Posted on Authorea 15 May 2020 | CC-BY 4.0 | https://doi.org/10.22541/au.158955456.63626060 | This a preprint and has not been peer reviewed. Data may be preliminary. Ts ult oto,adtacigo ertdpoen Scs Fii at,Uln ile,2017; Nielsen, & Uhlen, folding, Gatto, synthesis, (Feizi, the (SecPs) with proteins tasked secreted the products of with al., gene trafficking associated et >575 and (Gutierrez complexities includes control, production the machinery quality protein However, pathway production recombinant PTMs, dominant secretory in 2008). Murphy, the The bottleneck Wurm, (Jenkins, are function a & 2020). and cells remains safety Baldi, Mammalian machinery drug Hacker, for secretory required 2024. Matasci, mammalian are by that 2008; (USD) PTMs Tyther, perform billion therapeutics to & $36 ability protein their by plasma to grow the due autoimmu- system to example, cancers, For expected including disorders. is diseases, genetic diverse market and treating diseases, for infectious important nity/inflammation, increasingly are proteins Therapeutic Introduction during pathway secretory the of mechanisms its molecular on key relies synthesis of which understanding supporting SERPINC1, production. our interactions of down protein refined transient secretion Knock recombinant and the decreased stability. proteins the identified ER recombinant to successfully the secreted led labeling maintaining change, of Proximity-dependent association fold and strongest highest folding bonds. the the the proper disulfide showed with with in extensive interactor enzymes correlated roles measured and folding critical positively a oxidative model, their were ERP44, others, statistical supporting of N-glycosylation Among Bayesian formation, and a proteins. bond formation using model disulfide proteins four bond with secreted the disulfide of the (BioID) folding, identification features of protein biotin corresponding features in proximity-dependent specific involved the with proteins using SERPINC1, associated motifs found (SERPINA1, PPIs structural proteins identified secretion and recombinant We protein different PTMs recombinant various method. four supporting with of machinery SeAP) networks and the and (PPI) assembly, identify interaction SERPING1 Each folding, to protein-protein proper secrete. the aimed to for we measuring difficult machinery by Here are different they requires (PTMs). since and modifications patients properties to post-translational physicochemical inaccessible unique remain has drugs protein protein recombinant many potential, therapeutic their Despite Abstract 2020 15, May 6 5 4 3 2 1 Campos Rosa Alexandre Schinn Song-Min Samoudi Mojtaba recombinant for enzymes interactions therapeutic protein of detection situ In nvriyo aiona a Diego San California, of University Biosustain DTU T¨ubingen University Karls Eberhard Denmark of Institute University Discovery Technical Medical Prebys Burnham Diego Sanford San California of University 1 1 tfnKol Stefan , hhCugKuo Chih-Chung , 2 n ahnLewis Nathan and , 3 iu Weiss Linus , 1 aes Robinson Caressa , 4 aaPtre Bj Petersen Sara , 6 1 1 mShams-Ud-Doha Km , ø rn 5 Bj , ø nVoldborg rn 2 , 5 , Posted on Authorea 15 May 2020 — CC-BY 4.0 — https://doi.org/10.22541/au.158955456.63626060 — This a preprint and has not been peer reviewed. Data may be preliminary. rti) isnasml a mlydt uetesga etd fSRIC eet h -emnlof N-terminal the to gene SERPINC1 bait of any software peptide (without SnapGene signal BioID2 were the by secretable which fuse obtain overhangs, designed to To overlapping were employed the (NEB). long was Mix primers simplify with assembly Master vectors assembly Gibson to Assembly and protein), Gibson Gibson C-terminal fragments model using corresponding at assembled genome. integration each the then tag targeted host amplify of allows 3XFLAG to the C-terminal which used a into (Invitrogen), and the pcDNA5/FRT and transgenes to into between) the expression inserted BioID2 added of The were fusing ORFs assembly. linker isothermal by detection. glycine-serine Gibson immuno constructed and a were PCR (with by bait-BirA, protein constructed named were study hereafter this ORFs, lines in used cell plasmids stable All of generation and cloning Molecular Methods and manner. specific recombinant Materials will client the a PPIs of in these expression engineering of the pathway Identification during secretory identified for functions we feature. pathway targets protein model, novel secretory each introduce statistical the and with Bayesian how acid proteins correlated of a amino positively understanding Using different vary our are have proteins motifs. refine and that These modifications) local PPIs (ALPP). residue different critical Type and the form Placental bond consequently Phosphatase, disulfide that Alkaline glycosylation, sequences phosphatase of (e.g., alkaline PTMs form embryonic their secreted truncated and in a treatment angioedema) is hereditary SER- (SERPINA1: which of and attacks proteins deficiency, (SeAP), acute antithrombin SERPIN-family for Hereditary to three treatment BioID2 for 2016), Specifically, with treatment PING1: MacNeill, interact 1). SERPINC1: Varnait˙e & deficiency, that (Fig. 2016; Alpha-1-antitrypsin proteins SecMs al., for therapeutic pore identify et secreted we nuclear to different (Kim Here 2016), employed for BioID . vary al., was for involved 2019) et Nicchitta, SecMs ligase & the (Dong biotin Zheng, how and adhesions smaller Chen, explore centrosomes improved focal (Hoffman, human an ribosomes 2015), at membrane-bound BioID2, al., ER interactions radius used and et protein labeling 2014) Gupta mapped al., nanometer-scale 2015; et has in (Kim approach Stearns, interactors promis- this & a proximal example, (Firat-Karalar to For the fused cilia biotinylate 2014). interest subcellular interac- can al., of and protein et that stable networks a (Kim PPI (BirA) with expressing characterize ligase on along PPIs to biotin relies intracellular used cells, BioID cuous of been living 2016). detection has in MacNeill, systematic and (Varnait˙e APEX & interactions compartments for organization and cellular transient approach 2012) various Burke, high-throughput and in & a weak Raida, occurring offers identify Kim, BioID proximity Roux, can