REFERENCES AND NOTES

1. J. J. Linz, A. Stepan, Problems of Democratic Transitionand Consolidation: Southern Europe, South America, andPost-Communist Europe (Johns Hopkins Univ. Press,Baltimore, 1996).

2. L. Diamond, Developing Democracy: Toward Consolidation(Johns Hopkins Univ. Press, Baltimore, 1999).

3. E. Fehr, K. Hoff, Econ. J. 121, F396–F412 (2011).4. A. Alesina, E. L. Glaeser, Fighting Poverty in the US

and Europe: A World of Difference (Oxford Univ. Press,Oxford, 2004).

5. J. Henrich et al., Science 327, 1480–1484 (2010).6. A. Alesina, G.-M. Angeletos, Am. Econ. Rev. 95, 960–980

(2005).7. R. Bénabou, J. Tirole, Q. J. Econ. 121, 699–746 (2006).8. T. Piketty, Q. J. Econ. 110, 551–584 (1995).9. A. Alesina, N. Fuchs-Schündeln, Am. Econ. Rev. 97, 1507–1528

(2007).10. E. F. P. Luttmer, M. Singhal, Am. Econ. J. Econ. Policy 3,

157–179 (2011).11. U. Malmendier, S. Nagel, Q. J. Econ. 126, 373–416 (2011).

12. T. Persson, G. Tabellini, Am. Econ. J. Macroecon. 1, 88–126(2009).

13. R. Inglehart, Polit. Sci. Politics 36, 51–57 (2003).14. R. Inglehart, C. Welzel, Comp. Polit. 36, 61–79 (2003).15. M. Bratton, J. Democracy 15, 147–158 (2004).16. M. G. Marshall, K. Jaggers, Polity IV Project: Dataset Users’

Manual (Center for Global Policy, George Mason University,Fairfax, VA, 2005).

17. A small number of deviating cases, because of dataconstraints, are described in the supplementary materials.Note that because we include country or even country-yearfixed effects in all specifications and thus identify thecoefficients only through changes within a country (or acountry-year), the choice of the start year is in fact innocuous.

18. We follow standard specifications in the literature and includesome basic demographic characteristics of the respondentsas controls, namely variables related to age, gender, andeducation. Because our focus lies on establishing a causalrelationship, we omit likely endogenous attitudinal variables[as analyzed in, e.g., (20)].

19. A. Karatnycky, J. Democracy 14, 100–113 (2003).20. R. Mattes, M. Bratton, Am. J. Polit. Sci. 51, 192–217 (2007).

ACKNOWLEDGMENTS

Supported by the European Research Council under starting grant262116 (N.F.-S.) and by the research cluster “Formation ofNormative Orders” at Goethe University Frankfurt. Data used in theanalysis are described in the supplementary materials. All dataused for this study can be downloaded from publicly availablewebsites. World Values Survey data are available fromwww.worldvaluessurvey.org, Afrobarometer survey data fromwww.afrobarometer.org, Polity IV data from www.systemicpeace.org/polity/polity4.htm, and Freedom House data from www.freedomhouse.org. Statistical programs to replicate the analysisare archived in Dataverse (doi: 10.7910/DVN/29151).

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/347/6226/1145/suppl/DC1Materials and MethodsFig. S1Tables S1 to S6References (21–24)

16 October 2014; accepted 29 January 201510.1126/science.aaa0880

NUCLEAR PORES

Architecture of the nuclear porecomplex coatTobias Stuwe,1* Ana R. Correia,1* Daniel H. Lin,1 Marcin Paduch,2 Vincent T. Lu,2

Anthony A. Kossiakoff,2 André Hoelz1†

The nuclear pore complex (NPC) constitutes the sole gateway for bidirectionalnucleocytoplasmic transport. Despite half a century of structural characterization, thearchitecture of the NPC remains unknown. Here we present the crystal structure of areconstituted ~400-kilodalton coat nucleoporin complex (CNC) from Saccharomycescerevisiae at a 7.4 angstrom resolution. The crystal structure revealed a curved Y-shapedarchitecture and the molecular details of the coat nucleoporin interactions forming thecentral “triskelion” of the Y. A structural comparison of the yeast CNC with an electronmicroscopy reconstruction of its human counterpart suggested the evolutionaryconservation of the elucidated architecture. Moreover, 32 copies of the CNC crystalstructure docked readily into a cryoelectron tomographic reconstruction of the fullyassembled human NPC, thereby accounting for ~16 megadalton of its mass.

