Probing complex architecture with PNAS PLUS proximity-dependent biotinylation

Dae 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 Medical Research Institute, La Jolla, CA 92037; cInstitut Jacques Monod, Unité Mixte de Recherche 7592, Centre National de la Recherche Scientifique, Université Paris Diderot, 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 for been shown to be an extremely stable structure, with many of its identifying associations that occur in vivo. By fusing a constituents exhibiting long residence times (9, 10) and low promiscuous biotin ligase to a protein of interest expressed in turnover (11, 12). Anchored within the NE, NPCs mediate the living cells, BioID permits the labeling of proximate during nucleocytoplasmic trafficking of numerous cellular components. a defined labeling period. In this study we used BioID to study the NPCs are composed of multiple copies of ∼30 distinct proteins human nuclear pore complex (NPC), one of the largest macromolec- (nucleoporins or “Nups”) arranged with eightfold radial sym- ular assemblies in eukaryotes. Anchored within the nuclear envelope, metry, leading to an assembly of 500–1,000 proteins with an es- NPCs mediate the nucleocytoplasmic trafficking of numerous cellular timated mass of ∼125 MDa in vertebrates. The mammalian NPC components. We applied BioID to constituents of the Nup107–160 has a core structure composed of two outer membrane-proximal complex and the Nup93 complex, two conserved NPC subcomplexes. rings (built up by Nup107–160 scaffold complexes) that enclose A strikingly different set of NPC constituents was detected depending a central spoke ring containing the Nup93 complex. Interactions on the position of these BioID-fusion proteins within the NPC. By of these scaffold Nups with integral membrane proteins con- applying BioID to several constituents located throughout the ex- tribute to the anchoring of the NPC within the pore membrane. tremely stable Nup107–160 subcomplex, we refined our understand- Tethered by this membrane-embedded central framework, pe- ing of this highly conserved subcomplex, in part by demonstrating ripheral NPC components (notably a subset of Nups containing

a direct interaction of Nup43 with Nup85. Furthermore, by using the Phe-Gly repeats, FG-Nups) extend into the central pore channel CELL BIOLOGY extremely stable Nup107–160 structure as a molecular ruler, we de- and into the cytoplasm and the , where they form fined the practical labeling radius of BioID. These studies further our cytoplasmic filaments and the nuclear pore basket respectively understanding of human NPC organization and demonstrate that (reviewed in refs. 13–16) (Fig. 1A). BioID is a valuable tool for exploring the constituency and organiza- The metazoan Nup107–160/yeast Nup84 complex is a con- tion of large protein assemblies in living cells. served and extensively characterized NPC building block (reviewed in ref. 17). In vertebrates, this complex consists of nine subunits: he refined characterization of protein assemblies is a prereq- nucleoporin Nup133, nucleoporin Nup107, nucleoporin Nup96, Tuisite for understanding functional protein networks. Proximity- nucleoporin Nup85, nucleoporin Nup160, protein Sec13 homolog dependent biotin identification (BioID) is an approach recently Sec13, nucleoporin Seh1L, nucleoporin Nup37, and nucleoporin developed to address this problem. BioID is based on expression Nup43 (10, 18, 19), with the nucleoplasmic protein Elys sometimes B of a “bait” protein fused to a promiscuous biotin ligase (BirA*) considered a 10th member of the complex (see Fig. 1 ,refs.20and that will generate a history of the bait’s proximity-dependent 21, and references therein). Biochemical and structural analyses in various species have revealed the precise arrangement of these associations over a period by the biotinylation of interacting or “ ” neighboring “prey” proteins (1). BioID biotinylates proteins in Nups into Y-shaped complexes (hence the name Y-complex ) (reviewed in refs. 16 and 21; also see Fig. 1B). Photobleaching situ before their solubilization and subsequent purification and identification. Issues related to bait (and prey) protein solubility and the stability and/or duration of their interaction are thus Significance overcome. BioID has been used successfully to screen for con- stituents of the relatively insoluble mammalian nuclear lamina (1), Proximity-dependent biotinylation (BioID) is a readily accessible the trypanosome bilobe (2), cell junction complexes (3–5), and method for identifying protein associations that occur in living centrosomes (6, 7). The method also has been used to screen for cells. Fusion of a promiscuous biotin ligase to a bait protein for proteins involved in the Hippo signaling pathway (8). expression in live cells enables covalent biotin labeling, and thus Biotinylation by BioID is a mark of proximity and not evidence identification, of proteins proximate to the bait. Here we used for physical interaction. An outstanding issue concerning this BioID to probe the organization of the nuclear pore complex, a method is the radius of biotinylation. Previous application of large structure that regulates molecular transport between the BioID to lamin A (LaA) suggested that a majority of the can- nucleus and cytoplasm. These studies enhance our understand- didates resided within ∼20–30 nm of nuclear envelope (NE)- ing of major subcomplexes within the nuclear pore complex and associated LaA (1). Significantly, distinct subsets of BioID can- demonstrate the utility of BioID for studying the organization of didates were identified when BirA* was fused to the N versus the large protein assemblies. Additionally, we have measured the labeling radius of BioID, thus enabling the rational application C terminus of the cell-junction protein ZO-1 (3). These studies of this method and more meaningful data interpretation. suggested that BioD has a limited nanometer-scale (<20 nm)

