmethodbyusingtheClustalWcomputerprogram.Thescalebar
representsageneticdistanceof0.2aminoacidsubstitutionspersite.TheGenBankTMaccessionnumbersareasfollows:hMARCH-I,NM_017923;hMARCH-II,
NM_016496;hMARCH-III,NM_178450;hMARCH-IV,NM_020814;rMARCH-IV,XP_001074008;hMARCH-V,AB191202;hMARCH-VI/TEB4,AB011169;hMARCH-
VII/axotrophin,NM_022826;hMARCH-VIII/c-Mir,BC025394;hMARCH-IX,NM_138396;rMARCH-IX,AB048842;hMARCH-X,Q8NA82;rMARCH-XI,AB048841;
Kir7.1potassiumchannel,NP_002233,asanunrelatedoutgroup.C,Northernblotanalysisof20(cid:2)goftotalRNAsamplesfromrattissueswasperformedwith
32P-labeledcDNAprobesforMARCH-XI(top)and(cid:4)-actin(bottom).kb,kilobase(s).D,reversetranscription-PCRanalysisofMARCH-XI(top)and(cid:4)-actin(bottom)
wasperformedontotalRNAsfromindicatedculturedcellsandrattissues.E,membranelysatesofrattestis(400(cid:2)gofprotein)wereimmunoprecipitated(IP)
withanti-Mar11(Im;lanes1and3)orpreimmuneserum(Pre;lanes2and4).TheimmunoprecipitateswereanalyzedbyWesternblotting(WB)withanti-Mar11
(Im;lanes1and2)orpreimmuneserum(Pre;lanes3and4).Arrowheadanddouble-arrowheadindicatethebandscorrespondingtoMARCH-XIandIgGheavy
pchHa1in1,.5re(slapneecsti5veanlyd.F6,)t,ootra1l%mTermitbonraXn-e1s0o0f(ClaOnSe7s7cealnlsdt8ra)nfosrfe3c0temdinwoitnhicMeA.SRaCmHp-XleIswweereretrseuabtjeedctwedithtoPaBhSi(glahnsepse1eadncden2t)r,i1fuMgNataioCnl(alatn1e0s03,0a0n0d(cid:4)4g),,0a.n1dMrNesau2lCtiOn3g,
supernatants(S)andpellets(P)wereanalyzedbyWesternblottingwithanti-Mar11.
inserted into the EcoRI-BamHI sites of pGBKT7, yielding using a vacuum. The membranes were blocked with T-TBS
pGBKT7-Mar11 and pGBKT7-Y367A, respectively. These containing 5% nonfat milk followed by incubation with His-
C
constructs were transformed into the yeast strain AH109 Mar11 orHis-Mar11 (cid:3)in2%TritonX-100overnightat4°C.
C C
(MATa)withtheFast-yeasttransformationkit(GenoTechnol- The membrane was probed with anti-His or anti-GST anti-
6
ogy). AH109 cells were transformed with pGADT7–53 and body(1:3000or1:1000inT-TBS)followedbyanti-mouseIgG
pGBKT7-TorwithpGADT7andpGBKT7,whichwereusedas secondary antibody conjugated with alkaline phosphatase or
apositiveornegativecontrol,respectively.Thedoubletrans- horseradishperoxidase,respectively(1:3000;JacksonImmuno-
formantsweregrownonSDagarplateslackingTrpandLeu, ResearchLaboratories).
withexceptionofthepGADT7-(cid:2)1-pGBKT7-Mar11 andpos-
C RESULTS
itive control clones which were grown on plates lacking Trp,
Leu,andHisfor3–10daysat30°C.Colonieswerepickedand IdentificationofaNovelMemberoftheMARCHFamily—The
restreakedonTrp(cid:2)/Leu(cid:2)/His(cid:2)platesforselectionofinteract- sequencesofanovelmammalianMARCHmemberwerefound
ingclonesandassayedfor(cid:4)-galactosidaseactivity.For(cid:4)-galac- by Blast searches of the GenBankTM expression sequence tag
tosidaseassay,asinglecolonywasgrownovernightat30°Cin2 (EST) databases (GenBankTM accession numbers AW070319,
mlofTrp(cid:2)/Leu(cid:2)media.Aportionofeachculturewascentri- AA868748,andAI046596).AcorrespondingcDNAfragmentwas
fuged,andtheyeastpelletwasresuspendedtoanopticaldensity obtainedbyPCRamplificationfromratbrainandusedtoscreena
of 0.2 at 600 nm in 0.1% bovine serum albumin and 25 mM rat testis cDNA library. Positive clones contained a single open
HEPES,pH7.5.Onehundredmicrolitersoftheyeastsamples readingframeencodingaproteinof398aminoacidresidueswith
were used to test (cid:4)-galactosidase activity with the Beta-Glo acalculatedmolecularmassof44.1kDa(Fig.1A).Aphylogenetic
assaysystem(Promega). analysis revealed that the cloned protein is the newest 11th
Dot-blot Assay—Various amounts of GST and GST fusion MARCHmembercloselyrelatedtoMARCH-IVand-IX(seeFig.
