Báo cáo khoa học: Two separate regions essential for nuclear import of the hnRNP D nucleocytoplasmic shuttling sequence ppt

Two separate regions essential for nuclear import of the hnRNP D nucleocytoplasmic shuttling sequence Maiko Suzuki*, Megumi Iijima*, Akira Nishimura*, Yusuke Tomozoe, Daisuke Kamei and Michiyuki Yamada Graduate School of Integrated Science, Yokohama City University, Yokohama, Japan In eukaryotic cells, molecules all move into and out of the nucleus through nuclear pore complexes (NPCs) which span the nuclear envelope. Small molecules diffuse passively through the NPCs, while molecules of more than about 60 kDa are transported by an energy- dependent process. Most proteins are transported into and out of the nucleus by nuclear transport receptors and directionality is determined by high and low Ran- GTP concentrations in the nucleus and cytoplasm, respectively, generated by a RanGTPase system [1–5]. Many of nuclear proteins contain classical nuclear localization sequences (NLS) consisting of one or two clusters of basic amino acids termed basic type mono- partite or bipartite NLS, respectively. They are impor- ted into the nucleus by the nuclear import receptor importin b with or without an adaptor importin a. Other groups of nuclear RNA binding proteins, such as hnRNP A1, SR proteins and HuR, are imported into the nucleus by an mRNA synthesis-dependent process and they shuttle continuously between the nucleus and the cytoplasm [6–10]. Their NLS is bound by the nuc- lear transport receptor transportin (Trn), but not recog- nized by importin a ⁄ b [11–13]. These NLSs also serve as a nuclear export sequence (NES) [2]. hnRNP A1 has the best characterized nucleocytoplasmic shuttling sequence M9, which is a 38 amino acid sequence in the C-terminal domain [11,14,15]. M9 mutational analysis has provided information on a consensus Trn)1 inter- action motif [16]. There are various Trn-1 binding pro- teins, such as TAP, poly(A)-binding protein II, and Keywords AUF1; hnRNP D; nucleocytoplasimic shuttling sequence; nuclear transport; transportin Correspondence M. Yamada, Graduate School of Integrated Science, Yokohama City University, 22–2 Seto, Kanazawa-ku, Yokohama 236–0027, Japan Fax: +81 45 787 2413 Tel: +81 45 787 2214 E-mail: myamada@yokohama-cu.ac.jp *These authors contributed equally to this work. (Received 16 April 2005, revised 6 June 2005, accepted 14 June 2005) doi:10.1111/j.1742-4658.2005.04820.x Heterogeneous nuclear ribonucleoprotein (hnRNP) D ⁄ AUF1 functions in mRNA genesis in the nucleus and modulates mRNA decay in the cyto- plasm. Although it is primarily nuclear, it shuttles between the nucleus and cytoplasm. We studied the nuclear import and export of the last exon-enco- ding sequence common to all its isoforms by its expression as a green fluor- escent protein-fusion protein in HeLa cells and by heterokaryon assay. The C-terminal 19-residue sequence (SGYGKVSRRGGHQNSYKPY) was identified as an hnRNP D nucleocytoplasmic shuttling sequence (DNS). In vitro nuclear transport using permeabilized cells indicated that nuclear import of DNS is mediated by transportin-1 (Trn-1). DNS accumulation in the nucleus was dependent on Trn-1, Ran, and energy in multiple rounds of nuclear transport. Use of DNS with deletions, alanine scanning muta- genesis and point mutations revealed that two separate regions (the N-ter- minal seven residues and the C-terminal two residues) are crucial for in vivo and in vitro transport as well as for interaction with Trn-1. The N- and C-terminal motifs are conserved in the shuttling sequences of hnRNP A1 and JKTBP. Abbreviations DAPI, 4¢,6-diamino-2-phenylindole; DNS, hnRNP D ⁄ AUF1 nucleocytoplasmic shuttling sequence; EGFP, enhanced green fluorescent protein; GST, glutathione S-transferase; mt, mutant type; NES, nuclear export sequence; NLS, nuclear localization sequence; NPC, nuclear pore complexes; PAD, peptidylarginine deiminase; RU, resonance unit; SPR, surface plasmon resonance; Trn-1, transportin 1; wt, wild type. FEBS Journal 272 (2005) 3975–3987 ª 2005 FEBS 3975 HuR, but no obvious consensus Trn-1 binding sequence has been found in these naturally occurring proteins [12,17–19]. hnRNP D ⁄ AUF1 consists of two RNA binding domains (RBDs) and has a high content of glycine in the C-domain, like hnRNP A1 [20,21]. Four isoforms, D01 ⁄ p37, D02 ⁄ p40, D1 ⁄ p42 and D2 ⁄ p45, are formed by alternative splicing and are found in many tissues and various types of cultured cells [22–24]. They func- tion in trans-acting transcriptional factors, alternative splicing factors in the nucleus and in modulation of AU-rich element-directed mRNA decay in the cyto- plasm [21,25–28]. They are found mainly in the nuc- leus, but rapidly shuttle between the nucleus and the cytoplasm [22,29–31]. Besides, their subcellular distri- bution in cells changes in response to environmental stimuli such as temperature shift, cell differentiation and mRNA synthesis inhibition [26,30–32]. It has been shown by protein–protein blotting that hnRNP D2 binds to Trn-1 at the C-terminal 112 amino acid sequence [13]. Recently, the C-terminal 50 amino acid and 35 amino acid