labeling radius. However, its precise range remained uncertain. Author contributions: D.I.K., V.D., and K.J.R. designed research; D.I.K., B.K., W.Z., and K.M. Certain parameters are needed to analyze the range of bio- performed research; D.I.K., W.Z., K.M., V.D., and K.J.R. analyzed data; and V.D. and K.J.R. tinylation by BioID in live cells more carefully. An ideal test wrote the paper. would involve a stable multiprotein structure, preferably with The authors declare no conflict of interest. known dimensions that extend beyond 20 nm. Protein stability This article is a PNAS Direct Submission. within this complex is essential to generate accurate measure- 1To whom correspondence should be addressed. E-mail: [email protected]. ments. Ideally the complex also should be relatively stable in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cells. In nondividing cells the nuclear pore complex (NPC) as 1073/pnas.1406459111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1406459111 PNAS Early Edition | 1of9 Downloaded by guest on September 23, 2021 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 are depicted by rectangles. Nup43 is drawn with a dashed line because its localization was unknown at the time these studies were performed. The dotted line and 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.

studies of mammalian Y-complex Nups (Y-Nups) have revealed mAb414 (which detects a subset of Nups containing FXFG their extreme stability within the NPCs, with half-time recoveries repeats) (31) and anti-Nup153 (Fig. 2A and Fig. S1B). In addi- exceeding 35 h (22). By applying BioID to proteins that reside tion, the BioID-Nups (but not BioID-LaA) and biotinylated at distinct points within the elongated (33–39 nm long) human proteins also localized within cytoplasmic structures stained by Y-complex (21), we aimed to define the practical labeling radius mAb414 but not by anti-Nup153; these structures likely corre- of BioID. spond to populations of endoplasmic reticulum-associated nu- To improve our understanding of both BioID specificity and clear pores called “annulate lamellae” (9, 31, 32) (Fig. 2A and NPC architecture, we also applied BioID to nucleoporin Nup53, Fig. S1B). Immunoblot (IB) analysis revealed biotinylation of the a small dimeric membrane-associated protein that belongs to the BioID-Nups as well as variable levels of unidentified endogenous Nup93 complex, a distinct NPC scaffold complex that links the proteins (Fig. 2B). To identify the proteins biotinylated by each pore membrane with the Nup62 complex that resides within of the BioID-fusion proteins, material isolated from large-scale the central pore channel (refs. 23–27 and references therein; BioID pull-downs was analyzed by MS (1, 33) (Materials and reviewed in ref. 28). Taken together these distinct BioID datasets, Methods and Dataset S1). The identities and relative abundance encompassing both the Y- and Nup93 complexes, allowed us to of the candidates (Materials and Methods) associated with the define a practical labeling radius and hence the resolution of the NPC and NE are listed in Table 1. BioID technique, at the same time demonstrating the value of the technique in defining both the constituency and organization BioID-Nup Analyses Reveal Restricted Specificity. Immediately obvi- of large protein complexes. ous in the Nup BioID data of Fig. 2B and Table 1 is the lack of widespread protein biotinylation. Instead, 47–94% of the detected Results and Discussion candidates are proteins associated with the NPC. In contrast, Nups Identification of Proteins Biotinylated in Cells Stably Expressing represented only 15% of the prey proteins associated with LaA BioID-Nups. As a prelude to our studies we generated HEK293 (predominantly nuclear basket) and 2.1% of the prey proteins as- cell lines that constitutively express BirA*-tagged members of sociated with BirA*-only (Datasets S1–S3). Nup43 was unique in the Y-complex (Nup160, Nup133, Nup107, Nup85, and Nup43), its detection of a prominent non-NPC– or non-NE–associated BirA*-tagged Nup53, or LaA for comparison (Fig. 1C). To protein, namely t-complex protein 1 subunit theta (CCT8). CCT8 is minimize artifacts associated with fusion proteins targeting to a component of the chaperonin complex that mediates folding sites other than NPCs, care was taken to choose subclones of of proteins containing a WD-repeat, of which Nup43 is a member cells that expressed low levels of the fusion protein. Because (reviewed in ref. 34). BirA*-Nup133 was expressed at lower levels NPCs disassemble at mitosis, a stage when several Nups associ- than the other baits, and fewer peptides were detected by BioID ate with other structures [notably at kinetochores in the case of with Nup133. However, the percentages of those candidates that Y-Nups (29, 30)], was arrested for 72 h in low-serum were constituents of the Y-complex and NPC were similar to the medium before the induction of biotinylation in all of our percentages when the other baits were used. experiments,. Immunofluorescence analyses revealed that bio- When candidates from the BioID Y-complex and BioID-Nup53 tinylation catalyzed by each of the BioID-Nups was largely experiments are compared, it is clear that, in large part, they coincident with the NPCs, as revealed by colocalization with detected spatially distinct populations of proteins (Table 1).

