The Structure of the Nuclear Pore Complex

BI80CH26-Hoelz ARI 16 May 2011 12:36

The Structure of the NuclearPore ComplexAndre Hoelz,1 Erik W. Debler,2

and Gunter Blobel2,3

1Division of Chemistry and Chemical Engineering, California Institute of Technology,Pasadena, California 91125; email: [email protected] of Cell Biology and 3Howard Hughes Medical Institute, The RockefellerUniversity, New York, NY 10065

Annu. Rev. Biochem. 2011. 80:613–43

First published online as a Review in Advance onApril 12, 2011

The Annual Review of Biochemistry is online atbiochem.annualreviews.org

This article’s doi:10.1146/annurev-biochem-060109-151030

Copyright c© 2011 by Annual Reviews.All rights reserved

0066-4154/11/0707-0613$20.00

Keywords

α-helical solenoid, binding promiscuity, β-propeller,nucleocytoplasmic transport, nucleoporin, membrane coats

Abstract

In eukaryotic cells, the spatial segregation of replication and transcrip-tion in the nucleus and translation in the cytoplasm imposes the require-ment of transporting thousands of macromolecules between these twocompartments. Nuclear pore complexes (NPCs) are the sole gatewaysthat facilitate this macromolecular exchange across the nuclear enve-lope with the help of soluble transport receptors. Whereas the mobiletransport machinery is reasonably well understood at the atomic level,a commensurate structural characterization of the NPC has only be-gun in the past few years. Here, we describe the recent progress towardthe elucidation of the atomic structure of the NPC, highlight emerg-ing concepts of its underlying architecture, and discuss key outstandingquestions and challenges. The applied structure determination as wellas the described design principles of the NPC may serve as paradigmsfor other macromolecular assemblies.

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NPC: nuclear porecomplex

EM: electronmicroscopy

Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . 614Nucleoporin Family. . . . . . . . . . . . . . . . 614Functions of the Nuclear Pore

Complex . . . . . . . . . . . . . . . . . . . . . . . 615NUCLEAR PORE COMPLEX

ARCHITECTURE BYELECTRON MICROSCOPY . . . . . 617

NUCLEOPORIN STRUCTURESOF THE SYMMETRIC CORE . . . 619Coat Nucleoporins: Members of the

Heptameric Nup84 Complex . . . . 620Common Evolutionary Origin of the

Nuclear Pore Complex andOther Membrane Coats . . . . . . . . . 624

Adaptor Nucleoporins: Membersof the Heteromeric Nic96Complex . . . . . . . . . . . . . . . . . . . . . . . 625

Channel Nucleoporins: Members ofthe Heterotrimeric Nsp1Complex . . . . . . . . . . . . . . . . . . . . . . . 626

TRANSPORT FACTORFG-REPEATINTERACTIONS . . . . . . . . . . . . . . . . 626β-Karyopherin FG-Repeat

Interactions. . . . . . . . . . . . . . . . . . . . . 626RanGDP Import Factor

NTF2-FG-RepeatInteractions. . . . . . . . . . . . . . . . . . . . . 628

mRNA Export FactorTAP-p15-FG-RepeatInteractions. . . . . . . . . . . . . . . . . . . . . 628

STRUCTURES OF ASYMMETRICNUCLEOPORINS . . . . . . . . . . . . . . . 629Cytoplasmic Filament Nucleoporins

and Associated mRNA ExportFactors . . . . . . . . . . . . . . . . . . . . . . . . . 629

Nuclear Basket Nucleoporins . . . . . . . 631MODELS OF THE SYMMETRIC

NUCLEAR PORE COMPLEXCORE . . . . . . . . . . . . . . . . . . . . . . . . . . . . 631

INTRODUCTION

A eukaryotic cell is subdivided into spatiallyand functionally distinct, membrane-enclosed

compartments that enable it to concomitantlyperform numerous cellular tasks in specializedmicroenvironments. This subcellular divisionis achieved by intricate intracellular membranesystems. The double membrane of the nuclearenvelope segregates the nucleoplasm contain-ing the genetic material from the cytoplasm.Whereas the nucleus is the site of transcription,the synthesized RNA molecules have to leavethe nucleus to be translated into proteins byribosomes in the cytoplasm. In addition toprotecting the DNA from harmful effectsin the cytoplasm, the spatial separation oftranscription and translation imposes anotherlevel of regulation in the flow of informationfrom DNA to protein.

Nuclear pore complexes (NPCs) are em-bedded in pores of the nuclear envelope andconstitute large aqueous transport channelsthat mediate and regulate the bidirectionalexchange of macromolecules between the nu-cleus and cytoplasm. The NPC represents oneof the largest and most complex proteinaceousassemblies in the eukaryotic cell. Since itsdiscovery more than half a century ago, thestructure of the NPC has been extensivelyinvestigated by electron microscopy (EM).Atomic-resolution analysis of the entire NPCby X-ray crystallography has been hindered byits sheer size, dynamic and flexible nature, andthe difficulties in purifying sufficient amountsof homogeneous material. More recently, thecharacterization of the molecular compositionof the NPC and the establishment of its mod-ular architecture have enabled the structuredetermination of individual domains, proteins,and their subcomplexes at atomic resolutionby X-ray crystallography, which delineates a“divide-and-conquer” approach toward thecomplete atomic structure of the NPC.

Nucleoporin Family

The NPC is composed of a set of approx-imately 30 different proteins, collectivelytermed nucleoporins, that are conserved inevolutionarily distant eukaryotes ranging fromyeast to human (1–4). Reflecting a high degree

614 Hoelz · Debler · Blobel

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Nucleoporins:a family of ∼30evolutionarilyconserved proteinsthat construct theNPC. Most of themare termed Nupfollowed by theirmolecular mass

POM: an integralmembrane protein ofthe pore membranedomain of the nuclearenvelope

Nuclear basket:filaments that areattached to the nuclearface of the symmetricNPC core and that arebundled by a distalring

Cytoplasmicfilaments: filamentsattached to thecytoplasmic face of thesymmetric NPC corethat serve as dockingsites for transportfactors

Phenylalanine-glycine (FG) repeats:repeats in unstructurednucleoporin regionsthat form thepermeability barrierand serve as dockingsites for karyopherins

β-propeller domain:a protein fold that iscomposed of 4–8circularly arrangedstructural units calledblades that arecomposed of 4antiparallel β-strands

Karyopherins (Kaps):transport factors thatrecognize nuclearimport and exportsignal sequences incargo and facilitatetransport through theNPC

of internal symmetry, each nucleoporin occursin multiple copies, resulting in ∼500–1,000protein molecules in the fully assembledNPC. Using analytical ultracentrifugation, amolecular mass of ∼66 MDa was determinedfor isolated intact NPCs of the invertebrateSaccharomyces cerevisiae (5), whereas a molecularmass of ∼112 MDa was deduced for the NPCof the vertebrate Xenopus laevis from scanningtransmission EM (6). The ratio of the differentnucleoporins within a single NPC has been es-timated by semiquantitative methods. Becausethe results have been largely inconsistent,most likely owing to technical limitations,the absolute stoichiometry of all nucleoporinsremains unknown (2, 3, 7). Most nucleoporinsare denoted “Nup” followed by a number thatin most cases refers to their molecular mass.Because of the molecular mass differencesin various species, a uniform nomenclaturefor nucleoporins does not exist. However,based on their approximate localization withinthe NPC, the nucleoporins can be classifiedinto six categories: (a) integral membraneproteins of the pore membrane domain ofthe nuclear envelope (POMs), (b) membrane-apposed coat nucleoporins, (c) adaptornucleoporins, (d ) channel nucleoporins,(e) nuclear basket nucleoporins, and ( f ) cyto-plasmic filament nucleoporins (Figures 1 and2) (8).

Homology modeling suggests that nucle-oporins are primarily constructed from oneor more of the following structural units: α-helical regions, β-propellers, and unstructuredphenylalanine-glycine (FG) repeats (Figure 3)(9). β-propellers are ubiquitous disk-shapeddomains with an overall diameter of ∼70 Aand a thickness of ∼40 A (10). The canonicalβ-propeller core is generated by four to eightblades that are circularly arranged. Each bladeconsists of four antiparallel β-strands, whichby convention are termed A to D from theinside to the outside of the β-propeller. Sofar, atomic models for six of the nine predictedβ-propellers in yeast nucleoporins have beenexperimentally determined (Figure 3) (8,11–20). Whereas the β-propeller core provides

a sturdy structural scaffold, the structuresuncovered numerous unexpected decorativefeatures that play a central role in protein-protein interactions within the NPC. Thestructural characterization of 8 of the 25 pre-dicted α-helical regions of yeast nucleoporinsrevealed a set of diverse and novel folds withsurprising properties that could not have beenanticipated by homology modeling (Figure 3)(8, 14–19, 21–25). These domains are almostexclusively composed of α-helices arranged ina zigzag fashion and feature diverse topologies,which strikingly differ from the canonicalsuperhelical solenoids typically observed intransport receptors (26, 27).

Functions of the NuclearPore Complex

The principle function of the NPC is thefacilitation of nucleocytoplasmic traffic, whileat the same time generating a diffusion barrierto separate the cytoplasm from the nuclearcompartment. Diffusion channels with a cal-culated diameter of ∼90–100 A allow the freepassage of macromolecules of up to ∼40 kDa,whereas larger cargoes with a diameter ofup to ∼390 A require active translocation bytransport receptors (Supplemental Movies 1and 2) (28–30). (Follow the SupplementalMaterial link from the Annual Reviews homepage at http://www.annualreviews.org.) Thediffusion barrier is formed by extended nativelyunfolded nucleoporin segments that containnumerous FG repeats. The exact nature ofthe permeability barrier remains a heavily de-bated topic (31–39). Because of the intrinsiclyunstructured nature of the FG repeats, X-raycrystallography can only contribute to a minorextent to resolve this issue.

