pifƒ IWHQT pifƒ vetters RIQ @IWWUA IHW”IIR

sn vitro mem˜r—ne integr—tion of le—der peptid—se depends on the ƒe™

m—™hinery —nd —nioni™ phospholipids —nd ™—n o™™ur postEtr—nsl—tion—lly

—Y — —

‡im v—n ulompen˜urg BD enj— xFtFeF ‚idder D enne vFtF v—n ‚——lte D

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entoinette tF uilli—n D qunn—r von reijne D fen de uruij¡

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hep—rtment of fio™hemistry of wem˜r—nesD gentre for fiomem˜r—nes —nd vipid inzymologyD snstitute of fiomem˜r—nesD

tre™ht niversityD €—du—l——n VD QSVR gr tre™htD „he xetherl—nds

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hep—rtment of fio™hemistryD errhenius v—˜or—toryD ƒto™kholm niversityD ƒEIHT WI ƒto™kholmD ƒweden

‚e™eived S tune IWWUY revised version re™eived PU tune IWWU

tion ‘IP“F enioni™ phospholipidsD whi™h represent —˜out PS7 e˜str—™t e ™ellEfree system ˜—sed on — lys—te —nd mem˜r—ne

of the mem˜r—ne lipids of iF ™oliD —re —lso involved in tr—nsE vesi™les from is™heri™hi— ™oli is used to study ™h—r—™teristi™s of

lo™—tion ‘IQ“F „hey stimul—te the e„€—se —™tivity of ƒe™e ‘IR“ the mem˜r—ne integr—tion re—™tion of the polytopi™ mem˜r—ne

—nd —re ™—p—˜le of inter—™ting with sign—l peptides ‘IS“F purE protein le—der peptid—se @vepAF sntegr—tion into inverted inner

thermoreD e¤™ient in vivo tr—nslo™—tion requires the presen™e mem˜r—ne vesi™les w—s dete™ted ˜y p—rti—l prote™tion —g—inst

extern—lly —dded prote—seF sntegr—tion is most effi™ient when of — proton motive for™e @€wpA ‘IT“ —s well —s the presen™e of

™oupled to tr—nsl—tion ˜ut ™—n —lso o™™ur postEtr—nsl—tion—lly —nd two mem˜r—ne proteins @ƒe™h —nd ƒe™pA whi™h m—y ˜e inE

depends on the —™tion of the protein—™eous ƒe™ m—™hinery —nd volved in m—int—ining €wp during tr—nslo™—tion ˜ut the ex—™t

—v—il—˜ility of —nioni™ phospholipidsF vep is the first ex—mple of —

roles of whi™h —re unknown ‘IU“F efter tr—nslo™—tion the sigE

mem˜r—ne protein without ™le—v—˜le sign—l sequen™e whi™h

n—l sequen™es —re removed ˜y the —™tion of sign—l peptid—ses

requires —nioni™ lipids for integr—tion in vitroF

su™h —s vep ‘IV“F „r—nslo™—tion of some pre™ursors requires

z IWWU peder—tion of iurope—n fio™hemi™—l ƒo™ietiesF

other ™ytosoli™ ™omponents like the qroiv ™h—perone ‘IW“ or

prok—ryoti™ ƒ‚€ homologues ‘PH“F uey wordsX ve—der peptid—seY sn vitro tr—nslo™—tionY

sn vitro tr—nslo™—tion systemsD employing r—diol—˜elled preE wem˜r—ne proteinY is™heri™hi— ™oli

™ursors —nd puri¢ed inner mem˜r—ne vesi™lesD h—ve m—de —n

import—nt ™ontri˜ution to the ™urrent knowledge —˜out preE

™ursor protein tr—nslo™—tion ˜e™—use they —llow system—ti™

IF sntrodu™tion investig—tion of the f—™tors involvedF sn this study we est—˜lish

—n in vitro system for mem˜r—ne integr—tion of vep whi™h

ve—der peptid—se @vepA from is™heri™hi— ™oli is —n integr—l involves mem˜r—ne p—ss—ge of the peripl—smi™ dom—inF „he

inner mem˜r—ne protein whi™h sp—ns the mem˜r—ne with two requirements for this pro™ess —re investig—ted ˜y using v—rious

hydropho˜i™ segments whi™h —re ™onne™ted ˜y — short ™ytoE types of vesi™les —nd ˜y —dding —gents to the —ssem˜ly re—™E

pl—smi™ loop ‘I“F „he se™ond tr—nsmem˜r—ne segment preE tions whi™h ˜lo™k the fun™tion of ™omponents possi˜ly inE

™edes — l—rge peripl—smi™ dom—in whi™h during —ssem˜ly h—s volved in —ssem˜lyF

to p—ss the inner mem˜r—ne ‘P“F sn re™ent ye—rs vep h—s ˜een

extensively used —s — model to study mem˜r—ne protein inserE PF w—teri—ls —nd methods

tion in vivo —nd it —ppe—red th—t the me™h—nism for tr—nsE

PFIF w—teri—ls lo™—tion of peripl—smi™ loops depends on loop lengthF voops

„rinu™leotides @sodium s—ltsAD „R lig—se —nd the restri™tion endonuE

sm—ller th—n TH —mino —™id residues p—ss the mem˜r—ne ˜iE

™le—ses ƒm—s —nd ƒ—ls were o˜t—ined from €h—rm—™i—F polini™ —™id

l—yer in —n —pp—rently spont—neous pro™essD while tr—nslo™—E @™—l™ium s—ltAD phospho™re—tine @diE„ris s—ltA polymyxin f sulf—teD

tion of longer loops like the peripl—smi™ dom—in of vep reE phenyl methyl sulfonyl £uoride @€wƒpA —nd —mino —™ids were from