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Fig. 2. Characterization of cells stably expressing NPC BirA*-fusion proteins. (A) Immunofluorescence analyses of HEK293 cells stably expressing Y-complex members or Nup53 fused to BirA*. The biotin signal generated by the BirA*-fusion proteins is detected with fluorescently labeled streptavidin (green). and NPCs 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.

Y-complex Nups represented 14–24% of the total BioID candi- Mapping the Position of Nup43 Within the Y-Complex. At the time dates that were found when a Y-Nup-BirA* was used as a bait. In these studies were performed, there was no information as to contrast, few Y-Nups were found when Nup53 was used as bait (6%, the location of Nup43 within the Y-complex. Although our largely Nup96) or when with BirA*-LaA was used (2.7%, largely Nup43-BioID results suggested that this WD-repeat domain ELYS). Instead, 38% of the proteins identified by BioID-Nup53 protein is proximate to Nup96, Nup43 was specifically detected correspond to predicted nearest-neighbor Nups, namely, com- by BioID-Nup85. To assess biochemically how Nup43 integrates ponents of the Nup93 complex, their transmembrane Nup partners into the Y-complex, we immunoprecipitated exogenous epitope- nucleoporin Ndc1 and nuclear envelope pore membrane protein tagged complex members and asked if other Nups were coimmu- Pom121, and the Nup62 complex that is anchored by Nup93 (refs. noprecipitated. Assuming that, like the other small β-propeller – 35 37 and references therein). These Nups were detected only folded Y-Nups, Nup43 most likely interacts directly with one – rarely in Y-Nup BioID samples. A more global comparison of of the larger proteins, we performed immunoprecipitation (IP) – BioID Y-Nups versus BioID-Nup53 outcome reveals that, although of Nup43-HA and asked if it coimmunoprecipitated with GFP- some candidates overlap, BioID specifically detects different pop- tagged Nup133, Nup107, Nup96, Nup85, or Nup160. We found ulations of Nup proteins within the larger NPC assembly depending that Nup43-HA consistently pulled down GFP-Nup85, and, to ontheresidenceofthebait(Table1). a much lesser extent, GFP-Nup160, but inconsistently or never None of the BioID–Y-Nups detected all other members of pulled down GFP-Nup107, -Nup133, or -Nup96 (Fig. 4A). As a the Y-complex, although seven of its 10 constituents (including control, we reprobed these blots with anti-Nup107 and observed Elys) are biotinylated by at least one of the BioID–Y-Nups. that a fraction of endogenous Nup107 was identified in all co-IPs, Only Sec13 and Nup37, two small β-propeller Y-Nups (Fig. 1B), indicating that our lysis conditions permitted isolation of the en- were never identified. Endogenous Nup85 was detected only A modestly, even though it showed substantial expression and dogenous Y-complex (Fig. 4 ). However, the relative amount of biotinylation of other Y-Nups when fused to biotin ligase (Fig. endogenous Nup107 that was coprecipitated was significantly 2B and Dataset S1). This result suggests that Nup85 may not be less substantial than for GFP-Nup85, indicating that the trans- able to be efficiently biotinylated. By the more sensitive IB fected Nup43 and Nup85 likely interact independently of their analysis, low levels of endogenous Nup85 were detected in incorporation into the Y-complex or the NPC. BioID pull-downs for Nup160, Nup107, and Nup43 but not for To validate this result, we turned to an in vitro transcription Nup133 (Fig. 3). These results are not surprising, because and translation assay that revealed that Nup43 interacts with BioID-Nup85 detected Nup107 and, to a lesser extent, Nup160 Nup85 but with none of the other larger scaffold Y-Nups (Fig. and Nup43, suggesting proximity to these proteins. As a control 4B). Together these studies biochemically demonstrate a direct we reprobed these samples for Nup107 and observed detection interaction between Nup43 and Nup85. Thus, by demonstrating of this protein consistent with the MS results. These data reveal that Nup43 and Nup85 can associate with each other independently one limitation of BioID, namely, that not all proteins are bio- of their integration within the entire NPC, these results strengthen tinylated with similar efficiency. As in any large-scale experiment, a recent report that identified Nup43 residing in close proximity to negative results should be treated with caution. Nup85 and Seh1 by XL-MS (21).

