R E V I E W T H E O R G A N I Z A T I O NH U MA N R E D B L O O DARevi ew OF P R O T E I N S I N C E L L ME MB R A N ETHE T HE ODOR E L. S T E C K FromtheDepartmentsofBiochemistryandMedicine,TheUniversityof Chicago,Chicago, lllnois 60637 I.I NTRODUCTI ON Theel uci dat i onof themol ecul ararchi t ect ureofcell membranesis acent ralgoalfor cell biology,as structurelies atthehear t of function.Theeryt hro- cyt epl asmamembr anehaslongprovidedafa- voredtestinggroundforthisinquiry.Humanred bl oodcellsarereadi l yavai l abl e, relatively homo- geneous,andrel evant tomedicine.Thei rpl asma membranescanbeeasily isolated intactandessen- t i al l yfreeofcont ami nat i onfromot hercells, organelles, andcyt opl asmi ccontents.Thi smem- braneiscompl exenoughtobei nt erest i ngand, to some degree,representative,yet it is simple enough tobeanalyzedasawhole.Theseci rcumst ances makeitlikelyt hat thehumanredcellpl asma membr anewillbethefirstwhosemol ecul aranat - omyisknowninanydegreeof satisfying detail.Thel i t erat ureconcerningtheprot ei nsof eryt h- rocyt emembr anesandmembr anesingeneralhas beenthesubjectofrepeat edreview(19).This art i cl ewillfocusonthel ocal i zat i on andmodesof associ at i onof i ndi vi dual maj or pol ypept i des within t he humanred cell membr ane.II. THEPROTEI NF RACTI ON Redcellmembranescanbepurifiedaft erosmot i c hemol ysi sindilute, mi l dl yal kal i nebufferby repeat edwashinguntilthe"ghost s"(or" s t r o ma ' )arepear l ywhite(cf.10-12).Thehemogl obi n-free ghost hasamassof 11-12x10 - I sg(10).Taki ng themembr anedensi t yas1.15g/ cm 3 (13)andits surfacear eaas140~tm z (14),at hi cknessof 75A maybecal cul at ed, ingoodagreement withelec- t r onmi croscopestudies(15). Theprot ei nscanbeseparat edfromt helipids by ext ract i ngmembr aneswithpol ar organi csolvents (10,16-18).Afewpercentoftheprot ei nis l ostto theorgani cphase,suggestingt hepresenceofhydr ophobi cprot ei nsorprot eol i pi dcomplexes (16,18). Appr oxi mat el y52%of themembr ane massisprot ei n, 40%islipid, and8%iscarbohy- dr at e(10,18,19). Onl yabout 7%ofthissugaris carri edbygl ycosphi ngol i pi ds(20);therestt akes theformof smal l ol i gosacchari desl i nkedviaO- andN-gl ycosi di cbondstoserine(threonine)and asparagi ne residues in the membr anegl ycoprot ei ns (cf.19,21). Theami noacidcomposi t i onoftheer yt hr ocyt e membr aneprot ei nfract i onisnotdistinctively differentfromt hat det ermi nedforot hermem- branesor, indeed,formi xt uresof soluble prot ei ns (4,22).However, i ndi vi dual proteins, and, mor e par t i cul ar l y, t opogr aphi cal l y specified domai nsofcert ai npol ypept i des,havecharact eri st i ccomposi - t i onswhichrel at etot hei rdi sposi t i oninthe THEJOURNALOFCELLBIOLOGYVOLUME62, 1974p a g e s 1 - 1 9 1 on June 26, 2015jcb.rupress.orgDownloaded from Published July 1, 1974membr ane(4,9,19,23-25). Thereisadistinct excessofacidic(21%)overbasic (12%)residuesin theprot ei nfraction(18),consistentwithanappar -entneti soel ect ri c poi nt of pH4- 5(16,17). While cysteinerepresentsonlyabout 1%of ami noacid residues,thepar t i cul ar functionalsignificance and chemi cal react i vi t yof itssulfhydrylgroupinsitu havebeenthesubjectof severalinvestigations(cf. 26,27). I I I . THEPOLYPEPTI DES Whenredcellmembr anesaredissolvedinan excessofsodiumdodecyl sulfate(SDS), thepol y- pept i desaredenat ur edandseparat edfromthe lipidsandfromoneanot her, asdemonst rat edby pol yacr yl ami degelel ect rophoresi s(11,28,29). Despitethesi mpl i ci t y,sensitivity, andhighresolu- tionofthistechnique,itisstillsubjecttocertain l i mi t at i onsandanomal i es(cf.4).Inaddition,archi t ect ural andfunct i onal i nformat i onislost uponSDSt r eat ment , unlesssuchfeaturesare identifiedbeforemembr anedi ssol ut i on, asillus- t r at edlater.Fig.1 Ais adensi t omet ri cscanof theCoomas-siebl ue-st ai nedghostpol ypept i des onapol yacryl - ami degelaft erel ect rophoresi sinSDS. Tabl eI listsest i mat esofthemol ecul arweightandmass fract i onofeachmaj or membranecomponentderivedfromsuchprofiles.Whi l ethesevaluesare, atbest,appr oxi mat i ons, t heyaregeneral l ysup- port edbyindependentevidence.For exampl e,band6,thegl yceral dehyde3-Pdehydrogenase ( G3PD) pr ot omer (30),isknownbyot hermeans tohaveamol ecul arweightof~35, 000(31)andto const i t ut e4- 7%of themembr aneprot ei n(I 1,30, 32).Bands1,2,and5havebeenselectively eluted fromthemembr aneandshowntocompri se 25-30%of itsprot ei n(1 I,33-35). Gelfi l t rat i on chr omat ogr aphyof ghostpol ypept i des dissolved in 6Mguani di ne- HCl hasprovi dedi ndependentsupport ofthemol ecul arweightscal cul at edfrom SDS- gel mobi l i t i es (29,36).Gelel ect rophoresi s in ot her denat uri ngdet ergent sandatot herpHvalues (37)andtheuseofot herprot ei nstains(1 i )give si mi l arprofiles. Thereisgoodevidencet hat (with oneexception, see below) thecomponent slisted in Tabl eIarenotaggregat esorcomplexes, since t heycannot bereducedinsizebyavarietyof dis- sociatingagents;however,theyarequitevulner- abl etoprot eol yt i cdegr adat i on(11,38).Ghost s preparedbyseveraldifferentmet hodsallyield si mi l arel ect r ophor et ogr ams(39).Thepol ypep- t i deprofilesfromavari et yof mammal i aneryt h- rocyt esourcescloselyresembl ethehumanl the gl ycoprot ei npat t ernsaremorediverse(33,40-45).Someghost -associ at edprot ei nsmaybelost duri ngeryt hrocyt emat ur at i on, atl east int he r abbi t (46). TheCoomassi eblue-stainedpol ypept i des listed inTabl eIaccountforabout t hree-quart ersofthe st ai nedprofile. Therearealso manymi norcompo-nents(suchasbands2. 1-2. 3, andthebr oad,complexzonedesi gnat ed4.5),someofwhichare obscuredby t hemaj or gel component s. For exam- ple,t hereareonlyafewhundredNa +, K +- st i mul at edATPasesitespercell(47),whose 82pO4-acylphosphate i nt ermedi at e comi grat es with thevastexcessofband3pol ypept i des (48)andis t hereforenotvisualizedinstainedgels. Still ot hercomponent s, theheavily gl ycosyl at ed gl ycoprot ei ns,arenotreadi l y det ect ed by Coomas-sieblueandot herconvent i onal prot ei nstains(4, 11),butaremadevisiblebytheperi odi cacid- Schi ff( PAS) stainingtechnique(Fig. 1B).Thei rel ect rophoret i cmobi l i t i esmaybeanomal ouswith respecttonongl ycosyl at edreferenceprot ei ns,maki ngt hei rmol ecul ar weightassi gnment sam- bi guous (cf.49. 