Ato o orsodne([email protected]) correspondence for *Author Sweden. Stockholm, 91 SE-106 University, Stockholm Biophysics, Korea. ugjnJung Sung-jun the membrane of of translocation C-terminus facilitates Sec62–Sec63 The ARTICLE RESEARCH ß 4270 2014 July 14 Accepted 2014; March 21 Received post-translational yeast, In state al., 2000). 1 et translocatable Zimmermann Rapoport, 1988; unfolded, and al., et Plath an Deshaies 1988; 1988; in al., et keeping chains (Chirico in involved polypeptide are the chaperons cytosolic and translationally, hydrophobic of human cytosol. aggregation the its premature in prevents domains membrane second proteins are this of ER the as the majority proteins into The and translocated 2007). co-translationally Rapoport, yeast are 2004; that al., in et Berg that den is Sec61p/Sec61 where name homolog), first complex (the heterotrimeric Sec61p/Sec61 a is of machinery translocon translocation composed This main membrane. ER the nascent the translocon, the in Sec61 targets the SRP nascent the to the a Then, chain the of sequence. recognizes N-terminus signal from a the (SRP) called in chain, emerges particle residues recognition hydrophobic chain post-translational of signal stretch nascent a or a and the co-translational co- ribosome as the to In pathway, a targeted 2007). translocation first by (Rapoport, are pathway (ER) subcellular translocation membrane reticulum the plasma for endoplasmic the destined or proteins organelles membrane and Secretory INTRODUCTION Yeast Topology, Sec61, Co-translational translocation, ER, reticulum, Endoplasmic WORDS: KEY cell. in eukaryotic proteins membrane the of topogenesis Sec62– regulating the These in of translocon function proteins. Sec63 unappreciated an membrane is of there that C-terminus suggest results insertion the membrane that in of defects translocation to show impair and lead Sec62 and yeast we Sec63 with of interaction domain segments], its cytosolic N-terminal (TM) the in transmembrane mutations of and residues orientation charged flanking hydrophobicity, differing [with context sequence broad with proteins membrane multi-spanning analysis and systematic single of By translocon. Sec62–Sec63 the endoplasmic are by handled ER, the the not into targeted into co-translationally mainly are proteins membrane which that proteins, thought secretory been has primarily it Therefore, yeast. in of (ER) reticulum subset a of translocation post-translational mediates complex Sec62–Sec63 The ABSTRACT colo ilgclSine,SolNtoa nvriy eu 5-4,South 151-747, Seoul University, National Seoul Sciences, Biological of School 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,47–28doi:10.1242/jcs.153650 4270–4278 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. aysceoypoen,hwvr r agtdpost- targeted are however, proteins, secretory Many 2 etrfrBoebaeRsac,Dprmn fBohmsr and Biochemistry of Department Research, Biomembrane for Center 1 iEnHn Kim Hani Eun Ji , a Sbh1p/Sec61 , a stepr-omn uui (van subunit pore-forming the is 1 oansH Reithinger H. Johannes , b n Sss1p/Sec61 and c oitrc ihtengtvl hre -emnldmi of 2003). domain al., et C-terminal Willer charged 2000; al., negatively et shown the (Wittke been Sec63p has with and Schekman, interact residues charged cytosolic and to positively N-terminal in (Deshaies The rich is 1990). cytosol domain Schekman, C- the and and in Deshaies N- 1989; the located Mu both are 2000; and domains, al., membrane terminus (TM) integral et an transmembrane Tyedmers is two Sec62p 2000; (Rothblatt essential with 30-kDa eukaryotes al., The are 2010). all et al., Sec63p in Meyer et found and 1989; also al., Sec62p are et and 2011). yeast al., in proteins et Schekman, and Lyman Harada 1995; al., 1997; et (Panzner Sec71p subunits Sec63p, Sec72p Sec62p, complex, and of Sec61 consisting trimeric complex, the Sec62–Sec63 to the addition in requires, translocation ope sivle ntpgnsso ebaepoen nthe in proteins membrane C- of the topogenesis ER. in of Sec62–Sec63 involved the translocation is that interaction complex impair suggesting proteins, the and membrane of Sec63, disrupt yeast terminus and Sec62 the Sec62 of between domain in N-terminal proteins cytosolic, membrane the cerevisiae of multi-spanning Saccharomyces effects engineered the and of tested insertion and single- membrane mutants and translocation Sec62 proteins, on of membrane mutations of set biogenesis a the prepared in role we of general more a stage has it early pathway. be translocation not an post-translational might the complex Sec62–Sec63 at to findings the limited these of function membrane Therefore, signal the 2013). that imply the al., hydrophobic et moderately (Reithinger into translocation of proteins insertion anchor mediates (Meyer Sec62 Mu translocation co-translational 2000; in al., implying involved domain, et be ribosome-binding could a it Sec62 contain that translocation mammalian to the found protein Furthermore, been membrane 1992). has al., post-translational rescue et could (Green screening complex, defects and genetic that Sec62–Sec63 from proteins discovered co- the first for the were and of Sec72p 2001), both and components However, al., Sec71p et 1996). non-essential for Young 1995; al., two al., et essential et well (Brodsky (Ng translocation is is proteins recognize of complex might Sec63p sequences complex Sec62–Sec63 Sec62–Sec63 signal the the has the it that process, of proposed translocation been post-translational action the In of understood. mode the -emnldmiso e6 Fg B.Teblyhydrophobic bulky Sec62 The 1B). of the (Fig. set To or Sec62 a N-terminal of 1A). transmembrane, domains prepared (Fig. the C-terminal we in cytosol mutations Sec62, the with of mutants N- in function large the domains its investigate soluble with protein C-terminal membrane and double-spanning a is mutants Sec62 Sec62 viable of Selection RESULTS 2 ofrhrcaatrz h oeo e6 n oass whether assess to and Sec62 of role the characterize further To nor translocation post-translational of mechanism the Neither n ynKim Hyun and le ta. 00.Rcnl,w aesonthat shown have we Recently, 2010). al., et ¨ller 1, * u eut hwta uain nthe in mutations that show results Our . ¨ller