FG repeats also serve as docking sitesfor transport receptors, collectively termedkaryopherins (kaps) (also known as importinsand exportins), that “ferry” the cargo throughthe permeability barrier (26, 27, 40–47). Nu-cleocytoplasmic transport is dictated by shortsequence elements in cargo molecules, whichare recognized by karyopherins. A nuclear

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a

65°

FG repeats

POMs

Coat nucleoporins

Adaptor nucleoporins

Channel nucleoporins

Cytoplasmicfilaments

Nuclear basket

Nuclearenvelope

b

Cytoplasmic view

25°

Side view

Cargo intransit

Figure 1Overall structure of the nuclear pore complex (NPC). (a) Cryo-electron tomographic reconstruction of theDictyostelium discoideum NPC [Electron Microscopy Data Bank (EMDB) code 1097, Reference 81]. Thecytoplasmic filaments, the symmetric core, and the nuclear basket are colored in cyan, orange, and purple,respectively. (b) A schematic model of the NPC. The four concentric cylinders are composed of integral poremembrane proteins (POMs), coat nucleoporins, adaptor nucleoporins, and channel nucleoporins. Nativelyunfolded phenylalanine-glycine (FG) repeats of a number of nucleoporins make up the transport barrier inthe central channel and are indicated by a transparent plug.

Ran: a small GTPasethat is a keycomponent for thegeneration of thedirectionality ofnucleocytoplasmictransport

localization sequence is responsible for import,whereas a nuclear export sequence is used forexport. β-karyopherins (β-kaps) interact withcargo molecules either directly or indirectlyvia an adaptor karyopherin termed α-kap. Thedirectionality of transport is governed by aconcentration gradient of RanGTP, which ismaintained at a high level inside the nucleus

but at a low level in the cytoplasm (26, 45,47). Ran can adopt two distinct conformationsthat depend on the bound nucleotide (GTP orGDP). RanGTP not only disassembles importcomplexes upon entrance into the nucleus,but also promotes the assembly of exportcomplexes inside the nucleus. Upon arrival atthe cytoplasmic side of the NPC, these export


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complexes are disassembled by removal ofRanGTP with the help of proteins thatare tethered to the cytoplasmic filaments(Supplemental Movie 2).

The mobile transport machinery, the assem-bly and disassembly of transport complexes, aswell as cargo recognition have been extensivelystudied (26, 27, 40, 45–47). By contrast, thepassage of large cargo, such as preribosomalparticles, or the import of integral membraneproteins that are destined for the inner nu-clear membrane, are less well understood(Supplemental Movies 3 and 4). (Followthe Supplemental Material link from theAnnual Reviews home page at http://www.annualreviews.org.) Clearly, the NPC is notmerely a static conduit with a permeabilitybarrier but a dynamic transport organellethat must undergo substantial structural rear-rangements to permit these transport events(30, 48–51).

In addition to enabling nucleocytoplasmictransport, the NPC and its components areinvolved in numerous other cellular functions,such as chromatin organization, replication-coupled DNA repair, and regulation of gene ex-pression (52–61). In eukaryotes with open mito-sis, some nucleoporins have a well-documentedrole during cell division (62). Duringprometaphase, the NPC dismantles into dis-tinct subcomplexes from which it reassembles atthe completion of telophase (1, 63–65). Similarsubcomplexes can also be dissected biochem-ically from fully assembled interphase NPCsand from NPCs of eukaryotes with closed mi-tosis (66, 67). The best-characterized exampleof an NPC component with a cell cycle–specific function is the vertebrate Nup107–160complex that is targeted to kinetochores duringmitosis, where it functions in spindle assembly(63, 65, 68–70). On the basis of the central anddiverse roles of the NPC in cellular physiology,it is not surprising that defects in the NPC orits components are linked to a diverse set ofdiseases, including hematological neoplasms,heart arrhythmia, and primary biliary cirrhosis(71–73).

Symmetric nucleoporins Asymmetric nucleoporins

Yeast Human

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Gp210POM121

NDC1

Nup62

Nup58Nup45

Nup93Nup205Nup188

Nup107Nup133

Seh1Nup75Nup160Sec13Nup96

Nup37Nup43

Nup54

- - -- - -

Nup155

Nup35

POM152POM34NDC1

Nsp1Nup57

Nic96Nup192Nup188Nup157Nup170Nup53Nup59

Nup84Nup133

Seh1Nup85Nup120Sec13Nup145C

- - -- - -

- - -- - -

Nup49

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Yeast Human

Gle1Dbp5

Nup2Nup1

Nup60

Nup82Nup159

Nup100Nup145N

Nup116

Nup42

Mlp1Mlp2

Gle2

- - -

- - -

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Ddx19Gle1

Nup50Nup153

Nup88

CG1

Nup98

Nup358

ALADIN

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Nup214

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TPR}Nuc

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Figure 2Molecular composition of the yeast and human nuclear pore complexes(NPCs). Symmetric nucleoporins form the core region and are equallydistributed on the cytoplasmic and nucleoplasmic halves of the NPC.Asymmetric nucleoporins form the nuclear basket and the cytoplasmicfilaments that serve as docking sites for transport factors and include associatedmRNA export factors. The nucleoporin classification is as described inFigure 1b. POM, an integral membrane protein.

NUCLEAR PORE COMPLEXARCHITECTURE BY ELECTRONMICROSCOPY

Since the discovery of pores in the nuclearenvelope in 1950 (74) and of the embeddedNPCs thereafter (75), the overall architectureand characteristics of the NPC have been ex-tensively investigated by EM. Initially observedas cylindrical formations that penetrate thenuclear envelope with a diameter of ∼1,000 A(75), a more detailed study established an eight-fold rotational symmetry of NPCs along theirnucleocytoplasmic axes (76). Occasionally,


Supplemental Material

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~760

974 1,012

α/β-region RRM

RanBD

Gle11 538

N C

B α/β-region

U1 726460

N CNup84 α-helical solenoid 507

Nup159

Nup192 N C1 1,683

α-helical

Nup1881 1,655

N Cα-helical

Nup821 713452490

N Cβ-propeller α-helicalU

FG repeats αN CNup421 380 430

1 570 814 959FG repeats U CTDN CNup100

1 605209 458Nup145N FG repeats APDUN C

1 686115 166 1,113967N CFG repeats UGNup116

Seh1 N C1 349

β-propeller

1 57044 101 744N CNup85 DIM α-helical solenoid α-helicalU

N Cβ-propeller Insert α-helical domain α-helicalUNup1201 729382 1,037487460

Sec13 N C1 297

β-propeller

1 555125 183 711N CDIMU α-helical solenoid α-helicalNup145C

1 1,15752056N Cβ-propeller α-helical domainUNup133

Nic96 N1 839190

Cα α-helical domain

1 1,391~680Nup157 N Cβ-propeller α-helical

1 1,502~650 979~180Nup170 N CU β-propeller α-helical domain

Nup53 N C1 475

Nup59 N C1 528

Nsp18235911 631

N CFG repeats α-helicalU

Nup49 N Cα-helicalFG repeats2381 472

2561 541Nup57 N Cα-helicalFG repeats

2491 655α-helicalNDC1 N C

2991 147POM34 N C

1,3371 208POM152 N Cβ-strand region

1 48271 296NDbp5 CNTE Domain 1 Domain 2

N1 1,4601,103 1,177876387

β-propeller α-helicalFG repeats U DID C

Nup11,0761 ~400

N Cα/β-region FG repeats

Nup2 FG repeatsN C7201 ~245 ~55051

Mlp1 N1,8751

C

Mlp21,6791

N C

Nup605391

N Cα/β-region

α-helical

α-helical

β-propellerN CGle21 365

Cyto

plas

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filam

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Nuc

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POM

sCh

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α-helical

355251

B

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U α-helical

U

α/β-region

RRM271 397

α-helical region

β-propeller/β-strand region

Unstructured (U)

FG repeat region

Transmembrane helices

Fragments whose crystal structuresare experimentally determined:

Yeast

α/β-region

α/β-region

MammalianYeast and mammalian


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Pore membranedomain: the sectionof the nuclear envelopewhere inner and outernuclear membranesare fused to form thenuclear pores

Symmetric NPCcore: the NPC corehas an eightfoldrotational symmetryalong thenucleocytoplasmic axisand twofold rotationalsymmetryperpendicular to it

NPCs with nine- and tenfold rotationalsymmetry have been observed, suggesting asomewhat flexible and modular assembly (77).Three-dimensional (3D) reconstructions to∼90 A, computed from Xenopus NPCs em-bedded in nuclear envelopes, identified severaldiscrete units within the NPC (78): two coaxialrings with one on the cytoplasmic peripheryand the other on the nuclear periphery; eightelongated structures termed spokes connectingthe two rings; and a spherical particle termedcentral plug in the transport channel, whichwas subsequently shown to represent cargo intransit (79–81). Apparent twofold axes perpen-dicular to the octad axis suggest a symmetriccore of the NPC that is constructed fromtwo equal but oppositely facing halves (78).Attached to the symmetric core are cytoplasmicfilaments and a basket-like structure on the nu-cleoplasmic face, both of which provide bindingsites for cargo (79, 82–85). In vertebrate NPCs,peripheral or lateral channels with an averagediameter of ∼100 A were described (86), butwere significantly less pronounced in a cryo-EM reconstruction of detergent-extracted andmembrane-associated NPCs (87). Moreover,the latter study identified a lumenal ring, whichwas proposed to be responsible for anchoringthe NPC within the nuclear envelope pore, anddescribed an intrinsic conformational flexibilityof the spokes (87). Structural plasticity andflexibility, which are thought to be criticalaspects of NPC function, were also uncoveredin another analysis of Xenopus NPCs (88).