proteins were blotted onto Immobilon-P PVDF membranes 1BandsupplementalFig.S1forsequencealignment).Therefore,
AUGUST24,2007•VOLUME282•NUMBER34 JOURNALOFBIOLOGICALCHEMISTRY 24809
ANovelMARCHProteininSpermiogenesis
wedesignatedthisnovelprotein“MARCH-XI.”Ahydropathyplot
analysispredictedthatMARCH-XIhasatypicalMARCHmem-
branetopologywithN-andC-terminalcytoplasmictailsflanking
twotransmembranespans.AsshowninFig.1A,theN-terminal
tailcontainsproline-richsequences(dots)andaRING-CHfinger
(underline),andtheCterminuscontainsaPDZbindingsequence
(wavyline;theconsensussequenceE(S/T)X(V/I))(30).
NorthernblotanalysisofrattissuesshowedthatMARCH-XI
mRNA ((cid:6)1.7 kilobases) is predominantly expressed in testis
andweaklyinbrain(Fig.1C).Inaddition,reversetranscription-
PCRexperimentsrevealedthatMARCH-XIisalsoexpressedin
rat pituitary and the mouse pituitary corticotroph tumor cell
lineAtT20/D16v(Fig.1D).Toassesstheproteinexpressionof
MARCH-XI, we generated rabbit and rat polyclonal antisera
specifictoMARCH-XI(anti-Mar11,seesupplementalFig.S2).
Whenmembraneextractsofrattestisweresubjectedtoimmu-
noprecipitation followed by Western blot analysis with anti-
Mar11,asinglebandwasdetectedat(cid:6)48kDacorrespondingto
endogenous MARCH-XI protein (Fig. 1E, arrowhead).
MARCH-XIwasextractedfrommembranesbyTritonX-100
treatmentbutnotwithhighsaltoralkalinecarbonatesolu-
tion (Fig. 1F), indicating that MARCH-XI is a transmem-
braneprotein.
To examine whether the RING-CH finger of MARCH-XI
possessesanE3ubiquitinligaseactivity,invitroubiquitination
assays were performed using an established method with
recombinant GST fusion proteins of the MARCH-XI
RING-CHfinger(GST-RING)oritsmutantcontainingpoint
mutations in the conserved cysteine residues (C169S, C182S,
andC184S;GST-RINGmut).AsshowninFig.2A,GST-RING FIGURE2.UbiquitinligaseactivityofMARCH-XI.A,reactionmixturescon-
but not GST-RINGmut catalyzed the formation of polyubiq- tainingE1,E2(UbcH2,UbcH5B,UbcH5C,orUbcH7),andubiquitinwereincu-
batedinthepresenceofGSTfusionproteinsoftheMARCH-XIRINGfinger
uitinatedproductsinthepresenceoftheE2ubiquitin-conju-
(GST-RING)oritsmutant(GST-RINGmut)for4hat30°C.Ubiquitinatedmate-
gating enzymes UbcH5B and UbcH5C. Next, we sought to rialsweredetectedbyWesternblottingwithanti-ubiquitinantibody(top).
address whether the full-length MARCH-XI protein exhibits BottomshowsCoomassieBrilliantBluestainingoftheE2proteins(1(cid:2)g)used
inthisassay.B,plasmidsencodingHA-ubiquitin(0.6(cid:2)g)andFLAG-CD4(1(cid:2)g)
an E3 activity. A previous report has demonstrated that
wereco-transfectedintoCOS7cellstogetherwitheitheranemptypcDNA3
MARCH-IV down-regulates CD4 in a manner dependent on plasmid(pcDNA3;1.4(cid:2)g),MARCH-XI(MARCH-XI;1.4(cid:2)g),oritsRING-finger
mutant(RINGmut;1.4(cid:2)g).AftertreatmentwithconcanamycinA,membrane
thelysineresiduesinthecytoplasmictailofCD4(18),suggest-
lysatesweresubjectedtoimmunoprecipitation(IP)withanti-FLAGbeadsfol-
ing that CD4 is likely an E3 substrate for MARCH-IV. The lowedbyWesternblottingwithanti-HA(topleft),anti-FLAG(topmiddle),or
structuralsimilarityofMARCH-XItoMARCH-IVallowedus anti-Mar11antibody(topright).BottompanelsshowtheresultsofWesternblot-
tingofthelysates(10(cid:2)g;10%oftheinput)withanti-FLAG(left)oranti-Mar11
to determine whether MARCH-XI has ability to ubiquitinate
antibody(right).Arrowheadsandasteriskindicatethebandsofubiquitinated
CD4. FLAG-tagged CD4 (FLAG-CD4) was coexpressed in FLAG-CD4andofIgGheavychainusedforimmunoprecipitation,respectively.
