HnRNP I contains a novel type of bipartite nuclear localization signal NLS at the N-terminus of the protein that we have previously named nuclear determinant localization type I NLD-I..
Trang 1Importin a binds to an unusual bipartite nuclear localization signal
in the heterogeneous ribonucleoprotein type I
Maria G Romanelli and Carlo Morandi
Department of Mother and Child, Biology and Genetics, University of Verona, Italy
The heterogeneous nuclear ribonucleoprotein (hnRNP)
type I, a modulator of alternative splicing, localizes in the
nucleoplasm of mammalian cells and in a discrete
peri-nucleolar structure HnRNP I contains a novel type of
bipartite nuclear localization signal (NLS) at the N-terminus
of the protein that we have previously named nuclear
determinant localization type I (NLD-I) Recently, a neural
counterpart of hnRNP I has been identified that contains a
putative NLS with two strings of basic amino acids separated
by a spacer of 30 residues In the present study we show that
the neural hnRNP I NLS is necessary and sufficient for
nuclear localization and represents a variant of the novel
bipartite NLS present in the NLD-I domain Furthermore,
we demonstrate that the NLD-I is transported into the nuc-leus by cytoplasmic factor(s) with active transport modality Binding assays using recombinant importin a show an inter-action with NLD-I similar to that of SV40 large T antigen NLS Deletion analysis indicates that both stretches of basic residues are necessary for binding to importin a The above experimental results lead to the conclusion that importin a acts as cytoplasmic receptor for proteins characterized by a bipartite NLS signal that extends up to 37 residues Keywords: heterogeneous ribonucleoprotein-I; polypyrimi-dine tract-binding protein; PTB; nuclear localization signal; importin a
Transport of proteins and RNA into and out of the nucleus
occurs through nuclear pore complexes (NPCs), which are
plugged through the double membrane of the nuclear
envelope [1,2] Small molecules and ions may pass the NPC
passively, while macromolecules larger than 40–45 kDa are
actively transported through the NPC The active nuclear
import and export of proteins is mediated by specific
amino-acid sequences that are referred as nuclear localization
signals (NLSs) [3,4] and nuclear export signal (NESs) [5] At
least two different types of classical NLSs have been
defined: a short stretch of basic amino acids, exemplified by
the SV40 large T antigen NLS (T-ag PKKKRKV) [6] and a
bipartite NLS composed of two stretches of basic amino
acids separated by a spacer of 10–12 amino acids,
exempli-fied by nucleoplasmin (KRPAATKKAGQAKKKK) The
two sets of basic residues of bipartite-type NLS are required
for sufficient nuclear localization, while the spacer is
mutant-tolerant in sequence [7] NLSs are usually
recog-nized by the heterodimeric import receptor complex
com-prising importins a and b, also named karyopherins [8,9]
Importins a contain the NLS-binding site and importins b
are responsible for the docking of the importin–substrate complex to the cytoplasmic side of the NPC and its subsequent translocation through the pore Transfer through the pore of importin–NLS protein complex requires two additional soluble proteins, RanGTPase and nuclear transport factor-2 (NTF2) [1] Once inside the nucleus, Ran-GTP binding to importin b causes the disso-ciation of the import complex and release of the cargo [10,11] The directionality of the nuclear import is conferred
by an asymmetric distribution of the GTP and GDP-bound forms of Ran between the cytoplasm and the nucleus, with the GTP-form predominant in the nucleus [12,13] Based on the similarities of their primary structures, the importins a have been separated into three subfamilies, each of which shows distinct substrate specificity and differential expres-sion [14–16] Importins a consist of two structural and functional domains, a short basic N-terminal importin b binding (IBB) domain, and a large NLS-binding domain comprising armadillo (Arm) repeats [17–19] Crystal struc-tures of karyopherins a complexes with NLS peptide have revealed the determinants of specificity for the binding of NLS sequences [20–22]
A number of NLS sequences that do not conform to the classical NLS consensus motif have also been identified, such as the M9 sequence, present in the hnRNP A1, which is recognized by transportin (karyopherin-b2), rich in glycine rather than basic residues [23–25] A unique signal, called KNS, which allows nuclear transport via a mechanism independent of soluble factors has also been described in the hnRNP type K [26] These findings indicate that the most important level of control for nuclear protein transport is the targeting sequence
We have previously identified a novel bipartite NLS in the hnRNP type I protein [27], also known as polypyri-midine tract binding protein (PTB) [28,29], a member of the large set of RNA binding proteins, known as hnRNPs, that
Correspondence to M G Romanelli, Department of Mother and
Child, Biology and Genetics, University of Verona,
Strada le Grazie 8, 37134 Verona, Italy.
