Probing nuclear pore complex architecture withproximity-dependent biotinylationDae In Kima, Birendra KCa, Wenhong Zhub, Khatereh Motamedchabokib, Valérie Doyec, and Kyle J. Rouxa,d,1

aSanford Children’s Health Research Center, Sanford Research, Sioux Falls, SD 57104; bSanford-Burnham Proteomics Facility, Sanford-Burnham MedicalResearch Institute, La Jolla, CA 92037; cInstitut Jacques Monod, Unité Mixte de Recherche 7592, Centre National de la Recherche Scientifique, Université ParisDiderot, Sorbonne Paris Cité, F-75205 Paris, France; and dDepartment of Pediatrics, Sanford School of Medicine, University of South Dakota, Sioux Falls, SD 57105

Edited by Joseph G. Gall, Carnegie Institution of Washington, Baltimore, MD, and approved May 14, 2014 (received for review April 8, 2014)

Proximity-dependent biotin identification (BioID) is a method foridentifying protein associations that occur in vivo. By fusing apromiscuous biotin ligase to a protein of interest expressed inliving cells, BioID permits the labeling of proximate proteins duringa defined labeling period. In this study we used BioID to study thehuman nuclear pore complex (NPC), one of the largest macromolec-ular assemblies in eukaryotes. Anchored within the nuclear envelope,NPCs mediate the nucleocytoplasmic trafficking of numerous cellularcomponents. We applied BioID to constituents of the Nup107–160complex and the Nup93 complex, two conserved NPC subcomplexes.A strikingly different set of NPC constituents was detected dependingon the position of these BioID-fusion proteins within the NPC. Byapplying BioID to several constituents located throughout the ex-tremely stable Nup107–160 subcomplex, we refined our understand-ing of this highly conserved subcomplex, in part by demonstratinga direct interaction of Nup43 with Nup85. Furthermore, by using theextremely stable Nup107–160 structure as a molecular ruler, we de-fined the practical labeling radius of BioID. These studies further ourunderstanding of human NPC organization and demonstrate thatBioID is a valuable tool for exploring the constituency and organiza-tion of large protein assemblies in living cells.

The refined characterization of protein assemblies is a prereq-uisite for understanding functional protein networks. Proximity-

dependent biotin identification (BioID) is an approach recentlydeveloped to address this problem. BioID is based on expressionof a “bait” protein fused to a promiscuous biotin ligase (BirA*)that will generate a history of the bait’s proximity-dependentassociations over a period by the biotinylation of interacting orneighboring “prey” proteins (1). BioID biotinylates proteins insitu before their solubilization and subsequent purification andidentification. Issues related to bait (and prey) protein solubilityand the stability and/or duration of their interaction are thusovercome. BioID has been used successfully to screen for con-stituents of the relatively insoluble mammalian nuclear lamina (1),the trypanosome bilobe (2), cell junction complexes (3–5), andcentrosomes (6, 7). The method also has been used to screen forproteins involved in the Hippo signaling pathway (8).Biotinylation by BioID is a mark of proximity and not evidence

for physical interaction. An outstanding issue concerning thismethod is the radius of biotinylation. Previous application ofBioID to lamin A (LaA) suggested that a majority of the can-didates resided within ∼20–30 nm of nuclear envelope (NE)-associated LaA (1). Significantly, distinct subsets of BioID can-didates were identified when BirA* was fused to the N versus theC terminus of the cell-junction protein ZO-1 (3). These studiessuggested that BioD has a limited nanometer-scale (<20 nm)labeling radius. However, its precise range remained uncertain.Certain parameters are needed to analyze the range of bio-

tinylation by BioID in live cells more carefully. An ideal testwould involve a stable multiprotein structure, preferably withknown dimensions that extend beyond 20 nm. Protein stabilitywithin this complex is essential to generate accurate measure-ments. Ideally the complex also should be relatively stable incells. In nondividing cells the nuclear pore complex (NPC) as

been shown to be an extremely stable structure, with many of itsconstituents exhibiting long residence times (9, 10) and lowturnover (11, 12). Anchored within the NE, NPCs mediate thenucleocytoplasmic trafficking of numerous cellular components.NPCs are composed of multiple copies of ∼30 distinct proteins(nucleoporins or “Nups”) arranged with eightfold radial sym-metry, leading to an assembly of 500–1,000 proteins with an es-timated mass of ∼125 MDa in vertebrates. The mammalian NPChas a core structure composed of two outer membrane-proximalrings (built up by Nup107–160 scaffold complexes) that enclosea central spoke ring containing the Nup93 complex. Interactionsof these scaffold Nups with integral membrane proteins con-tribute to the anchoring of the NPC within the pore membrane.Tethered by this membrane-embedded central framework, pe-ripheral NPC components (notably a subset of Nups containingPhe-Gly repeats, FG-Nups) extend into the central pore channeland into the cytoplasm and the nucleoplasm, where they formcytoplasmic filaments and the nuclear pore basket respectively(reviewed in refs. 13–16) (Fig. 1A).The metazoan Nup107–160/yeast Nup84 complex is a con-

