ae,Switzerland. Basel, [email protected]) ([email protected]; correspondence for *Authors USA. 02139, MA Cambridge, Switzerland. iaJunne Tina translocon translocation Sec61/SecYEG protein inhibiting the product at natural new a Decatransin, ARTICLE RESEARCH ß eevd6Nvme 04 cetd1 aur 2015 January 15 Accepted 2014; November 6 Received 6 5 1 of and (TM1–TM10), domains transmembrane ten its with channel protein-conducting Sec61 a forming respectively, or eukaryotes, The and SecY 2011). bacteria of Hegde, consists and translocon Shao 2012; core translocon Rapoport, SecY/Sec61 and conserved (Park the complex endoplasmic by the eukaryotes or bacteria of of or reticulum membrane across plasma translocated the are into proteins inserted membrane and secretory Most INTRODUCTION Cotransin identification, Target profiling, Haploinsufficiency Translocation inhibition, reticulum, Endoplasmic SEC61, WORDS: KEY inhibitor. translocation decadepsipeptide new as this ‘decatransin’ for suggest name We the homolog. chemotypes, translocon Sec61 bacterial SecYEG, inhibit the both also to found were ribosome. chemotypes translating both interestingly, by a Most of independent post- mechanism substrates protein and a demonstrating co- translocated of both of inhibition structural reticulum translationally endoplasmic of confirmed the absence into analysis translocation an despite Biochemical affected chemotypes, mutations similarity. these both of by all, not inhibition but Most, resistance. conferred in profiling Chemogenomic from isolated was tulasneorum decadepsipeptide cyclic new A ABSTRACT avr eia col 4 ogodAeu,Bso,M 21,USA. 02115, MA Boston, Avenue, Longwood 240 School, Medical Harvard 4 ogodAeu,Bso,M 21,USA. 02115, MA Boston, Avenue, Longwood 240 oiatmttosi e6p(es)o Sec61 or (yeast) Sec61p in mutations dominant identified cells mammalian and fungal both in sequencing full-genome by followed mutagenesis genome-wide mammalian Unbiased, translocon. the Sec61 at translocation cotranslational inhibit previously to had shown that been cotransin) and HUN-7293 distinct a (including of chemotype heptadepsipeptides cyclic The of and target. those to the translocation similar is were profiles reticulum, protein endoplasmic the for at insertion machinery membrane the complex, translocon hpne Bhullar Bhupinder oii Hoepfner Dominic vnSchuierer Sven hlp Krastel Philipp oatsIsiue o iMdclRsac,20MsahstsAvenue, Massachusetts USA. 250 06033, Research, CT BioMedical Glastonbury, for Road, Institutes Farm Novartis Gates 60 LLC, Congenomics, Basel, 4056 50/70, Klingelbergstrasse Basel, of University Biozentrum, 05 ulse yTeCmayo ilgssLd|Junlo elSine(05 2,11–29doi:10.1242/jcs.165746 1217–1229 128, (2015) Science Cell of Journal | Ltd Biologists of Company The by Published 2015. 2 oatsIsiue o iMdclRsac,Nvri aps 4056 Campus, Novartis Research, BioMedical for Institutes Novartis ihptn iatvt nmmainadyatcells. yeast and mammalian on bioactivity potent with 1 oneWong Joanne , 3 oadHge eia nttt,HradMdclSchool, Medical Harvard Institute, Medical Hughes Howard 2 2 ioa Melin Nicolas , rn Petersen Frank , 1,2, 2 oetBruccoleri Robert , * .cerevisiae S. 2 hita Studer Christian , 2 dadJ Oakeley J. Edward , 2 4 niae htteSec61 the that indicated onA Tallarico A. John , eateto elBiology, Cell of Department a Sc1 nyat in yeast) in (Sec61p 5 a ,Ju mmas that (mammals) 1 Chaetosphaeria gEichenberger rg ¨ 2 hmsAust Thomas , 2 6 ua Oberer Lukas , o .Rapoport A. Tom , acn-hi–iooecmlxt h R eetri the in receptor the SRP bring the to to (SRP) is particle sequence complex signal recognition hydrophobic nascent-chain–ribosome signal a SecYEG/Sec61 by case, six the former recognized of to the are post-translationally proteins In ring or Substrate complex. cavity. co- constriction luminal either central targeted or periplasmic a that helix the plug by blocks lumenal a closed and residues hydrophobic is predominantly pore potential arsne l,20;Mcinne l,21) h rgnof 2006). al., origin et The (Harant 2014). clear not al., is et inhibition 2005; heptadepsipeptide of MacKinnon al., specificity 2005; cytosolic et cyclic signal (Besemer al., the blocked these et in is Garrison insertion by translocon signal but the unaffected inhibitors, with and are signal ribosome, the vestibule, the of of binding and interaction targeting SRP-dependent that of isolation by in Using confirmed was mutations which resistance 2007), al., al., Sec61 et and et identified (MacKinnon Westendorf has VCAM1 2011; labeling al., of Photoaffinity et 2011). translocation (Maifeld substrates cotranslational specific simplified been have other inhibit a 2005) al., to and et including 2005) (Garrison found cotransin al., called inhibitors thereof et version heptadepsipeptide (Besemer of group CAM741 a cyclic HUN-7293, Furthermore unknown. related is step closely blocked the been but has translocation 2009), metabolite, cotranslational (Cross cyanobacterial inhibit cells a to mammalian A, shown Apratoxin in 2009). translocon receptor al., the et SRP–SRP to the the complex of targeting from transfer the most complex preventing chemical and nascent-chain–ribosome A translocon a the processes. eeyarestatin, to binding complex is likely inhibitor of translocation mechanisms general molecular the elucidate translocation. initiate character clear gasket share hydrophobic not only general a that is a sequences as primary It diverse act 2011). with peptides Rapoport, that signal how and residues (Park constriction polypeptide the the around and plug pushing pore, the the the In into away 2014) inserted al., is et sequence Voorhees hydrophilic 2014; the al., process, et Park 2014; al., et (Gogala TM7 and pos- TM2 between SecY/Sec61 in translocon of helix a as intercalates the and translocon the to with Sec62–Sec63– reach engages sequence the signal the and bind involves Somehow complex. Sec66–Sec72 eukaryotes cytosol in to which, the translationally, ribosome into the SecY/Sec61synthesized of allowing loops cytoplasmic membrane appropriate w eihrlyatce ige otil-pnigsbnt SecG subunits Sec61 triple-spanning or to single- attached peripherally two 2 ai Estoppey David , hmclihbtr aefeunl enueu ol to tools useful been frequently have inhibitors Chemical 2 eeitW Bauer W. Benedikt , nvitro in b Shp,adSc rSec61 or SecE and (Sbh1p), 2 ap Riedl Ralph , rnlto–rnlcto sas thsbe shown been has it assays, translation–translocation a hc omaltrlgt notelpdbilayer lipid the into gate lateral a form which , 3,4 atnSpiess Martin , 2 ioeHartmann Nicole , SEC61A1 2 ulem Roma Guglielmo , 3,4 atnBeibel Martin , a lentvl,sbtae are substrates Alternatively, . Mcinne l,2014). al., et (MacKinnon c 1, Ssp.I t desae the state, idle its In (Sss1p). and * 2 nvitro in rtaKnapp Britta , a 2 , 2 stetarget the as , Lue al., et (Liu 1217 2 ,

Journal of Cell Science 1.Teegnsaelkl novdi ieoi cdand decadepsipeptide. and the NRPS of acid synthesis the the pipecolic He, identified in involved analysis 2004; genes in sequence accessory al., summary, involved et In likely (Field 2006). respectively are biosynthesis, genes homoleucine Table These material supplementary to S1). 1C; close (Fig. found gene also megasynthetase this were synthase 2-isopropylmalate L-pipecolate supported encoding and Genes oxidase 1C) the family. thus depsipeptide (Fig. is and the which bond, of 1, NRPS hallmark ester intermolecular position an the at through closure acid of peptide 2-hydroxy-amino neighborhood a of the incorporation in dehydrogenase 2-hydroxy-acid a gene of presence The 2012). al., et (Gao EERHARTICLE RESEARCH 1218 IC at of carcinoma cells colon kidney inhibition human monkey COS-1 growth HCT116 and as cells potent such lines, showed cell compound mammalian action the of site Although the as Sec61 complex the translocon identifies profiling chemogenomic Yeast the of compounds fungus identified cells saprophyte inhibition carcinoma growth human for HCT116 origin of natural of compounds new Screening from decadepsipeptide tulasneorum new a of Isolation RESULTS hne.W hssget‘earni’a h aefrti new this Sec61 for name the the through inhibitor. as translocation ‘decatransin’ translocation decadepsipeptide suggest all thus We blocks thechannel. compound of the indicated cells characterization mammalian that conserved and Biochemical yeast endoplasmic the both in cells. mechanism as the inhibition eukaryotic Sec61 identified in component assays target translocon All (ER) yeast in cells. reticulum yeast clones resistant used mammalian of we sequencing and and target, selection its by mutagenesis,followed identify genome-wide unbiased To and profiling growth. chemogenomic cell inhibits that atrs ial,tesnhts a emnlcondensation terminal a had synthetase (C domain the at respectively N-methylation Finally, present observed the the and homoleucines, with patterns. consistent were to 10, 9, and N-methyltransferases 7 and corresponding 5, and addition, 4, positions 7, acids 6 In and 2, 1A,B). pipecolic (Fig. 4 positions of 3, at positions positions domains at A with DAWTYGVA NRPS the single in a identified DPFMYLGI uniquely 1999) al., et selectivity of (Stachelhaus A-domain pattern define was The that modules. sequences compound signature ten eight-residue contained the three NRPSs, Of N-methyltransferases. predicted of four the and biosynthesis modules ten the contain natural to formation,expected for microbial responsible bond predict NRPS NPRSs of peptide genome multiple the to 2009), in identified and Ravel, were software and thioesters (Bachmann pathways a product as Using acids respectively. and amino (PCP) protein transferof of, carrier activation the for peptidyl responsible domains, (C) (A), domains, large condensation catalytic adenylation 7, are of the and organization NRPSs namely 4 modular (NRPS). 