Architecture of the nuclear pore inner ring complex

Tobias Stuwe

1,4

Christopher J. Bley

1,4

Karsten Thierbach

1,4

Stefan Petrovic

1,4

Sandra

Schilbach

1,†

Daniel J. Mayo

Thibaud Perriches

Emily J. Rundlet

Young E. Jeon

Leslie N. Collins

Ferdinand M. Huber

Daniel H. Lin

Marcin Paduch

Akiko Koide

Vincent Lu

Jessica Fischer

Ed Hurt

Shohei Koide

Anthony A. Kossiakoff

, and

André

Hoelz

1,*

California Institute of Technology, Division of Chemistry and Chemical Engineering, 1200 East

California Boulevard, Pasadena, CA, 91125, USA

Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL

60637, USA

Biochemistry Center of Heidelberg University, 69120 Heidelberg, Germany

Abstract

The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic

transport. We present the reconstitution and interdisciplinary analyses of the ~425-kDa inner ring

complex (IRC), which forms the central transport channel and diffusion barrier of the NPC,

revealing its interaction network and equimolar stoichiometry. The Nsp1•Nup49•Nup57 channel

nucleoporin hetero-trimer (CNT) attaches to the IRC solely through the adaptor nucleoporin

Nic96. The CNT•Nic96 structure reveals that Nic96 functions as an assembly sensor that

recognizes the three dimensional architecture of the CNT, thereby mediating the incorporation of a

defined CNT state into the NPC. We propose that the IRC adopts a relatively rigid scaffold that

recruits the CNT to primarily form the diffusion barrier of the NPC, rather than enabling channel

dilation.

One of the great hallmarks of eukaryotic evolution is the enclosure of genetic information in

the nucleus. The spatial segregation of replication and transcription in the nucleus from

translation in the cytoplasm imposes the requirement of transporting thousands of

macromolecules between these two compartments. Nuclear pore complexes (NPCs) are

massive transport channels that allow bidirectional macromolecular exchange across the

nuclear envelope (NE) and thus function as key regulators of the flow of genetic information

from DNA to RNA to protein (

Correspondence: hoelz@caltech.edu (A.H.).

these authors contributed equally to this work

†

present address: Max-Planck-Institute of Biophysical Chemistry, Molecular Biology, Am Fassberg 11, 37077 Göttingen, Germany

The authors declare no financial conflicts of interest.

Supplementary Materials:

Materials and Methods

Figures S1–S38

Tables S1–S9

Movies S1–S4

References (

42–77

)

HHS Public Access

Author manuscript

Science

. Author manuscript; available in PMC 2016 April 11.

Published in final edited form as:

Science

. 2015 October 2; 350(6256): 56–64. doi:10.1126/science.aac9176.

Author Manuscript

NPCs are formed by multiple copies of ~34 distinct proteins, termed nucleoporins (nups)

(

). The docking of the yeast coat nup complex (CNC) crystal structure into a cryo-electron

tomographic (ET) reconstruction of the intact human NPC revealed its organization into two

sixteen-membered CNC rings on the nuclear and cytoplasmic faces of ~100 nm NE pores

(Fig. 1A) (

). This arrangement established that the doughnut-shaped inner ring is

composed of adaptor and channel nups (

Anchoring of the inner ring in the NE pore is mediated by the membrane recruitment

complex, composed of adaptor nups Nup53 and Nup170, and transmembrane nup Ndc1 (

–

). Recently, Nup53 was shown to harbor distinct binding sites for Nic96, Nup170 and

Nup192, allowing the Nup53•Nup170 hetero-dimer to interact with either Nic96•Nup192 or

Nic96•Nup188 (

The inner ring harbors the central transport channel and diffusion barrier of the NPC,

preventing macromolecules larger than ~40 kDa to freely diffuse across the NE (

). The

channel nups Nsp1, Nup49, and Nup57 constitute part of the central transport channel and

form the diffusion barrier with their disordered phenylalanine-glycine (FG) repeats (