Fax: + 39 045 8027180, Tel.: + 39 045 8027182,
E-mail: mromanelli@univr.it
Abbreviations: NLS, nuclear localization signal; NLD-I, nuclear
localization determinant type I; T-ag, SV40 large tumor antigen;
hnRNP, heterogeneous nuclear ribonucleoprotein; FITC,
fluorescein isothiocyanate; NPC, nuclear pore complex; GST,
glutathione S-transferase; PTB, polypyrimidine tract-binding
protein; GFP, green fluorescent protein.
(Received 10 January 2002, revised 10 April 2002,
accepted 18 April 2002)
Trang 2have been implicated in mRNA maturation and transport
[30,31] In mammalian cells, hnRNPI/PTB functions as a
splicing repressor [32,33], and mediates exon skipping of
several genes, such as a- and b-tropomyosin premRNAs
[34,35], neuron-specific exon in the c-src, c-aminobutyric
acid A c2 receptor, clathrin light chain B, N-methyl-D
-aspartate premRNA [36,37] Interestingly, a PTB
homo-logue, abundantly present in the brain and in some neural
cell lines, known as neural PTB (nPTB), has been recently
identified [38] Such a protein interacts with neuron-specific
RNA binding proteins that participate in the control of
alternative splicing in neurons [39] nPTB is 74% identical in
amino-acid sequence to PTB and contains four unusual
RNA recognition motif (RRM) domains, and a putative
bipartite NLS near the N-terminus
HnRNP I localizes in HeLa cells both in the nucleoplasm
and in a discrete perinucleolar structure [29,41] We
previously reported that the N-terminal sequence of hnRNP
I/PTB contains a 60-amino-acid sequence that is both
necessary and sufficient to target the protein to the nucleus
[27] The sequence, named nuclear localization determinant
type I (NLD-I), is characterized by a NLS containing two
clusters of basic amino acids (KRand KKFK) that
resemble the nucleoplasmin bipartite signal, but are
separ-ated by an unusually long stretch of 30 amino acids
Compared to that of hnRNP I, the nPTB bipartite NLS
conserves the basic stretches whereas 14 residues differ in the
30-amino-acid spacer sequence
In the present work, we have characterized the NLD
domain present in the neural counterpart of hnRNPI/PTB
and investigated whether the hnRNPI/PTB import involves
the karyopherin a/b pathway We show that the hnRNP I
NLD-I domain displays significant binding to importin a,
which is diminished or eliminated by mutations in both
basic stretches The present data support the model for the
recognition of bipartite NLS derived by crystallographic
analysis
E X P E R I M E N T A L P R O C E D U R E S
Plasmid construction
A plasmid containing the full length cDNA of human nPTB
[39] was kindly provided by D L Black (Howard Hugnes
Medical Institute, Los Angeles, CA, USA) To generate
fusion constructs with the green fluorescent protein (GFP),
the nPTB entire coding region, or its fragments (Fig 2),
were obtained by PCRfrom the original cDNA The
forward and reverse primers used for PCRincluded SalI
and BamHI restriction enzyme site at the 5¢ and 3¢ ends,
respectively The PCRproducts were digested with SalI and
BamHI and cloned in frame with a 5¢-GFP coding sequence
into mammalian expression vector pEGFP-C1 (Clontech)
To introduce deletions into the NLD-I sequence of nPTB
we used a two-step PCRmethod In the first step we used
the following oligonucleotides: forward primer D11–13
(5¢-ATGGACGGAATCGTCACTGAAGTTGCAGTTA
reverse primer R1 (5¢-ATTGGATCCTTATACACGAGA
AGGAGCACC-3¢) to generate the pnPTB-NLD-I D11-13
mutant; forward primer F1 (5¢-GGCAGGCATTCAGTC
GACATGGACGGAATCGTCACT-3¢) and reverse
pri-mer D45-47 (5¢-TACACGAGAAGGAGCACCATCCA