3, a synthetase with and multienzymes peptide 9, non-ribosomal and by synthesized been 6 a cyclic had 2, compound the positions a that indicated at respectively, homoleucine of and as discovery acid well the and 1A). as Fig. to purification 1, Methods (Compound led and decadepsipeptide isolation, materials) Materials cultivation, (see supplementary elucidation up structure Scaled activity. nti td eietfe e iatv cyclodepsipeptide bioactive new a identified we study this In h rsneo h o-rtioei mn cd pipecolic acids amino non-proteinogenic the of presence The T aayigtecciaino RSpout nfungi in products NRPS of cyclization the catalyzing ) ihptn ilgclactivity biological potent with heoparatulasneorum Chaetosphaeria heoparatulasneorum Chaetosphaeria 50 Chaetosphaeria 010n,i also it nM, 30–140 ihpotent with .The W2 ivle nRAslcn) a on ocnana S2). contain Fig. material to (supplementary found phenotype the was for the library, splicing), in original mutation RNA frame-shift the heterozygous of in strain (involved HIP S1B). CWC21 hypersensitive 1 Fig. other material compound only Dose–response (supplementary with result The inhibitor. HIP strains the the validated individual of fully using target experiments Sec61– the growth the is that indicated translocon strongly This Sec63 HOP). 2A, (Fig. complex ta. 05 Fg B.Wt nIC as an With characterized 2B). Garrison (Fig. previously 2005; 2005) al., al., been et et (Besemer have inhibitors derivatives, translocon which its mammalian of and cotransins, HUN-7293 structure to the identical the almost However, is scaffolds 2 chemotypes. compound the distinct that indicating comparing hits, constitute relevant only other they any or when 2004)] Glen, 0.26 reveal and (Bender and not 2B,C). molecule, did (Fig. coefficient [Tanimoto entire 1 2 compound compound 2 to similarity with any compound a searches of similarity denoted profile HOP Structural and heptadepsipeptide, HIP the cyclic with similarity striking a identified ugsiganwgntcln ewe h e6–e6 translocon Sec61–Sec63 the metabolism. between lipid and link genetic new a the deleted, suggesting been have and to synthase) phosphorylceramide where conserved (mannosylinositol interesting decadepsipeptide decadepsipeptides the strains of is the profiles new HOP were It the in thus the hits chemotype. prominent that and note cotransin of higher and hits action HIP this heptadepsipeptide conserved of of at profiles comparison mechanism confirmed but the HOP Pairwise respectively, 2F) and and (Fig. 100, 2D). 2 (Fig. and HIP compound 30 concentration similar of than very factor yeast produced a wild-type by on decadepsipeptide effective less rwhdfcsatrhmzgu eeino h he non- three the of deletion synthetic homozygous as genes well after essential as HIP), defects 2A, compound growth (Fig. the ( complex of the core components produced all significance Sec61–Sec63 for decadepsipeptide haploinsufficiency by cyclic of revealed that The profiles relative strains measure Methods). the and a individual Materials plotting versus by of (sensitivity) visualized HOP reduction or are whereas factors growth results compensating compound, The identifies the and pathways. effects by synthetic or affected reveals proteins indicates directly HIP 1999). 2008). pathways al., al., et profiling (Hoon et collections homozygous deletion on (Giaever based and are pathways (HOP) (HIP) to or profiling profiling chemogenomic proteins Haploinsufficiency apply target to us identify enabling thus 1A), (Fig. eitne niaigta h neligmttoswere mutations underlying the the of the maintained sequencing Sanger clones that Direct heterozygous dominant. indicating the and resistance, cells, wild-type uain xlsvl in exclusively mutations SEC63 in inhibitors 30 resistant colonies the to 45 chemical obtained to we yeast, resistance drug-efflux-compromised genome-wide for for unbiased, selection parallel orthogonal and performed an using mutagenesis inhibitors we the of target approach, direct the identify To resistance in confer mutations that identifies Sec61 yeast in mutagenesis Genome-wide niie rwho h yeast the of growth inhibited oprn h eaesppiepoiet rhvddatasets archived to profile decadepsipeptide the Comparing m ora fCl cec 21)18 2712 doi:10.1242/jcs.165746 1217–1229 128, (2015) Science Cell of Journal opud1 hs eitn el eemtdwith mated were cells resistant These 1. compound M eelc eeld1 ifrn igeaioacid single-amino different 13 revealed loci gene .cerevisiae S. SBH1 .cerevisiae S. IPT1 , SEC66 n o amla C16cls In cells. HCT116 mammalian for and SEC61 ioiopopornfrs)or (inositolphosphotransferase) and .cerevisiae S. eeoyosadhomozygous and heterozygous 30 Fg A o;Tbe1.The 1). Table top; 3A, (Fig. SEC72 f200 of SEC63 fteSec61–Sec63 the of m SEC61 ,HN79 was HUN-7293 M, tIC at eeresponsible gene , , z soe see -score, SEC62 .5frthe for 0.25 50 , z -scores 2 SUR1 m and M

Journal of Cell Science EERHARTICLE RESEARCH eutn el eealval n eandrssac equivalent resistance retained of and copy viable all wild-type were drug-efflux-compromised the cells into resulting replacing introduced cells, were wild-type alleles mutant C condensation; C, adenylation; A, shown. is NPRS matching the of line assembly proposed and organization domain the addition, In S1. decadepsipeptide. Fig. bioactive material novel by a produced of decadepsipeptide biosynthesis new and Structure 1. Fig. tahda hosest h C oan.()Mdlsadseiiiysgaue fteAdmi idn okt ntemthn PS C Genomic (C) S1. NPRS. Table matching material the supplementary in in pockets synth provided 2-isopropylmalate binding are Orf5, A-domain dehydrogenase; sequences the 2-hydroxyacid inter protein Orf4, of peptide and oxidase; signatures The pipecolate DNA specificity structures. Orf2, NRPSs. (Orf3): and the other decadepsipeptide in Modules the Orf6, A–D (B) for labelled NRPS domains. the are PCP of methylations the context Backbone to methylation. thioesters M, as protein; attached carrier peptidyl PCP, condensation; terminal heoparatulasneorum Chaetosphaeria SEC61 oersos uvsfrHT1 n es el r hw nFg Badsupplementary and 4B Fig. in shown are cells yeast and HCT116 for curves Dose–response . The . A tutr,mlclrms n rwhihbto oec fcmon ,a 1, compound of potency inhibition growth and mass molecular Structure, (A) eeecddteciia agto opud1 Several 1. compound of IC target the critical increased the the that mutations demonstrating encoded colonies, resistant gene original the to ora fCl cec 21)18 2712 doi:10.1242/jcs.165746 1217–1229 128, (2015) Science Cell of Journal 50 au ymr hn100-fold, than more by value s;Of and Orf1 ase; eitsare mediates SEC61 1219 T ,