–

). Transport factors ferry cargo across the NE by binding to FG repeats in the central

transport channel (

). Furthermore, the central transport channel seems to be permanently

occupied by transport factors even after biochemical purification of NPCs (

). In

addition to their N-terminal FG repeats, the three channel nups are predicted to possess C-

terminal coiled-coil regions (

). The knockout of any of the three channel nups is lethal

S. cerevisiae

, whereas the deletion of any two N-terminal FG-repeat regions can be

tolerated, but reduces transport rates, suggesting an essential function of the coiled-coil

regions (

–

Arguably, the most important questions about the inner ring architecture pertain to the

recruitment and positioning of the channel nups, because of their essential function in

forming the diffusion barrier and providing binding sites for transport factors. Native

purifications from

S. cerevisiae

and mammalian cells showed that the channel nups co-

purified with Nic96, suggesting the evolutionary conservation of a hetero-tetrameric

Nsp1•Nup49•Nup57•Nic96 complex (

). Subsequent biochemical reconstitution

attempts yielded channel nup complexes with inconsistent stoichiometries that resisted

structural analysis (

–

). Reconstitutions employing channel nup fragments revealed

dynamic interactions and generated a series of crystal structures with various homomeric

and heteromeric assembly states (

). Biochemical analysis of these structures led to

heavily contested models that have resisted physiological validation, including the proposal

that the reversible karyopherin-mediated transition between homo- and hetero-oligomers

facilitates the constriction and dilation of the central transport channel (

–

Despite more than half a century of research, our understanding of the inner ring architecture

remains rudimentary. The prevalent assumption is that the inner ring of the NPC is

composed of multiple copies of a single NPC subcomplex, but such a complex has remained

elusive. Here, we present an in-depth characterization of the NPC’s inner ring. Starting with

the reconstitution of a ~425 kDa hetero-hexameric inner ring complex (IRC), we

demonstrate its scaffolding function by showing that it interacts with additional peripheral

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nups. In dissecting the underlying protein interaction network of the IRC, we determined an

equimolar stoichiometry for its six components and identified Nic96 as the sole NPC

attachment site for the channel nup hetero-trimer (CNT). Nic96 is essential for CNT

recruitment and, through its interaction with Nup192, for proper CNT positioning within the

inner ring scaffold

in vivo

. Structural and functional analyses of the intact CNT reveal a

defined coiled-coil domain architecture that is specifically recognized by Nic96. Our results

differ dramatically from previous characterizations of channel nup fragments and an

associated model for a flexible transport channel that is capable of constriction and dilation

of up to ~400 Å (

–

). We propose a model for the inner ring architecture in which

sixteen copies of the IRC form a relatively rigid scaffold. In the proposed arrangement, the

central transport channel would be filled with the channel nup FG repeats to establish the

diffusion barrier of the NPC and to provide binding sites for cargo•transport factor

complexes.

Reconstitution and dissection of the inner ring complex (IRC) and binding

of peripheral nups

To reconstitute and uncover the architectural principles of a putative inner ring complex

(IRC), we developed expression and purification protocols for the channel, adaptor, and

cytoplasmic filament nups from

C. thermophilum

on the milligram scale. All purified nups

lacked FG-repeat regions to facilitate soluble protein expression (Fig. 1A). We refer to all

nups in the remainder of the text according to the

C. thermophilum

nomenclature and

indicate nups from other species with a prefix.

Using recombinant purified nups, we reconstituted a monodisperse hetero-hexameric IRC

containing the adaptor nups Nup192, Nic96 and Nup145N, and the Nsp1•Nup49•Nup57

channel nup hetero-trimer (CNT) (Fig. 1, A and B and fig. S1A). The ~425 kDa measured

molecular mass of the IRC is consistent with an equimolar stoichiometry (Table S1). No

higher-order oligomers formed at concentrations up to ~5 μM. Our reconstitution omitted

Nup170 because of its low solubility in standard buffer conditions. However, the interaction

of Nup170 with a C-terminal region of Nup53, directly adjacent to the membrane-binding

motif, established its proximity to the pore membrane (

Further analyses showed that the reconstituted IRC is a

bona fide

NPC scaffold complex,

capable of binding the membrane recruiting Nup53, and the peripheral cytoplasmic filament

complex (CFC), forming a stable hetero-nonamer (Fig. 1C and fig. S1B). The measured

masses for both complexes, ~100 kDa lower than expected, indicated their dynamic nature at

the tested concentrations. The reconstitution and structure determination of a core IRC-CFC

attachment complex composed of the CFC nups Nup82

NTD

and Nup159

and the

autoproteolytic domain (APD) of the IRC component Nup145N revealed the molecular

details of this inter-subcomplex linkage (Fig. 1J and figs. S2, A and B and Table S2). A

structural comparison with its

S. cerevisiae

homolog showed that the interactions of the core

CFC•Nup145N

APD

complex are conserved, despite low sequence conservation (

This analysis supports the hypothesis that nup interactions in the NPC are evolutionary

conserved (fig. S3).