Journal of Cell Science 28,P1AadN2A opeetdthe complemented N220A, and P219A F218A, EERHARTICLE RESEARCH oiin ntemdlpoen htealdu odistinguish to various us enabled at model that engineered proteins These model were domains. the proteins in sites TM membrane positions N-glycosylation three model from Efficient derived or of membrane were series two and proteins a translocation one, of a with containing insertion out various assay carried and the insertion in we translocation membrane strains, ER in mutant the into changes proteins the membrane determine assay To insertion membrane and Translocation chose We at residues. a charged residues because to negatively (KKL 35–37, charged 103–105 residues with al., at positively or et 103DDD) 35DDD) the Willer DDD; DDD; to 2000; replaced (RQG 35–37 al., of we positions formation et thus, the (Wittke for 2003); complex important domain A be Sec62–Sec63 N-terminal to 185AAA). the the shown and in been FGI) residues previously (157AAA (185–187, has charged residues TM2 positively in Ala of cluster and with VSI) replaced (157–159, were TM1 in residue residues starting the and sequences the 23 with at indicated days carrying are 3 plasmid for residues a plate Mutated with agar strains. transformed (FOA) were mutant acid strains 5-fluoroorotic Sec62 JRY4 on of sequences. grown viability mutated the and by Sec62 followed in number, mutations of strains. mutant Positions Sec62 (B) of Viability 1. Fig. blt ocomplement (WT) to wild-type the ability encoding plasmid variants in Sec62 Ura-bearing a (FPN) All JRY4, (218AAA). 218–220 into cell Ala transformed residues by for were thus replaced essential were 2000), be domain al., to this et shown C-terminal (Wittke been the the has viability TM2 cytosolic in We but C-terminal following the unknown. 263–265 addition, domain domain, In far positions (263DDD). so Asp C-terminal at with is domain residues the domain Lys in this replaced post-translational of found impair significance also functional to charged positively are of shown cluster A residues been 2000). al., has et (Wittke translocation Asp to mutated 29 hwdagot eeta 37 at defect except growth variants, a Although Sec62 1B). showed all (Fig. WT that P219A the replace found functionally could We 218AAA, 1B). (Fig. shuffling o h is pt h olwn pt ee1-odsral diluted. serially 10-fold were spots following The spot. first the for sec62-1 .coli E. sec62 eeinwstse yplasmid by tested was deletion uatsri ihrsde37 residue with strain mutant A ceai ersnaino e6 n e6.Tecagddmis + n ( and (+) domains, charged The Sec62. and Sec63 of representation Schematic (A) eeinsri,cnann a containing strain, deletion edrppiae(LepB). peptidase leader ˚ ,tesnl mutants single the C, sec62 ˚ SEC62 ,30 C, deletion. ˚ n 37 and C sec62 The . ˚ .OD C. oan htaems estv npoigasuccessful and/or a These yeast in probing insertion. O used successfully in membrane been vitro TM have sensitive or proteins hydrophobic Ala most model marginally non-insertion and are for (hydrophobic) translocation, orientation. Leu that select of one to domains fine- number was residues than the domains (neutral) varying TM more the by with of tuned hydrophobicity proteins the Furthermore, model between oe rtiswt h -emn opstoso orLuand triply Leu protein. four a of translocated and compositions completely H-segment lumen), the ER a with the proteins is Model in (3G) is protein C-terminus glycosylated the cytosol, the in Fg A.A ercnl eotdta the that various reported our recently in TM we one protein) As with proteins 2A). model membrane (Fig. model (H1 of set domain a tested first We signal with proteins assay anchor insertion membrane and Translocation h -emnsi nteE ue,teCtriu si the in the is that C-terminus indicates N the (2G) an lumen, in protein inserted glycosylated ER protein doubly the a in cytosol), is N-terminus the rti,asnl lcsltdpoen(G niae htthe that indicates N an (1G) in membrane model protein the the into glycosylated inserted non-targeted of protein singly a topology indicates a (0G) membrane not protein, assess protein also to unglycosylated us an but allows glycosylation protein: that model targeting domain the H1 TM ER the of The only of 2A). distribution side (Fig. either asymmetric on domain sites an TM have a as and proteins both (H-segment) functions sequence segment H-segment signal This hydrophobic a residues. a Leu and with Ala Lep carrying of domains also TM Sec62 moderately of regions other effect. of same in we the mutations 2013), insertion had whether al., see et to membrane (Reithinger wanted proteins anchor and signal hydrophobic targeting impairs ¨ 600 eame l,21;Rihne ta. 2013). al., et Reithinger 2012; al., et jemalm 1mdlpoen eemd yrpaigtetoN-terminal two the replacing by made were proteins model H1 5 .0/lcls( cells 0.600/ml amla irsm ytm Lni ta. 2008; al., et (Lundin systems microsome mammalian ora fCl cec 21)17 2047 doi:10.1242/jcs.153650 4270–4278 127, (2014) Science Cell of Journal SEC62 , 3 6 To h indicated the or WT 10 in 7 -C el)wr iue 110 n 10 and (1:100) diluted were cells) out retto ie h -emnsis N-terminus the (i.e. orientation 2 sec62 ,aeshmtclyindicated. schematically are ), out uat.Teclswere cells The mutants. -C in sec62-1 retto (i.e. orientation sec62 m a used was l mutants mutant 4271 in

Journal of Cell Science eerdoaee t25 at radiolabeled were E ceai ersnaino TSc.Tesga nhrsqec sidctd(o)adteaddo uae eiusaesoni odo hdd D shaded. or bold in shown are residues mutated or added the and (box) indicated is 3 sequence C-terminal anchor a signal contains The Spc3 DT-Spc3. of representation Schematic (E) niae stre w n n ildcrls epciey n nugyoyae rdc sidctda noe ice C rnlcto effici Translocation (C) circle. [(1G+2G+3G)/total as open an calculated as was indicated analysis is blotting product western unglycosylated and an autoradiography and by respectively, both ( circles, determined filled as one proteins and model two three, as indicated SEC62 EERHARTICLE RESEARCH 4272 [S with whole-cell pulse- with after autoradiography blotting labelling and in western immunoprecipitation by expressed or either lysates, were detected H1-5L) and and strains, (H1-4L Leu five N a with proteins membrane single-spanning of insertion Membrane 2. Fig. erae oprdt h T niaigadfc ntargeting in defect products a with indicating glycosylated WT, of the treated amount to compared