COS7cellswithHA-taggedubiquitinintheabsenceorpresenceof
MARCH-XIoritsRING-fingermutant.Immunoprecipitationof (Fig. 3 and supplemental Fig. S3). No signal was observed in
FLAG-CD4 followed by Western blotting for HA-ubiquitin spermatogonia,spermatocytes,orsomaticcells(i.e.peritubu-
revealedincreasedlevelsofFLAG-CD4ubiquitinationinthecells lar,Leydig,andSertolicells).Ratspermiogenesisissubdivided
expressing wild-type MARCH-XI but not the RING-finger into19stepsonthebasisofthesizeandshapeofthenucleus,
mutant(Fig.2B).Inaddition,Westernblottingoftheanti-FLAG acrosome,andcellbodyofspermatids(1).MARCH-XIexpres-
immunoprecipitates revealed that both wild-type and mutant sionbecameprominentinearlyroundspermatidsatstep4(Fig.
MARCH-XIwereco-immunoprecipitatedwithFLAG-CD4,indi- 3B), remained until step 11 (Fig. 3, B–D), decreased at steps
cating that MARCH-XI acts as a ubiquitin ligase through its 12–15(Fig.3,EandF),anddiminishedafterstep16(Fig.3,A
RING-fingerdomain,targetingCD4forubiquitination.Together, andB).Suchstainingwasalsodetectedwithantigen-purified
theseresultsindicatethatMARCH-XIisatransmembraneubiq- antibodies but not detected with preimmune sera or antisera
uitinligasehighlyexpressedintestis. absorbedwiththeantigen(supplementalFig.S3).Theseresults
Stage-specific Expression in Rat Spermatids—To determine indicate that MARCH-XI is expressed in early developing
celltypesthatexpressMARCH-XI,immunofluorescencehis- spermatids.
tochemistryofrattesticularsectionswasperformedwithanti- SubcellularLocalizationinRoundSpermatids—Toidentify
Mar11. Strong punctate staining was observed in the middle structuresofearlyspermatidstowhichMARCH-XIassociates,
partoftheseminiferoustubules,wherespermatidsarelocalized immunoperoxidase electron microscopy was performed with
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FIGURE4.ImmunoelectronmicroscopyofMARCH-XIinratroundsper-
matids.Roundspermatids(steps4–6)werestainedwithrabbitanti-Mar11
FIGURE3.ImmunofluorescencestainingofMARCH-XIinrattestis.Sec-
antiserumbythepre-embeddingimmunoperoxidaseprocedure.Notethat
tionsofadultrattestiswerestainedwithrabbitanti-Mar11antiserum(green)
heavyimmunostainingforMARCH-XIwasdetectedinvesiclesoftheGolgi
andTO-PRO3(magenta)andobservedwithaconfocalmicroscope.StagesII–III
medulla(A)andtheMVB(B).N,nucleus;AG,acrosomalgranule;AV,acrosomal
(A),IV–VI(B),IX(C),XandXI(D),XII(E),andXIV–XVI(F)ofseminiferoustubulesare vesicle;G,Golgi;GM,Golgimedulla;M,mitochondrion.Bars,1(cid:2)m.
shown.Arabicnumbersindicatethestepsofthespermatids.Bar,20(cid:2)m.
anti-Mar11onrattesticularsections.Electronmicrographsof found to be colocalized with TGN38 (Fig. 5D). Furthermore,
roundspermatids(steps4–6;Fig.4andsupplementalFig.S4) whendoublestainingwasperformedwithanti-ubiquitinanti-
revealed that strong immunostaining of MARCH-XI was body FK1, which recognizes only polyubiquitinated proteins
detected in several vesicles in the Golgi medulla, a concave (33),theFK1signalwasmainlyobservedintheMARCH-XI-
regionnearthetrans-Golgistacks(Fig.4A).Densestainingwas positivecompartments(Fig.5E).Theseresultsestablishthat,in
alsofoundinmembranesoftheMVBs(Fig.4B).Nosignalwas roundspermatids,MARCH-XIislocalizedtotheTGN-derived
observedintheGolgioracrosomalvesiclemembranes. vesiclesandMVBs,bothofwhichcontainAP-1andpolyubiq-
To characterize the MARCH-XI-positive vesicles, double uitinatedproteins.