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Because Nup145N acts as an adaptor that attaches the CFC, we probed its interaction with

the other IRC components. Nup145N formed a stoichiometric complex with Nup192,

elicited weak CNT association, and barely interacted with the Nic96 solenoid (SOL) (fig.

S2, C to E). However, Nup145N was incapable of linking the CNT to Nup192, suggesting

mutually exclusive interactions (fig. S2F).

The IRC reconstitution enabled us to identify how the CNT is incorporated into the IRC by

defining a minimal CNT attachment complex. We found that Nup145N, Nic96

SOL

and the

N-terminal and middle domains of Nup192 were dispensable for CNT attachment, because a

minimal stoichiometric Nup192

TAIL

•Nic96

R1-R2

•CNT attachment complex could be

reconstituted (Fig. 1D and E and figs. S1, C and D). Nic96

R1-R2

is an adaptor linking the

CNT with Nup192

TAIL

(Fig. 1, F and G and figs. S1, E and F, and S4, A and B).

Consistently, no interaction between Nup192

TAIL

and the CNT was observed (fig. S4C).

Most importantly, an IRC lacking Nic96

failed to incorporate the CNT, demonstrating that

Nic96

is the sole IRC attachment site for the CNT (Fig. 1H and fig. S1G). We tested

whether Nup53 could rescue the deletion of Nic96

by providing an additional CNT

binding site. However, the CNT did not incorporate into the IRC in the presence of Nup53

(Fig. 1I and fig. S1H).

These data established an equimolar stoichiometry of the six different IRC components and

demonstrated that the CNT is solely attached to the adaptor nups through its interaction with

Nic96

The Nic96-Nup192 interaction positions the CNT in the central transport

channel

Consistent with previous findings that Nup192 and Nup188 form mutually exclusive

complexes (

), we were able to reconstitute an architecturally equivalent

Nup188

TAIL

•Nic96

R1-R2

•CNT hetero-pentamer (fig. S5, A and B). However, salt stability

and competition experiments showed that Nup192

TAIL

interacts more tightly with Nic96

than Nup188

TAIL

does (figs. S5, C and D and S6, A and B).

To gain insight into the molecular details of the Nic96

interaction with Nup192 and

Nup188, we determined the crystal structures of the TAIL domains of Nup192 and Nup188

(Table S2). Nup192

TAIL

and Nup188

TAIL

share a similar crescent-shaped architecture,

composed of ARM and HEAT repeats with overall similar surface conservation and

electrostatic properties. However, Nup188

TAIL

contains an additional evolutionarily

conserved C-terminal ARM repeat (Fig. 2A–E and figs. S7 to S11) (

). When docked into

the electron microscopy (EM) reconstruction of

S. cerevisiae

Nup192, Nup192

TAIL

was

located at the bottom of the question mark-shaped map (Fig. 2G) (

). Based on the

structural and biochemical results and previous findings that Nup192, but not Nup188, is

essential for viability in yeast, we focused our further analyses on the IRC containing

Nup192 (

To identify the Nic96

interaction surface in Nup192

TAIL

, we tested alanine mutants of 15

conserved surface residues and identified two adjacent residues, Phe1735 and Ile1730, that

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abolished binding and decreased the interaction with Nic96

, respectively (Fig. 2F to H and

figs. S12 and S13). Both residues are located in a hydrophobic pocket at the bottom of the

Nup192 molecule (Fig. 2G).

To evaluate the physiological relevance of the identified Nup192-Nic96 interaction for NPC

function, we analyzed the identified Nup192 mutants in

S. cerevisiae

. Whereas the F1735A

point mutant (Y1679A in yeast) displayed no significant defects in growth and ribosomal or

mRNA export, the removal of the entire TAIL domain resulted in a substantial growth defect

at all temperatures, and substantial mRNA and ribosomal export defects, at 30 ºC and 37 ºC,

respectively (Fig. 2I, K and L). Despite the severity of the ΔTAIL phenotypes, Nup57-GFP

yielded strong NE staining (Fig. 2J), consistent with our biochemical analyses that

Nup192

TAIL

is expendable for CNT attachment.