sequences of D01 and D02 were found as an NLS [29,30]. However, there is no clear evidence for the nucleocytoplasmic shuttling activity of NLS and the involvement of Trn-1 in the nuclear import. The C-terminal 21 amino acid sequence enco- ded by the hnRNP D last exon 8 is noted to be homologous with the 25 amino acid shuttling sequence in the JKTBP C-terminal tail [33]. In this study, we attempted to determine whether the nuclear import and export sequences are located in the same region. We identified the hnRNP D nucleocytoplasmic shut- tling sequence (DNS) as a 19 amino acid sequence and found that the N- and C-terminal portions of DNS are important for the nuclear import mediated by Trn-1. Results Determination of hnRNP D NLS The exon 8 of hnRNP D encodes the 21 amino acids sequence common to all the isoforms D01, D02, D1, and D2 (Fig. 1A) [23]. To examine whether the exon-8 encoding sequence has NLS activity, we prepared plas- mid constructs encoding the D02 C-terminal 25 amino acids (282–306) and mutants of this sequence with increasing N- and C-terminal deletions as fusion pro- teins with the C-end of a composite EGFP-GST-PAD protein ( 69 kDa) (Fig. 1B). These plasmids were used to transfect HeLa cells and after their expression, their subcellular localization in the cells were examined by fluorescence microscopy (Fig. 1C). Fluorescence micrographs of the cells revealed that the empty vector-encoding composite GFP-GST-PAD protein, used as a control, was exclusively present in the cyto- plasm (panel a), indicating that it is larger than the passive diffusion protein. The N-terminal deletion D02 mutants 282–306, 288–306, and 292–306 were present only in the nucleus (Fig. 1C, panels b, c and d). The shorter mutants, 293–306, 294–306, and 295–306, were found mainly in the nucleus but also slightly in the cytoplasm (Fig. 1C, panels e, f and g). However, a one residue shorter 11-residue mutant 296–306 and an eight-residue mutant 299–306 were found exclusively in the cytoplasm like the control with an empty vector (Fig. 1C, panels h and i and a). The nuclear localiza- tions of C-terminal deletion mutants were also studied in the same way (Fig. 1D). The C-3 and -6 amino acids deletion mutants 288–303 and 288–300 were found only in the cytoplasm like the control (Fig. 1D, panels c, d and a), while the 19-residue mutant 288– 306 was found in the nucleus (panel b). Immunoblot- ting of the cell lysates using anti-GFP confirmed the expression of mutant fusion proteins of the expected size of  70 kDa (data not shown), indicating that the cytoplasmic fluorescent signal was not that of a degra- ded protein. These results indicated that D02 NLS is mapped to the C-terminal 19 amino acids (288–306) encoded by exon 8. Role of amino acid residues of a D02 NLS in nuclear import To determine the role of amino acids of the C-terminal 19 residue NLS in nuclear import, we performed alan- ine scanning mutagenesis experiments. Mutants mt1- mt5 were prepared using the construct encoding an EGFP-GST-PAD-D02 NLS (288–306) fusion gene (wt) as a template by sequential consecutive three amino acid replacements by a cluster of three alanines (Fig. 2A). These constructs were examined for nuclear import in the same way as described above. As shown in Fig. 2B, mt3 was located in the nucleus as the wt (Fig. 2B, panels e and b), whereas mt1, mt2 and mt4 were mostly located in the nucleus, but also signifi- cantly in the cytoplasm (Fig. 2B, panels c, d and f). In contrast, the C-terminal three amino acid substitution mutant mt5 was located exclusively in the cytoplasm, likely the control with an empty vector (Fig. 2B, panels a and g). This prompted us to test the two last amino acid substitution mutants mt6–9 for nuclear import (Fig. 2C). The mutants mt6 (P305A ⁄ Y306A), mt7 (P305A), mt8 (Y306A) and mt9 (Y306D) were located in the cytoplasm (Fig. 2C, panels b–e), while the wt was imported into the nucleus (panel a). These results indicated that both C-terminal residues PY are hnRNP D ⁄ AUF1 nucleo-cytoplasmic shuttling sequence M. Suzuki et al. 3976 FEBS Journal 272 (2005) 3975–3987 ª 2005 FEBS A B C D Fig. 1. The C-terminal location of an NLS in hnRNP D. (A) hnRNP D ⁄ AUF1 isoforms. Boxes 2 and 7 show alternative splicing exons 2 and 7 enco- ding 19 and 49 amino acid residues, respectively. Box 8 denotes the exon 8-encoded sequence. The first and last amino acid residue numbers are shown under the corners of boxes. (B) Plasmid constructs for nuclear transport of the D02 C-terminal sequence (282–306) and its N-terminal and C-terminal deletion mutant sequences. 