50). ThePASprofi l ebearsaclose resembl ancetothedi st ri but i onofsialic acidinthe gel,as det ermi nedbydirectchemi calanal ysi s (11, 51,52)and, mor epart i cul arl y,by ther adi oact i vi t y profileof membraneprot ei nsgentlyreact edwith peri odi c acid (soas tooxidize only si al at e residues) andthenreducedby t r i t i at ed bor ohydr i de (53,54). Themaj or si al ogl ycoprot ei n cont ai ns64%car- bohydrat e, including 28%N- acet yl neur ami ni c acid (19);ithasbeenidentifiedwiththebindinglocus forinfluenza virus, certainbl ood groupi soant i bod- ies,andcert ai npl ant hemaggl ut i ni ns(cf.9,19,55, 56).Theest i mat eof 500,000si al ogl ycoprot ei n mol ecul espercell(Tabl eI)isinexcellentagree- ment withtheprevalenceof theselectinbinding sitesonhumaneryt hrocyt es(55,57).Itnow appearst hat bandsPAS- I andPAS- 2act ual l y representi nt erconvert i bl e formsofthesamemol e- cule(58).Thisunusualcaseshouldremi ndust hattheSDS-gel el ect rophoresi s systemisnot ani dealone(cf.reference4). Thereareot hermembr anegl ycoprot ei nswhich arenotwellrepresentedbythePAStechnique, presumabl ybecausetheyhavealowsialicacid content.Band3(Fig. 1A)isoneexampl e: it cont ai nsappr oxi mat el y7%car bohydr at e(1,59) andcont ri but esroughly10%of thet ot al mem- branesugar, yet itisvisualized inthePASprofile,atbest,onlyasasmal l shoulderonthet rai l i ng 2THEJOURNALOFCELLBIOLOGY-VOLUME62,1974 on June 26, 2015jcb.rupress.orgDownloaded from Published July 1, 19741.2 E0.8 o t O < 0.4 0 0.4 E E 0 ' , 0 t O < 0.2 , 2 A .1 4>2 4.1 t IPAS-1 LI PI D TD PAS-2 2'0' 4 ' 0 6 ' 0 ' 8 ' 0MI GRATI ON, mm FIGURE1Erythrocyte membranepolypeptides andglycoproteins.(A) a densitometric scanof a gel stained forproteinwith Coomassie blueafter electrophoresis of10 #1 of packedghosts(40 #gprotein).(B) asimilar gel stainedfor carbohydratewithPASreagent.Proceduresandnomenclatureare describedin references11 and118.H,polypeptidechainsfromhemoglobin;TD,inkedneedlestabrecordingthepositionofthe trackingdye.See TableIfordata. edgeof t hePAS- 1peak(Fig. 1B).Si mi l ar l y,t r eat i ngme mbr a ne s withgal act oseoxi daseplus t r i t i at edbor ohydr i dest r ongl yl abel sseveral ot her -wisei ndi st i nct component s int hepoor l yst ai ned regi onof PAS- 4andPAS- 2(orzone4.5)(54,60, 61).Suchdat ama ke itcl ear t hat apreci se enumer at i onof t heredcel l me mbr a ne pol ypep-t i desisani mpor t ant , unfi ni shedt ask.IV. AS YMME T R I C AL DI S T R I B UT I ON Some model s of me mbr a ne mol ecul ar or gani za-t i onhavepost ul at edt hat pr ot ei nscanservea st r uct ur al (i.e.st abi l i zi ngor spacefilling)rol ein t hemembr ane, cont r i but i ngt oitsfabri cr at hert hanitsf unct i on(cf.62,63).Suchf or mul at i ons do not r equi r et hat t hepr ot ei nsbespeci fi cal l yar- rangedwithrespect tot heme mbr a ne ' s t wosur- faces. Aconver sevi ewpoi nt r ecogni zest hat ever y bi ol ogi cal me mbr a ne isi nt er posedbet weent wo di st i nct l ydi f f er ent aqueous spacesandact sdi ffer- ent i al l yupont hem. Anas ymmet r yint hel ocusofatl east someme mbr a ne pr ot ei nscanbeant i ci -pat edont hi sbasis, r ef l ect i ngt hef unct i onalspeci al i zat i onof t het wome mbr a ne surfaces. A T.L.STECKOrganizationofProteinsintheHumanRedBloodCellMembrane3 on June 26, 2015jcb.rupress.orgDownloaded from Published July 1, 1974TABLEI TheMajorErythrocyteMembranePolypeptidesandGlycoproteins Percent of st ai nedPol ype pt i de s / Referencesfor purification, characteri- Componentmol wt*protein~Ghostzation, and other designations A.Polypeptides 1240,00015.1216,000[Spectrin (110112) TektinA (34) 2215,00014.7 I I 235,00011Myosin-like polypeptides (35) 388,00024.1940,000Component a (51) ProteinE (59) Minor glycoprotein (1) 4.178,0004.2 4.272,0005.0 543,0004.5 635,0005.5 729,0003.4 B.Glycoproteins PAS-1 PAS-2~55,000** 180,000 238,000 359,000Actin-like polypeptide (35) 540,000G3PD (30) 403,000 6. 7~(2.8)500,000 Glycophorin (23) Sialoglycoprotein (19) ABH andMNisoantigen (23, 56) * Valuesfromreference123; comparable values foundin referencesI 1, 29,35. ~: Average values fromalaboratory exercise where21medical studentsdeterminedthe distributionof Coomassie blue stainongelsof theirownredcell membranesaccordingtotheprocedureof Fairbanksetal.(1 I); comparablevalues arefoundinreferencesI 1,35. Calculatedassuming5.710 -1mgofproteinperhemoglobin-free ghost(I 1). I[ Perhapsslightlyoverestimatedbecauseof overlapwithband2.1(seeFig.1 A). Valueforpeakof thestainedband;tailextendsto~[05,000daitons. ** Provisionalvaluebasedonreferences23,49,56.PAS-IandPAS-2aretakenasthesamespecies(58). ~Calculatedassumingthisglycoprotein carries70%of themembranesialic acid(11,50,54)andthat1.6% of the membranemassissialic acid(11,18,19,76,86). Parentheticentryisforproteinportionalone(19). cent r al quest i onis,t her ef or e, towhat ext ent andin what ma nne r arei ndi vi dual pr ot ei ns(andot herconst i t uent s) as ymmet r i cal l yor i ent edint hemem-brane.Asi mpl eappr oacht ot heani s ot r opyof t he er yt hr ocyt eme mb r a n e hasbeentoexposet he i nt act redcellt opr obemol ecul espr esumednot t o passt hr ought hemembr ane. Theme mbr a ne con- st i t uent swhichr eact ar ej udgedt obeconf i nedt o itsout er sur f ace(asinr ef er ences64- 66) . Forexampl e, acet yl chol i nest er asecanbei nact i vat ed byexposi ngi nt act er yt hr ocyt es t opr ot eases(cf. 52,67,68),andsi al i caci dcanbequant i t at i vel y r el easedf r omredcellsbyt r eat i ngt hemwith si al i dase(69).Pr oof t hat t hesecomponent s ar e conf i nedtot heout er surfaceof t hemembr ane,however , callsfordemons t r at i ont hat t hesame r esponsedoesnot occur whent hecyt opl as mi c sur f aceissubj ect edt oani dent i cal t r eat ment . Such cor ol l ar ydat awillrul eout t hepossi bi l i t i est hat t he c ompone nt canmovebackandfort hbet weent he me mb r a n e sur f aces(ashasbeensuggest edatt i mes forme mbr a ne phosphol i pi ds[70]andpr ot ei ns[71, 72]),t hat t hepr obehasaccesst obot hsurfaces,t hat s omecopi esof t hecomponent ar eact ual l y pr esent at eachsurface, etc. Theinaccessibilityofa me mb r a