served and extensively characterized NPC building block (reviewedin ref. 17). In vertebrates, this complex consists of nine subunits:nucleoporin Nup133, nucleoporin Nup107, nucleoporin Nup96,nucleoporin Nup85, nucleoporin Nup160, protein Sec13 homologSec13, nucleoporin Seh1L, nucleoporin Nup37, and nucleoporinNup43 (10, 18, 19), with the nucleoplasmic protein Elys sometimesconsidered a 10th member of the complex (see Fig. 1B, refs. 20 and21, and references therein). Biochemical and structural analyses invarious species have revealed the precise arrangement of theseNups into Y-shaped complexes (hence the name “Y-complex”)(reviewed in refs. 16 and 21; also see Fig. 1B). Photobleaching

Significance

Proximity-dependent biotinylation (BioID) is a readily accessiblemethod for identifying protein associations that occur in livingcells. Fusion of a promiscuous biotin ligase to a bait protein forexpression in live cells enables covalent biotin labeling, and thusidentification, of proteins proximate to the bait. Here we usedBioID to probe the organization of the nuclear pore complex, alarge structure that regulates molecular transport between thenucleus and cytoplasm. These studies enhance our understand-ing of major subcomplexes within the nuclear pore complex anddemonstrate the utility of BioID for studying the organization oflarge protein assemblies. Additionally, we have measured thelabeling radius of BioID, thus enabling the rational applicationof this method and more meaningful data interpretation.

Author contributions: D.I.K., V.D., and K.J.R. designed research; D.I.K., B.K., W.Z., and K.M.performed research; D.I.K., W.Z., K.M., V.D., and K.J.R. analyzed data; and V.D. and K.J.R.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: kyle.roux@sanfordhealth.org.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1406459111/-/DCSupplemental.

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studies of mammalian Y-complex Nups (Y-Nups) have revealedtheir extreme stability within the NPCs, with half-time recoveriesexceeding 35 h (22). By applying BioID to proteins that resideat distinct points within the elongated (33–39 nm long) humanY-complex (21), we aimed to define the practical labeling radiusof BioID.To improve our understanding of both BioID specificity and

NPC architecture, we also applied BioID to nucleoporin Nup53,a small dimeric membrane-associated protein that belongs to theNup93 complex, a distinct NPC scaffold complex that links thepore membrane with the Nup62 complex that resides withinthe central pore channel (refs. 23–27 and references therein;reviewed in ref. 28). Taken together these distinct BioID datasets,encompassing both the Y- and Nup93 complexes, allowed us todefine a practical labeling radius and hence the resolution of theBioID technique, at the same time demonstrating the value ofthe technique in defining both the constituency and organizationof large protein complexes.

Results and DiscussionIdentification of Proteins Biotinylated in Cells Stably ExpressingBioID-Nups. As a prelude to our studies we generated HEK293cell lines that constitutively express BirA*-tagged members ofthe Y-complex (Nup160, Nup133, Nup107, Nup85, and Nup43),BirA*-tagged Nup53, or LaA for comparison (Fig. 1C). Tominimize artifacts associated with fusion proteins targeting tosites other than NPCs, care was taken to choose subclones ofcells that expressed low levels of the fusion protein. BecauseNPCs disassemble at mitosis, a stage when several Nups associ-ate with other structures [notably at kinetochores in the case ofY-Nups (29, 30)], cell growth was arrested for 72 h in low-serummedium before the induction of biotinylation in all of ourexperiments,. Immunofluorescence analyses revealed that bio-tinylation catalyzed by each of the BioID-Nups was largelycoincident with the NPCs, as revealed by colocalization with

mAb414 (which detects a subset of Nups containing FXFGrepeats) (31) and anti-Nup153 (Fig. 2A and Fig. S1B). In addi-tion, the BioID-Nups (but not BioID-LaA) and biotinylatedproteins also localized within cytoplasmic structures stained bymAb414 but not by anti-Nup153; these structures likely corre-spond to populations of endoplasmic reticulum-associated nu-clear pores called “annulate lamellae” (9, 31, 32) (Fig. 2A andFig. S1B). Immunoblot (IB) analysis revealed biotinylation of theBioID-Nups as well as variable levels of unidentified endogenousproteins (Fig. 2B). To identify the proteins biotinylated by eachof the BioID-fusion proteins, material isolated from large-scaleBioID pull-downs was analyzed by MS (1, 33) (Materials andMethods and Dataset S1). The identities and relative abundanceof the candidates (Materials and Methods) associated with theNPC and NE are listed in Table 1.