the decreased strains, were mutant 2B. 103DDD Fig. samples in example the in band, shown as (EndoH), H endoglycosidase (0G) unglycosylated fN of -emnladtoCtria lcslto ie,idctda .Fle ice niaemdfe lcslto ie.Tesqecswt their with sequences in The expressed sites. were glycosylation constructs modified H1-5L indicate and circles H1-4L Filled (B) Y. shown. as ( indicated insertion sites, of glycosylation energy C-terminal two and N-terminal E.coli 30 n 5 ˚ hnH-LadH-Lwr xrse nte3DDand 35DDD the in expressed were H1-5L and H1-4L When n muorcpttduigat-Aatbd.Smlswr nlzdb D-AEadvsaie yatrdorpy 14 n 15 xrse in expressed H1-5L and H1-4L autoradiography. by visualized and SDS-PAGE by analyzed were Samples antibody. anti-HA using immunoprecipitated and C ) D h eaieaon fN of amount relative The (D) 3). in -C e-ae 1mdlpoenhsasnl oeta Msget(e)wt ayn ubr fluie()adaaie()rsde.I seupe ihone with equipped is It residues. (A) alanine and (L) leucine of numbers varying with (red) segment TM potential single a has protein model H1 Lep-based out Twr rae iho ihu no odtc h nlcsltdfr.Til 3) oby(G n igy(G lcsltdpout are products glycosylated (1G) singly and (2G) doubly (3G), Triply form. unglycosylated the detect to EndoH without or with treated were WT omwscluae s[2G/(1G+2G) as calculated was form D G ˚ .AW apewstetdwt rwtotEd ,adgyoyae rdcsaeidctda ildcrls(ih) h eaieamount relative The (right). circles filled as indicated are products glycosylated and H, Endo without or with treated was sample WT A C. ausi clmlwr rdce iha with predicted were kcal/mol in values 35 Mt(i.2–) oietf the identify To 2B–D). (Fig. ]Met 6 Atg F TSc a xrse in expressed was DT-Spc3 (F) tag. HA in -C out omo h aesto aai i.2 a acltda [2G/(1G+2G) as calculated was 2C Fig. in data of set same the of form 6 0] eut r mean are Results 100]. sec62 SEC62 D G mutant To h niae uatsris rtiswr ailbldwt [S with radiolabeled were Proteins strains. mutant indicated the or WT SEC62 rdco)o h 1mdlpoen arigteHsgetcmoiin f3 o6 are 6L to 3L of compositions H-segment the carrying proteins model H1 the of predictor) 6 ...( s.e.m. Tor WT in oprdt h T h eaieaon fpoen nN in proteins of amount relative the WT, the 103DDD to and Compared 35DDD Furthermore, sec62 2B,C). (Fig. Other translocation 2B,C). (Fig. 5D tan.Sc a oiidt netwt ultopology and dual WT with in insert to tested modified also protein, was was Spc3 anchor (Spc3), strains. signal 3 35DDD complex Another peptidase 2D). signal (Fig. respectively H1-5L, sec62 C n -C sec62 out 5 out 3). ebaetplg a eue n3DDad103DDD and 35DDD in reduced was topology membrane retto srdcdin reduced is orientation uat y1%ad3%frH-L n 4 n 1 for 21% and 14% and H1-4L, for 32% and 18% by mutants 5D tanadvsaie yatrdorpya nB xetcells except B, in as autoradiography by visualized and strain 35DDD uat fetdtpgnsso 1mdlproteins. model H1 of topogenesis affected mutants ora fCl cec 21)17 2047 doi:10.1242/jcs.153650 4270–4278 127, (2014) Science Cell of Journal sec62 6 sec62 uat eandW eesof levels WT retained mutants 0] eut r mean are Results 100]. -emnlmtn strains. mutant N-terminal 6 0] eut r mean are Results 100]. 35 6 Mtfr5mnat min 5 for ]Met ...( s.e.m. paetfree apparent nyo H1 of ency 6 n s.e.m. 5 A An (A) 3). in T- -

Journal of Cell Science Fg A.Bcuete23D uatsri i o hwany show not did strain mutant 263DDD the Because 3A). (Fig. h ai fD-p3isrigi h N the H1 in inserting the 35DDD DT-Spc3 the of with in ratio the expressed agreement when model of In data, domain H1 protein TM 2E). model the the (Fig. flank to asymmetrically sites glycosylation Similar DT-Spc3 two 2013). and one al., protein, et (Reithinger (DT-Spc3) ARTICLE RESEARCH uatsriswr osrce (35DDD+103DDD, constructed these in and expressed H1-4L were were Then, H1-5L 103DDD+263DDD). and double strains additive, 35DDD+263DDD were Sec62 in mutant mutations 103DDD and strains 35DDD mutant 103DDD N- indispensable. the are and in similar Sec62 residues 35DDD of charged a the positively terminus to that in suggesting decreased 3B,C), seen was (Fig. that membrane ER to the level into in proteins expressed H1 were Then, H1-5L) residues. and 35AAA at (H1-4L Ala the in residues proteins neutral anchor residues above, with signal defect charged replaced H1 observed were positively the 35–37 of for positions charged deletion responsible negatively is the of Sec62 or introduction acids the indispensable amino are whether Sec62 distinguish of N-terminus To the in residues Basic emnlctslcdmi fSc2rdc h fiinyof efficiency the reduce Sec62 of domain 2F). cytosolic (Fig. WT terminal the to compared fivefold about by decreased ela ebaeisrini h N the in insertion membrane as as proteins anchor well signal hydrophobic moderately of translocation ihorerirfnig ihthe with 2013). al., findings et earlier our with utemr,t hc hte h eet asdb the by caused defects the whether check to Furthermore, hs eut niaeta h uain nrdcdi h N- the in introduced mutations the that indicate results These sec62 uatsri Fg A.Mmrn neto of insertion Membrane 3A). (Fig. strain mutant sec62 in sec62-1 -C in -C out obemtn strains mutant double out retto,consistent orientation, ebaetopology membrane uat(Reithinger mutant sec62 mutant, eiusi h -emnso e6 r rca o h folding charged the for with positively interaction crucial proper and/or are that Sec63p. domain Sec62 cytosolic N-terminal of suggest the N-terminus of cases, the results all in strains In residues These mutant mutations. single the 3B,C). single of that (Fig. to 103DDD to defect similar or was of efficiency 35DDD level insertion similar the a of exhibit to double that 35DDD other expected the in be all defects would additive, the not mutants are if mutants proteins, double 103DDD model and H1 for defect targeting niaigasal ope omto ihSc3(i.4). (Fig. co-precipitated, Sec63 with were formation 263DDD complex and stable a WT 1% indicating with Both anti-HA HA-tagged with solubilized immunoprecipitation containing antibodies. to isolated, subjected cells and were X-100 in Triton membranes expressed Crude interaction were Sec63. the 103DDD 263DDD the 35AAA, was disrupt 35DDD, whether and assay WT, Sec62 Sec62 co-immunoprecipitation investigate FLAG-tagged a out. of To carried Sec63, 2003). and region Sec62 al., this between (Wittke et in complex Willer Sec62–Sec63 substitutions 