Kim et al. PNAS Early Edition | 3of9 Downloaded by guest on September 23, 2021 Table 1. Summary of the proteins detected by BioID-Nups, BioID-LaA. and BirA*-only BirA* fusion protein baits

Identified candidates Nup160 Nup133 Nup107 Nup85 Nup43 Nup53 LaA BirA*

Nup107 complex (Y-complex + Elys) NUP160 Bait 0.1 0.1 NUP107 4.3 Bait 1.1 4.3 NUP133 Bait 7.0 0.4 0.2 NUP96 6.4 8.9 4.5 14.8 17.8 6.0 0.3 NUP85 Bait X NUP43 0.4 Bait SEH1L 0.6 7.7 X ELYS 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 0 Nup93 complex NUP53 Bait NUP93 0.1 NUP155 2.7 0.1 NUP205 0.3 NUP188 X 5.2 Nup62 complex NUP62* 5.6 17.2 NUP58/45 7.2 NUP54 1.6 Transmembrane nups POM121 1.8 1.4 0.5 0.2 2.6 1.4 NDC1 1.1 Percent of total that are expected Nup53 partners 210603820 Cytoplasmic nups NUP88 X 1.9 1.5 NUP214 0.9 8.7 12.5 <0.1 GLE1 0.4 CG1 5.3 DDX19B/DBP5 0.8 Cytoplasmic filament nups RANBP2/Nup358 5.1 6.5 25.1 13.5 4.0 4.7 0.8 RANGAP1 2.6 2.2 0.1 Nuclear pore basket NUP153 17.7 23.1 23.3 20 15.2 8.9 5.1 0.1 NUP50 1.2 2.8 11.7 16.5 5.4 1.3 4.4 TPR 11.7 1.2 SENP1 1.2 1.0 1.3 SENP2 6.7 1.3 5.2 NUP98 0.4 0.5 6.0 0.4 Import/export KPNB1 0.3 0.1 XPO1 0.3 Percent of total that are NPC-associated 47 61 92 94 54 86 15 2 Nuclear envelope constituents TMPO beta 25.9 28.9 4.3 12.7 1.3 LEMD3 3.5 0.6 3.7 <0.1 EMD 1.5 8.9 SYNE1 0.0 0.0 TMEM201 0.3 0.3 TMPO alpha 0.6 LBR 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 in bold indicate proteins identified by XL-MS on isolated Y complex or intact NPC. X, proteins identified by XL-MS but not BioID. BioID pull-down for LaA was performed with asynchronous cells.

Y-Nups as Macromolecular Rulers to Define the Practical Labeling Nup107, Nup85, and Nup43 (Fig. 5A). In contrast, the strong Radius of BioID Accurately. When analyzed in the context of detection of Nup96 in the case of Nup133 and the weaker iden- a monomeric Y-complex, the major BioID Y-Nup candidates tification of Y-Nups distantly positioned from the various baits appear positioned at 10–20 nm from the baits for Nup160, suggest a much larger radius (Table 1). However, a recent study