1, a control empty vector pEGFP-GST-PAD encoding a GFP-labeled composite protein (604 amino acids); 2–11, plasmid constructs encoding D02 (282–306) and its N-terminal and C-terminal deletion mutant sequences, respectively, represen- ted as a fusion protein linked to the C-end of the composite protein. (C) Subcellular localizations of D02 (282–306) and N-terminal deletion mutants expressed as GFP-fusion proteins in HeLa cells. HeLa cells were transfected with the plasmids described above and incubated on coverslips for their expression for 24 h and then were studied by fluorescence microscopy. Panels a–i, fluorescent signals of the cells expressing GFP-labeled proteins shown on the top of each panels; panels j–r, nuclear DNA stained with DAPI of the cells in the same views as in panels a–i, respectively. (D) Subcellular localizations of C-terminal deletion mutants described on the top of each panel were studied as described in (C). Pan- els a–d, fluorescent signals of cells; panels e–h, nuclear DNA stained with DAPI of the cells in the same view as in panels a–d, respectively. Plus and minus signs on the right of (B) show, respectively, positive and negative signals for nuclear import and nuclear export described below. M. Suzuki et al. hnRNP D ⁄ AUF1 nucleo-cytoplasmic shuttling sequence FEBS Journal 272 (2005) 3975–3987 ª 2005 FEBS 3977 essential for the nuclear import. Results on mt9 suggested that phosphorylation of the tyrosine residue is not related to the nuclear import. Then, the role of these two residues PY in nuclear import of a full-length D02 (1–306) was examined by mutation. Full-length D02 and the mutants were expressed as EGFP fusion proteins but not as EGFP-GST-PAD fusion proteins, and were studied in the same way as above (Fig. 3). Mutants D02 (1–306) P305A ⁄ Y306A and Y306A showed cytoplasmic localization and their signals appeared as numerous speckles around the nuclear periphery (Fig. 3, panels c and d), while a control of EGFP was seen throughout the cells and wild type D02 was seen exclusively in the nucleus (Fig. 3, panels a and b). Mutant D02 (D288-291), with a four amino acid SGYG (288–291) deletion, was also A B C Fig. 2. Effects of amino acid-substitutions of an hnRNP D NLS on nuclear import. (A) A wild type (wt) NLS (D02 288–306) and various mutant types (mt1–9) with replacements by Ala and Asp in the indicated sites were represented as a fusion protein linked to the C-terminal end of a composite protein GFP-GST-PAD in the pEGFP-GST-PAD vector described in Fig. 1B. Dashed lines indicate the same amino acids in the sequence as shown at the top. Plus and minus signs on the right show, respectively, positive and negative signals for nuclear import described below. (B) Subcellular localizations of NLS Ala scan mutant fusion proteins (wt and mt1–5) in cells. HeLa cells were transfected with the above plasmid constructs and grown for 24 h for expression. The cells were studied by fluorescence microscopy. Panels a–g, fluor- escent signals of the cells expressing GFP-labeled proteins shown on the top of each panel; panels h–n, nuclear DNA stained with DAPI of the cells in the same views as in panels a–g. (C) Subcellular localization of NLS C-terminal end mutants (mt6–9). Panels a–e, fluorescent signals of the cells expressing GFP-labeled proteins shown on the top of each panel; panels f–j, nuclear DNA stained with DAPI of the cells in the same views as in the panels a–e, respectively. hnRNP D ⁄ AUF1 nucleo-cytoplasmic shuttling sequence M. Suzuki et al. 3978 FEBS Journal 272 (2005) 3975–3987 ª 2005 FEBS located in the nucleus (panel e). Immunoblotting of cell lysates confirmed the expression of intact mole- cules (data not shown). These results taken together indicated that the N-terminal seven residues and the last two C-terminal residues PY are essential for nuc- lear import of the D02 C-terminal segment (288–306). Identification of an hnRNP D NLS as a nucleocytoplasmic shuttling sequence To investigate whether the above described NLS has nucleocytoplasmic shuttling activity, we used hetero- karyon assay. The constructs encoding NLS in pEG- FP-GST were expressed as GFP-tagged proteins in HeLa cells for 22 h to label the nucleus, and the cells were then fused to nontransfected murine 3T3 cells in the presence of a protein synthesis inhibitor, cyclohexi- mide, and further incubated for 1 h to see whether GFP- labeled protein migrated from the HeLa nucleus to the 3T3 nucleus in the heterokaryons. JKTBP2 served as a control for a nuclear retention protein remaining in the original HeLa nucleus (Fig. 4, panel a) and a full length D02 was used as a control positive for shuttling and was found in the nucleus of both HeLa and 3T3 cells (Fig. 4, panel b; arrow shows the position of the mouse nucleus) as expected [30,33]. Of the three NLS segments, D02 (282–306) and D02 (288–306) became located in the nucleus of both HeLa and 3T3 cells (Fig. 4, panels c and d). However, the four-residue shorter D02 (292–306) was found in the HeLa but not the 3T3 nucleus (Fig. 4, panel e), indica- ting that deletion of the four N-terminal residues SGYG (288–291) of D02 (288–306) has a more deleteri- ous effect on nuclear export than on import. It is note- worthy that the substitution of GHQ (298–300) by these alanines of mt3 did not affect shuttling activity so much as nuclear import activity (Fig. 4, panel f). These results indicated that the D02 C-terminal 19 residue sequence (288–306) constitutes the hnRNP D nucleo- cytoplasmic shuttling sequence. This was termed DNS. Trn-1-dependent import of hnRNP D We examined whether the nuclear import of DNS ⁄ D02 (288–306) was mediated by Trn-1. Nuclear import sub- strates were prepared as GST-GFP fusion proteins