immunofluorescence staining was performed for isolated rat Association with Fucose Glycoproteins—Given the fact that
round spermatids in smear samples. Confocal microscopy duringacrosomalbiogenesis,fucose-containingglycoproteins
revealed that MARCH-XI-specific immunoreactivity was aretransportedtotheMVBsfromtheTGN(4,6),weexamined
detected as a perinuclear fuzzy staining and several spherical whetherfucoseglycoproteinsarelocalizedtotheMARCH-XI
punctathroughoutthecytoplasm(Fig.5,leftandsupplemental positive compartments. Rat round spermatids were stained
Fig.S5).Thefuzzystructureappearedaclusterofsmallvesicles, with anti-Mar11 and AAL that binds specifically to an (cid:5)1,6-
asshownathighermagnification(Fig.5A,right,inset)andwere fucose residue attached to the asparagine-linked N-acetyl-D-
consistentlysurroundedbysignalsfortheTGNmarkerprotein glucosamine (34). As shown in Fig. 6A, confocal imaging
TGN38 (31) (Fig. 5A, asterisk). In this structure MARCH-XI revealedthatoverlapbetweenMARCH-XIandAALstainingin
was colocalized with (cid:3)-adaptin, a subunit of the heterotet- both the TGN region (arrow) and peripheral puncta (arrow-
rameric adaptor protein complex (AP)-1 associated with the head),raisingthepossibilitythatMARCH-XIformscomplexes
vesicles in the Golgi medulla (32) (Fig. 5B, arrowhead). The with them. We assessed this possibility by immunoprecipita-
colocalizationwasalsodetectedinsomesphericalMARCH-XI tion.Themembraneextractsofrattestiswereimmunoprecipi-
puncta,mostlikelycorrespondingtotheMVBs(Fig.5B,arrow). tatedwithanti-Mar11orpreimmuneserum,andtheimmuno-
In contrast, MARCH-XI was clearly distinguished from precipitates were separated by electrophoresis followed by
anothermedullacomponentGOPC(11)(Fig.5C),whichwas transfertoaPVDFmembrane.ThefilterwasblottedwithAAL
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ANovelMARCHProteininSpermiogenesis
FIGURE6.AssociationofMARCH-XIwithfucoseglycoproteins.A,twotop
andbottomleftpanels,ratroundspermatidsinsmearpreparationswereincu-
batedwithrabbitanti-Mar11antiserumandbiotinylatedAALfollowedby
AlexaFluor546-conjugatedanti-rabbitIgGandAlexaFluor488-conjugated
anti-biotinantibodies.ThenucleuswasstainedwithHoechst33342.Theflu-
orescence signals for MARCH-XI (magenta), AAL (green), and the nucleus
(blue)weredetectedwithaconfocalmicroscope.Arrowandarrowheadsindi-
cateco-stainingofMARCH-XIandAALintheGolgimedullaandperipheral
FIGURE5.ImmunofluorescenceconfocalmicroscopyofMARCH-XIin puncta,respectively.Bottomrightpanel,asanegativecontrolofAALstaining,
ratroundspermatids.Ratroundspermatidsinsmearpreparationswere the spermatids were stained with Alexa Fluor 488-conjugated anti-biotin
stained with rabbit anti-Mar11 (A, B, and E; magenta), rat anti-Mar11 antibodies(green)followedbyHoechst33342(blue).Bar,2(cid:2)m.B,membrane
(C;magenta),orrabbitanti-GOPCantiserum(D;magenta)followedbymouse lysatesofrattestiswereimmunoprecipitatedwithanti-Mar11(lanes2,5,and
monoclonalantibodiestoTGN38(AandD;green),(cid:3)-adaptin(B;green),or 8)orpreimmuneserum(lanes3,6,and9).Theimmunoprecipitates(IP)were
ubiquitin(cloneFK1;E;green)orrabbitpolyclonalantiserumtoGOPC(C;green). analyzedbylectinblottingwithAAL(leftpanel)orbyWesternblotting(WB)
NucleiwerestainedwithHoechst33342(blue).Thefluorescencesignalswere withanti-ubiquitin(cloneUbi-1)antibody(topright)orwithanti-Mar11anti-
detectedwithaconfocalmicroscope.TheinsetinAshowsahighermag- serum(bottomrightpanel).Thelysate(32(cid:2)g;4%oftheinput)wasrunin
nificationimageoftheMARCH-XI–positivestructureintheGolgimedulla parallel(lanes1,4,and7).Themiddlepanelshowsaprolongedexposureof
(asterisk).ArrowheadsandarrowsindicatecolocalizationofMARCH-XIand theleftpanel.NotethatsignalsforAALandanti-ubiquitinareseeninthe
markerproteinsintheGolgimedullaandperipheralpuncta,respectively. anti-Mar11immunoprecipitates(asterisk).
NotethatMARCH-XIisnotcolocalizedwithTGN38(asterisk)orGOPC(dou-
blearrowheads).Bar,2(cid:2)m.
