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In Vitro Membrane Integration of Leader Peptidase Depends on The

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In Vitro Membrane Integration of Leader Peptidase Depends on The pif IWHQT pif vetters RIQ @IWWUA IHWIIR sn vitro memrne integrtion of leder peptidse depends on the e mhinery nd nioni phospholipids nd n our postEtrnsltionlly Y im vn ulompenurg BD enj xFtFeF idder D enne vFtF vn lte D entoinette tF uillin D qunnr von reijne D fen de uruij¡ heprtment of fiohemistry of wemrnesD gentre for fiomemrnes nd vipid inzymologyD snstitute of fiomemrnesD treht niversityD duln VD QSVR gr trehtD he xetherlnds heprtment of fiohemistryD errhenius vortoryD tokholm niversityD EIHT WI tokholmD weden eeived S tune IWWUY revised version reeived PU tune IWWU tion IPF enioni phospholipidsD whih represent out PS7 estrt e ellEfree system sed on lyste nd memrne of the memrne lipids of iF oliD re lso involved in trnsE vesiles from isherihi oli is used to study hrteristis of lotion IQF hey stimulte the ese tivity of ee IR the memrne integrtion retion of the polytopi memrne nd re ple of interting with signl peptides ISF purE protein leder peptidse @vepAF sntegrtion into inverted inner thermoreD e¤ient in vivo trnslotion requires the presene memrne vesiles ws deteted y prtil protetion ginst externlly dded proteseF sntegrtion is most effiient when of proton motive fore @wpA IT s well s the presene of oupled to trnsltion ut n lso our postEtrnsltionlly nd two memrne proteins @eh nd epA whih my e inE depends on the tion of the proteineous e mhinery nd volved in mintining wp during trnslotion ut the ext vilility of nioni phospholipidsF vep is the first exmple of roles of whih re unknown IUF efter trnslotion the sigE memrne protein without levle signl sequene whih nl sequenes re removed y the tion of signl peptidses requires nioni lipids for integrtion in vitroF suh s vep IVF rnslotion of some preursors requires z IWWU pedertion of iuropen fiohemil oietiesF other ytosoli omponents like the qroiv hperone IW or prokryoti homologues PHF uey wordsX veder peptidseY sn vitro trnslotionY sn vitro trnslotion systemsD employing rdiolelled preE wemrne proteinY isherihi oli ursors nd puri¢ed inner memrne vesilesD hve mde n importnt ontriution to the urrent knowledge out preE ursor protein trnslotion euse they llow systemti IF sntrodution investigtion of the ftors involvedF sn this study we estlish n in vitro system for memrne integrtion of vep whih veder peptidse @vepA from isherihi oli is n integrl involves memrne pssge of the periplsmi dominF he inner memrne protein whih spns the memrne with two requirements for this proess re investigted y using vrious hydrophoi segments whih re onneted y short ytoE types of vesiles nd y dding gents to the ssemly reE plsmi loop IF he seond trnsmemrne segment preE tions whih lok the funtion of omponents possily inE edes lrge periplsmi domin whih during ssemly hs volved in ssemlyF to pss the inner memrne PF sn reent yers vep hs een extensively used s model to study memrne protein inserE PF wterils nd methods tion in vivo nd it ppered tht the mehnism for trnsE PFIF wterils lotion of periplsmi loops depends on loop lengthF voops rinuleotides @sodium sltsAD R ligse nd the restrition endonuE smller thn TH mino id residues pss the memrne iE leses ms nd ls were otined from hrmiF polini id lyer in n pprently spontneous proessD while trnsloE @lium sltAD phosphoretine @diEris sltA polymyxin f sulfteD tion of longer loops like the periplsmi domin of vep reE phenyl methyl sulfonyl £uoride @wpA nd mino ids were from igm @ eAF T xe polymerseD retine phosphokinseD txeD quires the tion of the soElled e mhinery QDRF puromyin dihydrohloride nd proteinse u were purhsed from yriginllyD e mhinery ws found to e essentil for ws puri¢ed s desried efore foehringer @qermnyAF vep@risA T trnslotion of periplsmi nd outer memrne proteins PIF ee nd vEltmse ntiodies were gift from hrF rF de ross the inner memrne @for review see SAF nlike vepD gokD treht niversityF these periplsmi nd outer memrne proteins re syntheE PFPF trins nd growth onditions sized s preursors with xEterminl extensions lled signl iF oli strin wi THH PP ws used for the prodution of EIQS sequenesF hey re often mintined in trnslotion omE lyste nd grown in qiston roth t QU³g until erly exponentil petent stte y the tetrmeri ef protein TVF hese ef phseF eferene vesiles were isolted from strin h IP PQ whih preursor omplexes hve high ¤nity for ee whih is ws grown t QU³g in vfF g PWUU PR ws used s soure of ts vesilesF gells were grown overnight t QH³g in vf with e found in the ytosol nd in multiple onformtions in the IH gGml tetryline nd t RP³gD Q h prior to vesile isoltionF inner memrne where it ouples e hydrolysis to the progE esiles with low ontents of nioni lipids were isolted from ress of trnslotion WIIF ogether with eeD eED Ei rhvII PSD whih ws