and tested for in vitro nuclear import activity using digito- nin-permeabilized HeLa cells supplemented with either reticulocyte lysates or a reconstituted mixture of Trn-1, Ran mix (RanGDP, NTF2 and RanGAP) and an energy-regenerating system. DNS 288–306 was effect- ively imported into the nucleus at 30 °C but not at 4 °C in the presence of reticulocyte lysates during a 30- min incubation period (Fig. 5A, left, panels a and b). This import into the nucleus was inhibited almost com- pletely by the addition of a 40-molar excess of hnRNP A1 (1–320), but not significantly by a shortened form (1–196) of hnRNP A1 ⁄ UP1 lacking the M9 domain (right panels a–c). This M9-mediated inhibition sugges- ted that DNS nuclear import is mediated by a nuclear transport receptor of Trn, but not other importins. Use of the reconstituted transport mixture instead of reticu- locyte lysates indicated that DNS accumulation in the nucleus was dependent on Ran mix, an energy-regener- ating system and Trn-1 (Fig. 5B). Ran and energy were required only when high substrate and low Trn-1 con- centrations were used in the assay. Next, to compare the NLS activity in vivo and in vitro, the DNS N- and C-terminal deletion mutants described above were tested for nuclear import activity in the presence or absence of Trn-1 (Fig. 5C). DNS ⁄ 288–306 was imported into the nucleus in a Trn- Fig. 3. Importance of C-terminal residues of a full-length hnRNP D02 for subcellular distribution. Plasmid constructs carrying a full length D02 wild type, D02 mutant (P305A ⁄ Y306A), D02 mutant (Y306A) and D02 deletion mutants (D288-291) gene linked in frame to the 3¢ end of the EGFP gene in pEGFP vector were used to transfect HeLa cells. After 24 h expressions the cells were studied for subcellular localizations by fluorescence microscopy. Panels a–e, fluorescent signals of cells expressing the GFP-labeled mutants shown on the top of each panel; panels f–j, nuclear DNA stained with DAPI of the cells in the same views as in panels a–e. M. Suzuki et al. hnRNP D ⁄ AUF1 nucleo-cytoplasmic shuttling sequence FEBS Journal 272 (2005) 3975–3987 ª 2005 FEBS 3979 1-dependent manner as efficiently as full D02 (1–306) (Fig. 5C, panels a–d). Nuclear imports of the DNS N- deletion mutants 292–306 and 293–306 were decreased to a low level but significantly higher levels than the levels in the absence of Trn-1 (Fig. 5C, panels e–h). However, the shorter N-deletion mutant 296–306 showed no nuclear import activity (Fig. 5C, panels i and j). The C-deletion mutant (288–303) lacking the last three residues of DNS showed no nuclear import activity (Fig. 5C, panels k and l). These results indica- ted that the C-terminal 19-residue sequence of D is necessary and sufficient for the in vitro nuclear import which is mediated through a Trn-1 system. DNS NLS (292–306) and (293–306) revealed that the nuclear import was much lower in vitro than in vivo (compare Fig. 5C, panels e–h, with Fig. 1C, panels d and e). Direct interaction of DNS NLS with Trn-1 We analyzed the interaction of DNS N-and C-terminal deletion NLS mutants with Trn-1 by GST pull-down assay and surface plasmon resonance (SPR) (Fig. 6). GST-tagged NLS mutant proteins immobilized to glutathione-beads were incubated with HeLa cell extracts at 4 °C for 4 h. NLS interacting proteins iso- lated by the beads were probed for Trn-1 by immuno- blotting using anti-Trn-1 (Fig. 6A, upper panel). Protein blots stained with Amido Black 10B indicated excess amounts of GST-NLS mutant proteins (Fig. 6A, lower panel). DNS (288–306) bound considerable Trn-1 (lane 3), while the N-deletion mutants 292–306, 293– 306, 294–306, and 295–306 bound Trn-1 slightly (lanes 4–7). However, the even shorter mutants 296–306 and 299–306 revealed no Trn-1 binding, like GST as a con- trol (lane 8 and 9 and 2). This is consistent with the finding that the minimum 12-residue C-terminal sequence (295–306) can be transported into the nucleus in vivo (Fig. 1C, panel g). Then, the DNS Ala scan mutants mts1–5 and DNS C-two residue single or dou- ble substitution mutants mts 6–8 described in Fig. 2A were studied for interaction with cellular Trn-1 in the same way (Fig. 6B). Ala scan mutants mt1 and mt3 showed weaker interaction with Trn-1 than wtDNS (lanes 3, 4 and 6), but mt2, mt4 and mt5 showed no A B Fig. 4. Identification of hnRNP D nucleocytoplasmic shuttling sequence. (A) Nucleocytoplasmic shuttling of hnRNP D NLS mutants in hetero- karyons. HeLa cells transfected with pEGFP-C constructs encoding JKTBP2 and D02 as a GFP fusion protein and with pEGFP-GST con- structs encoding D02 (282–306), D02 (288–306), D02 (292–306) and mt3 as a GFP-GST fusion protein were expressed for 22 h. The cells were then fused with 3T3 cells and cultured for another 1 h in the presence of cycloheximide. Arrows point out the murine nucleus. Panels a–f, fluorescent signals of the GFP- or GFP-GST-tagged proteins indicated at the top of each panel; g–l, nuclear morphology upon staining with Hoechst 33342; m–r, images of heterokaryons merged upon nuclear staining (dashed lines show approximate outlines). A heterokaryon in the same column is in the same view. (B) Alignment