Classical NLS Proteins from Saccharomyces cerevisiae - UKS · criteria allow one to distinguish...

doi:10.1016/j.jmb.2008.04.038 J. Mol. Biol. (2008) 379, 678–694

Available online at www.sciencedirect.com

Classical NLS Proteins from Saccharomyces cerevisiae

Silvia Hahn, Patrick Maurer, Stefanie Caesar and Gabriel Schlenstedt⁎

Medizinische Biochemie undMolekularbiologie, Universitätdes Saarlandes, Geb. 61.4,66421 Homburg, Germany

Received 16 January 2008;received in revised form12 April 2008;accepted 15 April 2008Available online22 April 2008

*Corresponding author. E-mail addrPresent addresses: S. Hahn, NeoL

7, 69123 Heidelberg, Germany; P. MUniversität Kaiserslautern, MikrobioKaiserslautern, Germany.Abbreviations used: cNLS, classic

signal; Impα, importin α; Impβ, imppore complex; GTP, guanosine 5′-trinuclear localization signal; SV40, simarmadillo; IBB, Impβ binding; SPR,resonance; GFP, green fluorescent pglutathione-S-transferase; GDP, guaDAPI, 4′,6′-diamidino-2-phenylindovirus; FITC, fluorescein isothiocyanaserum albumin.

0022-2836/$ - see front matter © 2008 E

Proteins can enter the nucleus through various receptor-mediated importpathways. One class of import cargos carries a classical nuclear localizationsignal (cNLS) containing a short cluster of basic residues. This pathwayinvolves importin α (Impα), which possesses the cNLS binding site, andimportin β (Impβ), which translocates the import complex through thenuclear pore complex. The defining criteria for a cNLS protein from Saccha-romyces cerevisiae are an in vivo import defect in Impα and Impβ mutants,direct binding to purified Impα, and stimulation of this binding by Impβ.Weshow for the first time that endogenous S. cerevisiae proteins Prp20, Cdc6,Swi5, Cdc45, andClb2 fulfill all of these criteria identifying them as authenticyeast cNLS cargos. Furthermore, we found that the targeting signal of Prp20is a bipartite cNLS and that of Cdc6 is a monopartite cNLS. Basic residuespresent within these motifs are of different significance for the interactionwith Impα. We determined the binding constants for import complexescontaining the five cNLS proteins by surface plasmon resonance spectro-metry. The dissociation constants for cNLS/α/β complexes differ consider-ably, ranging from 1 nM for Cdc6 to 112 nM for Swi5, suggesting that thenuclear import kinetics is determined by the strength of cNLS/Impαbinding. Impβ enhances the affinity of Impα for cNLSs approximately 100-fold. This stimulation of cNLS binding to Impα results from a faster asso-ciation in the presence of Impβ, whereas the dissociation rate is unaffectedby Impβ. This implies that, after entry into the nucleus, the release of Impβby the Ran guanosine triphosphatase (RanGTPase) from the import complexis not sufficient to dissociate the cNLS/Impα subcomplex. Our observationthat the nucleoporin Nup2, which had been previously shown to release thecNLS from Impα in vitro, is required for efficient import of all the genuinecNLS cargos supports a general role of Nup2 in import termination.

© 2008 Elsevier Ltd. All rights reserved.

Edited by J. Karn
Keywords: nuclear import; classical NLS; importin; Nup2; Ran
ess: [email protected] GmbH, Rischerstr.aurer, Technischelogie, 67663

al nuclear localizationortin β; NPC, nuclearphosphate; NLS,ian virus 40; ARM,surface plasmonrotein; GST,nosine diphosphate;le; TEV, tobacco etchte; BSA, bovine

lsevier Ltd. All rights reserve

Introduction

Transport of molecules between nucleus and cyto-plasm occurs through nuclear pore complexes (NPCs),which form channels that perforate the nuclear enve-lope. Although the channels are in principle largeenough for free diffusion of small macromolecules,they pose a permeability barrier that prevents trans-location of small proteins or RNAs across the nuclearenvelope. To reach their site of function, most nuclearproteins are translocated through the NPC by specifictransport factors. Nucleocytoplasmic transport fac-tors are able to recognize their macromolecular cargoin the appropriate cellular compartment, to migratewith it through the NPC, and to release it uponreaching the other side of the nuclear envelope.1–4

The majority of transport factors belong to thefamily of importin-β (Impβ)-like proteins. Whereas

d.

679Yeast Classical NLS Proteins

N20 Impβ-related receptors have been identified invertebrates, the yeast Saccharomyces cerevisiae ex-presses 14 family members, including 10 importinsand 4 exportins.3,4 These soluble receptors interactwith specific import or export signals present in theircargos, with components of the NPC (nucleoporins),and with Ran-guanosine 5′-triphosphate (Ran-GTP).The small GTPase Ran imparts asymmetric direc-tionality to nuclear import and export processes.The asymmetry of the system is generated by thecompartment-specific localization of Ran regula-tors, the nuclear guanine nucleotide exchange factor(RanGEF), and the cytoplasmic GTPase-activatingprotein (RanGAP). This results in a steep gradient ofRan-GTP with a high concentration in the nucleusand a low concentration in the cytoplasm. The Ran-GTP gradient is sensed by transport factors andregulates the interactionwith their respective cargos.Importins associate with their cargo in the cytoplasmin the absence of Ran-GTP, and the import complex isdissociated upon binding of Ran-GTP in the nucleus.Importins usually bind directly to a nuclear local-

ization signal (NLS) present within the cargo mo-lecule. NLSs are often short stretches that have anoverall basic character enriched in lysine residues,but differing motifs, such as the M9 domain recog-nized by transportin, have also been described.5

Impβ, the first transport receptor identified, recruitscargo usually by binding of an adapter molecule.It is thought that the majority of Impβ transportsubstrates are recognized by the adapter moleculeimportin α (Impα).6 These substrates contain theso-called classical nuclear localization signal (cNLS),a short sequence of basic amino acid residues (mono-partite cNLS) or two basic clusters separated byapproximately 10–12 residues (bipartite cNLS).7–10

Table 1. Classical NLS proteins characterized in the yeast S.

ProteinImport defect inImpα/β mutants

Direct bindinto Impα

Gln3 srp1cs Cofractionatiwith Impα

Proteasome precursorcomplexes

srp1–49(Pre6, Ump1, Rpn1, Rpn11)

Cofractionatiwith Impα/

Sto1/Cbc2 n.d. Cofractionatiwith Impα/

Sto1 n.d. +

Swi6 n.d. Binding toImpαΔIBB

Clb2 rsl1-3, srp1–31 n.d.Gcn4 rsl1-4, srp1–31 n.d.Upf3 srp1–31 n.d.

Prp20 rsl1–1, srp1–54 +

Cdc6 rsl1-1, srp1–31,srp1–54

+

Cdc45 rsl1-1, srp1–31,srp1–49, srp1–54

+

Clb2 rsl1-1, srp1–31,srp1–49, srp1–54

+

Swi5 rsl1-1, srp1–31, srp1–54 +

n.d., not determined; NMD, Nonsense-mediated mRNA decay.

The prototype of a monopartite cNLS is the sequencemotif PKKKRKV (basic residues are underlined) ofthe simian virus 40 (SV40) large T-antigen, whereasthe prototypical bipartite cNLS is that of nucleo-plasmin (KRPAATKKAGQAKKKKL).11,12 Impαforms a superhelix consisting of 10 so-called arma-dillo (ARM) repeats. The ARM repeat domain formsthe cNLS-binding groove,1,13 whereas the flexibleImpβ-binding (IBB) domain at the N-terminus ofImpα associates with the receptor Impβ.14–16 Thecrystal structure of cargo-free Impα reveals that theN-terminal IBB domain forms an intramolecularinteraction with the cNLS-binding pocket.17 Furtherstudies showed that the IBB domain is inhibitorywith respect to cNLS binding and that the auto-inhibition is relieved by binding of Impβ.18,19

Impβmediates the translocation of the trimeric NLSprotein/Impα/Impβ complex through the NPC. Theimport complex dissociates in the nuclear compart-ment upon Ran-GTP binding to Impβ.20,21 Impβ isrecycled to the cytoplasm in complex with Ran. Impαis transported back to the cytoplasm by its specificexportin Cse1/CAS, together with Ran-GTP.22–25 Themechanism of dissociation of the cNLS cargo/Impαcomplex is unclear. One model indicates that it dis-sociates spontaneously and the IBB domain inhibitsrebinding of cargo.17,26,27 According to a second me-chanism, Cse1/CAS and Ran-GTP release Impα fromthe cargo.26,28 A third model proposes that nucleo-porins facilitate the import termination step.26,28–30