n e component att hecyt opl asmi csur f ace pr ovi desst rongevi dencef or itsexcl usi vel ocal i za-t i onatt heext er nal surface.Ani ndi rect appr oacht ot heanal ysi sof t he cyt opl as mi csurfaceof t heredcel l me mbr a ne has beent oc ompa r e t her eact i vi t yoft heme mbr a ne in t hei nt act cellwitht hat of t hei sol at ed, unseal ed ghost ; t hedi f f er encebet weent het wopr epar at i onsisascr i bedtot heaccessi bi l i t yof t hecyt opl as mi c surfaceint hel at t er (e.g.50,73,74).However ,t her ear emor edi rect andsel ect i veappr oaches t o t hecyt opl as mi csurface. Advant agema ybet aken of t hef act t hat redcellme mbr a ne s canbeatl eastpar t i al l yreseal ed, af t er t heyareos mot i cal l ylysed,byappr opr i at emani pul at i onof t hei oni cmi l i eu (cf.12,75).Pr obest hesizeof pr ot ei nsandeven smal l sol ut escant husbeequi l i br at edi nt ot he i nt er i or spaceof ghost sdur i ngor af t er hemol ysi s 4THEJOURNALOFCELLBIOLOGYVOLUME62,1974 on June 26, 2015jcb.rupress.orgDownloaded from Published July 1, 1974andthent r appedinsidebyresealing.Washi ngthe resealedghostsremovestheprobefromtheexter- nalspace,sot hat thecyt opl asmi csurfacecanbe subsequentlyexami nedbyactivatingtheprobe.Anot her st rat egemfortheselective expl orat i on of thecyt opl asmi c surface is to generat eandpurify highlyi mper meabl einside-out(orinverted)vesi- clesfromunsealedghosts(7579).I mper meabl e ri ght -si de-out vesiclescanbepreparedbyclosely relatedprocedures(75,77-79).1Separ at i onof i nsi de-out fromri ght -si de-out specieshasbeen effectedont hebasisofphysi cochemi cal differ- encesbetweenthetwomembr anesurfacesby free-flow el ect rophoresi s (79)andt wo-phaseaque- ouspart i t i on(75). Fi nal l y, cyt ochemi cal andelectronmi croscope techniquesper mi tvisual det ect i on ofspecific labels atthe twosurfaces of unsealed ghosts (e.g.8085). Whi l eeachst rat egyhasitsl i mi t at i ons,thedat a fromallareingoodmut ual agreement andin strongsupport of asimple,farreachinghypot he- sis:namel y, t hat t hereis anabsol ut e asymmet r y in thedi st ri but i onof everyprot ei nconst i t uent be- tweent hetwosurfacesoftheredcellmembrane.That is,nocomponent issymmet r i cal l yrepre- sentedatbothmembr anefaces. Thispremi seisi l l ust rat edinTabl eII, which summari zesthesidednessof avari et yof eryt hro- cyte membr anemarkers. Al lof thespecies listed as out ersurfacecomponent sarereactive tononpene- t r at i ngprobesini nt act eryt hrocyt es, resealed ghosts,a nd/ or ri ght -si de-out vesicles.Mor ecriti- cally, noneis accessible ini nsi de-outvesicle prepa- rations, beyondasmal l fraction( ~10%)at t r i but a-bletocont ami nat i on, untiltheper meabi l i t ybar- rierisdest royedwithdet ergent s(75).Conversel y,theinner-surfacecomponent sarefullyaccessible ininside-out vesicles butarenotdet ect edinintact cells,sealedghosts,orri ght -si de-out vesicles,ex- cept foralowlevelcompat i bl ewithcont ami na- tion(75).Al l mar ker sarefullyaccessiblein unsealedghosts, butel ect ronmi croscopeanalysis ofthedi st ri but i onofelectron-densestainshas cor r obor at edt hei rasymmet r i cdi sposi t i oninthis case. Thecomponent slistedinTabl eI1makeup perhapsonlyasmal l fract i onofthet ot al mem- braneprot ei nmass,sot hat aseparat einquiry into ~lnonestudy(86; seealsoreference5)confusionof inverted andnormally orientedvesicles led to inappropri- ateconclusions,which subsequentanalysishasclarified (60). TABLE II MarkersofErythrocyteMembraneSidedness Outersurface Acetylcholinesterase(52, 60,67,68,78) Sialic acid(60, 69,76,78,79) Nicotinamide adeninedinucleotidase(158,159) Ouabainbindingsite(160,161) Carbohydrates (54, 60,83) Innersurface NADHdiaphorase (32, 60,78,82) G3PDandits bindingsite(32, 60,139) CyclicAMPbindingsite(106,139) Proteinkinase(106) ATPase (80, 162) thesidedness of themaj ormembranepol ypept i des wasundert akeninseverall aborat ori es. I nt acthumanredcellswereexposedtoenzymessuchas prot eases(52,60,65,8689),l act operoxi dase plus 125I-andH202(64,74,90,91),andgal act ose oxidaseplusBaH, (54,60,61),ortosmall,pr esumabl ynonpenet rat i ngcovalentl i gandssuch as[asS]diazoniumbenzenesulfonate(52,66,87), [ s~S] f or myl met hi onyl sul fonemet hyl phos phat e (51),t ri ni t robenzenesulfonate(60,92,93),and di sul foni cstilbenederi vat i ves(94,95).Themem- braneswere thenisolated andtheaffected pol ypep- tides anal yzed by gel electrophoresis.Theseseveral approachesdemonst r at edt hat twomaj or mem- branegl ycoprot ei ns, bands3andPAS- I , and several poorl y resolved gl ycoprot ei ns werereactive inthei nt act cells.Whenunsealedghostswere exposedtot hesameagents, al l of t hemaj ormembr anepol ypept i des seemedtobereactive.No component intheseghostsappearedinertorhidden, al t houghthevari ouspol ypept i des di dnot react equal l ywithallprobes.Asimplei nt erpret at i onhasbeendrawnfrom these dat a: component s3,PAS- 1, andanumberofmi nor gl ycoprot ei nsarepresentattheout ersur- faceof themembr ane, while component s1,2,2.1, 2.2,2.3,4.1,4.2,5,6,7,andseveralt race pol ypept i desareconfinedtothecyt opl asmi csur- face. Cer t ai nobj ect i onstothelabelingstudiesmustbeconsidered. Fi rst , howcanwebesuret hat a peakofradi oact i vi t y whichcoincides withamaj orst ai nedgel bandiscarri edbyt hat component and not byacomi grat i ngmi norspecies? For exampl e,band3hadsomet i mesbeenequat edi ncorrect l y withPAS- I andwithaNa+,K- ATPas epol y- pept i debecauseofsi mi l ari t yint hei rmobi l i t i es.Thi sprobl emisespeci al l y t r oubl esomewherethe T.L.STECKOrganizationofProteinsintheHumanRedBloodCellMembrane5 on June 26, 2015jcb.rupress.orgDownloaded from Published July 1, 1974amount ofl abel i ncorporat edisonl yasmal l mole fractionoftheput at i vet ar get pol ypept i de present (e.g.references51,74).Oneanswertothisprob- l em, toisolatethei ndi vi dual labeledcomponent s inpureform, issure,butdifficult.Asasimple al t ernat i ve, certainconst i t uent shavebeenidenti- fiedt hroughtheselectiveal t er at i onof theirrela- tiveel ect rophoret i c mobi l i t y bycross-linking (95), prot eol ysi s (73,89),or modi fi cat i on of el ect ropho- ret i ccondi t i ons(49,50).Arelatedprobl emwith labelingtechniquesist hat t heymaynotcl earl y demonst r at ewhetherallcopiesofagivencompo- nentareaccessibleat asinglesurfaceoronlya fraction(e.g.one-half).Cont rol l edprot eol ysi s can usuallycl earl ydemonst r at ewhichgelcomponent s arebeingat t acked, andtowhatextent;ithas provi dedaval uabl ecount erpart tot helabeling studies.