2000; the is al., of Sec63 et of formation C-terminus interaction the for and electrostatic important Sec62 an of N-terminus that the indicated between have studies Previous interaction disrupt Sec62 Sec63 of with N-terminus the in Mutations yooi,Ntria oano e6 blse t proper its abolishes Sec62 the in of Sec63. residues with results domain charged interaction These positively N-terminal 4). on of (Fig. cytosolic, replacement migrated others that than 103DDD suggest in slower detected 4). slightly not (Fig. SDS-PAGE were samples however, 103DDD, immunoprecipitated and 35AAA 35DDD, ora fCl cec 21)17 2047 doi:10.1242/jcs.153650 4270–4278 127, (2014) Science Cell of Journal C h eaieaon fN of amount relative The (C) mean are Results plotted. acltda [(1G+2G+3G)/total was as efficiency calculated Translocation (B) an circle. as open indicated is product an unglycosylated one and and respectively, two circles, three, filled as indicated are glycosylated products singly and doubly western Triply, by blotting. visualized Sec62 were in strains expressed mutant H1-5L and variants. H1-4L Sec62 (A) in model proteins H1 anchor with signal assay Translocation 3. Fig. mean [2G/ as calculated was (1G+2G) proteins model H1 of 6 ...( s.e.m. 6 0]adpotd eut are Results plotted. and 100] n 5 3). 6 ...( s.e.m. in -C 6 out 0]and 100] n orientation 5 3). 4273

Journal of Cell Science hl-ellstswr nlzdb D-AEadwsenbotn.Mmrn neto fiinywscluae si i.4.Rslsare Results 4B. Fig. in as calculated was efficiency insertion Membrane blotting. western and SDS-PAGE by mean analyzed were lysates Whole-cell rwtotEd .Mmrn neto fiinyo h 2sgetwscluae s[2+Gcevdpout/1+Gcevdproduct+2G+2G cleaved product)/(1G+1G membrane cleaved not [(2G+2G is as t segment calculated were H2 samples was WT the segment 2008). indica when al., H2 is only et the (Lundin product detected of A) unglycosylated is in efficiency an and (arrowhead insertion product) cleavage and cleavage Membrane peptidase respectively, cleaved unknown signal H. circles, an from Endo filled resulted from without that one resulted domain or and that C-terminal two domain glycosylated a as C-terminal **, indicated unglycosylated inserted. are an in products expressed *, were glycosylated circle; constructs resid (1G) glycosylati open H2-3L (A) glycosylated singly and alanine indicate H2-2L and and circles (B) (L) (2G) Filled shown. leucine Y. Doubly are of as segment number indicated H2 the are the changing which of by sites, predictor) 3 controlled glycosylation C-terminal was C-terminal a segment and contains H2 N- protein the with model of equipped Hydrophobicity is red. protein in model indicated H2 is segment) (H2 segment TM i.4 uain nteNtria oano e6 irp the disrupt Sec62 of domain N-terminal the in Mutations 4. Fig. ARTICLE RESEARCH 4274 strains. mutant Sec62 in proteins membrane double-spanning of insertion Membrane 5. Fig. anti- or Input. Sec63HA) In, (rabbit, antibody. anti-HA Sec62FLAG) either (rabbit, SDS-PAGE with to FLAG blotting subjected were western Samples by epitope. followed HA against an (mouse) to antibody conjugated anti-HA Sec63 an with (IP) immunoprecipitated and X-100 Sec63. with interaction 6 ...( s.e.m. n 5 6 4). 0] eut r mean are Results 100]. rd ebae eeslblzdwt %Triton 1% with solubilized were membranes Crude 6 Aeioetg h eune ihteraprn reeeg fisrin( insertion of energy free apparent their with sequences The tag. epitope HA 6 ...( s.e.m. n 5 ) C 21 n 2KL osrcswr xrse in expressed were constructs H2-K1LG and H2-1L (C) 5). eepesda 2mdlpoenta a w Mdmisin single-spanning original domains the TM of contains two protein segment only model has TM H2 that This protein strains. not mutant model Sec62 H2 proteins, complex an of membrane expressed multi-spanning Sec62–Sec63 we also insertion but defective proteins, membrane the membrane whether affects determine domain To the TM of second insertion hydrophobic inhibit moderately mutants 103DDD and 35DDD P) L Lad5 Fg A eeepesdin expressed were 5A) (Fig. 5L and 3L 2L, (P2), domains TM of insertion protein. 2008) the membrane model and H2 al., flanking targeting the protein et sites the of H-segment as (Lundin assessment glycosylation an allow residues segment) Two Ala and (H2 5A). and ER, (Fig. domain Leu the of TM numbers to downstream varying efficiently of comprised protein the targets h 2mdlpoen ihH emn opstoso 0L of compositions segment H2 with proteins model H2 The ora fCl cec 21)17 2047 doi:10.1242/jcs.153650 4270–4278 127, (2014) Science Cell of Journal SEC62 (A) To uatsris ape eeaaye si i.2B. Fig. in as analyzed were Samples strains. mutant or WT .coli E. E.coli e-ae 2mdlpoen h oeta second potential The protein. model H2 Lep-based e steusra Mdmi,which domain, TM upstream the as Lep D SEC62 G ausi clmlwr rdce with predicted were kcal/mol in values Tor WT sec62 5D uatstrains. mutant 35DDD SEC62 nsts h H2 The sites. on etdwith reated e san as ted Tand WT ues. D G

Journal of Cell Science 5D n 0DDmtns(7 n 3,respectively) 23%, and (27% mutants in strains 103DDD reduction in small C-terminus 103DDD and a the of and 35DDD showed translocation was also 35DDD and H2-3L however, insertion in membrane addition, H2-2L, In of reduced targeting 5B). segment twofold (Fig. H2 ER than the translocation of efficient more unglycosylated consequently, C-terminus little and, the indicating very insertion of Membrane or detected, 5B). no (Fig. were strains, all products In strains. mutant ARTICLE RESEARCH i.6 ebaeisrino ut-pnigmmrn rtisi e6 uatstrains. mutant Sec62 in proteins membrane multi-spanning of insertion Membrane 6. Fig. C-terminus the of a translocation indicating and glycosylated, insertion membrane doubly in were N- defect in 16% whereas were only glycosylated, that doubly strain, In was residues 35DDD 5A). H2-K1LG Ala Fig. of (H2-K1LG, 54% 18 segment residues cells, and WT H2 Lys three Leu the by 1 with flanked of terminally protein (O composed model segments strains. was H2 that an TM defective influence prepared the We domain Sec62 2012). of TM the in orientation flanking membrane residues residues charged flanking Positively charged with TM hydrophobic marginally segments. of C- insertion the membrane that of show and translocation proteins terminus impairs model complex and/ though two Sec62–Sec63 domain