TTTTATCTTCTCCTTTACTATCATTACCATTGGCT GT-3¢) to generate the pnPTB-NLD-I D45–47; forward primer D11–13 and reverse primer D45–47 to generate the pnPTB-NLD-I D11–13; 45–47 mutant (the numbers indicate the residues deleted within nPTB amino-acid sequence) In the second PCRstep, all the fragments were amplified with primers F1 and R2 that introduced the SalI and BamHI sites, respectively, and cloned in the pEGFP-C1 vector
The glutathione S-transferase (GST) fusion system was used to generate chimeric proteins [42] HnRNP I-NLD I, and mutants D11–13, D45–47, and D11–13; 45–47 frag-ments, previously cloned in a pA1-CAT vector [27,43], were amplified by PCRwith oligonucleotides that introduced EcoR I and HindIII restriction sites, and ligated into a modified pGEX-5X-1 vector (Amersham Pharmacia Bio-tech) where the XhoI had been mutated in a HindIII site
Cell culture and transfection Adherent HeLa cells were maintained in exponential growth
in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum Cells were grown on glass coverslips and transfected using Lipofectamine (Life Tech-nologies) according to manufacturer’s instructions Forty-eight hours after transfection, cells were washed with NaCl/Piand fixed in 2% paraformaldehyde in NaCl/Pi
at room temperature for 20 min Cellular localization of the nPTB–GFP fusion proteins was examined under a fluores-cent microscope
Expression of fusion proteins inEscherichia coli The pGEX-NLD-I, pGEX-NLD-I D11–13, pGEX-NLD-I D45–47 and pGEX-NLD-I D11–13; 45–47 constructs were transformed into the Escherichia coli strain BL 21(DE3) Cell culture and batch purification of the GST fusion proteins were performed essentially according to the manufacturer’s instructions (Amersham Pharmacia Biotech) All the proce-dures were carried out at 4C; 1 mM EGTA and 2 mM
dithiothreitol were included in the buffers throughout the purification procedures Recombinant GST proteins, after elution, were dialyzed at 4C against binding buffer (20 mM
Hepes, 150 mMKOAc, 2 mMMg (OAc)2, 2 mM dithiothre-itol) The concentration of proteins was determined by the method of Bradford [44] using the Bio-Rad dye reagent (Bio-Rad) and BSA as standard Protein samples were aliquoted, quick frozen in liquid N2and stored at)80 C
The E coli strain BLRcontaining GST fusions of a functional SV40 large T antigen nuclear localization signal (Tag NLS) or an inverse version of Tag NLS (Tag NLSinv) were cultured, and the proteins were purified as described previously [45]
Nuclear import assay Digitonin permeabilized HeLa cells were prepared essen-tially as described by Adam et al [46] Cells grown on coverslips were permeabilized with 55 lgÆmL)1 digitonin (Sigma) in transport buffer (TB: 20 mM Hepes pH 7.3,
110 mM potassium acetate, 5 mM sodium acetate, 2 mM
magnesium acetate, 1 mM EGTA, 2 mM dithiothreitol,
1 lgÆmL)1each of aprotinin, leupeptin, and pepstatin A)
Trang 3A standard 50-lg nuclear import assay was performed
in transport buffer containing an energy-regenerating
system (1 mM ATP, 0,5 mM GTP, 10 mM creatine
phos-phate, and 0.4 UÆmL)1 creatine phosphokinase), 5 lg of
GST fused proteins, and 30 lg of rabbit reticulocyte
lysate (Promega) as a cytosol source The reaction was
allowed to proceed for 45 min at 30C Where indicated,
wheat germ agglutinin (WGA); Sigma) at 50 lgÆmL)1, or
hexokinase at 100 UÆmL)1 and glucose at 10 mM, were
included Import assays were terminated by washing the
cells in cold NaCl/Pi followed by fixation in 2%
paraformaldehyde for 30 min
Immunofluorescence staining
Fixed cells were permeabilized for 3 min in)20 C acetone,
washed with NaCl/Pi and incubated for 40 min with
primary antibody diluted in 3% BSA/NaCl/Pi (1 : 100
monoclonal anti-GST Ig, Santa Cruz Biotechnology),
washed in NaCl/Pi and incubated for 40 min in 1 : 50
FITC-conjugated goat anti-(mouse IgG) Ig Cells were
finally washed with NaCl/Pi, coated with 90% glycerol in