(Innocasehascont rol l edprot easet reat - ment beenfoundtodi sruptt hemembr anepermea- bilitybarri er. ) Amor eser i ousquest i onconcer nst he i mper meabi l i t yoft hemembr anetothesmal lcovalentligandmolecules.Several workershave shownt hat reagent sbelievedtoreadi l ycrossthe membr anebar r i erexhibitthesameselective label- ingof component s3andPAS- I asdothepur- port ednonpenet rat i ngprobes(92,96,97).The inferenceist hat theselectivity ofprot ei nlabeling inintactmembr anesmaynotdi rect l yreflect membr aneasymmet r y, andt hat theenhanced labelingofallprot ei nsinisolated, unsealedghosts couldresultfromal t er at i ons int hei r conformat i on or localenvi ronment , r at her thanincreasedacces- sibility. (Therehavebeenot herreportswhich suggestedt hat amembr ane' smol ecul arorgani za- tionmayhealteredbyitsisolation[98101]) Bret scher(102)hasrespondedtotheseobj ect i ons bysuggesting t hat , underthecondi t i ons empl oyed,thepHatthe cyt opl asmi c surfaceof themembrane inintactcellswouldbelowert haninunsealed ghosts,thusreducingthelabelingofinnersurface prot ei nsbythepermeabl ereagents.However,grant i ngBret scher' s argument still doesnotpermi tustoconcludet hat thesmal l probesdiscussed abovecannot crossthemembr ane. Fur t her mor e,evenext ernal l act operoxi dasemaycat al yzethe i odi nat i onof hemogl obi nwithinintactredcells underunt owardcondi t i ons, presumabl ybythe releaseof someformof membr ane- per meat i ng activeiodine(103). Nevertheless, selective exami nat i onof the exter- nalandcyt opl asmi cmembr anesurfacesandthe useof specificmacr omol ecul ar probeshavesup- port edthethesisstatedabove.Reseal edghosts closelyresembl etheintacter yt hr ocyt eint hei r responsiveness(25,60,88),sot hat neitherthe removal of thecyt opl asmi c contentsnorthestress of osmot i clysiscausesageneral enhancement in thereact i vi t y ofthepol ypept i des.(Thesameis nott rueof theredcelllipids[cf.reference98].)It is possi bl e t hat resealing could healnot j ust thehol es intheghostbutwhateverot herhypot het i calar chi t ect ur alderangement saccompanyhemolysis.However, el ect ronmi croscopestudiesonunsealed ghostsshownolossoftheasymmet r i c l ocal i zat i on ofmembranecar bohydr at es(83),ATPas e(80),or spectrin(84).Fur t her mor e, thereisnosignificant differenceinreact i vi t ybetweenthei nt act cell membr aneandsealedri ght -si de-outvesicles which arederivedfromunsealedghosts(60). Whenselectiveradi oi odi nat i onofthecyt opl as- mic surface was performedby t rappi ng l act oper ox-idaseinsideresealedghosts, severalpol ypept i de specieswerelabeledwhichwereunreact i vein i nt act cells(90,104).Fi nal l y,membr anepol ypep- tidesnotreactiveini nt act cellsarecleavedby prot easesinsealedinside-outvesicles(60).Si nce out ersurfaceconst i t uent ssuchasthoselistedin Tabl e11arenotaccessibleinthesevesicles,itis cl eart hat thereact i ont ranspi resatthecyt opl as- mi csurface. Ani mpor t ant conclusionofseveralofthese studiesist hat atl east twogl ycoprot ei ns, compo-nents3andPAS- I , spanthemembranet hi ckness asymmet ri cal l y.Sinceitis cl ear t hat someport i on oftheseprot ei nsisaccessible attheout er surface, theargument underl yi ng t hi sthesisist hat anot her domai nofeachisdemonst r abl eonlyatthe cyt opl asmi csurface.Bretscher(50,73)showed t hat mor epept i defragment sfromeachofthese twoprot ei ns werelabeled bypS] f or myl me t hi onylsulfonemet hyl phosphat einunsealedghostst han inintactcell membranes. Heat t ri but edthisdiffer- encetouniqueport i onsofeachpol ypept i de being confinedtothecyt opl asmi c sideofthemembr ane.(However, ithasbeenpointedoutt hat evenmi norchangesintheenvi ronment orconformat i onoftheseprot ei nsduri ngghost pr epar at i oncoul d accountfortheincreased labeling, sincethereis no di rect evidencet hat thet agisact ual l yaffixedat theinnersurface.)Segrest etal.(25)haveshown t hat whiletheN- t er mi nal port i onof thesialo- prot ei n(whichbearsthecar bohydr at e) isaccessi- bletol act operoxi daseinintactcellsandresealed ghosts, t heC- t er mi nal regionisi odi nat edby l act operoxi daseonlyinunsealedghosts. Thei r 6THEJ OURNALOFCELLBI OLOGYVOLUME62,1 9 7 4 on June 26, 2015jcb.rupress.orgDownloaded from Published July 1, 1974sequencedat aalsosupport amodel whichplaces thehydrophi l i cN-andC- t er mi nal domai nsof gl ycophori n, respectively,at theout erandinner membr anesurfaces,connectedbyaroughly30- residuehydrophobi csegmentwhichwouldspan theapol arcore.ReichsteinandBlostein (90) found t hat l act operoxi dasesequesteredwithinfreshly resealedghostsl abel edband3butnotband PAS- I , whilebot hwerelabeled, ofcourse,atthe out ersurface.Mor erecently, Morri sonetal. (104) havereport ed t hat l act operoxi dase doesindeedt ag PAS- I , PAS- 2, andPAS- 3whensealedinto well-washedandresealedghosts.Thefailureofot herworkerstolabelthesi al ogl ycoprot ei nsin si mi l ar experi ment s(90)isascri bedtothepersist- enceof t r appedcat al aseandgl ut at hi oneperoxi- daseint hei rresealedghosts,sincethesecyt opl as- micenzymeswouldconsumetheH~O2requiredin thei odi nat i onreact i on(104). That bot hcomponent s3andPAS- I extendto thecyt opl asmi csurfacehasbeendemonst r at ed usingi nsi de-out vesicles.ThePAS- I sialoglyco- prot ei nisdigestedbyprot easesinbothnor mal l y ori ent edandinverted membranes, but t hesuscepti- bility pat t ernsare di st i nct l y different foreach(60). Radi oi odi nat i onoftheami no- t er mi nal regionof PAS-1proceeds preferent i al l y insealedright-side- out vesicles,whileinsealedi nsi de-out vesicles labelingoft hecar boxy- t er mi nal port i onisen- hanced(I. Kahane, V.T.Marchesi , andT.L. Steck. 