The first NPC reconstruction determinedby cryo-electron tomography (cryo-ET) wasperformed on native NPCs from Xenopusand revealed the “spongy” symmetric coreframework (80). NPCs of intact nuclei from

Dictyostelium discoideum show structural rear-rangements of the NPC scaffold in responseto cargo translocation (Figure 1a) (81). A newcryo-ET image-processing strategy yielded thehighest resolution (58 A) of the NPC to date(51). Tomograms recorded in the presence ofgold-labeled cargo outlined the trajectory ofimport cargo (51). The latest cryo-ET on theXenopus NPC revealed a fused concentric ringarchitecture and provided refined overall NPCdimensions, i.e., ∼1,250 A for the diameter,∼950 A for the height, and ∼550 A for thediameter of the central channel (89).

In comparison to the vertebrate NPCs, theNPC of S. cerevisiae is considerably smaller,with a comparable outer diameter (∼960 A), butwith a height that is less than half of its verte-brate counterpart (∼350 A) (90). This discrep-ancy correlates with the determined thicknessof the yeast and vertebrate nuclear envelopes of∼300 A (90) and ∼600 A (87), respectively. No-tably, lateral channels have not been observedin the more compact architecture of the yeastNPC (90). Despite these differences, a compar-ison of NPC structures, as determined by EMacross vertebrates and invertebrates, suggeststhat the overall architecture is well conserved(48, 86, 90).

NUCLEOPORIN STRUCTURESOF THE SYMMETRIC CORE

The pore membrane domain of the nuclear en-velope harbors three POMs that anchor thesymmetric NPC core (Figure 1b). In additionto their transmembrane helices, they containlarge regions that extend toward the lumenaland pore sides of the membrane. In NDC1

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 3Domain architecture of yeast nucleoporins. Domain borders are indicated by residue numbers. NTE, N-terminal extension;U, unstructured; FG repeat, phenylalanine-glycine (FG) repeat; DID, dynein light-chain interacting domain; CTD, C-terminaldomain; G, Gle2-binding sequence (GLEBS); APD, autoproteolytic domain; DIM, domain invasion motif; RRM, RNA-recognitionmotif; B, karyopherin-binding domain; RanBD, Ran-binding domain; POM, an integral membrane protein. The bars above thedomain organizations mark fragments whose crystal structures are experimentally determined (black, yeast; red, mammalian; green, yeastand mammalian). Notably, the nucleoporins Nup1 and Nup2 do not follow the common nomenclature because the numbers 1 and 2 donot refer to their molecular weight.


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α-helical solenoid:a protein fold that iscomposed of α-helicesarranged in a zigzagfashion

and POM34, these regions are located primar-ily on the pore side, whereas the major partof POM152 reaches into the lumen of the nu-clear envelope. Most recently, a fourth trans-membrane nucleoporin termed POM33 wasdiscovered (91). The remainder of the sym-metric core is composed of seven coat nucle-oporins, seven adaptor nucleoporins, and threechannel nucleoporins (Figure 2). The coat andadaptor nucleoporins are mostly composed ofβ-propellers and α-helical domains, whereasthe channel nucleoporins contain extensive na-tively unfolded FG-repeat regions. Altogether,the symmetric NPC core comprises ∼20distinct proteins.

Coat Nucleoporins: Members of theHeptameric Nup84 Complex

In yeast, the seven coat nucleoporins assembleinto the well-defined Nup84 complex, termedafter one of its components. This subcom-plex consists of Nup84, Nup120, Nup85,Nup145C, Sec13, Seh1, and Nup133 (92–95).Negative-stain two-dimensional EM of theintact heptamer, either assembled from recom-binant proteins or isolated and purified fromyeast cells, revealed an ∼400-A-long Y-shapedarchitecture (93, 94). The reconstitutionof the heptamer from dimeric and trimericpieces uncovered its modular anatomy. Two-dimensional EM of these fragments coupledwith biochemical analyses established therelative positions of its members (Figure 4b).Nup120 is capable of interacting with bothSec13·Nup145C and Seh1·Nup85 (94). All

members of the yeast heptamer are wellconserved; however, the vertebrate complexcontains two additional members, Nup37 andNup43, forming the nonameric Nup107–160complex (64, 96–98). During postmitoticNPC assembly, this complex is recruited tochromatin by MEL-28/ELYS (99). Thus, thisprotein may represent a tenth member of theNup107–160 complex (100). The Nup84 com-plex is localized close to the pore membrane andis suggested to serve as a “membrane-curvingmodule,” similar to the members of the COPI,COPII, and clathrin coats (2, 101). Consistentwith a key structural role of the Nup84 com-plex, deletion or immunodepletion of any of itsmembers has dramatic consequences for the ar-chitecture and function of the NPC, as evidentby the clustering of NPCs in one patch of thenuclear envelope and accumulation of PolyARNA within the nucleus (92, 93, 96, 102–107).

The first crystal structure of a Nup84 com-plex component was the N-terminal domainof human Nup133 (residues 76–478), abbre-viated as hNup13376−478 (throughout the text,the prefixes h, m, and r refer to human, mouse,and rat, respectively; all other proteins are fromyeast. Residue ranges refer to fragments), whichrevealed a seven-bladed β-propeller with twoα-helical insertions and a disordered 3D4Aloop (Figure 4a) (12). This loop was sug-gested to harbor a membrane curvature-sensingmotif termed the ArfGAP1 lipid-packing sen-sor, which forms an amphipathic membranecurvature-sensing α-helix in vitro (108).

The crystal structure of the hNup107658−925·hNup133934−1156 complex shows a compact,

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 4Structural characterization of the heptameric Nup84 complex. The prefix h refers to human; protein nameswithout a prefix refer to yeast. (a) Crystal structures of coat nucleoporins and complexes thereof:hSec13·Nup145C [Protein Data Bank (PDB) codes 3BG1, 3BG0], Seh1·Nup85 (3F3F, 3F3G, 3F3P,3EWE), Nup120NTD (N-terminal domain) (3F7F, 3H7N, 3HXR), Sec13·Nup145C·Nup84NTD (3IKO,3JRO), hNup107CTD·hNup133CTD (C-terminal domain) (3I4R, 3CQC, 3CQG), and hNup133NTD

(1XKS). For those structures with several PDB codes, the first one refers to the displayed structure.(b) Docking of crystal structures into the electron microscopy (EM) envelope of the heptameric Nup84complex ( first panel ). A 90◦-rotated view is shown (second panel ). The EM envelope of the secondreconstructed conformation of the heptamer in which the two hinge regions are completely extended (thirdpanel ). Superposition of the two determined Nup84 conformations ( fourth panel ). The hinge region at theNup145C·Nup84 interface is indicated and was used for the structural alignment.


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hydrophobic interface that is predominantlycomposed of two α-helices of each hNup107and hNup133. Both C-terminal fragmentsform α-helical domains with novel folds (24).

Subsequently, the same hNup107 fragmentwas crystallized in complex with the entireα-helical domain of hNup133 (residues 517–1156) revealing an extended α-helical solenoid

Nup120NTD

hNup133NTD

a

b

90°

Conformation 1 Conformation 2 Superposition

Seh1•Nup85

Sec13

Nup145C

Nup120NTD

Nup84NTD

hNup107CTD

hNup133NTD

hNup133CTD Nup145C•Nup84interface(hinge 1)

Hinge 2

hNup107CTD•hNup133CTD

hNup107CTD

hNup133CTD

hSec13•Nup145C

Nup145C

hSec13

Nup145CDIM

Seh1•Nup85

Nup85

Seh1

Nup85DIM

Sec13•Nup145C•Nup84NTD

Sec13

Nup145C

Nup84NTD

Nup145CDIM


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fold composed of 28 antiparallel α-helices(Figure 4a) (25).

The next advance was the structuraland functional analysis of the hSec131−316·Nup145C125−555 nucleoporin pair (Figure 4a),the centerpiece of the Nup84 complex (8).Sec13 provides six blades to a β-propellerdomain, to which Nup145C contributes anadditional seventh blade to yield a seven-bladed β-propeller. The complementation ofthe β-propeller fold by a domain invasionmotif (DIM) was unprecedented and thus farhas only been observed in complexes con-taining either Sec13 or Seh1 (8, 14, 15,18, 19, 109, 110). The remainder of theNup145C125−555 fragment forms a U-shaped α-helical solenoid domain with a novel fold. Inter-estingly, two different crystal forms harboredidentical hSec131−316·Nup145C125−555 hetero-octameric assemblies that are the result ofhomotypic dimerizations of both hSec131−316

and Nup145C125−555 (Figure 5a). This hetero-octameric assembly can be envisioned to form avertical pole in a cylindrical coat for the nuclearpore membrane (NPC coat) (Figure 5) (8). Insolution, the Sec13·Nup145C pair exists in adynamic equilibrium with various oligomers,including a hetero-octameric species, whichsupports the physiological relevance of the crys-tallized hetero-octamer (8).

