Structural Basis for Cell-Cycle-Dependent Nuclear Import Mediated by the Karyopherin Kap121p

Structural Basis for Cell-Cycle-Dependent Nuclear Import Mediated by the Karyopherin Kap121p

Junya Kobayashi1 and Yoshiyuki Matsuura1,2

1 - Division of Biological Science, Graduate School of Science, Nagoya University, Japan 2 - Structural Biology Research Center, Graduate School of Science, Nagoya University, Japan

Correspondence to Yoshiyuki Matsuura: [email protected] http://dx.doi.org/10.1016/j.jmb.2013.02.035 Edited by R. Huber

Abstract Kap121p (also known as Pse1p) is an essential karyopherin that mediates nuclear import of a plethora of cargoes including cell cycle regulators, transcription factors, and ribosomal proteins in Saccharomyces cerevisiae. It has been proposed that the spindle assembly checkpoint signaling triggers molecular rearrangements of nuclear pore complexes and thereby arrests Kap121p-mediated nuclear import at metaphase, while leaving import mediated by other karyopherins unaffected. The Kap121p-specific import inhibition is required for normal progression through mitosis. To understand the structural basis for Kap121p-mediated nuclear import and its unique regulatory mechanism during mitosis, we determined crystal structures of Kap121p in isolation and also in complex with either its import cargoes or nucleoporin Nup53p or RanGTP. Kap121p has a superhelical structure composed of 24 HEAT repeats. The structures of Kap121p–cargo complexes define a non-conventional nuclear localization signal (NLS) that has a consensus sequence of KV/IxKx1-2K/H/R. The structure of Kap121p–Nup53p complex shows that cargo and Nup53p compete for the same high-affinity binding site, explaining how Nup53p binding forces cargo release when the Kap121p-binding site of Nup53p is exposed during mitosis. Comparison of the NLS and RanGTP complexes reveals that RanGTP binding not only occludes the cargobinding site but also forces Kap121p into a conformation that is incompatible with NLS recognition. © 2013 Elsevier Ltd. All rights reserved.

Legend: The crystal structure of the karyopherin Kap121p bound to its nuclear import cargo provides mechanistic insights into how Kap121p recognizes a non-conventional nuclear localization signal. This advances understanding of transport carrier–cargo interaction in general.

0022-2836/$ - see front matter © 2013 Elsevier Ltd. All rights reserved.

J. Mol. Biol. (2013) 425, 1852–1868

Kap121p-Mediated Nuclear Import and its Regulation

Introduction In all eukaryotes, precise regulation of the bidirectional transport of macromolecules between the nucleus and the cytoplasm is an essential aspect of many cellular processes, including gene expression, signal transduction, and cell cycle progression. Nuclear transport occurs through nuclear pore complexes (NPCs) that form aqueous channels across the nuclear envelope. 1 In Saccharomyces cerevisiae, NPCs are built from approximately 30 different proteins collectively called nucleoporins (Nups) with a combined mass of 50 MDa. 2 NPCs pose permeability barriers that prevent diffusion of inert objects N 5 nm in diameter 3 and yet mediate active transport of large macromolecules. The Nups can be roughly classified into those forming the structure of the pore and those mediating transport through the NPC. The active transport pathways through the NPCs are mediated by soluble transport receptors, most of which belong to the karyopherin-β (Kapβ) superfamily. There are more than 20 Kapβs in human cells and 14 in budding yeast. Each Kapβ recognizes a unique set of proteins or RNAs, thus creating multiple nuclear transport pathways. Karyopherins responsible for importing cargoes are generally called importins, and exporters are called exportins. The direction of transport for each karyopherin is dictated by its differential interactions with cargoes and the small GTPase Ran. 1 Like other Ras-family GTPases, Ran cycles between GTP- and GDPbound states. 4 In the nucleus, Ran is kept primarily in its GTP-bound form by the Ran guanine nucleotide exchange factor (RCC1) that is bound to chromatin. In contrast, the Ran GTPase-activating protein (RanGAP) is localized to the cytoplasm, and Ran in the cytoplasm is primarily in the GDP-bound form. This RanGDP–RanGTP gradient from the cytoplasm to the nucleus determines the directionality of transport mediated by importins and exportins. Importins bind their cargoes in the cytoplasm and release cargoes in the nucleus upon RanGTP binding. By contrast, exportin binding to their cargoes is stabilized in the nucleus by the formation of a trimeric complex that includes RanGTP, and the ternary export complex is disassembled in the cytoplasm, where Ran GTPase is activated. Proteins synthesized in the cytoplasm that are destined for the nucleus carry targeting signals, generally termed nuclear localization signals (NLSs), that are recognized by importins. The mechanisms of NLS recognition and RanGTP-induced cargo release have been structurally characterized for import pathways mediated by Kapβs such as Kapβ1 (importin-β) and Kapβ2 (transportin). 5–8 The NLSs can be either short stretch of peptide sequence or folded domains. There are two types of well-characterized NLSs that belong to the former

1853 category: classical NLSs (cNLSs) and PY-NLSs. The cNLSs contain either one (monopartite cNLS) or two (bipartite cNLS) clusters of positively charged amino acids that are recognized by Kapα, which is an adaptor for Kapβ1. 9,10 The PY-NLSs are recognized by Kapβ2. The PY-NLSs are larger and more complex than cNLSs and have weak consensus motifs composed of a loose N-terminal hydrophobic or basic motif and a C-terminal RX2-5PY motif. 11 The NLSs for many other Kapβs remain uncharacterized and have yet to be discovered. The various nuclear transport pathways can be differentially regulated by diverse range of mechanisms, such as the modulation of the accessibility and affinity of target signal recognition by Kapβs, with phosphorylation/dephosphorylation of cargoes as a major mechanism. 12 Altering the structure of NPCs is emerging as another important mechanism to control nuclear transport, particularly in the context of cell cycle progression. In budding yeast, the potential for NPC-regulated nuclear import was discovered in the context of an essential karyopherin Kap121p and a nucleoporin Nup53p. 13 Kap121p (also known as Pse1p) mediates nuclear import of a range of cargoes including cell cycle regulators, transcription factors, and ribosomal proteins. 14–22 In each of these cases, Kap121p binds to a nonconventional NLSs rich in lysine residues, although diversity of NLS sequences and lengths precludes definition of clear consensus sequence. 22 The Kap121p-mediated nuclear import is inhibited during mitosis. In contrast to the FG repeats present on numerous Nups, which bind somewhat indiscriminately to multiple Kapβs with low affinity, Nup53p contains a segment that binds specifically and with high affinity to Kap121p. Previous biochemical and cell biological studies suggested that, during interphase, Nup170p occludes the Kap121p-binding segment of Nup53p but, during mitosis, molecular rearrangements of the NPC alter the interactions of Nup53p with its neighbors, exposing the Kap121pbinding site on Nup53p. 13 This allows Nup53p to bind Kap121p specifically during mitosis, slowing its movement through the NPC and inducing cargo release, while leaving import mediated by other importins unaffected. 13 The M-phase-specific regulation of Kap121p-mediated nuclear import is required for normal progression through mitosis. The inhibition of Kap121p-mediated transport appears to be restricted to periods of mitosis prior to chromosome segregation, and interestingly, the inhibition of Kap121p-mediated nuclear import is triggered by spindle assembly checkpoint signaling, indicating that the regulation of Kap121p-mediated import contributes to control the nuclear environment of the spindle. 23 Here we report X-ray crystal structures of Kap121p in isolation and also in complex with either its import cargoes or Nup53p or RanGTP that provide compre-