These data established that the Nic96-Nup192 interaction is dispensable for CNT

incorporation into the NPC, but required for proper NPC function, suggesting that the

Nic96-Nup192 interaction is required for correct CNT positioning, and, in turn, for the

proper placement of the FG-repeat meshwork.

Nic96 is the sole NPC attachment site for the CNT

in vivo

To characterize the adaptor function of Nic96, we carried out a mutational analysis of the

conserved R1 and R2 regions of Nic96, which interact with the CNT and Nup192,

respectively (Fig. 3, A to C and fig. S13). Because the Nic96

-CNT interaction is stable at

increased salt concentrations but sensitive to C-terminal truncations, we focused our analysis

on conserved hydrophobic residues in the C-terminal region of Nic96

(figs. S14 and

S15A). Indeed, mutating four leucines located at the C-terminal end of R1 to alanines

(LLLL mutant) abolished the Nic96

-CNT interaction (Fig. 3B and figs. S13 and S15B).

Similarly, because the Nic96

-Nup192

TAIL

interaction is also salt stable and was disrupted

by mutating a hydrophobic pocket on Nup192

TAIL

, we focused our mutational analysis on

evolutionarily conserved hydrophobic residues of Nic96

(figs. S5C and S13). We

identified two mutations, I294A and F298A, which reduced and abolished Nup192

TAIL

binding, respectively (Fig. 3C and fig. S16).

Next, we carried out functional analyses of the identified Nic96 mutants in

S. cerevisiae

(fig.

S17). Nic96 fragments lacking either Nic96

SOL

(ΔSOL) or Nic96

R1-R2

(SOL) were unable

to rescue the previously identified lethal

nic96Δ

phenotype and were not analyzed further

(Fig. 3D) (

). All remaining mutants were viable and yielded NE staining, consistent with

NPC incorporation (Fig. 3E). A Nic96 mutant lacking the Nup192-interacting region R2

(ΔR2) displayed severe growth and mRNA export defects yet failed to affect Nup57-GFP

localization (Fig. 3D to F). Nic96 mutants lacking the CNT-interacting region R1 (R2-SOL)

and R1-R2 linker (ΔLINKER) also had severe growth defects accompanied by a marked

decrease and no detectable NE staining for Nup57-GFP, respectively (Fig. 3D to F).

Additionally, ΔR2 and ΔLINKER displayed a mild ribosome export defect (Fig. 3G). The

milder LLLL and F159A mutants failed to yield significant phenotypes (Fig. 3D to G).

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Together, these experiments demonstrated that the channel nup Nup57 is solely anchored to

the NPC through its interaction with Nic96

in vivo

, in agreement with our biochemical

studies.

Channel nup fragments do not capture the solution behavior of the intact

CNT

A secondary structure analysis revealed that the channel nups Nsp1, Nup49, and Nup57

possessed evolutionarily conserved domain architectures with an N-terminal FG-repeat

region of varying length, followed by three predicted

-helical coiled-coil segments,

CCS1-3 (fig. S18A). Nup57 contained an additional evolutionarily conserved

region,

which preceded the three coiled-coil regions.

Lacking a fully assembled CNT structure, we attempted to obtain structural insight from

short channel nup fragments. Using fragments of Nic96 and the three channel nups from

three different species, we carried out a systematic interaction and crystallization analysis,

which led to the structure determination of multiple channel nup fragments (figs. S19 to

S21). This approach yielded six different crystal structures of homomeric and heteromeric

channel nup assemblies with different stoichiometries (figs. S18B, S22 and S23). Two

structures revealed novel assembly states (figs. S18B and S24). The remaining four were

almost identical to previously determined

R. norvegicus

structures (

). These

assembly states constituted the basis for the proposal of a dilating transport channel, whose

diameter is modulated by karyopherin-mediated transitioning between these homomeric and

heteromeric assembly states (

–

). Despite the remarkable degree of sequence

conservation between human and rat fragments with identical domain boundaries, we

observed different assembly states that behaved inconsistently when mutated or further

truncated (figs. S25 and S26).

In contrast, only two states were detected for the intact CNT in solution, corresponding to

monodisperse equimolar monomeric and dimeric species (fig. S18C). At concentrations up

to ~30 mg/ml, the predominant species was the CNT monomer. Both the CNT monomer and

dimer were capable of forming monodisperse hetero-tetrameric complexes with Nic96

solution (fig. S18D).

To identify which of the three channel nups form a specific interaction with Nic96

, we

carried out a GST-pull down interaction assay. We found that GST-Nic96

did not interact

with separately purified Nsp1 or Nup49•Nup57 in isolation, but formed a complex in the

presence of both (fig. S18E).