The yeast nucleoporin Nup2, which predominantlylocalizes to the nuclear side of the NPC, efficientlydissociates the import complex by binding to Impαwith high affinity, displacing the cNLS protein.29

Nup50, the eukaryotic counterpart of Nup2, plays asimilar role.31

cerevisiae

g Stimulationby Impβ Comment Reference

on n.d. Shuttling transcription factor 37

onβ

n.d. Core, base, and lid complexesare imported separately

38,39

onβ

n.d. Heterodimeric cap-bindingcomplex

40

+ Fragment containingputative cNLS

26

n.d. Shuttling transcription factor 41

n.d. Shuttling cyclin 42n.d. Transcription factor 43n.d. Shuttling protein required

for NMD44

+ Ran-specific guanineexchange factor

29 andThis study

+ Cell cycle regulator This study

+ Transcription factor This study

+ Cyclin B2 This study

+ Transcription factor This study

680 Yeast Classical NLS Proteins

Whereas vertebrates possess several closely relatedvariants of Impα,32,33 baker's yeast contains only onesingle essential gene, SRP1 (also known as KAP60).34

Similar to its vertebrate ortholog, it mediates thenuclear import of cNLS proteins together withKap95, the S. cerevisiae Impβ.35,36 Reporter proteinscontaining the SV40 large T-antigen cNLS are com-monly used when the classical NLS import pathwayis explored in yeast. Although a large number ofpotential genuine yeast cNLS proteins exist,9 none ofthe proposed NLSs were unambiguously character-ized as cNLS. The defining criteria for a cNLS proteinare an in vivo import defect in Impα/β mutants, di-rect and specific binding to purified Impα/β in vitro,and stimulation of Impα's binding by Impβ. Thesecriteria allow one to distinguish between a classicalNLS cargo and a cargo of the other nine importins.Most candidate cNLS proteins fulfill only one or twoof these criteria (Table 1). It was shown solely for thetranscription factor Gln3 and for some proteasomeprecursor proteins that they accumulate in the cyto-plasm of Impα mutants and cofractionate withImpα,37–39 but it is unknownwhether Impβ enhancestheir binding to Impα.In this study, we systematically studied the

nuclear import of five authentic yeast cNLS proteins.Their nuclear import is impaired in Impα, Impβ,and nup2-null mutants; they bind to Impα as recom-binant proteins synthesized in Escherichia coli, andthis binding is enhanced by Impβ. We determinedthe binding properties of these cNLS proteins forImpα by surface plasmon resonance (SPR) spectro-metry and discuss these results with respect to theimport mechanism. Furthermore, we intensively in-vestigated the cNLSs of Prp20, Ran-specific GEF,and Cdc6, a GTPase that regulates the initiation ofDNA synthesis. A mutant analysis revealed the pre-sence of a bipartite cNLS in Prp20 and amonopartitecNLS in Cdc6.

Results

The nuclear uptake of selected nuclear proteinsis inhibited in cNLS import mutants

We analyzed the nuclear import of putative cNLSproteins in vivo. We chose nuclear proteins of theyeast S. cerevisiae whose nuclear targeting sequenceshad been characterized before, but it was unclearwhich of the importins is responsible for their import(Table 1). Prp20, the guanine nucleotide exchangefactor for Gsp1 (yeast Ran), contains an NLS at itsN-terminus45 and binds to Impα in vitro.29 Cdc6, aGTPase involved in DNA synthesis and cell cycleregulation, also contains an NLS at its N-terminus.46

Amino acid substitutions of basic residues within theNLS motifs of Prp20 and Cdc6 inhibit nuclear im-port,45–47 but it is unclear whether these proteinscarry a monopartite NLS or a bipartite NLS. Thetranscription factor Swi5 contains a C-terminal NLSbetween residues 636 and 655.48 Cdc45, another

transcription factor, possesses an NLS between resi-dues 209 and 228.49 The introduction of point muta-tions showed that both Swi5 and Cdc45 carry abipartite NLS.48,49 Clb2, a B-type cyclin, contains abipartite NLS motif between residues 183 and 200,but the deletion of this motif leads only to partialinhibition of import.42 Nevertheless, the nuclear up-take of Clb2 is defective in Impα and Impβ mutants.The residual import of the NLS deletion mutant isprobably due to complexation with Cdc28. Becausethere is no other sequence in Clb2 that resembles aclassical NLS, it is very likely that the motif betweenresidues 183 and 200 of Clb2 constitutes a bipartitecNLS.42

We constructed plasmids in which the coding se-quence of the green fluorescent protein (GFP) fusedto the coding sequences of the five genes is under thecontrol of the galactose-inducible GAL1 promoter.As a control, we employed the well-studied cNLS ofthe SV40 large T-antigen. All fusion proteins have asize of N50 kDa, the diffusion limit of the NPC. Tolocalize the GFP fusion proteins, wild-type cells andheat-sensitive importin mutants were transformedwith these plasmids. We analyzed the Impβ mutantrsl1-150 as well as three different Impα mutants.Impα contains 10 ARM repeats, where ARM repeats2–8 are involved in cNLS binding.13,51,52 The com-monly used mutant srp1–31 has a point mutation(Ser116Phe) within the first ARM repeat, whereassrp1–49 (Glu145Lys) and srp1–54 (Gly459Val) aremutated in ARM repeats 2 and 9, respectively.53

Western blot analysis demonstrated that proteins ofthe expected molecular mass were synthesized in allstrains (data not shown).Figure 1 shows that all five analyzed GFP fusion

proteins as well as the SV40 large T-antigen cNLS-glutathione-S-transferase (GST)-GFP reporter pro-tein22 are located in the nucleus of wild-type cells.The NLS proteins colocalized with DNA, which wasstained with 4′,6′-diamidino-2-phenylindole (DAPI;data not shown). All GFP fusion proteins accumu-lated in the cytoplasm of rsl1-1 mutants after incu-bation at the nonpermissive temperature of 37 °C for1 h, indicating that Impβ is required for nuclearimport. The reporter proteins also mislocalized insrp1 mutants at the nonpermissive temperature,albeit to varying degrees. We observed that the im-port defect of all examined proteins was very pro-nounced in srp1–54 mutants, less prominent insrp1–31 cells, and relatively weak in srp1–49 mu-tants. Therefore, the srp1–54 mutant is best suitedwhen studying the dependence of nuclear import onImpα. The mislocalization phenotypes of the rsl1-1and srp1–54mutantswere almost identical. For Swi5,Cdc6, and Prp20, no significant import defect insrp1–49 mutants was detectable. The nuclear locali-zation of Prp20 was even unaffected in srp1–31 cells.The cytoplasmic accumulation of Prp20 in all mu-tants was comparably less prominent, which sug-gests that the nuclear import of Prp20 might also bemediated by an alternative pathway perhaps viaanother Impβ-related transport receptor. This is inagreement with an earlier analysis of Prp20 import45

Fig. 1. The nuclear import of NLS proteins is defective in Impα, Impβ, and nup2 mutants. Plasmids encoding a GST-GFP fusion protein to the NLS of the SV40 large T-antigen, or GFP-Prp20, GFP-Cdc6, GFP-Swi5, GFP-Cdc45, GFP-Clb2,or GST-GFP fused to the IBB domain of Impα (αIBB) were transformed into the indicated yeast strains. The wild-typeand Δnup2 cultures were grown at 30 °C in liquid selective media containing raffinose and were supplemented with2% galactose for induction of the GFP fusion proteins. After 2 h, the cells were harvested and prepared forimmunofluorescence microscopy. The temperature-sensitive Impβ (rsl1-1) and Impα (srp1) strains were cultured at25 °C. Following galactose addition, the cells were grown for 1 h at 25 °C and then shifted for 1 h to 37 °C. All cellswere probed with GFP-specific antibodies and analyzed by immunofluorescence microscopy to visualize the GFPfusion proteins.


and a study reporting that RCC1, the mammalianhomolog of Prp20, can be imported into the nucleuseither with or without Impα.54

We reported previously that the import of an SV40large T-antigen cNLS reporter protein is inhibited incells deleted for the nucleoporin Nup2.29 Therefore,we also analyzed the nuclear import in nup2 knock-out cells. Figure 1 shows that the nuclear uptake ofPrp20, Cdc6, Swi5, Cdc45, and Clb2 was impaired innup2 mutants. An almost complete import defectwas observed for Cdc6. Taken together, the trans-port of these proteins into the nucleus in vivo re-quires Impα and Impβ, indicating the presence of acNLS within these proteins. Furthermore, efficientnuclear import also necessitates Nup2, which sug-gests that this nucleoporin constitutes a generalimport factor.As a control, we analyzed the nuclear import of a

fusion protein comprising the IBB domain of Impα,which contains the 71 N-terminal residues of Impα.The fusion protein is located exclusively in thenucleus of wild-type cells. Its import is not inhibitedin Impα or nup2 mutants, but the IBB domainseverely mislocalized to the cytoplasm in Impβmutants (Fig. 1). This indicates that, as expected, theimport of the IBB domain does not depend on Impαand Nup2, but requires Impβ.