BioID-Nup Analyses Reveal Restricted Specificity. Immediately obvi-ous in the Nup BioID data of Fig. 2B and Table 1 is the lack ofwidespread protein biotinylation. Instead, 47–94% of the detectedcandidates are proteins associated with the NPC. In contrast, Nupsrepresented only 15% of the prey proteins associated with LaA(predominantly nuclear basket) and 2.1% of the prey proteins as-sociated with BirA*-only (Datasets S1–S3). Nup43 was unique inits detection of a prominent non-NPC– or non-NE–associatedprotein, namely t-complex protein 1 subunit theta (CCT8). CCT8 isa component of the chaperonin complex that mediates foldingof proteins containing a WD-repeat, of which Nup43 is a member(reviewed in ref. 34). BirA*-Nup133 was expressed at lower levelsthan the other baits, and fewer peptides were detected by BioIDwith Nup133. However, the percentages of those candidates thatwere constituents of the Y-complex and NPC were similar to thepercentages when the other baits were used.When candidates from the BioID Y-complex and BioID-Nup53

experiments are compared, it is clear that, in large part, theydetected spatially distinct populations of proteins (Table 1).

Fig. 1. Organization of the mammalian NPC, Y-complex, and BirA*-fusion proteins. (A) Positioning of the Y-complex (blue) and Nup53–93 complex (green)within a simplified model of NPC organization. A full description of the members of each pore subcomplex is shown in Table 1, leftmost column. TM Nups,transmembrane Nups. (B) Model of the human Y-complex. Its many β-propeller domains are schematized by circles or bulges; alpha-solenoid folds aredepicted by rectangles. Nup43 is drawn with a dashed line because its localization was unknown at the time these studies were performed. The dotted lineand oval indicates possible residence of ELYS near Nup160 and Nup37, extrapolated from studies in yeast (63, 64). Gray disks represent the predicted lo-calization of the BirA*-ligase based on available structural data. (C) Linear model for the NPC proteins fused to BirA* for the BioID studies.

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Y-complex Nups represented 14–24% of the total BioID candi-dates that were found when a Y-Nup-BirA* was used as a bait. Incontrast, few Y-Nups were found when Nup53 was used as bait (6%,largely Nup96) or when with BirA*-LaA was used (2.7%, largelyELYS). Instead, 38% of the proteins identified by BioID-Nup53correspond to predicted nearest-neighbor Nups, namely, com-ponents of the Nup93 complex, their transmembrane Nup partnersnucleoporin Ndc1 and nuclear envelope pore membrane proteinPom121, and the Nup62 complex that is anchored by Nup93 (refs.35–37 and references therein). These Nups were detected onlyrarely in Y-Nup–BioID samples. A more global comparison ofBioID–Y-Nups versus BioID-Nup53 outcome reveals that, althoughsome candidates overlap, BioID specifically detects different pop-ulations of Nup proteins within the larger NPC assembly dependingon the residence of the bait (Table 1).None of the BioID–Y-Nups detected all other members of

the Y-complex, although seven of its 10 constituents (includingElys) are biotinylated by at least one of the BioID–Y-Nups.Only Sec13 and Nup37, two small β-propeller Y-Nups (Fig. 1B),were never identified. Endogenous Nup85 was detected onlymodestly, even though it showed substantial expression andbiotinylation of other Y-Nups when fused to biotin ligase (Fig.2B and Dataset S1). This result suggests that Nup85 may not beable to be efficiently biotinylated. By the more sensitive IBanalysis, low levels of endogenous Nup85 were detected inBioID pull-downs for Nup160, Nup107, and Nup43 but not forNup133 (Fig. 3). These results are not surprising, becauseBioID-Nup85 detected Nup107 and, to a lesser extent, Nup160and Nup43, suggesting proximity to these proteins. As a controlwe reprobed these samples for Nup107 and observed detectionof this protein consistent with the MS results. These data revealone limitation of BioID, namely, that not all proteins are bio-tinylated with similar efficiency. As in any large-scale experiment,negative results should be treated with caution.

Mapping the Position of Nup43 Within the Y-Complex. At the timethese studies were performed, there was no information as tothe location of Nup43 within the Y-complex. Although ourNup43-BioID results suggested that this WD-repeat domainprotein is proximate to Nup96, Nup43 was specifically detectedby BioID-Nup85. To assess biochemically how Nup43 integratesinto the Y-complex, we immunoprecipitated exogenous epitope-tagged complex members and asked if other Nups were coimmu-noprecipitated. Assuming that, like the other small β-propellerfolded Y-Nups, Nup43 most likely interacts directly with oneof the larger proteins, we performed immunoprecipitation (IP)of Nup43-HA and asked if it coimmunoprecipitated with GFP-tagged Nup133, Nup107, Nup96, Nup85, or Nup160. We foundthat Nup43-HA consistently pulled down GFP-Nup85, and, toa much lesser extent, GFP-Nup160, but inconsistently or neverpulled down GFP-Nup107, -Nup133, or -Nup96 (Fig. 4A). As acontrol, we reprobed these blots with anti-Nup107 and observedthat a fraction of endogenous Nup107 was identified in all co-IPs,indicating that our lysis conditions permitted isolation of the en-dogenous Y-complex (Fig. 4A). However, the relative amount ofendogenous Nup107 that was coprecipitated was significantlyless substantial than for GFP-Nup85, indicating that the trans-fected Nup43 and Nup85 likely interact independently of theirincorporation into the Y-complex or the NPC.To validate this result, we turned to an in vitro transcription