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1406459111 Kim et al. Downloaded by guest on September 23, 2021 but were largely absent from the BioID–Y-Nups results (Table PNAS PLUS 1). The identification of this subset of Nups connecting the pore membrane to the more central Nup62–Nup58–Nup54 complex is consistent with the proposed model of the inner pore complex of the NPC (23). Because of its flexibility (42, 43), the Nup62 complex may be further capable of sampling the membrane- proximate region where Nup53 resides. Also well represented in the BioID-Nup53 candidates are proteins reported to localize on the cytoplasmic side of the NPCs, nucleoporin Nup88 and nucleoporin Nup214, which are known to associate with each other (44–48), ATP-dependent RNA Ddx19/Dbp5, which is known to interact with Nup214 (49), and nucleoporin Gle1 and its binding partner nucleoporin hCG1/Npl1 (50, 51) (Fig. 5). The identification of these more distant partners might result from the intrinsic dynamics of Nup53, which has a reported residency half-time of ∼5 h in dividing cells (22), or from the existence of distinct population of the 32 copies of Nup53 within the NPC (52). However, these data also could reflect a more central 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 the Fig. 3. Nup85 is a poor substrate for BioID. IB analysis detects low levels of apparent bending of the Nup214–Nup88 complex toward the endogenous Nup85 (open arrowhead) in the Nup160, Nup107, and Nup43 central pore channel observed by cryo-ET (21). BioID pull-down samples and significant levels of the exogenous mycBirA*- Nup133 and Nup160 detected substantially more Pom121 Nup85 (arrowheads) in the Nup85 BioID pull-down. (Top) For clarity, total than any of the other Y-Nups that were tested. These two Nups lysates are shown at a lower exposure than the BioID samples. (Middle)For also were unique in detecting TMPO (also known as lamina- comparison, we detect similar levels of the BirA*-fusion proteins with anti- associated polypeptide 2, “Lap2”) and LEMD3 (also known as BirA in these same samples. (Bottom) Reprobing the same membrane with inner nuclear membrane protein, “Man1”), both of which are CELL BIOLOGY anti-Nup107 reveals levels of endogenous Nup107 (arrow) that correlate with the MS results, thus corroborating those results. Exogenous mycBirA*- transmembrane proteins located in the inner nuclear membrane Nup85 remains detected below the endogenous Nup107 (asterisk). of the NE. Detection of these candidates indicates the proximity of Nup133 and Nup160 to the NPC membrane, a property consistent with previously published data (21, 36, 54, 55). provides a compelling model for the mammalian NPC in which Conversely, Nup85 detected substantially more Nup214 than offset Y-complex dimers are arranged in a head-to-tail staggered any of the other tested Y-Nups, a result supported by a recent parallel fashion to form ring-like structures on the nucleoplasmic cryo-ET study (21). Moreover, BioID-Nup85 was unique among and cytoplasmic sides of the pore (21). With this organization it is the tested Y-Nups in its ability to detect nucleoporin Nup62 and clear that in some instances we are likely observing intercomplex nucleoporin Nup88. Nup62 is a constituent of a subcomplex rather than intracomplex labeling by BioID. Prime examples are located in the central channel of the pore, whose other con- the detection of Nup96 by BioID-Nup133 and of Nup107 by stituents (nucleoporin Nup58/45 and nucleoporin Nup54) are BioID-Nup85 or BioID-Nup43 (Table 1 and Fig. 5B). Reevaluat- not detected by BioID-Nup85. Although its detection may re- ing the BioID results from the Y-complex in the context of the veal distinct positioning of Nup62 compared with Nup58/45 and Nup54 (42), Nup62 also was proposed to associate with Nup88 whole NPC, we thus can restrict the practical labeling radius of Xenopus – BioID (defined as the ability to detect proteins by MS following and Nup214 in a distinct complex in egg extracts (56 58), as was demonstrated for its ortholog, Nsp1, in budding yeast BioID pull-down) to ∼10 nm (Fig. 5B). However, because not all (59). Our BioID results thus highlight the existence of a Nup62– Nups within 10 nm are labeled by a BioID-Nup bait, one again Nup88–Nup214 complex in human cells and indicate that Nup85 must view negative results with caution. likely projects toward the Nup88–Nup214–Nup62 complex. Finally, among the identified NPC-associated nuclear basket Refining NPC Organization with BioID. Keeping in mind the esti- constituents, the structural nucleoprotein Tpr (39) was detected mated labeling radius, we then analyzed the NPC constituents solely by BioID-Nup107, whereas the SUMO isopeptidase outside the stable Y-complex that were detected by BioID-Nups to sentrin-specific protease 2 (SENP2), previously reported to as- get insight into the whole NPC architecture (Fig. 6 and Table 1). sociate with the Y-complex (60), was strongly detected by Among the identified Nups, a few, most notably FG-Nups as- BioID-Nup133 and BioID-Nup43 and to a lesser extent by sociated with the cytoplasmic filaments (nucleoporin Nup358/ BioID-Nup107. Our study thus now enables us to position Tpr RANBP2) or the nuclear basket (nucleoporin Nup50 and nucleo- near Nup107 and SENP2 near the head-to-tail connections porin Nup153), were substantially detected by all Y-Nups. Among between Y-complex dimers (Fig. 5B). them, Nup153 was reported previously to associate with the Y-complex (18), and recent crosslinking-MS (XL-MS) and cryo- Conclusions electron tomography (cryo-ET) studies recognized the proximity Using a stable protein complex as a molecular ruler, we determined of Nup358 and the Y-complex (21). However, the identification the practical labeling radius of BioID in vivo to be ∼10 nm. We of these large, flexible, or dynamic Nups (22, 38–41) by BioID- also demonstrated that, when applied to proteins within distinct Nup53 suggests that these FG-Nups may associate with multiple regions of the NPC, BioID is capable of detecting distinct pop- Nups only transiently as they sample the NPC environment. ulations of candidate proteins. The recent studies on the human Nevertheless, most Nups were identified in a restricted subset Y-complex that use XL-MS provide some comparisons with BioID of BioID samples, thus validating or refining our actual knowl- (21) (Table 1). XL-MS permits the identification and precise edge of NPC organization. In particular, the more central NPC (amino acid resolution) mapping of extremely close interactions constituents, such as those in the Nup93 and Nup62 complexes (within a couple of nanometers), whereas BioID has a much larger and Ndc1, were all identified when we applied BioID to Nup53 radius and thus far does not allow the mapping of biotinylated