Journal of Cell Science EERHARTICLE RESEARCH aeilFg 3.Mtto ftesxrsde fteconstriction 1220 the of residues six (supplementary 2007) the 2 of al., and Mutation 1 et S3). compounds Fig. the (Junne to material in sensitivity topogenesis to isolated respect protein been hepta- with had membrane and indicates our that of deca- tested mutants This also context Sec61p the we of 3C). of addition, library Fig. In action existing Sec61. of 1; on cross- (Table inhibitors modes depsipeptide were distinct, 2 2. mutants were yet compound the which compound similar, and 2, to G430D, to compound HUN-7293 and resistant to A186T cross-resistance sensitive 3; G97D, completely for mutations heptadepsipeptide compound for the tested Except of we 2, target the cotransin), be (compound to shown translocon chemotype Sec61 been the that inhibitory also Given the 3B). had in Fig. increases 1; (Table moderate concentration caused only others whereas it because strain HIP SEC61 the heterozygous with the grouped to and due circle not is a strain with this labeled of is hypersensitivity YLR379w that ORF revealed dubious the The with prevalent. overlaps are substantially function translocon. Sec61–Sec63 translocon the Sec61–Sec63 inhibit to compounds the that suggest versus HOP sensitivity and HIP 2. Fig. needn xeiet ihcmon .()Cmaio fteatvte fcmons13b arws HIP by pair-wise demonstrated (HUN-7293) by is 3 1–3 profiling compound compounds HIP inhibitor of of translocation activities Reproducibility known (E) the related A. of closely for Comparison the described (F) of as 1. heptadepsipeptides and compound two 2 with the compound experiments of heptadepsipeptide independent profiles new HOP a and of HIP activity (C,D) and Structure (B) S2). z soe ryadbakdt ersn tan ihdltosi seta n o-seta ee,rsetvl.HPadHPsrisrelated strains HOP and HIP respectively. genes, non-essential and essential in deletions with strains represent dots black and Gray -score. SEC61 eeadisdlto fet ohOF.TeCC1sri smre iha seikbcueflo-panalysis follow-up because asterisk an with marked is strain CWC21 The ORFs. both affects deletion its and gene CWC21 M lorsle nsrn eitnet ohinhibitors. both of to deletion resistance and strong domain, in plug resulted the of also deletion TM2 full and partial ring, o opud2(i.4) ihteecpino n single however, one 1, of compound exception to cross-resistant the were With also IC 4A). they increased (Fig. 20-fold clone, 2 than The more compound the characterization. showed for clones further in the for of picked majority cultured new were colonies the were N-ethyl-N- growing 1 of of cells and availability presence limited mutagenized (EMS) to methanesulfonate decadepsipeptide, Owing (ENU). ethyl nitrosourea using HCT116 human of cells mutagenesis genome-wide performed also We supports conservation cells target mammalian in mutagenesis Genome-wide eeinbtt a to but deletion ora fCl cec 21)18 2712 doi:10.1242/jcs.165746 1217–1229 128, (2015) Science Cell of Journal A I O rfl ftedcdpietd opud1 plotting 1, compound decadepsipeptide the of profile HOP HIP (A) SEC63 m opud2 fe ek,1 strongly 12 weeks, 2 After 2. compound M akrudmtto splmnaymtra Fig. material (supplementary mutation background z soealignment. -score z soeaineto two of alignment -score . 50

Journal of Cell Science EERHARTICLE RESEARCH oto hmol a –-odicesdIC increased 2–3-fold a had only them of most exc notable to the resistance with high 2, compound to to Moderate resistant respectively. also 2, were compound They mutations. and all IC 1 for Resulting compound validated G430D. was for selected) and mutants A186T originally point were G97D, to Sec61p they newly resistant which indicated The Mutations (against the domains. respectively. 1 expressing below, compound transmembrane cultures and numbered liquid a occurrence) with (not in have of bar green resista they strains in frequency a be if 2 their to as compound red found (with to shown in were only above shown schematically that resistant are indicated is mutants 2 point are sequence and single mutations Sec61p 1 Sec61p resistance the studied compounds previously old middle, Below, the the panel. and In top the selected presented. in similarly stereo are in shown 2) 2006) Table al., et 30 inhibitors. (Junne to the complex resistance Sec61 to for selected resistant cells mutants yeast yeast drug-efflux-compromised mutagenized of Selection 3. Fig. pligteetrefles h eewt h ihs ubrof number highest the with gene samples. the parental filters, the three of these were analysis Applying that RNAseq genes to and in according Materials mutations expressed only (see included function we protein Finally, to Methods). not deleterious were be that to SNPs missense predicted in excluded mutations further nonsense We regions. and coding missense further For only excluded. considered were we sample analysis, sequencing parental (e.g. the metrics in quality Variants several depth). to according filtered andthen samples the of each for called were insertions/deletions (indels)] and (SNPs) polymorphisms sensitive nucleotide [single human variants two for and the genome, to libraries aligned and exon-enriched were reads prepare resulting clones The sequencing. to resistant Illumina used ten and samples, from parental in isolated was observed 4C). DNA was (Fig. taxol change compound no control the because to sensitivity specific was Resistance uvsfrrssatmtnssoni h oe ato r rsne nsplmnaymtra i.S3. Fig. material supplementary in presented are A of part lower the in shown mutants resistant for curves oietf h eitnecnern uain,tegenomic the mutations, resistance-conferring the identify To 50 prl and roag ( orange or ) r 2 ausaelse nTbe1 oe n ice niaeidpnetrpiaeeprmns Dose–response experiments. replicate independent indicate circles and Boxes 1. Table in listed are values prl 50 hntp,adi lei o.Mttosrssatol ocmon (not 1 compound to only resistant Mutations not. if blue in and phenotype, aus(i.4B). (Fig. values prl .Tewa uat r niae ylgtrsae.(,)Ds–epnecre fwl-yeyeast wild-type of curves Dose–response (B,C) shades. lighter by indicated are mutants weak The ). A hrendfeetsnl mn cdmttoswr dniidin identified were mutations acid amino single different Thirteen (A) m opud1o lts hi oiin r niae ntemdlo h yeast the of model the in indicated are positions Their plates. on 1 compound M uain was mutations 7Fcoe niae needn vns(al ) efurther We 3). (Table the events sequenced independent indicated two clones the of S71F patterns the whereas patterns, SNP similar clones highly showed D60E two and M65R from originated two clones clones The ten progenitors. independent ten the eight patterns, the SNP of the out on Based eight 4D). in (Fig. mutations with Sec61, yeast of ortholog umr,slcinfrrssatcoe dniisteSec61 the cells. In mammalian and hepta-depsipeptide identifies yeast resistance. and both deca- clones in of inhibitors of target conserved resistant the levels as for translocon different selection yielding summary, although differentspecies, in independently identified been even have Sec61 Sec61p in in S71F S72F mutations identical mutated the when Thus resistance produced 3). The also (Fig. which mutated Sec61p, 3). yeast (Table in Sec61 S72 mutations human and of R67 harbored S71 and also R66 D60, residues which of all mutagenesis, six (ENU) N-ethyl-N-nitrosourea by obtained clones ora fCl cec 21)18 2712 doi:10.1242/jcs.165746 1217–1229 128, (2015) Science Cell of Journal SEC61A1 SEC61A1 DAo i diinlrssatHCT116 resistant additional six of cDNA hc noe Sec61 encodes which , prl r hw nca,those cyan, in shown are ) a orsodt D61, to correspond 1 SEC61 a ,tehuman the 1, fchemically of both pinof eption a t(see nt 1and 1221

Journal of Cell Science h e6pmtnsioae srssatt opud1wr etdfrthe for tested were 1 compound to resistant as isolated mutants Sec61p The Blue Cyan A446T G430D ,sniie ,rssat(IC resistant R, sensitive; S, Blue S307F 28 Blue Cyan A298T A186T uainClrcode Color mutation Leu 9DCyan G97D amla O- el,a hw nFg Ffrthe for 5F Leu Fig. protein, of made membrane in signal-anchors II generic in shown type with a translocation derivatives as H1, and inhibited cells, receptor similarly asialoglycoprotein action COS-1 compounds the mammalian mammalian in both conservation of sequence target the signal cells, with independence specific agreement the In yeast. the from in inhibitors residues these underlines leucine of consecutive mechanism This 16 or 5E). 