Characterization of the NLSs of Prp20 and Cdc6

We intended to elucidate whether the NLSs ofPrp20 and Cdc6 are monopartite or bipartite. TheNLS of Prp20 is located at its very N-terminus.45 Theonly cluster of positive charges in the N-terminus ofPrp20 resides between residues 17 and 23. Thiscluster is separated by 12 residues from two basicresidues at positions 3 and 4 (Fig. 5b). To discrimi-nate between a monopartite cNLS and a bipartitecNLS, we introduced point mutations into Prp20fused to GFP, where the basic amino acids atpositions 3, 4, 17, 19, 20, and 23 were replaced bythreonine. Likewise, the Met residue at position 21was mutagenized to a Thr residue. The M21T muta-tion (data not shown) and the K19T mutation (Fig. 2)had no effect on import. The K3T, R4T, and K17Tmutations induced partial cytoplasmic accumula-tion in some, but not all, cells. The K23T mutationand the K3T R4T double mutations caused an im-port defect in approximately 50% of the cells. How-ever, nuclear import of Prp20 K20T was stronglydefective and was only slightly deteriorated byadditionally introducing the K19T mutation (Fig. 2).The mislocalization of Prp20 K20T was as strong asthe mislocalization of wild-type Prp20 in rsl1-1 orsrp1–54 mutants (Fig. 1). These data indicate that

Fig. 3. The amino acid residues 19–33 mediate thenuclear uptake of Cdc6. (a) Plasmids encoding GFP-Cdc6,GFP-Cdc6ΔN18, GFP-Cdc6ΔN33, GFP2-Cdc6-N50, orGST-GFP-Cdc6-N14 were transformed into wild-typecells. The synthesis of the fusion proteins was induced bythe addition of 2% galactose to liquid cultures grown at30 °C. After 2 h, the cells were harvested and analyzed byimmunofluorescence microscopy. The samples wereprobed with anti-GFP antibodies and FITC-labeled second-ary antibodies and stained with DAPI to visualize theDNA. (b) The Lys29 and Arg30 residues of Cdc6 arerequired for nuclear import. Plasmids coding for GFPfusion proteins to Cdc6 carrying the indicated pointmutations were transformed into wild-type cells. The cellswere treated as described in (a) and analyzed by immuno-fluorescence microscopy to visualize the GFP signal.

Fig. 2. Basic amino acid residues at the N-terminus ofPrp20 are required for nuclear import. Plasmids coding forGFP fusion proteins to Prp20 carrying point mutationswith the indicated amino acid substitutions were trans-formed into wild-type cells. The cultures were incubatedat 30 °C until they had reached the early logarithmicgrowth phase and then 2% galactose was added. The cellswere harvested and analyzed by immunofluorescencemicroscopy with anti-GFP primary antibodies and FITC-conjugated secondary antibodies.


Lys3, Arg4, Lys17, and Lys23 are required forefficient nuclear import in vivo and that the lysineat position 20 is a crucial residue. The importance ofthe Lys3 and Arg4 residues suggests that Prp20contains a bipartite cNLS at its N-terminus.According to the current model of Ran-dependent

nuclear transport, nuclear localization of the Ran-GEF is required to ensure that Ran is bound to GTPin the nucleus. To test the effect of the expression ofcytoplasmic Prp20, we transformed importinmutants with plasmids encoding GFP-Prp20 andstreaked them onto agar plates (Fig. S1a). All cellsgrew well on glucose plates, where the GAL1 pro-moter is suppressed and GFP-Prp20 is not synthe-sized. On galactose plates, however, the expressionof GFP-Prp20 caused a dominant lethal phenotypein rsl1-1 and srp1–54 mutants, which had accumu-lated Prp20 in the cytoplasm (Fig. 1). This is inagreement with the idea that cytoplasmic Ran-GTPdisrupts the Ran gradient in these mutants. Thetoxicity is explained by the inhibitory effect of Ran-GTP on the formation of import complexes in thecytoplasm. This result also confirms that the srp1–54mutant displays a stronger import defect phenotypethan the srp1–31 and srp1–49 mutants. We thentested the expression of Prp20 mutants in wild-typecells by this assay. A dominant lethal phenotype wasinduced only by the point mutant Prp20 K20T butnot by all other point mutants (Fig. S1b). Thisunderlines the importance of the lysine residue atposition 20 for nuclear import (Fig. 2). Furthermore,the import of a cNLS-GFP reporter is defective whenK20 mutants are expressed in wild-type cells (datanot shown; Ref. 45).

We next performed a deletion analysis to deli-neate the Cdc6 NLS. Similar to Prp20, Cdc6 containstwo basic clusters at its N-terminus. Four positivelycharged residues between amino acids 10 and 14 areseparated by 11 amino acids from five basic residuesbetween amino acids 26 and 32 (Fig. 5a). To deter-mine the significance of these two basic clusters, weexpressed the N-terminal truncation Cdc6ΔN18,lacking only the first basic cluster, or Cdc6ΔN33,lacking both basic clusters, in wild-type yeast cells(Fig. 3a). We observed that Cdc6ΔN18 localizedcompletely to the nucleus, whereas Cdc6ΔN33 was


cytoplasmic. In addition, the 50 N-terminal residuesdirect GFP2 to the nucleus, while the 14 N-terminalresidues fused to GST-GFP are completely cyto-plasmic (Fig. 3a). These data show that the regionbetween residues 19 and 50 is necessary andsufficient for nuclear import of Cdc6 in vivo, whereasthe N-terminal 18 amino acids containing four basicresidues are dispensable for the migration of Cdc6into the nucleus. These results point to the presenceof a monopartite cNLS near the N-terminus of Cdc6.To determine the importance of individual resi-

dues of the basic cluster (residues 29–32 of Cdc6) for

nuclear import, we introduced point mutations intoCdc6 fused to GFP where these residues were re-placed by a threonine residue. The K29T and theR30T mutations both cause a strong import defect.The K29T mutant showed a somewhat more severemislocalization than the R30T mutant (Fig. 3b). Incontrast, the K31T and K32T mutations do not leadto a significant inhibition of import, although theK32T mutant accumulates slightly in the cytoplasm.These data indicate that Lys29 and Arg30 are re-quired for efficient nuclear uptake and that the Lysresidues at positions 31 and 32 play a minor role inthe nuclear import of Cdc6 in vivo.