Kim et al. PNAS Early Edition | 5of9 Downloaded by guest on September 23, 2021 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 endogenous Nup107 (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.

residues within the prey proteins. However, because of sample additional insights into the assembly and function of NPC complexity, the identification by XL-MS of cross-linked peptides constituents. from complex assemblies, such as intact NPCs within purified NEs, appears quite challenging. This complexity explains the Materials and Methods rather low number of confidently assigned interactions (17 in Plasmids. Nup85, Nup107, Nup133, Nup160, Nup53, and Nup96 were amplified total, of which 11 involved Y-Nups or Nup53) in this XL-MS by PCR from human cDNA. The PCR products were digested (by XhoI and study (21). In contrast, BioID does not require prior purification BamHI for Nup85, Nup107, and Nup160; by XhoI and EcoRI for Nup133; by XhoI of organelles and is technically far less demanding. Thus BioID is and HindIII for Nup53; by XhoI and AflII for Nup96) and inserted into mycBioID pcDNA3.1 (35700; Addgene). Nup43 was amplified and digested with NheI and a useful tool for scientists interested in probing the protein EcoRI. The digested PCR product was inserted into BioID-HA pcDNA 3.1 (36047; constituency and mapping the organization of large structural Addgene). Human LaA was inserted into mycBioID pcDNA3.1 following di- protein assemblies. In this way it provides a complementary gestion with XhoI and AflII (1). Nup43-HA was PCR-amplified using a reverse approach to XL-MS. Future studies, including baits from other primer containing the HA-tag sequence and was inserted into pcDNA 3.1 NPC subcomplexes and evaluation of BioID candidates of the after NheI and PmeI digestion. GFP-Nup85, GFP-Nup107, GFP-Nup96, GFP- Y-complex during discrete stages of the cell cycle, should provide Nup133, and GFP-Nup160 and were used as previously reported (10).

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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-complex subunit 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-complex dimers are arranged in a head-to-tail fashion within the NPC (Left). The approximate positions of Y-Nups are labeled and schematized (Right)onthis map. (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 abundance of biotinylated Y-Nups for BioID-fusion proteins. The gray disks (dark: 5-nm radius; light: 10-nm radius) provide an approximation of the labeling radius of BioID.