1854

Kap121p-Mediated Nuclear Import and its Regulation

hensive structural delineation of the unique features of the NLS binding to Kap121p and its regulatory mechanisms. The structures of Kap121p–cargo complexes define a novel Kap121p-specific NLS that is distinct from cNLS or PY-NLS and identify an NLS-binding site on the inner surface of Kap121p. Moreover, the structure of Kap121p–Nup53p complex provides evidence that Nup53p directly competes with cargo for binding to Kap121p during mitosis. Finally, the structure of Kap121p–RanGTP complex shows how RanGTP displaces cargoes in the terminal step of Kap121p-mediated nuclear import.

Results and Discussion Structure determination Although our initial attempts to crystallize Kap121p–cargo complexes and Kap121p–Nup53p complex were unsuccessful, we obtained diffractionquality crystals of Kap121p–RanGTP complex and solved the structure at 2.7 Å resolution by combination of molecular replacement and selenomethionine (SeMet) single-wavelength anomalous dispersion (SAD) phasing. This structure allowed us to identify

Table 1. Crystallographic data and refinement statistics Crystal Unliganded Kap121p (wild type)

Unliganded Kap121p (mutant 7m)a

Unliganded Kap121p (mutant 3K)b

Kap121p bound to Ste12p

Kap121p bound to Pho4p

Kap121p bound to Nup53p

Kap121p bound to RanGTP

SPring-8, BL41XU 1.00000 P21

SPring-8, BL41XU 1.00000 P21

SPring-8, BL41XU 1.00000 P21

PF, BL-17A

PF, BL-5A

0.98000 P212121

SPring-8, BL41XU 1.00000 P212121

1.00000 P212121

SPring-8, BL41XU 0.97897 P3221

77.77 124.39 85.18 90.00 117.02 90.00 31.10–2.90 (3.06–2.90) 0.080 (0.66) 10.3 (2.1) 0.998 (0.692)

77.38 124.20 85.03 90.00 116.73 90.00 26.55–2.60 (2.70–2.60) 0.062 (0.72) 11.7 (1.8) 0.998 (0.586)

77.79 122.31 84.67 90.00 116.08 90.00 25.55–3.20 (3.42–3.20) 0.092 (0.50) 8.3 (2.3) 0.996 (0.794)

77.57 126.16 130.78 90.00 90.00 90.00 39.18–2.20 (2.25–2.20) 0.083 (0.76) 11.5 (1.7) 0.998 (0.608)

78.09 126.31 128.05 90.00 90.00 90.00 29.97–2.90 (3.08–2.90) 0.13 (0.56) 7.2 (2.3) 0.992 (0.729)

78.33 131.44 131.50 90.00 90.00 90.00 30.99–2.80 (2.94–2.80) 0.11 (0.64) 9.0 (1.7) 0.997 (0.689)

97.75 97.75 289.98 90.00 90.00 120.00 48.77–2.70 (2.79–2.70) 0.087 (0.92) 11.0 (1.9) 0.998 (0.609)

30,302 20.12 24.74

40,914 22.18 26.74

22,284 20.28 25.19

59,696 21.53 25.62

26,917 22.43 26.82

30,646 25.08 29.67

42,804 26.06 29.78

7845 0 0 0

8088 0 0 0

7841 0 0 0

7938 101 0 0

7849 0 0 0

7862 0 0 0

9109 4 32 1

0.013 1.8

0.013 1.8

0.010 1.5

0.016 1.9

0.011 1.5

0.011 1.6

0.013 1.6

96.8 0 3W3T

96.2 0 3W3U

96.7 0 3W3V

95.7 0 3W3W

96.3 0 3W3X

96.0 0 3W3Y

96.5 0 3W3Z

Data collection X-ray source Wavelength (Å) Space group Unit cell parameters a (Å) b (Å) c (Å) α (°) β (°) γ (°) Resolution (Å)c

Rmergec Mean I/σ(I)c Mean I half-set correlation CC(1/2)c 99.6 (99.7) 97.7 (84.3) 99.6 (99.8) 95.9 (75.5) 98.9 (96.9) 95.2 (79.2) 99.7 (100.0) Completeness (%)c 3.7 (3.7) 3.8 (3.4) 3.3 (3.3) 5.8 (3.9) 4.5 (3.7) 5.8 (3.5) 4.3 (3.3) Multiplicityc 119,358 161,850 78,232 365,367 126,735 187,468 365,031 No. of measured (17,024) (12,915) (13,974) (13,205) (16,507) (11,837) (27,505) reflectionsc 31,943 (4660) 43,084 (3838) 23,498 (4250) 62,953 (3397) 28,382 (4408) 32,341 (3419) 45,100 (4388) No. of unique reflectionsc Refinement No. of used reflections Rwork (%) Rfree (%) No. of atoms Protein Water GTP Mg2+ rmsd Bond lengths (Å) Bond angles (°) Ramachandran plotd (%) Favored Outliers Protein Data Bank code a b c d

Mutant 7m refers to the R349A/Q350A/D353A/E396A/N430K/D438A/N477A mutant of Kap121p. Mutant 3K refers to the D353K/E396K/D438K mutant of Kap121p. Values for the highest-resolution shell are shown in parenthesis. As defined by MolProbity.24