13 (Fig. of made than sequences CPY signal inhibition general, to In sensitive to membrane). more DPAPB. required plasma the of be that time to and reflects (the appeared wall likely 2 translocation compounds cell most the the compound dependence of cross 5C,D): time kinetics (Fig. This penetration time longer. even pre-incubation on required as a of well presence the of independent is protein and ribosome. of translating 2005), al., thus the et the for is and (Garrison concluded been of receptors from already action has SRP as cotransins increase of translocon, and the mechanism to an SRP targeting the and of post-translational that independent of glycosylated demonstrates Inhibition the 5A). translocation (Fig. of forms unglycosylated in reduction apparent is effectively as 2 a translocation post-translational compound and heptadepsipeptide co- post-translationally the inhibited decadepsipeptide and and the 1 co- Both compound established respectively. substrates, are SDS-PAGE. translocated and CPY and glycosylation and the immunoprecipitation on DPAPB after based assessed pattern was Y processing ER carboxypeptidase the into and (CPY) (DPAPB) B dipeptidylaminopeptidase al .Ihbtrsniiiyo es e6pmtnsslce yterrssac ocmon 1 compound to resistance their Sec61 by selected mutants Sec61p yeast of sensitivity Inhibitor 1. Table ARTICLE RESEARCH 1222 labeled metabolically were absence cells [ or yeast presence the compounds, with in these directly function of translocon test ER To the post-translational into and translocation co- protein both inhibit compounds The 8IBlue T87I 8LBlue P84L 7DRd+–309625()1409928(R) 2.8 0.999 1.4 (R) (R) 3 0.997 2.5 0.996 3.6 3 – – – + – + + + Red Red Red G81D Red E79K S72F A71D upeetr aeilFg S3; Fig. material supplementary 4DRd+–100978 R 83 0.977 100 – + Red – G47D Wild-type uain ofrigrssac oa es n opudcrepnst htue nFg A oe panel; lower 3A, Fig. in used that to corresponds compound one least at to resistance conferring mutations ohtpso niiosas ce nnnntrl generic non-natural, on acted also inhibitors of types Both as 5B) (Fig. concentration the on dependent was Inhibition 25 segments. 35 , ]ehoiefr5mn n rnlcto of translocation and min, 5 for S]methionine 0mnt ec aia ees n opud1 compound and levels, maximal reach to min 30 nvitro in a 50 2 2 2 2 2 2 2 prl 2 2 tlat5-odhge hnwl-ye;() oeaeyrssat(IC resistant moderately (R), wild-type); than higher 50-fold least at rnlto–rnlcto experiments translation–translocation c r 2 ausaesonweeasgodlcrecudb fitted. be could curve sigmoidal a where shown are values . .9 . R . .9 . (R) S 2.6 1 0.999 0.986 1.3 0.5 (R) (R) 2.3 3.1 0.995 0.996 2.7 3.7 – – – . .9 R . .9 . S 0.6 0.998 0.3 (R) 2 0.993 2.4 – – – CPY – defect translation – . .8 . .8 S 1 0.980 0.5 S 1 0.985 1.2 – . . . opud1Cmon 2 Compound 1 Compound . IC . ( . . m 0 – 200 0 – 200 0 – 200 0 – 200 0 – 200 0 – – 200 200 M) 50 b r 2c 13 or prl . . . . . ocnrlPhenotype control to IC . . 0 R 100 0 R 100 0 . .9 . S 0.6 0.997 0.3 R 100 0 R 100 0 R 100 0 R R 100 100 hntp n P rnlcto eet si un ta.(un ta. 2007). al., et (Junne al. et Junne in as defects translocation CPY and phenotype 50 ratio neetnl,dlto fteSc lgdmi conferred shift domain considerable a plug by curves. shown SecY the as of chemotypes the both to of resistance deletion Interestingly, n e aKno ta. 04.Pu uain have mutations Plug a 2014). cause to al., 4D, Fig. found et 3A; (Fig. been MacKinnon domain plug previously the see to close and or in conspicuously are concentrated mutations resistance-conferring identified The Most estimated with 5G) (Fig. model fashion 2014). pro-OmpA dose-dependent IC al., a a of et in by translocation (Bauer substrate inhibited targeted previously compounds described be performed Both as as We membranes can mutations lipid purified question 2. also on the fungal and conserved translocons experiments raised the 1 and bacterial species compounds on across whether homologs chemotypes resistance to Sec61p both confer of that human action and conserved The putative, site a binding of conserved detection allows activity Cross-species rnlcn n hrb aiiaepr pnn,rdcn the a the for tested reducing mutants We Sec61p translocon. opening, the resistant by new pore acceptance 1981). signal facilitate for al., stringency thereby et and (Emr translocon, mutations sequence prl signal of suppression htteihbtr idt h lsdsaeo h wild-type the of compounds. state the for closed affinity the binding reduced to bind In The translocon. inhibitors inhibitors. the the of that one the least between at correlation to resistant From all it. significantly 2), plug towards were the (Table pointing in TM1 collection in four older 5H). the (G47D) our plug, (Fig. one the and residues involving itself suppressed three mutations domain by carry them truncated indeed of was All peptide signal mutants the CPY of of resistant defect translocation isolated newly 50 uain pcfclydsaiietecoe tt fthe of state closed the destabilize specifically mutations f10 of prl ora fCl cec 21)18 2712 doi:10.1242/jcs.165746 1217–1229 128, (2015) Science Cell of Journal uain ofrrssac oSc1inhibitors Sec61 to resistance confer mutations m 50 o opud1ad90 and 1 compound for M prl tlattofl ihrta wild-type). than higher two-fold least at uat hssaei etblzd eutn in resulting destabilized, is state this mutants . . . . IC . ( . . . m 0 – 200 0 – 200 0 – 200 0 – 200 0 – 200 b 0 – – 200 200 – 200 M) 50 orsodn IC corresponding b prl .coli E. D hntp n eitnesuggests resistance and phenotype nwihtehdohbccore hydrophobic the which in 3 r 2c rnlcn eosiue into reconstituted translocons prl prl uat xetoe(I86T) one except mutants 50 hntp.Fv fte13 the of Five phenotype. prl ...... Phenotype control to IC uvsaesonin shown are curves 0 R 100 0 R 100 0 R 100 0 R 100 0 R 100 0 R R 100 R 100 100 nvitro in 50 m o opud2. compound for M hntp,ie the i.e. phenotype, ratio a h oo oeof code color The translocation

Journal of Cell Science h orni hmtp n o h rgnlihbtrHUN-7293 inhibitor original the 3). (compound for and of chemotype 2) cotransin (compound heptadepsipeptide the active was yeast profile new a same the for the of obtained HOP subunits Essentially core complex. and all translocon HIP identified heptameric 2014) high-resolution al., our et target: (Hoepfner platform fungal complex potential Sec61 heptadepsipeptide the the on as in home of rapidly to profiling chemogenomic group have inhibitors. translocon that a mammalian similarity as described molecular also cotransins, been name previously detectable Sec61/SecYEG The the without decatransin. compounds it the to name to alludes and across propose co- we inhibits in translocon, translocation compound the as this of well that analysis post-translational as Given biochemical cells, processes. and mammalian assays inhibited genomic and using yeast bacteria, in target conserved EERHARTICLE RESEARCH ee epeeta nertdsuyo e bioactive new a of the and genome- of by study organism sequencing identification producer the the integrated of and megasynthetase responsible isolation an its from present decadepsipeptide we th of Here, 2014). model al., stereo 201 et the al., (MacKinnon in DISCUSSION et al. shown (MacKinnon et are al. MacKinnon (HUN-7293)] et by 3 MacKinnon shown compound by mutations to mutations the related gray, identified closely mutation; Sec61 recently compound Below, double the respectively. a gray, the of 2011; and yellow, red and al., in 2 et in compound complex [Maifeld Sec61 to CT08 human resistance cotransin for to here resistance selected conferring mutations The shown. identified are with cells. clones human resistant in 12 mutants of resistant of Selection 4. Fig. h ciiyo earni nteyeast the on decatransin of activity The nsilico in nlsst h eemnto fthe of determination the to analysis SEC61A1 uain gis opud A n B,adaanttxl() h mean The (C). taxol against and (B), 1 and (A) 2 compounds against mutations .cerevisiae S. C16clswr uaeie n eetdfrrssac ocmon .Ds–epnevalidation Dose–response 2. compound to resistance for selected and mutagenized were cells HCT116 loe for allowed a ssona a ihistasebaedmisnmee.Rd h igemutations; single the Red, numbered. domains transmembrane its with bar a as shown is 1 ta. 02 ihee l,21;Sdihe l,21;Shimada 2013; al., acid et amino that Sadlish be might 2013; mechanism underlying al., The et towards 2013). al., and Richie et bias 2012; (Nyfeler sequenced a al., fungi and selected et is are mutants there in spontaneous drug-resistance if pleiotropic genes that in SNPs experiments revealed and aberrations of chromosomal have mutagenesis strategy cells our mammalian resistant Multiple to spontaneously due selecting clones. than be the rather sequencing. as might unambiguously mutations Sec61 inducing to yielded This RNA analysis failed candidate. the case, approach total best-scoring our In the targets. and of study the identify proteins, that pioneered proof binding resistance in documented have known However, as 2012) with spontaneous well substances al., performed two et using been for also (Wacker approach al. already this was et Wacker analysis had principle. this cells target previously, cotransin mammalian the Although mutagenesis cells. in mammalian genome-wide in identified target has unbiased, drug whole-genome-sequencing the an is by this Although where knowledge followed our protein. approach, study to yeast, binding first in history primary the long a their has approach this as Sec61 confirmed uaeei olwdb eunigo eitn clones resistant of sequencing by followed Mutagenesis ora fCl cec 21)18 2712 doi:10.1242/jcs.165746 1217–1229 128, (2015) Science Cell of Journal 6 ..o rpiaedeterminations triplicate of s.d. 1223 4) e

