From the Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037
Received for publication, February 3, 2003
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ABSTRACT |
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Molecular recognition of the importin Importin The crystal structure of importin As seen in the crystal structure of importin Using the three-dimensional structure of the importin Analysis of the Importin Site-directed Mutagenesis, Expression, and Purification--
All
mutants were derived from a full-length His-S-tagged importin Microtiter Plate Binding Assays--
Solid phase binding assays
were carried out on microtiter plates (Maxisorp) coated with 100 ng of
recombinant GST·IBB domain (residues 1-60). Assays were conducted as
described (4, 17), and binding data were analyzed with Prism 3.0 software. All curves were fitted using non-linear regression, and the
apparent dissociation constant was calculated using the GraphPad Prism program.
Nuclear Import Assay, Microinjection, and Immunofluorescence
Microscopy--
The nuclear import assay was carried out in NRK cells
permeabilized with digitonin and subsequently incubated with 10 µg/ml RanQ69L and 10 µg/ml RanBP1 for 15 min at 30 °C to deplete them of
endogenous importin Conserved Tryptophans of Importin
To understand the importance of the four conserved tryptophans in the
recognition of the IBB domain, we generated libraries of importin Binding of Importin Functional Characterization of Importin Translocation of Importin Cation-
To further investigate our predictions, we used the program CaPTURE
(Cation-
Based on our reexamination of the structure of the import adaptor
importin Population Selection and Binding--
The dynamics of the importin
The view of the IBB domain as a population of dynamically
interconverting conformers (31, 32) suggests that the recognition of
the IBB domain by importin
In this regard, the position of the tryptophans in importin
This view of tryptophan as a "stabilizer" of the IBB helical
folding agrees with the observation made by Burley and Petsko (34, 35)
that an aromatic residue such a tryptophan can engage in specific
energetically favorable interactions stabilizing protein structure. In
contrast, in the case of the Trp Conclusion--
Our data strongly suggest that the importin
-binding
(IBB) domain of importin
by importin
is critical for the
nuclear import of protein cargoes containing a classical nuclear
localization signal. We have studied the function of four conserved
tryptophans of importin
(Trp-342, Trp-430, Trp-472, and Trp-864)
located at the binding interface with the IBB domain by systematic
alanine substitution mutagenesis. We found that Trp-864 is a mutational hot spot that significantly affects IBB-binding and import activity, whereas residues Trp-342, Trp-430, and Trp-472 are mutationally silent
when analyzed individually. Interestingly, the combination of the hot
spot at residue Trp-864 with mutations in the other three tryptophans
gives rise to a striking synergy that diminishes IBB domain binding by
up to ~1000-fold and, in turn, abolishes import activity. We propose
that importin
uses the tryptophans to select and stabilize a
helical conformation of the IBB domain, which, in turn, conveys
specific, high affinity binding.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(also known as karyopherin
-1) represents the
prototype of the importin
-superfamily of nuclear transport
receptors (1-3). The interaction of importin
with import cargoes
involves recognition of the nuclear localization signal
(NLS),1 either directly, as
in the case of cargoes like parathyroid hormone-related protein (PTHrP)
(4), or via an adaptor. The adaptors importin
and snurportin, which
are involved in the import of classical NLS (cNLS) (1-3) containing
cargoes and trimethylguanosine (m3G)-capped U small nucleolar
ribonucleoprotein (snRNP) particles (5), respectively, interact
with importin
via an N-terminal basic importin
-binding (IBB) domain (1-3). The IBB domain represents the minimal structural region of importin
that binds to importin
with high affinity and can itself serve as a functional NLS (1-3).
For example, large cargoes such as
-galactosidase fused to the IBB
domain are specifically imported into the nucleus of permeabilized
cells by importin
(6, 7). Translocation of the import complex
through the nuclear pore complex (NPC) involves multiple rounds of
interaction of importin
with nucleoporins (8). An essential
cofactor is the small GTPase Ran, which, in its GTP-bound form,
dissociates importin
from nucleoporins and from the
adaptor·cargo complex (1-3).
complexed with the IBB domain of
importin
(residues 11-54) was solved by x-ray crystallography to a
resolution of 2.3Å (9). Importin
contains a modular structure
built of 19 tandem HEAT repeats arranged to form a superhelix. Each
HEAT repeat represents a secondary structure motif formed by two
helices connected by a loop. The HEAT repeat array contains two
structurally and functionally distinct surfaces. The convex outer
surface has nucleoporin-binding sites (10), and the concave internal
face binds Ran (HEAT repeats 1-8) (11) and cargoes. Whereas the IBB
domain binds mostly to the C terminus of importin
(HEAT repeats
7-19) (9), PTHrP interacts with the N-terminal HEAT repeats 2-11 in a
region of the protein that overlaps with the Ran-binding domain
(4).
complexed with the IBB
domain of importin
, residues 22-51 of the IBB domain form a
straight helix, whereas the N-terminal moiety (residues 11-21) adopts
a more extended conformation, interrupted between residues 13-15 by a
310 helix. The importin
·IBB domain-binding interface
involves an extended network of interactions, which accounts for over
40 specific bonds including electrostatic, hydrophobic, and Van der
Waals contacts (9). Likewise, the binding cavity of importin
is
highly enriched in acidic residues and affords an ideal environment for
the folding of a basic peptide. This has led to the idea that importin
may serve both as an import receptor and a chaperone to keep small
basic proteins from aggregating (12).