AssociationwithAP-1—AsequencealignmentoftheC-ter-
minal cytoplasmic tails of MARCH-IV, -IX, and -XI revealed
to detect fucose glycoproteins. The high molecular weight
thepresenceofaconservedtyrosine-basedmotif,YXXL,which
smearAALsignalwasdetectedintheanti-Mar11immunopre- isknowntoberecognizedbytheAP(cid:2)-subunits(12,35)(Fig.
cipitates(Fig.6B,lane2,asterisks)butnotinthecontrolpre-
1A,double-underline,andsupplementalFig.S1).Inadditionto
immune precipitates (Fig. 6B, lane 3), indicating that
thisfinding,thecolocalizationbetweenMARCH-XIandAP-1
MARCH-XIassociateswithfucoseglycoproteins.Noteworthy, led us to determine whether the AP-1 (cid:2)-subunit (cid:2)1-adaptin
asimilarstainingpatternwasobservedwhenWesternblotting
can recognize the tyrosine-based motif of MARCH-XI
was performed with anti-ubiquitin antibody (Fig. 6B, lane 5, (367YVLL370) using the yeast two-hybrid system. We con-
asterisk).Thisresulttogetherwithourobservationofthecolo- structed the pGADT7 plasmids encoding (cid:2)1-adaptin or
calization between MARCH-XI and ubiquitinated proteins (cid:2)2-adaptin (the AP-2 (cid:2) subunit) and the two-hybrid bait
(Fig. 5E) suggests that a certain portion of the MARCH-XI- pGBKT7plasmidsencodingthelast76-aminoacidresiduesof
associatedfucoseglycoproteinsareubiquitinated.Itispossible MARCH-XI or its mutant where Tyr367 was replaced by an
that these glycoproteins are in vivo substrates for the alanine residue. An interaction between the MARCH-XI tail
MARCH-XIubiquitinligase. and(cid:2)1-adaptinwasdetectedbygrowthonahistidine-deficient
24812 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER34•AUGUST24,2007
ANovelMARCHProteininSpermiogenesis
evidencefortheassociationbetweenMARCH-XIandAP-1in
vivo,membraneextractsofrattestisweresubjectedtoimmu-
noprecipitationwithanti-Mar11followedbyWesternblotting
withanti-(cid:3)-adaptinantibody.AsshowninFig.7D,(cid:3)-adaptin
wasco-immunoprecipitatedwithanti-Mar11(middlelane)but
notwithpreimmuneserum(rightlane).Togethertheseresults
suggestthatMARCH-XIassociateswithAP-1throughbinding
to(cid:2)1-adaptinviaitscytoplasmictyrosine-basedmotif.
InteractionwithVeliPDZProteins—Wepreviouslydemon-
stratedthatthePDZbindingmotifofMARCH-IIdirectlybinds
to the PDZ protein Veli-3 (25). Because Veli is known to be
expressedinrattestis(36,37),wedeterminedwhetherthePDZ
binding motif of MARCH-XI (395VTSV398) is recognized by
Velibyadot-blotanalysiswitharecombinantHis -taggedpro-
6
teincontainingthelast76-aminoacidresiduesofMARCH-XI
(His-Mar11 ).PurifiedGSTfusionproteinsofthePDZdomain
C
of Veli-3 (GST-Veli) were spotted on a PVDF membrane. In
parallel, GST as a negative control and the PDZ domain of
GOPC(GST-GOPC),awellcharacterizedspermatidPDZpro-
tein(11),werealsospotted.Themembranewasoverlaidwith
purifiedHis-Mar11 ,andthenthesignalsfortheHis tagwere
C 6
detected with anti-His antibody. A robust binding was
6
detected between His-Mar11 and the Veli PDZ domain,
FIGURE7.AssociationofMARCH-XIwiththeadaptinsubunitsofAP-1. C
whereasamuchweakerbindingaffinitywasobservedwiththe
A,AH109yeastcellswerecotransformedwithaplasmidencodingaGAL4
activatingdomainfusionproteinof(cid:2)1-adaptin((cid:2)1)or(cid:2)2-adaptin((cid:2)2)anda GOPCPDZdomain(Fig.8A,leftpanel).Nobindingwasobvi-
plasmidencodingaGAL4DNAbindingdomainfusionproteinoftheC-termi- ous with His-Mar11 lacking the PDZ binding motif (His-
nalregionofMARCH-XI(WT)oritsY367Amutant(Y367A).Thecellswere C
grownonplateswith((cid:5)His)orwithout((cid:2)His)histidine.Theemptyvectors Mar11C(cid:3);Fig.8A,middlepanel).Tofurtherexaminetheinter-
wereusedasanegativecontrol,andthevectorsencodingaGAL4activating action between MARCH-XI and Veli, COS7 cells were
domainfusionproteinofp53(p53)andaGAL4DNAbindingdomainfusion
transfected with expression plasmids encoding Myc-tagged
proteinofSV40largeTantigen(T)wereasapositivecontrol.B,fiveindepend-
entcloneswereselectedandgrowntoA valuesof0.2,and(cid:4)-galactosidase Veli-3 and either mock, MARCH-XI, or its deletion mutant
activitiesweremeasuredwiththeBeta6-0G0loassaysystem.(cid:4)-Galactosidase lackingthelastaminoacidVal398(MARCH-XI(cid:3)).Celllysates