1855

Kap121p-Mediated Nuclear Import and its Regulation

flexible regions of Kap121p and optimize the Kap121p construct for crystallization in complex with cargoes (Ste12p and Pho4p) or Nup53p, by deleting a flexible loop (residues 80–90) at the Nterminal region of Kap121p. The deletion mutant [Kap121p (Δ80–90)] complemented wild-type Kap121p in S. cerevisiae and, thus, retained the essential functions of Kap121p in vivo (Fig. S1a). Consistently, Kap121p (Δ80–90) was also functional in vitro and retained the ability to bind cargoes, Nup53p, and RanGTP (Fig. S1b). We obtained orthorhombic crystals of Kap121p (Δ80–90) bound to the Kap121p-binding domain of Ste12p, Pho4p, and Nup53p and determined the structures by molecular replacement at 2.2 Å, 2.9 Å, and 2.8 Å resolution, respectively. We also obtained monoclinic crystals of unliganded Kap121p (Δ80–90) and determined the structure by molecular replacement at 2.9 Å resolution. All of the structures had excellent stereochemistry and contained no Ramachandran outliers (Table 1). Overall structure of Kap121p The structure of unliganded Kap121p is described first (Fig. 1) as a reference to examine the effects of the binding of different partners. As commonly observed for the Kapβ family members, Kap121p is constructed from 24 tandem HEAT repeats

(a)

N

arranged into a right-handed superhelical solenoid (Fig. 1a). Each of the HEAT repeats consists of two antiparallel α-helices connected by loops of varying length. The folding topology of Kap121p is illustrated schematically in Fig. 1b. The repeats pack side by side in a parallel fashion, generally with a righthanded twist that generates a superhelical molecule with the A-helices forming outer convex surface whereas the B-helices form the inner concave surface. The regular right-handed twist between the HEAT repeats is interrupted at a prominent lefthanded twist between HEATs 8 and 9, which is stabilized by an acidic loop (connecting the helices of HEAT 8 and so referred to as H8 loop) packing against HEATs 9–12. Other deviations are at the first and the last HEAT repeats, which pack against the adjacent repeat in an almost perpendicular fashion to cap the N- and C-terminal ends of the superhelix. A unique feature of Kap121p, not observed in the other Kapβs, is an insertion between the helices of HEAT 15 (H15 insert), which is folded into a small protruding globular domain consisting of threestranded β-sheet flanked on one side by a distorted helix. In the unliganded Kap121p, the H15 insert is located close to the inner surface of HEAT 2, and two acidic residues of the H15 insert (Glu642 and Glu644) approach Arg72 and Lys73 of HEAT 2, making electrostatic interactions. Another uniquely long insert is found in HEAT 18. A long loop

H15 insert H8 loop

Kap121p 10A

H18 loop H8 loop

N 1A

C

1B

H15 insert

90

(b)

Left handed twist

Fig. 1. Structure of Kap121p. (a) Two orthogonal views, showing an overview of unliganded Kap121p. The A- and B-helices of each HEAT repeat are represented by orange and green cylinders, respectively. The H8 loop, H15 insert, and H18 loop are highlighted in blue, red, and black, respectively. (b) Schematic illustration of Kap121p structure and domain organization. Color coding is the same as in (a).

1856

Kap121p-Mediated Nuclear Import and its Regulation

(a) N

(b) N

p p Kap121p

(c) N

Kap121p

Kap121p

115

C

C

C

K409

K146

K611 H614

V144

K143

I609

K148 N150

K616

V407

K608 T145 S610

S606

A607

P612 L613

Ste12p

S408

T617 I615

S411

K406

N142

N147 A141

N410

S149

T412

Pho4p

Nup53p

F405

Fig. 2. Overall structures of Kap121p bound to (a) Ste12p, (b) Pho4p, and (c) Nup53p. The 2Fo − Fc electron density map for each ligand contoured at 1.5 σ is superposed and is enlarged at the lower panel.

connecting the helices of HEAT 18 (H18 loop) extends toward the N-terminal arch of Kap121p and five residues in the loop (residues 817–821)

(a)

Kap121p-Ste12p complex

(b)

packs intimately against HEATs 5–7. These interactions between the N- and C-terminal halves of Kap121p via two long inserts (H15 insert and H18

(c)

Kap121p-Pho4p complex

Kap121p-Nup53p complex E480

E480 E480

E396 N477

E396

K608

Q434 K611 G433

A474

A474 K143

D353

V14 4 H470

Q512

H470

10B

11B

9B

8B

12B

Q434

K409

A474 V40 C4297 Q512

9B

11B

Q350

N147

10B

C429 N430 K556

K406

S393

S392

H614

R349

D353 K148

I609

Q512

E396 N477

Q434 K146

N477

D338 H8 loop

8B

Q350 N430 N410

H470

10B 12B 11B

H8 loop

D338

9B

8B

K406 K608

K611

K409

K146

K143 K614

K148

Nup53p

Ste12p V144 I609

V407 N410

Pho4p N147

Fig. 3. Details of the Kap121p–ligand interactions at the NLS-binding site. (a) Kap121p–Ste12p complex. (b) Kap121p– Pho4p complex. (c) Kap121p–Nup53p complex. In the upper panel, a ball-and-stick model of each ligand is shown together with ribbon model of Kap121p, with key residues of Kap121p at the interface being shown as stick models. Broken lines indicate hydrogen bonds or salt bridges. In the lower panel, the electrostatic surface potential of Kap121p is shown together with the bound ligand represented in a ball-and-stick model. Solvent-accessible surface of Kap121p is colored by electrostatic potential: positively charged regions are colored blue, negatively charged regions are colored red, and neutral regions are colored white.

Kap121p-Mediated Nuclear Import and its Regulation Fig. 4. Schematic illustrations of Kap121p interactions with ligands. (a) Kap121p–Ste12p complex. (b) Kap121p–Pho4p complex. (c) Kap121p–Nup53p complex.

1857

1858

Kap121p-Mediated Nuclear Import and its Regulation

loop) may help stabilize the overall architecture of unliganded Kap121p.

or salt bridges with acidic residues of Kap121p (Asp353, Glu396, and Asp438). In addition to the side-chain interactions, the main-chain NH and CO groups of the Ste12p NLS make multiple hydrogen bonds either directly or via water molecules to the surface hydrophilic side chains of Kap121p (Arg349, Gln350, Asp353, Asn477, and Gln512). In particular, the side chain of Asn477 Kap121p forms hydrogen bonds to both of the main-chain NH and CO groups of Ile609 Ste12p, holding this residue at the appropriate position so that the side chain of Ile609 Ste12p fits into the nonpolar pocket. Thus, the Ste12p NLS is recognized by Kap121p through multiple hydrogen bonds, electrostatic interactions, hydrophobic interactions, and van der Waals contacts. The structure of Kap121p–Pho4p complex (Fig. 2b) shows that both Ste12p NLS and Pho4p NLS (residues 141–150) bind to the same NLSbinding site on Kap121p through similar interactions (Figs. 3b and 4b), burying a similar surface area (720 Å 2 for Ste12p; 678 Å 2 for Pho4p). The hydrophobic side chain of Val144 Pho4p fits into the nonpolar pocket on Kap121p in exactly the same way as Ile609 Ste12p does, and three basic residues of Pho4p NLS (Lys143, Lys146, and Lys148) interact with acidic residues of Kap121p, just like Lys608, Lys611, and His614 of Ste12p, respectively.