ƒigm— @ ƒeAF ƒ€T ‚xe polymer—seD ™re—tine phosphokin—seD t‚xeD quires the —™tion of the soE™—lled ƒe™ m—™hinery ‘QDR“F

puromy™in dihydro™hloride —nd protein—se u were pur™h—sed from

yrigin—llyD ƒe™ m—™hinery w—s found to ˜e essenti—l for

w—s puri¢ed —s des™ri˜ed ˜efore foehringer @qerm—nyAF vep@risA T

tr—nslo™—tion of peripl—smi™ —nd outer mem˜r—ne proteins

‘PI“F ƒe™e —nd vEl—™t—m—se —nti˜odies were — gift from hrF rF de

—™ross the inner mem˜r—ne @for review see ‘S“AF nlike vepD go™kD tre™ht niversityF

these peripl—smi™ —nd outer mem˜r—ne proteins —re syntheE

PFPF ƒtr—ins —nd growth ™onditions sized —s pre™ursors with xEtermin—l extensions ™—lled sign—l

iF ™oli str—in w‚i THH ‘PP“ w—s used for the produ™tion of ƒEIQS

sequen™esF „hey —re often m—int—ined in — tr—nslo™—tion ™omE

lys—te —nd grown in qiston ˜roth —t QU³g until e—rly exponenti—l

petent st—te ˜y the tetr—meri™ ƒe™f protein ‘T”V“F „hese ƒe™f” ph—seF ‚eferen™e vesi™les were isol—ted from str—in ƒh IP ‘PQ“ whi™h

pre™ursor ™omplexes h—ve — high —¤nity for ƒe™e whi™h is w—s grown —t QU³g in vfF €g PWUU ‘PR“ w—s used —s — sour™e of

ts

vesi™lesF gells were grown overnight —t QH³g in vf with ƒe™‰ found in the ™ytosol —nd in multiple ™onform—tions in the

IH ‡gGml tetr—™y™line —nd —t RP³gD Q h prior to vesi™le isol—tionF

inner mem˜r—ne where it ™ouples e„€ hydrolysis to the progE

†esi™les with low ™ontents of —nioni™ lipids were isol—ted from

ress of tr—nslo™—tion ‘W”II“F „ogether with ƒe™eD ƒe™E‰D Ei

rhvII ‘PS“D whi™h w—s grown in vf supplemented with IH ‡gGml

—nd Eq proteins ™onstitute the ˜—si™ m—™hinery for tr—nslo™—E tetr—™y™lineD PS ‡gGml ™hloro—mpheni™ol —nd SH ‡gGml k—n—my™inF

PFQF sn vitro tr—ns™riptionD tr—nsl—tion —nd —ssem˜ly

Bgorresponding —uthorF €resent —ddressX hep—rtment of wole™ul—r ƒEIQS ™ell extr—™ts —nd inverted inner mem˜r—ne vesi™les were preE

wi™ro˜iologyD qffD uerkl——n QHD WUSI xx r—renD „he xetherl—ndsF p—red from iF ™oli —™™ording to pu˜lished pro™edures ‘PT“F €l—smid

p—xX @QIA SHEQTQPISRF pƒ€vep w—s ™re—ted ˜y ™loning the ƒ—ls”ƒm—s fr—gment ™ont—ining

iEm—ilX wFklompen˜urgd˜iolFrugFnl lepf from — psxqI derived in vivo expression ve™tor to the in vitro

HHIRESUWQGWUG6IUFHH ß IWWU peder—tion of iurope—n fio™hemi™—l ƒo™ietiesF ell rights reservedF