Thenuclear pore complex (NPC) is composedof ~34 different proteins, termed nucleo-porins (Nups), that assemble in numerouscopies to yield a ~120MD transport channelembedded in the nuclear envelope (NE)

(1). To facilitate the extensive membrane curva-ture generated in each NE pore, NPCs require amembrane-bending coat. TheNPC coat is believedto be formed by an evolutionarily conserved coatNup complex (CNC), the Nup107/160 complex inhumansand theNup84complex in Saccharomycescerevisiae, the latter of which is composed ofNup120, Sec13, Nup145C, Seh1, Nup85, Nup84,and Nup133 (1, 2).We reconstituted a heterohexameric CNC con-

taining the yeast Nups Nup120, Sec13, Nup145C,

Seh1, Nup85, and the Nup84 N-terminal domain(NTD) (Fig. 1, A and B). Our reconstituted CNCdid not include Nup133 because this nup is con-formationally flexible and loosely associated(2–4). Because the initial crystals of this recon-stituted CNC diffracted poorly, we generated aseries of conformation-specific, high-affinity syn-thetic antibodies (sABs) and tested them as crys-tallization chaperones (5). This approach yieldedcrystals of the CNC in complexwith sAB-57, whichallowed us to solve the structure to 7.4 Å by mo-lecular replacement, using high-resolution crystalstructures of CNC components and the sAB scaf-fold (figs. S1 and S2) (6–10). The inclusion of asecond sAB (sAB-87) produced another crystalform, for which we collected anomalous x-raydiffraction data of Seleno-L-methionine and heavymetal–labeled crystals to confirm the placementof the CNC components (figs. S1 to S3). Becausethe coatNups in bothCNC•sAB complexes adoptedthe same arrangement, we focused our analy-sis on the better-ordered CNC•sAB-57 structure(figs. S4 to S6).

The CNC adopted a curved Y-shaped structurespanning ~250 Å in length and width, consistentwith previous negative-stain electronmicroscopy(EM) analyses (Fig. 1C and movie S1) (2–4, 11).The Seh1•Nup85 pair and Nup120 constitutedthe upper arms of the Y, which were connectedto the rest of the CNC through a central triskel-ion. Sec13•Nup145C•Nup84NTD formed the stalkat the bottom of the triskelion and would attachthe tail formed by Nup84CTD and Nup133, whichwere absent in the structure. Both arms curvedout so that the Nup120 b-propeller domain wasperpendicular to the plane of the Y. Nup145Corganized the CNC through four distinct inter-action surfaces contacting nearly every memberof the complex. sAB-57 bound at the Nup145C-Nup85 interface and formed crystal packing con-tacts (Fig. 2 and fig. S4).The C-terminal domains (CTDs) of Nup145C

(residues 553 to 712), Nup85 (residues 545 to 744),and Nup120 (residues 729 to 1037) convergedto form the CNC triskelion. Although we ob-served clear electron density that revealed theconnectivity of the three CTDs and their inter-actions (Fig. 2 and fig. S2), the sequence registerin the triskelion was only approximate because ofthe absence of side-chain density. Nup120CTDwassandwiched between Nup85CTD and Nup145CCTD,and no direct contacts were observed betweenNup85CTD and Nup145CCTD (Fig. 2, A and B). Theinteractions between Nup85CTD, Nup145CCTD, andNup120CTDweremediated predominantly by theirmost C-terminal helices. An additional interac-tion was made by an N-terminal Nup145C helixbound to a groove in theNup85CTD surface ~60Åaway from the triskelion center, an interactionthat was recognized by sAB-57 (Fig. 2C).Consistent with our structural data, we recon-

stituted a stoichiometric complex betweenNup120and Nup85CTD as monitored by size-exclusionchromatography interaction experiments (fig. S7A).Furthermore, Nup120 failed to interact withSec13•Nup145C in the absence of Nup145CCTD

(fig. S7, B and C). The interaction betweenSeh1•Nup85 and Sec13•Nup145C depended onthe presence of an N-terminal Nup145C fragment