and translation assay that revealed that Nup43 interacts withNup85 but with none of the other larger scaffold Y-Nups (Fig.4B). Together these studies biochemically demonstrate a directinteraction between Nup43 and Nup85. Thus, by demonstratingthat Nup43 and Nup85 can associate with each other independentlyof their integration within the entire NPC, these results strengthena recent report that identified Nup43 residing in close proximity toNup85 and Seh1 by XL-MS (21).

Fig. 2. Characterization of cells stably expressing NPC BirA*-fusion proteins. (A) Immunofluorescence analyses of HEK293 cells stably expressing Y-complexmembers or Nup53 fused to BirA*. The biotin signal generated by the BirA*-fusion proteins is detected with fluorescently labeled streptavidin (green). andNPCs are detected with anti-Nup153 (red). (Scale bar: 7 μm.) (B) Following SDS/PAGE of cell lysates, biotinylated proteins were detected with streptavidin-HRP.Asterisks indicate the location of the BirA*-fusion protein (detected with anti-BirA). Tubulin was used as loading control.

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Y-Nups as Macromolecular Rulers to Define the Practical LabelingRadius of BioID Accurately. When analyzed in the context ofa monomeric Y-complex, the major BioID Y-Nup candidatesappear positioned at 10–20 nm from the baits for Nup160,

Nup107, Nup85, and Nup43 (Fig. 5A). In contrast, the strongdetection of Nup96 in the case of Nup133 and the weaker iden-tification of Y-Nups distantly positioned from the various baitssuggest a much larger radius (Table 1). However, a recent study

Table 1. Summary of the proteins detected by BioID-Nups, BioID-LaA. and BirA*-only

Identified candidates

BirA* fusion protein baits

Nup160 Nup133 Nup107 Nup85 Nup43 Nup53 LaA BirA*

Nup107 complex (Y-complex + Elys)NUP160 Bait 0.1 0.1NUP107 4.3 Bait 1.1 4.3NUP133 Bait 7.0 0.4 0.2NUP96 6.4 8.9 4.5 14.8 17.8 6.0 0.3NUP85 Bait XNUP43 0.4 BaitSEH1L 0.6 7.7 XELYS 14.5 6.9 1.6 X 0.2 2.2

Percent of total that are Nup107 complex/Elys components 21 20 14 24 23 6 3 0Nup93 complex

NUP53 BaitNUP93 0.1NUP155 2.7 0.1NUP205 0.3NUP188 X 5.2

Nup62 complexNUP62* 5.6 17.2NUP58/45 7.2NUP54 1.6

Transmembrane nupsPOM121 1.8 1.4 0.5 0.2 2.6 1.4NDC1 1.1

Percent of total that are expected Nup53 partners 2 1 0 6 0 38 2 0Cytoplasmic nups

NUP88 X 1.9 1.5NUP214 0.9 8.7 12.5 <0.1GLE1 0.4CG1 5.3DDX19B/DBP5 0.8

Cytoplasmic filament nupsRANBP2/Nup358 5.1 6.5 25.1 13.5 4.0 4.7 0.8RANGAP1 2.6 2.2 0.1

Nuclear pore basketNUP153 17.7 23.1 23.3 20 15.2 8.9 5.1 0.1NUP50 1.2 2.8 11.7 16.5 5.4 1.3 4.4TPR 11.7 1.2SENP1 1.2 1.0 1.3SENP2 6.7 1.3 5.2NUP98 0.4 0.5 6.0 0.4

Import/exportKPNB1 0.3 0.1XPO1 0.3

Percent of total that are NPC-associated 47 61 92 94 54 86 15 2Nuclear envelope constituents

TMPO beta 25.9 28.9 4.3 12.7 1.3LEMD3 3.5 0.6 3.7 <0.1EMD 1.5 8.9SYNE1 0.0 0.0TMEM201 0.3 0.3TMPO alpha 0.6LBR 0.2

Percent of total that are NPC/NE-associated 76 90 92 94 54 92 41 4

Numbers are the percent of total adjusted peptides (excluding BirA*-fusion protein). Candidates listed as 0.0 are <0.1. Numbers inbold indicate proteins identified by XL-MS on isolated Y complex or intact NPC. X, proteins identified by XL-MS but not BioID. BioIDpull-down for LaA was performed with asynchronous cells.