of the hnRNP D shuttling sequence with those of JKTBP1, hnRNP A1 and consen- sus Trn)1 interaction motif. Identical amino acid and similar amino acid residues in a column were colored pink. J, Hydrophilic amino acid; Z, hydrophobic amino acid; X, any residue. hnRNP D ⁄ AUF1 nucleo-cytoplasmic shuttling sequence M. Suzuki et al. 3980 FEBS Journal 272 (2005) 3975–3987 ª 2005 FEBS interaction with Trn-1 (lanes 5, 7 and 8). Both the last two amino acid C-terminal mutants mts 6, 7, and 8 showed almost no interaction with Trn-1 (lanes 9–11). Next, the direct interactions between the two purified recombinant DNS NLS mutants and Trn-1 at 25 °C were studied by SPR. The response signal was monit- h r GFP DAPI 288-306 292-306 296-306 ++ + + ab c d efg kl mno p q 1-306 288-303 + ij st GFP DAPI Trn-1 DAPI GST-GFP-DNS ab cd ef Competitors hnRNP A1 UP-1 A B 30 o C4 o C C + Trn-1 Ran + energy + Ran energy + Apyrase + Ran + energy + Ran + energy Tr n- 1 293-306 + abc d GFP DAPI Trn-1 uvwx a e bcd hfg Fig. 5. Nuclear imports of full-length D02 and DNS deletion mutants in permeabilized HeLa cells. (A) The right panels show inhibition of the nuclear import of DNS ⁄ (288–306) by hnRNP A1. Permeabilized HeLa cells were incubated with 0.1 l M GST-EGFP-DNS fusion proteins in the presence or absence of a 40-fold molar excess of the competitors indicated on the top of each panels in transport buffer (10 lL) supplemen- ted with 4 lL of reticulocyte lysates at 30 °C for 30 min. The cells were stained with anti-GST IgG followed with goat anti rabbit IgG (H + L)- biotin conjugate and streptoavidin. Panels a–c, localization of DNS; panels d–f, nuclear DNA stained with DAPI in the same views as in panels a–c. The left panels show temperature dependent nuclear import of DNS. Permeabilized cells were incubated with 2 l M GST-EGFP-DNS fusion protein as described above at the indicated temperature of 30 °Cor4°C and studied for GFP fluorescent signal and nuclear DNA. Pan- els a and b, localization of DNS; panels c and d, nuclear DNA in the same views as in panels a and b, respectively. (B) Dependency of DNS nuclear import on Trn-1, RanGTP generating system and energy-regenerating system. Permeabilized cells were incubated with 4 l M GST- EGFP-DNS, 0.2 l M Trn-1, Ran mixture and an energy-regenerating system as described in the Experimental procedures, except that either Trn-1, Ran mixture or the energy-regenerating system was omitted and on omission of the latter apyrase (1 unit) was added to deplete resid- ual ATP and GTP as stated at the top of the panels. Note high substrate and low Trn-1. The cells were studied for GFP-fluorescent signals and nuclear DNA stain. Upper and lower panels in a column show the same view. (C) Permeabilized cells were incubated with 2 l M GST-EGFP- D02 (1–306) and -DNS N-terminal and C-terminal deletion mutants as transport substrates stated at the tops of the panels in the presence (+) or absence (–) of 2 l M Trn-1 in a nuclear import mixture containing Ran mixture and energy mixture and the cells were studied by fluores- cence microscopy. a and b, full-length D02; c and d, DNS ⁄ 288–306, e and f, g and h, and i and j, N-deletion DNS mutants 292–306, 293–306, and 296–306, respectively; k and l, C-deletion DNS mutant 288–303. Upper and lower panels in columns show the same view. M. Suzuki et al. hnRNP D ⁄ AUF1 nucleo-cytoplasmic shuttling sequence FEBS Journal 272 (2005) 3975–3987 ª 2005 FEBS 3981 ored for 2 min in flow of 100 nm Trn-1 over the various DNS NLS mutant-GST fusion proteins which had been immobilized on sensor chips. Figure 6C–E shows their sensorgrams. Figure 6D indicates that full-length D02 interacted with Trn-1 as well as DNS (curves 1 and 2). The DNS N-deletion mutants D02 (292–306) and D02 (293–306) interacted with Trn-1 much less well than DNS, but significantly more than the control GST-GFP (Fig. 6C, curves 1–3 and 5). However, the DNS C-dele- tion mutants D02 (288–303), D02 (288–300) and D02 (288–297) did not interact with Trn-1 at all, like the con- trol GST-GFP (Fig. 6D, curves 3–6 and Fig. 6C, curve 4). The Ala scan mutants mt3 and mt1 interacted with Trn-1 much less well than DNS (Fig. 6E, curves 1–3). Other Ala scan mutants, mts4, 2 and 5, and the C-two amino acid substitution mutants mts6, 7, 8 and 9 did not interact with Trn-1 significantly like GST (Fig. 6E, curves 4–11). These results substantiate the importance of the N-seven amino acids and the last two C-terminal amino acids PY in DNS for interaction with Trn-1. Affinities of D02, DNS and DNS mutants for Trn-1 Kinetic parameters of the association rate constants (k a ), dissociation rate constants (k d ) and dissociation constants (K D ) of D02, DNS, and DNS mutants for Trn-1 were determined at various concentrations of Trn-1 (2.5–40 nm)at25°C by SPR (Table 1). The K D A B C D E hnRNP D ⁄ AUF1 nucleo-cytoplasmic shuttling sequence M. Suzuki et al. 3982 FEBS Journal 272 (2005) 3975–3987 ª 2005 FEBS of D02 was 4.0 nm and the K D of DNS 9.2 nm, that is double that of D02. This larger K D was accounted for by the twofold larger k d of DNS than that of D02 and their nearly similar k a values. The N-four residue