activitiesinrelativeunitsarepresentedasmeanvalues(cid:7)S.E.(bars).The
asteriskindicatesasignificantdifferencebetweentheWT-(cid:2)1-andY367A-(cid:2)1- wereimmunoprecipitatedwithanti-Mar11followedbyWest-
transformedcells(p(cid:8)0.01,Student’sttest).NS,notsignificant.C,COS7cells ernblottingwithanti-Mycantibody.AsshowninFig.8B,Myc-
weretransientlytransfectedwithanexpressionplasmidfor3(cid:4)FLAG-tagged tagged Veli-3 was co-immunoprecipitated with wild-type
(cid:2)1-adaptinintheabsence(Mock)orpresenceofthatforMARCH-XI(WT)orits
mutant with the Y367A mutation (Y367A). Cell lysates were subjected to MARCH-XI(lane2)butnotwithMARCH-XI(cid:3)(lane3),con-
immunoprecipitation(IP)withanti-Mar11.Theanti-Mar11immunoprecipi- firmingtheVeli-MARCH-XIinteraction.Furthermore,toeval-
tates(toptwopanels)andthelysates(20(cid:2)g;20%oftheinput;twobottom
uatetheirassociationinvivo,Tritonextractsofrattestiswere
panels)wereanalyzedbyWesternblotting(WB)withanti-FLAGantibody
(FLAG) or anti-Mar11 (MARCH-XI). The asterisk indicates the bands of IgG immunoprecipitated with anti-Veli antibodies followed by
heavychainusedforimmunoprecipitation.D,membranelysatesofrattestis Westernblottingwithanti-Mar11.MARCH-XIwasdetectedin
wereimmunoprecipitated(IP)withanti-Mar11(middlelane)orpreimmune
serum(rightlane).Themembraneextracts(20(cid:2)g;5%ofinput;leftlane)and the anti-Veli immunoprecipitates (Fig. 8C, lane 2) but not in
immunoprecipitates(righttwolanes)wereanalyzedbyWesternblottingwith thoseofthecontrol(Fig.8C,lane3).Incontrast,MARCH-XI
anti-(cid:3)-adaptinantibody(top)oranti-Mar11antiserum(bottom).
wasnotcoimmunoprecipitatedwithGOPC(supplementalFig.
S6). Veli expression in round spermatids was confirmed by
plate(Fig.7A)andbythe(cid:4)-galactosidaseassay(Fig.7B).This immunofluorescenceconfocalmicroscopyshowingitsdiffuse
interactionwasabolishedbyaY367Amutation.Incontrast,no localization throughout the cytoplasm (Fig. 8D). Moreover,
interactionwasdetectedbetweenMARCH-XIand(cid:2)2-adaptin. subcellular fractionation analysis of rat testis revealed the
TheseresultsindicatethattheYVLLsequenceofMARCH-XI membraneassociationofVeli(Fig.8E).Thus,theseresultsindi-
isresponsibleforthespecificinteractionwith(cid:2)1-adaptin.To catethatMARCH-XIinteractswithVelithroughitsC-terminal
further examine the interaction between MARCH-XI and PDZbindingmotif.
(cid:2)1-adaptin in mammalian cultured cells, COS7 cells were
transfected with expression plasmids encoding 3 (cid:4) FLAG- DISCUSSION
tagged(cid:2)1-adaptinandeithermock,MARCH-XI,oritsmutant Inthisstudy,weperformedtheidentificationandcharacter-
with the Y367A mutation. Cell lysates were immunoprecipi- izationofMARCH-XI,anoveltransmembraneubiquitinligase
tatedwithanti-Mar11followedbyWesternblottingwithanti- that is predominantly expressed in round spermatids of rats.
FLAGantibody.AsshowninFig.7C,FLAG-tagged(cid:2)1-adaptin Thestage-specificexpression(Fig.3)suggeststhatduringearly
wasco-immunoprecipitatedwithwild-typeMARCH-XI(mid- spermiogenesis,thespecificubiquitinsystemshouldbeutilized
dlelane)butnotwithitsY367Amutant(rightlane),confirming foracertainpurpose.Themajoreventinearlyspermiogenesis
binding of (cid:2)1-adaptin to MARCH-XI. Finally, to obtain the is acrosomal formation, which is thought to be achieved by a
AUGUST24,2007•VOLUME282•NUMBER34 JOURNALOFBIOLOGICALCHEMISTRY 24813
ANovelMARCHProteininSpermiogenesis
thatubiquitinconjugatesaredetectedintheTGNareaatearly
spermiogenicstages(steps1–7)andincreasedintheMVBsat
laterstages(steps8–15)(17).Inaddition,Eps15,aubiquitin-
bindingadaptorproteinwitharoleinendocytosisandvesicular
trafficking,hasbeenfoundtobeassociatedwithAP-1-positive
vesiclesintheGolgimedulla(10).Itis,therefore,reasonableto
assumethatubiquitinmodificationcontrolstheselectivepro-
teinsortingtotheMVBpathway.WeshowedthatMARCH-XI
iscolocalizedwithubiquitinatedproteins(Fig.5E)andassoci-
atedwithfucoseglycoproteins,someofwhichappearedtobe
ubiquitinated(Fig.6B).PossiblyMARCH-XImaymediatethe
sortingoffucoseglycoproteinsdestinedtotheMVBsbyubiq-
uitinatingthem.Furtherstudiesarenecessarytodeterminethe
molecularidentityoftheinvivoE3substratesforMARCH-XI.