1974.Unpubl i shedobservations). Compo-nent3isdigestedinintactcellsbychymot rypsi n andPronase, butnottrypsin;however,trypsin at t ackst hi spol ypept i de ininside-out vesicles (60). Fur t her mor e, component 3canbecross-l i nked to adi merbydisulfidebondformat i onwhenthe oxidizingagent ispresent edtosealedinside-out but notri ght -si de-outvesicles (105).Fi nal l y,band 3provi des sites for G3PDbinding(32,105)andfor phosphoryl at i on(sectionVIandreference106) onl yatthecyt opl asmi cmembr anesurface.Thattheout ersurfaceport i onofthesetwoglyco- prot ei nsisnotavai l abl einthei nsi de-out vesicles hasbeenexplicitlydemonst r at edbyt hei rresist- ancetosi al i dase(76,77),gal act oseoxidase(54, 60),andcert ai nprot eases(60). Insummar y, eachmaj or pol ypept i decanbe assignedtoonesurfaceor theother,withthe penet rat i nggl ycoprot ei ns spanni ngthemembrane asymmet ri cal l y, sugar-sideout.Thedat aprovi de nosupport for(st ruct ural )prot ei nswhichare indifferenttovect ori al t opogr aphyorhavemor e t hanonedi sposi t i on.Thereis alsonoi ndi cat i on ofpol ypept i demovement t hroughthemembrane thicknessorrot at i onabout anaxis lying paral l elto thepl aneoft hemembrane.Purified ghostsandinside-out andri ght -si de-outvesiclesderivedt herefromconservethedet ai l ed asymmet r i cdi sposi t i onofconst i t uent sinferred fromprobi ngtheintactcell.Thispoi nt iscrucial,sinceitsupport stheassumpt i ont hat isolated membr anederivatives aresuitablefort opogr aphi c anal ysi sof theprot ei ns(butperhapsnotthelipids [reference98]).Fi nal l y,theevidence forasymmet - ricalpenet rat i onof somegl ycoprot ei nst hrough themembr anesuggests:(a)t hat theymightbe boundhydr ophobi cal l yinthelipidcore,and(b) t hat t heymi ght beinvolvedint r ansmembr ane (e.g.,t r anspor t ) functions.Thereconjecturesare consideredfurtherbelow. V.AS S OCI ATI ONS ANDDI SSOCI ATI ONS Everyprot ei nintheeryt hrocyt emembr aneap- pearstobeboundinaspecificandcharact eri st i c fashion.Manysolubilizing agentsdislodge cert ai n component squiteselectively,and, indoingso, revealsomet hi ngabout t hei rmodeofat t achmenttothemembr ane.A. HighIonicStrengthElution Ithasbeenwidelyobservedt hat nor mal l ysolu- blegl ycol yt i cenzymesarerecoveredoni sol at ed humanandbovineeryt hrocyt emembranes(cf. 30,32,107,108).Becausetheseenzymescanbe readi l yrel easedbyraisingtheionicst rengt hto physi ol ogi clevels,itcouldbearguedt hat they, l i kehemogl obi n(109),canbeadsorbedbyghosts invitrot hr oughnonspecificel ect rost at i cforcesin di l ut esal t solutions.However, recentevidence poi nt stospecificityintheassoci at i onofatl eastG3PD(band6)withthehumanredcellmem- brane. Thi senzymebi ndstothemembr ane(prob- abl yviacomponent 3[105])onlyatthecyt o- pl asmi csurface,fromwhichitcanbecompl et el y releasedbymi l l i mol ar levelsof certaingl ycol yt i c i nt ermedi at es(32,108). B.LowIonicStrengthElution Threepromi nent pol ypept i des,component s1, 2, and5,areselectivelyl i berat edwhenghostsare placedinalowionicstrength,mi l dl yal kal i ne medi umfreeof di val entcat i ons(11,34,110-113). Thesepol ypept i des(ori gi nal l ycalledspectrin [110])havebeenidentifiedwitht hefi bri l l arma- t er i alobservedattheinnersurfaceof ghosts, since T.L.STECKOrganization of Proteins inthe Human RedBlood Cell Membrane7 on June 26, 2015jcb.rupress.orgDownloaded from Published July 1, 1974(a)thosefibrilsdisappearduringtheelutionpro- cess,and(b)fibrilsformwhentheionicstrength of thespectrineluatesisincreased(110,114).The elutedproteinsbehavehydrodynamically as small, highlyasymmetricoligomers (34,112),sothatan electrostaticdisaggregationordepolymerization ofthefibrilspresumablyaccompaniesandmay actuallybringabouttheirrelease.Itcouldbe that bands1,2,and5actually bindvery weakly tothe membranebutremaininplacebecauseoftheir extensiveself-associationwithintheghost(see below). C.ElutionbyProteinPerturbants Avarietyofreagents,knowntodenatureor covalentlymodify proteins,selectively solubilize a certaingroupofmembranepolypeptides,leaving theremainderstillassociatedwithallofthelipid andcarbohydrateinamembranousresidue. Amongtheseagentsare 6Mguanidine hydrochlo- ride (115),hydrochloric (116),oraceticacid (117), 0.1NNaOH, 40mMlithiumdiiodosalicylate (LIS),succinicanhydride,dimethylmaleicanhy- dride,15mMp-chloromercuribenzoate(all,ref- erence118),and p-chloromercuribenzene sulfonate (27,118).Thepolypeptidesreleased,accounting forone-half ofthemembraneprotein,are,ineach case:bands1,2,2.1,2.2,2.3,4.1,4.2,5,and6, plusseveral minor,unnamedspecies (Fig. 2).(Not surprisingistheobservationthatthepolypeptides mostreadilyelutedbythevariousperturbing agentsarecomponentsI,2,5,and6;i.e.,those speciesreleasedbymeremanipulationof theionic milieu.)Retainedintheresidualmembranesare components3and7,thepoorlyresolvedzone termed4.5(Fig.2),andtheentirePASprofile. Theinferencedrawnfromtheseeffects(118)is thatthenonglycosylated half of theprotein mass is associatedwiththemembranebysite-specific bondingwhichisundonebyperturbationofpro- teinstructure.Theelutedspeciescouldbecalled "peri pheral "or"extrinsic"(2,3)inthesense that they aredissociated fromthelipid stratumwithout its dissolution; however, their selective release does notnecessarilyimplyloosebindingoranexoge- nousorigin.Itisnoteworthythatallofthe perturbant-elutedpolypeptidesareuniquelyasso- ciatedwiththecytoplasmicsurfaceofthemem- brane. Incontrast,themembraneglycoproteinsseem tobetightlyboundtothemembrane,sothat modificationordenaturationdoesnotextract them.Hydrophobicanchoring,asopposedto stereospecific binding,seemslikely, particularly in viewoftheirincreasedcontentofapolarresidues (118)andevidencethatatleasttwomembersof thisgroupspanthemembrane.Itisnoteworthy thatall of thepolypeptide species accessible atthe outersurfaceofthemembraneareinthisgroup. Onlyoneperturbant-resistantcomponentisfound uniquelyatthecytoplasmic surface:band7. D.ElutionbyNonionicDetergents Certainnonionicdetergentscanbringmem- branelip~dsintomicellar solutionanddisplace the lipid fromhydrophobic proteins withoutdenatura- FIGURE 2Selectivesolubilizationofpolypeptidesfrom ghostsbyproteinperturbantsandnonionicdetergents. Isolated ghostswere incubatedin5mMsodiumphos- phate buffer,pH8 (left); wateradjustedto pH12.0 with NaOH(middle);and0.5%TritonX-100 in56mM sodiumborate,pH8.0 (right).After centrifugation,ali- quotsofpellet(P)andsupernatant(S)fractionsde- rivedfrom10~tlofghostswereelectrophoresedand thegels stainedwithCoomassieblue.TD, tracking dye. (Reprintedbycopyright permissionfromJ.Supramol. Struct.1973, 1:220 and 233.) 