dele- tion mutant 292–306 of DNS and a one residue shorter mutant 293–306 had, respectively, about six and 14 times larger K D values than that of DNS. These larger K D values were largely accounted for by the decreased k a values. These results indicate that the DNS N-seven amino acids sequence contributes greatly to the associ- ation with Trn-1. The DNS Ala scan mutants mt1, 2 and 3 had about seven times larger K D values than DNS, largely accounted for by their lower k a values. Other mutants, including the C-terminal mutants, showed too weak bindings to determine as described above. For comparison, the K D , k a , and k d values of hnRNP A1 for Trn-1 were estimated to be 6.3 nm, 2.2 · 10 5 m )1 Æs )1 , and 1.4 · 10 )3 s )1 , respectively. The K D values of A1 M3 NLS (238–320) and TAP-NLS (61–102) for Trn-1 have been determined as 2.8 and 18.7 nm, respectively, by fluorescence titration and SPR [34]. This indicates that affinity of D for Trn-1 is in a similar order to those of A1 and TAP. Discussion In this study we identified the NLS and NES of D02 as an hnRNP D nucleocytoplasmic shuttling sequence (DNS), which is located at the C-terminal tail. The sequence identified was 19 amino acids long, SGYGKVSRRGGHQNSYKPY (residues 288–306), which is encoded by exon 8 common to all D isoforms. Mutational analysis of DNS indicated that two separ- ate regions in DNS, the N-terminal seven amino acids and the two C-terminal amino acids, are essential for nuclear import mediated by Trn-1. Heterokaryon assay indicated that DNS as well as hnRNP D rapidly shuttles between the nucleus and the cytoplasm. A DNS mutant lacking an N-terminal SGYG sequence was imported into the nucleus, but could not be exported from the nucleus. It would appear that nuclear export of DNS occurs in a facilita- ted manner but not diffusion. Nuclear export of hnRNP D has been shown to be insensitive to lepto- mycin B and therefore to be independent of a nuclear export receptor of CRM1 ⁄ exportin-1 [30]. Consistent with this finding, the DNS sequence has no similarity Table 1. Kinetic parameters of D02, DNS, and DNS N-deletion and Ala scan mutants interacting with Trn-1. GST-fusion forms of D02, DNS and DNS mutants (Figs 1 and 2) were bound as a ligand to an anti-GST Ig-immobilized sensor chip and sensorgrams were obtained by injecting various concentrations (2.5, 5, 10, 20 and 40 n M) of Trn-1 as an analyte at 25 °C and then were analyzed using BIACORE kinetic software. Ligand Trn-1 as an analyte k a (1 ⁄ M )1Æ s )1 ) k d (1 ⁄ s) K D (nM) D02 7.5 · 10 5 3.0 · 10 )3 4.0 DNS ⁄ 288–306 7.2 · 10 5 6.6 · 10 )3 9.2 292–306 1.4 · 10 5 3.5 · 10 )3 24.7 293–306 0.7 · 10 5 4.0 · 10 )3 56.2 mt1 1.2 · 10 5 3.6 · 10 )3 29.8 mt2 1.1 · 10 5 3.1 · 10 )3 29.2 mt3 1.4 · 10 5 4.3 · 10 )3 30.3 Fig. 6. Interaction between D02 NLS mutants and Trn-1. (A) Interaction of GST-tagged DNS N-deletion NLS mutants with cellular Trn-1. Var- ious D02 NLS mutants fused with the C-terminal end of GST in place of GFP-GST-PAD described in Fig. 1B were produced in Escherichia coli, and purified as glutathione-Sepharose bead-bound forms. The bead-bound GST-NLS fusion proteins were incubated with HeLa cell extracts for 4 h at 4 °C and then washed. Bead-bound proteins were eluted and analyzed by immunoblotting using anti-Trn-1. The upper panel shows the immunoblots; lane 1, 16 lg of cell extracts used as a source for pull-down assay; lanes 2–9, pull-down assays from 160 lg of cell extracts; 2, GST; 3, DNS ⁄ 288–306; 4, D02 (292–306); 5, D02 (293–306); 6, D02 (294–306); 7, D02 (295–306); 8, D02 (296–306); 9, D02 (299–306); lower panel: the same blots stained with Amide Black 10B. Only positively stained sections of the immuno and protein blots are shown. Arrows on the right show Trn-1 and GST-NLS. (B) Interaction of GST-tagged DNS Ala scan mutants and point mutation mutants with cellular Trn-1. DNS Ala scan mutants and C-terminal point mutation mutants described in Fig. 2 were allowed to express GST fusion protein and purified. They were subjected to GST pull-down with cell extracts as described above. The upper panel shows the immunoblots probed with anti-Trn-1. The lower panel shows the same blots stained for protein. Lanes; 1, one-tenth of cell extracts; 2–7, pull-down assays of cell extracts with GST-fusion proteins; 2, GST; 3, DNS (wt); 4, mt1; 5, mt2; 6, mt3; 7, mt4; 8, mt5; 9, mt6; 10, mt7; 11, mt8. (C) SPR ana- lysis of interaction of Trn-1 with DNS N-deletion NLS mutants. The purified Trn-1 and NLS mutant proteins were used as analyte and ligands, respectively. The response signal was monitored for 2 min at 25 °C on injection of Trn-1 (100 n M in HBS-EP) over the DNS various N-dele- tion mutant GST-GFP fusion proteins which had been immobilized on an anti-GST antibody-bound sensor chip and then HBS-EP. 1, DNS ⁄ 288–306; 2 (292–306); 3 (293–306); 4, C-deletion mutant (288–303); 5, GST-GFP. (D) SPR analysis of interaction of Trn-1 with DNS C-deletion NLS mutants. The response signal was monitored on a flow of Trn-1 over the DNS various C-deletion NLS mutants GST-GFP fusion protein-immobilized sensor chips as described above. 