Purified Impα and Impβ bind to Prp20, Cdc6,Swi5, Cdc45, and Clb2

We previously showed that Impα alone bindsweakly to Prp20 and that this interaction is stronglystimulated by Impβ.29 We now tested whether theN-terminal domain of Prp20 containing the cNLSalone is also able to bind to Impα and Impβ. GST-Prp20ΔN38 and GST-Prp20-N38 fusion proteinswere purified from Escherichia coli lysates, immobi-lized to glutathione Sepharose, and incubated withrecombinant Impα and Impβ, which had been puri-fied from bacterial extracts as 6-histidine fusionproteins. Figure 4a shows that the importins did notinteract with Prp20ΔN38 but efficiently bound to

Fig. 4. Impα and Impβ bind to various NLS sequencesin a cooperative manner. GST fused to the indicated full-length proteins, protein fragments, or point mutants ofPrp20, Cdc6, Cdc45, Swi5, and Clb2 were bound toglutathione Sepharose by incubation at 4 °C for 60 min.The immobilized proteins were incubated further withbuffer alone (−) or with the indicated factors for 60 min at4 °C. After three washing steps, the bound material waseluted with SDS sample buffer and examined by SDS-PAGE followed by Coomassie blue staining (a–c and f)and immunoblotting with antibodies directed againstImpα and Impβ (d–f). All proteins were purified asrecombinant factors from E. coli lysates. Where indicated,the loads (L) of the importins were also analyzed.Molecular mass markers (M) are given in kilodaltons. (a)Prebound GST fused to Prp20 or Prp20 fragments (8 μgper reaction) was incubated with buffer alone or with 8 μgof Srp1 (Impα) and 12 μg of Kap95 (Impβ). (b)Immobilized Prp20 or Prp20 point mutants (8 μg perreaction) were incubated with 10 μg of Gsp1 (Ran) loadedwith either GTP or GDP. (c) GST fused to Prp20 or Prp20point mutants (8 μg per reaction) was incubated with 8 μgof Impα alone (α), with 12 μg of Impβ alone (β), withImpα and Impβ together (αβ), or with 8 μg of ImpαΔN60(αΔIBB). (d) Immobilized Cdc6 (8 μg per reaction) wasincubated with Impα and/or Impβ. Complexes contain-ing Impα and Impβ bound to Cdc6 were reisolated bythree washes with buffer and further incubated with SV40NLS peptides (1000-fold molar excess), 4 μg of Nup2-N,12 μg of full-length Nup2, or 10 μg of Gsp1 (Ran) loadedwith GTP or GDP for 15 min at 4 °C. (e) GST fusions withCdc6 fragments (8 μg per reaction) were incubated withImpα and/or Impβ. (f) Immobilized Cdc45-N250, Swi5-C82, and Clb2-N249 (8 μg per reaction) were incubatedwith Impα and/or Impβ or with ImpαΔIBB.


Prp20-N38. Similar to full-length Prp20 (Fig. 4c),Prp20-N38 bound to the importins in a stronglycooperative manner. It did not bind to Impβ, and thebinding to Impα alone was weak and detectableonly by immunoblotting analysis (data not shown).We conclude that the N-terminal 38 residues ofPrp20 are necessary and sufficient for direct bindingto the Impα/β heterodimer in vitro.We then analyzed the Prp20 point mutants in pull-

down assays. To test their functionality, we incubatedthe immobilized Prp20 mutants with Gsp1 (yeastRan) loaded with either GTP or guanosine dipho-sphate (GDP; Fig. 4b). As expected for the RanGEF,we observed the binding of Ran-GTPase to wild-typePrp20. Because all analyzed mutants also bound toGsp1 (Fig. 4b; data not shown), we conclude thatamino acid changes at the Prp20 N-terminus do notaffect the overall conformation of the protein. Prp20also bound efficiently to ImpαΔIBB, a truncated formlacking the N-terminal 60 residues of Impα (Fig. 4c).This confirms that the deletion of the autoinhibitoryIBB domain overcomes the requirement for Impβfor high-affinity binding to the NLS.18,19 From theanalyzed point mutants, Prp20 K19T bound slightlyweaker to ImpαΔIBB or Impα/β in a still coopera-tive manner. The K23T mutation caused a significantinhibition of binding to Impα/β or ImpαΔIBB,whereas the binding of the K20T mutant or theK3T R4T double mutant was no longer detectable inCoomassie-blue-stained gels. This shows that theresidues K3, R4, K20, and K23 are required forefficient interaction with Impα. The extent of bindinginhibition in vitro correlated well with variousdegrees of import inhibition in vivo shown in Fig. 2.We then investigated the interaction of Cdc6 with

Impα and Impβ (Fig. 4d). GST-Cdc6 was preboundto glutathione beads and incubated with Impα.Surprisingly, Cdc6 bound efficiently to Impα alone,and this interaction was hardly stimulated by Impβ.This observation is in contrast to the binding char-acteristics of Prp20 and the SV40 NLS (Fig. 4c andRef. 29), which both bind to Impα and Impβ in acooperative manner. Nevertheless, after the beadshad been washed with buffer, excess amounts ofSV40 NLS peptides released most of Impα andImpβ, indicating that the Cdc6 NLS and the SV40NLS interact with the same binding site. Moreover,Nup2-N containing the Impα-binding domain ofNup229 or full-length Nup2 displaced Impα/β fromCdc6. This suggests that Nup2 is universally capableof dissociating cNLS import complexes by bindingto Impα.29 When the washed Cdc6/Impα/β com-plex was incubated with Ran, we observed that Ran-GDP, as expected, did not affect the stability of thecomplex. Remarkably, Ran-GTP released Impβ, butnot Impα, indicating that the Cdc6/Impα complex isstable during the incubation for 15 min. Accordingto these data, Ran-GTP is not sufficient to dissociatethe Cdc6/Impα complex in the nucleus, which sug-gests that Nup2 and Ran together disassemble NLSimport complexes in vivo.To examine whether the N-terminal NLS of Cdc6

interacts with Impα/β, we analyzed Cdc6-N50,

Cdc6ΔN18, and Cdc6ΔN33 in pull-down assays(Fig. 4e). Both Cdc6-N50 and Cdc6ΔN18 bound toImpα and Impα/Impβ, whereas Cdc6ΔN33 did not.Cdc6-N50 and Cdc6ΔN18 bound well to Impα, butin contrast to full-length Cdc6, this binding wasclearly enhanced by Impβ. We conclude that theregion between residues 19 and 33, which mediatesthe nuclear import of Cdc6, is also necessary andsufficient for the direct interaction with Impα/β. Thein vivo and in vitro results combined clearly prove thatthis stretch of 15 amino acid residues within Cdc6contains a classical Impα/β-dependent NLS.We next attempted to analyze whether Cdc45,

Swi5, and Clb2 interact with Impα/β in vitro. N- orC-terminal fragments of these proteins containing therespective NLS were produced in bacteria as GSTfusion proteins. GST fusions to Cdc45-N250, Swi5-C82, and Clb2-N249 were incubated with Impα andImpβ, as well as with ImpαΔIBB (Fig. 4f). Similarto what we observed for Prp20, Impα bound onlyweakly to all three analyzed proteins, and this inter-action was strongly stimulated by Impβ. The bindingstrength of ImpαΔIBB equaled that of Impα in thepresence of Impβ. Thus, these proteins bind to Impαand Impβ in a strongly cooperative manner. Thesedata, combined with the mislocalization phenotypein importin mutants (Fig. 1), characterize Cdc45,Swi5, and Clb2 as cNLS proteins.

The peptide spot method defines the Cdc6 cNLSas monopartite and the Prp20 cNLS asbipartite

To analyze the effect of amino acid substitutions,we incubated Impαwith immobilized NLS peptidesof Cdc6 and Prp20 synthesized on the surface ofcellulose membranes by the peptide spot method.55

The positively charged amino acid residues lysine orarginine in the N-termini of Cdc6 and Prp20 werereplaced by a threonine residue. In order tocircumvent the requirement for Impβ, ImpαDIBBwas used in this series of experiments. The SV40cNLS and an SV40 cNLS mutant, which cannotsupport nuclear import,56 served as positive andnegative controls. Our localization studies and pull-down assays indicated that the cNLS of Cdc6 islocated between residues 19 and 33. Five positivelycharged residues are found between positions 26and 32 (Fig. 5a). Peptides corresponding to residues23–34 of Cdc6 were synthesized onto cellulosemembranes and incubated with ImpαΔIBB. Figure5a shows that this region mediated the sameintensity of binding to ImpαΔIBB as the SV40NLS, as quantified by densitometry. The re-placement of the basic residues affected the bindingefficiency of ImpαΔIBB. The weakest inhibition wasobserved for the K31T replacement with 77% bind-ing compared to the wild-type peptide. The R26Tand K32T peptides still bound 56–58% of Impα-ΔIBB, whereas K29T peptides only bound 33% ofImpαΔIBB in comparison to wild-type peptidesand were comparable to the binding ability of theSV40 mutant peptides. The strongest inhibition was

