Single-molecule Structural and Functional Analyses of Nuclear Pore Complex

Shotaro Otsuka, Hirohide Takahashi and Shige H. YoshimuraGraduate School of Biostudies, Kyoto University

Kitashirakawa-oiwake-cho, Sakyo-ku, Kyoto, 606-8502Japan

Abstract:Macromolecular traffic between the nucleoplasm and thecytoplasm is governed by the mechanisms of interactionbetween cargo molecules and the nuclear pore complex (NPC).In the last decade, many of the molecular components of theNPC have been identified and considerable insights into thestructure and the biological functions of the NPC have beenobtained. However, the detailed molecular mechanism of thetransport event still remains unaddressed. By combiningatomic force microscopy (AFM), fluorescence observationtechnique, and molecular and cellular biology, we established asystem to investigate the molecular mechanism of the nucleartransport at the single-molecule level.

1. INTRODUCTIONBiological membranes prevent the passage ofmacromolecules and ions, and, thus, play a central role inestablishing various environments in cytoplasm and inintracellular membrane organelles. The efficient andselective transport of macromolecules across the cellularmembrane requires the assistance of membrane proteins,such as ion channels, pumps and transporters. The energyrequired for the transport is supplied from electrochemicalgradients of ions across the membrane, or other mechanismsinvolving ATP hydrolysis. Among these transport systems,the transport across the nuclear envelope (nuclear transport)has several unique characteristics. The nuclear transport ismediated by a large protein complex called nuclear porecomplex (NPC), which is embedded in the doublemembrane of the nuclear envelope (Fig. la). Water, ionsand proteins smaller than 40 kDa can pass through theNPC relatively freely, whereas proteins larger than 40 kDaand large protein complexes can not diffuse through theNPC and require additional transport mediators. The NPCmediates the transport of various transcription factors andribosomal proteins into the nucleus, and also of ribosome(the diameter is 20 nm) and mRNAs out of the nucleus.

[ 1,2]. However, despite this difference, the overallarchitecture of the NPC is well conserved among species.The study using electron microscopy (EM) revealed that theNPC has a conserved eightfold symmetric structure whichconsists of several distinct sub-structures; cytoplasmicfilaments, nuclear and cytoplasmic rings, a scaffold of largespokes, a central transporter region, and a basket of nuclearfilaments [3,4]. The proteomic analysis of the NPC revealedthat the single NPC is composed of 30 different proteinstermed nucleoporins (Nups) (Fig. lb) [5,6]. The most highlyconserved feature of the Nups is a phenylalanine-glycine(FG) repeat which is present in approximately one third ofthe Nups [7]. This repeat interacts with transport mediatorsinvolved in the protein import and export [8].

a ,,Nuclear EnvelopeInner nuclearcompartments

Nuclear Lamina

Chromatin /

'Nuclear Pore Complex

b

NupS8 Mumma

..................cample

107-

complex

m NupO

107-16

complex..

XX........fiX.........

..... .......

...........

..............

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Cytoplasm

...................................................................................................................................

Nucleoplasm

2. NPc, RAN AND KARYOPHERINS; KEY PLAYERS INNUCLEAR TRANSPORT

2.1 NPC is composed of Nucleoporins and mediates

karyopherin-dependent protein transportThe NPC is one of the biggest macromolecular

structures in eukaryotic cells, having the molecular mass

varying from 50 MDa in yeast to 125 MDa in vertebrates

Fig. 1 (a) The structure of cell nucleus. The cell nucleus is composed ofnuclear envelope, nuclear pore complex, nuclear lamina, chromatin(chromosome), nucleolus, and other inner nuclear compartments such asPML body and nuclear speckles. (b) The NPC is composed of -30different Nups. The localization of the Nups within the NPC isillustrated based on the previous report [5,6]. The Nups containingFG-rich domain (FG-Nups) is indicated in red.