8THEJOURNAL OF CELL BIOLOGY VOLUME 62,1974 on June 26, 2015jcb.rupress.orgDownloaded from Published July 1, 1974tion(cf.119-121).Wewouldant i ci pat e, therefore,t hat pol ypept i desanchoredinthemembrane t hroughapol ar associ at i onwithlipidscouldbe solubilized asdet ergent compl exes,leaving prot ei n t er t i ar yst ruct ureandquat ernaryassoci at i onsin- t act .Theselectiveactionof Tr i t onX-100onhuman ghosts(119)isnearl yreci procal t ot hat seenwith theper t ur bant s(Fig. 2).Rel easedinat rul y soluble,lipid-freeformarebands3,4.2,6,and (notshownhere)theentirePAS- st ai nedprofile. Somewhat mor ethanhal fofthemembr anephos- phol i pi d(pri nci pal l y t heglycerolipids) is alsosolu- bilized.Theremai ni ngpol ypept i des(pri mari l y bands1,2,4.1,5,and7)areret ai nedinghost- shapedresidueswhichappear inelectronmicro- graphst obecompri sedofadenseret i cul umof finelyfi l ament ousmat eri al studdedwithsegments oflipid( pr i mar i l ysphingolipids) (Fig. 3). Thisfi l ament ousmat r i xis probabl y at t r i but abl e tothe"spect r i n"pol ypept i des (bands1,2,and5), since(a)theycompri seatleasthal f of t heprotein intheresidues;(b)noTr i t onresidues areobserved ifthesecomponent sarepreviouslyselectively elutedbylowionicstrengtht reat ment ; and(c) spectrinhasprevi ousl ybeenidentifiedwithinner surfacefi bri l l armat er i al (110,111, 114).The spectrinfi l ament smaythusformasubmembr ane cont i nuumheldt oget her byself-association,rat her t hanbindingt othelipidpart ofthemembrane (119). Component s3,4.2,and6alsoseemtoretain specificquat ernaryassoci at i onsinTri t onX-100 solution(105,122).Band3behavesl i keadi mer,j ust asinthemembr ane(123),andband6remai ns t et rameri c, asj udgedbyitsunal t eredsedi ment a- tionpropert i esandG3PDactivity. Al lofband6is specificallybound, apparent l ycooperat i vel y, to component 3intheTr i t onX-100ext ract , two G3PDt et r amer sperband3di mer, asisalsothe caseinghosts(105). Fi nal l y, band4.2maybeintheformof a t et r amer , sinceformal dehyderat herspecifically cross-l i nks ittoa270,000-daltonform, bothwhile membr aneboundandinTri t onsolution(123). Thi spol ypept i dealsoappearst i ght l yboundto band3,sinceitcopurifieswiththel at t er ina vari et yofsystems, unlessper t ur bant sarefirst empl oyedtobr eakt hecompl ex(105,122).These dat amakecl earwhybands4.2and6areselec- tivelyel ut edwiththegl ycoprot ei nsbyTr i t on X-lO0(Fig. 2). E. De t e c t i ono f As s o c i a t i o n s byEl e c t r onMi c r o s c o p yAs est i mat edbyfreeze-fract ure el ect ronmi cros- copy, eachhumanredbl oodcellmembr anehas about 500,000i nt r amembr anepart i cl eswithan averagedi amet er ofappr oxi mat el y80A, which arebelievedtobecomposedof prot ei n(14).The part i cl es ineryt hrocyt es andfreshly i sol at ed mem- branesarel at eral l ydi spersedwithnoapparentorderi ng. Itisnotnowknowniftheyarefreeto moveorarefixedinplaceunderthesecondi t i ons.Tr eat ment ofghosts,butnoteryt hrocyt es, with digestive enzymes andcert ai nal t erat i ons in thepH orionicstrengthoft hemedi umcausesthepar t i -clestocl ust er(cf.14,124).Thisshiftindi st ri bu- tioncouldrepresent ei t herani ni t i at i onof part i cl e t r ansl at i onoranaggregat i onorpart i cl esal r eady inmot i on,It hasproveddifficulttoidentifythedi spersed i nt r amembr anepart i cl eswithaspecificprotein.However, aft ertheinductionofclustering, t he di st ri but i onof part i cl eaggregat esparal l el sthe pat t er nof bindingsitesfortheseligands:ant i - A bl oodgroupant i body(125),ki dneybeanphyt o-hemmagl ut i ni n(126),influenzavirus(126),and col l oi dalironhydroxi de(127).Theseactivities are allassoci at ed(atleastinpart )witht hemaj orsi al ogl ycoprot ei n(9,19,23,55),which,further- more, hasat r ansmembr anedi sposi t i onandan abundance(Tabl eI)appr opr i at etobeingthe i nt r amembr anepart i cl e. Ontheot herhand, the si al ogl ycoprot ei n(and, morepar t i cul ar l y, itshy- dr ophobi ct r ansmembr anesegment)appear st oo smal ltoaccount for t hesizeof thei nt r amembr ane part i cl e(24;however,seereference128).It has beensuggested,therefore, t hat thepart i cl es repre- sentgroupsof l at eral l yassoci at edmembr ane-penet rat i ngproteins, perhapsinvolvingallof the gl ycoprot ei nspecies(7,9).What isnot known, however,iswhetherthevari ousmembr anegl yco- prot ei nspeciesarenor mal l yl at eral l yassoci at ed,sincet hi sgroupi ngcouldresultfromthemem- braneper t ur bat i onsrequiredintheseexperi ment s.Twocross-l i nki ngstudiesonhuman(123)and bovine(129)redcellmembranesfailedtodet ectanycomplexesformedbythesi al ogl ycoprot ei ns;however,Ji(130)hasrecentlyreport edt hat new PAS- st ai nedgelbandsappear aft erdi met hyl adi- pi mi dat et r eat ment of ghosts.Fi nal l y, itshould benotedt hat band3,ofitself, coul d account for all thei nt r amembr anepart i cl es. Asspheri cal 180,000 T.L.STECKOrganizationofProteinsintheHumanRedBloodCellMembrane9 on June 26, 2015jcb.rupress.orgDownloaded from Published July 1, 1974FIGURE3Triton X-100-extracted ghosts. Ghosts were incubated in 5 mM sodium phosphate (pH 8.0)with orwithout 0.5% TritonX-100, thennegatively stained with aqueous I%uranyl acetate.