kDa 120 90

(c)

kDa 120 90

Kap121p

V144W, K146E

KV/IxKx1-2K/H/R

Wild type

Consensus sequence of Kap121p-specific NLS

I609W, K611E

(b) V603W, K605E

(a)

Wild type

In the Kap121p–Ste12p complex, residues 606– 617 of Ste12p bind in an extended conformation to line the concave surface of the central region of Kap121p (HEATs 7–12) (Fig. 2a). The structure of Kap121p in this complex is essentially identical with that of unliganded Kap121p (C α rmsd of 1.8 Å), and thus, the binding of Ste12p NLS is not associated with major conformational changes of Kap121p. The main chain of Ste12p runs in the opposite direction to that of Kap121p, reminiscent of Kapβ2–cargo complexes. 11,25 Figure 3 shows the details of NLS recognition and Fig. 4 illustrates the interactions schematically. Tracing from the N-terminus of Ste12p, Lys608 Ste12p makes a salt bridge with Glu480 Kap121p (Figs. 3a and 4a). The hydrophobic side chain of the next residue (Ile609 Ste12p) fits into an electrostatically neutral pocket formed by Kap121p residues (Asn430, Gly433, Gln434, His470, Ala473, and Ala474) on the B-helices of HEATs 10 and 11. In the C-terminal half of the Ste12p NLS, the side chains of two basic residues (Lys611 and His614) form multiple hydrogen bonds

V603W, K605E, K608E, I609W, K611E V603A, K605A, K608A, I609A, K611A

Mechanism of recognition of Kap121p-specific NLSs

Kap121p

70

70 50

GST-Ste12p (581-688)

50

(d)

Ste12p (V603W, K605E, K608E, Ste12p (wild- I609W, K611E)GFP2 type)-GFP2

Pho4p (wildtype)-GFP2

GST-Pho4p (101-200)

Pho4p (V144W, K146E)-GFP2

Fig. 5. Mutational analysis verified the contributions made by conserved residues of NLS. (a) A multiple alignment of Kap121p-specific NLS sequences. The consensus sequence is shown above the sequence alignment. (b) A pull-down assay using Ste12p mutants. Immobilized 8.5 μg GST-Ste12p (residues 581–688) was incubated with 16 μg Kap121p. (c) A pull-down assay using Pho4p mutants. Immobilized 8.4 μg GST-Pho4p (residues 101–200) was incubated with 16 μg Kap121p. (d) In vivo localization assay. The localization of NLS-GFP-GFP fusion proteins in yeast cells arrested in S phase was monitored by GFP fluorescence. The mutations of key residues of NLSs severely impaired nuclear import of cargoes.

1859

Kap121p-Mediated Nuclear Import and its Regulation

The interactions between Kap121p and the main chain of Pho4p are also similar to those between Kap121p and Ste12p. The Pho4p NLS makes additional interactions with Kap121p, which are formed by Asn142 Pho4p that makes hydrogen bonds with Gln512 Kap121p and Lys556 Kap121p and Asn147 Pho4p that is hydrogen bonded to Asp338 Kap121p and Gln350 Kap121p. Thus, Asp338 Kap121p, which is one of the acidic residues in the acidic H8 loop, is involved in Pho4p NLS recognition, although not involved in the case of Ste12p NLS recognition. As was the case with Ste12p NLS, the binding of Pho4p NLS is not associated with major conformational changes of Kap121p (C α rmsd of 1.6 Å when Kap121p–Pho4p complex is compared to unliganded Kap121p). A sequence alignment shows that the amino acid sequence of human importin-5 (also known as Kapβ3 or RanBP5) is similar to yeast Kap121p (65.2% similarity and 28.3% identity) (Fig. S2). Interestingly, the residues involved in the NLS recognition in Kap121p are highly conserved between Kap121p and importin-5 (Fig. S2). This is consistent with the observation that, like Kap121p, importin-5 mediates nuclear import of ribosomal proteins and core histones. 26–29 By contrast, some of the key residues in the NLS-binding site are not conserved in Kap123p, which is the major importer for many ribosomal proteins and histones H3 and H4 in yeast. 14,30,31 Although it is known that the

(a)

Kap123p import pathway overlaps with the Kap121p pathway, the divergent sequences indicate diversities in the cargo specificities and NLS recognition mechanisms, which might account for the fact that Kap121p is essential but Kap123p is not essential in yeast. 14 Mutational analyses of Kap121p–NLS interactions The similarities in the NLS recognition of the two cargoes (Ste12p and Pho4p) and the sequence alignment of Kap121p-specific cargoes suggest a consensus sequence of KV/IxKx1-2K/H/R, where x can be any amino acid (Fig. 5a). To evaluate the contributions made by the conserved NLS residues (one hydrophobic residue and three basic residues), we engineered NLS mutants and analyzed their binding to Kap121p by pull-down assays. A double mutant (I609W/K611E) of Ste12p was made to substitute Ile609 with much bulkier tryptophan that is too large to be accommodated in the nonpolar pocket of the NLS-binding site and also to reverse the charge of Lys611 to disrupt the electrostatic interactions with acidic residues of Kap121p. These double mutations weakened the binding to Kap121p as expected (Fig. 5b), but surprisingly, the mutational effects were not as dramatic as would be expected from the drastic changes of amino acid properties. We therefore re-examined the Ste12p sequence and

(c)

kDa 120 90

Kap121p

vector Kap121p wt

70 50

GST-Ste12p (581-688)

Kap121p K 121 3K Kap121p 7m

SC-UL (control)

SC-L + 5FOA(0.5 mg/ml)

(b) kDa

1 2 3 4 5

120 90 70

6 Kap121p GST-Pho4p

1: wild type 2: D353K 3: D353K, E396K 4: D353K, E396K, D438K : 3K 5: D353A, E396A, D438A 6: R349A, Q350A, D353A, E396A, N430K, D438A, N477A :7m

Fig. 6. Mutational analysis verified the contributions made by key residues of Kap121p at the NLS-binding site. (a and b) Pull-down assays using Kap121p mutants. Immobilized 8.5 μg GST-Ste12p (residues 581–688) or 8.4 μg GST-Pho4p (full length) was incubated with 16 μg Kap121p (wild type or mutant). (c) A plasmid shuffle assay. S. cerevisiae cells deleted for the endogenous Kap121p (ΔKAP121) but containing a Kap121p maintenance plasmid were transformed with vector alone (negative control) or a plasmid encoding wild-type Kap121p (positive control) or Kap121p mutant. Cells were plated on control plates or on plates containing the drug 5-FOA and grown at 30 °C.