grown in vf supplemented with IH gGml nd Eq proteins onstitute the si mhinery for trnsloE tetrylineD PS gGml hlorompheniol nd SH gGml knmyinF PFQF sn vitro trnsriptionD trnsltion nd ssemly Bgorresponding uthorF resent ddressX heprtment of woleulr EIQS ell extrts nd inverted inner memrne vesiles were preE wiroiologyD qffD uerkln QHD WUSI xx rrenD he xetherlndsF pred from iF oli ording to pulished proedures PTF lsmid pxX @QIA SHEQTQPISRF pvep ws reted y loning the lsms frgment ontining iEmilX wFklompenurgdiolFrugFnl lepf from psxqI derived in vivo expression vetor to the in vitro HHIRESUWQGWUG6IUFHH ß IWWU pedertion of iuropen fiohemil oietiesF ell rights reservedF ss HHIRESUWQ@WUAHHVVVEH pif IWHQT PPEIHEWU IIH F vn ulompenurg et lFGpif vetters RIQ @IWWUA IHWIIR expression vetor pTRF hxe ws puri¢ed using wizrd minipreps hxe puri¢tion resin @romegD eA nd used to diret the trnE sription of the vepf gene y T xe polymerse during QH min inution t RH³g following the mnufturer9s instrutionsF rnsE ltions were rried out t QU³g s desried PTF pive minutes fter initition of trnsltionD inverted inner memrne vesiles were dded to ¢nl onentrtion of R mgGml @proteinA nd the inution ws ontinued for PS min t QU³gF gorret ssemly of vep ws demonE strted y protetion of the P domin ginst the tion of externlly dded proteinse u @PHH gGml ¢nl onentrtionAF he protese tretment ws rried out for PH min t room temperture fter whih P mw wp ws ddedF he smples were nlysed y hEeqi nd exposure of the gel in hosphor smgerF o ssess the in£uene of dditions @ntiodies or polymyxinAD these were mixed with the vesiles prior to inution in the trnslotion mixtureF PFRF smmunopreipittion smmunopreipittions using ntiserum direted ginst vep were performed s desried PU using rotein eEoupled ephrose gvRf @hrmiD vufD wedenAF PFSF roympe trnslotion QS proympe ws puri¢ed s desried PV nd stored in V w QS proympe ws diluted SHEfold into PS l of trnslotion ureF u¡er @RH mw risEetteD pr VFHD IHFV mw mgnesium etteD PV mw potssium etteD P mw hD HFS mgGml feA ontining inverted inner memrne vesiles @HFRH mg proteinGmlAD S l EIQS ell extrt nd R mw e VF hen indited I l of polylonl ntiE serum direted ginst ee or vEltmse ws ddedF rnslotion ws llowed to proeed for PH min t QU³gD followed y nlysis s desried for the ssemly of vepF QF esults o llow in vitro synthesis of mxe y T xe polyE merseD the vepf gene ws loned into n in vitro expression vetorF wost expression plsmids rry the gene oding for pigF IF lsmid direted in vitro synthesis nd identi¢tion of vepF the seletle mrker vEltmse in the sme orienttion s eX lsmid mp of pvepF estriition sitesD genes @vepf nd flAD the T promoter nd the origin of replition @oriA re indiE the T promoter whih gives rise to sustntil mounts of tedF fX sn vitro protein synthesis from pTR without @lne IA or expression of this proteinF feuse of similr moleulr with n insert ontining the vepf gene @lnes PRAF efter synthesis weights of vep nd vEltmseD this would omplite the smples were put on ie @lne PA or treted with PHH gGml proteinE interprettion of the resultsF herefore we mde use of se u @lne QA or immunopreipitted using vep ntiserum @lne RAF pTR to lone the vepf gene in the opposite orienttion s ell smples were fter their respetive tretments nlysed y hE eqi followed y utordiogrphyF he position of nonElelled the vEltmse gene to result in pvep @pigF IeAF efter puri¢ed vep on the gel is inditedF trnsltion of the xe y memrneEfree lyste of iF oli QS methionineD the produts wi THH in the presene of were resolved y hEeqi nd visulised y utordiogrE PeD lne PA whih ould e immunopreipitted with vep ntiE phyF lsmid pTR without insert did not diret the synthesis odies @results not shownAF he QP kh nd ws not present of ny rdiolelled protein @pigF IfD lne IAD ut the plsmid in the sene of proteinse u @lne IA nd ws degrded rrying the vepf insert @lne PA gve rise to one rdiolelled when the vesiles were soluilized y EIHH @lne QA efore ndF e omprle onstrut with the gene oding for vElE protese tretmentF herefore it is onluded tht proteinE tmse reding in the sme diretion s vep gve rise to se u leves the I loop while the P domin is proteted seond nd diretly under the nd of vep @results not ginst proteolyti ttk inside the vesile @pigF PfAF sn this shownAF he nd in lne P hs n pprent moleulr weight otrnsltionl ssy PH7 of the synthesized vep gets inteE of QT kh nd runs t the sme position s puri¢ed unlelled grted in the vesilesF risEtgged vep @position inditedAF woreoverD fter proteiE he hrteristis of otrnsltionl memrne integrtion nse u tretment of the smpleD no rdiolelled protein ws of vep were studied using ntiodies ginst ee nd y oserved @lne QA showing tht there is no intrinsi protese using vesiles de¢ient in e funtionF he vlidity of the resistne in this proteinF he identity ws on¢rmed y imE ntiody pproh ws tested y studying the in£uene of munopreipittion
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