Antibodies. Mouse monoclonal anti-Nup153 (SA1) was used as previously arrest cell division, and promiscuous biotinylation by the BioID-Nups was reported (61). Rabbit anti-Nup107 was used as previously described (19). Rabbit induced by the addition of biotin to this cell-culture medium to a final polyclonal anti-HA (ab9110; Abcam), anti-myc (ab9106; Abcam), anti-GFP (ab290; concentration of 50 μM for 18 h. For the co-IP experiments shown in Fig. 3B, Abcam), anti-Nup85 (A303-977A; Bethyl Laboratories), chicken polyclonal anti- 2.4 × 106 HEK293 cells were cotransfected with equal amounts of HA-Nup43 BirA (ab14002; Abcam), mouse monoclonal anti-tubulin (T9026; Sigma) and and GFP-Y-Nups plasmids (1 μg each) 24 h before co-IP. mAb414 (MMS-120p-500; Eurogentek) were used as primary antibodies. Immunostaining. HEK293 cells were fixed in 3% (wt/vol) paraformaldehyde/

Cell Lines and Transfection. Human HEK293 cells were maintained in 5.0% CO2 at PBS for 10 min and permeabilized using 0.4% Triton X-100/PBS for 15 min 37 °C in DMEM (SH3024301; HyClone) supplemented with 10% (vol/vol) FBS. To followed by 0.5% SDS/PBS for 10 min. After fixation and permeabilization, generate cells stably expressing BioID fusion proteins, HEK293 cells were trans- cells were labeled with appropriate primary and secondary antibodies for 20 fected via Lipofectamine 2000 (Life Technologies) using the manufacturer’srec- min at 25 °C in 0.4%Triton X-100/PBS. Primary antibodies were detected with ommended protocols and subjected to G418 (700 μg/mL) selection. Subclones of Alexa-Fluor 568–conjugated goat anti-mouse (A11031; Life Technologies) cells that expressed low levels of the fusion protein were chosen to minimize po- or goat anti-rabbit (A11036; Life Technologies) secondary antibodies. tential artifacts associated with spill-over of fusion proteins to sites other Alexa-Fluor 488–conjugated streptavidin (S32354; Life Technologies) was than NPCs. Before all the analyses described in this report, cells were growth used to detect biotinylated proteins. DNA was detected with Hoechst dye arrested by incubation in DMEM supplemented with 0.1% FBS for 72 h to 33258. Coverslips were mounted in 10% (wt/vol) Mowiol 4–88 (17951;

Kim et al. PNAS Early Edition | 7of9 Downloaded by guest on September 23, 2021 Fig. 6. Biotinylation of NPC constituents generally correlates with the location of the fusion protein. The candidate Nups identified in this BioID studies are positioned within a simplified model of NPC organization that integrates data from the literature and extrapolations based on previous studies in budding yeast. 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 candidates correlates with the level of detection of candidates predominantly detected by the different types of BioID-fusion proteins (Table 1). In A, biotinylated Y-Nups are 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 multiple locations within the NPC and are unlikely to be biotinylated at that specific place.

Polysciences). Images were obtained using Nikon A1-confocal microscope beads–proteins suspension mix, and proteins were reduced at 40 °C for 30 min. (60×/1.49 oil APO TIRF Nikon objective) and a CCD camera (CoolSnap HQ; Then 8 μL of 0.5 M Iodoacetamide was added, and proteins were alkylated at Photometrics) linked to a workstation running NIS-Element software (Nikon). room temperature in the dark for 30 min. MS-grade trypsin (Promega) was added (1:20 ratio) for overnight digestion at 37 °C using an Eppendorf Ther- Immunoblot and Immunoprecipitation. For immunoblot of total cell lysates, momixer at 700 rpm. Digested peptides were separated from magnetic beads 1.2 × 106 cells were lysed in SDS/PAGE sample buffer, sonicated to shear by centrifugation and a GE Healthcare MagRack and were transferred to DNA, and boiled for 5 min. For co-IP analyses transiently transfected HEK293 a new tube. Formic acid was added to the peptide solution (to 2%), followed cells (2.4 × 106) were lysed in 1 mL of IP lysis buffer [50 mM Tris (pH 7.5), 150 by desalting by Microtrap (catalog no. 77720; Thermo) and then on-line