Tpr Tpr

The nuclear transport of large molecules (> 40 kDa)requires soluble transport mediators called karyopherins(Kaps) [9]. So far, 20 different Kaps have been identifiedin vertebrates and each of them is involved in the transportof different cargo proteins (for review [10]). The Kapsrecognize specific amino acid sequences called nuclearlocalization signal (NLS) and nuclear export signal (NES)within the cargo protein (Fig. 2a) [11,12]. The twowell-characterized NLSs are those in SV40 large T antigenand in nucleoplasmin [13,14]. They contain severalconserved basic amino acids and are specifically recognizedby a karyopherin complex, importin ac and importin P [15].Importin ac contains binding sites both for the NLS andimportin f, and, thus, bridges the NLS-containing proteinand importin P.

2.2 A small GTPase, Ran, is a key regulator of thenuclear transport cycle

In addition to the NPC and the Kaps, a small G-proteincalled Ran plays important roles in the nuclear transport

a

b

a

Cyoplasm L

Nucleoplasm

Fig.3 Karyopherins and small G protein, Ran, regulate the nucleartransport. (a) Transport signal sequences. Nuclear localization signals(NLSs) from SV40 large T antigen and nucleoplasmin are composed ofseveral basic amino acids. The highly conserved amino acids areunderlined. The nuclear export signals (NESs) from HIV Rev andprotein A phosphorylation inhibitor are also indicated [11,12]. (b)Outlines of the selective transport mechanism mediated by karyopherins.(Left) Importin binds to the cargo in the cytoplasm, translocates throughthe NPC into the nucleus, and releases the cargo, which is induced bybinding of RanGTP. (Right) Exportin binds to the cargo protein andRanGTP in the nucleoplasm, goes through the NPC and finally releasesthe cargo, which is induced by the hydrolysis of RanGTP.

(Fig. 2b). The GTP-bound form of Ran (RanGTP)predominantly localizes in the nucleus and the GDP-boundform (RanGDP) localizes in the cytoplasm, resulting in a

large concentration difference across the nuclear envelope[16]. When the importin-cargo complex enters the NPC andreaches to the nucleoplasm, it encounters RanGTP andreleases the cargo protein into the nucleoplasm. Thus,RanGTP functions as a "releasing factor" of the importincargo complex in the nucleoplasmic side of the NPC. On theother hand, exportin binds to the cargo protein in thepresence of RanGTP in the nucleus and the exportin-RanGTP-cargo complex goes through the NPC and isdisassembled in the cytoplasm where RanGTP is hydrolyzedto RanGDP by Ran GTPase-activating protein (RanGAP).After releasing the cargo, the importin and the exportinreturn back to the cytoplasm and nucleoplasm respectively,to mediate another round of transport (for review, [17]).

The experiment using permeabilized HeLa cellsindicated that the concentration gradients of RanGTP andRanGDP across the nuclear envelope determine thedirection of the transport. It was previously demonstratedthat the conversion of the concentration gradient of RanGTPacross the nuclear envelope resulted in the oppositetransport direction [18]. When the RanGTP concentration ishigh in the nucleoplasm, which is physiological condition inmost of the cells, a NES-bearing protein was exported out ofthe nucleus by exportin, CRM1. On the other hand, whenthe RanGTP concentration is high in the cytoplasm, theNES-bearing protein was imported into the nucleus. Theseresults indicate that the concentration gradient of RanGTPacross the nuclear envelope is a determinant of the directionof the transport.

Is the energy derived from GTP hydrolysis directlyinvolved in the transport process? The previous report usingpermeabilized cells demonstrated that the substitution ofGTP with non-hydrolysable analogue (GMP-PNP) did notabolish the importin ac/p-mediated nuclear import [19].Thus, it seems likely that the protein translocation throughthe NPC itself is an energy-independent process and is notdriven by GTP hydrolysis, although RanGTP-RanGDPcycle plays an important role in the regulation of thetransport.