1, full length D02; 2, DNS; 3–6 * merging lines: 3 (288–303); 4 (288–300); 5 (288–297); 6, GST-GFP. Note no measurable difference in 3–6. (E) SPR analysis of interaction of Trn-1 with DNS Ala scan mutants and C-terminal mutants. Response signals were monitored over the various DNS Ala scan mutants and single point mutants described in (B) as described above. 1, DNS; 2, mt3; 3, mt1; 4–11 *, from upper line to lower line: 4, mt4; 5, mt2; 6, mt7; 7, mt5; 8, mt6; 9, mt9; 10, mt8; 11, GST. M. Suzuki et al. hnRNP D ⁄ AUF1 nucleo-cytoplasmic shuttling sequence FEBS Journal 272 (2005) 3975–3987 ª 2005 FEBS 3983 to a leucine-rich NES which is contained in many CRM1-mediated nuclear export proteins [35]. In addi- tion, DNS N-deletion mutants suggested that N-ter- minal four residues are important for nuclear export, but not necessarily essential for nuclear import. To understand whether export sequence is only limited to an N-terminal region of DNS or contains a whole DNS sequence, further investigation is needed. Two smaller isoforms (D01 and D02) were found to contain an exon 6 and 8 encoded 35 amino acid NLS, but not NES [30]. It has been suggested that larger iso- forms (D1 and D2) contain inactive NLS with an exon 7 insertion containing an NES and their associations with a smaller isoform are involved in nuclear import and consequently D shuttling occurs [30]. However, in this work, mutational analysis of DNS in the D02 molecule suggested that D02 is able to shuttle alone between the nucleus and the cytoplasm. This discrep- ancy remains to be understood. The DNS is rich in hydrophilic amino acids and glycine, and differs from the classical basic type NLS. Successive deletions of up to seven residues from the N-terminal portion of DNS gradually reduced the nuclear import activity in vivo and in vitro and also similarly decreased its binding to Trn-1. In contrast, deletion of the last three C-terminal amino acids KPY, alanine substitution and even point mutation of either of the last two C-terminal amino acids PY completely abolished the in vivo and in vitro nuclear import activity as well as the binding to Trn-1. Ala- nine scanning mutagenesis of the sequence linking the two motifs had moderate effects on nuclear import and binding to Trn-1. Thus, the N- SGYGKVS (288– 294) and C-PY (305–306) motifs in DNS are more crucial for nuclear import than the internal 10-residue sequence separated by the two motifs. Interestingly, these two motifs are conserved in JKTBP1 and ABBP1 C-terminal tail sequences and an N-terminal portion (271–289) of M9 when they are aligned (Fig. 4B and [33]) The N-SGYGKVS motif is also conserved in the C-portion of the consensus Trn)1 interaction motif (12 residues), which is derived from randomized M9s and is necessary for both the import and export activity of M9 [16]. However, the PY motif is located 10 residues C-terminal of the consen- sus Trn interaction motif (Fig. 4B). Some differences between the nuclear imports in vivo and in vitro of DNS N-deletion and substitution mutants were observed (compare Fig. 1C with Fig. 5C). Nuclear import in vivo appeared to plateau within 1 day. However, it is not known at what time point plateau was reached. Therefore, the results of in vivo and in vitro could not be directly compared. The D1 C-terminal 112 amino acid sequence on a blot has been shown to bind to Trn-1 [13]. In vitro transport assay provided convincing evidence that DNS nuclear import is mediated through NPC by Trn-1. Ran and energy were required for nuclear import at a low concentration of 0.1–0.2 lm Trn-1 with a high concentration of 4 lm DNS, but not at a high concentration of 1 lm Trn-1. This indicates that the translocation of the substrate–Trn-1 complex through NPCs to the nucleoplasmic side is independent of both Ran and energy and that RanGTP and GTP energy are required only for the release of substrate from the substrate Trn-1 complex and for multiple rounds of Trn-1-mediated nuclear import, as was found in M9 Trn-1-mediated nuclear transport [36–38]. SPR analysis provided clear evidence that DNS interacts directly with Trn-1. In DNS, both the N-terminal seven amino acid sequence SGYGKVS (288–294) and the last two C-terminal residues PY are essential for binding to Trn-1. The shorter, import-deficient N- and C-terminal deletion mutants, which also show no ability for in vitro nuclear import, do not bind Trn-1. DNS N-deletion and Ala scan mutants, which have reduced ability for nuclear import, revealed 6–14 times larger K D values with a decreased k a and fairly invaried k d for Trn-1 as compared with DNS. Whether these DNS mutations affect release of substrate from the substrate–Trn com- plex on binding to RanGTP remains to be studied. Structural studies on the Trn-1 ⁄ karyopherin b2A- RanGppNp complex indicated that the structural change of Trn-1 upon binding RanGTP in its N-ter- minal arch is transmitted through a long internal acidic loop to the substrate–Trn complex in its C-terminal arch concomitantly with release of the sub- strate [34,39]. As the C-terminal tail of D is predicted to have no secondary structure, the extended DNS conformation might be stabilized at the N- and C-ter- minal residues by binding to the C-terminal arch of Trn-1, as found in basic type bipartite NLS importin a complexes [40,41]. Trn-1 recognizes various kinds of nuclear proteins and the nuclear pore complex proteins nup98 and nup153 as substrates [3,11,12,17–19,33]. DNS and its mutants will aid in understanding such a broad recognition of Trn-1 on the basis of the crystal structure of the Trn-1-substrate complex. Experimental procedures Clonings of cDNAs for hnRNP D02, Trn-1, RanGAP and Ran A human full length hnRNP D02 cDNA was isolated from a human monocytic SKM-1 cDNA library using a RBD1 hnRNP D ⁄ AUF1 nucleo-cytoplasmic shuttling sequence M. Suzuki et al. 3984 FEBS Journal 272 (2005) 3975–3987 ª 2005 FEBS [...]