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
Trang 1hnRNP 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 bimono-partite 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-terN-ter-minal 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.
Trang 2HuR, 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 transcripfunc-tional 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
Trang 3B
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; panPan-els e–h, nuclear DNA stained with DAPI of the cells in the same view as in panPan-els 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.
Trang 4essential 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.
Trang 5located 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 30C but not at
4C 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 mediasugges-ted 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.
Trang 61-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 4C 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.
Trang 7interaction 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 25C were studied by SPR The response signal was
monit-h
r
GFP
DAPI
288-306
q
1-306
288-303
+
GFP
DAPI
Trn-1
DAPI
GST-GFP-DNS
Competitors hnRNP A1 UP-1
A
B
30 oC 4 oC
C
+ Trn-1
Ran + energy
+ Ran energy + Apyrase
+ Ran + energy
+ Ran + energy
Trn-1
293-306
+
d
GFP
DAPI
Trn-1
a
e
h
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 C or 4 C and studied for GFP fluorescent signal and nuclear DNA Pan-els a and b, localization of DNS; panPan-els c and d, nuclear DNA in the same views as in panPan-els 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.
Trang 8ored 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 (ka), dissociation rate constants (kd) and dissociation constants (KD) of D02, DNS, and DNS mutants for Trn-1 were determined at various concentrations of Trn-1 (2.5–40 nm) at 25C by SPR (Table 1) The KD
C
D
E
Trang 9of D02 was 4.0 nm and the KDof DNS 9.2 nm, that is
double that of D02 This larger KDwas accounted for
by the twofold larger kdof DNS than that of D02 and
their nearly similar kavalues 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 KDvalues than that of DNS These larger
KDvalues were largely accounted for by the decreased
ka 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 KD values than
DNS, largely accounted for by their lower ka values
Other mutants, including the C-terminal mutants,
showed too weak bindings to determine as described above For comparison, the KD, ka, and kd values of hnRNP A1 for Trn-1 were estimated to be 6.3 nm, 2.2· 105 m)1Æs)1, and 1.4· 10)3s)1, respectively The
KD 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
ka(1 ⁄ M )1Æs)1) k
d (1 ⁄ s) KD(n M )
3.5 · 10)3 24.7
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.
Trang 10to 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 vitrocould 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 KD values with a decreased ka and fairly invaried kdfor 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-terC-ter-minal 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