2.3 A number of FG-Nups form a barrier within theNPC and prevent the diffusion of large moleculesApproximately one-third of the Nups carry FG-rich domainsand are thought to play important roles in forming thecentral channel of the NPC (Fig. lb). The studies usingX-ray crystallography and other biophysical analysesincluding atomic force microscopy (AFM) demonstratedthat the FG-rich domains of the FG-Nups do not have any

particular secondary or tertiary structures [20-22]. Therecent study using single-molecule force measurementrevealed that the FG-rich domain of Nup153 is fully

Cargo protein Sequence

SV40 large T antigen PKKKRKV

NLS

nucleoplasmin KRPAATKKAGQAKKKK

EHV-1 Rev LPPLERLTL

NESprotein A LALKLAGLDIN

phosphoiryltioun inh,ibitor LALKLGLD

t

1-110

extended and forms a brush-like conformation throughintermolecular hydrophobic interaction [23]. Since a singleNPC contains 100 FG Nups, it can be speculated that theFG-rich domains fill the central channel of the NPC to forman entropic barrier and prevent non-NLS proteins frompassing through.

Although most of the FG-Nups are distributedsymmetrically along the pore, several FG-Nups showasymmetric localizations within the NPC, and, thus, havebeen considered as possible key players in the directionaltransport. However, when FG-rich domains of theasymmetric FG-Nups (Nup42 and Nup 159 in thecytoplasmic side, and Nup 1, Nup2 and Nup6O in thenucleoplasmic side of the NPC) were deplete in yeast cells,the NPC still retained the nuclear import and exportactivities [24]. Similarly, when Nup358 and Nup214, bothof which localize in the cytoplasmic filaments inmammalian cells, were depleted from the Xenopus eggextract, the reconstituted NPC was still able to facilitate theKap-mediated nuclear import [25]. These results suggestthat the asymmetric FG-Nups are not necessary for theKap-mediated nuclear transport.

2.4 Fluorescence-based microscopic observationrevealed the fast process of Kap-dependent transport

The nuclear transport of various cargo proteins hasbeen analyzed extensively by fluorescence microscope.Fluorescently-labeled nuclear transport factor 2 (NTF2),which can directly interact with the FG-Nups, was appliedto the permeabilized cells and the fluorescence signalaccumulated in the nucleus was measured by time-lapsefluorescence microscopy [28]. This experimentdemonstrated that NTF2 dimer (-29 kDa) passed throughthe NPC 120 times faster than GFP (-28 kDa). Thetransport rate of the NTF2 was 2500 molecules per NPCper second when the cytoplasmic concentration was 100[M. Another study showed that the export rate ofNTF2 was50 times larger than that of GFP [29]. These resultsindicated that the NPC strongly facilitates the transport ofproteins which can interact with Nups.

The transport rate varies depending on the type of theKaps and the size of the cargo. Transportin (one of theKaps, 100 kDa), transportin-cargo complex (-630 kDa)and NTF2 dimer (-29 kDa) were imported at 65, 28 and 250molecules per NPC per second, respectively, when thecytoplasmic concentration was 1 [tM [28]. Thus, althoughthe Kaps and the Kap-cargo complex can directly interactwith the FG-Nups, the movement of the Kaps in the NPC isstill restricted by the entropic barrier of the FG-Nups, whichresults in the size-dependent transport rate.

How can the Kaps overcome the entropic barrier ofthe FG-Nups and obtain such a fast transport rate? Recentstudy demonstrated that the transport rate of the Kapsthrough the NPC is as high as the diffusion rate of the same

molecule in the aqueous cylinder [28]. The previous EMobservation combined with protein-coated gold particlesrevealed that the NPC can transport a molecule up to 39nm in diameter [30]. Based on this result and other EMobservations, the central channel of the NPC could beapproximated to be a cylinder of 40 nm in diameter and40 nm in length. Then, the rate of NTF2 (-2.5 nm in

diameter) passing through this cylinder from one end to theother end by simple diffusion was calculated [28].Surprisingly, when the cylinder is filled with a physiologicalsolution, the diffusion rate becomes close to theexperimental result of the NTF2 transport rate. These resultsindicate that though the FG-Nups form a steric barrier in thecentral channel of the NPC and prevent the diffusion ofnon-cargo proteins, Kaps can go though this barrier withsame rate as the simple diffusion in an aqueous phase.Although the molecular mechanism of this fast transportremains unclear, weak interactions between the Kaps andthe FG-Nups are supposed to play important roles.