1860 found that residues 602–608 of Ste12p (602SVGKSAK-608), which is immediately upstream of the NLS residues (608-KISKPLH-614) identified from crystallographic electron density map, conform to the Kap121p-specific consensus NLS sequence motif except for the first residue, which is not lysine as found in the crystallographically identified NLS. We hypothesized that Ste12p has two redundant NLSs (residues 602–608, NLS1; residues 608–614, NLS2), either of which can similarly bind the NLSbinding site (although NLS2 binds more strongly than NLS1), and that this is the reason why only the two mutations (I609W and K611E) were not sufficient to abolish the binding of Ste12p to Kap121p. To test this idea, we engineered a Ste12p mutant in which additional three mutations (V603W/K605E/ K608E) were introduced. The five mutations (V603W/K605E/K608E/I609W/K611E) abolished the Kap121p binding as expected, supporting the idea that both NLS1 and NLS2 contribute to Kap121p binding (Fig. 5b). The observation that V603W/K605E mutations alone were not effective in weakening the Kap121p binding (Fig. 5b) supports the idea that NLS2, but not NLS1, is the major binding site and verifies the interpretation of electron density map observed in the co-crystal. A milder mutant of Ste12p, in which the five residues (Val603, Lys605, Lys608, Ile609, and Lys611) were mutated into alanine, also showed undetectable binding to Kap121p (Fig. 5b). We also examined the importance of the key residues in Pho4p NLS and found that double mutations (V144W/K146E) of Pho4p dramatically weaken the binding to Kap121p, firmly supporting the crystal structure (Fig. 5c). We used the structure-based mutants of Ste12p and Pho4p to evaluate how NLS mutations affect nuclear import in vivo by observing the localization of NLS-GFP (green fluorescent protein) fusions in living yeast cells (Fig. 5d). Consistent with the in vitro binding assays, the five mutations (V603W/ K605E/K608E/I609W/K611E) of Ste12p and two mutations (V144W/K146E) of Pho4p severely impaired nuclear import of the NLS-GFP fusions in S. cerevisiae cells under the conditions (cells arrested in S phase) in which Kap121p normally imports wildtype NLS cargoes (Fig. 5d). We also verified functional significance of the Kap121p–NLS interactions by engineering Kap121p mutants in which key residues involved in the NLS recognition are mutated (Fig. 6). In a pull-down assay, reverse-charge mutations (D353K/E396K/ D438K) of Kap121p abolished the binding to both Ste12p and Pho4p, confirming the crucial role played by the interactions between these acidic residues of Kap121p and the conserved basic residues of NLSs (Fig. 6a and b). The mutational effects in weakening Ste12p binding became severer as progressively more charge reversals were introduced, indicating that all of the three residues

Kap121p-Mediated Nuclear Import and its Regulation

(Asp353, Glu396, and Asp438) contribute to NLS recognition (Fig. 6a). Milder triple mutations (D353A/ E396A/D438A) were less effective in weakening Ste12p binding, but the binding was abolished by introducing additional four mutations (R349A/ Q350A/N430K/N477A) into Kap121p (Fig. 6a). This confirms that not only the acidic residues but also other residues at the NLS-binding site are important for NLS recognition. Furthermore, we used the mutants of Kap121p to see how the mutations affect the essential function of Kap121p required for viability of yeast cells (Fig. 6c). Consistent with the results of in vitro binding assay, two mutants of Kap121p (D353K/E396K/D438K, referred to as mutant 3K, and R349A/Q350A/D353A/E396A/ N430K/D438A/N477A, referred to as mutant 7m) showed lethal phenotype in vivo and did not complement wild-type Kap121p in yeast (Fig. 6c). However, to advance our understanding of the structure–function relationship of Kap121p, it was important to establish that the loss of function of Kap121p was due directly to the changes introduced into the residues mutated and had not arisen indirectly through the mutations producing a more global change in the structure of Kap121p. We therefore crystallized the mutants 3K and 7m and determined the structures by molecular replacement at 3.2 Å and 2.6 Å resolution, respectively. The crystals of the mutants were isomorphous to that of wild type, and the structures showed that the mutations introduced negligible changes in the structure of Kap121p (Fig. S3). The overall C α rmsd was 0.38 Å and 0.28 Å, when the structure of wild-type Kap121p was superposed with the structures of mutants 3K and 7m, respectively. Evidence for direct competition between Nup53p and import cargoes for the binding to Kap121p The structure of Kap121p–Nup53p complex showed that a pseudo-NLS sequence of Nup53p (residues 405–412) binds to the NLS-binding site of Kap121p (Fig. 2c), although the NLS-like sequence of Nup53p lacks the basic residue at the C-terminus of the consensus NLS sequence (Fig. 5a). The binding of Nup53p to Kap121p is very similar to that of Pho4p. Lys406, Val407, Lys409, and Asn410 of Nup53p bind to Kap121p in the same manner as Lys143, Val144, Lys146, and Asn147 of Pho4p bind to Kap121p (Figs. 3c and 4c). The main chains and side chains of Ste12p, Pho4p, and Nup53p when bound to Kap121p superpose well (Fig. 7a). The structure of Kap121p complexed with Nup53p is essentially identical with that of unliganded Kap121p (overall C α rmsd of 1.8 Å), as was the case with the NLS complexes. Mutational analyses verified the contributions made by key residues (Fig. 7b). Triple mutations (K406A/V407A/K409A) of Nup53p weakened the binding to Kap121p, and more drastic

1861

Kap121p-Mediated Nuclear Import and its Regulation

(a)

Lysine

Lysine Basic amino acid

W, K409E K406E, V407W

kDa

Wild type

(b)

A, K409A K406A, V407A

Valine/Isoleucine

120 90

Asparagine

(c)

Ste12p Pho4p Nup53p

cytoplasm cargo

cargo

Kap121p Kap121p

70

Kap121p 50

GST-Nup5 3p (401-448)

30

NPC

Fig. 7. Nup53p directly competes with NLSs for binding to the same site on Kap121p. (a) A superposition of Ste12p NLS, Pho4p NLS, and Nup53p bound to Kap121p. (b) A pull-down assay. Immobilized 9.4 μg GST-Nup53p (residues 401–448, either wild type or mutant) was incubated with 16 μg Kap121p. (c) A schematic model illustrating the competition between NLS and Nup53p during mitosis.