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1Division of Chemistry and Chemical Engineering, CaliforniaInstitute of Technology, 1200 East California Boulevard,Pasadena, CA 91125, USA. 2Department of Biochemistry andMolecular Biology, University of Chicago, Chicago, IL 60637, USA.*These authors contributed equally to this work.†Corresponding author: E-mail: hoelz@caltech.edu

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(residues 75 to 125) (fig. S7, D and E). Furthermapping identified a region of Nup145C (resi-dues 75 to 109) that was sufficient for Nup85CTD

binding (fig. S7F), confirming that this fragmentbridged the two subcomplexes. Nup120CTD andNup85CTD were essential for the formation of theCNC (figs. S8 and S9). These data were consistentwith published CNC cross-linking data of threedifferent species, more so than the models gen-erated by coarse-grained analysis (3, 11, 12). Lastly,to validate their placement in the structures, weconfirmed the interactions of sAB-57 and sAB-87with Seh1•Nup85•Nup145C1–123 and Nup120NTD,respectively (fig. S10).Next, we compared the CNC structure to pre-

viously determined EM reconstructions of theyeast and human CNCs (3, 4). Although the EMreconstruction of the yeast CNC recapitulates itsoverall shape, significant deviations were appar-ent (fig. S11 and movie S2). No density was ob-served in the EM structure for the U-shaped tipof Nup85. The overall shape of the crystal struc-ture was also consistent with the human CNC EMreconstruction, which contains the crystallizedevolutionarily conserved core as well as two addi-tional human components, Nup37 and Nup43(Fig. 3A and movie S3). Our crystal structuredid not account for additional EM density di-rectly adjacent to the Seh1•Nup85 and Nup120arms, which reportedly accommodate Nup43 andNup37, respectively (Fig. 3A) (3). In the humanCNC, Nup43 appears to bind to the same site assAB-57 on the Seh1•Nup85 armof the Y. Themajordifference between the CNC crystal structure andboth EM reconstructions is the curvature ofthe arms of the Y, and thus the orientation of

the Nup120 b propeller was substantially differ-ent in the crystal structure (Fig. 3A). The flatnessof both EM reconstructions suggests that thesedeviations may be a result of EM sample prepa-ration. Despite this, the degree of similarity be-tween the yeast CNC crystal structure and thehuman CNC EM reconstruction suggested sub-stantial evolutionary conservation of the CNCarchitecture.The higher-order arrangement of CNCs in a

fully assembled NPC has been debated, with sev-eral models proposed based on various struc-tural, biophysical, or computational approaches(3, 7, 13, 14). Given the evolutionary conservationof the CNC architecture, we tested whether ourcrystal structure could be docked into the ~32 Å–resolution tomographic reconstruction of anintact human NPC (3). Indeed, an unbiased six-dimensional searchcombinedwithacross-correlationanalysis confidently docked 32 copies of the CNCcrystal structure in the tomographic reconstruc-tion, yielding a model for the NPC coat (Figs. 3Band 4A and fig. S12). These results agreed withthe stoichiometry and approximate localizationpreviously proposed based on cross-linking massspectrometry and the docking of the humanCNCEM reconstruction (3). However, the crystalstructure fit the tomographic reconstruction sub-stantially better than the human CNC EM recon-struction (Fig. 3B).The NPC coat was formed by 32 copies of the

CNC arranged in four eight-membered rings(Fig. 4, A and B, and movie S4). The eight CNCsin each ringwere oriented horizontally, with theirlong axis positioned parallel to the surface of theNE in a head-to-tail fashion. On each side of the