Yang et al. successfully observed the movement of asingle protein transported through the NPC by fluorescencemicroscopy. A fluorescently-labeled protein bearing theNLS was applied to the permeabilized HeLa cells togetherwith importin a and importin P and observed byfluorescence microscope with the frame rate of 3 ms [31].The detail analysis of the fluorescence signal revealed thatthe dwell time of the cargo protein within the NPC was 10ms. Considering that the net transport rate is 1000 moleculesper NPC per second [28], this result indicates that 10 cargomolecules reside within a single NPC at a given moment.The similar result was obtained with NTF2 and transportin1, in which more than 10 molecules of these Kaps reside ina NPC at a given moment of the transport [32].

3. SINGLE-MOLECULE FORCE MEASUREMENT BETWEENIMPORTIN a AND IMPORTIN ,B

3.1 Single-molecule approachAFM has been utilized to observe nano-scale

structures of a variety of biomolecules and cellulararchitectures including the NPC. AFM can visualizespecimen's surface topology in the physiological bufferwithout fixation and fluorescence labeling. The AFMobservation of the nuclear envelope of Xenopus oocyterevealed that the nuclear basket of the NPC becomes openin the presence of calcium, and close in its absence [33]. Aconformational change of the NPC induced by a syntheticsteroid, dexamethasone, was also observed by AFM [34].Although AFM can visualize non-fixed specimen in aphysiological buffer, it is still difficult to capture thetransport event of the NPC in real time, because thetransport event is much faster than the scanning rate ofAFM (several seconds per frame). The possible solution of

this problem may be brought by the recently-developedfast-scanning AFM, which can take nano-scale images atseveral frames per second [35,36]. The application of thisfast-scanning AFM will be able to provide a detailedmolecular mechanism of the transport.

The AFM has also been used to measure the forcegoverning various inter- and intra-molecular interactions atpN level. Since a number of interactions between the cargoprotein and the FG-Nups govern the transport kinetics, thecharacterization of these interactions at a single moleculelevel will provide a large amount of information on themolecular mechanism of the nuclear transport. The recentprogress in chemical modifications of the AFM cantileverenabled a wide variety of single-molecule forcemeasurement using AFM [37-40].

Here, we developed a novel method to attachGST-fusion protein to an AFM cantilever by covalentlyattaching glutathione to the cantilever via flexible PEGlinker. By using this method, the interaction betweenimportin a and importin f3 was measured.

3.2 MethodsAFM cantilever made of silicon nitride,

OMCL-TR400PSA (Olympus Co. Ltd.), was treated with3-aminopropyltriethoxysilane (APTES) (Sigma) andincubated with heterobifunctional cross linker (5 mg/mlmaleimide-dPEG12-NHS ester (Quanta BioDesign Ltd.))which was dissolved in chloroform containing 0.7 %triethylamine for 2 hours at room temperature. ThePEG-modified cantilever was then incubated with 10 mMglutathione in PBS (pH 7.5) for 1 hour at room temperature.The unreacted maleimide groups were blocked by freecystein (1 mM in PBS). For the measurement of protein-protein interaction, glutathione-coupled cantilever wasimmersed in the solution of glutathione- S-transferase(GST) or GST-importin P for 1 hour at 4 °C, washed withPBS and immediately used in the experiment.

Purified GST or importin a ( 60 rtM) was mixed withSATP (final concentration; 500 [tM in PBS) and incubatedfor 30 min at room temperature. The mixture was thenpassed through PD-10 Column (GE Healthcare) to removefree SATP. The eluted fraction was mixed with PBScontaining 500 mM Hydroxylamine and 25 mM EDTA (pH7.4), and incubated for 2 hours at room temperature. A cleancover glass (18 mm x 18 mm) was functionalized withamino group by Amino Coat Kit (Nippon Sheet Glass) byfollowing the manufacturer's protocol.m-maleimidobenzoyl- N-hydoxysuccinimide ester (1 mM inPBS) was dropped on the amino-coated cover glass andincubated for 30 min at room temperature. After washing byPBS (pH 7.2), the SATP-treated protein was dropped on theamino-coated cover glass and incubated for 30 min at roomtemperature.