Furthermore, New 2014; 2013) al., tions. processes. et al., biological (Kim of manner. et BioID basis client-specific as (Rhee molecular a such the in methods deciphering kinds labeling for dependent different efforts tools expression of invaluable biology the given are proteins synthetic support networks any expressing mammalian that PPI guide of support SecMs can capable regulated to the This cells elucidate is needed enhanced features. to SecMs SecMs engineer specific need several the to with a Unfortunately, proteins of is pro- recombinant there expression pathway. secreted different Thus, that secretory of protein of unknown. diversity suggesting the human remain the showed 2017), in protein support also SecPs secreted al., to client the study et pattern (Feizi facilitate support previous tissue-specific to modifications to A a adapted their in been proteins. and diversity, expressed not recombinant teins this are has most handle genes cells for pathway to producing desired secretory machinery modifica- protein secretion secretion recombinant and titer customized of structures high machinery a the Dickson, secretory in necessitate & variability the could importantly, Maldonado-Agurto, but proteins (Hussain, More recombinant quality adap- 2011). of and in Robinson, quantity tions resulting & protein homeostasis, Yuraszeck, both and Young, functionality impair in 2014; its can proteins impact that heterologous could effi- of responses system and Overexpression tive glyco- complex pathway. precision and and regulated machinery secretory isomerases The tightly secretory disulfide the this these protein 2002). within of (e.g., transporters Simpson, effort enzymes and & coordinated modifying syltransferases), the Reynaud chaperones, from 1981; including results (SecMs) pathway Schekman, secretory components & mammalian Ferro, the Novick, of ciency 2017; al., et Lund 2 Posted on Authorea 15 May 2020 — CC-BY 4.0 — https://doi.org/10.22541/au.158955456.63626060 — This a preprint and has not been peer reviewed. Data may be preliminary. h c n meitl ujce oEIA lglvl eeqatfidfo ertdbi-iAfusion bait-BirA secreted from quantified were levels Flag -80 ELISA. of out to taken subjected were immediately cultures transfected and esiRNA ice from the supernatants clarified of Aliquots For used. 3% was ratio. by Substrate 1:2000 ELISA blocked at ECL Western was buffer Clarity blocking membrane same in The the diluted streptavidin bands, earlier. 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Western and were algorithm metrics Coloc ball threshold Above using rolling background. determined Fiji’s Manders, for were using 2004; adjust Thresholds (Li, subtracted 0.5) to (ROI). was channels to used Coefficients intensity interest -0.5 two Colocalization pixel of range: of Manders’ Background region (IQC, Quotient 1.0), a colocalization 1993). Correlation to Intensity Aten, intensity Li’s -1.0 & and pixel 2015). (range: 1.0), Verbeek, evaluating to Eliceiri, Coefficient 0 Pearson’s for & range: as Hiner, report (MCC, such Rueden, comprehensive methods (Schindelin, Coloc various a channels 1.52p) by (Streptavidin) generates (ImageJ 594 Fiji’s tool and Colocalization using This (anti-flag) Deconvolution. images at antifade 650 deconvolved Lightning minutes using the with the 30 slide between Confocal for for the SP8 on cells performed mounted Leica fixed was DAPI, using with quantification with imaged incubated counterstained and and washed, mountant, respectively then vectashield buffer, were goat blocking Cells in normal were temperature. 1:1000 5% proteins, the room biotinylated and and min targeting the 1:300 BSA 10 (Thermofisher), targeting at for 1% conjugate (Thermofisher), PFA diluted with 650 conjugate 4% cells antibody-Dylight 594 containing streptavidin-DyLight fixed monoclonal PBS and incubating in mouse bait-BirA, by fixed Anti-flag then PBST. performed with were was in supplemented Cells Blocking serum medium confluent. complete temperature. 70% in room until grown coverslips at on were biotin fusions uM BioID2 50 expressing cells HEK293 Recombinant expression for screened Immunofluorescence and up scaling of 10% Blotting. further Western density containing for by plates a DMEM proteins 24-well fusion at in in the seeded plates grown and of were well pooled, isolated, cells 6 were Lipofectamine 150 in transfection, colonies and using seeded from PenStrep, plasmid recovery were 1% FBS, pOG44 After cells 293 and directions. 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Caae lxneso,&Lvne,21) esu otae(ynv o,21)wsepoe for employed was Normalyzer 2018) into Cox, integrated data & (Tyanova and 2005) of software processing (Smyth, ratio Perseus for sample the method allowed 2014). as of LOESS was Levander, calculated steps the cleavages & various were using Alexandersson, missed during purifications normalized (Chawade, two affinity introduced were identification streptavidin biases of dataset protein in systematic maximum and generation, proteins the a spectrum of remove and for Enrichment To filter mode intensity. modification, 1%. (FDR) specific fixed to rate set discovery a a was false as in modifications. target-decoy-based searched variable trypsin The as was searched charge to searching. were cysteines ion set N-terminal precursor of protein was maximum of Carbamidomethylation acetylation The Enzyme and Da. 7. methionines 0.5 of was search, oxidation tolerance main searching while to the mass for for set a and was with used completed was searched tolerance state recalibration MS/MS were mass mass 2018) ions initial Precursor where January 1.5.5.1. Product search contaminants). (version respectively. version first the database protein 2016) for sequence known 4.5ppm al., protein (commonly and 20ppm Uniprot et sequences (Tyanova sapiens cRAP software Homo GPM the MaxQuant and against with searched analyzed were were spectra 20 spectra of mass duration All a with enabled Analysis was triggered, data exclusion were MS Dynamic cycle duty window 25. per isolation of an spectra 5e4, energy MS2 of in 12 collision target to AGC operated normalized an Up a was and m/z. sec. 17,500 and spectrometer of 1700 AGC m/z mass resolution to an 1.6 a The 350 70,000, with of of from acquired resolution 300nL/min. range and a