mutations (K406E/V407W/K409E) abolished the binding (Fig. 7b). Thus, the structural and biochemical data suggest that Nup53p competes with cargoes for the binding to the same site on Kap121p (Fig. 7c). We used surface plasmon resonance to analyze the ligand binding to Kap121p quantitatively. An analysis of the binding curves using a simple 1:1 binding model yielded an apparent KD = 3.0 × 10 − 8 M for Pho4p and 4.2 × 10 − 8 M for Nup53p (Table 2). Thus, the Kap121p-specific NLS and Nup53p bind to Kap121p with comparable affinity, suggesting that the binding of Nup53p is strong

enough to displace cargoes when the pseudo-NLS sequence of Nup53p is exposed during mitosis due to molecular rearrangements of the NPC. The binding data of Ste12p fitted poorly to the simple 1:1 binding model and we were unable to estimate a reliable value of KD, presumably because Ste12p has two redundant NLSs. The KD values in the nanomolar range are commonly observed for binding of functional NLSs to their import receptors, 25,32 suggesting that the nanomolar affinity is strong enough to maintain the integrity of importin–cargo complexes during the translocation across the NPC. Mechanism of RanGTP-mediated dissociation of cargoes from Kap121p

Table 2. Kinetic parameters of Kap121p interacting with ligands Analyte

Ligand

ka (M

−1

−1

s )

Kap121p Pho4p 8.8 × 104 Kap121p Nup53p 3.2 × 105

−1

kd (s )

KD (M)

χ

2

2.6 × 10−3 3.0 × 10− 8 2.4 1.3 × 10−2 4.2 × 10− 8 1.7

The binding of RanGTP to Kap121p in the nucleus is important to terminate nuclear import. Ran has a Ras-like fold with two switch regions (switch I and switch II) that interact with bound GTP and undergo conformational changes when GTP is hydrolyzed. 4

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Kap121p-Mediated Nuclear Import and its Regulation

(c) 6B

E244 K142

T284

E251

K141

P280 7B N143

(b) 1A 1B

2B L66

E31

R27

P22

T283 E287 R354 H347

3B

T62 I81

Y79

Q350 K121

Q82

N24

R140

K73 V111 R110

V45

E340

R161 E113

D77

V47

8B

Y147

H120

D346 K134 L357

K159

H8 loop

Switch II

Switch I

N

Kap121p

Switch II

(a)

130

K71 E644

N648

Q651

H15 insert

Y652

Mg2+

Y39 GTP

K38

RanGTP

Switch I

C Fig. 8. Structure of Kap121p–RanGTP complex. The overall structure is shown as a ribbon model, together with closeup views of the interactions between Kap121p (green) and RanGTP (pink). The views show the interactions at switch I (blue) (a), switch II (yellow) (b), and the basic patch (c) of Ran. GTP is shown as space-filling spheres.

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Kap121p-Mediated Nuclear Import and its Regulation

(a)

Kap121p 7B 6B

8B

9B 10B

11B 12B

Ste12p GTP

RanGTP

Kap121p-RanGTP Kap121p-Ste12p

(b)

H24

H1

H8 H9 H7 NLS binding site

H10 H11 H12

Fig. 9 (legend on next page)

1864 The structure of Kap121p–RanGTP complex provides structural explanation for how RanGTP promotes cargo dissociation in the nucleus (Fig. 8). In the Kap121p–RanGTP complex, Ran binds to the inner surface of the N-terminal half of Kap121p and is clamped by HEATs 1–8 on one side and the H15 insert (a small globular domain) on the other side. Both of the two switch regions of Ran are involved in the binding to Kap121p. The switch I interacts with the H15 insert through hydrogen bonds and van der Waals contacts between Ran (Gly20, Lys38, Tyr39, and Lys71) and Kap121p (Glu644, Asn648, Gln651, and Tyr652) (Fig. 8a). The switch II packs against the inner surface of HEATs 1 and 2 through numerous van der Waals contacts, hydrophobic interactions, and electrostatic interactions (Fig. 8b). These interactions are possible only when both switch regions adopt the GTP-bound conformation, explaining the ability of Kap121p to discriminate between GTP- and GDP-bound Ran. Ran makes additional contacts with the inner B-helices of HEAT 3 and HEATs 6–8, with the residues on the basic patch of Ran (Arg140, Lys142, and Lys159) making hydrogen bonds or electrostatic interactions with the acidic residues of Kap121p (Glu244, Glu248, Glu287, Glu340, and Gln350) (Fig. 8c). Thus, RanGTP is almost fully encircled by Kap121p. Importantly, the RanGTP-binding site partially overlaps with the NLS-binding site at HEATs 7 and 8, and RanGTP sterically clashes with NLS when the structures of Kap121p–RanGTP complex and Kap121p–NLS complex are superposed (Fig. 9a). This explains why the binding of NLS and RanGTP are mutually exclusive. Although the binding of NLSs to Kap121p does not require conformational changes of Kap121p, the binding of RanGTP is associated with “opening” of the Kap121p superhelix (Fig. 9b). The overall C α rmsd is as large as 13 Å when Kap121p–RanGTP complex is compared with Kap121p–Ste12p complex. Interestingly, the large-scale movement required to open up the space to accommodate RanGTP is caused by conformational changes along the central regions of Kap121p (HEATs 8– 12), where the NLS-binding site is located. The conformations of the other regions of Kap121p remain mostly unchanged, and the Kap121p Nterminal regions (HEATs 1–8) have a similar structure (C α rmsd is only 1.3 Å) and so do the Cterminal regions (HEATs 13–24; C α rmsd is only 1.4 Å) when the structures of RanGTP and Ste12p complexes are compared. Thus, the NLS-binding site formed on the inner surface of HEATs 7–12 is

Kap121p-Mediated Nuclear Import and its Regulation

the major site where conformational changes occur upon RanGTP binding. Taken together, the structure of Kap121p–RanGTP complex and its comparison with NLS complexes suggest that the RanGTPmediated dissociation of cargoes in the nucleus involves not only direct competition between Ran and NLS for the binding to the same site but also a global conformational change that locks Kap121p into a cargo-incompatible conformation. In summary, our structural data reveal the precise mechanisms of how Kap121p recognizes import cargoes, how Nup53p competes with cargoes during mitosis, and how RanGTP terminates nuclear import mediated by Kap121p. The proposed mechanisms are firmly supported by biochemical and cell biological analyses using structure-based mutants. As a short linear sequence motif of nuclear-targeting sequence, the Kap121p-specific NLS characterized in detail in this study is distinct from cNLS and PYNLS. Thus, this study has advanced understanding of the diversity of nuclear import pathways and how various pathways can be differentially regulated.