Force measurement was performed with MolecularForce Probe 3D (MFP-3D; Asylum Research). Themodified cantilever with a spring constant of 0.02 N/m wasroutinely used. The actual spring constant of each cantileverwas determined by thermal method. The force measurementwas performed in PBS. The obtained data was analyzed bythe software accompanying with the AFM imaging module(Asylum Research).

3.3 Single-molecule force measurement betweenimportin a and importin P3.

The most important issue to be considered in thesingle-molecule force measurement is how to attach themolecule of interest to the cantilever. Here, we utilizedspecific interaction between glutathione and GST, anddeveloped a method to covalently bind glutathione to anAFM cantilever via PEG linker and attach GST-fusedprotein to this cantilever.

Glutathione contains a cystein residue in the middle ofthe tri-peptide (NH2-Gln-Cys-Gly-COOH). We decided touse this reactive group to attach glutathione to an AFMcantilever. Since this thiol-mediated coupling has also beenused in commercially available glutathione beads(glutathione Sepharose 4B, GE Healthcare), it does nothinder the binding of GST. To avoid the steric hindrancefrom the cantilever surface and to confer enough flexibility,a PEG linker (dPEG12) was inserted between glutathioneand the cantilever surface. An amino-functionalizedcantilever made of silicon nitride was incubated withNHS-PEG12-maleimide and then with glutathione (for thedetail procedure, see section 3.2).

aa N50PNL

p Lp

o extension

|50 pNL10nml

piezo position

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0 50 1Ma 150Force (pN)

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piezo position

3o I

10

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200 0 50 100 150

Force (pN)

200

Fig. 3 Force measurement between glutathione and GST (a, b) andGST-importin f3 and importin a (c, d). The force between glutathione onthe cantilever and GST on the glass substrate (a, b) and GST-importin f3on the cantilever and importin a on the glass surface (c, d) weremeasured by AFM. (a, c) Typical single-molecule force curves. Theforce was plotted against the piezo position. The force-extension curveof this measurement was shown in inset. (b, d) Histograms of the ruptureforce obtained in the force measurement.

The rupture force between glutathione and GST wasmeasured by AFM. GST was expressed in bacteria, purifiedwith glutathione-Sepharose 4B beads, and then dialyzedagainst a glutathione-free buffer. This dialyzed GST wasable to re-bind to the glutathione-beads with more than90 % efficiency. The purified GST was covalently attachedto a glass surface to avoid the detachment from the surfaceduring the force measurement. Fig. 3a shows a typicalforce-extension curve of the glutathione-GST interaction.The extension curve well fits to the worm-like-chain modelof the PEG molecule [41]. The interaction betweenglutathione and GST was ruptured at around 100 pN. Thestatistical analysis revealed that there are the two peaks inthe histogram of the rupture force. The mean value of eachpeak is 105.9+13.8 pN (n=10) and 176.7+20.1 pN (n=6)(Fig. 3b). The larger one is supposed to be a double-tethering and 100 pN is the rupture force for the singleglutathione-GST interaction. An addition of free glutathionein the measurement solution completely abolished theinteraction, indicating that this 100 pN force is specific tothe glutathione-GST interaction

We then applied the glutathione-coupled cantilever forthe force measurement of protein-protein interaction.Importin oc and importin f3 have been known to interact witheach other and play an important role in the proteintransport from the cytoplasm to the nucleoplasm [42]. Theamino-terminal domain of importin oc directly binds to thecarboxyl terminal domain of importin f3 [43]. To avoid asteric effect, GST was fused to the amino-terminus ofimportin f3 and this fusion protein was attached to theglutathione-coupled cantilever. Importin oc was alsoexpressed as a GST-fusion protein in bacteria and attachedto the glass surface after separated from the GST moiety bysite-specific protease digestion.