... hnRNP D NLS and Trn-1 A GST-pull down assay was performed as described previously [33] GST -hnRNP D0 2 and GST-NLS mutant fusion protein were purified as a glutathione bead-bound form About 20 lL of a 50% slurry of the packed beads was mixed with HeLa cell extract (5–8 mg) in a volume of 1.5 mL binding buffer at 4 °C for 4 h Bound proteins were eluted from the beads with SDS sample buffer and analyzed... 30 °C for 30 min 1 U apyrase (grade VI, Sigma) was added to the reaction mix without an energy-regenerating system to deplete residual 3985 hnRNP D ⁄ AUF1 nucleo-cytoplasmic shuttling sequence ATP Without transport factors, reticulocyte lysates (Promega, Madison, WI, USA) was used After the reaction, the cells were fixed with 4% (v ⁄ v) paraformaldehyde for 30 min at 4 °C and were studied for the nuclear. .. recorded with a cooled CCD camera For heterokaryon assay, 3.8 · 104 HeLa cells transfected with various plasmids were grown on a 0.1% (w ⁄ v) gelatincoated glass coverslip for 24 h First the slides were rinsed and overlaid with 5 · 104 Balb ⁄ C NIH3T3 cells and cultured for 3 h, and then for 15 min with 20 lgÆmL)1 cycloheximide The cells were then rinsed and fused by exposure to 100 lL of 50% (w ⁄ v) polyethylene... accomplished with 10 mm glycine ⁄ HCl, pH 2.2 ka, kd and KD were determined at various concentrations of Trn-1 with biaevaluation version 3.0 (with a global fitting program) Acknowledgements We are grateful for Dr Gideon Dreyfuss (University of Pennsylvania School of Medicine, PA) for a gift of antibody D4 5 to Trn-1 and to Dr Ichiro Tanaka for the use of a fluorescence microscope We thank Manabu Takahashi and... role for hnRNP D in the in vivo mRNA destabilization directed by the AU-rich element Genes Dev 13, 1884–1897 FEBS Journal 272 (2005) 3975–3987 ª 2005 FEBS hnRNP D ⁄ AUF1 nucleo-cytoplasmic shuttling sequence 33 Kawamura H, Tomozoe Y, Akagi T, Kamei D, Ochiai M & Yamada M (2002) Identification of the nucleocytoplasmic shuttling sequence of heterogeneous nuclear ribonucleoprotein d- like protein JKTBP and... primer designed for the defined N-terminal amino acid sequence of NLS and a 3¢ SalI-attached primer designed for 3¢-UTR The amplified cDNAs were subcloned at an EcoRI ⁄ SalI site in appropriate expression vectors: the bacterial expression vectors used were pGEX-6P-1 and pGEX-6P-3-EGFP-C composite vector encoding a GST-EGFP fusion protein; the mammalian expression vectors were pEGFP-C, pEGFP-GST encoding... 1000-fold diluted Trn-1 monoclonal antibody D4 5 Protein concentrations were determined by the Bradford method, using bovine serum albumin as a standard [46] SPR analysis was performed at 25 °C with a BIACORE type 3000 instrument Anti-GST IgG was fixed to a sensor chip CM5 at a concentration of 16820 resonance units (RU) by amine coupling according to instructions of the supplier (BIAcore BR-10023 and BR-100–50)... fusion protein, and pEGFP-GST-PAD encoding a tripartite protein EGFPGST-PAD (PAD is an N-terminal 104 residues sequence of a NLS deficient full-length PAD IV ⁄ V(1–663) [43]) For the preparation of C-terminal deletion NLS mutants, alanine scanning mutants and substitution mutants, synthetic oligomers (30–35-mer) were used as PCR primers, containing a stop codon or codons for defined single or triple... of JKTBP1 cDNA as a probe in the course of JKTBP cloning [42] Its entire coding sequence was amplified by PCR with a set of 5¢ and 3¢ EcoRI-attached primers (0.5 lm) and 6% (v ⁄ v) dimethylsulfoxide, and subcloned into an EcoRI site of pGEX-6P-1 (Amersham BioScience, Inc., Piscataway, NJ, USA) and pEGFP-C vectors (Clontech, Mountain View, CA, USA) The entire coding regions of human Trn-1, RanGAP and... Imasaki for their help in this work and Hidenobu Kawamura and Dr Mamoru Sato for discussion and advice This work was partly supported by grants-in-aids for Promotion of Research at Yokohama City University References 1 Gorlich D & Kutay U (1999) Transport between the cell ¨ nucleus and the cytoplasm Annu Rev Cell Dev Biol 15, 607–660 3986 M Suzuki et al 2 Nakielny S & Dreyfuss G (1999) Transport of proteins . Trn-1. Results Determination of hnRNP D NLS The exon 8 of hnRNP D encodes the 21 amino acids sequence common to all the isoforms D0 1, D0 2, D1 , and D2 (Fig. 1A) [23] that of D0 2. This larger K D was accounted for by the twofold larger k d of DNS than that of D0 2 and their nearly similar k a values. The N-four residue dele- tion