€ss ƒHHIRESUWQ@WUAHHVVVEH pifƒ IWHQT PPEIHEWU

IIH ‡F v—n ulompen˜urg et —lFGpifƒ vetters RIQ @IWWUA IHW”IIR

expression ve™tor pƒ€TRF hxe w—s puri¢ed using wiz—rd minipreps

hxe puri¢™—tion resin @€romeg—D ƒeA —nd used to dire™t the tr—nE

s™ription of the vepf gene ˜y ƒ€T ‚xe polymer—se during — QH min

in™u˜—tion —t RH³g following the m—nuf—™turer9s instru™tionsF „r—nsE

l—tions were ™—rried out —t QU³g —s des™ri˜ed ‘PT“F pive minutes —fter

initi—tion of tr—nsl—tionD inverted inner mem˜r—ne vesi™les were —dded

to — ¢n—l ™on™entr—tion of R mgGml @proteinA —nd the in™u˜—tion w—s

™ontinued for PS min —t QU³gF gorre™t —ssem˜ly of vep w—s demonE

str—ted ˜y prote™tion of the €P dom—in —g—inst the —™tion of extern—lly

—dded protein—se u @PHH ‡gGml ¢n—l ™on™entr—tionAF „he prote—se

tre—tment w—s ™—rried out for PH min —t room temper—ture —fter whi™h

P mw €wƒp w—s —ddedF „he s—mples were —n—lysed ˜y ƒhƒE€eqi

—nd exposure of the gel in — €hosphor sm—gerF „o —ssess the in£uen™e

of —dditions @—nti˜odies or polymyxinAD these were mixed with the

vesi™les prior to in™u˜—tion in the tr—nslo™—tion mixtureF

PFRF smmunopre™ipit—tion

smmunopre™ipit—tions using —ntiserum dire™ted —g—inst vep were

performed —s des™ri˜ed ‘PU“ using €rotein eE™oupled ƒeph—rose

gvRf @€h—rm—™i—D vufD ƒwedenAF

PFSF €roympe tr—nslo™—tion

QS

ƒ“proympe w—s puri¢ed —s des™ri˜ed ‘PV“ —nd stored in V w ‘

QS

ƒ“proympe w—s diluted SHEfold into PS ‡l of tr—nslo™—tion ure—F ‘

˜u¡er @RH mw „risE—™et—teD pr VFHD IHFV mw m—gnesium —™et—teD

PV mw pot—ssium —™et—teD P mw h„„D HFS mgGml fƒeA ™ont—ining

inverted inner mem˜r—ne vesi™les @HFRH mg proteinGmlAD S ‡l ƒEIQS ™ell

extr—™t —nd R mw e„€ ‘V“F ‡hen indi™—ted I ‡l of poly™lon—l —ntiE

serum dire™ted —g—inst ƒe™e or vEl—™t—m—se w—s —ddedF „r—nslo™—tion

w—s —llowed to pro™eed for PH min —t QU³gD followed ˜y —n—lysis —s

des™ri˜ed for the —ssem˜ly of vepF

QF ‚esults

„o —llow in vitro synthesis of m‚xe ˜y ƒ€T ‚xe polyE

mer—seD the vepf gene w—s ™loned into —n in vitro expression

ve™torF wost expression pl—smids ™—rry the gene ™oding for

pigF IF €l—smid dire™ted in vitro synthesis —nd identi¢™—tion of vepF

the sele™t—˜le m—rker vEl—™t—m—se in the s—me orient—tion —s eX €l—smid m—p of pƒ€vepF ‚estri™ition sitesD genes @vepf —nd

fl—AD the ƒ€T promoter —nd the origin of repli™—tion @oriA —re indiE the ƒ€T promoter whi™h gives rise to su˜st—nti—l —mounts of

™—tedF fX sn vitro protein synthesis from pƒ€TR without @l—ne IA or expression of this proteinF fe™—use of simil—r mole™ul—r

with —n insert ™ont—ining the vepf gene @l—nes P”RAF efter synthesis

weights of vep —nd vEl—™t—m—seD this would ™ompli™—te the

s—mples were put on i™e @l—ne PA or tre—ted with PHH ‡gGml proteinE

interpret—tion of the resultsF „herefore we m—de use of —se u @l—ne QA or immunopre™ipit—ted using vep —ntiserum @l—ne RAF

pƒ€TR to ™lone the vepf gene in the opposite orient—tion —s ell s—mples were —fter their respe™tive tre—tments —n—lysed ˜y ƒhƒE

€eqi followed ˜y —utor—diogr—phyF „he position of nonEl—˜elled the vEl—™t—m—se gene to result in pƒ€vep @pigF IeAF efter

puri¢ed vep on the gel is indi™—tedF

tr—nsl—tion of the ‚xe ˜y — mem˜r—neEfree lys—te of iF ™oli

QS

ƒ“methionineD the produ™ts w‚i THH in the presen™e of ‘

were resolved ˜y ƒhƒE€eqi —nd visu—lised ˜y —utor—diogr—E PeD l—ne PA whi™h ™ould ˜e immunopre™ipit—ted with vep —ntiE

phyF €l—smid pƒ€TR without insert did not dire™t the synthesis ˜odies @results not shownAF „he QP kh— ˜—nd w—s not present

of —ny r—diol—˜elled protein @pigF IfD l—ne IAD ˜ut the pl—smid in the —˜sen™e of protein—se u @l—ne IA —nd w—s degr—ded

™—rrying the vepf insert @l—ne PA g—ve rise to one r—diol—˜elled when the vesi™les were solu˜ilized ˜y „ˆEIHH @l—ne QA ˜efore

˜—ndF e ™omp—r—˜le ™onstru™t with the gene ™oding for vEl—™E prote—se tre—tmentF „herefore it is ™on™luded th—t proteinE

t—m—se re—ding in the s—me dire™tion —s vep g—ve rise to — —se u ™le—ves the €I loop while the €P dom—in is prote™ted

se™ond ˜—nd dire™tly under the ˜—nd of vep @results not —g—inst proteolyti™ —tt—™k inside the vesi™le @pigF PfAF sn this

shownAF „he ˜—nd in l—ne P h—s —n —pp—rent mole™ul—r weight ™otr—nsl—tion—l —ss—y PH7 of the synthesized vep gets inteE

of QT kh— —nd runs —t the s—me position —s puri¢ed unl—˜elled gr—ted in the vesi™lesF

risEt—gged vep @position indi™—tedAF woreoverD —fter proteiE „he ™h—r—™teristi™s of ™otr—nsl—tion—l mem˜r—ne integr—tion

n—se u tre—tment of the s—mpleD no r—diol—˜elled protein w—s of vep were studied using —nti˜odies —g—inst ƒe™e —nd ˜y

o˜served @l—ne QA showing th—t there is no intrinsi™ prote—se using vesi™les de¢™ient in ƒe™‰ fun™tionF „he v—lidity of the

resist—n™e in this proteinF „he identity w—s ™on¢rmed ˜y imE —nti˜ody —ppro—™h w—s tested ˜y studying the in£uen™e of