NE, a pair of inner and outer CNC rings emergedup to ~210 Å (Fig. 4A). These rings were sepa-rated by a ~280 Å gap, yielding a total height of~700 Å. The diameters of the outer CNC ringswere slightly larger than those of the inner CNCrings, spanning ~1200Å and ~1050Å, respectively(Fig. 4B). Although the CNCs in both rings werearranged with the same directionality, each CNCfrom the outer ring was offset from its mate inthe inner ring by ~120 Å in a clockwise direction(Fig. 4B). Moreover, the tandem CNC rings onthe nuclear and cytoplasmic side of the NE pos-sessed the same handedness and were related bytwofold rotational symmetry (Fig. 4Aand fig. S12D).The unambiguous placement and orientation

of the coat Nups and their conserved surfaces al-lowed for an investigation into the details of theirinteractions in the assembledNPC coat. Each CNCwas situated on top of the NE andwas oriented sothat the plane of the Ywas nearly perpendicular tothemembrane, with the Nup120 and Seh1•Nup85arms pointed at or away from the membrane, re-spectively (Fig. 4A). Only two interfaces appearedto be responsible for oligomerization of individ-ual CNCs into the NPC coat. The inner and outerrings interacted only where the top of the tris-kelion of each inner ring CNC met the bottom ofthe Nup84-Nup145C interface of each outer-ringCNC (Fig. 4C). Nup120 was oriented so that itsNup133 binding site was directly adjacent to thedensity assigned to the N terminus of Nup133,which is consistent with our previous findingsthat this interaction is responsible for CNC ringformation and critical for NPC assembly (Fig. 4D)(9). This Nup120 orientation also pointed the apexof its b propeller directly toward the NE, which

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Fig. 1. Overall architecture of the CNC. (A) Domain structures of the yeast coat Nups and sAB-57. Black lines indicate the crystallized fragments. U, unstructured;D, domain invasion motif; VH, heavy chain variable region; CH, heavy chain constant region; VL, light chain variable region; CL, light chain constant region.(B) Reconstitution of the yeast CNC•sAB-57, lackingNup133. Elution profiles fromaSuperdex 200 10/300 columnare shown forNup120•Seh1•Nup85 (trimer 1),Sec13•Nup145C•Nup84NTD (trimer 2), CNC, and CNC•sAB-57 (left). An SDS–polyacrylamide gel electrophoresis gel of the reconstituted CNC•sAB-57 used forcrystallization is shown (right). (C) Cartoon and schematic representations of the yeast CNC•sAB-57 crystal structure viewed from two sides.

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was the onlymembrane contact that we observedin our model (Fig. 4D). This region of the Nup120b-propeller domain also contains a conserved sur-face patch on its side (9) that may serve as a NEanchor point for the entire NPC coat, either

through a direct interaction with the membraneor via a membrane-anchored Nup, as previouslyreported (15).The NPC coat architecture is dissimilar to that

of other structurally characterized membrane

coats. Whereas the latter are generated by homo-typic vertex elements (16, 17), the NPC coat isformed by heterotypic interactions of its asym-metric CNC protomers. Given the location of theCNC rings above and below the NE, other Nups

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Fig. 2. Architecture of the CNC triskelion. Car-toon representation of the triskelion formed byNup120, Nup85, and Nup145C. Insets (A) to (C)depict magnified views of the interactions be-tween (A) Nup120CTD, Nup85CTD, and Nup145CCTD;(B) Nup120CTD, Nup85CTD, and the N-terminalNup145C helix; and (C) Nup145C, Nup85CTD, andsAB-57.The density-modified electron density mapis contoured at 1.0s.

Fig. 3. Comparison of yeast and human CNCs. (A) Fit of the yeast CNC crystalstructure into the human CNC negative-stain EM reconstruction (gray) (3).Arrows indicate density accounted for by the additional human coat NupsNup37 or Nup43. (B) Comparison of the quality of fit for the yeast CNC crystalstructure and human CNC EM reconstruction (cyan) into the intact human NPCcryoelectron tomographic reconstruction (gray) (3). Arrows indicate regionswhere the human CNC EM reconstruction protrudes from the cryoelectrontomographic reconstruction.

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are likely to play a role in generating the complexcurvature of the NE pores. Although the place-ment of the CNCs in the NPC coat did not directlyaddress the organization of the central trans-port channel (fig. S13), it accounted for ~16 MDof the total mass of the NPC, bridged the reso-lution gap between low-resolution EM analysesand high-resolution crystallographic studies, andsuggested the evolutionary conservation of itsarchitecture.