NaCl/Pi, and observed under a Leitz Orthoplan microscope
with an epifluorescence attachment
In vitro binding assay
The plasmid pRSET-hSRP1 containing a cDNA of human
importin a [47] was used to produce a [35
S]methionine-labeled protein using the Promega TNT T7 Quick Coupled
Translation System Fifteen micrograms of GST–NLD-I or
NLD-I mutants and 7 lg of GST–Tag NLS or GST–
TagNLSinv were incubated with 40 lL of
glutathione-agarose beads (Amersham Pharmacia Biotech) in 0.5 mL of
binding buffer (20 mM Hepes, pH 6.8, 150 mM KOAc,
2 mMMg (OAc)2, 2 mMdithiothreitol, 0.1% Tween 20 for
2 h at 4C The beads were collected and washed three times
with binding buffer After washing, one-twentieth of the
beads were removed and the amount of immobilized GST
fusion proteins were analyzed by SDS/PAGE The rest of
the beads were incubated with 90 lL of in vitro translated
importin reaction mixture for 4 h at 4C The beads were
then washed six times in binding buffer, boiled in 30 lL of
sample buffer, and the immobilized proteins were resolved
on a SDS/10% polyacrylamide gel The35S-labeled importin
bound to the GST fusion proteins was detected by fluoro-graphy using Amplify Reagent (Amersham Pharmacia Biotech)
R E S U L T S
The NLD-I of neuronal PTB is capable of targeting
a heterologous protein to the nucleus
In a previous study, we identified the sequence implicated
in the nuclear transport of the human hnRNPI/PTB at the N-terminal of the protein [27] This region, which we called NLD-I, extends in the first 60 amino acids, upstream to the RRM1 (Figs 1A) The sequence contains two short basic sequences (KRand KKFK) that resemble the SV40 T-ag NLS, separated by an unusual long spacer sequence of 30 amino acids Both short basic sequence were necessary for transport and, taken alone, were not able to target the protein to the nucleus Sequence comparisons among NLD-I and the N-terminal region
of neural PTB and PTB homologues isolated from pig, rat, mouse and Xenopus show that the basic stretches are highly conserved in all the hnRNPI/PTB homologues, whereas the sequence of the spacer, that may vary from
29 to 33 amino acids, is less conserved (Fig 1B) The nPTB NLD-I sequence is identical in human and mouse, whereas the variations in NLD-I sequence between human nPTB and hnRNPI/PTB far exceed that among PTBs from different vertebrates
In order to characterize the novel type of bipartite NLS
we first examined the ability of the NLD-I motif of the neuronal PTB to target a heterologous protein to the nucleus GFP–nPTB fusion proteins were constructed in which NLD-I motif was deleted from nPTB or was the only sequence fused to GFP Following transfection with GFP constructs, HeLa cells were fixed and visualized by direct fluorescence (Fig 2) GFP is a small protein (30 kDa) that can passively diffuse through the nuclear pores and does not produce a subcellular localization bias (Fig 2A), whereas fusion of the entire nPTB ORF to GFP led to a peptide that accumulates exclusively in the nucleus (Fig 2B) The first 60 amino acids corresponding to the NLD-I domain are necessary and sufficient to localize the fusion protein completely in the nucleus, as demonstrated by the pGFP-nPTBD60 construct that was confined to the cytoplasm,
Fig 1 Functional domains of the human hnRNPI/PTB protein.
four RNA recognition motifs (RRM1–4) and the nuclear localization determinant type I (NLD-I) (B) Amino acid alignment of the hnRNP-I NLD-I domain with homologous domains from human and mouse nPTB, and from mouse, rat, pig and Xenopus PTB The basic clusters are in bold letters Alignment was performed by program Asterisks and dots show identical and similar amino acids, respectively.