HCD, mass of by with a fragmented rate measured and were flow 1e6 spectra a of MS1 target at 2 mode. B using acquisition mm, solvent separated data-dependent 500 were positive 2-28% x Peptides of 0.075 Scientific). Scientific) 0.1% column gradient Fisher Fisher PepMap acetonitrile, linear (Thermo (Thermo Acclaim system spectrometer 2% C18 SpeedVac nanoLC mass with analytical a EASY Plus an Proxeon in reconstituted Q-Exactive a removed were a using was to LC-MS/MS peptides solvent coupled by organic Dried analyzed the and analysis. and acid, LC-MS/MS formic cartridges to C18 70 AssayMap prior at using cartridges concentrator WI) platform Trypsin/Lys- streptavidin Madison, Bravo grade AssayMap spec the (Promega, by mass using in with enzyme quantified (Agilent) digestion digestion and on-cartridge platform to Rapid debris biotinylated AssayMap subjected C were of cellular Bravo proteins purification remove a bound bicarbonate Affinity the to in ammonium and (Agilent), recommendations. g 50mM out manufacturer urea, x carried per 8M was 14,000 70% as in proteins at 50 Scientific) approximately Hz) centrifuged (Thermo with (20 to were assay shaking triplicate) h proteins BCA vigorous in 24 extracted with replicate for lysed buffer, biological incubated PBS, lysis per cold then plate in and twice (one media washed plates complete mm in 245 confluence in grown were Cells experiments. further Spectrometry for harvested, were -80degC Mass supernatants at culture stored h, 72 and and aliquoted Tar- 48 then for guidelines. centrifugation, occur manufacturer’s low-speed to the Lipofectamine allowed by was using to clarified control esiRNA according positive by as knockdown performed esiRNA gene were KIF11 geted transfections or control All negative (Invitrogen). a at as RNAiMAX seeded esiRNA were EGFP SERPINC1-BirA or expressing esiRNA cific cell HEK293 Sigma. from 0.6X10 ordered was ERP44 targeting esiRNA experiment each knockdown below). of (see RNAi control control negative negative the as with PDIA’s comparison esiRNA of in EGFP effect measured with was The transfected proteins triplicate. All line model in Chemicals). cell the instructions (Cayman of manufacture Kit secretion the ELISA on to Detection knockdown Flag-Tag according the performed using were ELISA measurements anti-flag competitive by proteins 5 el/eli 4wl ltswt opeemdu n ees rnfce ih7 go R4 spe- ERP44 of ng 72 with transfected reverse and medium complete with plates 24-well in cells/well 4 º o h ietdppie eete desalted then were peptides Digested 2h. for C μ atce Tem cetfi)i 93-min a in Scientific) (Thermo particles m μ itn el eehretdand harvested were Cells biotin. M Posted on Authorea 15 May 2020 — CC-BY 4.0 — https://doi.org/10.22541/au.158955456.63626060 — This a preprint and has not been peer reviewed. Data may be preliminary. n nitrco.T ute con o ePadfauepoiciy eipeeta siaeo the of estimate ? an frequency interaction implement the we promiscuity, modeled We feature interact. and to SecP g for and account f further for To tendency interactor. an ? and frequency, interaction effective This f number The (f p SecP a effective in the occurs feature calculate this we times features, of number protein the with counting protein and interacting data each (? of frequency total associations such quantify To framework modeling Bayesian 2014)) al., 2017). et (Liberzon al., (Croft (mSigDB et Reactome pathways 2017), (Thul L Morishima, biological L localizations & curated & Sato, subcellular & Tanabe, into Furumichi, (Blatch and (Blatch (Kanehisa, aggregated motifs KEGG phosphosite be 2011), structural al., and also et shared can Uniprot pathway interactors include secretory from pathway the properties PTM secretory of common of components The between These sites according SecM. interdependencies known pooled predict reveal given products. be to can a can their SecPs we and interactors the pathway and SecPs interactors, of secretory the their properties the between and shared and interactions to SecPs samples the BirA-fused the in the between between implicated interactions interactions features proteins the aggregated structural Given we important interactors, the their features. and several SecPs on individual between based synergy interactions the interaction generalize further of To estimation and to interactions robust of are Aggregation cutoffs optimal the set suggesting cutoffs components, the changes, pathway around chosen. fold secretory peaked negative standards known also with gold interactors. of sets interactors significant the set gene the all gene pathway-related removed among the secretory components and pathway independent through 0.1 two secretory at known for FDR enrichment of for enrichment as The the components cutoffs pathway optimizes the secretory this set known as We the using standard. determined gold was a interactions significant for threshold The interactions significant of Detection differentially identify proteins. to t-test intense employed Student’s lower data. was A 0.05 to transformed of biased log2 distribution. FDR are using shifted an proteins dataset for from (Zhang expressed the correction package draws in permutation-based random DEP values multi-sample a using missing The with whether performed explore abundance. was to protein imputation used differential Left-censored was of 2018) computation al., and et filtering, preparation, data p saddt h oa rqec,? frequency, total the to added is f,g fitrcin ewe ahfauegn ar(,)b on hog vr ePi our in SecP every through going by (f,g) pair feature-gene each between interactions of ) f,g ae scoe oetmtn neato ffiiybtenafeature a between affinity interaction estimating to closer us takes , f,g nywe n neat(hn? (when interact g and p when only , 5 sl,19;Hrbc ta. 05.The 2015). al., et Hornbeck 1999; ¨ assle, p f,g . ) ihaPisndistribution. Poisson a with interact sl,1999), ¨ assle, pg 1). = (p,g) Posted on Authorea 15 May 2020 — CC-BY 4.0 — https://doi.org/10.22541/au.158955456.63626060 — This a preprint and has not been peer reviewed. Data may be preliminary. rbblt fafeature a of probability SecM) esrstetnec o secM a synergy for tendency the measures SecMs of (number promiscuity feature of sum the by parameterized is distribution g Poisson the for mean The aeinpir r l omlydsrbtdaon ,wt