These data show that the coiled-coil architecture of the intact CNT cannot be elucidated by a

reductionist approach that involves channel nup coiled-coil fragments, because of their

inconsistent behavior in solution and their inability to capture the correct stoichiometry of

the intact CNT.

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Crystal structure of CNT•Nic96

Our biochemical and

in vivo

data established that the CNT architecture can only be

elucidated by a structure of the intact CNT and that the physiologically relevant state is the

CNT•Nic96

attachment complex. However, extensive crystallization attempts for the

CNT•Nic96

yielded crystals that diffracted to ~10 Å resolution, at best. To improve the

diffraction quality, we generated a series of conformation-specific, high-affinity monobodies

(MBs) and synthetic antibody Fab fragments (sABs) by phage display selection methods as

crystallization chaperones. Up to two different MBs or sABs could bind to the intact IRC

and CNT•Nic96

, forming monodisperse stoichiometric complexes (figs. S27 and S28).

These data established that access to the CNT was sterically unrestricted by its incorporation

into the IRC, suggesting that the CNT protrudes from the IRC.

Crystals of CNT•Nic96

•sAB-158 diffracted to a 3.77 Å resolution, facilitating structure

determination by single anomalous dispersion (SAD) (fig. S29 and Table S4). The crystal

contacts were primarily mediated by sAB-158, demonstrating the effectiveness of

chaperoning reagents in aiding the crystallization of difficult structural targets (fig. S30).

The three channel nups formed a parallel, three-stranded, hetero-trimeric coiled-coil

structure with two sharp kinks that divided the CNT into three left-handed coiled-coil

domains (CCD1-3), with an overall resemblance to the numeral “4” and maximum

dimensions of ~145 Å × ~80 Å × ~60 Å (Fig. 4, A and B and movies S1 and S2). CCD1

formed the ~145 Å long stalk and was composed of the N-terminal coiled-coil segments

(CCS1) of Nsp1, Nup49, and Nup57, each containing twelve heptad repeats. Analogously,

CCS2 and CCS3 were each composed of five heptad repeats that were ~70 Å in length. The

three CCDs were connected by unstructured linker regions, of varying length, between the

three coiled-coil segments of each channel nup. CCS1 of Nup57 was interrupted close to the

base of the stalk by a 56-residue insertion forming a novel fold with two parallel

-sheets

and one

-helix, termed the

insertion domain (Fig. 4B).

A salient feature of the structure was the extensive interaction of Nic96

with all three

CCDs of the CNT, located in the triangular opening at the apex of the “4” (Fig. 4B and

movie S3). Nic96

formed two 11-residue

-helices,

1 and

2, which were linked by a

sharply kinked 16-residue connector (Fig. 4C). One face of helix

1 recognized CCD1 by

binding the Nup57-Nup49 surface, while the opposite face of helix

1 interacted with the

Nsp1-Nup57 surface of CCD3. The kinked loop inserted as a wedge between the top of the

long stalk-forming CCD1 and CCD2. Residues at the N- and C-termini of the loop formed

additional electrostatic contacts with the Nup57-Nup49 surface of CCD1 and extensive

hydrophobic contacts with the Nup49-Nsp1 surface of CCD2, respectively. Finally, helix

which harbors our LLLL mutant, bound exclusively to the hydrophobic Nsp1-Nup49 surface

of CCD3. Not only did Nic96

recognize the proper assembly of all three CCDs by

forming specific interactions with composite CCD surfaces that are each formed by two

different channel nups, but it also locked the CNT into a specific conformation. Overall,

~4,150 Å

of surface area are buried in the Nic96

-CNT interface, involving 38 and 64

residues of Nic96

and the CNT, respectively. The evolutionary conservation of these

interface residues further corroborates the evolutionary conservation of the CNT•Nic96

architecture (fig. S13, S31 to S34).

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CNT•Nic96

•sAB-158 forms a cross-handshake dimer in the asymmetric unit of the crystal

that is generated by two identical dimerization interfaces, involving the Nup57

insertion

domain and CCD3 (Fig. 4D and movie S4). The CNT•Nic96

dimer buried ~2,600 Å

surface area. The N-termini of all channel nups in the dimer point in the same direction,

which would permit the FG repeats to be projected towards the central transport channel.