·IBB domain
complex as a guide, we have analyzed the molecular basis for the
recognition of the IBB domain of importin
. Through systematic alanine substitution mutagenesis, we have targeted four tryptophans of
importin
located at the binding interface. We have identified a
single tryptophan that is essential for efficient binding to the IBB
domain and severely diminishes the binding affinity after mutation to
alanine. Moreover, we have demonstrated that this hot spot can
synergize with mutations in other tryptophans at the binding interface,
which only marginally affect the interaction when analyzed alone. Based
on these data, we propose a model for recognition of the IBB domain by
importin
that involves the selection of a population of distinct
helical IBB conformers by the receptor.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
·IBB Domain-binding
Interface--
The crystal structure of the importin
·IBB domain
complex (9) (Protein Data Bank accession number 1QGK) was extensively analyzed using the program O (13). All ribbon and sticks-and-ball models were produced using the Bobscript (14) and RASTER3D (15) programs.
plasmid (16) and recloned in pTYB4 vector (New England Biolabs). This
plasmid was used as a template for site-directed mutagenesis using the
QuikChange mutagenesis kit (Stratagene), as directed by the
manufacturer. Wild type and Trp
Ala importin
mutants were
expressed in the Escherichia coli ER1003 strain, and
purification was performed on chitin beads followed by gel filtration
on a Superose-12 column (Amersham Biosciences). After purification, all
Trp
Ala mutants and wild type importin
were extensively
dialyzed against transport buffer (20 mM Hepes, pH 7.3, 110 mM KOAc, 2 mM MgOAc, 2 mM
dithiothreitol, and 1 µg/ml pepstatin, leupeptin, and aprotinin) and
subsequently concentrated to ~25 mg ml
1 using a
Millipore concentrator (molecular mass cutoff, 10 kDa). Recombinant importin
, RanQ69L, NTF2, RanBP1, and GST·IBB were expressed and purified as described previously (7, 16).
(18-19). The cells were then preincubated on
ice with importin
mutants or with an equivalent volume of transport
buffer, and nuclear import was carried out and quantified by flow
cytometry as described (19). Microinjection experiments in NRK cells
were carried out as described previously (20). Microinjected cells were
fixed after 1, 5, 30, or 60 min in 4% formaldehyde and permeabilized
with 0.2% Triton X-100. The localization of the various injected
importin
constructs was determined by indirect immunofluorescence
microscopy using a polyclonal goat anti-S antibody (Amersham
Biosciences).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
at the Binding Interface with
the IBB Domain--
One of the intriguing features of the importin
·IBB-binding interface is the presence of four conserved
tryptophans in importin
, Trp-342, Trp-430, Trp-472, and Trp-864
(Fig. 1a), which are located
in close proximity (3.8-4.2 Å) to four highly conserved IBB residues
(9), namely Lys-18/22 (Fig. 1, b and c) and
Arg-13/51 (Fig. 1, d and e). Whereas the indole
rings of Trp-430, Trp-472, and Trp-864 point directly toward the
-amino-terminal groups of IBB-Lys18/22 and the guanidinum group of
IBB-Arg-51, respectively, W342 is oriented more toward the backbone of
IBB-Arg-13. The extended conformation adopted by IBB-residues Lys-18/22
and Arg-13/51 exposes the polar guanidinium and
-amino-terminal
groups to engage in bidentate and monodentate interactions with at
least other six residues of importin
and four residues from the IBB
domain (Fig. 1, b-e).
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Fig. 1.
Importin tryptophans at the IBB domain binding interface.
a, worm representation of importin
bound to the IBB
domain (depicted as red ball-and-sticks). The four
tryptophans Trp-342, Trp-430, Trp-472, and Trp-864, located at the
binding interface, are shown as space-filled objects. b-e,
close-up views of the molecular interactions engaged by the four
importin
tryptophans (in yellow). IBB-residues directly
contacted by the tryptophans are in red, whereas other
importin
and IBB-residues forming the binding pockets are show in
gray and red, respectively. Nitrogen, oxygen, and
sulfur atoms are colored in blue, red, and
green, respectively.
point mutants (Table I) in which each
tryptophan was replaced by an alanine, a residue that is compatible
with
-helical folding. A first generation library consisted of the four Trp
Ala single point mutants. Additional tryptophans in these
single point mutants were then mutated to produce six double and four
triple Trp
Ala mutants, representing the second and third
generation mutant libraries, respectively. In total, fourteen Trp
Ala importin
mutants were expressed in E. coli and
purified to homogeneity. Similar to wild type importin
, all the Trp
Ala mutants displayed high solubility under physiological salt conditions (see "Experimental Procedures") and were eluted as a
monomer by gel filtration chromatography (data not shown). In addition,
circular dichroism analysis revealed that the Trp
Ala mutations did
not alter the secondary structure content of the mutants (data not
shown). Together, these data indicate that the point mutations did not
disrupt the overall folding of importin
.