munopre™ipit—tion with vep —nti˜ody @l—ne RAF st w—s thereE ƒe™e —nti˜odies on the tr—nslo™—tion of puri¢ed proympeF

fore ™on™luded th—t pƒ€vep spe™i¢™—lly dire™ts the synthesis €roympe is the pre™ursor form of —n outer mem˜r—ne proE

of vepF tein of iF ™oli —nd its tr—nslo™—tion is dependent on — fun™E

„o study mem˜r—ne integr—tion of vep in the s—me dire™E tion—l ƒe™ m—™hineryF „r—nslo™—tion w—s performed —s in ƒe™P

tion —s in whole ™ellsD inverted inner mem˜r—ne vesi™les from —nd —nti˜odies were —dded to the vesi™les prior to the tr—nsE

str—in ƒhIP were —dded during tr—nsl—tionF €rotein—se u w—s l—tion”tr—nslo™—tion re—™tionF ƒin™e pre™ursor pro™essing is

—dded —fterw—rds to investig—te put—tive lumen—l lo™—lis—tion most often not fully fun™tion—l in these in vitro systemsD tr—nsE

of p—rts of vepF „his reve—led one single ˜—nd of QP kh— @pigF lo™—tion w—s de¢ned —s the —mount of prote—se prote™ted pifƒ IWHQT PPEIHEWU

‡F v—n ulompen˜urg et —lFGpifƒ vetters RIQ @IWWUA IHW”IIR III

des™ri˜ed ˜efore for preproteinsF vep —ssem˜ly into these

vesi™les w—s redu™ed to IFS þ I7 @n a QA @l—nes U —nd VAF „his

very minor integr—tion —™tivity ™ould ˜e due to residu—l fun™E

tion—l ƒe™‰ mole™ulesF st is therefore ™on™luded th—t the in

vitro integr—tion re—™tion of vep requires — fun™tion—l ƒe™

m—™hineryF

i¤™ient tr—nslo™—tion of pre™ursor proteins depends on

—nioni™ phospholipids in the mem˜r—ne ‘IQDPS“F „o investig—te

the in£uen™e of —nioni™ phospholipids on the —ssem˜ly of vepD

vesi™les were isol—ted from — lipid ˜iosyntheti™ mut—nt str—inD

rhvIIF sn this str—inD the pgse gene is pl—™ed under ™ontrol

of the l—™ operonF „his gene is responsi˜le for the synthesis of

the m—jor neg—tively ™h—rged phospholipid in iF ™oliD phosE

ph—tidylgly™erolF sn the —˜sen™e of the indu™er s€„qD the

—nioni™ phospholipid ™ontent of the inner mem˜r—ne is ˜elow

IH7 ‘PS“D whi™h is PFS times lower th—n in wildEtype ™ellsF vep

—ssem˜les into vesi™les isol—ted from rhvIID whi™h w—s

grown in the —˜sen™e of s€„qD with e¤™ien™ies ˜elow I7

@l—nes W —nd IHAF „he import—n™e of —nioni™ phospholipids

for the integr—tion pro™ess w—s —lso tested ˜y shielding the

neg—tive ™h—rges ˜y — positively ™h—rged —gentF „he poly ™—tE

ioni™ —nti˜ioti™ €olymyxinf spe™i¢™—lly inter—™ts with —nioni™

phospholipids ‘PW“F †esi™les with wildEtype lipid ™omposition

were in™u˜—ted with di¡erent ™on™entr—tions of polymyxinf

prior to the integr—tion experimentF pigF R shows th—t integr—E

tion e¤™ien™ies de™re—se with in™re—sing polymyxin ™on™entr—E

tionsF €olymyxinf @PH ‡wA ™—uses — h—lf m—xim—l ˜lo™k of the

integr—tion re—™tionF

por pre™ursor proteins it w—s shown th—t tr—nslo™—tion

™ould o™™ur —fter ™ompletion of tr—nsl—tionF sing the in vitro

system the possi˜ility of postEtr—nsl—tion—l integr—tion of the

integr—l mem˜r—ne protein vep ™ould ˜e investig—tedF sn our

in vitro systemD synthesis of vep re—™hes — m—ximum within

PS”QH min @results not shownA —nd to ensure —rrest of synE

thesisD puromy™in w—s —dded to disso™i—te the ri˜osomesF

€ostEtr—nsl—tion—l integr—tion w—s —ss—yed —s followsX PV min

—fter st—rt of tr—nsl—tionD PH ‡w puromy™in w—s —ddedF efter

P min vesi™les were —dded @pigF SD l—nes P”SA or not @l—ne IAF

e„€ w—s —dded to one in™u˜—tion mixture @l—ne RA to m—ke

sure th—t energy w—s not in short supplyF efter PH min €u w—s pigF PF gotr—nsl—tion—l —ssem˜ly of in vitro synthesized vep in inE

verted inner mem˜r—ne vesi™lesF eX en—lysis of the —ssem˜ly pro™E —dded @l—nes Q —nd RAF foth in —˜sen™e —nd presen™e of extr—

essF pive minutes —fter st—rt of tr—nsl—tion vesi™les were —dded —nd e„€D Q þ I7 integr—tion w—s o˜servedD whi™h is S”IH times

the tr—nsl—tion w—s prolonged for PS minF efter this s—mples were

less th—n in the ™otr—nsl—tion—l experimentF ‡hen „riton

either not tre—ted @l—ne IA or tre—ted with PHH ‡gGml protein—se u in

ˆEIHH w—s —dded ˜efore prote—se tre—tmentD —ll protein w—s the —˜sen™e @l—ne PA or presen™e @l—ne QA of „riton ˆEIHHF fX yrienE

degr—ded @l—ne SAF st w—s ™he™ked th—t under the integr—tion t—tion of vep in inverted mem˜r—ne vesi™lesF