REFERENCES AND NOTES

1. A. Hoelz, E. W. Debler, G. Blobel, Annu. Rev. Biochem. 80, 613–643(2011).

2. M. Lutzmann, R. Kunze, A. Buerer, U. Aebi, E. Hurt, EMBO J. 21,387–397 (2002).

3. K. H. Bui et al., Cell 155, 1233–1243 (2013).4. M. Kampmann, G. Blobel, Nat. Struct. Mol. Biol. 16, 782–788 (2009).5. M. Paduch et al., Methods 60, 3–14 (2013).6. E. W. Debler et al., Mol. Cell 32, 815–826 (2008).7. K. C. Hsia, P. Stavropoulos, G. Blobel, A. Hoelz, Cell 131,

1313–1326 (2007).8. V. Nagy et al., Proc. Natl. Acad. Sci. U.S.A. 106, 17693–17698

(2009).9. H. S. Seo et al., Proc. Natl. Acad. Sci. U.S.A. 106, 14281–14286

(2009).10. S. S. Rizk et al., Nat. Struct. Mol. Biol. 18, 437–442 (2011).11. K. Thierbach et al., Structure 21, 1672–1682 (2013).12. Y. Shi et al., Mol. Cell. Proteomics 13, 2927–2943 (2014).13. F. Alber et al., Nature 450, 695–701 (2007).14. S. G. Brohawn, N. C. Leksa, E. D. Spear, K. R. Rajashankar,

T. U. Schwartz, Science 322, 1369–1373 (2008).15. J. M. Mitchell, J. Mansfeld, J. Capitanio, U. Kutay,

R. W. Wozniak, J. Cell Biol. 191, 505–521 (2010).

16. S. C. Harrison, T. Kirchhausen, Nature 466, 1048–1049(2010).

17. E. W. Debler, K. C. Hsia, V. Nagy, H. S. Seo, A. Hoelz, Nucleus 1,150–157 (2010).

ACKNOWLEDGMENTS

We thank C. J. Bley, W. M. Clemons, A. M. Davenport,O. Dreesen, A. Patke, D. C. Rees, S. O. Shan, P. Stravropoulos,and K. Thierbach for critical reading of the manuscript; K. Katoand K. Kato for their contributions at the initial stages of thisproject; L. N. Collins for technical support; D. King for massspectrometry analysis; E. Hurt and P. Loppnau for material;S. Koide for providing the phage display library; S. Gräslund forthe pSFV4 vector; P. Afonine for advice regarding structurerefinement in PHENIX; and J. Kaiser and the scientific staffof Stanford Synchrotron Radiation Laboratory (SSRL) Beamline12-2 and the National Institute of General Medical Sciences andNational Cancer Institute Structural Biology Facility (GM/CA) at the

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Fig. 4. Architecture of the NPC coat. (A) Thirty-two copies of the yeastCNC, shown in cartoon representationwith a representative subunit colored asin Fig. 1, docked into the cryoelectron tomographic reconstruction of the intacthumanNPC (3), shown as a gray surface.The outer and inner cytoplasmic andnuclear CNC rings are highlighted in orange, cyan, pink, and blue, respectively.(B) Cartoon representations of 16 yeastCNC copies from the cytoplasmic sideof the NPC coat. Schematics indicating the positions assigned to Nup84CTD

and Nup133,which were not crystallized, are shown. (C) Interface between the

inner and outer CNC rings.Two views of the yeast CNC and its mate from theinner ring are shown. (D) Orientation of the Nup120 b propeller relative toneighboring coat Nups and the membrane. Portions of two CNCs from thecytoplasmic outer ring are shown in cartoon representation. Green and cyanshading indicate the positioning of Nup84CTD and Nup133, respectively. Thecyan line represents the N-terminal unstructured segment of Nup133 thatbinds to Nup120 (9). A schematic representation of the ring-forming Nup120-Nup133 interaction is shown below.

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Advanced Photon Source (APS) for their support with x-raydiffraction measurements. We acknowledge the Gordonand Betty Moore Foundation, the Beckman Institute, and theSanofi-Aventis Bioengineering Research Program for theirsupport of the Molecular Observatory at the California Instituteof Technology (Caltech). The operations at the SSRL and APSare supported by the U.S. Department of Energy and theNational Institutes of Health (NIH). GM/CA has been funded inwhole or in part with federal funds from the National CancerInstitute (ACB-12002) and the National Institute of GeneralMedical Sciences (AGM-12006). T.S. was supported by aPostdoctoral Fellowship of the Deutsche Forschungsgemeinschaft.