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provides a compelling model for the mammalian NPC in whichoffset Y-complex dimers are arranged in a head-to-tail staggeredparallel fashion to form ring-like structures on the nucleoplasmicand cytoplasmic sides of the pore (21). With this organization it isclear that in some instances we are likely observing intercomplexrather than intracomplex labeling by BioID. Prime examples arethe detection of Nup96 by BioID-Nup133 and of Nup107 byBioID-Nup85 or BioID-Nup43 (Table 1 and Fig. 5B). Reevaluat-ing the BioID results from the Y-complex in the context of thewhole NPC, we thus can restrict the practical labeling radius ofBioID (defined as the ability to detect proteins by MS followingBioID pull-down) to ∼10 nm (Fig. 5B). However, because not allNups within 10 nm are labeled by a BioID-Nup bait, one againmust view negative results with caution.

Refining NPC Organization with BioID. Keeping in mind the esti-mated labeling radius, we then analyzed the NPC constituentsoutside the stable Y-complex that were detected by BioID-Nups toget insight into the whole NPC architecture (Fig. 6 and Table 1).Among the identified Nups, a few, most notably FG-Nups as-sociated with the cytoplasmic filaments (nucleoporin Nup358/RANBP2) or the nuclear basket (nucleoporin Nup50 and nucleo-porin Nup153), were substantially detected by all Y-Nups. Amongthem, Nup153 was reported previously to associate with theY-complex (18), and recent crosslinking-MS (XL-MS) and cryo-electron tomography (cryo-ET) studies recognized the proximityof Nup358 and the Y-complex (21). However, the identificationof these large, flexible, or dynamic Nups (22, 38–41) by BioID-Nup53 suggests that these FG-Nups may associate with multipleNups only transiently as they sample the NPC environment.Nevertheless, most Nups were identified in a restricted subset

of BioID samples, thus validating or refining our actual knowl-edge of NPC organization. In particular, the more central NPCconstituents, such as those in the Nup93 and Nup62 complexesand Ndc1, were all identified when we applied BioID to Nup53

but were largely absent from the BioID–Y-Nups results (Table1). The identification of this subset of Nups connecting the poremembrane to the more central Nup62–Nup58–Nup54 complex isconsistent with the proposed model of the inner pore complexof the NPC (23). Because of its flexibility (42, 43), the Nup62complex may be further capable of sampling the membrane-proximate region where Nup53 resides. Also well represented inthe BioID-Nup53 candidates are proteins reported to localizeon the cytoplasmic side of the NPCs, nucleoporin Nup88 andnucleoporin Nup214, which are known to associate with eachother (44–48), ATP-dependent RNA helicase Ddx19/Dbp5,which is known to interact with Nup214 (49), and nucleoporinGle1 and its binding partner nucleoporin hCG1/Npl1 (50, 51)(Fig. 5). The identification of these more distant partners mightresult from the intrinsic dynamics of Nup53, which has a reportedresidency half-time of ∼5 h in dividing cells (22), or from theexistence of distinct population of the 32 copies of Nup53 withinthe NPC (52). However, these data also could reflect a morecentral positioning of these “cytoplasmic” Nups, a feature com-patible with the reported interaction between Gle1 and nucleo-porin Nup155 (a direct Nup53 binding partner) (53) and with theapparent bending of the Nup214–Nup88 complex toward thecentral pore channel observed by cryo-ET (21).Nup133 and Nup160 detected substantially more Pom121

than any of the other Y-Nups that were tested. These two Nupsalso were unique in detecting TMPO (also known as lamina-associated polypeptide 2, “Lap2”) and LEMD3 (also known asinner nuclear membrane protein, “Man1”), both of which aretransmembrane proteins located in the inner nuclear membraneof the NE. Detection of these candidates indicates the proximityof Nup133 and Nup160 to the NPC membrane, a propertyconsistent with previously published data (21, 36, 54, 55).Conversely, Nup85 detected substantially more Nup214 thanany of the other tested Y-Nups, a result supported by a recentcryo-ET study (21). Moreover, BioID-Nup85 was unique amongthe tested Y-Nups in its ability to detect nucleoporin Nup62 andnucleoporin Nup88. Nup62 is a constituent of a subcomplexlocated in the central channel of the pore, whose other con-stituents (nucleoporin Nup58/45 and nucleoporin Nup54) arenot detected by BioID-Nup85. Although its detection may re-veal distinct positioning of Nup62 compared with Nup58/45 andNup54 (42), Nup62 also was proposed to associate with Nup88and Nup214 in a distinct complex in Xenopus egg extracts (56–58), as was demonstrated for its ortholog, Nsp1, in budding yeast(59). Our BioID results thus highlight the existence of a Nup62–Nup88–Nup214 complex in human cells and indicate that Nup85likely projects toward the Nup88–Nup214–Nup62 complex.Finally, among the identified NPC-associated nuclear basket

constituents, the structural nucleoprotein Tpr (39) was detectedsolely by BioID-Nup107, whereas the SUMO isopeptidasesentrin-specific protease 2 (SENP2), previously reported to as-sociate with the Y-complex (60), was strongly detected byBioID-Nup133 and BioID-Nup43 and to a lesser extent byBioID-Nup107. Our study thus now enables us to position Tprnear Nup107 and SENP2 near the head-to-tail connectionsbetween Y-complex dimers (Fig. 5B).