Fig. 5. The peptide spot method reveals the importance of positively charged amino acid residues within the NLSs ofCdc6 and Prp20. Peptides containing the NLS sequences of the SV40 large T-antigen, Cdc6, and Prp20 with the indicatedsubstitutions of basic amino acids by threonine were synthesized onto a cellulose membrane. Sequences are given insingle amino acid letter codes. The membranes were incubated with 14C-labeled Srp1ΔN60 (ImpαΔIBB). After boundImpαΔIBB had been washed, it was detected by autoradiography. At the bottom, the N-termini of Cdc6 and Prp20 areshown, and the residues relevant for efficient binding to Impα are indicated. (a) The SV40 NLS peptide and a Cdc6peptide (residues 23–34) with arginine and lysine residues replaced by threonine residues were analyzed for binding toImpαΔIBB. The amount of bound Impα was quantified by densitometry. (b) The SV40 NLS peptide, as well as Prp20fragments corresponding to residues 1–12, 15–26, and 1–25 with single or multiple amino acid substitutions, wereanalyzed for binding to ImpαΔIBB. The amount of bound Impα was quantified by densitometry.


observed for the R30T replacement, which dec-reased the binding intensity to 8%. Double replace-ments further reduced the binding of ImpαΔIBB,and multiple replacements resulted in a completeloss of interaction with Impα. These results definethe region between residues 26 and 32 of Cdc6 as amonopartite cNLS, with all positively charged resi-dues contributing to Impα binding.Three regions of the Prp20 N-terminal 26 residues

were analyzed separately, and basic amino acidresidues were again replaced by a threonine residue(Fig. 5b). No binding of ImpαΔIBB was detected inresidues 1–12 of Prp20 containing the lysine andarginine residues at positions 3 and 4. The peptidecorresponding to residues 15–26 of Prp20 bound toImpαΔIBB with a lower binding efficiency com-pared to the SV40 NLS. When the basic residues atpositions 17, 19, 20, and 23 were replaced, thebinding efficiency was diminished, indicating thatall of these residues contribute to recognition byImpα. The introduction of multiple replacementsbasically abolished the binding to Impα. The bindingof ImpαΔIBB to peptides 15–26 of Prp20 was lessefficient than to a peptide ranging from residues 1 to25. Thus, the basic residues between positions 17 and23 are not sufficient for proper binding to Impα. Thereplacement of residues 3 and 4 by Thr significantlydecreased the binding of ImpαΔIBB, indicating thatthese residues also contribute to efficient Impα bind-ing. We conclude that Prp20 contains a bipartite

cNLS and that the residues at positions 3, 4, 17, 19, 20,and 23 are important sequence determinants of thiscNLS.

Determination of the binding affinities of cNLSproteins for Impα

The binding properties of the identified cNLSproteins were analyzed by SPR spectrometry. TheGST-cNLS fusion proteins served as ligands thatwere immobilized to the surface of a sensor chip in aflow cell via cross-linked anti-GST antibodies. By thisapproach, the antibody, and not the GST fusion pro-tein, is covalently linked to the matrix of the sensorchip. This ensures the best possible presentation of theimportin-bindingdomains. To further reduce artificialinteractions, we used full-length cNLS proteins orlarge domains of these proteins containing the cNLS.Impα, Impβ, and ImpαΔIBB served as analytes andwere passed over the surface of the sensor chip atdifferent concentrations. Unspecific interactionswere minimized by using GST alone in a referencecell with a second sensor chip. After injection of theanalytes into the flow cells, the injection of bufferinitiated the dissociation phase. The differences inthe resonance signal between the sample flow celland the reference cell were monitored and used tocalculate the binding constants by using appropriateBIAcore software programs. A sensorgram containsall measurement cycles for a specific ligand–analyte

Fig. 6. Sensorgrams of the interaction of NLS proteins with Impα. A monoclonal GST-specific antibody was covalentlycoupled onto a BIAcore CM5 chip. GST fusion proteins to Prp20 (a–c), Cdc6-N50 (d), Clb2-N249 (e), and the SV40 NLS (f)were bound to the chip. Impα, ImpαΔIBB, and Impβ at the indicated concentrations were then injected into the sampleflow chamber (association phase). Subsequently, the dissociation phase was initiated by washing with buffer. Thesensorgrams show the SPR difference (in resonance units) between the sample flow chamber and the reference flowchamber, which contained immobilized GST only.


pair. The recorded sensorgrams are shown in Fig. 6and Figs. S2 and S3. Table 2 summarizes for eachligand–analyte pair the resulting dissociation con-stant Kd, the association constant Ka, the rateconstants for dissociation kd (off-rate constant), therate constants for association ka (on-rate constant),and the χ2 value, which is an indicator of the meandeviation of the experimental data from the calcu-lated curves.

Figure 6a–c shows the sensorgrams for the inter-action of full-length Prp20 with Impα, Impα+Impβ(preincubated at a 1:1 molar ratio), and ImpαΔIBB.The equilibrium constants of the Prp20 cNLS differsignificantly depending on the presence of Impβ.The dissociation constants for binding to ImpαwereKd=1.52×10

−6 M in the absence of Impβ, butKd=2.26×10

−8 M in the presence of Impβ (i.e., thedissociation constant decreased by 67-fold with

Table 2. SPR spectrometry data for the interaction between NLS proteins and Impα, Impα+Impβ, or ImpαΔIBB

Ligand Analyte Kd (nM) Ka (×106 M−1) kd (×10−3 s−1) ka (×10

3 M−1 s−1) χ2 Sensorgram

SV40 NLS Impα 280.0 3.5 61.0 2.2 0.7 Fig. S2aImpα+Impβ 6.1 160 2.6 430 2.5 Fig. S2bImpαΔIBB 9.0 110 3.3 360 1.5 Fig. 6f

Prp20 Impα 1520.0 0.66 2.9 1.9 1.6 Fig. 6aImpα+Impβ 22.6 44 5.9 260 1.8 Fig. 6cImpαΔIBB 19.3 52 3.9 200 2.4 Fig. 6b

Prp20-N96 Impα+Impβ 17.0 58 6.5 380 1.3 Fig. S3eImpαΔIBB 11.0 94 2.7 250 1.1 Fig. S3f

Prp20 K3T R4T ImpαΔIBB 10,300.0 0.097 74.0 7.2 1.3 Fig. S3aPrp20 K19T ImpαΔIBB 150.0 6.7 13.6 91 1.5 Fig. S3bPrp20 K20T ImpαΔIBB 7400.0 0.14 118.0 16 0.5 Fig. S3cPrp20 K23T ImpαΔIBB 2200.0 0.46 3.3 1.5 0.9 Fig. S3dCdc6-N50 Impα 171.0 5.8 6.0 35 2.7 Fig. S2c

Impα+Impβ 1.0 1000 7.2 7200 1.6 Fig. S2dImpαΔIBB 1.8 790 4.4 3500 1.2 Fig. 6d

Clb2-N249 Impα+Impβ 37.0 28 5.6 150 2.0 Fig. S2gImpαΔIBB 57.0 17 5.3 92 2.5 Fig. 6e

Swi5-C82 Impα+Impβ 112.0 9.0 46.0 410 0.5 Fig. S3hImpαΔIBB 160.0 6.3 15.0 93 0.9 Fig. S3g

Cdc45-N250 Impα+Impβ 93.0 11 12.3 130 0.5 Fig. S2fImpαΔIBB 55.0 18 11.3 210 1.4 Fig. S2e


Impβ). The Kd value for the binding to ImpαΔIBBwas 1.93×10−8 M, which is very close to the valuemeasured for the binding of Impα+Impβ. This con-firms that the deletion of the autoinhibitory IBBdomain leads to an Impα molecule that does notrequire Impβ for high-affinity cNLS binding. Re-markably, the off-rate constants for the cNLS inter-action with Impα, Impα+Impβ, or ImpαΔIBB wereall in the same range (kd=2.9×10

−3 to 5.9×10−3 s−1).On the other hand, the on-rate constants differed byN100-fold for Impα (ka=1.9×10

3 M−1 s−1) comparedto those for Impα+Impβ (ka=2.6×10

5 M−1 s−1) orImpαΔIBB (ka=2.0×105 M

−1 s−1). The low ka valuefor Impα alone indicates that the IBB domain pre-vents rapid binding to the cNLS. The nearly iden-tical kd values reveal that once the cNLS is bound toImpα, the stability of that complex is independent ofImpβ. Thus, only the difference in the associationdynamics is responsible for the lower affinity ofImpα in the absence of Impβ.The Prp20-N96 fragment showed very similar

dissociation and association constants compared tofull-length Prp20 (Table 2 and Fig. S3e and f). ThePrp20 point mutants had to be incubated withhigher concentrations of ImpαΔIBB (Table 2 and Fig.S3). Both association and dissociation rates were re-duced, resulting in dissociation constants of Kd=1.5×10−7 M for Prp20 K19T, Kd=2.2×10