Journal of Cell Science EERHARTICLE RESEARCH 1224 2005; as decatransin, al., that et show experiments (Besemer Our 2005). translocon al., mammalian et ribosome–nascent-chain Garrison the the to of binding complex and targeting dependent under phenotype spontaneous detrimental no against conditions. have or laboratory that bias components efflux alterations drug a copy to contrast gene is in a genes, there of these in absence mutations pressure the in selective Thus outcompeted. strong rapidly deleterious, are be often cells can these target and primary essential, the in mutations 5. Fig. thsbe hw rvosyta h ornisalwSRP- allow cotransins the that previously shown been has It e etpg o legend. for page next See htctasnpeet h inlsqec rmisrigit the into inserting from sequence signal showed the recently prevents have cotransin 2014) al., that et (MacKinnon al., et MacKinnon h rsneo iooebudt h rnlcncmlxi not is complex translocon also the Thus to bound essential. but ribosome substrates. a of presence translocation, (SRP-independent) the post-translational (SRP-dependent) specific not inhibits is co-translational action the Their to translocation. to limited bacterial not similarly and act are fungal but family, translocation, on mammalian cotransin or the human of of inhibition member a as well yextensive By ora fCl cec 21)18 2712 doi:10.1242/jcs.165746 1217–1229 128, (2015) Science Cell of Journal nvitro in rslnigo retdnsetchains, nascent arrested of crosslinking

Journal of Cell Science eune rgnrchdohbcsqecscmoe fLeu signal-anchor of natural composed its sequences from with hydrophobic derived H1, generic protein protein or a membrane sequence, H1*, II (E) type shown. mammalian are the experiments independent two of ae nqatfcto ftreeprmns H e6pmtnsisolated mutants 30 Sec61p to (H) resistance experiments. their three by of mean quantification are on curves based and dose–response electrophoresis gel presented by substrate The followed the autoradiography. treatment K of proteinase Translocation compound by (–ATP). measured indicated without was the or of ATP presence with the concentrations in incubated fusion was translocated protein) post-translationally (a proOmpA-DHFR Radiolabeled ( 60–74 residues ( residues cysteine lacking [full-length ybl o opud1 n ildsmosfrcmon .Temean open The CPY, 2. for compound for circles symbols DPAPB, filled for and used 1, compound are for Squares symbols inhibition C. translocation of in Quantification as (D) experiments min. 0–30 fixed of a times preincubation using by above, analyzed 10 as was CPY, of in respectively, inhibition and concentration DPAPB DMSO, of of with time-course or labeling The without (C) metabolic labeling inhibitors. preincubation indicate of after 0* absence 2 and the or 0 1 min. compound 30 above, of as for labeling concentrations translocation metabolic indicated by of the analyzed Dose-dependence using was (B) CPY indicated. and DPAPB standards is for mass kDa inhibition molecular in of weight and position their glycosylated The with DPAPB. the lumen of indicate ER forms u the unglycosylated and in g proCPY preproCPY. glycosylated untranslocated indicate and and pp electrophoresis and gel p1 immunoprecipitation, autoradiography. by analyzed and compounds, ieto.Tefato ftasoae rdcsi niae sthe as (mean indicated total is (endoH) the products H of translocated endoglycosidase percentage by of the deglycosylated fraction control, was The a lane digestion. As first lumen, cytosolic. the ER is in the form material into preproCPY translocation unglycosylated indicates the forms whereas p1 the to autoradiography. Glycosylation and electrophoresis, of SDS-gel labeling immunoprecipitation, metabolic analyzing CPY by expressing residues) cells apolar three CPY lacking of sequence defect translocation the suppress analyze and To in immunoprecipitated (G) translocation were autoradiography. of derivatives and inhibition its electrophoresis and gel H1 by 2. analyzed or 1 compound of opud2(m2 oafnlcnetaino 100 of concentration final [ a with without to or heptadepsipeptide (Cmp2) with cotransin 2 DMSO or compound 1% (Cmp1) with 1 min compound 30 decadepsipeptide for substrates, preincubated translocated were co-translationally respectively) and and human, post- yeast, (typical the DPAPB by translocons. translocation Sec61/SecYEG inhibit bacterial compounds The 5. Fig. ARTICLE RESEARCH oteitro ftetasoo ntecoe tt,teinhibitor Inhibitor conformation. the open state, an closed in the directly in there translocon bind point only the residues could of affected interior the gate constitutes the that structure lateral to plug Given 2014) the the of site. to part of cotransin-binding al., concentration this the end that conspicuous recently suggested et lumenal gate The lower the TM3. (MacKinnon and CT08 to and and al. TM2 cotransin plug helices et the to to MacKinnon localize by five resistance The published mutations. causing resistance the mutations identified blocks a inhibitor we the insertion how in understand signal To and 2014). al., et end (MacKinnon Sec61 residues C-terminal TNF cysteine into with its – engineered conformation of helical by a suggesting signal-anchor preferentially pattern – The crosslinked pore. translocation generic by replaced was signal-anchor Leu in its of derivatives of sequences or core were H1 hydrophobic cells receptor the COS-1 asialoglycoprotein which To cells, the (F) mammalian express respectively. in to 3, translocation transfected and three-fold on 2 and effect two- 0, the the by analyze and indicated unglycosylated, by are The translocation A. forms of in glycosylated inhibition as 2 for and analyzed 1 and compounds yeast in expressed were [ 35 ]ehoiei h rsneo bec fteidctdconcentrations indicated the of absence or presence the in S]methionine 35 ]ehoiei h otne rsneo bec fthe of absence or presence continued the in S]methionine D 13 07 and 60-74 m rLeu or opud1o opud2 n h indicated the and 2, compound or 1 compound M D r sacnrl P o i,floe by followed min, 5 for CPY control, a as or, 3 m opud1wr nlzdfrteraiiyto ability their for analyzed were 1 compound M a 25 .coli E. D el eelbldfr3 i with min 30 for labeled were Cells . ttectslctpo h aea gate lateral the of top cytosolic the at 6 lg]wr eosiue noproteolipsomes. into reconstituted were Plug)] s.d.; uiidSc n eYGderivatives SecYEG and SecA purified , n D 5 y n T rwt eeino plug of deletion with or WT) and Cys 3). A es el xrsigCYor CPY expressing cells Yeast (A) D CYwt uatsignal mutant a with (CPY 3 m ,lbldfr5min 5 for labeled M, a 13 ol be could 6 rLeu or s.d. 6 s.d. 16 eit h upeso fsga etd eet,the defects, peptide signal of suppression the mediate or deca- mutations. many cyclic by compact resistance cause a to a by effects allosteric over covered or conformational distributed be suggesting hepta-depsipeptide, are could mutations than some yeast area S71F and larger the In yeast However, in region. (S72F human). exchange gate acid in amino same and the case one with plug in even mutated, were the residues homologous same to the instances, localize also to and/or Several mutations action. resistant of were decatransin mechanisms residues similar indicating different chemotypes, to both 15 in resistance mutations 16 conferring cotransins; residues the different contact all to point likely residues not plug are directly. affected residues they inhibitor various different directions, the three different that of very Given total in K). a and R66 to domain. G studies plug (I, the both in in all mutated region, The same was the entering. to Sec61 localize from human also study in signal partially independently the the identified and mutations preventing five plug thereby the stabilize gate, to opened proposed thus was binding irr hc eeae ,7,7 ed iha vrg edlnt of length read average shotgun an with one reads prepared, Newbler 1,277,077 were generated Roche/454 libraries which the sequencing library and Two al., was v.2.6. sequencing genome et assembler The Roche/454 Germany. (White in using debris sequencing leaf determined maple to Spacer from