the defective Even from TM a results upstream length), 2008). the loop of the al., presence or of the et context (e.g. the (Lundin sequence on the depending segment differ might TM hydrophobicity absolute first the insertion of 50% for residues Leu less ( require proteins is model not insertion) H2 where 5L, membrane was proteins (50% model H2-5L hydrophobicity H1 threshold segment, to the Compared H2 shown). not hydrophobic (data more affected The 5B). (Fig. noe ice 34 xrse nSc2W a rae iho ihu noHt eetteugyoyae om fiinyo h -emnltransloc C-terminal the of Efficiency form. unglycosylated the detect to H Endo product C-termi unglycosylated without the an or and at with respectively, 3G) HA-tag circles, treated + an filled was [3G/(2G two and WT Y, and as Sec62 three as calculated in as indicated indicated was expressed in are are H3-4L expressed which products ( circle. sites glycosylated were insertion open (2G) glycosylation constructs doubly an of C-terminal H3-5L and energy and (G) and free Triply N- H3-4L 2B. with apparent (B) Fig. equipped their glycans. is with indicate protein sequences circles model the Filled H3 and The red shown. in are indicated predictor) is segment) (H3 segment , et eivsiae ebaeisrino Mdomain TM a of insertion membrane investigated we Next, L3) n hsdfeec slkl ob u otepresence the to due be to likely is difference this and 2L–3L), 6 0] eut r mean are Results 100]. ¨ eame al., et jemalm 6 ...( s.e.m. n , 5 sec62 4). 4L– nyosre o Lad5 osrcsbtntfrless for not but constructs 5L and 4L for observed only muto Gfr erae nte3DDad103DDD and 35DDD was the in segment decreased relative H3 form The 3G the (3G). of translocated amount forms; or distinct (2G) membrane-inserted two either show differing proteins inserted the were model In of products were 6B). glycosylated (Fig. and singly segments segment) detected WT a and unglycosylated in TM by little membrane (H3 very followed ER two the residues, segment into first Ala efficiently 12 TM The and hydrophobicity. third Leu 7 potential with domain TM 0DDmtns epciey hs eut niaeta the that indicate and results 24% 35DDD These and in C H3-5L 28% respectively. for a 13% mutants, was and 103DDD 22% there and WT; H3-4L, the for decrease to compared strains mutant yrpoi Mdmi u loaT oanwt charged with domain TM Sec62–Sec63 a moderately defective also a residues. only but the flanking not domain that of TM insertion hydrophobic show membrane data impairs complex These 5C). (Fig. eue in reduced rgnlfrtT oanof domain TM first protein TM (O model last original IV This the Lep three 6A). from when (Fig. derived carrying cytosol inserted is membrane the protein is facing segment model C-terminus TM the another with mutant Sec62 tested segments, Sec62 in defective in we is C- impaired insertion strains, the membrane were of or translocation protein end the terminal whether model distinguish the H2 To of strains. the mutant insertion in membrane segment and C-terminus H2 the of Translocation C The SEC62 out ebaeoinaincmae oteC the to compared orientation membrane out ora fCl cec 21)17 2047 doi:10.1242/jcs.153650 4270–4278 127, (2014) Science Cell of Journal To h niae uatsris ape eeaaye sin as analyzed were Samples strains. mutant indicated the or WT retto srdcdi e6 uatstrains mutant Sec62 in reduced is orientation sec62 (A) .coli E. uatsris ti fnt htti fetwas effect this that note of is It strains. mutant e-ae 3mdlpoen h oeta hr TM third potential The protein. model H3 Lep-based D G ¨ ausi clmlwr rdce with predicted were kcal/mol in values eame l,21) hc otisan contains which 2012), al., et jemalm SEC62 .coli E. Tsri,H-LadH3-5L and H3-4L strain, WT e,ahdohbcsecond hydrophobic a Lep, sec62 in retto was orientation sidctdas indicated is uat,as mutants, D sec62 G 4275 ation nus.

Journal of Cell Science yrpoi Msget srdcdi 5D n 103DDD and 35DDD in reduced is sec62 segments TM hydrophobic hwta h C the that show be segments. might TM segment two protein, H3 first model the the H1 of of is the independent insertion that to membrane segment similar the H3 is suggesting the mutations Sec62 of to hydrophobicity not sensitive of (data range constructs (7L) The hydrophobic shown). more or (2L) hydrophobic ARTICLE RESEARCH 4276 signal the of prepro- residues of derivative the sequence site-specific into lysine incorporated elegant photoreactive site-specifically an a was using When binding approach. sequence cross-linking membrane signal ER of and ER. the mode the in translocation to those proteins in of test targeting complex than the rather of impaired Sec62–Sec63 the shown). insertion to the not due membrane are (data of study co-translational single- this that in function SRP-dependent more observed observed Spc3 defects we the is the model and Hence, indeed H3 by proteins and and H1 H2 targeted model translocation, that of likely are H3 is segments proteins it and proteins, H2 TM membrane spanning of than segment co- hydrophobic the single- to TM that subject Given Spc3 2013). are first a al., and proteins et with (Reithinger these strain model translocation yeast that translational H1 a suggesting in of SRP, impaired defective targeting is proteins study the membrane earlier spanning that Our proteins. shown in membrane defects has multi-spanning single- to as of lead well segments and TM as hydrophobic Sec63 moderately Our with of of interaction translocation orientations). domain the cytosolic N-terminal disrupt membrane the Sec62 flanking in different mutations segments, that show and TM results of residues various number (e.g. charged hydrophobicity, context membrane sequence of diverse and degrees with translocation proteins mediate of mutants insertion Sec62 the assessed In these and details. Sec62 how mechanistic mutagenized and systematically we molecular study, of present lack but a to yeast, been limited in has being proteins there as secretory regarded of been translocation long post-translational has Sec62 of function The DISCUSSION membrane. rtisi e6 eetv tan N ta. 96 could 1996) N al., as et orientate correctly to peptide (Ng failing signal Sec62 strains defective the previously by defective explained the be Sec62 results, alternatively if our translocated in surprising post-translationally of be proteins of not light defect