−6 M forPrp20 K23T, Kd=7.4×10

−6 M for Prp20 K20T, andKd=1.03×10

−5 M for Prp20 K3T R4T, which corres-pond to a reduction of affinity for Impα by factors of8 (K19T), 114 (K23T), 383 (K20T), and 534 (K3T R4T).The degree of increase of the Kd values correlateswell with the various effects of the point mutationson nuclear import in vivo and on the binding cap-ability in our pull-down assays.The Kd value of 1.71×10−7 M for binding of the

Cdc6 cNLS to Impα alone was relatively low. Never-theless, the affinity increased by a factor of 170 or 95when Impα+Impβ or ImpαΔIBB was used as ana-

lyte. As observed for the Prp20 cNLS, only a loweron-rate constant, but not a change in the off-rate rateconstant, is responsible for the decreased Kd value inthe absence of Impβ or in the presence of the IBBdomain. The stimulation of binding by Impβ wasalso observed for the classical NLS of the SV40 largeT-antigen: a Kd value of 2.8×10−6 M was monitoredfor Impα alone and was decreased by 46-fold or31-fold for Impα+Impβ or ImpαΔIBB, respectively.The dissociation constants measured for Impα+Impβ(6.1 nM) and ImpαΔIBB (9 nM) are comparable tovalues obtained before by SPR spectrometry (35 nMfor Impα+Impβ and 16 nM for ImpαΔIBB)19 or byfluorescence anisotropy (18 nM for Impα+Impβ and9 nM for ImpαΔIBB).57,58 The dissociation constantsfor the binding of Impα+Impβ to the cNLSs of Clb2,Swi5, and Cdc45 were 3.7×10−8, 1.12×10−7, and9.3×10−8 M, respectively, and were similar to the Kdvalues of ImpαΔIBB binding. Thus, the affinitiestowards Impα vary significantly between differentcNLSs. The largest divergence in the Kd values is 112-fold for Cdc6 versus Swi5, suggesting that cNLSstrength affects the import efficiency of a particularnuclear protein.

Discussion

The remarkable diversity of nuclear import path-ways makes it difficult to assign a certain importcargo to its specific import receptor. More than 10importins transport their cargo into the nucleus inparallel pathways. Due to this diversity, the knowl-edge about classical NLS motifs has been fragmen-tary, particularly in yeast. We unequivocally identi-fied five genuine yeast proteins as cNLS proteinswhose import is dependent on Impα and Impβ.According to the stringent defining criteria for cNLSproteins, these five proteins are the first cNLS pro-teins identified in S. cerevisiae. These criteria—a


transport defect of the NLS protein in Impα andImpβ mutants in vivo, direct binding to Impα invitro, and stimulation of this binding by Impβ—apply for Prp20, Cdc6, Swi5, Cdc45, and Clb2altogether. Other plausible examples are Gln3 andproteasome precursor proteins, in particular Rpn2and Rpt2.37–39 However, it has not been shown forthese cargos that they bind to Impα and Impβ asbacterially purified proteins and that Impβ stimu-lates their binding to Impα.We employed different temperature-sensitive

Impα mutants to show in vivo mislocalization ofcNLS proteins. Cytoplasmic accumulation of cNLScargo was best detectable using the srp1–54 mutant,whereas srp1–31 showed a weaker phenotype andsrp1–49 displayed the least import defect. Therefore,the so far disregarded srp1–54mutant is the strain ofchoice for studying cNLS import in living cells. Theobservation that the nup2-null mutant is defective inthe nuclear import of all cNLS proteins investigateddefines Nup2 as a general factor required forefficient import of both monopartite and bipartiteNLS substrates.The trimeric cNLS import complex can be dis-

sociated by Ran-GTP binding to Impβ in the nuclearcompartment. It was hypothesized that dissociationof the residual cNLS/Impα complex is promoted byCse1 and Ran-GTP.26,28 However, because the cargo-bound form of Impα cannot interact with Cse1,22 theImpα-specific exportin and Ran are rather requiredfor efficient recycling, which most likely also in-volves Nup2.26,29,30Our SPR spectrometry data and the liquid bind-

ing/release assays with Cdc6 rule out a mechanismby spontaneous dissociation of the cNLS/Impαcomplex. The biosensor measurements show thatthe dissociation rate of the cNLS from Impα isindependent of the presence or the absence of Impβ.Thus, after release of Impβ from the import complexby Ran, the cNLS/Impα subcomplex will remainstable. We recorded Impβ-independent off-rateconstants for the Prp20 and the Cdc6 cNLS. Incontrast, the SV40 cNLS reporter protein showed a20 times slower dissociation rate in the presence ofImpβ or in the absence of the Impα IBB domain(Table 2). One explanation is that the reporterprotein contains only seven amino acid residues ofthe SV40 large T-antigen (PKKKRKV). It wassimilarly observed for the NLS of Sto1, the largesubunit of the cap-binding protein, that the dis-sociation of the cNLS/Impα complex occurs 10times slower in the presence of Impβ. Similar to ourSV40 reporter protein, the construct used in thatstudy contains only a small fragment (30 residues) ofthe protein,26 whereas our GST fusion proteins withyeast cNLS proteins compose a larger fragment ofCdc6 or full-length Prp20. Because it is known thatprotein context contributes to cNLS recognition,59,60

it seems likely that the reduction to a small proteinsegment containing only the cNLS core leads to adecrease of the stability of complexes with Impα.Obviously, efficient dissociation of the cNLS/

Impα complex is mediated primarily by Nup2,

which binds to Impα via its N-terminal Impα-binding domain and thereby displaces Impα fromthe cNLS protein.29 It seems likely that the trimericimport complex is disassembled first by Nup2already in the basket region of the NPC. Then,Ran-GTP releases Impβ from the Nup2-boundImpα.29 Alternatively, Ran-GTP removes Impβfrom the cargo/α/β complex first, and the cNLS/Impα subcomplex is then dissociated by Nup2. Inany case, both Nup2 and Ran-GTP complementeach other to ensure an efficient recycling of theimportins. A role similar to that of Nup2 is appa-rently played by Nup1,26,29 a nucleoporin related toNup2, and by the eukaryotic Nup2 orthologNup50.31,61 We hypothesize that Nup2 becomesespecially important for cNLS proteins possessing avery high affinity for Impα. One such case is Cdc6,which shows the lowest measured Kd value of 1 nMfrom all cNLSs investigated. Full-length Cdc6 is ableto bind with high affinity to Impα in an Impβ-independent fashion (Fig. 4). Because the binding ofboth Cdc6ΔN18 and Cdc6-N50 to Impα is clearlystimulated by Impβ, it can be assumed that thesequences both N-terminal and C-terminal to thecNLS contribute to a strong binding to Impα,bypassing the inhibitory effect of the IBB domain.Nevertheless, the import of Cdc6 still requires Impβ(Fig. 1), but Impα cannot be released from Cdc6 byRan-GTP binding to Impβ. However, the importcomplex can be disassembled by Nup2 (Fig. 4), andthe importance of Nup2 for Cdc6 import issubstantiated by an exceptionally strong importdefect in nup2-null cells (Fig. 1).Crystallization studies showed that cNLSs bind in

an extended conformation to two binding sites ofImpα (P1′–P4′, the minor binding site; P1–P6, themajor binding site).13,51 Although monopartitecNLSs interact with both binding sites (see Table 3),only the binding to the major site appears to bephysiologically relevant.13 Early studies on the SV40large T-antigen NLS showed that the basic residue(usually Lys) at position P2 is of crucial importance,whereas position P3 is occupied by a Lys or Argresidue. The presence of basic residues at positionsP4 and P5 is less critical, and a basic residue atposition P1 seems not to be required for NLSfunction.56,62,63 These findings were confirmed bymore recent work showing that a basic residue atposition P2 is strictly required for Impα binding andnuclear import18,57 because it offers the mostenergetically advantageous interaction in the ImpαcNLS-binding pocket, both in monopartite and inbipartite cNLSs.10,13,51,52 The energetic contributionto the Impα/cNLS interaction decreases in the orderP2NP3NP5NP4.