™onditions no protein synthesis t—kes pl—™eF por this purposeD

tr—nsl—tion w—s ™—rried out in the presen™e of unl—˜elled meE

proympe —nd ympeF sn the —˜sen™e of —nti˜odies @pigF QeD thionine for PV minF ƒu˜sequently the tr—nsl—tion mixture w—s

l—ne PA PH7 tr—nslo™—tion w—s o˜servedD while the presen™e of in™u˜—ted with PH ‡w puromy™in for P minD —fter whi™h r—E

ƒe™e —nti˜ody prevented tr—nslo™—tion of proympe ™omE dio—™tively l—˜elled methionine w—s —dded —nd the in™u˜—tion

pletely @pigF QeD l—ne QAF es — ™ontrolD tr—nslo™—tion w—s prolonged for PH minF xo r—dio—™tive m—teri—l w—s o˜served

studied in the presen™e of the vEl—™t—m—se —nti˜odies @pigF —t the position of the fullElength vep protein @pigF SD l—ne TAF st

QeD l—ne RA whi™h did not in£uen™e tr—nslo™—tion e¤™ien™yF should therefore ˜e ™on™luded th—t under the ™onditions deE

ƒin™e the ƒe™e —nti˜odies ˜lo™k ƒe™Edependent tr—nslo™—tionD s™ri˜ed —˜oveD integr—tion ™—n o™™ur postEtr—nsl—tion—lly —l˜eit

this pro™edure w—s used to est—˜lish whether ƒe™e is required very ine¤™ientF

in the in vitro —ssem˜ly re—™tion of vepF sn the —˜sen™e of

—nti˜odies vep —ssem˜les into sw†s with —n e¤™ien™y of RF his™ussion

PH þ I7 @n a RA @pigF QfD l—nes I —nd PAF sn the presen™e of

ƒe™e —nti˜ody this e¤™ien™y drops ˜elow I7 @l—nes Q —nd RA sn this p—per — ™ellEfree tr—ns™ription”tr—nsl—tion system is

while no in£uen™e of the —ddition of vEl—™t—m—se —nti˜odies des™ri˜ed to synthesize le—der peptid—se whi™h ™—n ˜e used to

w—s me—sur—˜le @l—ne S —nd TAF „he requirement for ƒe™‰ w—s study mem˜r—ne integr—tionF sn the present system vep is

tested using vesi™les from — temper—ture sensitive str—in —s w—s produ™ed in — ƒEIQS lys—te to study ™oE —nd postEtr—nsl—tion—l pifƒ IWHQT PPEIHEWU

IIP ‡F v—n ulompen˜urg et —lFGpifƒ vetters RIQ @IWWUA IHW”IIR

pigF QF ‚equirements for mem˜r—ne —ssem˜ly of vepF eX „r—nslo™—tion of puri¢ed proympe ™—n ˜e ˜lo™ked ˜y —nti˜odies —g—inst ƒe™eF „he

—mount of tr—nslo™—ted ympe —nd proympe were ™omp—red to PH7 of the —dded pre™ursor @l—ne IAF „r—nslo™—tion w—s ™—rried out in the

presen™e of ƒe™e —nti˜odies @l—ne QAD vEl—™t—m—se —nti˜odies @l—ne RA or in the —˜sen™e of —nti˜odies @l—ne PAF fX essem˜ly of vep in wildEtype

vesi™les @l—nes I —nd PAD in vesi™les tre—ted with —nti˜odies —g—inst ƒe™e @l—nes Q —nd RA or with —nti˜odies —g—inst vEl—™t—m—se @l—nes S —nd TAD

vesi™les depleted of fun™tion—l ƒe™‰ @l—nes U —nd VA or vesi™les redu™ed in —nioni™ phospholipid ™ontents @l—nes W —nd IHAF „he —mount of proE

te—se prote™ted vep @even num˜ered l—nesA w—s ™omp—red to PH7 of the vep synthesized @odd num˜ered l—nesA in presen™e of vesi™lesF

mem˜r—ne integr—tionF huring — ™otr—nsl—tion—l in™u˜—tion integr—te in the mem˜r—nes of mut—nt str—ins in whi™h the

with wildEtype vesi™les PH7 of the synthesized vep ˜e™—me ƒe™Edependent pre™ursor tr—nslo™—tion is imp—ired ‘R“F ynly

integr—ted in the vesi™lesF „his seems re—son—˜le for in vitro

systems sin™e tr—nslo™—tion of pre™ursor proteins in simil—r

systems is usu—lly in the s—me r—ngeD typi™—lly PS”RH7

‘PTDQH“F sn — previous p—per ‘PI“D the n—tive popul—tion of

vep mole™ules in inverted inner mem˜r—ne vesi™les w—s

studiedF €rote—seu tre—tment of inverted mem˜r—ne vesi™lesD

followed ˜y ƒhƒE€eqi —nd ‡estern ˜lotting with vep —ntiE

˜ody yielded —lso — ˜—nd of QP kh— ‘PI“F „his not only ™orE

ro˜or—tes the —ssignment of the o˜served QP kh— fr—gment in

the present studyD ˜ut —lso shows th—t the in vitro synthesized

vep resides in the s—me orient—tion in inverted inner memE

˜r—ne vesi™les —s the n—tive popul—tionF

yn the ˜—sis of in vivo experiments the integr—tion of memE

˜r—ne proteins is thought to follow one of two possi˜le p—thE

pigF RF i¡e™ts of polymyxin f on integr—tion e¤™ien™yF €rior to the w—ysF €roteins with l—rge peripl—smi™ loopsD su™h —s vepD deE