(aandb) control ghosts; (c and d) extracted ghosts. Calibration barisI ~m throughout. (Reprinted by copyright permission from J.Supramol.Struct.1973, 1:233.) daltondimers,thiscomponentwouldcontribute ~5l0 s particlesof75-Adiametertotheplane offracture. Twolaboratorieshaverecentlypresentedevi- dencethatthelateralmotionofthesialoglyco- proteins and the membrane-penetrating particles is constrainedbythespectrinpolypeptides(bands1, 2,and 5).Nicolson andPainter ( 131,132) reported thattreating ghosts withantiserapreparedagainst spectrinpromotedredistributionofthecolloidal iron hydroxidebinding patternwhichidentifies the sialoglycoproteins attheexternalmembranesur- face.ElgsaeterandBranton(124)havefound that gentlyperturbing spectrinbyincubating ghostsin lowionicstrength,mildlyalkalinesolutionis associatedwithanincreasedclusteringofthe 10THEJOURNALOFCEL[.BIOLOGYVOLUME62,1974 on June 26, 2015jcb.rupress.orgDownloaded from Published July 1, 1974intramembraneparticlesduringsubsequentexpo- sureof themembranestomediafavoringparticle aggregation(e.g.,lowpHorhighionicstrength). Thepresumptionisthatspectrinfilamentsare interactingwithmembrane-penetratingproteins at thecytoplasmicsurface.Theseauthorsfavoran interpretationinvolvingadirectassociationof penetratingproteinswithspectrin.However,itis perhapsmorelikelythatthepenetratingproteins areentrappedwithintheintersticesofthedense filamentousreticulumwithoutbeingspecifically bound,sincetheglycoproteins canbeeasilysepa- ratedfromthespectrinbymild nonionic detergent treatmentwhich doesnot destroy other quaternary proteinassociations (119).Thesealternative mech- anismsmightbedistinguishedbyasearchfor directbindingof solubilized bands3andPAS- Ito spectrinisolates. VI.PHOSPHORYLATI ONS Whenintacthumanerytbrocytesareincubatedin thepresenceof3~PO~, themembraneproteinand lipidfractionsbothbecomelabeled(133).The sameistrueforisolatedghostmembranesincu- batedwith7-[32P]ATP(48,134.-138).Theidentity ofthelabeledpolypeptideshasbeeninvestigated usingpolyacrylamide gelelectrophoresisinSDS. AvruchandFairbanks(48)havedetecteda 105,000-daltonpolypeptideinwhichthebound 32PO4turnedoverrapidly.Theyprovidedstrong evidencethatthiscomponentis theacylphosphate intermediateoftheNa +,K+-ATPase.Williams (134)hasalsofounda~2P-labeledgelbandof approximately98,000daltonswhichshowedcer- tainfeaturesascribable toaphosphorylatedinter- mediateoftheATPase,yetdifferedfromthe aforementionedcomponent(48)incertainimpor- tantrespects. Stablephosphoserineandphosphothreonine linkagestoproteinshavealsobeendemonstrated afterincubationofghostsin3,-[32P]ATP(137, 138).Mostheavilylabeledisagelpeakwhich appears tocorrespond toband2(215,000daltons); a90,000-daltoncomponent(presumablyband3) is taggedlesswellanda50,000-daltonpeakisonly slightly labeled.Mi cromol arlevels of cyclic AMP greatlystimulatethelabeling of the50,000-dalton peak;increaseinthe90,000-daltonpeakis moder- ate,whilethelabelingof the215,000-daltoncom- ponentisnotstimulated(135,137,138).Acyclic AMP-st i mul at edproteinkinaseactivitycanalso be demonstrated in humanerythrocyte membranes using exogenoushistonesasasubstrate(136,137). Theenzymeappearstoresideexclusivelyatthe cytoplasmic sideof themembrane(106),afurther argumentforthepresenceofthephosphorylated proteinsatthatsurface.WhetherthecyclicAMP bindingsite,whichhasalsobeenascribedtothe innersurfaceoftheerythrocytemembrane(106, 139),isthesameastheregulatorysiteofthe proteinkinaseisuncertain,sincethecyclicAMP concentrationrequiredforhalf-maximalsatura- tionofthebindingsite(3nM)is10100times lower thanthatrequiredforhalf-maximal stimula- tionofphosphorylation(135,136,139).Photoaf- finitylabelingwasrecentlyusedtodemonstrate thatthe 50,000-daltonpolypeptide whose phospho- rylationismoststimulatedbycyclic AMPproba- blycorrespondstothiscyclicAMPbindingsite (140). Arethemembraneproteinsnormallyphospho- rylated insitu?Several workers ( 16-18)havefound residualphosphorusinthemembraneprotein fractionafterexhaustivelipidextraction.While thiscouldbeascribedtotightlyboundphospho- lipid, it couldalso representphosphoproteins,with approximatelyonetotwophosphatesper50,000 daltonsof protein.Since thislevel of phosphoryla- tiongreatlyexceedstheextentofincorporation achievedin vitro, it may be thatthelatter is limited by preexisting specific phosphoprotein complexes. VII.CONCLUSI ONSANDCONJECTURES Fig.4summarizessomeofthedatadiscussed above.Itisintendedtodepictthetopographyof thenineortenprincipalpolypeptides whichcom- priseabout80%oftheproteinmass.Itignores theircharacteristic functions,molecularsizes,and abundance.Eachpolypeptidecanbeassignedto onesurfaceofthemembraneortheother.Two polypeptideswhichseemtoextendacrossthe membranedosoanisotropically, withtheircarbo- hydrateportionsdetectableonlyattheouter surface.All of theoutersurfaceproteins seemvery firmlyanchored,presumablybypenetrationinto or throughthehydrophobic core of themembrane. Theconstituentsconfinedtotheinnersurfaceare (withtheexceptionofband7)boundbymore readily reversedassociation,presumablywithspe- cificsites.Oligomersamonglikepolypeptides are present,whichin turnformcomplexes with certain otherproteinsatthecytoplasmicsurface. Thisschemeisnecessarily provisionalandinac- curate.Thenumberandidentityoftheminor glycoprotein species is uncertain.Manyor evenall ofthemcouldspanthemembrane(cf.104),buta T.L.STECKOrganizationofProteinsintheHumanRedBloodCellMembrane11 on June 26, 2015jcb.rupress.orgDownloaded from Published July 1, 1974INSIDE FIGURE 4A possible arrangement for the major erythrocyte membrane polypeptides. The lipid permeabil- itybarrierisshaded.Theglycoproteins penetrateintoorthrough thehydrophobic zone, with alltheir carbohydrates exposed to the extracellular space. The other polypeptides are, in general, less firmly anchored at the cytoplasmic face, where they participate in specific interprotein associations. See text for details and reservations. rigorousidentification ofwhichspeciesextendto thecytoplasmic surfaceispresently wanting.Fur- thermore,itisnotknownwhetherband4. 2and band6(G3PD)tetramerscanbind independently toband3monomersorevendimers,particularly since G3PDbinding tothedimers appears cooper- ative (105).Finally, therepresentation of bands 1, 2,and5isnotintendedtoconveyamodel,but ratherourpresentignorance of theirassociations insi t u. Itisknownthatbands1and2canbe coupledby covalent cross-linking reagents,appar- ently eachtoitself aswellastooneanother,both onghosts(123,141) andinsolution (9,34,123); however,band 5 appears not to participate in these complexes.Indeed,theformertwopolypeptides canbeseparatedfromthelatteraftergentle elution (34,124), although Guidotti hasarguedfor theirpersistentassociationinsolution(35).As discussedbelow,thereis diverse evidence favoring theview thatbands 1, 2, and 5 could be depicted as anactomyosinsystem. Thusfar,allredcellmembranecarbohydrates --bot hglycoprotein andglycolipid--appear local- izedtotheexternalsurface;furthermore,allthe majoroutersurfaceproteinsbearsugar(cf.54). NADaseand acetylcholinesterase should therefore beexaminedforthepresenceofboundcarbohy- dratetofurthertestthishypothesis.Similarly, we