ConclusionsUsing a stable protein complex as a molecular ruler, we determinedthe practical labeling radius of BioID in vivo to be ∼10 nm. Wealso demonstrated that, when applied to proteins within distinctregions of the NPC, BioID is capable of detecting distinct pop-ulations of candidate proteins. The recent studies on the humanY-complex that use XL-MS provide some comparisons with BioID(21) (Table 1). XL-MS permits the identification and precise(amino acid resolution) mapping of extremely close interactions(within a couple of nanometers), whereas BioID has a much largerradius and thus far does not allow the mapping of biotinylated

Fig. 3. Nup85 is a poor substrate for BioID. IB analysis detects low levels ofendogenous Nup85 (open arrowhead) in the Nup160, Nup107, and Nup43BioID pull-down samples and significant levels of the exogenous mycBirA*-Nup85 (arrowheads) in the Nup85 BioID pull-down. (Top) For clarity, totallysates are shown at a lower exposure than the BioID samples. (Middle) Forcomparison, we detect similar levels of the BirA*-fusion proteins with anti-BirA in these same samples. (Bottom) Reprobing the same membrane withanti-Nup107 reveals levels of endogenous Nup107 (arrow) that correlatewith the MS results, thus corroborating those results. Exogenous mycBirA*-Nup85 remains detected below the endogenous Nup107 (asterisk).

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residues within the prey proteins. However, because of samplecomplexity, the identification by XL-MS of cross-linked peptidesfrom complex assemblies, such as intact NPCs within purifiedNEs, appears quite challenging. This complexity explains therather low number of confidently assigned interactions (17 intotal, of which 11 involved Y-Nups or Nup53) in this XL-MSstudy (21). In contrast, BioID does not require prior purificationof organelles and is technically far less demanding. Thus BioID isa useful tool for scientists interested in probing the proteinconstituency and mapping the organization of large structuralprotein assemblies. In this way it provides a complementaryapproach to XL-MS. Future studies, including baits from otherNPC subcomplexes and evaluation of BioID candidates of theY-complex during discrete stages of the cell cycle, should provide

additional insights into the assembly and function of NPCconstituents.

Materials and MethodsPlasmids.Nup85,Nup107, Nup133, Nup160, Nup53, andNup96were amplifiedby PCR from human cDNA. The PCR products were digested (by XhoI andBamHI forNup85,Nup107, andNup160; byXhoI andEcoRI forNup133; byXhoIandHindIII for Nup53; by XhoI and AflII for Nup96) and inserted intomycBioIDpcDNA3.1 (35700;Addgene). Nup43was amplified anddigestedwithNheI andEcoRI. The digested PCR product was inserted into BioID-HA pcDNA 3.1 (36047;Addgene). Human LaA was inserted into mycBioID pcDNA3.1 following di-gestion with XhoI and AflII (1). Nup43-HA was PCR-amplified using a reverseprimer containing the HA-tag sequence and was inserted into pcDNA 3.1after NheI and PmeI digestion. GFP-Nup85, GFP-Nup107, GFP-Nup96, GFP-Nup133, and GFP-Nup160 and were used as previously reported (10).

Fig. 4. Nup43 interacts with Nup85. (A) Anti-HA co-IP from lysates of HEK293 cells cotransfected with Nup43-HA (Middle, arrowhead) and GFP–Y-Nups (Top)indicates that GFP-Nup85 is pulled down most efficiently by Nup43-HA. (Bottom) Reprobing the samples with anti-Nup107 reveals low levels of endogenousNup107 (arrow) in all the Nup43-HA pull-down samples. (B) Co-IPs from in vitro transcription/translation reactions in reticulocyte lysates using Nup43-HA(Lower, arrowhead) alone or with mycBirA*-tagged Y-Nups (Upper). Only mycBirA*-Nup85 is detected in the Nup43-HA pull-down fraction.

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Antibodies. Mouse monoclonal anti-Nup153 (SA1) was used as previouslyreported (61). Rabbit anti-Nup107 was used as previously described (19). Rabbitpolyclonal anti-HA (ab9110; Abcam), anti-myc (ab9106; Abcam), anti-GFP (ab290;Abcam), anti-Nup85 (A303-977A; Bethyl Laboratories), chicken polyclonal anti-BirA (ab14002; Abcam), mouse monoclonal anti-tubulin (T9026; Sigma) andmAb414 (MMS-120p-500; Eurogentek) were used as primary antibodies.