isolated Transcribed was related 1990), Internal closely by strain determined sequencing fungal genome full A and isolation strain Producer METHODS AND MATERIALS un ta. 07 mt ta. 05 n es Jnee al., et (Junne yeast and 2005) al., et Smith 1981; al., 2007; 2007). et al., (Emr et bacteria Junne in described phenotype localization’) ol loitreewt inletyadmgtntb viable. be not might and entry signal binding with inhibitor interfere block According directly also that depsipeptides. would mutations mutations the the hypothesis, release this block Destabilizing rapidly to to might translocon signal. the state in incoming This phase. the an lateral (including lipid the for into the intercalate translocon contact to the helix and to as extended up such gate an step, – of next do the formation signals prevents the structure natural circular the as their part with the manner where engage point similar in they a that sequences, (although in conceivable is translocon hydrophobic It signal acids. of amino unusual) oligopeptides Like eukaryotic are specific two conserved inhibitors interactions. are the there that in unlikely between conservation inhibitor–protein appears sequence thus identity no It plug. with 17% domain), the SecY, plug bacterial than the and of less sequences residues 16 is of 11 there (including man and as yeast and evolution Sec61 in the opening Although distant channel as the the of translocons to state of insertion. bind signal the early affinity preventing inhibitors thereby an of the it, binding or stabilize that ‘flexibilization’ the translocon suggest closed reduce by might This to explained inhibitors. surfaces this be interaction showed that might fact gate The domain, positions. inhibition lateral other plug all the at mutations the almost of as well or of as phenotype, residues Mutations peptide ring of translocation. constriction initiation and and entry insertion signal facilitating translocon, the ait fdfeetmttoshv rvosybe on to found been previously have mutations different of variety A 21 in mutations 22 of number higher a identified we yeast, In ti tiigta ohdctasnadctasn inhibit cotransins and decatransin both that striking is It prl ora fCl cec 21)18 2712 doi:10.1242/jcs.165746 1217–1229 128, (2015) Science Cell of Journal uain pcfclydsaiietecoe tt of state closed the destabilize specifically mutations prl prl uat hwdsm ee frssac to resistance of level some showed mutants uat)mgtalwfrsfiin flexibility sufficient for allow might mutants) a uui hrsams 0 dniybetween identity 60% almost shares subunit heoparatulasneorum Chaetosphaeria .coli E. es n man. and yeast , prl a (‘protein nour in 1 1225 ,as

Journal of Cell Science o ecito fcln eoyeaayi,seMtrasadMethods. and Materials see analysis, genotype colony of description For 6I1;R6 ATC R66K I41D; 16 5I1;R6 ATC R66K I41D; 15 4S1 TCT S71F 14 3S1 TCT S71F 13 hs e6 uat aepeiul encaatrzdb un ta.(un ta. 06 un ta. 07 un ta. 2010). al., et Junne 2007; al., et Junne 2006; al., et (Junne al. et Junne by characterized been previously have mutants Sec61 These 2S1 TCT S71P 12 rnlcto eet eeaaye yJnee l Jnee l,20) ,sniie S yesniie(IC hypersensitive HS, sensitive; S, 2007). al., et (Junne al. et Junne by analyzed Blue were defects translocation M450K oeGnm-ie40609 21 0.98 12.15 0.99 1.04 0.6 7 4 Genome-wide Genome-wide TCT S71P None None 11 10 9 40 – – M400K P292S 7FTCT S71F 8 24 Green K284E 20 – Blue P200L T185K 6GGAC D60G 7 18 – D168A 6EGAC D60E 6 11 rne+ Orange S161T 6EGAC D60E 5 11 rne+ Orange L131P 6RATG AGA M65R R66I 4 3 9R– Q96R 6RATG M65R 2 mutation 9R– – Q93R I91T Sec61 mutants Sec61 described previously of sensitivity Inhibitor 2. Table ARTICLE RESEARCH T––––01 .900 0.97 0.05 0.99 0.14 – 1226 – TCT – S71F – 1 WT colonies HCT116 2-resistant compound in mutations SEC61A1 Identified 3. Table assembler 2.6 Newbler the peak using a assembled with library were reads paired-end paired Sequences bp 953,342 29. The 2768 of of bp. of total depth 692 a average yielding of distance twice, deviation sequenced pair standard was with distance pair library a paired-end and a and bp, 676 6CRd++ + Red R67C 8T–+ + – Red I86T E79G 6NRd+ + Red Red L63N D61N opudcrepnst htue nFg A oe panel; lower 3A, Fig. in used that to corresponds compound Colony# 3R– W35R D D D 6 6 6 6 R,mdrtl eitn (IC resistant moderately (R), imi uv ol efitted. be could curve sigmoid hr imia uv ol efitted. be could curve sigmoidal a where – Wild-type 6 6 6 6 M ..+ + n.d. (+) + + – Red Red TM2 tip plug e (+) (+) + + + Red Red Red Red W S G A code Color Sec61 in Mutation a a oo hneSqecn Cluster Sequencing change Codon 1 50 22 22 2 62 2. 62 22 22 22 22 2 prl 22 6 22 tlattofl ihrta idtp) .. o determined. not n.d., wild-type); than higher two-fold least at . .8 .3S0809911 S 1.14 0.979 0.8 S 1.63 0.981 1.3 + 2 2 . .7 .8S0208402 HS 0.29 0.804 0.2 S 1.38 0.972 1.1 + CPY 2 2. defect translation 2 2. 2. (+) ...... A;AGA AAC; A;AGA AAC; T E6A – SEC61A1 TTT T E6A – SEC61A1 TTT C E6A – SEC61A1 CCT C E6A 0.55 – SEC61A1 CCT T eoewd .709 .60.99 8.56 0.98 0.37 2 Genome-wide TTT T eoewd .509 22 0.99 22.28 0.99 0.25 1 Genome-wide TTT G eoewd 0.81 5 Genome-wide AGG G eoewd 0.79 5 Genome-wide AGG G eoewd 6.22 6 Genome-wide GGC A eoewd .809 .10.99 8.71 0.99 0.48 3 Genome-wide GAA A eoewd .209 .10.99 7.31 0.99 0.42 3 Genome-wide GAA T eoewd .809 .30.99 7.63 0.99 0.18 8 Genome-wide ATA . .9 R . .6 .4(R) 3.14 0.968 2.2 (R) 4 0.893 3.2 . .9 . .9 S 1 0.999 0.7 S 1 0.991 0.8 . .7 .8S1609722 (R) 2.29 0.977 1.6 S 1.38 0.971 1.1 . .8 .8S0809811 S 1.14 0.998 0.8 S 1.38 0.981 1.1 .7 .5S0409205 S 0.57 0.982 0.4 S 1.25 0.976 1 . .6 .3S 0.63 0.967 0.5 . .7 .5S 0.75 0.974 0.6 . .9 . .9 S 1 0.997 0.7 S 1 0.993 0.8 .8 .5S0409405 S 0.57 0.974 0.4 S 1.25 0.986 1 opud1Cmon 2 Compound 1 Compound . .9 .8S0409905 S 0.57 0.989 0.4 S 1.88 0.991 1.5 IC . .7 .3(R) 3.63 0.978 2.9 . [ . .9 . .7 .1S 0.71 0.975 0.5 S 1 0.994 0.8 . .9 . .9 S 1 0.998 0.7 S . . . 1 . . 0.995 . 0.8 m . . 0 – 100 0 – – 100 100 0 – 100 0 – – 100 – 100 – 100 – 100 – 100 – 100 100 M] 50 A E6A – SEC61A1 AAA A E6A .109 06 0.99 10.61 0.99 0.11 – SEC61A1 AAA * b orsodn IC corresponding r 2 ** a orsodn IC Corresponding . . . IC ocnrlPhenotype control to ...... 0 R 100 0 R R 100 100 0 R 100 0 R R R 100 R 100 R 100 R 100 R 100 100 100 50 50 ratio iewti hs cfod a 2,2 p 5 cfodcni size contig contig average scaffold (the N50 bp average bp; 2,374,876 124,629 an of was with N50 scaffolds four generated, an these and were and within scaffolding scaffolds bp size 843,301 the 50 of specifying of size total for A except processors. options, default with uvsaesoni upeetr aeilFg S3; Fig. material supplementary in shown are curves ora fCl cec 21)18 2712 doi:10.1242/jcs.165746 1217–1229 128, (2015) Science Cell of Journal a 50 h oo oeo uain ofrigrssac oa es one least at to resistance conferring mutations of code color The uvsaesoni i.4A,B; Fig. in shown are curves 50 ...... IC . [ ...... m tlattofl oe hnwl-ye;R resistant; R, wild-type); than lower two-fold least at 0 – 100 0 – 100 0 – 100 0 – 100 0 – – 100 100 0 – 100 0 – – 100 – 100 – 100 – 100 – 100 – 100 100 M] 50 –––– –––– –––– –––– opud1Cmon 2 Compound ( IC 1 Compound m b M) 50 a r 2c . . . . . r 2b .91.20.98 12.12 0.99 0.99 0.99 0.99 0.99 b r 2 ...... Phenotype control to IC ausaesonweea where shown are values 0 R 100 0 R 100 0 R 100 0 R 100 0 R R 100 100 0 R 100 0 R R R 100 R 100 R 100 R 100 R 100 100 100 50 ratio prl . . . . ( IC m c r 0– 30 0– 30 0– 30 0– 30 M) hntp n CPY and phenotype 50 2 ausaeshown are values a r 2b