mightenquirewhetheraglycoproteinsubunitof theredcellNa+, K+-ATPase(ifanalogousto othertissues;references72and142) isexposedto theexternal surface (whereit couldbind ouabain), sincethenonglycosylatedpolypeptidesubunitis phosphorylatedatthecytoplasmicside.Thereis nowanampleandrapidlygrowingliterature indicatingthatoutersurfaceantigens,enzymes, receptors,andnumerousproteinsofpresently unknown function areglycosylatedinavariety of cells.Clearlythetrend,ifnottherule,isthat proteinsintrinsictotheoutersurfaceofplasma membranesgenerallybearcarbohydrate,while thoseconfinedtothecytoplasmicsidedonot(cf. alsoreference85). Thestrictasymmetry in thesidedness of thered cell membrane proteins must reflect their function. Someenzymes attheexternalsurfacepresumably servetheneedsoftheorganismratherthanthe cell.For example, theouter surface acetylcholines- terase can destroy theneurotransmitter circulating intheplasmawithoutitbeing transportedacross theimpermeableerythrocytemembrane.Exter- nallyorientedNADase,likethe5'-nucleotidase found onthesurfaceof othercells(143),might be partofamechanism generating permeantnucleo- sidesfromimpermeantnucleotides forintracellu- larsalvage.Conversely,innersurfaceenzymes (TableII)wouldappeartoaddressintracellular homeostasis. Polypeptideswhichspanthemembraneare logicalcandidatesfortransportsites.However,it isconceivablethatoligomericproteincomplexes couldalsoplaysucharolewithoutanysingle polypeptideconstituentbeingaccessibleatboth membranesurfaces.Na+, K+-ATPase(cf.142) andcytochromeoxidase(144)might beexamples. Ontheotherhand,adeeperunderstanding ofthe structureofproteinswithinthelipidcoremight makeitclearthattheassemblyofatransmem- branepermeationmechanismoutofmorethana single polypeptideisunfavorable. 1] ThEJOURNALOFCELLBIOLOGYVOLUME62,1974 on June 26, 2015jcb.rupress.orgDownloaded from Published July 1, 1974CabantchikandRothstein(89,94,95)have recentlyshownaclosecorrelationbetweenthe inhibitionofthefacilitateddiffusionofCI-and SO4 ~ throughtheredcell membranebyaradioac- tivestilbenederivativeanditsselectivecovalent labelingofband3.Completeinhibitionoccurred when3105moleculesofinhibitorwerebound percell,attheoutersurface. Thereisalsoindirectevidenceconsistentwith thepremisethatband3provides~3105 sites to facilitatethediffusionofD-glucoseacrossthe membrane(145-147).Tospeculatebriefly,there areenoughband3pol ypept i des--and evendimers - - t oaccountforthesevariouscapacities.That band3iscomprisedofmorethanonemolecular formissuggestedbyitscomplexstainingprofile (Figs.1 and2).This hypothesis is also favored by a recentreportthatseveral dansylatedaminoacids, presumedtorepresentamino-terminalresidues, canberecoveredfromtheband3regionof polyacrylamidegels(148).However,Tannerand Boxer(59)foundnofreeN-terminusinpurified band3sothatthedansylatedresidues(148)could havederivedfromotherpolypeptidesofsimilar mobility(e.g.,PAS-I). Theband3polypeptide behaveshomogeneouslywithregardtocross-link- ing (123),proteolytic cleavage (52,60,73,88),and otherfeatures(59).Ithasbeensuggestedthatthe dispersion in itsstained electrophoregram could be causedbyavariationinitscarbohydratecomple- ment(54).At present,therefore, we cannot dismiss anyof thesepossibilities: (a)theprimarypolypep- tidestructureofband3ishomogeneous,yetit facilitatesthetransportofmorethanonesolute (butcf.149);(b)band3isafamilyofclosely relatedpolypeptides,specializedtofacilitatedif- ferentsolutes across themembrane;and(c) band3 isnotthesourceofspecificityinthefacilitated diffusionof thesesolutes.If band3doesfacilitate thediffusionofsolutes,itsdimerization,itsphos- phorylation,andbindingofband6(G3PD)and band4.2takeonaddedinterest. Wehaveconsidereddatawhichindicatethat bands1,2,and5constituteadenselyself- associated reticulumof microfilaments adherent to thecytoplasmic surfaceof theredcellmembrane. Thismatrixcouldofferthetoughandinextensible skeletal supportwhich the delicate anddeformable fluidlipidbilayer(withitsintegratedproteins) mightrequiretowithstandtherigors of prolonged shearstressinthecirculation(cf.also35).The "t wo-pl y"structurecreatedbytheappositionof thefibrousmeshworktothesupplelipidstratum mighthelptoaccountfor thecomplexmechanical properties of themembranesobservedinavariety ofbiophysicalstudies(cf.14,150-152).Thisfila- mentoussystemcoulddefineandcontrolcell shape,andperhapsrespondtocytoplasmic or even extracellularsignalsviaenergy-dependentstruc- turalmodification. Insupportoftheseconceptsaretheseobserva- tions:(a)Selective elutionofbands1,2,and5is accompaniedbythebreakdownofghostsvia endocyticvesiculation,implyingaspecificde- stabilizationofthemembrane(76,l l 0). (b)Gui- dotti,havingreviewedliteraturepointingtothe presenceofactin-andmyosin-likeproteinsat othercell surfaces (1),suggested(35) thatbandsI, 2,and5maybe"erythrocyteactomyosin,"by virtueoftheirmolecular weight,aminoacidcom- position, morphol ogy, andassoci at edCa +- dependentATPaseactivity (114),While thepreci- sionofthisanalogyremainstobedefined,recent studies haveindeedindicated thatpurified compo- nent5canbepolymerizedathighionicstrength intofibrilswhichresembleactinintheircapacity tostimulatemyosinATPaseactivityandintheir patternofarrowheaddecorationbyheavymero- myosin(M.Sheetz,R.G.Painter,andS.J. Singer,personalcommunication).(c)Mg-ATP stimulates the phosphorylation of band 2(135,137, 138),thebreakdownof bovine ghosts by endocytic vesiculation(153),andtheemergenceinghost preparationsofaninfraredspectrumidentified withtheanti-parallel betaconformation(154),all of whichcouldconceivably relate tochanges in the stateof thespectrinsystem.(d)Strikingincreases intherigidityoferythrocytesoccurafterATP depletionandCa uptake,whicharereversed by Ca ++removalandincreasedlevelsofMg-ATP;isolatedghostssharesomeoftheseproperties(8, 155).(e)Finally, Jacobetal. (156)have implicated submembranemicrofilamentousproteinsinthe abnormalities of shapeandturgorseeninerythro- cytes frompatientssuffering fromhereditary sphe- rocytosisanderythrocytes treatedwithcoichicine, vincristine,andstrychnine.Seemanetal.(157), however,haveprovidedevidencethatthesealka- loidscouldbeaffectingthelipidportionofthe membraneinstead. 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