Cell Lines and Transfection. HumanHEK293 cellsweremaintained in5.0%CO2at37 °C in DMEM (SH3024301; HyClone) supplemented with 10% (vol/vol) FBS. Togenerate cells stably expressing BioID fusion proteins, HEK293 cells were trans-fected via Lipofectamine 2000 (Life Technologies) using the manufacturer’s rec-ommended protocols and subjected to G418 (700 μg/mL) selection. Subclones ofcells that expressed low levels of the fusion protein were chosen to minimize po-tential artifacts associated with spill-over of fusion proteins to sites otherthan NPCs. Before all the analyses described in this report, cells were growtharrested by incubation in DMEM supplemented with 0.1% FBS for 72 h to

arrest cell division, and promiscuous biotinylation by the BioID-Nups wasinduced by the addition of biotin to this cell-culture medium to a finalconcentration of 50 μM for 18 h. For the co-IP experiments shown in Fig. 3B,2.4 × 106 HEK293 cells were cotransfected with equal amounts of HA-Nup43and GFP-Y-Nups plasmids (1 μg each) 24 h before co-IP.

Immunostaining. HEK293 cells were fixed in 3% (wt/vol) paraformaldehyde/PBS for 10 min and permeabilized using 0.4% Triton X-100/PBS for 15 minfollowed by 0.5% SDS/PBS for 10 min. After fixation and permeabilization,cells were labeled with appropriate primary and secondary antibodies for 20min at 25 °C in 0.4%Triton X-100/PBS. Primary antibodies were detected withAlexa-Fluor 568–conjugated goat anti-mouse (A11031; Life Technologies)or goat anti-rabbit (A11036; Life Technologies) secondary antibodies.Alexa-Fluor 488–conjugated streptavidin (S32354; Life Technologies) wasused to detect biotinylated proteins. DNA was detected with Hoechst dye33258. Coverslips were mounted in 10% (wt/vol) Mowiol 4–88 (17951;

Fig. 5. Biotinylation of Y-Nups in the context of the whole NPC defines a practical labeling radius. (A) For each BioID-fusion, a model of a single Y-complexsubunit is used to depict the relative abundance of Y-Nups detected following BioID pull-down. The red circles depict the approximate position of the BirA*ligase. Gray disks (10-nm radius) provide an approximation of the labeling radius of BioID. (B) Structural model from Bui et al. (21) in which offset Y-complexdimers are arranged in a head-to-tail fashion within the NPC (Left). The approximate positions of Y-Nups are labeled and schematized (Right) on thismap. (Modified from ref. 21.) (C) BioID data were applied to the dimer model of Y-complex. The color code in A is used to depict the relative abundanceof biotinylated Y-Nups for BioID-fusion proteins. The gray disks (dark: 5-nm radius; light: 10-nm radius) provide an approximation of the labeling radiusof BioID.

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Polysciences). Images were obtained using Nikon A1-confocal microscope(60×/1.49 oil APO TIRF Nikon objective) and a CCD camera (CoolSnap HQ;Photometrics) linked to a workstation running NIS-Element software (Nikon).

Immunoblot and Immunoprecipitation. For immunoblot of total cell lysates,1.2 × 106 cells were lysed in SDS/PAGE sample buffer, sonicated to shearDNA, and boiled for 5 min. For co-IP analyses transiently transfected HEK293cells (2.4 × 106) were lysed in 1 mL of IP lysis buffer [50 mM Tris (pH 7.5), 150mM NaCl, 2.5 mM MgCl2, 1 mM DTT, 1% Triton X-100, and 1× proteinaseinhibitor (1861278; Thermo Scientific)]. Lysates were passed througha 21-gauge needle 10 times and centrifuged at 16,500 × g for 10 min at 4 °C.The supernatants were rotated overnight at 4 °C with 20 μL of protein ASepharose beads (20365; Thermo Scientific) and 2 μg of rabbit anti-HA an-tibody. Samples were washed thoroughly three times with the IP lysis bufferand twice with wash buffer [50 mM Tris (pH 7.5) and 50 mM NaCl] at 4 °C.Proteins were solubilized in 25 μL SDS/PAGE sample buffer and boiled for5 min. Proteins were separated on 8% SDS/PAGE and were transferred tonitrocellulose membrane (Bio-Rad), which subsequently was blocked [10%(vol/vol) adult bovine serum, 0.2% Triton X-100, 1× PBS] and incubated withappropriate primary antibodies overnight at 4 °C. After washes withblocking buffer, blots were incubated with HRP-conjugated anti-mouse(F21453; Life Technologies), anti-rabbit (G21234; Life Technologies), oranti-chicken (A9046; Sigma) antibodies to detect proteins following en-hanced chemiluminescence. To detect biotinylated proteins, High Sensi-tivity Streptavidin-HRP (21130; Thermo Scientific) was used as previouslydescribed (1, 33). The in vitro transcription and translation was performed byusing TnT Quick Coupled Transcription/Translation Systems (Promega) with themanufacturer’s recommended protocol. For 20-μL reactions, 2 μL was reservedfor total protein analysis. The remaining volume was added to 0.5 mL of the IPlysis buffer. IP and IB steps were performed as described above.