Trang 4whereas when the first 60 amino acids are fused to GFP
(pGFP–nPTB60), the protein localized completely into the
nucleus (Fig 2D,C, respectively)
Deletions of three amino acids, including a lysine in the
first basic stretch (deletion Gly-Val-Lys in pGFP–
nPTBD11–13) abolished the nuclear localization and left
the fusion protein to diffuse passively through the nuclear
pore (Fig 2E) A similar cellular distribution was observed
when a serine and two lysines were deleted in the second
basic stretch (pGFP–nPTBD45–47) or when both type of
deletions were present in the basic stretches (construct
pGFP–nPTBD11–13; 45–47) (Fig 2F,G) The above
obser-vations indicated that the two basic motifs in NLD-I of
nPTB are interdependently required for full nuclear
local-ization of the chimeric protein Similar results were
previ-ously obtained with the NLD-I present in hnRNP I/PTB
that differ from that of nPTB essentially in the spacer
sequence (Fig 1) These data show that the intervening
sequence does not contribute to the nuclear localization and
may be modified without effect on localization, as it has
been shown for the bipartite NLS of nucleoplasmin [7]
Taken together, these results indicate that the identified
NLS motif is a bona fide nuclear import signal for PTB like
proteins The NLS contained in the NLD-I is a new variant
of bipartite NLS sequences
Nuclear import of GST–NLD-I is an energy-dependent process that requires soluble cytoplasmic factors and is inhibited by WGA
Due to the fact that NLD-containing NLSs motifs
imported by different mechanisms [48], we have undertaken
a study on the identification of the receptor pathway that mediates nuclear import of the PTB NLS An in vitro nuclear transport assay was used in which the plasma membrane of HeLa cells was permeabilized with the weak nonionic detergent digitonin that leaves the nuclear envel-ope intact [46] NLD-I motif was fused to a GST protein, that itself does not accumulate into the nucleus [49] (Fig 3A) Nuclear transport of GST–NLD-I was examined
in the presence of a transport buffer containing rabbit reticulocyte lysate as a source of cytosolic proteins and an ATP-regenerating system, to provide energy for transloca-tion The subcellular distribution of the GST–NLD-I was determined by indirect immunofluorescence microscopy using an antibody against GST In such experiments, we
Fig 2 The NLD-I domain of nPTB directs the nuclear import of a heterologous protein (A) Schematic representation of the nPTB regions used to produce GFP fusion proteins Structural domains are diagrammed with shaded boxes representing the RRMs Black boxes or black boxes interrupted by a white strip represent the basic stretches or the dele-ted sequences in the basic stretches, respect-ively, at the N-terminus of the protein (B) Plasmids expressing the native GFP (a), GFP fused to the entire nPTB (b), the 60-amino-acid region at N-terminus of nPTB (c),
or nPTB deleted of the first 60 amino acids (d), were transiently transfected, expressed in HeLa cells and visualized by fluorescent microscopy Likewise, GFP fused to the 60-amino acid region containing deletions at amino acids 11–13 (e), or 44–47 (f), or both type deletions (g) were also expressed in HeLa cells.