hi aine aaeeie ytehprpriors their hyper promiscuities, the the by parameterized regularize variances approach better their this To with work, 0, previous 2017). around In distributed al., ? chance. normally et random all (Shen by are than intensity priors more Bayesian epistasis interact estimated g and correctly f has which to degree the quantifies C au o05o ero’ au ooedmntae eedn rti tiigptenbetween pattern staining Li’s protein (closer dependent proteins a proximal demonstrates and the one S1), tag to Fig. to value channels). and system R green 1 different Pearson’s and labeling (Table calculated red or BioID WT we the 0.5 the the colocalizations, through to to of value compared the specificity BirA and ICQ quantify biotinylation the by images To increased confirmed interactors the show results 3). from the not the methods) did of (Fig. (see WT labeling conditions metrics while studied colocalization experimental successful bait-BirA, then same demonstrated and partners was the proteins results actual proteins under biotin-labeled The are biotinylated the proteins the of biotinylated proteins. and colocalization whether model proteins proteins test specific the bait-BirA to tagged the of microscopy successfully is BioID2 of co-immunofluorescence cells that multicolor Colocalization bait-BirA suggesting the by while 2b), of proteins, western cells. (Fig. specific profile by pathway WT the of biotinylation biotinylation for profile The secretory within increased observed biotinylation substantial biotin. is the with the change of culture the enter obvious verified absence the change no to they and also added not presence We that is the did biotin expected in when secretion. fusion different line is for BirA cell packaged proteins it each the and bait-BirA for and Thus, blot processed of proteins, are 2a). secretion model they successful (Fig. the where observed blot of We Western localization alters by secretion. reciprocally intracellular evaluated which the integration lines supernatant, in random cell culture some involved by in into network caused response ensure level PPIs protein to unfolded system mRNA active locus the Flp-In in genomic as the the Variations same such using the response transgene. adaptive into cells measured trigger each transgenes HEK293 be can the of in can of expressed rates integration SecMs was transcription targeted bait-BirA supporting comparable for each their methods) this, and and do materials SecP To (see each method. between BioID proteins PPIs the intracellular using secreted if with investigated first colocalized We proteins tag successfully can BioID Results coefficients. the used We distribution. f once oafeature) a to connected ? , g n ? and μ s h neato yeg fgada necp aibe h etr promiscuity feature The variable. intercept an and sf,g synergy interaction the , g f,g s between hc olwa xoeta itiuin h intercept The distribution. exponential an follow which f and f opraei nitrcinwt n eM.Lkws,teitrco promiscuity interactor the Likewise, secMs. any with interaction an in partake to μ rethinking g neatrpoiciy(ubro eP ihafeature a with SecPs of (number promiscuity interactor , f uniyn h eret which to degree the quantifying , g oitrc ihayfaue.Tececeto neet h interaction the interest, of coefficient The features. any with interact to akg MErah 00 ocntuttemdladsml the sample and model the construct to 2020) (McElreath, package R 6 f and a g sprmtrzdb tnadnormal standard a by parameterized is neatmr hnb admchance, random by than more interact f neatn iha with interacting μ φ unie the quantifies μ γ Posted on Authorea 15 May 2020 — CC-BY 4.0 — https://doi.org/10.22541/au.158955456.63626060 — This a preprint and has not been peer reviewed. Data may be preliminary. h orsodn ertr ahnr sn aeinmdln rmwr semtos.Aogthe Among methods). (see with framework associations modeling their Bayesian analyzed a and proteins using model machinery all secretory aggre- across we corresponding pathway, properties secretory structural establish the To the and pref- with synthesis. PTM would interactions PTM various structural PTM product-specific the gated around specific in for affinity connection a responsible higher differ comprehensive requiring components have more proteins machinery proteins features a secretory that shared bait-BirA the hypothesize with with the we proteins in interact specifically, bait-BirA As erentially upregulated More if se- consistently wondered interactors. the 2017). we are specific across 6), PDIs al., for PTMs example, (Fig. et For PTMs certain (Feizi Prodromou, and linked enzymes. proteins & composition has responsible Pearl disulfide-rich their analysis 1997; secreting of al., co-expression tissues et expression Incidentally, Ikawa (Boni- the 1994; proteins 2019). to Scheller, secreted cretome al., & of et Peterson, transportation Watanabe Bennett, and Calakos, 2006; modification 2004; folding, Glick, the model & mediate facino pathway of secretory in features PPIs structural and post-translational proteins interactions reflect private these interactors if Private remained protein. question interactions, model shared the each the Thus, when of from promiscuous substantially. properties for Apart highly differ unique enriched 2010). are to protein significantly (Mayer, chaperones correspond model flexibility are molecular inherent each products Indeed, their for in gene to PPIs detected 5b). SERPIN due PPIs all folding (Fig. Probable protein among folding assisting shared protein proteins. model hits in co-secreted all involved and between for proteins (n=19) hits crosstalk proteins 300 the top model than pathway the secretory all products 5a among the between their enriched interactions (Fig. frequent more and samples more are machinery suggesting protein components proteins, model co-secreted machinery machinery, other all pathway secretory than 3 secretory across The proteins from the PPIs S3). compiled for probable enrichment standards Fig. among significant and enrichment gold proteins a pathway-related greater saw co-secreted secretory We and or on methods). secretory-resident 3-fold based (see interactions) having sets significant gene interactors all independent as for PPIs 5b probable Fig. p-value identified adjusted we an protein and secreted each