A typical force-extension curve was shown in Fig. 3c.As is the case for the glutathione-GST interaction, astretching of the PEG linker was observed before theinteraction was ruptured. The averaged rupture force was43.92 +10.4 pN (n=28) (Fig. 3d), much smaller than that ofthe glutathione-GST interaction, indicating that theGST-glutathione interaction was tight enough. The sameexperiment was performed with the glutathione-coupledcantilever without GST-importin P3. However, no specificinteraction was detected between glutathione and importincc.

4. INTERACTION BETWEEN NPC AND IMPORTIN ,B

4.1 IntroductionAs described in section 2.3, the characteristic of the

nuclear transport is largely dependent on the interactionbetween transport cargo (Kaps) and the Nups in the NPC.

Several in vitro experiments using purified proteins revealedthe dissociation constant values of various protein-proteininteractions including Nups, Kaps and Ran [44]. Theinteraction between importin and FG-Nups are affected byRanGTP or RanGDP. To further characterize the interactionbetween importin and the NPC, we performed single-molecule force measurement between importin f3 and theentire molecule ofNPC.

4.2 MethodsFull-grown oocytes were obtained from an adult

female Xenopus Laevis and a nucleus (geryminal vesicle) wasisolated with micro needles in nuclear isolation medium(NIM, 10 mM NaCl, 90 mM KCl, 2 mM MgCl2, 1.1 mMEGTA, 10 mM HEPES-KOH). Chromatin was separatedfrom the nuclear envelope (NE) and the NE was spread on aglass surface coated with poly-L-lysine. The NE waswashed twice with pure water and dehydrated in air formore than four hours. The NE was then rehydrated in NIMand immediately used for the AFM imaging and forcemeasurement. For imaging, the 100 ptm long cantilever witha spring constant of 6 pN/nm was used (Bio-Lever,Olympus). The images were taken in contact mode with ascan rate of 1-2 Hz. The importin [3-coupled cantilever wasprepared and the force measurement was performed asdescribed in section 3.2.

4.3 The unbinding force between importin ,B and NPC isdifferent in the cytoplasmic and nucleoplasmic sides ofthe NPC.

ba

C

0C)ci0)crL-

d

C)

ci)IL

Force (pN) Force (pN)

Fig.4 AFM image of the NPC of Xenopus laevis (a, b) and forcemeasurement between importin P and the NPC (c, d) The NE wasisolated from Xenopus laevis and scanned by AFM in contact mode inliquid. The cytoplasmic side (a) and nucleoplasmic side of the NPC (b)was observed. The enlarged image is shown in inset. Scale bar, 300 nm.(c, d) Histograms of the rupture force between importin P and thecytoplasmic and the nucleoplasmic sides of the NPC, respectively.

The NE dissected out from Xenopus oocyte wasspread and attached on a glass surface. By a finemicromanipulation, both cytoplasmic and nucleoplasmicfaces of the NE could be prepared. The AFM observation ofthese specimens revealed a clear difference in the structure(Fig. 4a, b). The nuclear face of the NE showed nuclearlamina and the NPC, whereas the cytoplasmic face showedonly NPC. The size (outer and inner diameters) of the NPCwell matched to the previous reports ( 120 nm). Thus,

After the imaging, the same specimen was used forthe single-molecule force measurement. The importin f3-coupled cantilever was prepared and used in the experiment.The unbinding force between importin P and thecytoplasmic side of the NPC was 39.71 + 10.2 pN (n=53),whereas the unbinding force between importin f3 andnucleoplasmic side of the NPC was 50.09 + 13.7 pN (n=51)(Fig. 4c, d). These results indicated that the interactionbetween importin f3 and the nuclear face of the NPC isslightly stronger than cytoplasmic face of the NPC, andsuggest that this difference might play an important role inthe importin [3-dependent protein import.

5. ACKNOWLEDGMENTS

This work was supported by the Japanese Ministry ofEducation, Culture, Sports, Science and Technology(Grant-in-Aid for Scientific Research on Priority Areas forS.H.Y.), Japan Society for the Promotion of Science(Grant-in-Aid for Young Scientists (A) for S.H.Y.) and NewEnergy and Industrial Technology DevelopmentOrganization (NEDO for S.H.Y.).

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