Notably, sAB-158 did not participate in CNT•Nic96

dimer formation, as it recognized a

composite Nup57-Nup49 surface in the middle of the CCD1 stalk.

The N-terminal regions of Nsp1, Nup49, and Nup57 in the three-stranded CCD1 stalk

appeared to contain seven-residue signature motifs akin to coiled-coil trigger sequences that

were previously shown to facilitate proper three-stranded coiled-coil assembly (fig. S35A)

(

). An N-terminal truncation of Nup57, Δ

, which lacked four helical turns of CCS1 and

the

insertion domain, transformed the equimolar and monodisperse CNT into a

polydisperse mixture (fig. S35, B to E). These data provided a molecular explanation for the

observed heterogeneous stoichiometries and dynamic nature of previous mammalian CNT

reconstitutions, which also lacked this region (

). In addition to removing the

trigger sequence, Nup57 Δ

exposed a hydrophobic surface of approximately four

unpaired heptad repeats (~50 Å in length). Thus, misfolding and aggregation would be

expected.

We conclude that the CNT adopts a robust coiled-coil domain architecture with a single

defined assembly state that is specifically recognized by Nic96

to ensure NPC

incorporation of only properly assembled CNT. None of the interactions from previously

determined channel nup fragment crystal structures occured in the intact CNT. Thus, these

data strongly disagree with the model in which the CNT undergoes dynamic rearrangements

to facilitate NPC constriction and dilation, as previously proposed based on the dynamic

nature of various channel nup fragment interactions (

Nic96 is an assembly sensor for the properly assembled CNT

Next, we identified channel nup mutants that disrupted the Nic96

-CNT interaction. As we

deemed alanine scanning mutagenesis unlikely to disrupt the extensive CNT-Nic96

interface, we characterized a previously identified quintuple

S. cerevisiae

Nup49 mutant

(EVPIP; K376E, I390V, I391P, V398I, and L449P) (

). The five Nup49 EVPIP mutations

mapped to two Nic96

interfaces located in CCD2 and CCD3 (Figs. 4C and 5A). The

corresponding

C. thermophilum

CNT

EVPIP

mutant failed to interact with the minimal IRC in

SEC-MALS experiments (Fig. 5A and fig. S36A). Further analysis showed that CNT

EPP

was sufficient to abolish binding and CNT

severely reduced binding, but no single mutant

alone affected complex formation (Fig. 5B and fig. S36, B to F). The severity of the required

mutations demonstrated the robustness of the CNT-Nic96

interaction.

Next, we tested the effects of these Nup49 mutations in

S. cerevisiae

. Whereas, the single

mutants displayed no phenotypes, PP displayed a temperature-sensitive growth defect,

which was intensified in EPP and EVPIP, consistent with their decreased thermostability

(Fig. 5C and fig. S37). In line with the growth defects, PP showed wild type levels of

mCherry-Nup49 and Nup57-GFP NE staining at 30 °C, but barely detectable levels at 37 °C

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(Fig. 5D). The temperature-sensitive localization defect for both nups was escalated in EPP

and EVPIP, which showed severe reduction and complete loss of NE staining at 30 °C and

37 °C, respectively (Fig. 5D). Interestingly, despite an almost complete loss of Nup49 and

Nup57 from the NE, we only observed a mild effect on mRNA export (Fig. 5E). Regardless,

the EPP and EVPIP mutants resulted in a ribosomal export defect at 30 °C. (Fig. 5F).

These data established that NE recruitment of Nup57 and Nup49 are co-dependent,

consistent with our biochemical and structural analyses that Nic96

only interacts with the

intact CNT, validating that the CNT•Nic96

structure represents the physiologically

relevant state in the assembled NPC.

Conclusions

Through reconstitution and systematic structural and functional dissection of the IRC, we

established the equimolar stoichiometry of its six components and uncovered the

physiologically relevant underlying interaction network. We showed that Nup53 and the

CFC could be attached to the IRC, facilitating membrane attachment and functionalization at

the cytoplasmic face, respectively. Surprisingly, none of the previously determined dynamic

channel nup fragment assemblies occur in the intact CNT•Nic96

structure. In fact, the

CNT adopts a robust parallel three-stranded coiled-coil domain architecture resembling the

numeral 4. This organization guarantees that the N-terminal FG repeats of the three channel

nups emanate from a single site on the CNT surface. Because Nic96 harbors an assembly

sensor for this specific CNT state, the NPC incorporation of the three channel nups is co-

dependent and thereby enables the concomitant generation of the diffusion barrier and

cargo•transport factor complex docking sites. Additionally, we demonstrated that efficient

nucleocytoplasmic transport also requires proper CNT positioning, which is achieved by the