D.H.L. was supported by a NIH Research Service Award(5 T32 GM07616). A.A.K. was supported by NIH awards U01GM094588 and U54 GM087519 and by Searle Funds at TheChicago Community Trust. A.H. was supported by Caltechstartup funds, the Albert Wyrick V Scholar Award of theV Foundation for Cancer Research, the 54th MallinckrodtScholar Award of the Edward Mallinckrodt Jr. Foundation, and aKimmel Scholar Award of the Sidney Kimmel Foundation forCancer Research. The coordinates and structure factors havebeen deposited with the Protein Data Bank with accession codes4XMM and 4XMN. The authors declare no financial conflictsof interest.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/347/6226/1148/suppl/DC1Materials and MethodsFigs. S1 to S13Tables S1 and S2References (18–28)Movies S1 to S4

18 September 2014; accepted 27 January 2015Published online 12 February 2015;10.1126/science.aaa4136

PROTEIN TARGETING

Structure of the Get3 targeting factorin complex with its membraneprotein cargoAgnieszka Mateja,1 Marcin Paduch,1 Hsin-Yang Chang,1 Anna Szydlowska,1

Anthony A. Kossiakoff,1 Ramanujan S. Hegde,2* Robert J. Keenan1*

Tail-anchored (TA) proteins are a physiologically important class of membrane proteinstargeted to the endoplasmic reticulum by the conserved guided-entry of TA proteins(GET) pathway. During transit, their hydrophobic transmembrane domains (TMDs) arechaperoned by the cytosolic targeting factor Get3, but the molecular nature of thefunctional Get3-TA protein targeting complex remains unknown. We reconstituted thephysiologic assembly pathway for a functional targeting complex and showed that itcomprises a TA protein bound to a Get3 homodimer. Crystal structures of Get3 bound todifferent TA proteins showed an α-helical TMD occupying a hydrophobic groove that spansthe Get3 homodimer. Our data elucidate the mechanism of TA protein recognition andshielding by Get3 and suggest general principles of hydrophobic domain chaperoning bycellular targeting factors.

Integral membrane proteins contain hydro-phobic transmembrane domains (TMDs) thatmust be shielded from the cytosol until theirinsertion into the lipid bilayer. Whereas mosteukaryoticmembrane proteins are cotransla-

tionally targeted to the endoplasmic reticulum(ER) by the signal recognition particle (SRP) (1),tail-anchored (TA) membrane proteins are post-translationally targeted by the cytosolic factorGet3 (2–7). This conserved adenosine triphos-phatase (ATPase) changes conformation in anucleotide-regulated manner (8–12) to bind TMDsin the cytosol and release them at its ER mem-brane receptor (6, 13–16).Assembly of the Get3-TA targeting complex

requires “pretargeting” factors that mediate load-ing onto Get3 (17, 18). This pathway begins withTA protein in complex with the chaperone Sgt2.The Get4-Get5 scaffolding complex then recruitsSgt2 via Get5, while Get4 recruits ATP-boundGet3 (19). A hand-off reaction within this com-plex results in transfer of TA protein from Sgt2 to

Get3. TA substrate-loaded Get3 then dissociatesfrom Get4 (20–22), resulting in a targeting com-plex whose architecture and stoichiometry havebeen debated (8–12, 20–23).To define the physiologically relevant Get3

targeting complex, we recapitulated its assemblyin vitro, using purified recombinant factors atin vivo concentrations (Fig. 1A). Translation ofradiolabeled TA protein in the presence of SGTA(the mammalian homolog of Sgt2) produced astable complex detectable by chemical cross-linking(fig. S1). The TA protein remained associatedwith SGTA upon addition of either Get4-Get5 orGet3, but released efficiently when both factorswere added (Fig. 1B). Correspondingly, Get3 effi-ciently acquired substrate from SGTA only whenGet4-Get5 was present.The transfer reaction was rapid and unidirec-