10

Cdc6 is an important regulator that functions inthe formation of DNA replication complexes.64,65 Itis mainly cytoplasmic, enters the nucleus in a cell-cycle-dependent manner, and is completely nuclearduring mitosis.46 We circumvented the regulation ofnuclear import of Cdc6 by using a galactose-inducible promoter. We show here that Cdc6possesses a monopartite cNLS with the sequence

Table 3. Sequence alignment of yeast cNLSs and predicted interactions of these cNLSs with the binding pockets of Impα

The cNLSs of nucleoplasmin and SV40 large T-antigen are shown for comparison. Their interactions with specific binding pockets ofImpα had been shown previously.13,51 The consensus sequence for bipartite cNLSs is taken from Ref. 10. Relevant basic residues arehighlighted in bold, and the helix-breaking proline and glycine residues are in italics.


26RPLKRKK32, which is in agreement with thepreviously found consensus sequence BBBB orPXXBBB (where B is a base and X is any residue)for monopartite cNLSs.8,66 The mutation of allpositively charged residues individually betweenpositions 26 and 32 reduced the binding to Impα,but the residues R26, K31, and K32 showed amoderate effect when mutated separately. This is inagreement with the finding that the oligopeptide27PLKRKKL33 of Cdc6 is able to target β-galactosi-dase to the nucleus, which is disrupted by a K29mutation.46 We show here that the residues K29 andR30 are both required for efficient import in vivo,whereas K31 and K32 mutations are reconcilablewith nuclear import. Our data indicate that residuesK29 and R30 of Cdc6 correspond to positions P2 andP3 (Table 3), and that residues R26, K31, and K32contribute to binding to Impα to a lesser extent.Prp20 possesses a bipartite cNLS at its N-termi-

nus. RCC1, the vertebrate homolog of Prp20, alsocontains a bipartite N-terminal NLS where the basicresidues are found at almost identical positions,54,67

suggesting that the N-terminal cNLS has beenconserved during evolution. N-terminal truncationsof Prp20 lacking the cNLS are still imported into thenucleus, even though this import is incomplete (Ref.45; data not shown). Furthermore, we found that theimport defect of Prp20 in cNLS mutants is compar-ably weak (Fig. 1). These two observations hint of analternative Impα-independent import pathway aswas shown for RCC1.54 A previous study reported amislocalization of Prp20 in srp1–31 mutants.45

However, Fig. 2 in Akhtar et al. showed only anegligible import defect,45 which corresponds wellto our findings. Nevertheless, Prp20 significantlyaccumulates in the cytoplasm of srp1–54, rsl1-1, andΔnup2 mutants, indicating that efficient import ofPrp20 requires the cNLS import pathway. Weobserved a remarkable correlation with respect tothe various in vivo and in vitro phenotypes of thePrp20 cNLS point mutants. The degree of increase inthe Kd values matches well with the effects of the

point mutations on nuclear import and on thebinding capability in pull-down assays. Importantresidues are K3, K4, R17, K23, and especially K20,but K19 is dispensable. The K19T mutant boundwell to Impα and Impβ in pull-down assays. Thedissociation constant of Prp20 K19T was 150 nM,and although this is 10-fold higher than that of wild-type Prp20, it is similar to that of the Swi5 cNLS andcaused no import defect in vivo. The critical thresh-old for a nonfunctional cNLS is around Kd=2 μM forbinding to Impα/β, which approximates the valuemeasured for Prp20 K23Tand is in the same range ofwhat was estimated before.57

We found that the K20 residue of Prp20 is abso-lutely required for cNLS function. The vital impor-tance of K20 is supported by an earlier study, whichshowed that a Prp20 K20Q mutant inhibited thegrowth of yeast cells upon overexpression.45 Simi-larly, we found that overexpressed Prp20 K20Tcauses a dominant lethal phenotype. The expressionof wild-type Prp20 had the same toxic effect whenanalyzed in the rsl1-1 and srp1–54 mutants. Thisresult is in good agreement with the model indicat-ing that cytoplasmic Ran-GTP is detrimental to cellsbecause it disassembles import complexes beforethey reach the NPC. A similar dominant lethalphenotype is induced by the expression of a Ranmutant that is defective in GTP hydrolysis.68

All analyzed cNLSs contain at least one commonlyoccurring helix-breaking residue such as prolineprior to the basic core (Table 3), which facilitates thebinding to the cNLS-binding pocket of Impα.13

Bipartite cNLSs usually contain a linker of 10–12residues that is not important for nuclear import,69

although linker residues contribute to Impα bin-ding.10,51 The consensus for bipartite cNLSs wasdefined as BBX10–12BBB, KRX10–12KRXK, or KRX10–12KRRK.7,8,10,12 The cNLS of Prp20 does not exactlymatch these consensus sequences. Because K19 ofPrp20 is dispensable for NLS function, whereas K20and K23 are not, our data suggest that K20 evidentlycorresponds to the crucial P2 position (Table 3). The


Prp20 cNLS is unusual in two ways. First, it containsan exceptionally long linker of 15 residues, and,second, it apparently includes aMet residue at the P3position. Interestingly, a Met residue at the P3position of the SV40 large T-antigen NLS is partiallyfunctional.63 The outlined consensus rules allowedus to predict the interaction of individual residueswithin the cNLSs of Cdc45, Clb2, and Swi5 with thebinding pockets of Impα (Table 3). These predictionscorrespond well with the previously proposedconsensus sequence for bipartite cNLSs.10

The affinities of the various cNLSs for Impα differremarkably. Cdc6 has a 112 times stronger cNLS thanSwi5. The lower Kd values of high-affinity cNLSs areprimarily based on a faster on-rate of binding toImpα (Table 2), indicating that these complexes willform much faster. This suggests that the bindingstrength of a given cNLS to Impα will directly affectthe import rate into the nucleus. That this is indeedthe case was shown earlier for Impα-dependent

Table 4. Plasmids used in this study

pGS number Encoded protein

267 6His-Impα311 z-Nup2418 GST-SV40 NLS422 SV40 NLS-GST-GFP468 6His-Gsp1 Q71L722 6His-Impβ726 6His-ImpαΔIBB815 GST-Nup2-N817 GST-Prp20831 GST-Cdc45848 GFP-Clb2850 GFP-Cdc45876 GFP-Swi51113 GST-Prp20-N381114 GST-Prp20-N961116 GST-Prp20ΔN381308 GST-Cdc6ΔN181309 GST-Cdc6ΔN331310 GST-Cdc6-N501311 GFP-Cdc61312 GFP-Cdc6ΔN18 YC1313 GFP-Cdc6ΔN33 YC1314 GFP-GFP-Cdc6-N50 Y1338 GFP-Prp201356 GFP-Prp20 K3T Y1357 GFP-Prp20 R4T1358 GFP-Prp20 K3T R4T1359 GFP-Prp20 R17T1360 GFP-Prp20 K19T1361 GFP-Prp20 K20T1362 GFP-Prp20 M21T1363 GFP-Prp20 K23T1364 GFP-Prp20 K19T K20T1365 GST-Prp20 K3T R4T1366 GST-Prp20 K19T1367 GST-Prp20 K20T1368 GST-Prp20 K23T1525 GST-Swi5-C821527 GST-Clb2-N2491528 GST-Cdc45-N2501922 GST-GFP-ImpαIBB YCp1923 Cdc6-N14-GST-GFP YCp1928 GFP-CDC6 K29T Y1929 GFP-CDC6 R30T Y1930 GFP-CDC6 K31T Y1931 GFP-CDC6 K32T Y

import of mutated cNLS reporter proteins70,71 andfor various Impα-independent NLS cargos.72

Materials and Methods

Plasmid constructions

For expression in E. coli or yeast, the complete ortruncated coding regions of the respective genes wereamplified by polymerase chain reaction with the proof-reading Pwo polymerase (Roche) and inserted into thefollowing vectors: pQE9 (Qiagen), pGEX-4T-1 (AmershamPharmacia), pGEX4-TEV (pGS804),29 YCpGAL-SGFPCEN LEU2 (pGS372),73 YCpGAL-GFP-GFP CEN LEU2(pGS519),74 and YCpGAL-GST-SGFP CEN URA3(pGS846).29 The PRP20 point mutations were introducedby polymerase chain reaction. The CDC6 point mutationswere generated with the QuikChange site-directed muta-genesis kit (Stratagene). All mutations were verified by