pro- For co-secreted and components pathway BirA secretory system for teins biotinylation for substrate the enriched potential show are to a Interactors evidence is as protein considered is bait cell. and the the enzyme within because the properly (Fig. of expected working (LFC) vicinity differentially is is change closest top log-fold observation the the highest in the This Among the located showed using biotinylation. proteins TableS1). endogenous bait Thus, and of the 4 result samples, each bait-BirA a believed. in in likely detected previously proteins interactions were out biotinylated that filtered than we sample background, pervasive samples, bait-BirA as protein sample more model WT model the other be general from detected in the may partners the detected interaction which from biotinylation proteins to isolated interacting fide endogenous proteins extent with bona problematic biotinylated overlap the suggesting is the with considerable Although quantified, which showed along systematically carboxylases sample 2015). endogenously as been WT al., such biotinylated not et implemented cofactor has are biotinylation a (Tytgat When that endogenous as detection 2018). proteins biotin protein Cox, bind identified streptavidin-based & that we with (Tyanova proteins line, Perseus include samples using cell These in WT control interactors. proteins the then WT were biotinylated to a proteins the compared biotinylated in were Differentially and sample proteins each LC-MS/MS, 2016). Purified Cox, in to & streptavidin. identified Temu, subjected (Tyanova, using MaxQuant were protein. using purified bait peptides mapped were each were and proteins with interactions biotinylated trypsin, the and by identify lysed, digested to were aimed we cells proteins this, proximal For the of tagging successful landscape After biotinylation endogenous revealed cells WT < .,i oe rtiscmae oW oto Fg 2fraltrsod and thresholds all for S2 (Fig. control WT to compared proteins model in 0.1, 7 Posted on Authorea 15 May 2020 — CC-BY 4.0 — https://doi.org/10.22541/au.158955456.63626060 — This a preprint and has not been peer reviewed. Data may be preliminary. h dnicto fteeoioeuts nye ihihsteiprac fE eo oesai in proteins. homeostasis misfolded redox of ER consequences of the from importance its cells the 2017). alters protecting Sevier, highlights which and & enzymes formation oxidation Wang, oxidoreductase Pareja, bond cysteine by (Siegenthaler, disulfide these HSAP5 folding conditions of reverse protein folding identification oxidative can suboptimal with The with SIL1 cope and catabolism to P4HB ER- 2013), peroxide activity reoxidizes were chaperone hydrogen (Zito, efficiently SIL1 couples peroxide ERO1LB and PRDX4 hydrogen is PRDX4 formation. reducing 2001), ERO1LB, secretory bond addition, al., disulfide when In with et particularly associated (Mezghrani bond. formation, were disulfide bond that suggests dominant enzymes disulfide ERp44 with of localized in proteins association retention strong the the thiol-mediated with Hence, loaded the 2003). of al., bond importance et disulfide (Anelli the disulfides oxidoreductase the mixed the reversible of of of localization secretion formation ER the in mediates role ERp44 Lamb, (LFC SERPING1. Ero1 & important association Watowich, and strongest SERPINC1 (Ng, their i.e. the lumen showed proteins highlighting ER enriched ERp44 the 2000), PDI, in proteins of control The Haslam, co-chaperone The quality proteins. a & secretion. and enriched DNAJB11, folding protein Haslam, and protein HSAP5 of Yu, in chaperones role efficiency 1992; ER key the a major limit play with that interact can HSAP5, also enzymes that folding bonds oxidative stability breakage disulfide the maintaining more formation, of with and importance the the folding highlights catalyze M protein PDIs (Kozlov, enzymes various in domains of These thioredoxin-like identification the The 7a). through 2010). with Gehring, bonds interacted disulfide (Fig. significantly of PDIA6 bonds rearrangement and disulfide and PDIA4 PDIA3, more P4HB, containing including family model-BirAs secretion PDI the protein of in members Several rate-limiting is glycans. N-linked formation with and bond suggest proteins findings chaperones Disulfide secreting these molecular and glycoproteins synthesizing these misfolded for of the critical involved most are to of glycosyltransferase pathways that degradation Man8GlcNAc2 control Given a mediated from quality ERAD is the trimming 2014). in mannose al., 7a), involved catalyzing are et by enzymes (Ninagawa (Fig. glycoproteins N-glycans another proteins of in EDEM3, containing degradation Man7GlcNAc2 2014). the mediated N-glycan Kaufman, of ERAD & the component in Kodali, with a (Ferris, (ERAD) is associated HSPA5 degradation and interactor substitutions, ER-associated bait-BirA. acid for glycan-containing amino with them (Fig. N-linked HSPA5 glycoproteins targets with recognizes as which such interact activity, system (GQC) chaperone to and quality-control with glycoprotein found 2014) Proteins et (Sakono also Ito, 2015). structure Calnexin/Calreticulin-PDIA3 were Molinari, & polypeptide & Takeda, 7a), native Hebert, control. the Seko, Pisoni, attain quality Tannous, (Sakono, to 2014; and polypeptides bonds al., peptidyl-prolyl nascent folding of of isomerization glycoprotein promotes folding in ERp29 oxidative baites role the N-glycosylated or with their promote of refolding association and complexes suggesting showed and PDIA3 part family, also recognizes ER 7a) PDI misfolded calreticulin/calnexin, the the which with of to the (Fig. complex members UGGT1, recycling Two a near for with form 2013). CALR which N-glycans Kaufman, interacted by ERp29, & single Arvan, also recognized Molinari, then reglucosylates They Jaber, (Ferris, are and degradation proteins 1995). Reglucosylated defects Clark, protein. folding glycoproteins the & minor a of McCormick, (CALR), control with calreticulin quality (Nauseef, glycoproteins with and ER assembly, interacted oligomeric baits the folding, glycosylated in promotes heavily the more that example, for For chaperone processes calcium-binding 7a). related and (Fig. cycle proteins Calnexin/Calreticulin the glycosylated path- in interactions control significant quality detected