OurdataprovideevidenceforanovelroleoftheMARCHfam-
ilyintheregulationofspermiogenesis.
TheGolgimedullaofroundspermatidscontainsnumerous
vesicularandtubularmembranestructures,includingclathrin-
coated vesicles (4, 31, 38). MARCH-XI should be sorted to
AP-1/clathrin-coatedvesiclesbecause1)MARCH-XIiscolo-
calizedwith(cid:3)-adaptinneartheTGN(Fig.5B),2)theC-termi-
nalYVLLsequenceofMARCH-XIisspecificallyrecognizedby
(cid:2)1-adaptin (Fig. 7, A–C), and 3) MARCH-XI associates with
(cid:3)-adaptin(Fig.7D).Inearlyspermatids,clathrin-coatedbuds
havebeenfoundontheTGNmembrane(4,31,38),implying
FIGURE8.InteractionofMARCH-XIwithVelithroughthePDZbinding that the spermatid AP-1 mediates MARCH-XI sorting at the
motif.A,dot-blotanalysis.Variousamounts(1,3,15,and30pmol)ofpurified
GST,GST-Veli,andGST-GOPCwereblottedonPVDFmembranes.Themem- TGN,couplingwiththeformationofclathrin-coatedvesicles.
braneswereincubatedwitheitherHis-Mar11 (left)orHis-Mar11 (cid:3)(middle) Inyeastandmammaliansomaticcells,ithasbeenassumedthat
C C
followedbyanti-His antibody.ToconfirmthepresenceoftheGSTproteins,
6 AP-1isutilizedintheTGN-to-endosometrafficking.However,
themembranewasincubatedwithanti-GSTantibody(right).B,COS7cells
were transiently transfected with an expression plasmid for Myc-tagged thestudiesusingAP-1-deficientcellshaveindicatedthatAP-1
Veli-3intheabsence(lanes1and4)orpresenceofthatforMARCH-XI(lanes2 more likely mediates the retrograde endosome-to-TGN traf-
and5)orMARCH-XI(cid:3)(lanes3and6).Celllysatesweresubjectedtoimmuno-
ficking (39–41). Thus, another possibility is that AP-1 is
precipitation(IP)withanti-Mar11.Theanti-Mar11immunoprecipitates(lanes
1–3)andthelysates(20(cid:2)g;4%oftheinput;lanes4–6)wereanalyzedby involved in retrieval of MARCH-XI back to the TGN region
Westernblotting(WB)withanti-Mar11(top)oranti-Mycantibody(bottom). fromtheMVBs,allowingMARCH-XItoescapethelysosomal
ThearrowheadandasteriskindicatethebandsofMARCH-XIandofIgGheavy
chainusedforimmunoprecipitation,respectively.C,membranelysatesofrat degradation.AnywayitcouldbearguedthatAP-1wouldfunc-
testiswereimmunoprecipitatedwithanti-Veli(lane2)orcontrolIgGbeads tionintheTGN-MVBpathwayinearlyspermatids.However,
(lane3).Themembraneextracts(20(cid:2)g;1%ofinput;lane1)andimmunopre-
takingaccountofthefactthatclathrinandGolgi-residentpro-
cipitates(lanes2and3)wereanalyzedbyWesternblottingwithrabbitanti-
Mar11antiserum(top),correspondingpreimmuneserum(middle),andanti- teinshavebeendetectedontheacrosomalmembrane(7,8,42),
Veliantiserum(bottom).Thearrowheadindicatesthebandcorrespondingto we cannot rule out the possible role of AP-1 in retrieval of
MARCH-XI.D,ratroundspermatidswerestainedwithratanti-Mar11anti-
mislocalizedGolgiproteins.
serumfollowedbyrabbitanti-Veli(top)orantigen-preabsorbedanti-Velianti-
serum(bottom).SignalsforVeli(leftandgreeninright),MARCH-XI(magentain AdditionalfindingofthisstudyisthatMARCH-XIinteracts
right),andnucleistainedwithHoechst33342(blueinright)weredetected
withthePDZproteinVeli.ThreeVeliisoforms(Veli-1,-2,and
withaconfocalmicroscope.Bar,2(cid:2)m.E,homogenatesofrattestiswere
subjectedtoahighspeedcentrifugation(25,000rpmonanSW28rotor).The -3)arerecognizedinvariousmammaliantissues(36).Allthree
resultingsupernatant(S)andmembranepellet(P)wereanalyzedbyWestern isoforms are likely to be expressed in rat testis because our
blottingwithanti-Mar11(top)andanti-Veliantiserum(bottom).