tional: Once substrate released from SGTA, it didnot rebind (fig. S2). Likewise, substrate preloadeddirectly on Get3 (fig. S3) did not effectively trans-fer to SGTA (Fig. 1B). Structure-guided muta-tions disrupting either the SGTA-Get5 interaction[SGTA(C38S)] (24) or the Get4-Get3 interaction[Get3(E253R)] (20) abolished substrate releasefrom SGTA (Fig. 1C). Targeting complex producedvia Get4-Get5 supported TA protein insertion intoyeast ER microsomes (Fig. 1D), while an identical

reaction containing SGTA(C38S) showed reducedinsertion (Fig. 1D). Thus, the recombinant assem-bly system requires all factors and interactions ofthe early GET pathway and produces insertion-competent Get3-TA protein targeting complex.Three lines of evidence suggested that func-

tional targeting complex assembled via pretar-geting factors consists of dimeric Get3 bound toTA protein. First, the targeting complex, contain-ing a small (~10 kD) TA protein, had the samenative size as purified Get3 dimer, and was clear-ly distinguishable from higher-order Get3 com-plexes (Fig. 1E). Such higher-order complexes,often seen when Get3 is coexpressed with TAprotein inEscherichia coli (fig. S5) (8, 22, 23), werenot observed even when the loading reactioncontained 10-fold excess Get3 (fig. S4A). Second,titration of Get3 into the loading reaction showedno evidence of cooperativity (fig. S4B), arguingagainst its higher-order assembly during target-ing complex formation. Third, size-exclusion chroma-tography and multiangle laser light scattering(SEC-MALLS) indicated that prior to loading, asingle Get3 dimer is bound by two copies of theGet4-Get5 complex (fig S4C). Thus, TA protein isloaded onto dimeric Get3 to form a functionaltargeting complex.To gain insight into how the TA protein is

shielded by Get3 in this targeting complex,we sought to determine its structure. Duringphysiologic targeting complex assembly, Get4 pre-ferentially recruits and stabilizes adenosine 5´-triphosphate (ATP)–bound Get3 (19, 20, 22). Tomimic this during recombinant expression inE. coli, we biased Get3 to the ATP-bound statevia the D57N hydrolysis mutant (10). Coexpres-sion of this mutant with TA protein resulted in atargeting complex that was homogeneously di-meric for Get3 by SEC-MALLS (fig. S6A) andcomigratedwith in vitro–assembled targeting com-plex on sucrose gradients (fig. S6B).To facilitate crystallization, we generated a

high-affinity synthetic antibody fragment (sAB)(25) that recognizes the closed (ATP-bound) con-formation of Get3. Kinetic analysis revealed thatthis sAB binds with subnanomolar affinity tonucleotide-bound Get3, both in the presence andabsence of TA protein (fig. S7). Thus, rather thaninducing a large conformational change in Get3,the TA protein binds to a preorganized confor-mation that closely resembles the closed (ATP-bound) state.Using this sAB, we crystallized Get3(D57N) in

complex with the TMD of the yeast TA protein

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1Department of Biochemistry and Molecular Biology, TheUniversity of Chicago, 929 East 57th Street, Chicago, IL 60637,USA. 2MRC Laboratory of Molecular Biology, Francis CrickAvenue, Cambridge CB2 0QH, UK.*Corresponding author. E-mail: rhegde@mrc-lmb.cam.ac.uk(R.S.H.); bkeenan@uchicago.edu (R.J.K.)

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Architecture of the nuclear pore complex coatTobias Stuwe, Ana R. Correia, Daniel H. Lin, Marcin Paduch, Vincent T. Lu, Anthony A. Kossiakoff and André Hoelz

originally published online February 12, 2015DOI: 10.1126/science.aaa4136 (6226), 1148-1152.347Science 

, this issue p. 1148Sciencerings and revealed the details of higher-order CNC oligomerization at the near-atomic level.

stackedtomography reconstruction of the human NPC established the presence of 32 copies of the CNC arranged in four in the presence of an engineered antibody fragment. Docking the crystal structure into an electroncerevisiae

Saccharomyces describe the crystal structure of the intact ç400-kD coat nucleoporin complex (CNC) of et al.Now, Stuwe cytoplasm and the nucleus, has been difficult to ascertain owing to the size and complexity of this subcellular structure.

The precise molecular architecture of the nuclear pore complex (NPC), which mediates traffic between theA closeup view of the nuclear pore's coat

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