Plasmid name Source

pQE9-SRP1 22pQE70-z-NUP2 29

pGEX-5G-SV40 NLS 29YCpGAL-NLS-GST-GFP CEN LEU2 22

pQE9-GSP1 Q71L 75pQE9-KAP95 74

pQE9-SRP1ΔN60 This studypGEX-4T-TEV-NUP2-N173 29

pGEX-2TEV-PRP20 29pGEX-2TEV-CDC45 This study

YCpGAL-SGFP-CLB2 CEN LEU2 This studyYCpGAL-SGFP-CDC45 CEN LEU2 This studyYCpGAL-SGFP-SWI5 CEN LEU2 This study

pGEX-4T-PRP20-N38 This studypGEX-4T-PRP20-N96 This studypGEX-4T-PRP20ΔN38 This studypGEX-4T-CDC6-ΔN18 This studypGEX-4T-CDC6-ΔN33 This studypGEX-4T-CDC6-N50 This study

YCpGAL-SGFP-CDC6 CEN LEU2 This studypGAL-SGFP-CDC6-ΔN18 CEN LEU2 This studypGAL-SGFP-CDC6-ΔN33 CEN LEU2 This studyCpGAL-2GFP-CDC6-N50 CEN LEU2 This studyYCpGAL-SGFP-PRP20 CEN LEU2 This studyCpGAL-SGFP-PRP20-K3T CEN LEU2 This study

YCpGAL-SGFP-PRP20-R4T This studyYCpGAL-SGFP-PRP20-K3T R4T This studyYCpGAL-SGFP-PRP20-R17T This studyYCpGAL-SGFP-PRP20-K19T This studyYCpGAL-SGFP-PRP20-K20T This studyYCpGAL-SGFP-PRP20-M21T This studyYCpGAL-SGFP-PRP20-K23T This study

YCpGAL-SGFP-PRP20-K19T K20T This studypGEX-4T-PRP20-K3T R4T This studypGEX-4T-PRP20-K19T This studypGEX-4T-PRP20-K20T This studypGEX-4T-PRP20-K23T This studypGEX-4TEV-SWI5-C82 This studypGEX-4T-CLB2-N249 This studypGEX-4T-CDC45-N250 This study

GAL-GST-SGFP-SRP1-N71 CEN URA3 This studyGAL-CDC6-N14-GST-SGFP CEN URA3 This studyCpGAL-SGFP-CDC6 K29T CEN LEU2 This studyCpGAL-SGFP-CDC6 R30T CEN LEU2 This studyCpGAL-SGFP-CDC6 K31T CEN LEU2 This studyCpGAL-SGFP-CDC6 K32T CEN LEU2 This study


DNA sequencing. The plasmids used in this study arelisted in Table 4.

Protein purification and pull-down assays

Following the induction of recombinant proteins, E. colicell lysates were prepared as described.73,75 GST fusionproteins were purified by affinity chromatography onglutathione Sepharose (Amersham Pharmacia). Fusionsof transport factors to an N-terminal 6-histidine tag werepurified with nickel–nitrolotriacetic acid agarose (Qiagen)from bacterial extracts. 6His-Srp1, 6His-Srp1ΔIBB lackingthe 60 N-terminal residues of Srp1, and 6His-Kap95 werefurther purified by Mono Q (Amersham Pharmacia)chromatography. 6His-Gsp1 Q71L was purified on aMono S column, which separates Gsp1-GTP from Gsp1-GDP.75 Nup2-N was purified after cleavage of GST-Nup2-N by tobacco etch virus (TEV) protease asdescribed.29 In pull-down assays, GST fusion proteinswere bound to glutathione Sepharose in PBSKMT buffer(25 mM sodium phosphate, 150 mM NaCl, 3 mM KCl,1 mM MgCl2, and 0.1% Tween 20, pH 7.3) by incubationfor 1 h at 4 °C.75 After three washes with 1 ml of PBSKMTbuffer, 6His fusion proteins were added to a volume of500 μl, and the reactions were further incubated for30 min at 4 °C. After bound proteins had been washedthree times with PBSKMT buffer, they were eluted withSDS sample buffer. Aliquots of the samples were loadedon SDS gels containing 11% polyacrylamide and furtheranalyzed by Coomassie staining or immunoblotting.Western blotting was carried out with the enhancedchemiluminescence kit (Amersham Pharmacia) usingaffinity-purified antibodies against Srp1 or Kap95 andhorseradish-peroxidase-conjugated goat anti-rabbit IgG(Sigma) as secondary antibody.

Cell culture and microscopy

Yeast cell cultures were grown at 30 °C in media lackinguracil or leucine with 2% raffinose to a density of 107 cells/ml. Temperature-sensitive strains were cultured at 25 °C.All cells examined by fluorescence microscopy were alsoanalyzed by immunoblotting with anti-GFP antibodies toshow that fusion proteins of the expected size had beensynthesized. For localization studies, we used the follow-ing strains: wild-type GSY154,68 srp1–31 MATα ura3 leu2trp1 his3 ade2 (GSY202),53 srp1–49MATα ura3 leu2 trp1 his3ade2 (GSY203),53 rsl1-1 (GSY468),50 and Δnup2 (JLY506/GSY432).76 The strain srp1–54 MATa ura3 leu2 his3 trp1ade2 can1 (NOY672/GSY664) was a kind gift of Dr.Masayasu Nomura (University of California, Irvine). Weemployed only Impα mutants with chromosomal allelesof srp1–31, srp1–49, and srp1–54 because plasmid-contain-ing mutants display a weaker phenotype. The cells wereharvested 2 h after the addition of 2% galactose. Indirectimmunofluorescence microscopy using affinity-purifiedpolyclonal antibodies against GFP and fluorescein iso-thiocyanate (FITC)-labeled secondary antibodies, andstaining of the cells with the DNA dye DAPI wereperformed as described before.22,73

Peptide spot method

For radioactive labeling, 20 μg of Srp1ΔIBB was dilutedinto 180 μl of 100mM sodium phosphate (pH 7.4) solution.Then, 10 μl of 1% sodium cyanoborohydride solution and10 μl of [14C]formaldehyde (1 mCi/ml; Amersham

Pharmacia) were added.77 After incubation for 30 min at30 °C, the protein was gel-filtrated using a NAP5 column(Amersham Pharmacia). Oligopeptides were synthesizedusing an Auto-Spot Robot ASP222 (Abimed) on thesurface of cellulose membranes.55 The peptides werecoupled to the membrane by their C-terminal ends to β-alanine as the primary linker. The membranes wereincubated with binding buffer (2% bovine serum albumin(BSA), 25 mM sodium phosphate, 650 mM NaCl, 3 mMKCl, and 1 mM MgCl2, pH 7.3) for 30 min at 4 °C,incubated in binding buffer supplemented with 40 μl of[14C]Srp1ΔIBB for 30 min at 4 °C, washed three times withbinding buffer, and washed twice with washing buffer(blocking buffer without BSA). The membranes were thendried and exposed to X-ray films.

SPR spectrometry

SPR measurements were carried out with a BIAlitebiosensor and the GST kit for fusion capture in accordancewith the guidelines of BIAcore. A goat monoclonal anti-GSTantibody was covalently coupled onto a CM5 chip viaamino groups. The concentration of the GST fusionproteins (ligands) during the binding reaction to thesensor chip was approximately 0.5 mg/ml. The proteinconcentration was determined by measuring the absor-bance at 280 nm and by calibration with a BSA standard.The reference flow chamber was loaded with recombinantGST at a concentration of 0.2 mg/ml. The various ligandswere immobilized such that 100–300 resonance units wereregistered. The analytes (Impα, ImpαΔIBB, andImpα+Impβ preincubated for 30 min at 4 °C at equimolarconcentrations) were diluted into PBSKMT buffer, whichalso served as washing buffer. The data were analyzedwith the BIAcore X and BIAevaluation 3.1 softwareprograms based on the Longmuir 1:1 model. After datacollection, the chips were regenerated with 0.2 M glycine(pH 2.2).

Acknowledgements

We thank Wolfgang Nastainczyk for preparationof the peptide spot membranes, and Sandra Helfen,Karsten Mayr, and Silke Guthörl for expert technicalassistance. We thank Masayasu Nomura (Universityof California, Irvine) for the mutant strain contain-ing the genomically integrated srp1–54 allele. Thiswork was supported by the Deutsche Forschungs-gemeinschaft.

Supplementary Data

Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2008.04.038

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