Nic96-Nup192 interaction. The conclusion that the inner ring of the NPC adopts a relatively

rigid architecture with a transport channel filled with channel nup FG repeats, is also

supported by a channel nup FG repeat-coated artificial nanopore, which mimicked the

NPC’s transport selectivity (

NPC recruitment of a single robust CNT assembly state rules out the possibility that channel

nups dynamically rearrange into different assembly states with various stoichiometries to

facilitate karyopherin-assisted dilation and constriction of the central transport channel, as

previously proposed (

–

). Indeed, dilation is unnecessary as the central transport channel

of the human NPC can easily accommodate even one of the largest cargoes, the pre-60S

ribosomal particle (fig. S38) (

Apart from its role in the CNT, Nsp1 forms a mutually exclusive complex with the C-

terminal coiled-coil relions coilded of Nup82 and Nup159 at the cytoplasmic face of the

NPC (

). The CNT•Nic96

structure suggests that Nsp1•Nup82•Nup159 might adopt a

similar three-stranded coiled-coil domain architecture. More generally, our biochemical and

structural analyses of the three channel nups instruct that utmost caution is required when

analyzing heteromeric coiled-coil interactions.

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The finding that the IRC and the IRC•CFC•Nup53 hetero-nonamer are monomeric in

solution is not unexpected. The CNC is also monomeric in solution, but organizes into

densely packed, sixteen-membered peripheral rings in the intact NPC (

). The cryo-ET

reconstruction of the human NPC suggests that the inner ring is large enough to

accommodate sixteen copies of the IRC, and we propose that they are recruited to the

nuclear pore membrane by their association with the membrane anchored

Nup53•Nup170•Ndc1 complex (Fig. 6) (

). Stabilization of the inner ring scaffold could

occur through multiple weak interactions between adjacent IRCs, including the dimerization

of neighboring CNTs, the formation of a FG-repeat hydrogel in the central transport channel

(

), and/or transport factor•cargo complex binding to such a FG-repeat meshwork. A

sixteen-membered inner ring scaffold accounts for ~10-MDa of the NPC mass. Our results

represent a major step forward towards the

in vitro

reconstitution of the entire NPC, which is

essential for the development of

in vitro

assays to quantitatively characterize

nucleocytoplasmic transport.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.

Acknowledgments

We thank W. M. Clemons, A. Correia, G. Mobbs, A. Patke, D. C. Rees, S. O. Shan, and E. Stuwe for critical

reading of the manuscript, M. Budd for help with yeast experiments, J. Herrmann, R. Kunze, and L. Zhang for

technical support, and J. Kaiser and the scientific staff of the Stanford Synchrotron Radiation Laboratory (SSRL)

Beamline 12-2, the National Institute of General Medical Sciences and National Cancer Institute Structural Biology

Facility (GM/CA) at the Advanced Photon Source (APS), and the Advanced Light Source (ALS) beamline 8.2.1 for

their support with x-ray diffraction measurements. We acknowledge the Gordon and Betty Moore Foundation, the

Beckman Institute, and the Sanofi-Aventis Bioengineering Research Program for their support of the Molecular

Observatory at the California Institute of Technology (Caltech). The operations at the SSRL, ALS, and APS are

supported by the U.S. Department of Energy and the National Institutes of Health (NIH). GM/CA has been funded

in whole or in part with federal funds from the National Cancer Institute (ACB-12002) and the National Institute of

General Medical Sciences (AGM-12006). T.S. was supported by a Postdoctoral Fellowship of the Deutsche

Forschungsgemeinschaft. S.P. and D.H.L are Amgen Graduate Fellows, supported through the Caltech-Amgen

Research Collaboration. F.M.H. was supported by a PhD student fellowship of the Boehringer Ingelheim Fonds.