Journal of Cell Science eoedt ihRAe olwn h uutsRAe tutorial. the RNAseq Augustus supplemented the We following generated. RNAseq 80 with were of data total reads A genome paired-end platform. HiSeq2000 prep 76-bp Illumina RNA kit the million Illumina mini using an plant sequenced using RNeasy and prepared the parallel, kit, were In using genes. libraries fungus was (Parra be spliced the RNA-seq software of from (Qiagen). set genome CEGMA must isolated training the was initial Augustus RNA used the an total provide we 2011). to and of 2007) al., fungus, al., et new et prediction each (Keller on and trained Augustus modeling using undertaken Gene bp). 594,413 ARTICLE RESEARCH ni ny5%o h el omdclne.Attlo 2 of amino containing total synthetic glucose) without 2% with mixture, A Base acid dishes Nitrogen amino colonies. CSM 30 Yeast 14-cm MPbio formed g/l Difco two 0.79 g/l cells acids, on (0.7 plated medium the complete were of cells 50% mutagenized only until BY4743 Strain TIBCO resistant in of Selection fit curve regression logistic Strain a v3.2.1. Spotfire applying and 96-well measurements compound. in the 150 number of cultures was dilutions cat. volume serial log-phase with assay (OpenBiosystems, medium testing The YPD collections and in strains plates HOP YSC1055) microtiter individual the and and picking HIP YSC1056 by validated the were from profiles HOP and HIP curves Growth using determined was substances test wild-type of potency HOP growth-inhibitory and The HIP – profiling Chemogenomic glucose g/l, 2 protein culture soy g/l, main MgSO 1.6 ml g/l, extract 60 malt 20 with g/l, flasks 2 shake extract (yeast 200-ml medium in cultivated was strain The elucidation and structure purification compound conditions, clusters. Fermentation gene these. metabolite AntiSMASH Non-Redundant secondary annotated. from find NCBI were to the sequences the predicted used predicted and was the in were Swissprot files, both data sequences transcripts with protein Blast protein putative NCBI and Using the sample, identify mRNA to frames reading (http://ccb. cufflinks.cbcb.umd.edu) 2.0.4 open Tophat used all we Finally, jhu.edu/software/tophat/index. genome. identified the within we acids amino addition, In sequences. ltswt ecinvlm f150 of 96-well volume in reaction assayed a were with a recorded dilutions with was serial plates medium Twelve-point rich system. in robotic cultures growing exponentially of ouin otiigDS eenraie o2.IC 2%. to normalized were DMSO containing Solutions ehd tutrladseta aafrcmon a efudi the in found spectroscopic be S2). can Table The 1 material compound (supplementary experiments. for materials supplementary data and 2D-NMR spectral normal mass and and structural by by methods extract 1D- determined isolated yeast was and were g/l, structure Compounds spectroscopy 1 The g/l). (agar chromatography. 15.6 preculture phase extract reversed 6-day malt a of g/l, 4 ml 1.5 with inoculation siaino eevraiiyfrom the variability as gene of computed estimation was compound combination. each Sensitivity concentration for score and (MADL) 2014). logarithmic deviation al., absolute TIBCO median et by (Hoepfner generated previously fits Inc.). curve Software (TIBCO regression v3.2.1 logistic Spotfire using calculated omlzdt % uvswr acltdb aigte1 OD h 11 the taking by calculated were Curves 2%. to normalized epnecre nYDmdu ihsra iuin fcmons1or 1 compounds of dose– dilutions recording serial 200 at with and 2 medium cells YPD in BY4743 curves fresh response into mutated mutations to corresponding due Resistance optda ecie ndti npeiul Hefe ta. 2014). al., et (Hoepfner previously in detail in described as computed sn h eepeitosfo uuts epeitdprotein predicted we Augustus, from predictions gene the Using I,HPadmcora nlsswr efre sdescribed as performed were analysis microarray and HOP HIP, m opud1 fe as 5rssatclne eeisolated. were colonies resistant 45 days, 4 After 1. compound M m aiu ocnrto n 1sra iuin.DS was DMSO dilutions. serial 11 and concentration maximum M .cerevisiae S. 4 D /,KH g/l, 2 Hefe ta. 02 a nuae ih25 EMS 2.5% with incubated was 2012) al., et (Hoepfner 8 HO/YDL228C Y73 h pia est t60n (OD nm 600 at density optical The BY4743. 2 .cerevisiae S. PO 4 m /)a 28 at g/l) 2 /el tr OD start l/well, SEC61 z hm)adCflns202(http:// 2.0.2 Cufflinks and shtml) soe r ae narbs parametric robust a on based are -scores . a sda h idtp reference. wild-type the as used was 00dfeetpoiig n were and profilings different 3000 a ofre ycoigthe cloning by confirmed was cells m ˚ n 0 p o asafter days 8 for rpm 200 and C ;tesatOD start the l; 600 a .1 MOwas DMSO 0.01, was 30 600 auswere values a 0.05. was . 6 500 600 10 600 7 ) 04.Alwr lndit R46wt P rmtr es cells Yeast promoter. GPD a 150 were with al., al. proteins et pRS426 et (Goder substrate into al. Junne et expressing cloned Goder by by were derivatives described All and were H1* 2004). CPY and tags 2007), and al., epitope et HA (CPY), (Junne triple Y C-terminal carboxypeptidase with (DPAPB), B aminopeptidase SEC61 yrtsigteclsfrgot nadlto eisfo n et to tenth one from series IC dilution a the in times growth hundred for cells one confirmed the days. was Resistance retesting 3–4 weeks. every by 2–4 changed within was appeared medium colonies and Resistant confluent, 50–80% were they gln ueeetX atr lgncetdsfloigteNuGen the and following blockers oligonucleotides NuGen capture then of were XT combination Libraries SureSelect a kit. using Agilent library Fluorometric were captured ultralow and libraries DR DNA Qubit multiplexed NuGen The sequencing the bp. Illumina-compatible by 300 using of prepared and length quantified using average end-repaired, an fragmented to was was were ultrasonicator DNA E210 DNA/ ng Covaris and a 100 Prep ALL Technologies). 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Journal of Cell Science n ailbldwt [ with radiolabeled and c.Tasoainwste ntae yicbto o i t37 at min 5 for on incubation s by 30 for initiated DMSO then or 2 was or Translocation 1 compounds ice. of concentrations different with yaei ufrcnann 0m ee-aHp .,5 MKland reticulocyte KCl mM the 50 from 7.5, MgCl pH 1:50 mM Hepes-NaOH diluted mM 5 50 pOA-DHFR containing buffer and in ATP lysate mM 5 SecA, and (Bauer previously described 2009). as Rapoport, (Promega) lysate reticulocyte rabbit oano eY(eius6–4 a eee eeprfe.The purified. and were proOmpA of containing deleted acids proteolipsomes was into coli 60–74) reconstituted E. were (residues complexes SecY SecYEG of domain Bacterial 1997). Spiess, and as (Wahlberg previously described labeled as were in and H1Leu25 added and were transfected, Inhibitors 2003). ( grown, Spiess, DMSO and labeling were (Goder metabolic described cells for previously acid COS-1 5-fluororotic using experiments. eliminated was plasmid his3-11,-15 ARTICLE RESEARCH 1228 A. T. Rapoport, and W. B. Bauer, J. Ravel, and O. B. Bachmann, Reference at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.165746/-/DC1 online available material Supplementary months. material 6 after Supplementary the release of for fellowship PMC Ph.D. in a Deposited Medical by Fonds. Hughes supported Ingelheim Howard was Boehringer a of Bauer is Institutes T.A.R. B. National T.A.R. Investigator. the to Institute from GM052586] [grant grant number Foundation a [grant Science and Health National M.S.]; Swiss to the 31003A-125423 by number supported was work This Funding and experiments, analyzed and and manuscript. designed the performed M.S. J.W. wrote and and T.J. D.H. experiments. experiments. M.B., analyzed analyzed experiments. performed S.S. designed C.S. J.T. G.R., and and L.O., R.R., T.A.R. F.P., R.B., E.O., N.H., N.M., Bhullar, P.K., B. B.K., experiments. D.E., J.E., Bauer, B. T.A., contributions interests. Author of financial employee or an competing is no R.B. declare Novartis. authors of other employees The R.R., are L.O., Congenomics. D.H. E.O., N.M., and P.K., J.T. F.P., B.K., N.H., S.S., D.E., G.R., J.E., Bhullar, B. M.B., T.A., C.S., J.W., interests Competing compounds the study. of this team characterization in Unit and used Products isolation, Natural Extended fermentation, Novartis supported the that acknowledge to like would We Acknowledgements His residues C-terminal cysteine all a with and 1–831 residues (residues serine SecA by 2014). replaced al., et (Bauer coli Escherichia vitro In rnlcto fpADF a etdb rtiaeKtreatment K proteinase by autoradiography. tested and SDS-PAGE was by pOA-DHFR followed of Translocation wt l ytiersde elcdb eiersde n ihaN- a with and residues serine by replaced His residues terminal cysteine all (with CEN eee,J,Hrn,H,Wn,S,Oehue,B,Mrurt . Foster, K., Marquardt, B., Oberhauser, S., Wang, H., Harant, J., C. Besemer, R. Glen, and A. Bender, A. T. Rapoport, and Y. Chen, T., Shemesh, W., B. Bauer, fmcoilplktd n orbsmlppieboytei ahasfrom pathways biosynthetic peptide data. nonribosomal sequence and DNA polyketide microbial of eAAPs uigtranslocation. during ATPase SecA .A,Shenr .P,d re,J . ace-ae,C n ide,I J. I. Lindley, and C. Dascher-Nadel, E., J. Vries, de P., E. Schreiner, A., C. secretory informatics. of molecular translocation ATPase. SecA sequence-insensitive the by allows proteins mechanism slide’’ 20805. rtoiooe otiig0.1 containing Proteoliposomes containing ) oa ii xrc.pADF,afso ftefrt15amino 175 first the of fusion a pOA-DHFR, extract. lipid polar # %o h eim uigtelbln eid 1 H1Leu13, H1, period. labeling the during medium) the of 1% nvitro in , rnlcto saswt uiidcmoet from components purified