BioID, On-Bead Protein Digestion, and Identification by 1D LC-MS/MS. Large-scale (4 × 107 cells) BioID pull-downs for MS analysis were performed aspreviously described with the exception that pooled lysates were incubatedin a 15-mL conical tube overnight at 4 °C before washing. Ninety percent ofeach sample was used for MS analysis, and 10% was reserved for IB analysis.Sample volume was adjusted to 200 μL with 50 mM ammonium bicarbonate.Then 4 μL of 0.5 M Tris(2-carboxyethyl)phosphine was added to 200 μL of the

beads–proteins suspension mix, and proteins were reduced at 40 °C for 30 min.Then 8 μL of 0.5 M Iodoacetamide was added, and proteins were alkylated atroom temperature in the dark for 30 min. MS-grade trypsin (Promega) wasadded (1:20 ratio) for overnight digestion at 37 °C using an Eppendorf Ther-momixer at 700 rpm. Digested peptides were separated from magnetic beadsby centrifugation and a GE Healthcare MagRack and were transferred toa new tube. Formic acid was added to the peptide solution (to 2%), followedby desalting by Microtrap (catalog no. 77720; Thermo) and then on-lineanalysis of peptides by high-resolution, high-mass accuracy liquid chroma-tography tandem MS (LC-MS/MS) consisting of a Michrom HPLC, a 15-cmMichrom Magic C18 column, a low-flow ADVANCED Michrom MS source, anda LTQ-Orbitrap XL (Thermo Fisher Scientific). A 120-min gradient of 10–30% B(0.1% formic acid, 100% acetonitrile) was used to separate the peptides. Thetotal LC time was 140 min. The LTQ-Orbitrap XL was set to scan precursors inthe Orbitrap followed by data-dependent MS/MS of the top 10 precursors.Raw LC-MS/MS data were submitted to Sorcerer Enterprise (Sage-N ResearchInc.) for protein identification against the ipi.HUMAN.vs.3.73 protein database,which contains semitryptic peptide sequences with the allowance of up to twomissed cleavages. Differential search included 16 Da for methionine oxidation,57 Da for cysteines to account for carboxyamidomethylation, and 226 Dafor biotinylation of lysine. Search results were sorted, filtered, staticallyanalyzed, and displayed using PeptideProphet and ProteinProphet (In-stitute for Systems Biology). The minimum Trans-Proteomic Pipeline (TPP)probability score for proteins was set to 0.95 to assure a TPP error rate lowerthan 0.01. The relative abundance of each of the identified proteins in dif-ferent samples was analyzed by QTools, an open-source tool developed in-housefor automated differential peptide/protein spectral count analysis (62). Proteinsdetected in the control sample (cells lacking BirA*) and common BioID back-ground proteins (BirA*-only) (Dataset S2) were subtracted from the results unlesstheir abundance was threefold more than in the BirA*-only. For all datasets, thetotal spectral counts for each protein then were normalized to account for thetotal length of the protein in amino acids. The relative abundance of each preyfinally was expressed as percentage of the sum of all of the adjusted spectralcounts except those of the BioID-fusion protein within a given BioID sample.

ACKNOWLEDGMENTS. We thank Brian Burke and Benoit Palancade forhelpful discussions and advice. These studies were supported by GrantsRO1GM102203, RO1GM102486, and RO1EB014869 (to K.J.R.) from the NationalInstitutes of Health; Sanford Research startup funds (K.J.R.); French National

Fig. 6. Biotinylation of NPC constituents generally correlates with the location of the fusion protein. The candidate Nups identified in this BioID studies arepositioned within a simplified model of NPC organization that integrates data from the literature and extrapolations based on previous studies in buddingyeast. The baits (bold text) used are shaded yellow. Intensity of (A) blue- (BioID-Y-Nups), (B) green- (BioID-Nup53), or (C) red- (BioID-LaA) shaded candidatescorrelates with the level of detection of candidates predominantly detected by the different types of BioID-fusion proteins (Table 1). In A, biotinylated Y-Nupsare not shaded blue for clarity (Fig. 5). The asterisks next to Nup62 in A and next to Nup133, Nup96, and Nup98 in C represent candidates with multiplelocations within the NPC and are unlikely to be biotinylated at that specific place.

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Research Agency Grant ANR-12-BSV2-0008-01 (to V.D.); and Fondation ARCpourla Recherche sur le Cancer (V.D.). This project used the Imaging Coreand Protein Biochemistry Core at Sanford Research, which are supported

by Institutional Development Awards from the National Institute of GeneralMedical Sciences and the National Institutes of Health under GrantsP20GM103548 and P20GM103620.

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