Trang 5found that GST–NLD-I was clearly visible within the nuclei
in cells were the plasma membrane was permeabilized by
digitonin, followed by incubation at 30C for 45 min with
rabbit reticulocyte lysate and an ATP energy-regenerating
system (Fig 3C) A similar distribution was observed for
GST–TagNLS, which is transported to the nucleus by the
conventional NLS-mediated nuclear protein import
path-way utilizing Ran and the importin ab/heterodimer [45]
(Fig 3B) This accumulation is ATP dependent as no
nuclear accumulation was observed when the permeabilized
cells were incubated with hexokinase and glucose to deplete
residual ATP, and when import assay was performed in the
absence of an ATP-regenerating system (Fig 3D) As
expected, the transport reaction was also
temperature-dependent; if the assay was carried out at 4C, no transport
was observed (Fig 3E), confirming the necessity of ATP
hydrolysis for nuclear protein import When permeabilized
cells were preincubated in transport buffer containing
50 lgÆmL)1WGA, a lectin that associates with glycosylated
nucleoproteins and inhibits nuclear pore complex function,
the nuclear accumulation of NLD-I was inhibited (Fig 3F)
To characterize the amino-acid sequence requirement for
nuclear import, we examined whether amino-acid deletions
into NLD-I would perturb import When we used GST–
NLD-ID11–13, GST–NLD-ID45–47, or GST–NLD-ID11–
13;45–47 as the cargo in different import assays, no
transport in the nucleus was seen (Fig 3G,H,J)
NLD-I domain binds to importin a
The previous data suggest that the import of the PTB NLS
might be mediated by cytosol receptor like the importin a/b
complex The crystal structure of importin a reveals two
NLS peptide binding pockets and the distance between the
two binding sites allows a 10-residue spacer to link the two
peptide segments [20,50] Importin a binding to a bipartite signal with a spacer sequence longer than 30 amino acids have not been tested thus far The unusual sequence of the PTB NLS raised the possibility that it might not be recognized directly by importin a
To test if NLD-I would bind to importin a, we first performed a binding assay using in vitro translated importin
a and recombinant GST–NLD-I fusion protein The GST fusions with SV40 tag NLS (GST–NLS) and an inverse version of Tag NLS (GST–NLSinv) served as positive and negative controls, respectively, for the importin a binding
As shown in Fig 4A, importin a is able to bind NLD-I (lane GST–NLD-I) To ascertain whether the amino acid that impaired nuclear transport in NLD-I deletion mutants, mediate the importin a binding, we tested importin a binding to NLD-I D11–13, NLD-ID45–47, or NLD-ID11– 13;45–47, expressed as GFP-fusions All mutants showed no detectable or very weak binding to importin a (Fig 4B) These results indicate that importin a binds to NLD–I by interaction to the residues of the two short basic stretches, when both basic stretches are present in the NLS sequence
D I S C U S S I O N
In this report, we have demonstrated that nuclear translo-cation of nPTB and hnRNPI/PTB occurs via a polybasic NLS sequence present in the N-terminus NLD-I motif This sequence functions in the nuclear import of PTB-like proteins via an active energy-dependent process and binds
to the importin a PTB NLS shares common features with the known bipartite type NLSs; in fact, it contains two clusters of basic amino acids, a smaller one of two basic amino acids (KR) and a larger one with three basic amino acids in a group of five (KKFKG); both are essential for full nuclear localization and importin a binding However, it
Fig 3 Nuclear import of NLD-I is an
energy-dependent process inhibited by WGA
Digito-nin-permeabilized HeLa cells were incubated
with GST, GST–TagNLS (a control for the
conventional importin a/b mediated nuclear
import pathway directed by SV40 NLS),
GST–NLD-I, or mutated NLD-I and
visual-ized by indirect immunofluorescence, using a
GST monoclonal antibody In vitro nuclear
transport (see Material and methods) was
carried out in the presence of cytosol and an
ATP-regenerating system (A–J) The effects
on nuclear transport of the ATP-regenerating
system omission (D), or 4 C incubation (E),
were examined Transport studies were also
carried out after preincubation with WGA
(F) Images are representative of at least three
independent experiments.