integr—tion re—™tion vesi™les were in™u˜—ted with the indi™—ted ™onE

pend fully on the —™tivity of the ƒe™ m—™hineryD while sm—ller

™entr—tions polymyxin f sulf—teF „r—nslo™—tion w—s ™—rried out —nd

peripl—smi™ loops ™—n p—ss the inner mem˜r—ne independent

—n—lysed —s des™ri˜ed under ƒe™tion PF we—n v—lues of tr—nslo™—tion

of the ƒe™ m—™hineryF roweverD ƒe™Eindependen™e is most e¤™ien™ies —re depi™ted —nd the error ˜—rs indi™—te st—nd—rd devi—E

tionF often only oper—tion—lly de¢ned —s the —˜ility to su™™essfully pifƒ IWHQT PPEIHEWU

‡F v—n ulompen˜urg et —lFGpifƒ vetters RIQ @IWWUA IHW”IIR IIQ

in in vitro systems —nd stimul—ted ˜y the presen™e of the

mole™ul—r ™h—perone qroiv ‘QR“F

sn this p—per two methods were used to study the in£uen™e

of —nioni™ lipids on integr—tion of vepF †esi™les from the lipid

˜iosyntheti™ mut—nt str—in rhvII whi™h ™ont—in less th—n

IH7 —nioni™ phospholipids were employed —nd the poly™—tE

ioni™ —nti˜ioti™ polymyxin f w—s used to shield neg—tive

™h—rges on vesi™les with wildEtype lipid ™ompositionF sn ˜oth

experimentsD the integr—tion e¤™ien™ies were de™re—sed in

™omp—rison to the ™ontrol situ—tionF ƒtr—in rhvII w—s

used ˜efore in sever—l studies —imed —t the e¡e™ts of redu™ed

—nioni™ lipid on protein tr—nslo™—tion —nd ™oli™in —™tionF

pigF SF ess—y to study postEtr—nsl—tion—l mem˜r—ne integr—tion of ‘PSDQS“ st w—s shown thereD th—t in this p—rti™ul—r str—in low

le—der peptid—seF ƒee text for det—ilsF „r—nsl—tion w—s ™—rried out

—nioni™ lipid ™on™entr—tions did not —¡e™t intern—l e„€ ™onE

for PV min in the presen™e of r—diol—˜elled @l—nes I”SA or nonEl—E

™entr—tion nor the mem˜r—ne potenti—l ‘QS“F st w—s therefore

˜elled methionine @l—ne TA —fter whi™h s—mples were in™u˜—ted with

™on™luded th—t the e¡e™ts of —nioni™ lipids —re not ™—used ˜y — PH ‡w puromy™in for P minF iither e„€ —nd l—˜elled methionine

@l—ne TA or inverted inner mem˜r—ne vesi™les were —dded —nd the inE deEenergized mem˜r—neF €olymyxin whi™h ™—rries ¢ve positive

™u˜—tion w—s prolonged for IS minF essem˜ly w—s determined —s ™h—rgesD w—s used previously to study the —nioni™ lipid deE

des™ri˜ed in ƒe™tion PF

penden™y of pre™ursor proteins in vitro ‘QT“F st w—s reported

th—t polymyxin did not —¡e™t the mem˜r—ne potenti—l or ™—use

—ggreg—tion of vesi™les ‘QT“F sn our studies — h—lfEm—ximum

in vitro systems —llow to ¢rmly —ddress this pointF sn our inhi˜itory e¡e™t w—s found —t —pproxim—tely PH ‡w whi™h

system the integr—tion of vep w—s dependent on the ƒe™ m—E ™orresponds to the presen™e of I polymyxin mole™ule in the

™hinery just —s w—s o˜served in vivoF €reviouslyD —nother in —ss—y per R”S —nioni™ phospholipid mole™ules whi™h is ™lose to

vitro system w—s des™ri˜ed to study mem˜r—ne integr—tion of — ™h—rge”stoi™hiometri™ ™omplexF st is ™on™luded th—t in vitro

vepD whi™h w—s fo™ussed on identifying segments within vep mem˜r—ne insertion of vep depends on —nioni™ lipidsF „his is

th—t were required for —ssem˜ly ‘QI“F „he in vitro system from the ¢rst ex—mple of — mem˜r—ne protein without ™le—v—˜le

liter—ture di¡ers in two points from the present systemF yur sign—l sequen™e whi™h requires —nioni™ phospholipids for inE

system does not require puri¢ed er—g protein —nd it m—kes tegr—tionF

use of — ƒEIQS lys—te inste—d of — ƒEQH lys—teF „here —re —t le—st two di¡erent w—ys in whi™h —nioni™ phosE

yur results indi™—te th—t the integr—l mem˜r—ne protein vep pholipids ™ould —¡e™t the integr—tion of vepF yne possi˜ility is

™—n integr—te postEtr—nsl—tion—lly in inverted mem˜r—ne th—t the de™re—sed tr—nslo™—tion e„€—se —™tivity of ƒe™e

vesi™les using —n in vitro systemF „he o˜vious question is upon lowering the —mount of —nioni™ phospholipids ‘IR“ —™E

then whether this ™—n —lso h—ppen in living ™ellsF endersson ™ounts for h—mpered pre™ursor tr—nslo™—tion or mem˜r—ne