We with associated are glycosylation ways increased corresponding with addition, the Proteins bond with disulfide proteins glycosylation, of with secretion associated help affinity interactions to higher folding. detected known demonstrated protein the are and analyzed glycans that we N-linked Thus, 7a) and PTMs. bonds (fig. disulfide interactions with specific towards proteins bait-BirA PTMs, studied α a xdrdci htroiie 4Bt nbeadtoa onso iufiefrain hog the through formation) disulfide of rounds additional enable to P4HB reoxidizes that oxidoreductin (an 8 > ,Fg 4 ihdsld bond disulfide with S4) Fig. 8, ¨ a ¨ att nn hms & Thomas, ¨ anen, Posted on Authorea 15 May 2020 — CC-BY 4.0 — https://doi.org/10.22541/au.158955456.63626060 — This a preprint and has not been peer reviewed. Data may be preliminary. rtis eas yohszdta eM rfrnilyitrcigwt atBr rtista carry SecMs that linking evidence proteins While bait-BirA proteins. those with of model secretion interacting the the of preferentially for features essential SecMs the be may and and that features SecMs responses therefore hypothesized structural the adaptive and certain between stability, also in ER associations involved restoring we the for actively Given crucial proteins, enrichment are are proteins. the PDIs which recombinant to bonds showed the response disulfide results yielding dominant in these with level evidence proteins Together, expression clear of family are secretion sites. 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MacNeill, to & (Varnait˙e used systems been has BioID stabilize help Discussion can it that suggests St-turn with predominant NUCB1 a the stabilize with of which protein interaction of features of enriched N-termini capping more folding the the helix with Thus, at as proteins 1998). frequently regarded our Rosee, occur are aggregation with turns and protein strongly ST 2008) prevents more Thornton, that interacted to 7b). Henrick, protein 2017), 1992). peptide (Fig. & Rickert, binding Graham, the & ST-turns amyloid (Golovin & of Unno, chaperone-like Sakmar, motif ability Shannon, The a Barbash, Imperiali, the asx family. (Bonito-Oliva, 1992; NUCB1, on the Rickert, HSP70 example, depending & the in another Shannon, sites, of depleted (Imperiali, As Asn-X-Thr/Ser chaperone motif proteins of molecular Asx-turn a occupancy an model adopt DNAJB1, N-glycan example, with impacts For interact motif to features asx structural 7b). tendency SecP higher between showed (Fig. associations 2008) found SecMs proteins framework modeling the bait Bayesian and the on PTMs, motifs to addition structural In with post-transfection, associated three are bond and disulfide PPIs two the Identified day of at secretion All SERPINC1 on ELISA. of retention by secretion Thiol-mediated the the SERPINC1-BirA in of protein. cells of enriched reduction SERPINC1-BirA importance secretion in 41% the knockdown the and supporting ERP44 respectively, 44% showed measuring results the for The to post-transfection duplicate. led experiments in esiRNA h repeated of were 72 supernatants quantifications The and respectively. ERP44, controls, 48 against negative/positive esiRNA collected as with Habermann, Franke, transfected KIF11 were Heninger, and SERPINC1-BirA (Kittler, EGFP expressing using knockdown with cells SERPINC1-BirA, gene HEK293 expressing productivity target cells 2005). on to the Buchholz, change, in esiRNAs, & ERP44 fold approach, down highest RNAi knocked the we orthogonal showed bonds an which disulfide interaction, more with ERp44 proteins the of of importance the validate To α- eia eodr structure. secondary helical 9 α- eie Di,Saly aatu,& Macarthur, Stapley, (Doig, helices α- eie npoen Arr & (Aurora proteins in helices nsitu, in and Posted on Authorea 15 May 2020 — CC-BY 4.0 — https://doi.org/10.22541/au.158955456.63626060 — This a preprint and has not been peer reviewed. Data may be preliminary. oioOia . abs,S,Ska,T . rhm .V 21) uloidn1bnst multiple to binds 1 Nucleobindin (2017). V. W. fibrillization. Graham, inhibits and & P., amyloid pre-fibrillar T. of Sakmar, Fusion. types and S., Budding Barbash, Vesicle A., of Bonito-Oliva, Mechanisms The (2004). S. B. https://doi.org/10.1016/s0092-8674(03)01079-1 Glick, 153–166. & S., J. Bonifacino, capping. Helix 939. (1998). interaction. L D. Thiol- & L., G. G. (2003). Blatch, R. Rosee, Sitia, & . . https://doi.org/10.1002/pro.5560070103 . R., ERp44. S., of Camerini, Aurora, role G., the Bertoli, reticulum: L., endoplasmic Bergamelli, the 5015–5022. in A., retention Bachi, protein M., mediated Alessio, T., Anelli, References interests. competing no declare authors The assays. pilot Interest on of support for Conflict Wulff Tune Center NIGMS thank the to from to like and provided also BGV) Foundation would (NNF10CC1016517, Nordisk authors Denmark Novo The of NEL). the University GM119850, from Technical (R35 funding the at generous Biosustainability by for supported was work This manner. specific client a Acknowledgments in proteins and diverse of synthesize be quality can that and hosts titer interactions production such the of biotherapeutic protein improve identification wherein to The the biology, pathway. engineered synthetic identify secretory rationally mammalian product-specific for to avenues the approach define opens thus machinery an and here proteins, secreted demonstrate support we endomembrane summary, the of and In labeling TurboID proximal et when ligase, (Branon method efficiency effective biotin desired. an greater as of much is considered easily mutants with organelles be even be can promiscuous therefore labeling not two and proximity BioID2 may 2020) recently, catalyze supplementation al., to More biotinylation developed 2016) interactions. where been al., 2019). systems have luminal et miniTurbo, al., new (Kim expected et to protein introduced proximate (Sears many insurmountable be of accomplished an identified to labeling not enhanced BioID successfully is labeling exhibits for challenge reducing construct and allowing this thus supplementation, pathway, showed BioID2 biotin secretory we the less the can Here in requires and that 2016). accessible cells Roux, as of in & cytoplasm be (Kim the issue, not compartment into may biotin- that imported it that actively acknowledge in but we is nucleus, efficacy for proteins, Biotin the association secreted to well. of diffuse novel as synthesis freely limitations the a some studying have represent for methods well turn based works BioID ST show and we While motif extended For asx bind sequence 2013). and to certain Argon, study. evolved chaperones & further with family between Stevens, interact HSP70 (Gidalevitz, identified selectively pathways the associations folding chaperones of particular molecular chaperones the many favor example, to lacking, elements is structural motifs and structural the to iEsy:Nw n eiw nMlclr ellradDvlpetlBiology Developmental and Cellular Molecular, in Reviews and News BioEssays: sl,M 19) h ertioetd eet tutrlmtfmdaigprotein-protein mediating motif structural a repeat: tetratricopeptide The (1999). 