Trang 6represents a novel member of bipartite signal because there
are differences between the critical basic residues of NLS
and the consensus sequences of other bipartite signals, and
because the spacer between the basic motifs is unusually
longer (30 amino acids) than that of other well characterized
bipartite NLSs (Table 1) Searching SWISSPROT and
standard databases by PROSITE, and analyzing data
deposited at the PredictNLS server (http://maple.bioc
columbia.edu/predictNLS/) [56], we find that functional
bipartite NLSs are characterized by an intervening region of
different lengths, but not longer than 18 amino acids, like
the human androgen receptor NLS motif [57] In transfected
cells, it has been shown that the segment spacer of nucleoplasmin bipartite NLS can be replaced by a sequence
up to 20 alanine residues, without disrupting efficient nuclear import [8] Recently, a bipartite NLS, containing a spacer between the basic motifs of 32 amino acids, has been functionally characterized in hypoxia inducible factors 1a [58] We indicate a consensus sequence for bipartite long type NLS as KRx(30–32)K[K/R]xK, according to Cokol
et al [56] Searching the PredictNLS database using this motif, we found 61 proteins, with a true nuclear protein percent of 83.6
Nuclear import of most proteins requires both impor-tins a and b, with importin a as the adapter between importin b and the cargo protein Some proteins undergo nuclear import via direct binding to importin b without involvement of importin a Our results suggest that nuclear import of PTB-like proteins, and probably of all the proteins that contain the NLD-I type NLS, is an energy-dependent process mediated by importin a Deletions in the basic regions within the N-terminal domain of hnRNP I and nPTB inhibit nuclear import and/or accu-mulation and reduces importin a binding This conclusion fits with the in vitro binding studies, which showed that the NLD-I domain binds strongly to importin a and the bind-ing is diminished or eliminated even if only one of the two basic stretches are mutated The affinity of the importin-targeting sequence interaction is a critical parameter in determining transport efficiency [5] This is the first study
to demonstrate that importin a recognizes and binds an NLS sequence that extends up to 37 residues The crystal structures of importin a [20–22,50] clearly reveal two distinct binding sites that can accommodate both essential elements of the bipartite NLS The larger binding site is structured optimally for the recognition of five lysine or arginine residues, while the smaller binding site allows specific recognition of two basic residues, and the interac-tion is simultaneous at both sites The distance between the two binding sites allow a 10-residue spacer to link the two peptide segments, while a shorter linker would impair the simultaneous binding of the two clusters The smaller basic cluster is required to be upstream of the larger cluster The
Table 1 Bipartite type nuclear localization signal The single-letter amino acid code is used The bold letters indicate the two arms of basic residues
of the bipartite NLS aa, amino acids.
Bipartite short type NLSs
Bipartite long type NLSs
a CAP-binding protein 80 b Xenopus laevis phosphoprotein c S cerevisiae transcription factor d Hypoxia inducible factor 1a.
Fig 4 Binding of importin a to wild-type or mutated NLD-I (A)
Wild-type GST–NLD-I, or GST–NLS and GST–NLSinv, immobilized on
glutathione sepharose 4B, were incubated with 90 lL of in vitro
translated protein a labeled with [ 35 S]methionine for 4 h at 4 C The
proteins were separated by SDS/10% PAGE gel, and bound importin
a was analyzed by fluorography (upper panel) An amount
repre-senting 1/20th of the beads incubated with GST fusion proteins,
extensively washed, was resolved by SDS/PAGE and stained with
Comassie-blue (lower panel, CB staining) (B) Binding of importin a
was also analyzed using immobilized GST–NLD-I peptides mutated
by deletions The results are representative of two independent
experiments.
Trang 7spacing between a defined number of binding sites acts as
molecular ruler that sets further constraints on target
specificity Binding properties of NLD-I type NLS to
importin a are consistent with crystallographic structure of
interaction with bipartite signal and further characterize
the bipartite signals, confirming that the linker sequence
does not contain a consensus, and may be as long as 30
amino acids
Mammalian paralogs of importin a have recently been
discovered and six genes for importin a have been found in
human The importin a that we have used in our binding
experiments (hSRP1a) represents one of the three
sub-families of importins a [15] Several experiments clearly
support the hypothesis that importins a might be specialized
in their efficiency to transport different nuclear proteins [59]
It will be interesting to analyze the binding specificity of the
different types of importin a to NLD-I NLS and to the
putative NLD-I type NLSs present in other nuclear
proteins
A C K N O W L E D G E M E N T S
We wish to thank Pamela Lorenzi for excellent technical help We
thank Michael F Rexach for his generous gift of GST–NLS and GST–
NLSinv constructs and Karsten Weis for his generous gift of
pRSET-hSRP a plasmid This work was supported by grants from Ministery of
Scientific Research and Technology (ex 60%).
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