—nd von reijne performed in vivo integr—tion experiments in protein integr—tion —t de™re—sed —nioni™ lipid ™ontentsF yn

whi™h the mem˜r—ne p—ss—ge of €P of vep is ˜lo™ked ˜y the other h—ndD —lso dire™t inter—™tions ˜etween —nioni™ lipids

—ddition of the un™oupler ggg€F efter in—™tiv—tion of this —nd vep —re fe—si˜leF

™ompoundD tr—nslo™—tion of the €P dom—in ™ould pro™eed „here —re — few ex—mples illustr—ting th—t h—mpered ƒe™e

‘QP“F sn —dditionD geneti™—lly engineered mem˜r—ne proteins fun™tion is —t le—st not the only f—™tor expl—ining de™re—sed

with four mem˜r—neEsp—nning segments inserted in vivo in tr—nslo™—tion e¤™ien™ies —t low —nioni™ lipid levelsF ixperiE

the mem˜r—ne in — f—shion whi™h w—s not ™omp—ti˜le with ments using ™himeri™ proymppEvpp mole™ules with —rti¢™i—l

— simple line—r xE to gEintegr—tion pro™essF ƒu™h — line—r sign—l sequen™es reve—led th—t e¡e™ts of —nioni™ phospholipids

integr—tion w—s expe™ted if insertion w—s to h—ppen ™omE on tr—nslo™—tion depended on the —mino —™id ™omposition of

pletely ™otr—nsl—tion—lly ‘QQ“F st should therefore ˜e ™on™luded the sign—l sequen™eD ˜ut not on the ƒe™e dependen™y of tr—nsE

th—t integr—tion of mem˜r—ne proteins ™—n o™™ur postEtr—nsE lo™—tionF „r—nslo™—tion of €roymppEvpp mole™ules with long

l—tion—llyF por pre™ursor proteins it w—s shown th—t their hydropho˜i™ polyleu™ine stret™hes in their sign—l sequen™e w—s

tr—nslo™—tion ™ould —lso o™™ur —fter ™ompletion of tr—nsl—tionF independent of —nioni™ phospholipids ˜ut still required ƒe™e

V7 of the pre€hoi whi™h w—s synthesized in —n in vitro sysE for tr—nslo™—tion ‘QUDQV“F sn —ddition it w—s shown th—t ƒe™e

tem tr—nslo™—ted into the lumen of — vesi™le in — postEtr—nsE independent integr—tion of the wIQ pro™o—t mem˜r—ne proE

l—tion—l tr—nslo™—tion experiment while during ™otr—nsl—tion—l tein h—d — simil—r requirement for —nioni™ phospholipids —s —

in™u˜—tionsY PS7 of the synthesized m—teri—l w—s tr—nslo™—ted ™onstru™t in whi™h the peripl—smi™ dom—in w—s enl—rged to

‘PT“F sn our experimentsD postEtr—nsl—tion—l mem˜r—ne integr—E ˜e™ome ƒe™e dependent ‘QW“F „hese studies demonstr—te th—t

tion of vep is even less e¤™ient @Q7A when ™omp—red to ™oE —nioni™ lipids ™ould —lso inter—™t dire™tly with pre™ursors —nd

tr—nsl—tion—l experiments in whi™h PH7 w—s integr—tedF „he mem˜r—ne proteinsF por the wIQ pro™o—t protein it w—s o˜E

rel—tively low e¤™ien™y of postEtr—nsl—tion—l integr—tion of served th—t positively ™h—rged —mino —™yl residues in the ™yE

vep m—y ˜e expl—ined ˜y properties of the proteinF vep ™onE topl—smi™ regions £—nking the two hydropho˜i™ segments

t—ins three hydropho˜i™ stret™hesD of whi™h two —ssume — were required for insertion ‘RH“ —nd the results of ˜inding

tr—nsmem˜r—ne ™on¢gur—tionF st ™—n ˜e envis—ged th—t these studies with lipid vesi™les indi™—ted th—t ele™trost—ti™ inter—™E

segments h—ve — strong tenden™y to —ggreg—te —nd require — tions ˜etween these positively ™h—rged residues —nd —nioni™

™h—perone for postEtr—nsl—tion—l insertionF ‚e™entlyD it w—s phospholipids —re involved in initi—tion of insertion‘RH“F st is

reported th—t postEtr—nsl—tion—l mem˜r—ne insertion of the fe—si˜le th—t ˜inding of positive ™h—rges to the neg—tive surE

hydropho˜i™ mem˜r—ne protein l—™tose perme—se w—s possi˜le f—™e of the mem˜r—ne f—™ilit—tes the insertion of hydropho˜i™ pifƒ IWHQT PPEIHEWU