Seh1·Nup85, another tightly-associatednucleoporin pair of the heptamer, is ho-mologous to Sec13·Nup145C (8, 94). In-deed, the crystallographic analysis of Seh1·Nup851−570 uncovered a heterodimer that bearsremarkable resemblance to the hSec131−316·Nup145C125−555 pair (Figure 4a) (8, 15).In detail, Seh1·Nup851−570 recapitulates a

seven-bladed β-propeller that is formed by sixblades of Seh1 and complemented by the DIMof Nup85. The α-helical region of Nup85forms a U-shaped α-helical solenoid domainof topology similar to Nup145C. In threedifferent crystal structures, Seh1·Nup851−570

dimerizes into identical heterotetramers that inturn assemble into related, elongated higher-order structures (Figure 5a). Two adjacentSeh1·Nup851−570 tetramers form an elongatedhetero-octamer that closely resembles thehSec131−316·Nup145C125−555 hetero-octamerwith respect to its curvature, symmetry, anddimensions (Figure 5a). This finding suggeststhat this complex forms an additional verticalpole in the proposed NPC coat (8, 15). Com-parison of the higher-order Seh1·Nup851−570

structures in different crystal forms revealsa flexible joint between adjacent heterote-tramers, which may portray conformationalchanges of the pole in the NPC during nucleo-cytoplasmic transport (15). A low-resolutionstructure of Seh1·Nup851−564 obtained froma fourth crystal form confirms the overallarchitecture of two interacting heterodimers(14). However, a conclusive analysis of thisstructure is hampered by the partial characterof the model and the presence of sequenceregister shifts (for details, see Reference 15).

The crystal structure of Nup1201−729

displays a β-propeller and an α-helical domainrepresenting a novel fold with a leucinezipper-like hydrophobic core (Figure 4a) (16,17). The seven-bladed β-propeller domaincontains several insertions, most notably afour-helix bundle, forming a small subdomain.Biochemical analyses uncovered a previouslyunknown interaction of Nup120 with Nup133;

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 5Model for the architecture of a “coat for the nuclear pore membrane.” (a) Structures of the hSec13·Nup145C and Seh1·Nup85hetero-octamers. (b) Binding promiscuity of Nup145C. Nup145C·Nup145C homodimerization in the hSec13·Nup145C hetero-octamer ( panel a, left) and Nup145C·Nup84 heterodimerization in the Sec13·Nup145C·Nup84NTD heterotrimer (right).(c) Schematic representation of the heptameric Nup84 complex and the approximate localization of its seven nucleoporins (left). Eightheptamers are circumferentially arranged in a head-to-tail fashion in four stacked rings, thereby forming a cylindrical scaffold (right).(d ) Two alternative states of the coat for the nuclear pore membrane. Black lines indicate interactions that have not been described aspromiscuous. In one state (left), Nup145C heterodimerizes with Nup84 (red lines). In another state (right), Nup145C homodimerizes(red lines) and forms a vertical hetero-octameric pole. Note that large protein rearrangements would not be necessary to convertbetween the two states.


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~300

Å

Nup84

Nup145C

Nup120

Nup133

Nup85

Sec13

Seh1

Sec13Nup145C

pole

Seh1Nup85

pole

Eightfold

Twofold~400

Å

~1,000 Å

c

Sec13•Nup145C

Seh1•Nup85

Nup120

Nup84•Nup133

a b

hSec13•Nup145Chetero-octamer

Nup145C

hSec13

hSec13

hSec13

hSec13

Nup145C

Nup145C

Nup145C

Seh1•Nup85hetero-octamer

Seh1

Nup85

Seh1

Seh1

Seh1

Nup85

Nup85

Nup85

Nup145C•Nup145Chomodimerization

Nup145C•Nup84heterodimerization

d

Nup84

Nup133

Nup84

Nup133Nup84

Nup133

Nup145C

Sec13Nup120

Nup85

Seh1

Nup84

Nup133Nup84

Nup133

Nup84

Nup133

Nup120Seh1

Nup85

Nup145C

Sec13

Sec13

Nup145C

Sec13

Nup145C

Seh1

Nup85

Nup85

Seh1

Seh1

Nup85

Seh1

Nup85

Sec13

Nup145C

Sec13

Nup145CNup120

Nup120

Nup120

Nup120

1/8 1/8

Nup84

Nup133

Nup145C

Sec13Nup120

Nup85

Seh1

Nup84

Nup133

Nup145C

Sec13Nup120

Nup85

Seh1

Sec13

Nup145C

Sec13

Nup145C

Nup84

Nup133

Nup84

Nup133

Nup84

Nup133

Nup84

Nup133

Nup84

Nup133Nup84

Nup133

Nup84

Nup133

Nup84

Nup133

Seh1

Nup85

Nup145C

Sec13

Sec13

Nup145C

Nup145C

Sec13

Sec13

Nup145C

Seh1

Nup85

Nup85

Seh1

Seh1

Nup85

Nup85

Seh1

Seh1

Nup85

Nup120

Nup120

Nup120 Nup120

Nup120Nup120

Nup145C

Sec13Nup120

Nup85

Seh1

Nup145C

Sec13Nup120

Nup85

Seh1

1/8 1/8

Nup145C•Nup84heterodimerization

Nup145C•Nup145Chomodimerization

90° 90°


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the disruption of this interaction causes asevere mRNA export defect as well as an NPCclustering phenotype in vivo (17). Becausemapping of the individual components in theNup84 complex places Nup120 and Nup133at opposite ends of the heptamer (Figure 5c),these findings are consistent with a head-to-tailarrangement of elongated Nup84 complexes inthe assembled NPC. Interestingly, the attach-ment site for Nup120 lies at the very end of anextended unstructured N-terminal region inNup133 (17). The common occurrence of suchunstructured terminal segments in nucleo-porins (Figure 3) suggests that this anchor-likemechanism of linking two nucleoporins maybe a general feature in the assembled NPC.In cells with open mitosis, these regions maynot only provide flexible tethers betweennucleoporins, but also may be ideal sites forposttranslational modifications, which maytrigger the disassembly of the NPCs (17). Infact, the unstructured segments of the ver-tebrate nonameric Nup107–160 complex arehyperphosphorylated, mainly in N-terminalregions, exclusively during mitosis (65).

The most recent crystallographic ad-vance refers to the structure of theSec13·Nup145C125−555·Nup841−460 complex(Figure 4a) (18). Nup841−460 forms a U-shapedα-helical solenoid domain, topologically simi-lar to two other nucleoporins of the heptamer,Nup145C and Nup85. The interaction be-tween Nup84 and Nup145C is mediated bya hydrophobic interface, located in the kinkregions of the two solenoids, that is reinforcedby additional interactions of two long Nup84loops. The Nup84 binding site partiallyoverlaps with the homodimerization interfaceof Nup145C, suggesting alternative, promis-cuous binding events (Figure 5b). A secondstructure of this complex at significantly lowerresolution corroborates the overall topology,but precludes a detailed analysis owing tosequence register shifts in Nup84 (19).

A 3D EM reconstruction of the Nup84complex at ∼35-A resolution identifies twospecific hinge regions at which the heptamershows great flexibility (Figure 4b) (111). With

the use of tagged proteins, the positioning ofsome of the members was experimentally veri-fied, and the available crystal structures couldbe docked into the maps of the two con-formers (18, 94, 111). Notably, the elongatedZ-shaped Sec13·Nup145C125−555·Nup841−460

heterotrimer was only consistent with one ofthe two heptamer conformers, indicating thatstructural changes occur at the promiscuousNup145C-Nup84 interface (Figures 4b and5b) (18).

The combination of X-ray crystallographicand EM data have yielded an atomic modelfor ∼85% of the Nup84 complex (Figure 4b).The last missing piece of the heptamer per-tains to the triskelion part, which links the threearms of the Y and consists of the C-terminalα-helical regions of Nup120, Nup145C, andNup85 (Figure 3). The structural characteriza-tion of the Nup84 complex serves as a paradigmfor other NPC subcomplexes and illustrates theenormous demand for achieving even higher-resolution EM structures. The relatively sim-ple fold composition and the resulting similarshapes of the building blocks of the heptamersubstantially complicate the docking of crystalstructures into EM maps at current resolutions.

Common Evolutionary Origin of theNuclear Pore Complex and OtherMembrane Coats

According to the “protocoatomer hypothesis”(101), the NPC shares a common evolutionaryorigin with coat protein assemblies (9, 101,112). The recent advances in determiningthe structure of the coat nucleoporins havenot only strengthened this hypothesis butalso unveiled several parallels that extendbeyond the original predictions. Hallmarksof these coat protein complexes are DIMsand topologically similar U-shaped α-helicalsolenoid domains, alternatively termed an-cestral coatomer elements 1 (8, 14, 15, 109).Although the arrangement of coat nucleoporinswithin the NPC is not known at present, theelongated and slightly curved hetero-octamericassemblies of Sec13·Nup145C and


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Seh1·Nup85 bear remarkable similaritiesto the established architecture of Sec13·Sec31in the COPII cage (8, 15, 109, 113, 114). Thebasic repeating unit of the lace-like COPII coatis an elongated Sec13·Sec31 hetero-octamerthat is built from homotypic interactions of theN-terminal β-propeller and the C-terminalα-helical domain of Sec31 (112). Similardesign principles were observed in the recentstructures of Sec13·Sec16, which is thought tobe a template for COPII coat assembly, andof coatomer proteins of the COPI cage (110,115, 116). Notably, pseudoatomic structuresof entire coat cages have only been determinedfor the mammalian clathrin and COPII coatsassembled in vitro (113, 114, 117). By contrast,in vitro assembly systems for the COPI vesiclecoat and the symmetric NPC core havenot been developed thus far, hampering theelucidation of their 3D organization.