anti-VeliantiserumdetectedtriplexbandsbyWesternblotand
immunoprecipitationanalyses(Fig.8,CandE).Itisunknown
continuous fusion of Golgi-derived vesicles to the immature whichisoformisexpressedandinteractswithMARCH-XIin
acrosomal vesicle (1). In addition to this acrosomal pathway, roundspermatids,whereasthedot-blotanalysisdemonstrated
previouscytochemicalstudiesreportedthatnewlysynthesized thedirectbindingofVeli-3(Fig.8A).Althoughnopreciserole
fucoseglycoproteinsaredeliveredtotheMVBs(4,6),suggest- hasbeenassignedtoeachVeliisoform,theyplaycrucialrolesin
ingthatatleasttwoanterogradetransportpathwaysexistfrom 1)thedockingandtransportofsynapticvesiclestothesynaptic
theGolgi.Themembraneflowtotheendosomalsystemshould plasmamembranesofneurons(43,44)and2)thesortingand
be selective because some endosomal and lysosomal proteins stable localization of basolateral transmembrane proteins in
(i.e.mannose6-phosphatereceptorsandlysosome-associated polarizedepithelialcells(45–49).Additionally,wehaveprevi-
membrane proteins (LAMPs) are segregated from the acro- ouslydemonstratedthatthePDZbindingmotifsofMARCH-II
some (5, 32). The subcellular localization of MARCH-XI and-IIIareimportantfortheirefficientexitfromtheendoplas-
stronglysuggeststhatMARCH-XIalsofollowstheTGN-MVB mic reticulum (24, 25). Thus, the interaction with Veli may
pathway.Apreviouselectronmicroscopicstudydemonstrated facilitatethetransportandsubcellularlocalizationofMARCH-
24814 JOURNALOFBIOLOGICALCHEMISTRY VOLUME282•NUMBER34•AUGUST24,2007
ANovelMARCHProteininSpermiogenesis
XI.WealsoshowedthattheGOPCPDZdomaincanbindtothe 17. Haraguchi, C. M., Mabuchi, T., Hirata, S., Shoda, T., Hoshi, K., and
PDZ binding motif of MARCH-XI, albeit with much lower Yokota,S.(2004)J.Histochem.Cytochem.52,1393–1403
18. Bartee,E.,Mansouri,M.,HoveyNerenberg,B.T.,Gouveia,K.,andFru¨h,
affinity than Veli-3 (Fig. 8A). Nevertheless, it is unlikely that
K.(2004)J.Virol.78,1109–1120
MARCH-XIinteractswithGOPCinvivobecausetwoproteins
19. Goto,E.,Ishido,S.,Sato,Y.,Ohgimoto,S.,Ohgimoto,K.,Nagano-Fujii,
werenotcolocalized(Fig.5C)orcoimmunoprecipitated(sup-
M.,andHotta,H.(2003)J.Biol.Chem.278,14657–14668
plementalFig.S6). 20. Lehner,P.J.,Hoer,S.,Dodd,R.,andDuncan,L.M.(2005)Immunol.Rev.
WedemonstratedthatMARCH-XImediatesCD4ubiquiti- 207,112–125
nationinCOS7cells(Fig.2B)butthereisnoevidenceforCD4 21. Bartee,E.,McCormack,A.,andFru¨h,K.(2006)PLoSPathog.2,975–988
22. Hoer,S.,Smith,L.,andLehner,P.J.(2007)FEBSLett.581,45–51
expression or ubiquitination in spermatids (50). It is evident
23. Ohmura-Hoshino,M.,Matsuki,Y.,Aoki,M.,Goto,E.,Mito,M.,Uematsu,
thatMARCH-XIwouldshareoverlappingE3-substraterecog-
M.,Kakiuchi,T.,Hotta,H.,andIshido,S.(2006)J.Immunol.177,341–354
nition as well as the characteristic sequence motifs with 24. Fukuda,H.,Nakamura,N.,andHirose,S.(2006)J.Biochem.(Tokyo)139,
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are structurally and functionally relevant with each other. In 25. Nakamura,N.,Fukuda,H.,Kato,A.,andHirose,S.(2005)Mol.Biol.Cell
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26. Mansouri,M.,Bartee,E.,Gouveia,K.,HoveyNerenberg,B.T.,Barrett,J.,
sion throughout human tissues (18). Interestingly, when
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ectopically expressed, MARCH-IX is localized to the TGN,
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colocalizing with AP-1 (18), and to the late endosomes and 27. Nakamura,N.,Kimura,Y.,Tokuda,M.,Honda,S.,andHirose,S.(2006)
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