IIR ‡F v—n ulompen˜urg et —lFGpifƒ vetters RIQ @IWWUA IHW”IIR

‘IW“ fo™hk—rev—D iFƒFD vissinD xFwF —nd qirshovi™hD eFƒF @IWVVA x—E segments into the hydropho˜i™ ™ore of the mem˜r—neF gh—rge

ture QQTD PSR”PSUF

inter—™tions ˜etween —nioni™ phospholipids —nd positively

‘PH“ vuirinkD tF et —lF @IWWPA x—ture QSWD URI”URQF

™h—rged —mino —™ids ™—n pl—y — gener—l role in mem˜r—ne

‘PI“ †—n ulompen˜urgD ‡F et —lF @IWWSA wolF wemF fiolF IPD QRW”

protein insertionF QSQF

‘PP“ g—mm—™kD uFeF —nd ‡—deD rFiF @IWTSA fio™hemF tF WVD TUI”TVHF

‘PQ“ ƒhi˜uy—D sFD wiy—z—kiD gF —nd yht—D eF @IWVSA tF f—™teriolF ITID

‚eferen™es

IHVT”IHWPF

‘PR“ ƒhi˜—D uF et —lF @IWVRA iwfy tF QD TQI”TQSF

‘I“ ‡olfeD €FfFD ‡i™knerD ‡F —nd qoodm—nD tFwF @IWVQA tF fiolF

‘PS“ uustersD ‚FD howh—nD ‡FeF —nd de uruij¡D fF @IWWIA tF fiolF

ghemF PSVD IPHUQ”IPHVHF

ghemF PTTD VTSW”VTTPF

‘P“ h—l˜eyD ‚FiF —nd ‡i™knerD ‡F @IWVUA ƒ™ien™e PQSD UVQ”UVUF

‘PT“ he †rijeD „FD „omm—ssenD tF —nd de uruij¡D fF @IWVUA fio™himF

‘Q“ veeD tFsFD uuhnD eF —nd h—l˜eyD ‚FiF @IWWPA tF fiolF ghemF PTUD

fiophysF e™t—F WHHD TQ”UPF

WQV”WRQF

‘PU“ †on reijneD qF @IWVWA x—ture @vondFA QRID RST”RSVF

‘R“ enderssonD rF —nd von reijneD qF @IWWQA iwfy tF IPD TVQ”TWIF

‘PV“ xouwenD xFD de uruij¡D fF —nd „omm—ssenD tF @IWWTA €ro™F x—tlF

‘S“ ƒ™h—tzD €FtF —nd fe™kwithD tF @IWWHA ennuF ‚evF qenetF PRD PIS”

e™—dF ƒ™iF ƒe WQD SWSQ”SWSUF

PRVF

‘PW“ il w—sh—kD iFwF —nd „o™—nneD tFpF @IWVHA fio™himF fiophysF

‘T“ uum—motoD gFeF —nd fe™kwithD tF @IWVSA tF f—™teriolF ITQD PTU”

e™t— SWTD ITS”IUWF

PURF

‘QH“ ghenD vFD ‚ho—dsD hF —nd „—iD €FgF @IWVSA tF f—™teriolF ITID WUQ”

‘U“ uum—motoD gFeF @IWWIA wolF wi™ro˜iolF SD IW”PPF

WVHF

‘V“ uustersD ‚F et —lF @IWVWA tF fiolF ghemF PTRD PHVPU”PHVQHF

‘QI“ wooreD uFiFD h—l˜eyD ‚FiF —nd ‡i™knerD ‡F @IWVVA tF f—™teriolF

‘W“ r—rtlD pF F et —lF @IWWHA gell TQD PTW”PUWF

IUHD RQWS”RQWVF

‘IH“ yliverD hFfF —nd fe™kwithD tF @IWVPA gell QHD QII”QIWF

‘QP“ enderssonD rF —nd von reijneD qF @IWWRA iwfy tF IQD PPTU”

‘II“ i™onomouD eF —nd ‡i™knerD ‡F @IWWRA gell UVD VQS”VRQF

PPUPF

‘IP“ „okud—D rF @IWWRA pifƒ vettF QRTD TS”TVF

‘QQ“ q—fvelinD qF —nd von reijneD qF @IWWRA gell UUD RHI”RIPF

‘IQ“ de †rijeD „F et —lF @IWVVA x—ture QQRD IUQ”IUSF

‘QR“ fo™hk—rev—D iFƒF et —lF @IWWTA tF fiolF ghemF PUID PPPST”PPPTIF

‘IR“ villD ‚FD howh—nD ‡F —nd ‡i™knerD ‡F @IWWHA gell THD PUI”PVHF

‘QS“ †—n der qootD pFqF et —lF @IWWQA iurF tF fio™hemF PIQD PIU”PPIF

‘IS“ uellerD ‚FgFeFD uilli—nD tFeF —nd de uruij¡D fF @IWWPA fio™hemE

‘QT“ he †rijeD „F et —lF @IWVWA iurF tF fio™hemF IVHD QVS”QWPF

istry QID ITUP”ITUUF

‘QU“ €hoenixD hFeF et —lF @IWWQA tF fiolF ghemF PTVD IUHTW”IUHUQF

‘IT“ f—kkerD iF€F —nd ‚—nd—llD vFvF @IWVRA iwfy tF QD VWS”WHHF

‘QV“ €hoenixD hFeF et —lF @IWWQA pifƒ vettF QPRD IIQ”IITF

‘IU“ erkowitzD ‚FeF —nd ‡i™knerD ‡F @IWWRA iwfy tF IQD WSR”WTQF

‘QW“ uustersD ‚F et —lF @IWWRA tF fiolF ghemF PTWD ISTH”ISTQF

‘IV“ h—l˜eyD ‚FiF —nd von reijneD qF @IWWPA „rends fio™hemF ƒ™iF IUD

‘RH“ q—llusserD eF —nd uuhnD eF @IWWHA iwfy tF WD PUPQ”PUPWF

RUR”RUVF pifƒ IWHQT PPEIHEWU