Adaptor Nucleoporins: Members ofthe Heteromeric Nic96 Complex

The detailed arrangement of the sevenyeast adaptor nucleoporins Nic96, Nup157,Nup170, Nup188, Nup192, Nup53, andNup59 as well as the overall architecture ofthis subcomplex are not established at present.Structural information of this subcomplex isparticularly relevant for the anatomy of theNPC as the adaptor nucleoporins associate withall other subcomplexes in the symmetric NPCcore. Nup53 and Nup59 engage in interactionswith several other nucleoporins and, hence,appear to be central for this complex. Towardthe pore membrane, Nup53 and Nup59 bindto NDC1 of the transmembrane nucleoporincomplex composed of POM34, POM152, andNDC1 (118–120). Furthermore, Nup53 andNup59 interact with Nup170 and Nup157(120), and the latter weakly binds to the coatnucleoporin Nup120 (95). Nic96 binds tothe large structural nucleoporins Nup188 andNup192 and interacts with Nsp1 of the chan-nel nucleoporins (121–125). The homologousvertebrate Nup93·Nup188·Nup205 complexwas identified in Xenopus oocyte extracts (126).

The human homolog of Nup53 associates withthe Nic96 homolog Nup93 (118). Adaptornucleoporins are primarily α-helical, with onlyNup157 and Nup170 containing a predictedβ-propeller (Figure 3). Nup53 and Nup59differ from other nucleoporins in that they arepredicted to be α/β-proteins that harbor a so-called RNA-recognition motif (RRM) (126).Like the coat nucleoporins, the adaptor nucle-oporins do not contain FG-repeat regions.

The structures of two similar Nic96 frag-ments (Nic96186−839 and Nic96190−839) reveala block-shaped domain that is composed of 32antiparallel α-helices (Figure 6) (22, 23). Con-trary to the regular stacking of the antiparallelhelices in HEAT, Armadillo, or TPR repeatsthat generally result in superhelical solenoids,the α-helices of Nic96 are arranged in anirregular fashion forming a domain with anoverall J-like topology. Strikingly, the Nic96α-helical domain displays a dichotomy of sur-face potentials (23). Whereas the kink regionis highly positively charged, the remainder ofthe domain features a negative electrostatic

Nic96CTD Nup170CTD mNup35 RRM

homodimer

Figure 6Crystal structures of adaptor nucleoporins: Nic96CTD (C-terminal domain)(2RFO, 2QX5), Nup170CTD (3I5P, 3I5Q), and the mNup35 RNA-recognitionmotif (RRM) homodimer (1WWH). The ribbon representations of Nic96CTD

and Nup170CTD are shown in rainbow colors from blue to red along thepolypeptide chain from the N to the C terminus. Protein Data Bank codes arein parentheses. The prefix m refers to mouse; protein names without a prefixrefer to yeast.


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potential, generating a strong dipole moment(23). A C-terminal fragment of Nup170979−1502

also displays an irregular α-helical stack com-posed of 26 α-helices that form an elongatedrod (Figure 6) (25). This domain can bedivided into three segments that remotelyresemble those of the Nup133 α-helicaldomain (Figure 3).

The structure of the RRM in mNup35,the murine homolog of Nup53, displays acharacteristic RRM fold, but noncanonicalribonucleoprotein 1 and 2 motifs, which areresponsible for RNA binding in other RRMdomains (Figure 6) (127, 128). Consistent withthis finding, the RRM domain of mNup35 isunable to bind RNA molecules. Indeed, thepositively charged residues that are typicallyinvolved in RNA binding are mutated to highlyconserved hydrophobic residues that engagein dimer formation, whereas canonical RRMmotifs are monomeric.

Channel Nucleoporins: Members ofthe Heterotrimeric Nsp1 Complex

The innermost cylindrical layer of the NPCscaffold comprises Nsp1, Nup49, and Nup57in yeast (123, 125, 129). These three proteinsshare a similar domain organization in whichan ∼300-residue α-helical region is flanked byFG repeats. The α-helical regions are believedto form the perimeter of the channel, whereasthe tentacle-like FG repeats project into thecentral transport conduit, contributing to thepermeability barrier. Interestingly, Nsp1 is amember of another subcomplex consisting ofthe cytoplasmic filament components Nup82and Nup159 (130, 131). Nsp1 binds to the α-helical region of Nup82 and the N-terminal α-helical region of Nic96 in a promiscuous andmutually exclusive manner (130, 132). The ho-mologous mammalian Nup62 subcomplex con-sists of Nup62, Nup54, Nup58, and the Nup58splice variant Nup45 (133–135).

The only available channel nucleoporinstructure pertains to a portion of the α-helical region of rat Nup58 (Figure 7a) (21).rNup58327−415 forms an α-helical hairpin that

associates in an antiparallel fashion with asecond hairpin via an extensive hydropho-bic interface. In turn, this rNup58327−415

dimer forms distinct tetramers via a polarinterface exclusively featuring large hy-drophilic invariant residues that can functionas hydrogen-bond donors, acceptors, or both.Various rNup58327−415 tetramers display lateraldisplacements of their dimeric building blocksalong the helical axes by up to ∼11 A, corre-sponding to two α-helical turns (Figure 7a).In the different tetramer states, the long sidechains that mediate the dimer-dimer associ-ation engage in remodeled hydrogen-bondnetworks and would act like bristles when the α-helices “slide” against each other. Importantly,the other channel nucleoporins Nup54 andNup62 also contain conserved, amphipathicα-helical regions that may permit their lateraldisplacements. A circular arrangement of thesesliding modules in the fully assembled NPCmay allow the dilation of the central channel inresponse to cargo translocation (Figure 7b,c).Such iris-like adjustments of the central chan-nel have been observed by EM (81, 88, 136).

TRANSPORT FACTORFG-REPEAT INTERACTIONS

The FG-repeat regions of 11 nucleoporins inyeast (Figure 3) form the permeability barrierof the NPC and serve as docking sites forvarious classes of transport receptors. Theinteractions between FG repeats and transportreceptors have been well characterized byX-ray crystallography.

β-Karyopherin FG-RepeatInteractions

β-karyopherins are α-helical proteins that arecomposed of ∼20 HEAT repeats. The ∼40residues of the HEAT motif fold into a pairof antiparallel α-helices that are arranged intandem to form a right-handed superhelicalfold. Cargo recognition by β-karyopherins aswell as their interaction with the small GTPaseRan has been extensively studied (26, 47).


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rNup58/45 state I rNup58/45 state II Superposition

Centralchannel

a

b c

Cargo

Cargo

Cargo

Channelnucleoporins

Figure 7Crystallographic analysis of the channel nucleoporin Nup58. (a) Ribbon representations of two tetramericrat rNup58 conformers (left and middle). Superposition of the two tetrameric rNup58 states (right) reveals alateral shift in which two dimers are offset by up to two helical turns. Note that the crystallized fragmentrefers to a shared region of Nup58 and its splice variant Nup45. (b) Schematic representation of the centralchannel shown along the nucleocytoplasmic axis. Eight rNup58 tetrameric assemblies are circularly arrangedto form a ring (light gray). Simultaneous helical sliding of the eight rNup58 tetramers would result in theoverall dilation of the central channel (dark gray). (c) Localized changes in the channel diameter by α-helicalsliding in response to cargo transport ( purple) across the central channel (orange).

β-karyopherins mediate translocation throughthe NPC via reversible binding to FG repeatsof nucleoporins.

FxFG and GLFG (x denotes any residue) aretwo common core motifs of FG repeats in nu-cleoporins (2). Both motifs were crystallized incomplex with hKap-β11−442 (Figure 8a) (137,138). The FxFG and GLFG motifs intercalateinto the same hydrophobic groove between thehelices of two neighboring HEAT repeats onthe convex surface of the transport receptor.The phenylalanine in the third position of eachmotif is deeply inserted into a hydrophobicpocket and constitutes the predominant bind-ing determinant, thereby providing a structuralbasis for the analogous binding mode of theFxFG and GLFG motifs. A second FxFG motifbinds in a groove adjacent to the primary site.

In both structures, the paucity of the contactsbetween transport factors and FG repeats isconsistent with the observation that their inter-actions are generally of low affinity, reflectingthe need for movement through rather thanstatic binding to the NPC (139). However, theregions between FG repeats can increase theaffinity by forming additional interactions withtransport factors, as illustrated in the structureof Kap95 in complex with a portion of theFG region of Nup1 (Kap95·Nup1963−1076)(140).

Because RanGTP and FG repeats bind todifferent sites on Kap-β1, RanGTP is thoughtto release Kap-β1 from FG nucleoporins byinducing a conformational change (137). Mu-tational and computational analyses suggestthat karyopherins possess significantly more


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hKap-β1•Nsp1FG hTAPUBA•Nsp1FG

a b c d

NTF2•Nsp1FG hTAPp15•hp15•hNup214FG

p15TAP

NTF2 NTF2

Figure 8Crystal structures of transport factors in complex with FG repeats: (a) hKap-β1·Nsp1FG (1F59, 1O6O,1O6P), (b) NTF2·Nsp1FG (1GYB), (c) hTAPp15·hp15·hNup214FG, and (d ) hTAPUBA ·Nsp1FG (1OAI). FGrepeats are illustrated in purple stick representation. Protein Data Bank codes are in parentheses. The prefixh refers to human; protein names without a prefix refer to yeast.

FG-repeat binding sites (up to ∼10) than thosecaptured in the crystal structures (141, 142).Consistent with multiple FG-repeat bindingsites, β-karyopherins are capable of condens-ing FG-repeat regions in single molecule stud-ies, whereas addition of RanGTP restores theirbrush-like extended conformation (36).