with assays translocation 2 6 trp1-1 h itr a pi noeulvlmsadincubated and volumes equal into split was mixture The . a nSc)a ela eiaiei hc h plug the which in derivative a as well as SecE) in tag SEC61 eepromdesnilya ecie previously described as essentially performed were , rnlcto assay translocation ade2-1 ehd Enzymol. Methods r.Boo.Chem. Biomol. Org. .coli E. }(oe ta. 04,adtewild-type the and 2004), al., et (Goder ]} 35 , ]ehoieby S]methionine 20) oeua iiaiy e ehiu in technique key a similarity: Molecular (2004). can1-100 20) hpe .Mtosfri iioprediction silico in for Methods 8. Chapter (2009). Cell 20) apn oyetd neatoso the of interactions polypeptide Mapping (2009). iyrflt euts,wssynthesized was reductase, dihydrofolate 157 m rc al cd c.USA Sci. Acad. Natl. Proc. eYGwr ie ih0.4 with mixed were SecYEG M 1416-1429. , , 458 sec61::HIS3 2 3204-3218. , 181-217. , nvitro in 6 YPa3 ( [YCPlac33 , a)adSecYEG and tag) 21) ‘uhand ‘‘push A (2014). rnlto with translation 106 20800- , SEC61 URA3 m ˚ C. M e M. He, aat . ete,N,Hfr . brasr . eVis .E n ide,I J. I. Lindley, and E. J. Vries, de B., Oberhauser, L., Hofer, N., Lettner, H., Harant, oaa . ekr . eti,B,Amce .P,Bri-aca C., Barrio-Garcia, P., J. Armache, B., Beatrix, T., Becker, M., Gogala, M. Spiess, and T. Junne, V., Goder, yee,B,Hefe,D,Plsrn,D,Kry .A,Wieed . u R., Yu, L., Whitehead, A., C. Kirby, D., Palestrant, D., Hoepfner, B., Nyfeler, iee,G,Somkr .D,Jns .W,Lag . izlr .A., E. Winzeler, M. H., Spiess, and Liang, V. W., Goder, T. Jones, D., D. Shoemaker, G., Giaever, J. Taunton, and S. R. Hegde, J., E. Kunkel, L., J. Garrison, cen,A,Hna M., Hanna, A., McKenna, R. Durbin, and H. Li, S. Waack, and M. Stanke, M., Kollmar, O., Keller, M. Spiess, and V. Goder, T., Schwede, T., Junne, E., M. Spiess, Fung, and M., L. Kocik, Miranda, T., Junne, S., Suresh, M., I. Wallace, M., A. Smith, S., Hoon, Brachat, I., Filipuzzi, S., Schuierer, H., Sadlish, B., S. Helliwell, D., T., Hoepfner, Aust, R., Riedl, C., Studer, S., C. Lim, W., C. McNamara, D., Hoepfner, a,X,Hye,S . ms .D,Wn,P,Ve,L . as,C .and T. C. Walsh, P., L. Vien, P., Wang, D., B. Ames, W., S. Haynes, X., Gao, R. Mithen, and G. Vancanneyt, J., Botterman, M., Traka, G., Cardon, B., Field, J. T. Silhavy, and S. Hanley-Way, D., C., S. Rabu, Emr, M., Piacenti, P., Roboti, C., A. Callan, C., McKibbin, C., B. Cross, cae,W,Picad . is . hn . lck .adCniga,F. Cunningham, and P. Flicek, Y., Chen, D., Rios, B., Pritchard, W., McLaren, J., E. Kunkel, A., Sharma, L., J. Garrison, L., A. MacKinnon, V., S. Maifeld, J. Taunton, and S. R. Hegde, A., Sharma, O., V. 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Daly, analyzing for S., framework data. Gabriel, MapReduce sequencing a D., DNA Toolkit: Altshuler, Analysis next-generation Genome K., The (2010). Garimella, A., Kernytsky, 1082-1088. alignments. sequence multiple protein employing Bioinformatics method prediction integration. membrane for Cell threshold Biol. hydrophobicity the defines translocon bioactivities. small-molecule reveals al. assays et Biol. W. genomic R. Davis, of C., Zhang, platform M., al. K. Shokat, M., et Proctor, pathways M. targets, Altorfer, reveals Y., eukaryote functions. model Abraham, gene a and of U., dissection Plikat, chemical resolution B., lysyl-tRNA Bhullar, S., falciparum plasmodium al. the cladosporin. et metabolite S. of secondary Schuierer, 11 fungal inhibition S., the by Meister, specific synthetase M., and D. Plouffe, Selective L., S. McCormack, odnainlk domain. Arabidopsis. condensation-like in Y. biosynthesis Tang, acid amino and Physiol. sequence. Glucosinolate signal defective (2004). al. a with et protein R. a of M. export restore Pool, L., endoplasmic S. the at Flitsch, translocation protein reticulum. R., Sec61-mediated inhibits Whitehead, I Eeyarestatin M., C. Wilson, 21) eiigtecneune fgnmcvrat ihteEsmlAPI Ensembl the with variants Predictor. genomic Effect SNP of and consequences the Deriving (2010). modulators. Sec61 promiscuous and selective by J. Taunton, inactivation and S. R. Hegde, the at helices of transmembrane gate. nascent inhibitor traps lateral inhibitor Sec61 cyclodepsipeptide allosteric An a (2014). of target the translocation. reveals cotranslational incorporation leucine translocation. cotranslational 76 preventing by pathway secretory transform. 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Journal of Cell Science ak .adRppr,T A. T. Rapoport, and E. Park, A. T. Rapoport, and E. Park, ARTICLE RESEARCH hmd,K,Flpzi . th,M,Hliel .B,Sue,C,Hefe,D., Hoepfner, C., Studer, B., S. Helliwell, M., Stahl, I., Filipuzzi, K., Shimada, ho .adHge .S. R. Hegde, and S. Shao, L., Chang, B., Bhullar, T., Aust, G., C. Paris, G., Galicia-Vazquez, H., Sadlish, Ro and R. Schekman, M., Pilon, I. Korf, and K. Bradnam, G., Parra, Me E., Park, ihe .L,Topo,K . tdr . rnl,V . ut . id,R., Riedl, T., Aust, C., V. Prindle, C., Studer, V., K. Thompson, L., D. Richie, ebr . owt,R,Mva .R n asr .M. S. Gasser, and R. N. Movva, R., Loewith, A., Seeber, membranes. across translocation protein translocation. protein bacterial during 242. molecules small for reticulum. complex. eIF4A-RNA the on Biol. al. site Chem. et binding R. rocaglamide Riedl, relevant B., Knapp, functionally D., Hoepfner, B., S. Helliwell, genomes. eukaryotic in genes 1067. core annotate translocation. protein W. of C. initiation Akey, and A. T. Rapoport, vlaino oe ctlcaesnhs niiosa niuglagents. antifungal as inhibitors synthase acetolactate Chemother. Agents novel Antimicrob. al. et T. of Zabawa, A., evaluation J. Sexton, for J., Tao, cytosol D., Estoppey, the to reticulum endoplasmic the from degradation. protein secretory misfolded ´ne nu e.Cl e.Biol. Dev. Cell Rev. Annu. 8 te,J . ubr,J . ute .J,L,W,Wyo,A., Whynot, W., Li, J., S. Ludtke, C., J. Gumbart, F., J. ´tret, MOJ. EMBO 1519-1527. , 16 21) ebaepoenisrina h endoplasmic the at insertion protein Membrane (2011). 4540-4548. , ic,K. misch, ¨ 21) ehnsso Sec61/SecY-mediated of Mechanisms (2012). 57 Nature 21) rsrigtemmrn barrier membrane the Preserving (2011). 21) tutr fteSc hne during channel SecY the of Structure (2014). 2272-2280. , 20) EM:appln oaccurately to pipeline a CEGMA: (2007). 27 25-56. , 506 nu e.Biophys. Rev. Annu. 19) e6pmdae xoto a of export mediates Sec61p (1997). 102-106. , Bioinformatics 21) dniiainand Identification (2013). 21) vdnefra for Evidence (2013). Nature 21) TORC2 (2013). 41 21-40. , 23 473 1061- , 239- , ACS ht,T . rn,T,Le .adTyo,J. Taylor, and S. Lee, T., Bruns, J., T. White, ores .M,Ferna A. M., R. M. Voorhees, Marahiel, and M. D. A. H. Flower, Mootz, and T., J. C. Stachelhaus, DeMars, Jr, M., W. Clemons, A., M. Smith, aleg .M n pes M. Spiess, and M. M. T. J. Kapoor, and Wahlberg, O. Elemento, R., B. Houghtaling, A., S. Wacker, etnof . cmd,A,Ci,I,Fret . iei,I,Zmai,D., Zampatis, I., Ridelis, J., Furkert, I., Coin, A., Schmidt, C., Westendorf, efn,J .SisyadT .Wie,p.3532 a ig,C:Academic CA: In Diego, San 315-322. Applications phylogenetics. pp. White), and for J. Inc. T. Methods and press genes Sninsky to J. RNA J. Guide Gelfand, ribosomal A fungal Protocols: of sequencing fboytei fhmnedtei eetrb h cyclodepsipeptide the by receptor B endothelin human of cotransin. biosynthesis of inln aha urnesgnm tblt ntefc fDAstrand DNA of face the in stability genome breaks. guarantees pathway signaling tutr ftemmainrbsm-e6 ope o34A 3.4 to complex ribosome-Sec61 mammalian the of Structure synthetases. peptide nonribosomal in domains Biol. complex. adenylation Chem. SecY of coli code Escherichia conferring the on mutations prl Bacteriol. of J. effects the Modeling retto fsga-nhrpoen:tetpgncrl ftehydrophobic the of role topogenic the proteins: domain. and signal signal-anchor action of drug orientation of mechanisms identify to resistance. sequencing transcriptome Using 157 uz . ise,B,Rsnhl . eemn,M tal. et M. Beyermann, W., Rosenthal, B., Wiesner, C., Rutz, 1632-1643. , ora fCl cec 21)18 2712 doi:10.1242/jcs.165746 1217–1229 128, (2015) Science Cell of Journal o.Cell Mol. .Bo.Chem. Biol. J. a.Ce.Biol. Chem. Nat. 6 187 493-505. , .Cl Biol. Cell J. 6454-6465. , 51 829-839. , ne,I . cee,S .adHge .S. R. Hegde, and H. S. Scheres, S., I. ´ndez, 286 137 8 35588-35600. , 235-237. , 555-562. , 19) utpedtriat ietthe direct determinants Multiple (1997). 19) mlfcto n direct and Amplification (1990). e.M .Ins .H. D. Innis, A. M. (ed. 19) h specificity- The (1999). ˚ 21) Inhibition (2011). resolution. (2014). (2005). (2012). 1229 PCR Cell

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