would translocation In it observed 1996), both. al., recognizes et Sec62 (Ng peptide signal TM those to of hydrophobic similar the are moderately proteins membrane and multi-spanning of of segments context moderate, structural is and sequence proteins of peptides Sec62–Sec71 translocated signal C- with of hydrophobicity post-translationally interacts that the Given sequence 1998). al., signal that et (Plath the pp suggesting of the Sec62–Sec71, end of terminal to end C-terminal crosslinked only the same derivative, sequence at lysine the located the phenylalanine than photo-probe when linker photoreactive the Interestingly, shorter a a 2004). has with that al., derivative performed (Plath et membrane was Plath the experiment of 1998; plane al., the to et sequence perpendicular signal helix the and a Sec62–Sec71, forms and Sec61 by formed is signal-sequence-binding the site the along that indicating occurred sequence, Sec62–Sec71 signal and entire Sec61 and sequence, signal ae oehr eut rmaltrest fmdlproteins model of sets three all from results together, Taken nerirsuyb lt ta.(lt ta. 98 eeldthe revealed 1998) al., et (Plath al. et Plath by study earlier An uatstrains. mutant a out fco (pp -factor ebaeoinaino marginally of orientation membrane a ,cosikn ewe h pp the between crosslinking ), in -C out o laaei h ER the in cleavage for a signal a R46E6) a pRS416SEC62), eune r nelnd Lntn ta. 98.TersligPCR resulting The 1998). W303-1 al., into transformed et was (Longtine fragment underlined) are sequences AAC-3 TGAAAAACTATACTAATCACTTATATC GGATCCCCGGGTTAATTAA-3 SEC63 histidine ura3 al., without trp1, plates et agar leu2, (Kitada on ( selected underlined) JRY6 were (–His). are colonies resultant sequences The 1995). (recombination plasmid pCgH ATAATGGAAGTGTTGTAAAACGACGGCCAGT-3 TGACC-3 GGGCTTTTATAATTGCAGTTGAA W303-1 of ATCCGAGGTTACAATATAGAAGG-3 the First, 5 recombination. homologous and primers plate agar 5FOA SEC62 a on shuffling ( JRY4 strains Yeast METHODS AND MATERIALS TAGGATAC-3 aesotrNtria al ihcagdrsde n r less are and residues charged signal with the tails that of N-terminal sequences signal shorter features have have the to TRAM- appear that 1996). proteins al., secretory shown et dependent (Voigt been Saurı dependency 2003; has TRAM al., dictate It et sequence McCormick 2007). 2000; Voigt al., 1996; al., al., et et et Heinrich Do 1994; 1996; al., al., et et Mothes 1993; al., et and High 1993; secretory some and (Go of translocation Sec61 proteins of to membrane stage proximity early close the in in found functions been membrane has Sec62–Sec63 translocating-chain-associating (TRAM) protein yeast Sec62– the mammalian Further, the the membrane proteins. multi-spanning whether to of insertion seen handles also be similarity complex that Sec63 to functional suggesting remains 2012), It a the al., complex. for et is defects (Lang translocation proteins there to secretory leads of Sec63 subset or Sec62 of depletion Sec62–Sec63 the of further biogenesis. role Sec63 protein and general membrane Sec62 in and with translocon possible complex highly a in a elusive, are suggests remains they insertion that multi-spanning protein fact of membrane the function of in the Sec72 Although translocation as and 1992). Sec71 discovered al., in et first (Green defects proteins were membrane rescued Sec72 and that Sec71 subunits. Sec72 and amla el.Tu,i em htteTA n TRAP and to TRAM resemblance yeast. the functional in translocon a that Sec62–Sec63 have the cells seems mammalian it in complexes in Thus, topogenesis protein cells. protein membrane translocon-associated mammalian in the involved that is (Sommer complex reported al. (TRAP) have et Sommer 2013) Recently, al., 1996). et al., signal et the by (Voigt involved cleavage peptidase for be correctly might sequence signal TRAM the that orienting in proposed was it thus hydrophobic, eimadrpiapae naYDG1 plate. YPD-G418 a on replica-plated and medium 5 HA procedureusedtomakeJRY4todeletethe The W303-1 The SEC62 into transformed underlined). was are plasmid pRS416 sequences recombinant (recombination recombination into homologous introduced was 9 -CGATACGGATACAGAAGCTGAAGATGATGAATCACCAGAA nmmaincls td yLn ta.hssonthat shown has al. et Lang by study a cells, mammalian In Sec71 non-essential the includes translocon Sec62–Sec63 The a n eetbemre o aayiewsapiidwt primers with amplified was kanamycine for marker selectable and tag HIS3 9 eewsrpae ihthe with replaced was eeicuigtesqecs15k ptemwsapiidusing amplified was upstream kb 1.5 sequences the including gene ihtrecpe fan of copies three with MAT ora fCl cec 21)17 2047 doi:10.1242/jcs.153650 4270–4278 127, (2014) Science Cell of Journal R7)fo h F6-H-aM6pamd(recombination plasmid pFA6a-3HA-kanMX6 the from (RP71) 9 a 9 R2)ad5 and (RP27) - eewsapiiduigpies5 primers using amplified was gene ( CGATAAGCTTGATATCGAATTCCTGCAGGGGTCTATC- a MAT ,sec62 MAT 9 R46E6)wsgnrtdb agn h genomic the tagging by generated was pRS416SEC62) , R2)ad5 and (RP25) a d2 a1 i3 e2 r1 ura3 trp1, leu2, his3, can1, ade2, , a SEC62 E6:H-aM sec62 SEC63::HA-KanM, , Sma D :I3 d2 a1 i3 e2 r1 ura3 trp1, leu2, his3, can1, ade2, ::HIS3, 9 - rihe l,19;Go 1992; al., et ¨rlich GAAGGTTTATACAGT I cevdpS1 n R46pamd by plasmids pRS416 and pRS415 -cleaved eeinsri,wsgnrtdb plasmid by generated was strain, deletion HA 9 HIS3 9 - GGCGGCCGCTCTAGAACTAGTGG- R7)ad5 and (RP70) ptp,adte olwn h same the following then and epitope, TGCAGTCACAGGAAACAGCTA- eeb oooosrecombination. homologous by gene a 9 n el eegono YPD on grown were cells and , R2)fo h eoi DNA genomic the from (RP26) SEC62 GAATTCGAGCTCGTTTA- D :I3 d2 a1 his3, can1, ade2, ::HIS3, 9 ee h asteo the of cassette The gene. -CTAAGAGCTAAAA- rihadRapoport, and ¨rlich AGAGCTATACAGG- 9 .Teapiidgene amplified The ). 9 - GGGAGAAGAGT- R2)fo the from (RP28) a hn genomic Then, . C- ´ ,