S.K. was supported by NIH Awards R01-GM090324 and U54-GM087519 and by the University of Chicago

Comprehensive Cancer Center (P30-CA014599). A.A.K. was supported by NIH awards U01-GM094588 and U54-

GM087519 and by Searle Funds at The Chicago Community Trust. A.H. was supported by Caltech startup funds,

the Albert Wyrick V Scholar Award of the V Foundation for Cancer Research, the 54th Mallinckrodt Scholar Award

of the Edward Mallinckrodt Jr. Foundation, a Kimmel Scholar Award of the Sidney Kimmel Foundation for Cancer

Research, a Camille-Dreyfus Teacher Scholar Award of The Camille & Henry Dreyfus Foundation, and NIH grant

R01-GM111461. The coordinates and structure factors have been deposited with the Protein Data Bank with

accession codes 5CWV (Nup192

TAIL

), 5CWU (Nup188

TAIL

), 4JQ5 (

Nup49

CCS2+3

*), 4JNV and 4JNU

(

Nup57

CCS3

*), 5CWT (Nup57

CCS3

*), 4JO7 (

Nup49

CCS2+3

*•hsNup57

CCS3

*, 2:2 stoichiometry), 4JO9

(

Nup49

CCS2+3

*•hsNup57

CCS3

*, 1:2 stoichiometry), 5CWW (Nup82

NTD

•Nup159

•Nup145N

APD

) and

5CWS (CNT•Nic96

•sAB-158).

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Fig. 1. Reconstitution and Dissection of the IRC

(

) Cross-sectional schematic representation of the NPC and domain structures of the

Chaetomium thermophilum

nucleoporins (nups). Black lines indicate regions used for

reconstitution. U, unstructured; T, TAIL; NTD, N-terminal domain; MID, middle domain;

SH3, Src-homology 3-like domain; L, linker domain; APD, auto-proteolytic domain; RRM,

RNA recognition motif; M, membrane-binding motif; FG repeats, phenylalanine-glycine

repeats; CCS, coiled-coil segment;

insertion domain; IRC, inner ring complex. (

) Pair-wise biochemical interaction analyses of various reconstituted nup complexes. Size-

exclusion chromatography coupled to multiangle light scattering (SEC-MALS) profiles of

nup or nup complexes are shown individually (red and blue) and after their preincubation

(green). Measured molecular masses are indicated for the peak fractions. (

) Structure of the

Nup82

NTD

•Nup159

•Nup145N

APD

cytoplasmic filament nup complex (CFC) is shown in a

cartoon representation. Nup145N

APD

(green), Nup159

(red), and Nup82

NTD

(blue), the

Nup82

NTD

helical 4CD (gray) and 6CD (orange) insertions and FGL loop (yellow), and the

conserved Nup145N

APD

K/R loop (purple) are illustrated.

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Fig. 2. Structural and functional analyses of the TAIL domains of Nup192 and Nup188

Cartoon representations of (

) Nup192

TAIL

(blue), (

) Nup188

TAIL

(red), and (

) a

superposition of the two structures is shown in two different orientations. (

D, E

) Schematic

representation of Nup192

TAIL

and Nup188

TAIL

structures. Positions of HEAT and ARM

repeats are indicated. (

) Domain structures of Nup192 and Nic96 are shown; black bars

indicate fragments used for interaction studies in (H). (

) Docking of Nup192

TAIL

and the

previously determined Nup192

NTD

crystal structure into the yeast Nup192 EM envelope (

). The

Inset

illustrates the position of Nup192

TAIL

and is expanded on the right, rotated by

90º. Surface representation of Nup192

TAIL

with the location of the analyzed mutations and

their effect on Nic96

binding indicated; no effect (green), decreased binding (orange),

abolished binding (red). (

) Summary of tested Nup192

TAIL

mutants and their effect on

SUMO-Nic96

binding; (-) no effect, (+) decreased binding, (+++) abolished binding. (

)

Growth analysis of

S. cerevisiae

strains carrying the indicated GFP-

NUP192

variants. Serial

dilutions of the respective cells were spotted onto YPD plates and grown for 2–3 days. (

)

Subcellular localization of mCherry-Nup192 variants (red) and Nup57-GFP (green) in a

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nup192Δ

/NUP57-GFP

strain. (

) Subcellular localization of the 60S ribosomal export

reporter Rpl25-mCherry (red) and GFP-tagged Nup192 variants (green) in a

nup192Δ

strain.

Representative images and quantification of nuclear Rpl25-mCherry retention are shown on

the right. (

) mRNA export assay in a

nup192Δ

strain carrying GFP-

NUP192

variants.

Representative images and quantification of nuclear poly(A)

RNA retention are shown.

Cells were analyzed at the indicated temperatures and incubation times. Error bars represent

the standard deviation. Scalebars are 5 μm.

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