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Manders’ value ICQ Li’s value R Pearson’s Cells )Scesu erto ftebi-iApoen notecluespraatwseautdby evaluated was supernatant culture the into proteins bait-BirA the of secretion Successful a) ehd nEnzymology in Methods irba Biotechnology Microbial h ora fBooia Chemistry Biological of Journal The ehd nMlclrBiology Molecular in Methods aueProtocols Nature p 3–6.https://doi.org/10.1016/b978-0-12-385928-0.00014-6 235–260. pp. , hr sltl orlto eotdfrteW,weesteeis there whereas WT, the for reported correlation little is There Proteomics Science , 11 1) 2301–2319. (12), , 8 , 1,164–168. (1), 356 14 , 16 aueProtocols Nature 64) https://doi.org/10.1126/science.aal3321 (6340). , 1) 2503–2518. (19), 1711 133–148. , , 275 nixdns&RdxSignaling Redox & Antioxidants 3) 24984–24992. (32), , 13 3,530–550. (3), aueCommunications Nature g ,tM r , ) 18 (13), , Posted on Authorea 15 May 2020 — CC-BY 4.0 — https://doi.org/10.22541/au.158955456.63626060 — This a preprint and has not been peer reviewed. Data may be preliminary. rtis(e ehd) nyitrcoshvn infiatascain ihmdlpoenfaue are features protein standardized model the with shows associations heatmap significant The model having all interactors across proteins. features Only structural model shown. or of methods). PTMs and (see features features. interactors proteins structural certain protein between (b) estimated with affinities and and interaction correlate PTMs hydrophobicity interactors (a) protein available Detected as specific valleys only such and are features 7. hills motifs predicted protein. Figure The entire structural whereas the and respectively. structure, for accessibility (b) PDB available solvent and are the disorder that by (a) covered content. Note panel structural regions in and features. for motifs PTM tertiary structural their the protein and and in indicate sites left diversity the show PTM on proteins known attributes. protein bait represent pathway model secretory The each The their for 6. on summarized interactions. Figure are based significant 6, top indicate on Fig. entries interactors in labeled shadowed between detailed are the interactions proteins, interactors Quantified where a model (b) is (y-axis), the (which of proteins protein). Signal-BirA features secreted model except compo- mammalian samples pathway the a all secretory along and for not the (top) (x-axis) hits of and set significant enrichment BirA most gene significant each secreted the showed for the lone among hits) hits of and proteins all 300 coverage co-secreted top for sets and The The S3 gene nents shown. pathway-related (Fig. change). are sample secretory fold with (bottom) BirA 3 by p-values interactors secretable enrichment of ordered added hypergeometric coverage (rank corresponding iteratively overall cutoffs their and the with significance first indicates the enrichment interactions y-axis x-axis iterative significant The the an most changes. performed the we fold included machinery. placentallower genes, we pathway pathway-related phosphatase, which secretory secretory Alkaline in for for of analysis enrich tag enriched form interactions to are truncated significant system proteins a if labeling Interacting fold is BioID highest SeAP 5. the Figure the of cells. showed capability live significantly the the indicating bait-protein within (ALPP). cases, of the type interactors all depicted expression vivo in As in after almost the WT biotinylation change. to fold increased compared and change significantly p-value show to experimental proteins according same of the proteins. Dozens under bait-BirA biotinylation 650 increased 4. show antibody-Dylight not Figure monoclonal did anti-flag WT with while (stained color), bait-BirA conditions. red and in color) illustrated green and Streptavidin-Dylight in with biotin-staining. illustrated (stained proteins with and biotin-labeled colocalized biotinylated594 the of are endogenously secreted colocalization intracellular proteins WT. few as the fusion in the demonstrated interactions A in Bait-BirA seen tags and not pathway. 3. system biotin are secretory Figure labeling of which the absence BioID (B+) the the through proteins in that trafficked of the appear demonstrates subset and to proteins This fused a synthesized was model of are biotin. domain biotinylation the BirA proteins of the the of absence to When profiling or led HRP-streptavidin. biotinylation WT addition with immunoblotting biotin cells proteins, The HEK293 in model b) control WT antibody. and HRP-anti-flag proteins using blot Western ocn ltsoigtedsrbto fteqatfidboiyae rtisb MS by proteins biotinylated quantified the of distribution the showing plot Volcano 15 neatr eeascae with associated were Interactors Co-Immunofluorescence a odetermine To (a) osadlines and Dots Posted on Authorea 15 May 2020 — CC-BY 4.0 — https://doi.org/10.22541/au.158955456.63626060 — This a preprint and has not been peer reviewed. Data may be preliminary. 100 150 250 10 15 20 25 37 50 75 KD a) – – – – – – – – – – Bait plasmids Bait - BioID2 BioID2 SeAP SerpinG1 SerpinC1 SerpinA1 BirA Bayesian statistical * model model expression Stable 100 150 250 10 15 20 25 37 50 75 KD b) – – – – – – – – – – 16 Biotin MS Identification ER - Golgi Streptavidin & Co Cell lysis Cytosol - IP with Posted on Authorea 15 May 2020 — CC-BY 4.0 — https://doi.org/10.22541/au.158955456.63626060 — This a preprint and has not been peer reviewed. Data may be preliminary. 17