RanGDP Import FactorNTF2-FG-Repeat Interactions

Nuclear export is mediated by heterotrimericcomplexes composed of cargo, export Kap-β,and RanGTP, which are dismantled by RanGTPase-activating proteins (RanGAPs) on thecytoplasmic face of the NPC (SupplementalMovie 2) (143). To replenish RanGTP de-pleted in the nucleus during export, RanGDPis reimported by nuclear transport factor2 (NTF2, also known as p10) (144–146).NTF2 forms a homodimer with two RanGDPbinding sites at opposite ends of the dimerand two binding sites for FG repeats at thedimer interface (Figure 8b) (147, 148). NTF2recapitulates the FxFG recognition of Kap-βin which the second phenylalanine in an overallsimilar conformation is deeply buried in ahydrophobic pocket (137, 147).

mRNA Export FactorTAP-p15-FG-Repeat Interactions

The export of mRNA into the cytosol oc-curs independently of Ran and karyopherins,and instead, it is mediated by a dedicated het-erodimeric mRNA export factor composed ofTAP and p15 in metazoans (Mex67 and Mtr2 inyeast). TAP comprises four domains arrangedin tandem: an N-terminal RNA-binding do-main, followed by a leucine-rich repeat do-main, a p15-binding domain, and a C-terminalubiquitin-associated (UBA) domain. Both theRNA-binding and leucine-rich repeat domainsare required for RNA cargo binding, whereasthe two C-terminal domains facilitate the asso-ciation with FG repeats.

The structure of the p15-binding domain ofTAP in complex with p15 and an FG-repeat re-gion of hNup214 closely resembles the NTF2homodimer, but unveiled only one FG-repeatbinding site located on TAP (Figure 8c) (149).Again, the second phenylalanine is bound in ahydrophobic pocket, and the peptide backboneassumes a conformation that is similar to theKap-β-bound FxFG motif. The p15-bindingdomain of TAP exclusively recognizes the FGmoiety (149), whereas the helical bundle of theC-terminal UBA domain intimately interacts


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with both aromatic residues of an Nsp1 FxFGcore region (Figure 8d ) (150).

STRUCTURES OF ASYMMETRICNUCLEOPORINS

The symmetric core of the NPC is decoratedwith cytoplasmic filaments and a nuclear basketstructure that are built from different setsof nucleoporins (Figures 1 and 2). Theseasymmetric nucleoporins are key componentsin establishing the directionality of nucleo-cytoplasmic transport. Several asymmetricnucleoporins contain FG repeats that serve asbinding sites for karyopherins. Altogether, ∼14proteins can be considered to be asymmetricnucleoporins (Figure 2).

Cytoplasmic Filament Nucleoporinsand Associated mRNA Export Factors

The yeast cytoplasmic filaments are primarilycomposed of Nup82 and Nup159 and providebinding sites for various mRNA export factorsthat are critical for mRNA ribonucleoproteinremodeling events. Because mRNA export fac-tors are constitutively attached to the cytoplas-mic filaments, they can be considered part of theNPC. The DEAD-box helicase Dbp5 (hDbp5)plays a central role in the remodeling machin-ery and is recruited to the NPC via Nup159(hNup214). The ATPase activity of Dbp5 isstimulated by Gle1 (hGle1) and the smallcoactivator molecule inositol hexakisphosphate(151, 152). Gle1 is recruited to the cytoplasmicface of the NPC via its interaction with Nup42.

The structure of Nup1591−387 reveals an un-usually asymmetric seven-bladed β-propeller(Figure 9a) (11). The structure of the corre-sponding domain of hNup214 (residues 1–450)is similar in its conserved asymmetric core butdeviates from its yeast counterpart by numerousextended loops and a ∼30-residue C-terminalpeptide segment, which folds back onto theβ-propeller and binds to its bottom face(Figure 9b) (13). The complex ofhNup2141−450 with hDbp5-ADP showsthat the association between the two proteins

is primarily mediated by the 6D7A loop ofthe β-propeller and the N-terminal RecA-likedomain of hDbp5 (Figure 9c) (153, 154).Strikingly, the interface is characterized bystrong opposing electrostatic surface poten-tials. The positively charged surface area ofhDbp5 that binds hNup214 is also utilizedfor its interaction with RNA, as visualizedin the structure of hDbp5 in complex withthe ATP analog AMPPNP and U6-RNA(Figure 9d ) (154, 155). These findings explainthe mutually exclusive binding events. Whereasthe N-terminal RecA-like domain of hDbp5interacts with hNup214, the C-terminal RecA-like domain binds the ATPase activator Gle1(156). The relative arrangement of the twoRecA-like domains is governed by the boundnucleotide (Figure 9e) (155). Altogether,the conformational changes and the iterativeswitch in binding partners of hDbp5 have beenproposed to be an integral part of a ratchetmechanism of mRNA export (42, 153, 154).

Nup145N (hNup98) is another mRNAexport factor, which localizes to the cytoplas-mic filaments via its interaction with Nup82.Nup145N and Nup145C (hNup96) are theN-terminal and C-terminal products of theevolutionarily conserved autoproteolytic cleav-age of the Nup145 (hNup98–96) precursorprotein, which is catalyzed by the C-terminaldomain of Nup145N (97, 157). Nup145C ispart of the Nup84 complex (92). The structureof hNup98676−920 reveals a novel α/β-fold andprovides mechanistic insight into the autopro-teolytic cleavage (Figure 9f ) (158–160). In ad-dition to catalysis, the autoproteolytic domainof hNup98 is responsible for its NPC localiza-tion. hNup98 is homologous to three proteinsin yeast, Nup145N, Nup100, and Nup116,which share a similar domain organization (161,162). Although Nup100 and Nup116 possessthe autoproteolytic domain, these nucleoporinsare not generated by autoproteolysis.

The autoproteolytic domain is predictedto be the only structured region of hNup98,whereas the remaining ∼700-residue re-gion contains numerous FG repeats and the57-residue Gle2-binding sequence (GLEBS,


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hNup214NTD hNup214NTD•hDbp5-ADP

hNup98APD•hNup96N

Nup98APD

Nup96N peptide

Nup159NTD

hDbp5-AMPPNP•U6-RNA

Rae1

Nup98GLEBS

hRae1•hNup98GLEBS

a

α-helical region1 830 1,301 1,848 2,142 2,439 2,771 3,040 3,224

I Zinc fingers II III E3 IV CycH1,171 1,355 2,012 2,309 2,631 2,911 3,062

j

b c

d e f

g

hDbp5-ADP

hSUMO•hRanGAP1CTD•hUbc9•hNup358E3

RanGAP1CTD

Ubc9

Nup358E3

Sumo

i

hRan-GMPPNP•hNup358RanBD1

Ran Nup358RanBD1

h

Figure 9Crystal structures of cytoplasmic-filament nucleoporins and associated mRNA export factors:(a) Nup159NTD (1XIP), (b) hNup214NTD (2OIT), (c) hNup214NTD·hDbp5-ADP (3FMO, 3FMP, 3FHC),(d ) hDbp5-AMPPNP·U6-RNA (3FHT, 3G0H), (e) hDbp5-ADP (3EWS), ( f ) hNup98APD·hNup96N

(1KO6), ( g) hRae1·hNup98GLEBS (3MMY), (h) hRan-GMPPNP·hNup358RanBD1 (1RRP), (i ) hSUMO·hRanGAP1CTD·hUbc9·hNup358E3 (1Z5S), and ( j) domain organization of hNup358. APD, autoproteolyticdomain; CycH, cyclophilin homology domain; CTD, C-terminal domain; E3, E3 ligase domain; GLEBS,Gle2-binding sequence; I−IV, Ran-binding domains; NTD, N-terminial domain; RanBD, Ran-bindingdomain. Protein Data Bank codes are in parentheses. The prefix h refers to human; protein names without aprefix refer to yeast.


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residues 157–213), which serves as the dock-ing site for the human mRNA export factorhRae1 (Gle2 in yeast) (Figure 3). This inter-action is essential for a normal morphologyof the NPC with respect to the surroundingouter nuclear envelope membrane. Among thethree yeast hNup98 homologs, only Nup116features a GLEBS motif. The structure ofhRae1·hNup98GLEBS reveals a seven-bladed β-propeller of hRae1 with several extensive sur-face loops that binds the GLEBS motif on itstop face (Figure 9g) (20).

Vertebrates contain an additional cyto-plasmic filament nucleoporin, Nup358, whichprovides binding sites for transport factors,Ran, and RanGAP1. These binding eventsplay important roles in the assembly anddisassembly of karyopherin-cargo transportcomplexes (163, 164). Nup358 contains four∼150-residue domains that facilitate binding toRan (RanBD1–4) (Figure 9j). The structuralanalysis of Ran bound to the nonhydrolysableGTP analog GMPPNP in complex withhNup358RanBD1 reveals that the GTPasedomain of Ran and its tethered C-terminalhelix embrace the pleckstrin-homology do-main of hNup358RanBD1 (Figure 9h) (165). A63-residue region in hNup358 provides thebinding site for SUMOylated RanGAP1 andwas visualized in the structure of the hSUMO·hRanGAP1419−587·hUbc9·hNup3582631−2693

complex (Figure 9i) (166).

Nuclear Basket Nucleoporins

The nuclear basket represents the least ex-plored part of the NPC and is composed ofNup1, Nup2, Nup60, Mlp1, and Mlp2 in yeast,whereas the vertebrate basket harbors onlythree nucleoporins, Nup153, Nup50, and TPR.In addition to the large coiled-coils of Mlp1/2(TPR), the nuclear basket nucleoporins con-tain α/β-regions and FG repeats (Figure 3).Furthermore, Nup153 contains four zinc fin-gers arranged in tandem that are not presentin its yeast homolog Nup1 and that facilitatebinding to Ran in both its GDP- and GTP-bound states. Notably, eight zinc fingers are

also present in Nup358. Together, the zinc fin-ger domains of these two nucleoporins appearto generate a high local Ran concentration atthe NPC, increasing the efficiency of nucleo-cytoplasmic transport (167).