Materials and Methods Expression and purification of proteins for crystallization Standard techniques were used for the manipulation of recombinant DNA. 33 To prepare SeMet-substituted Kap121p in complex with native RanGTP for crystallization, we expressed glutathione S-transferase (GST)Kap121p (S. cerevisiae, full length) and His/S-Ran (canine, residues 1–176) separately from pGEX-TEV and pET30a-TEV, 34 respectively, in the Escherichia coli host strain BL21-CodonPlus(DE3)RIL (Stratagene). We truncated the C-terminus of Ran (residues 177–216) because it is absent or disordered in other Kapβ–RanGTP complexes 5,35 and also because truncation of the Cterminus stabilizes the GTP-bound form of Ran. 36 SeMetsubstituted GST-Kap121p was expressed in the E. coli cells grown in M9 minimal media and starved before addition of SeMet as previously described. 37 The two sets of cells were mixed, resuspended in buffer A [30 mM Tris– HCl (pH 7.5), 150 mM NaCl, 10 mM imidazole, 5 mM Mg(OAc)2, 0.1 mM GTP, 7 mM 2-mercaptoethanol, and 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF)], and lysed by sonication on ice. All subsequent purification steps were performed at 4 °C. Clarified lysates were loaded onto Ni-NTA resin (Novagen), washed with buffer A containing 25 mM imidazole, and eluted with buffer A containing 250 mM imidazole. Tween-20 was added to the eluate to a final concentration of 0.05%. After incubating the Ni-NTA eluate with glutathione-Sepharose 4B resin (GE Healthcare), we

Fig. 9. Mechanism of RanGTP-mediated dissociation of NLSs. (a) Overlay of the NLS and RanGTP complexes suggests that RanGTP sterically clashes with NLS. (b) The binding of RanGTP is associated with opening of the Kap121p superhelix and induces conformational changes of the NLS-binding site at HEATs 7–12. The centers of consecutive HEAT repeats are represented by spheres. Blue, Kap121p in the Kap121p–Ste12p complex; green, Kap121p in the Kap121p– RanGTP complex.

1865

Kap121p-Mediated Nuclear Import and its Regulation

washed the resin with buffer B [10 mM Tris–HCl (pH 7.5), 120 mM NaCl, 5 mM Mg(OAc)2, 0.05% Tween-20, and 2 mM 2-mercaptoethanol]. The GST- and His/S-tags were cleaved off Kap121p and Ran by incubating the resin overnight with His-TEV protease (0.2 mg/ml) in buffer B containing 0.1 mM GTP and 0.2 mM AEBSF. The Kap121p–Ran complex, released from the resin, was finally purified by gel filtration over Superdex200 (GE Healthcare) in buffer B without Tween-20. GTP was added to the gelfiltration fractions to a final concentration of 0.1 mM and the complex was concentrated to 15 mg/ml using a Millipore concentrator (molecular weight cutoff: 10,000). To prepare native Kap121p–Ste12p complex and native Kap121p–Nup53p complex for crystallization, we expressed GST-Kap121p (Δ80–90; S. cerevisiae, full length except that residues 80–90 are deleted), His/S-Ste12p (S. cerevisiae, residues 581–649), and His/S-Nup53p (S. cerevisiae, residues 401–448) separately from the E. coli host strain BL21-CodonPlus(DE3)RIL (Stratagene). The residues 80–90 of Kap121p correspond to a loop connecting HEATs 2 and 3, which has no electron density in Kap121p–RanGTP complex. Truncation of this loop was vital to obtain crystals of Kap121p–cargo complexes, Kap121p–Nup53p complex, and unliganded Kap121p. Kap121p–Ste12p complex and Kap121p–Nup53p complex were purified using the same protocol as described above for Kap121p–RanGTP complex, except that buffers without Mg(OAc)2 and GTP were used. To prepare unliganded Kap121p and Kap121p–Pho4p complex for crystallization, we expressed GST-Kap121p (Δ80–90) from the E. coli host strain BL21-CodonPlus(DE3)RIL (Stratagene). After harvesting, we resuspended the cells in buffer C (phosphate-buffered saline, 2 mM DTT, and 1 mM AEBSF) and sonicated them on ice. Tween-20 was added to clarified lysate to a final concentration of 0.1%, and the clarified lysate was incubated with glutathione-Sepharose 4B resin (GE Healthcare) for 12 h. The resin was washed with buffer D [10 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.05% Tween20, and 2 mM 2-mercaptoethanol], and the GST-tag was cleaved off by incubating the resin overnight with His-TEV protease (0.2 mg/ml). Kap121p was finally purified by gel filtration over Superdex200 (GE Healthcare) in buffer D without Tween-20. The purified Kap121p was concentrated to 20 mg/ml using a Millipore concentrator (molecular weight cutoff: 10,000). To prepare Kap121p–Pho4p complex, we incubated a synthetic peptide of Pho4p (residues 140–166), purchased from Sigma-Aldrich Japan, with the purified Kap121p in a Kap121p:Pho4p molar ratio of 1:3.5 prior to set up of crystallization. Crystallization and data collection Crystals of Kap121p–RanGTP complex were obtained at 20 °C by streak seeding hanging drops containing 16 mg/ml proteins, 0.1 M 4-morpholineethanesulfonic acid (pH 6.5), 0.2 M CaCl2, and 10% polyethylene glycol (PEG) 20,000. Crystals were cryoprotected using mother liquor containing 12% PEG 20,000 and 18% glycerol and flashcooled in liquid nitrogen. SAD diffraction data at the Se absorption peak wavelength were collected at 100 K at SPring-8 beamline BL41XU. Crystals of Kap121p–Ste12p complex, Kap121p–Pho4p complex, Kap121p–Nup53p complex, and unliganded