Journal of Cell Science rw t30 at grown the introduced of (5 and conjugation were WT For SEC62 epitope Japan). to FLAG (Toyobo, tag FLAG kit the mutagenesis and 2008; site-directed Mutations al., a using et 2013). (Lundin al., described et previously Reithinger as recombination homologous by h uentn a etiue t28,000 at centrifuged was supernatant the osrcino plasmids of Construction (O bases IV 17 Lep Lep contained the of primer of 653–669 case reverse nucleotides the the complementing In (Lep-H3), interest. protein of model the gene truncated the of IV complementing end bases and 17–20 start and bases the recombination homologous complementing for the 30 2008) al., of et contained (Lundin sequences plasmid downstream primers and reverse upstream and Forward design Primer ARTICLE RESEARCH upeetr aeilaalbeoln at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.153650/-/DC1 online available material Supplementary material Supplementary Korea. of to Foundation C00048 Research number 2012- [grant National number Grant the Network [grant from Research Grant Global H.K.] Research a and Basic H.K.] a to by 0001935 supported was work This Funding experiments. the the wrote performed and experiments, J.K. the and analyzed S.J. and designed manuscript. H.K. and J.H.R. J.K., S.J., contributions Author interests. competing no declare authors The interests Competing O Karin and Heijne providing for von (Sweden) Gunnar University to Stockholm thanks special give Authors Acknowledgements 3000 at harvested were cells unit FLAG-tagged expressing cells JRY6 Co-immunoprecipitation (Reithinger previously described 2013). as al., out et carried were immunoprecipitation experiments and These pulse-labeling analysis, blot Western in found are sequences protein The S1. 2013). Fig. al., material supplementary et (Reithinger previously C-3 CAAGGACGACGATGACAAGTGAACTTCCATTATCCTGTATAG- from amplified were W303-1 of interest DNA of genomic the genes synthesized, oligonucleotide the Using hyrahda pia est t60n (OD nm 600 at density optical an reached they nte 150 Of min. Another 30 for 20 mM ice resuspension, 1 on NaCl, incubated the and mM cocktail) 100 inhibitor glycerol, protease 10% and X-100, PMSF Triton 200 1% in Tris-HCl, resuspended mM was (20 pellet The fraction. membrane eewse newt muorcptto ufrB(5m Tris-HCl, mM (15 B buffer immunoprecipitation with once washed were n 0 MNC) hn h ahdbaswr etdwt 50 with heated were Tris-HCl 65 beads at buffer washed mM sample the PAGE (15 Then, immunoprecipitation NaCl). C with mM buffer immunoprecipitation 100 twice with and NaCl), once and mM A, 100 buffer and X-100 Trition 0.4% D-AEadwsenblotting. western and SDS-PAGE eltwsslblzdwt 200 with solubilized was pellet n aoaoymmesfrciia edn ftemanuscript. the of reading critical for members laboratory and muorcptto ufrA(5m rsHl .%TiinX10and 3 X-100 NaCl), Trition mM 0.2% Tris-HCl, 100 mM (15 A buffer immunoprecipitation 0m DA H8 0m al 0 MSrio,1m MFand for PMSF beads mM glass 1 4 Sorbitol, prewashed at mM with min 300 vortexed 10 NaCl, and mM cocktail) 20 inhibitor 8, protease pH EDTA, mM 10 ed,te h itr a oae vrih t4 at overnight rotated was mixture the then beads, 9 -GTTTTGTTCGGCTTTTTCATTGATG-3 9 eeue.pK3 TScH a osrce sdescribed as constructed was DT-Spc3HA pHK131 used. were ) ˚ .Telst a etiue rel ormv eldbi,and debris, cell remove to briefly centrifuged was lysate The C. ˚ nmdu ihu ecn n itdn –e Hs until –His) (–Leu histidine and leucine without medium in C m ftersseso a ie ih500 with mixed was resuspension the of l m niH nioy n 25 and antibody, anti-HA l m fterato a ae u sa nu sample. input an as out taken was reaction the of l ˚ o 5mn n ape eete nlzdby analyzed then were samples and min, 15 for C a sec62 ycln C.Alpamd eeconstructed were plasmids All PCR. colony by m g flssbfe 2 MTi-C H8, pH Tris-HCl mM (20 buffer lysis of l uat npS1 lsi,pie RP98 primer plasmid, pRS415 in mutants n ahdwt itle H distilled with washed and SEC62 .coli E. g o 0mna 4 at min 30 for 600 9 Sma n P0 (5 RP100 and ) To mutant or WT e oe rti constructs, protein model Lep )of1.0.Atotalof10OD ¨ iei p424GPDHA a in site I eame l,2012). al., et jemalm m lofprotein-G–agarose ˚ .Teaaoebeads agarose The C. ¨ m eamin jemalm ˚ loflysisbuffer Ctoharvestthe 2 sec62 .Tecell The O. 9 -GACTA- m lSDS- m lof were 600 lt,K,Wlisn .M,Siln,C .adRppr,T A. T. Rapoport, and J. C. Stirling, M., B. Wilkinson, K., Plath, lt,K,Mte,W,Wlisn .M,Siln,C .adRppr,T A. T. Rapoport, and J. C. Stirling, M., B. Wilkinson, W., Mothes, K., Plath, A. T. Rapoport, and A. K. T. Plath, Rapoport, and S. Kostka, E., Hartmann, L., Dreier, S., Panzner, Mu O P. Walter, and D. J. Brown, T., D. Ng, and U. A. T. K. Rapoport, Kalies, and S., S. Prehn, Prehn, S., W., Mothes, Kostka, R., Kraft, H., Grau, A., H. Meyer, comc,P . io . ho . i,J n ono,A E. A. Johnson, and J. Lin, Y., Shao, Y., Miao, R. J., P. Schekman, McCormick, and K. S. Lyman, aaa . i . al .S,L,H n enr,W J. W. Lennarz, and H. Li, S., J. Wall, H., Li, Y., Harada, P. Walter, and H. Fang, N., Green, Go E. A. Johnson, Go and W. D. Andrews, J., Lin, D., Falcone, H., Do, udn . i,H,Nlsn . ht,S .advnHin,G. Heijne, von and H. S. White, I., Nilsson, H., Kim, C., Lundin, enih .U,Mte,W,Bunr .adRppr,T A. T. Rapoport, and J. Brunner, W., Mothes, U., S. Heinrich, and A. E. Craig, M., Werner-Washburne, D., B. Koch, J., R. Deshaies, R. Schekman, and J. R. Deshaies, R. G. Schekman, and Blobel, J. and R. Deshaies, G. M. Waters, J., W. Chirico, ag . eei,J,Fdls .V,Shr,S,Shra . Scha C., Schirra, S., Schorr, V., S. Fedeles, J., Benedix, S., Lang, Go B., Martoglio, S., High, ogie .S,MKni,A,3d eaii .J,Sa,N . ah A., Wach, G., N. Shah, J., D. Demarini, 3rd, A., McKenzie, S., M. Longtine, rdk,J . ocee,J n cemn R. Schekman, and J. Goeckeler, L., J. Brodsky, References iaa . aauh,E n rsw,M. Arisawa, and E. Yamaguchi, K., Kitada, ¨ eam . aln,K . iso,I n o eje G. Heijne, von and I. Nilsson, K., K. Halling, K., jemalm, lc,D,Pen . atan . ais .U n aoot .A. T. Rapoport, and U. K. Kalies, E., Hartmann, S., Prehn, D., rlich, ¨ lr . eEcuiz,M . aoe . hi,M,Jn,M,Mu M., Jung, M., Theis, P., Lajoie, D., M. Escauriaza, de L., ller, ¨ lc,D n aoot .A. T. Rapoport, and D. rlich, ¨ neatosbtenSccmlxadper-lh-atrduring prepro-alpha-factor and complex Sec between Interactions h es Rmembrane. across ER transport protein yeast posttranslational the in recognition endoplasmic sequence Signal the (1998). into transport their before reticulum. substrates posttranslational from complex Kar2p. purified and a proteins with Sec reconstituted yeast of in transport protein Posttranslational rfrne fnihoighlcscndieE neto famarginally a of insertion ER drive can helix. helices transmembrane neighboring hydrophobic of preferences membrane. reticulum