The structural analyses of individualrNup153 zinc finger motifs in complex withhRan-GDP demonstrate that each zinc fingermodule independently binds to Ran in anidentical fashion (Figure 10) (168–170).Another important function of the nuclearbasket is the termination of Kap-α-mediatedcargo import. The structures of Kap-α boundto nuclear localization sequence peptides orin complex with a short N-terminal sequencemotif of mNup50 (Nup2) show that the sameKap-α surface is utilized for both bindingevents, thereby providing a structural basisfor basket-mediated disassembly of importcomplexes (Figure 10) (171–173).

MODELS OF THE SYMMETRICNUCLEAR PORE COMPLEX CORE

The highest resolution of EM reconstructionsof the entire NPC obtained to date (∼60 A)does not allow the placement of individualcrystal structures (51, 89). Even the dockingof the entire ∼400-A-long Nup84 complex,which represents a major portion of the sym-metric NPC core, determined to ∼35-A res-olution has not yet been achieved. Therefore,the spatial arrangement and stoichiometry ofthe nucleoporins in the NPC core remain un-known. Moreover, neighboring nucleoporinswithin the intact NPC may potentially mod-ulate the interactions that are observed in 3DEM reconstructions of the isolated heptamerand in the crystal structures. Further compli-cating factors in the structural characterizationof the NPC are its flexible nature and dynamicchanges, such as the dilation of the central chan-nel during transport or the proposed lateralopening of the NPC scaffold during the importof integral membrane proteins (Figure 3c,d andMovies 3 and 4) (21, 88, 112, 136). An attractivescenario to explain this remarkable plasticitywould be the notion that nucleoporins change


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rNup153ZnF2•hRanGDP

Nup153ZnF2

RanGDP

mKap-α•mNup50N

Kap-α

Nup50N

646 876

C1,683

Zinc fingersUnstructured FG repeatsNup153 N1

a

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Zn2+

Mg2+

Figure 10(a) Crystal structures of basket nucleoporins: mKap-α·mNup50N (2C1M) and rNup153ZnF2·hRanGDP (3CH5, 3GJ3–8). The Zn andMg ions are represented as orange and red spheres, respectively. (b) Domain organization of hNup153. The red bar marks the fragmentwhose crystal structure is experimentally determined. Protein Data Bank codes are in parentheses. N, N-terminal domain; h, human;m, mouse; r, rat.

their interaction partners dynamically. In thisrespect, the symmetric NPC core can be re-garded to possess fluid-like properties. Supportfor this idea comes from crystal structures thatreveal promiscuous interactions (8, 18, 19, 21).Collectively, these considerations illustrate thedifficulty in arriving at a high-resolution struc-ture of the intact NPC. In the absence of high-resolution EM structures of the intact NPC,alternative indirect approaches have thereforebeen applied to formulate models for the archi-tecture of the NPC.

We proposed a model termed “coat for thenuclear pore membrane,” or “NPC coat,” thatwas deduced from several crystal structures,biochemical and in vivo analyses, homologiesbetween COPII components and nucleoporins,

as well as symmetry and size considerationsderived from EM studies (Figure 5c) (8, 15,112). Specifically, we made the assumption thatthe arrangement of the seven nucleoporins ofthe heptameric Nup84 complex corresponds totheir mapped positions in the isolated heptamerand that the observed hetero-octameric as-semblies of Sec13·Nup145C and Seh1·Nup85occur as vertical poles in the assembled NPC.These assumptions would result in a cylindricalscaffold that positions 32 copies of each of theseven nucleoporins in four rings, which arestacked in an antiparallel fashion, consistentwith the eight- and twofold rotational symme-tries of the NPC core. The overall dimensionsof this cylindrical NPC coat would be in accordwith the experimentally determined size of


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the yeast NPC (Figure 5c). The observedpromiscuity of Nup145C to interact witheither Nup145C or Nup84 suggests that alter-native interactions can occur, whereas othertight interactions, such as those within theSec13·Nup145C and Seh1·Nup85 pairs, arenot expected to break. In summary, the proteinswould maintain their relative position in thescaffold, whereas changes in their interactionpartners would confer a dynamic character tothe NPC (Figure 5d ). This NPC coat is envi-sioned to be sandwiched between the POM andthe adaptor cylinders and to contain gaps andholes, which would allow for interdigitationwith adjacent nucleoporins (Figure 1b).

In an alternative “computational model,” adiverse set of biophysical and proteomic data

was used as input to calculate a blueprint ofthe NPC (Figure 11a) (67, 174). Key assump-tions of this model refer to a fixed absolute stoi-chiometry of 30 different nucleoporins (456proteins in total), to a static single state of theNPC, and to the symmetry of the NPC estab-lished by EM. In the resulting model, Nup84complexes would form two separated rings onthe cytoplasmic and nucleoplasmic periphery,each containing eight copies of the heptamer.These peripheral outer rings would sandwichtwo inner rings containing Nup157, Nup170,Nup188, and Nup192. Attached onto this scaf-fold are the remaining nucleoporins.

The third model resembles the computa-tional model in that it contains two periph-eral outer rings each containing eight copies of

Cytoplasmicring

Inner ring

Nucleoplasmicring

b

aOuter rings Inner rings Symmetric NPC core

Nup133

Nup120

Nup

84

Nup85

Nup1

45C

Sec13

Seh1

Figure 11Alternative models of the architecture of the symmetric nuclear pore complex (NPC) core. (a) The “computational model” and (b) the“lattice model.” For details, see description in the text. Images are reprinted with permission from References 67 and 14, respectively.


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the heptameric Nup84 complex, but this modeldiffers in that it features only a single innerring composed of Nic96, Nup157, Nup170,Nup188, and Nup192 (Figure 11b) (14). Thekey difference from the other two models isthe assumption that the Nup84 complexes arenot arranged horizontally but vertically, withNup133 molecules facing toward the nucle-oplasm and cytoplasm, respectively. The fiveproteins of the inner ring are assumed to assem-ble into a structural unit that would bridge thetips of the Y-shaped heptamers at the equato-rial plane in a lattice-like fashion. Since the firstintroduction of the “lattice model,” four strik-ingly different arrangements were proposed forthis lattice (14, 175, 176).

Owing to the assumptions and uncertain-ties, the proposed models clearly have to beconsidered as working hypotheses that provide

an important framework for future experimentsand validations. The determination of the re-maining individual nucleoporin structures inthe near future will certainly help to improveand potentially modify the current modelsand continue to provide unexpected and spec-tacular insight into the complex architectureof the NPC. However, the greatest advancestoward an atomic structure of the NPC willprobably be the structure determinations oflarge multinucleoporin complexes by X-raycrystallography and/or EM. The challenge isthen to assemble an atomic-resolution mosaicof the entire NPC. With the rapid pace ofthe past five years, we are optimistic that thisgreat challenge will be overcome, resultingin the elucidation of the remaining mysteriesof the NPC, one of the largest proteinaceousassemblies of the eukaryotic cell.

SUMMARY POINTS

1. The nuclear pore complex (NPC) is embedded in pores of the nuclear envelope andconstitutes the portal for all transport events between the cytoplasm and the nucleus.The NPC consists of an evolutionarily conserved set of ∼30 different proteins, termednucleoporins, that are organized into several subcomplexes, each of which occurs inmultiple copies, resulting in ∼500–1,000 protein molecules in the fully assembled NPC.

2. The characterization of the NPC by electron microscopy (EM) revealed a doughnut-shaped central core with eightfold rotational symmetry that is decorated with cytoplasmicfilaments and a nuclear basket. The NPC is flexible and dynamic, as, for example, observedduring cargo translocation.

3. The permeability barrier of the NPC is formed by unstructured phenylalanine-glycine(FG)-repeat regions that also serve as docking sites for transport factor-cargo complexes.Whereas the mobile transport machinery is reasonably well understood at the atomiclevel, the precise nature of the permeability barrier remains an area of heavy debate.

4. The large size, flexible nature, and difficulty of obtaining sufficient quantities of puri-fied NPCs preclude the structure determination of the entire NPC with present X-raycrystallography technology. Therefore, a divide-and-conquer strategy has been applied,whereby crystal structures of NPC components are determined and then assembled intohigher-order structures with the help of EM and computational approaches.

5. The predominant folds present in nucleoporins are seven-bladed β-propellers and ir-regular α-helical zigzag domains. Surprisingly, two seven-bladed β-propellers contain ablade that is contributed in trans by another nucleoporin, an architectural feature that isalso observed in the COPII vesicle coat.


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6. The crystal structures of the components of the heptameric Nup84 complex could ap-proximately be fit into a 3D EM map of the entire heptamer. The Nup84 complexdisplays conformational flexibility, and some of its components can interact in a promis-cuous fashion, which perhaps provides a structural basis for the flexible nature of theNPC.

7. The structures of NPC components and of coat protein complexes in the endocytic andsecretory pathways uncovered striking similarities with respect to their folds, architec-tures, and associations, supporting their common evolutionary origin in a progenitorprotocoatomer.

8. Various architectures for the symmetric NPC core have been proposed on the basis ofdifferent approaches and assumptions that need to be validated in the future. Therefore,these models should be regarded as working hypotheses.

FUTURE ISSUES

1. The remaining structured regions of nucleoporins and further subcomplexes are expectedto be determined in the near future. Specifically, the heteromeric Nic96 complex needsto be characterized in a similar fashion as the heptameric Nup84 complex.