Kap121p were grown at 20 °C from 10 to 20 mg/ml proteins by hanging-drop vapor diffusion against 0.1 M Hepes (pH 7.0), 10% 2-propanol, and 24% PEG 20,000. Crystals were cryoprotected using mother liquor containing 24% PEG 20,000 and 10% glycerol and flash-cooled in liquid nitrogen. Native diffraction data for Kap121p–Ste12p complex were collected at 100 K at Photon Factory beamline BL-17A, those for Kap121p–Nup53p complex were collected at 100 K at Photon Factory beamline BL-5A, and those for Kap121p–Pho4p complex and unliganded Kap121p were collected at 100 K at SPring-8 beamline BL41XU. Structure determination and refinement Diffraction data were processed using MOSFLM and CCP4 programs. 38 The structure of Kap121p–RanGTP complex was solved by a combination of molecular replacement using Ran in the Cse1p–Kap60p–RanGTP complex 34 as a search model and SeMet SAD phasing using the program Phaser. 39 Density modification using the program Parrot 40 yielded an interpretable electron density map. Model building was carried out using the program Coot 41 and refinement was carried out using the program REFMAC5. 42 The structure of Kap121p–Ste12p complex was solved by molecular replacement using the program MOLREP 43 with the use of the structure of Kap121p in the Kap121p– RanGTP complex as a search model. The structures of unliganded Kap121p and Kap121p bound to Pho4p or Nup53p were solved by molecular replacement using the program MOLREP with the use of the structure of Kap121p in the Kap121p–Ste12p complex as a search model. The structures of unliganded Kap121p and Kap121p bound to Ste12p or Pho4p or Nup53p were refined using the programs Coot and REFMAC5. Structural figures were produced using PyMOL 44 and CCP4MG. 45 Expression and purification of proteins for biochemical assays All proteins were expressed in the E. coli host strain BL21-CodonPlus(DE3)RIL (Stratagene). GST-Kap121p, GST-Gsp1p, GST-Ste12p, GST-Nup53p, and GSTPho4p were expressed from pGEX-TEV. 34 GST-tagged proteins except for GST-Kap121p were purified over glutathione-Sepharose 4B (GE Healthcare) and gel filtration over Superdex75 (GE Healthcare) or Superdex200 (GE Healthcare). GST-Kap121p was initially purified over glutathione-Sepharose 4B (GE Healthcare). After removal of the GST-tag by His-TEV protease, Kap121p was finally purified over Superdex200. His/SRan (canine, full length) was expressed from pET30aTEV 34 and purified over Ni-NTA (Novagen) and gel filtration over Superdex75 (GE Healthcare). Mutants were created using the QuikChange system (Stratagene). All DNA constructs were verified by DNA sequencing. GST pull-down assay Pull-down assays were performed in binding buffer (phosphate-buffered saline, 0.1% Tween-20, 0.2 mM DTT,

1866 and 0.2 mM PMSF) as previously described. 34 GST fusions were immobilized on 10 μl of packed glutathioneSepharose 4B (GE Healthcare) beads and each binding reaction was performed by incubating the beads with reaction mixtures in a total volume of 50 μl for 1 h at 4 °C. The amounts of proteins used are indicated in the figure legends. Beads were then spun down and washed twice with 1 ml of binding buffer, and bound proteins were analyzed by SDS-PAGE and Coomassie staining. Surface plasmon resonance analysis All measurements were performed using a BIAcore2000 biosensor (GE Healthcare). Anti-GST IgG was immobilized onto a carboxymethyl dextran sensor (CM5) chip using Nhydroxysuccinimide/1-ethyl-N'-(3-dimethylaminopropyl) carbodiimide hydrochloride coupling. GST fusion proteins were captured by immobilized anti-GST IgG at a concentration of about 1000 resonance units. The samples for analyses were prepared at various concentrations in TNT buffer [10 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.005% Tween-20] and were injected over the sensor surface at a flow rate of 20 μl/min. Following the completion of the injection phase, we monitored dissociation in TNT buffer for 180 s at the same flow rate. Bound proteins were eluted, and the surface was regenerated between injections, using regeneration buffer [10 mM glycine (pH 2.2)]. Regeneration conditions did not denature the immobilized antigen as shown by equivalent signals upon reinjection of the ligand. The apparent association and dissociation rate constants were calculated using BIAevaluation version 4.1 (GE Healthcare). The binding curves were fitted to the simplest (Langmuir) model (A + B ↔ AB) of 1:1 binding. Dissociation constants were derived with both association and dissociation rates (KD = kd/ka). The goodness of the fit between experimental data and fitted curves was estimated by χ 2 analysis.

Kap121p-Mediated Nuclear Import and its Regulation

log phase, treated with 0.2 M hydroxyurea for 2 h to arrest in S phase, and the localization of the NLS-GFP fusion proteins was monitored using fluorescence microscopy on an Olympus FV1000 confocal microscope. Accession numbers Atomic coordinates and structural factors have been deposited in the Protein Data Bank with accession codes 3W3T (unliganded Kap121p wild type), 3W3U (unliganded Kap121p R349A/Q350A/D353A/E396A/N430K/D438A/ N477A mutant), 3W3V (unliganded Kap121p D353K/ E396K/D438K mutant), 3W3W (Kap121p–Ste12p complex), 3W3X (Kap121p–Pho4p complex), 3W3Y (Kap121p– Nup53p complex), and 3W3Z (Kap121p–RanGTP complex).

Acknowledgements We thank our colleagues in Nagoya, especially Masako Koyama and Hidemi Hirano, for assistance and discussion. We are also indebted to the staff of Photon Factory and SPring-8 for assistance during data collection. The X-ray diffraction data collection experiments at Photon Factory were performed with the approval of the Photon Factory Program Advisory Committee (Proposal No. 2012G664). The X-ray diffraction data collection experiments at the beamline BL41XU of SPring-8 were performed with the approval of the Japan Synchrotron Radiation Research Institute (Proposal Nos. 2010B1072, 2011A1093, and 2011B1083). This work was supported in part by the Inamori Foundation and the Kurata Memorial Hitachi Science and Technology Foundation.

In vivo functional analysis

Supplementary Data

The in vivo function of Kap121p mutants was assessed using a plasmid shuffle technique. 46 For the plasmid shuffle, plasmids (CEN, LEU2) encoding the wild-type Kap121p or Kap121p mutants expressed from the KAP121 promoter or a control vector (pRS315) 47 were each transformed into kap121Δ cells (MATa his3Δ1 leu2Δ0 ura3Δ0 kap121Δ;;kanMX4) containing a wild-type KAP121 maintenance plasmid (CEN, URA3). Single transformants were grown in SC-Ura-Leu medium for 1 day to saturation, serially diluted 1:6, and spotted on control plates lacking uracil and test plates containing 5fluoroorotic acid (5-FOA). 5-FOA eliminates the URA3 maintenance plasmid encoding wild-type Kap121p. Plates were incubated at 30 °C for 3 days. NLS-GFP fusion proteins were used to assess the impact of amino acid substitutions of NLS residues for nuclear import. Yeast cells [kap121Δ cells (MATa his3Δ1 leu2Δ0 ura3Δ0 kap121Δ;;kanMX4) containing a wild-type KAP121 maintenance plasmid (CEN, LEU2)] were transformed with plasmids (CEN, HIS3) encoding either Ste12p (residues 581–688; wild type or a mutant)-GFP-GFP or Pho4p (residues 101–200; wild type or a mutant)-GFP-GFP expressed from ADH1 promoter. Cells were grown in SC-Leu-His medium to early

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jmb.2013.02.035 Received 9 January 2013; Received in revised form 17 February 2013; Accepted 20 February 2013 Available online 28 March 2013 Keywords: Kap121p; nuclear import; NLS; nuclear pore complex; Nup53p Abbreviations used: AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride; 5-FOA, 5-fluoroorotic acid; NPC, nuclear pore complex; NLS, nuclear localization signal; GST, glutathione S-transferase; SeMet, selenomethionine; SAD, singlewavelength anomalous dispersion; PEG, polyethylene glycol.

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32.

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