mM NaCl, 2.5 mM MgCl2, 1 mM DTT, 1% Triton X-100, and 1× proteinase analysis of peptides by high-resolution, high-mass accuracy liquid chroma- inhibitor (1861278; Thermo Scientific)]. Lysates were passed through tography tandem MS (LC-MS/MS) consisting of a Michrom HPLC, a 15-cm a 21-gauge needle 10 times and centrifuged at 16,500 × g for 10 min at 4 °C. Michrom Magic C18 column, a low-flow ADVANCED Michrom MS source, and – The supernatants were rotated overnight at 4 °C with 20 μL of protein A a LTQ-Orbitrap XL (Thermo Fisher Scientific). A 120-min gradient of 10 30% B Sepharose beads (20365; Thermo Scientific) and 2 μg of rabbit anti-HA an- (0.1% formic acid, 100% acetonitrile) was used to separate the peptides. The tibody. Samples were washed thoroughly three times with the IP lysis buffer total LC time was 140 min. The LTQ-Orbitrap XL was set to scan precursors in and twice with wash buffer [50 mM Tris (pH 7.5) and 50 mM NaCl] at 4 °C. the Orbitrap followed by data-dependent MS/MS of the top 10 precursors. Proteins were solubilized in 25 μL SDS/PAGE sample buffer and boiled for Raw LC-MS/MS data were submitted to Sorcerer Enterprise (Sage-N Research 5 min. Proteins were separated on 8% SDS/PAGE and were transferred to Inc.) for protein identification against the ipi.HUMAN.vs.3.73 protein database, nitrocellulose membrane (Bio-Rad), which subsequently was blocked [10% which contains semitryptic peptide sequences with the allowance of up to two (vol/vol) adult bovine serum, 0.2% Triton X-100, 1× PBS] and incubated with missed cleavages. Differential search included 16 Da for methionine oxidation, appropriate primary antibodies overnight at 4 °C. After washes with 57 Da for cysteines to account for carboxyamidomethylation, and 226 Da blocking buffer, blots were incubated with HRP-conjugated anti-mouse for biotinylation of lysine. Search results were sorted, filtered, statically analyzed, and displayed using PeptideProphet and ProteinProphet (In- (F21453; Life Technologies), anti-rabbit (G21234; Life Technologies), or stitute for Systems Biology). The minimum Trans-Proteomic Pipeline (TPP) anti-chicken (A9046; Sigma) antibodies to detect proteins following en- probability score for proteins was set to 0.95 to assure a TPP error rate lower hanced chemiluminescence. To detect biotinylated proteins, High Sensi- than 0.01. The relative abundance of each of the identified proteins in dif- tivity Streptavidin-HRP (21130; Thermo Scientific) was used as previously ferent samples was analyzed by QTools, an open-source tool developed in-house described (1, 33). The in vitro transcription and translation was performed by for automated differential peptide/protein spectral count analysis (62). Proteins using TnT Quick Coupled Transcription/Translation Systems (Promega) with the detected in the control sample (cells lacking BirA*) and common BioID back- manufacturer’s recommended protocol. For 20-μLreactions,2μL was reserved ground proteins (BirA*-only) (Dataset S2) were subtracted from the results unless for total protein analysis. The remaining volume was added to 0.5 mL of the IP their abundance was threefold more than in the BirA*-only. For all datasets, the lysis buffer. IP and IB steps were performed as described above. total spectral counts for each protein then were normalized to account for the total length of the protein in amino acids. The relative abundance of each prey BioID, On-Bead Protein Digestion, and Identification by 1D LC-MS/MS. Large- finally was expressed as percentage of the sum of all of the adjusted spectral × 7 scale (4 10 cells) BioID pull-downs for MS analysis were performed as counts except those of the BioID-fusion protein within a given BioID sample. previously described with the exception that pooled lysates were incubated in a 15-mL conical tube overnight at 4 °C before washing. Ninety percent of ACKNOWLEDGMENTS. We thank Brian Burke and Benoit Palancade for each sample was used for MS analysis, and 10% was reserved for IB analysis. helpful discussions and advice. These studies were supported by Grants Sample volume was adjusted to 200 μL with 50 mM ammonium bicarbonate. RO1GM102203, RO1GM102486, and RO1EB014869 (to K.J.R.) from the National Then 4 μL of 0.5 M Tris(2-carboxyethyl)phosphine was added to 200 μLofthe Institutes of Health; Sanford Research startup funds (K.J.R.); French National

8of9 | www.pnas.org/cgi/doi/10.1073/pnas.1406459111 Kim et al. Downloaded by guest on September 23, 2021 Research Agency Grant ANR-12-BSV2-0008-01 (to V.D.); and Fondation ARC by Institutional Development Awards from the National Institute of General PNAS PLUS pourla Recherche sur le (V.D.). This project used the Imaging Core Medical Sciences and the National Institutes of Health under Grants and Protein Biochemistry Core at Sanford Research, which are supported P20GM103548 and P20GM103620.

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