2. The current highest resolution of EM reconstructions of the entire NPC is insufficientfor the docking of nucleoporin crystal structures. Pushing this resolution limit throughsignificant advances in EM techniques and a complete inventory of nucleoporin structuresmay ultimately yield a complete pseudoatomic model for the entire NPC and resolvemajor unresolved questions, such as its detailed architecture and stoichiometry.

3. The following key questions must be addressed: What is the stoichiometry of the ∼30nucleoporins in the assembled NPC? What is the basis for the substantial mass differencebetween vertebrate and yeast NPCs (∼112 versus ∼66 MDa)? Do nucleoporins interactwith each other in the assembled NPC in the same way as in isolated subcomplexes?

4. Can the structure of the NPC be assembled in a LEGO-like fashion from its individualparts? What is the molecular mechanism for the cell cycle–dependent reversible assemblyand disassembly in cells with open mitosis? Are there cellular factors that regulate theassembly and/or disassembly of the NPC? The development of an in vitro assemblysystem of the symmetric NPC core would certainly help in answering these questions.

5. Transport events such as the import of integral membrane proteins of the nuclear enve-lope destined for the inner nuclear membrane suggest that large-scale rearrangementsoccur in the NPC. However, the structural basis and molecular mechanisms are poorlyunderstood.

6. The promiscuous nature of the NPC complicates the in vivo analysis with current func-tional assays. An additional obstacle is the fact that nucleoporins have distinct functionsduring the cell cycle. Therefore, in vitro assays that quantitatively probe the NPC func-tion need to be developed.


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7. The ultimate goal of the structural characterization of the NPC is an atomic-resolutionmovie that depicts the dynamic changes of the NPC during nucleocytoplasmic transportof various cargoes and that integrates the many other functions of the NPC.

8. The advances in the structural characterization of the NPC will finally contribute to abetter understanding of the mechanisms of “nucleoporin diseases,” which in many casesremain enigmatic at present.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

We thank Kuo-Chiang Hsia, Jana Mitchell, Vivien Nagy, Alina Patke, Tobias Reichenbach, Hyuk-Soo Seo, Pete Stavropoulos, and Deniz Top for discussions and comments on the manuscript, andStephanie Etherton for help with editing the manuscript. E.W.D. is the Dale F. and Betty AnnFrey Fellow of the Damon Runyon Cancer Research Foundation, DRG-1977-08, and A.H. wassupported by a SCOR grant from the Leukemia and Lymphoma Society and by a V Scholar Awardfrom the V Foundation for Cancer Research. We apologize in advance to those investigators whosework was inadvertently overlooked or could not be included due to space restrictions.

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164. Matunis MJ, Coutavas E, Blobel G. 1996. A novel ubiquitin-like modification modulates the partitioningof the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex.J. Cell Biol. 135:1457–70

165. Vetter IR, Nowak C, Nishimoto T, Kuhlmann J, Wittinghofer A. 1999. Structure of a Ran-bindingdomain complexed with Ran bound to a GTP analogue: implications for nuclear transport. Nature398:39–46

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BI80-FrontMatter ARI 16 May 2011 14:13

Annual Review ofBiochemistry

Volume 80, 2011Contents

Preface

Past, Present, and Future Triumphs of BiochemistryJoAnne Stubbe � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �v

Prefatory

From Serendipity to TherapyElizabeth F. Neufeld � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Journey of a Molecular BiologistMasayasu Nomura � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �16

My Life with NatureJulius Adler � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �42

Membrane Vesicle Theme

Protein Folding and Modification in the MammalianEndoplasmic ReticulumIneke Braakman and Neil J. Bulleid � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �71

Mechanisms of Membrane Curvature SensingBruno Antonny � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 101

Biogenesis and Cargo Selectivity of AutophagosomesHilla Weidberg, Elena Shvets, and Zvulun Elazar � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 125

Membrane Protein Folding and Insertion Theme

Introduction to Theme “Membrane Protein Folding and Insertion”Gunnar von Heijne � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 157

Assembly of Bacterial Inner Membrane ProteinsRoss E. Dalbey, Peng Wang, and Andreas Kuhn � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 161

β-Barrel Membrane Protein Assembly by the Bam ComplexChristine L. Hagan, Thomas J. Silhavy, and Daniel Kahne � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 189

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Transmembrane Communication: General Principles and Lessonsfrom the Structure and Function of the M2 Proton Channel, K+

Channels, and Integrin ReceptorsGevorg Grigoryan, David T. Moore, and William F. DeGrado � � � � � � � � � � � � � � � � � � � � � � � � 211

Biological Mass Spectrometry Theme

Mass Spectrometry in the Postgenomic EraBrian T. Chait � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 239

Advances in the Mass Spectrometry of Membrane Proteins:From Individual Proteins to Intact ComplexesNelson P. Barrera and Carol V. Robinson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 247

Quantitative, High-Resolution Proteomics for Data-DrivenSystems BiologyJurgen Cox and Matthias Mann � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 273

Applications of Mass Spectrometry to Lipids and MembranesRichard Harkewicz and Edward A. Dennis � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 301

Cellular Imaging Theme

Emerging In Vivo Analyses of Cell Function UsingFluorescence ImagingJennifer Lippincott-Schwartz � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 327

Biochemistry of Mobile Zinc and Nitric Oxide Revealedby Fluorescent SensorsMichael D. Pluth, Elisa Tomat, and Stephen J. Lippard � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 333

Development of Probes for Cellular Functions Using FluorescentProteins and Fluorescence Resonance Energy TransferAtsushi Miyawaki � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 357

Reporting from the Field: Genetically Encoded Fluorescent ReportersUncover Signaling Dynamics in Living Biological SystemsSohum Mehta and Jin Zhang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 375

Recent Advances in Biochemistry

DNA Replicases from a Bacterial PerspectiveCharles S. McHenry � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 403

Genomic and Biochemical Insights into the Specificity of ETSTranscription FactorsPeter C. Hollenhorst, Lawrence P. McIntosh, and Barbara J. Graves � � � � � � � � � � � � � � � � � � � 437

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Signals and Combinatorial Functions of Histone ModificationsTamaki Suganuma and Jerry L. Workman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 473

Assembly of Bacterial RibosomesZahra Shajani, Michael T. Sykes, and James R. Williamson � � � � � � � � � � � � � � � � � � � � � � � � � � � � 501

The Mechanism of Peptidyl Transfer Catalysis by the RibosomeEdward Ki Yun Leung, Nikolai Suslov, Nicole Tuttle, Raghuvir Sengupta,

and Joseph Anthony Piccirilli � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 527

Amyloid Structure: Conformational Diversity and ConsequencesBrandon H. Toyama and Jonathan S. Weissman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 557

AAA+ Proteases: ATP-Fueled Machines of Protein DestructionRobert T. Sauer and Tania A. Baker � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 587

The Structure of the Nuclear Pore ComplexAndre Hoelz, Erik W. Debler, and Gunter Blobel � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 613

Benchmark Reaction Rates, the Stability of Biological Moleculesin Water, and the Evolution of Catalytic Power in EnzymesRichard Wolfenden � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 645

Biological Phosphoryl-Transfer Reactions: Understanding Mechanismand CatalysisJonathan K. Lassila, Jesse G. Zalatan, and Daniel Herschlag � � � � � � � � � � � � � � � � � � � � � � � � � � � 669

Enzymatic Transition States, Transition-State Analogs, Dynamics,Thermodynamics, and LifetimesVern L. Schramm � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 703

Class I Ribonucleotide Reductases: Metallocofactor Assemblyand Repair In Vitro and In VivoJoseph A. Cotruvo Jr. and JoAnne Stubbe � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 733

The Evolution of Protein Kinase Inhibitors from Antagoniststo Agonists of Cellular SignalingArvin C. Dar and Kevan M. Shokat � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 769

Glycan Microarrays for Decoding the GlycomeCory D. Rillahan and James C. Paulson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 797

Cross Talk Between O-GlcNAcylation and Phosphorylation:Roles in Signaling, Transcription, and Chronic DiseaseGerald W. Hart, Chad Slawson, Genaro Ramirez-Correa, and Olof Lagerlof � � � � � � � � � 825

Regulation of Phospholipid Synthesis in the YeastSaccharomyces cerevisiaeGeorge M. Carman and Gil-Soo Han � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 859

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Sterol Regulation of Metabolism, Homeostasis, and DevelopmentJoshua Wollam and Adam Antebi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 885

Structural Biology of the Toll-Like Receptor FamilyJin Young Kang and Jie-Oh Lee � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 917

Structure-Function Relationships of the G Domain, a CanonicalSwitch MotifAlfred Wittinghofer and Ingrid R. Vetter � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 943

STIM Proteins and the Endoplasmic Reticulum-PlasmaMembrane JunctionsSilvia Carrasco and Tobias Meyer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 973

Amino Acid Signaling in TOR ActivationJoungmok Kim and Kun-Liang Guan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1001

Mitochondrial tRNA Import and Its Consequencesfor Mitochondrial TranslationAndre Schneider � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1033

Caspase Substrates and Cellular RemodelingEmily D. Crawford and James A. Wells � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1055

Regulation of HSF1 Function in the Heat Stress Response:Implications in Aging and DiseaseJulius Anckar and Lea Sistonen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1089

Indexes

Cumulative Index of Contributing Authors, Volumes 76–80 � � � � � � � � � � � � � � � � � � � � � � � � � �1117

Cumulative Index of Chapter Titles, Volumes 76–80 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1121

Errata

An online log of corrections to Annual Review of Biochemistry articles may be found athttp://biochem.annualreviews.org/errata.shtml

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Documents

The Structure of the Nuclear Pore Complex