endoplasmic the to route humans. to yeast al. from Sec62 et protein J. membrane Cell Dudek, ER Biol. the L., for E. function Snapp, of gain M., Greiner, C., Burgard, the through translocation during membrane. protein ER secretory translocating a of environment Chem. Biol. J. E. Hartmann, ornltoa rti nerto noteE ebaei eitdb the by mediated proteins. is translocon membrane to ER chains nascent the of into binding integration protein BiP. by Cotranslational regulated is subcomplex translocon a to polypeptides rti rnlcto costeedpamcrtclmmmrn of membrane reticulum endoplasmic soluble and the into cerevisiae. across insertion Saccharomyces protein translocation membrane protein inhibit groups complementation and ribosomes with translocation. associated during is SECYp polypeptides and nascent SEC61p of homolog endoplasmic mammalian the of components purified membrane. reticulum from reconstituted proteoliposomes iia o (n-(u)adNot-(n rnmmrn helices. transmembrane is N(out)-C(in) membrane USA and reticulum Sci. endoplasmic Acad. N(in)-C(out) the in for insertion similar protein for code Molecular e6pcmlxmdae h nerto fammrn rti yallowing by protein domain. membrane transmembrane a the of of integration partitioning the lipid complex. mediates translocon complex Sec61p post-translational heptameric the Chem. of Biol. J. assembly the and is bilayer phospholipid process. the multistep into proteins a membrane of integration cotranslational polypeptides. precursor mitochondrial and secretory reticulum R. Schekman, endoplasmic yeast the of machinery. import component protein membrane-bound a Sec62p, reticulum. endoplasmic yeast the Biol. into Cell translocation J. protein for required protein microsomes. into translocation protein stimulate proteins h nolsi eiuu fmmaincells. al. mammalian et of reticulum J. endoplasmic Tatzelt, the S., Hassdenteufel, Sec61 of M., effects Greiner, C., Jalal, estl n cnmclPRbsdgn eeinadmdfcto in modification and deletion gene PCR-based cerevisiae. economical R. Saccharomyces J. and Pringle, and versatile P. Philippsen, A., Brachat, eurdfrbt o n otrnltoa rti rnlcto noteyeast the into translocation protein posttranslational and reticulum. co- endoplasmic both for required lbaaTP n I3gns n osrcino hi irpatsrisby strains disruptant their of construction transformation. and integrative genes, sequential HIS3 and TRP1 glabrata different contact TRAM sequence. signal and 26751. membrane-inserted Sec61p a al. that of et B. regions reveals Dobberstein, A., photocross-linking T. Rapoport, specific S., Prehn, E., Hartmann, ora fCl cec 21)17 2047 doi:10.1242/jcs.153650 4270–4278 127, (2014) Science Cell of Journal 21 .Cl Biol. Cell J. 691-703. , 109 20) amla e6 sascae ihSc2adSec63. and Sec62 with associated is Sec61 Mammalian (2000). 275 286 18) ufml fsrs rtisfclttstasoainof translocation facilitates proteins stress of subfamily A (1988). MOJ. 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Journal of Cell Science EERHARTICLE RESEARCH 4278 aoot .A. T. Rapoport, ydes . enr . is . ue,J,Sornk .H,Ha,I G., I. Haas, H., M. Skowronek, J., Dudek, C., E. Bies, Hartmann, M., Lerner, and J., M. Tyedmers, Spiess, U., K. Kalies, T., Junne, N., Sommer, R. Schekman, and Saurı G. Daum, L., S. Sanders, J., R. H. Deshaies, A., Kim, J. Rothblatt, and E. J. Kim, H., J. Reithinger, otrnltoa rti rnpr noteedpamcreticulum. endoplasmic 15 the into transport protein posttranslational fteyatSccmlxsbnt e6padSc3 r bnatpoen in proteins abundant are Sec63p and microsomes. Sec62p pancreas subunits dog complex Sec yeast the R. of Zimmermann, and J. Volkmer, W., Nastainczyk, N., Heim, membrane. ER mammalian Acta the Biophys. at Biochim. topogenesis protein membrane assists TRAP into proteins yeast. secretory in of reticulum insertion proper endoplasmic for the required are genes Multiple (1989). anchor signal (ER). hydrophobic reticulum endoplasmic moderately the of in orientation proteins and insertion membrane membranes. plasma bacterial and reticulum e6apaadTA r eunilyajcn oansetviral nascent a to adjacent integration. sequentially ER its are during protein TRAM membrane and Sec61alpha 1-10. , ,A,MCrik .J,Jhsn .E n igro I. Mingarro, and E. A. Johnson, J., P. McCormick, A., ´, 20) rti rnlcto costeekroi endoplasmic eukaryotic the across translocation Protein (2007). 1833 rc al cd c.USA Sci. Acad. Natl. Proc. 3104-3111. , .Cl Biol. Cell J. .Bo.Chem. Biol. J. .Ml Biol. Mol. J. Nature 21) e6 rti mediates protein Sec62 (2013). 109 450 2641-2652. , 97 663-669. , 7214-7219. , 366 288 366-374. , 20) Homologs (2000). 18058-18067. , o.Bo.Cell Biol. Mol. (2007). (2013). imran . asetr . ei,M .adPla,H R. H. Pelham, and J. M. Lewis, M., Sagstetter, R., Zimmermann, Du S., Wittke, J. C. Stirling, and P. B. Young, J., A. Jermy, M., Willer, on,B . rvn .A,Ri,P . ilr .adSiln,C J. C. Stirling, and M. Willer, J., P. Reid, A., R. Craven, P., B. Young, A. T. Rapoport, and E. Hartmann, B., E., Jungnickel, Hartmann, S., Y., Voigt, Modis, I., Collinson, Jr, M., W. Clemons, B., Berg, den van eiuoyelst tmlt moto 1 rca rti nomicrosomes. into from protein component procoat additional M13 of an J. import and EMBO stimulate proteins lysate shock reticulocyte heat Seventy-kilodalton complex Sec63 membrane. the of ER surface yeast cytosolic the the in at interactions protein-protein novel 25-35. e6padKrpaerqie o h rnlcto fSRP-dependent of translocation the for vivo. in reticulum required endoplasmic yeast are the into Kar2p precursors and multiple Sec63p contains machinery, Sec-complex. the translocation for sites protein binding reticulum endoplasmic of phases early during protein membrane. reticulum TRAM endoplasmic the the across transport of protein function sequence-dependent A. T. Rapoport, and channel. conducting C. S. Harrison, ora fCl cec 21)17 2047 doi:10.1242/jcs.153650 4270–4278 127, (2014) Science Cell of Journal 7 nad .adJhso,N. Johnsson, and M. nnwald, ¨ 2875-2880. , Nature 427 Yeast 36-44. , o.Bo.Cell Biol. Mol. 20 133-148. , 20) -a tutr faprotein- a of structure X-ray (2004). 20) e6p opnn fthe of component a Sec62p, (2000). 11 3859-3871. , 20) dniiainof Identification (2003). MOJ. EMBO .Cl Biol. Cell J. 19) Signal (1996). 20 262-271. , (1988). (2001). 134 ,

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