Posted on Authorea 15 May 2020 — CC-BY 4.0 — https://doi.org/10.22541/au.158955456.63626060 — This a preprint and has not been peer reviewed. Data may be preliminary.

ycosylation l O−linked.G b) a)

ycosylation l

N−linked.G Hypergeometric P−value Geneset Coverage hor c

GPI−an 0.0 0.1 0.2 0.3 1e−04 1e−03 1e−02 1e−01 1e+00

Disulfide bond Disulfide -Log(P- -Log(P-

der r diso

0

y h opat r yd h value) value)

0 MYPN

TPD52L2 100 SeAP−BirA

DNAJB1

NSFL1C 100 SeAP−BirA

GNS

UGGT1

200

TTC13

PCSK6 200 WT) LogFC

PCMT1

MVD LogFC Secretor LGMN

300 IKBIP

FUCA2 300

DNAJC10 (SerpinC1

0 COG6

(

COPE y Geneset 0 SeAP FKBP7

Ser

Non-significant Significant interactions LAMB1 100 PRKCSH Ser pinA1−BirA

Number ofinter 100

SYNGR2 - pinA1−BirA

ERC1 BirA CGREF1

-

200 ALPP vs BirA

200 T3 L YN D

vs WT) vs DDT

Secretor

CTSD

DNASE2 300 KDELC2

300

P4HA2 actors included(significancecutoff) PDE4DIP 0 y pathw

Secretory pathway-resident

SERPINC1 0 PDIA4

SerpinC1 False True

A6 P

HS Ser

A5 P

HS Ser 100 a

DNAJB11 y machine pinC1−BirA

100

O1LB R

E ALPP pinC1−BirA

GALNS

PDIA6

SIL1 200

TXNDC16 200 UGGT2 r

y

18

PDIA3

MCFD2

ERP29 300 -Log(P- -Log(P- CNPY2 300 COL6A1 Secretor

0

Secretory Function MANF value) value)

0 TXNDC12

Tr Protein f ERAD COPI/COPII

CNPY4 Ser afficking

y pathw Ser NUCB1 100

100 pinG1−BirA

RCN1 pinG1−BirA

olding P4HB

a OU1 Y H

y−resident

HSP90B1 200

200 EDEM3

CALR LogFC WT) LogFC

B2M TXNDC5

300

SUMF2 300

SDF4

RCN2 (SerpinA1 (SerpinG1

PLA2G12A 0

0

PDIA5 Co−secreted proteins

P4HA1

OS9

NUCB2 Signal−BirA 100 Signal−BirA

100 NENF

MESDC2

AP1 P LR DNAJC3

-

-

CNPY3 vs BirA WT) 200

BirA vs vs BirA 200 CALU

ERLEC1

ERP44

LAMC1

VL1 D 300 300 PRDX4 SerpinG1-BirA SerpinC1-BirA SerpinA1-BirA Signal-BirA SeAP-BirA Secretory pathway-resident Secretory Function SerpinA1 SerpinG1 Posted on Authorea 15 May 2020 — CC-BY 4.0 — https://doi.org/10.22541/au.158955456.63626060 — This a preprint and has not been peer reviewed. Data may be preliminary. a) b) −2 −2 −2 −2 Relative tertiary features −2 −2 −2 −2 −2 Relative tertiary features −2 0 2 0 2 0 2 0 2 0 2 0 2 0 2 0 2 0 2 0 2 0 0 200 200 Amino acidposition 19

400 400

pinC1−BirA r Se SeAP−BirA pinA1−BirA r Se Signal−BirA pinG1−BirA r Se pinC1−BirA r Se SeAP−BirA pinA1−BirA r Se Signal−BirA pinG1−BirA r Se Protein ter PTM Protein ter Str uctur acc disorder h Ubiquitination Phosphor O−link N−link GPI−anchor Disulfide bond acc disorder h Schellman loop ST motif Asx motif ST tur Asx tur Alpha−Beta motif ST staple ydropath ydropath al motif ed Glycosylation n ed Glycosylation tiar n tiar ylation y y y f y f eatures eatures Posted on Authorea 15 May 2020 — CC-BY 4.0 — https://doi.org/10.22541/au.158955456.63626060 — This a preprint and has not been peer reviewed. Data may be preliminary.

(a)

Secretory pathway-resident Secretory

Secretory Function Secretory

Phosphorylation

Ubiquitination

Disulfide bond Disulfide N−lin

TTC13 CALR PRDX4 IKBIP P4HB PDIA4 COL6A1 PDIA3 ERP29 UGGT1 CNPY2 MANF B2M TXNDC16 GALNS SIL1 RCN1 PDIA6 ER EDEM3 HSP HY CNPY4 DNAJB11 MYPN k ed Glycosylation ed OU1 O1LB A5 Secretory Function Secretory pathway-resident (b) Protein f ERAD False True

20 Secretory pathway-resident Secretory

olding

Secretory Function Secretory

Asx mofit Asx

ST turn ST

b Beta

Schellman loop Schellman ulge loop ulge NUCB1 CGREF1 DNAJB1 Interaction affinity −1 0 1