Binding of wild type and Trp Ala importin
mutant to the IBB
domain
(Trp
Ala) Mutants to the IBB
Domain--
We used a solid phase binding assay to study the effect of
the Trp
Ala mutations on the interaction of importin
with the IBB domain of importin
. The IBB domain fused to GST was adsorbed to
microtiter plates, and the binding of either wild type importin
or
the Trp
Ala mutants was analyzed in a concentration range of
0.01-100 nM (Fig. 2,
a-c). For wild type importin
we reproducibly measured an apparent dissociation constant
(Kd) for the IBB domain of about 1 nM
(Fig. 2a and Table I). Whereas the first generation importin
(Trp
Ala) mutants in residues Trp-342, Trp-430, and Trp-472 did
not show a significant decrease in their binding affinity for the IBB
domain, the mutation of Trp-864 reduced the dissociation constant for
the IBB domain by ~35-fold (Kd = 35.1 nM; Fig. 2a and Table I). This suggested that
Trp-864 is a mutational hot spot (21). Second generation Trp
Ala
mutants in which the Trp-864 was not mutated were still able to bind
the IBB domain with nearly wild type affinity (Fig. 2b and
Table I). In contrast, all second generation Trp
Ala mutants
containing a combination of a single silent mutation (Trp-342, Trp-430,
and Trp-472) and the hot spot Trp-864 showed a dramatic decrease in their binding affinity (~360-450-fold; see Table I). An even more
pronounced synergy of silent and hot spot mutations was observed for
the third generation Trp
Ala importin
mutants (Fig.
2c). In this case, the only triple mutant that showed
sufficiently strong binding to accurately measure a
Kd in the 0.01-100 nM importin
concentration was the triple mutant W342A/W430A/W472A. We therefore
repeated the binding assay using up to a 10-fold higher concentration
of importin
(0.01-1 µM range) for both second and
third generation mutants (Fig. 2d). From these measurements the apparent dissociation constant of second and third generation mutants containing the hot spot W864A was reduced to ~400 and 950 nM, respectively (Table I). However, because the binding isotherms did not reach saturation in these cases, these values should
be viewed as approximations.
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Fig. 2.
Isotherms depicting the binding of wild type
and importin Trp
Ala mutants to the IBB
domain of importin
. First generation
(a), second generation (b), and third generation
(c) importin
Trp
Ala mutants were analyzed in the
concentration range 0.01-1 nM. In panel
d, the second and third generation mutants were re-assayed
in a concentration range of 0.1-1 µM. The curves
obtained in each panel were obtained by averaging three independent
measurements carried out under identical conditions. The apparent
dissociation constants (Kd) calculated using the
GraphPad Prism program are presented in Table I. wt, wild
type.
Trp
Ala Mutants in
Nuclear Import--
To investigate whether the decreased binding
affinity of the importin
mutants for the IBB domain of importin
resulted in a reduced level of nuclear import of cNLS-cargo, we assayed the Trp
Ala mutants in a nuclear import assay involving
permeabilized HeLa cells reconstituted with recombinant transport
factors (18-19) (Fig. 3). Bovine serum
albumin (BSA) coupled to a cNLS peptide was used as the cargo for
import. In the presence of ATP, wild type importin
stimulated
import of BSA-cNLS by ~4-fold (designated as 100% in Fig. 3) with
respect to a control reaction wherein no recombinant importin
was
supplied to the cells. Whereas the single point mutants targeting
residues Trp-342, Trp-430, and Trp-472 supported nuclear import of
BSA-cNLS cargo at nearly wild type levels, the W864A mutant yielded
only ~60% of the level of import (Fig. 3). In contrast, the second
generation mutants yielded a significantly lower level of import, which
was most pronounced for constructs containing the hot spot W864A
mutation. For instance, the import levels with the W342A/W430A and
W342A/W472A double mutants were about 50% of wild type importin
,
and the levels with double mutants containing the T864A substitution
were only ~25-40% of the wild type (Fig. 3). Finally, the three
third generation mutants bearing the hot spot W864A mutation were
almost completely inactive in the nuclear import of NLS-BSA, whereas
the triple mutant carrying the three weaker mutations
(W342A/W430A/W472A) was only 50% reduced in import.
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Fig. 3.
Functional characterization of Trp Ala
mutants of importin
. Nuclear import
reactions in digitonin-permeabilized cells were conduced with wild type
(w.t.) importin
and 14 Trp
Ala mutants. Each import
reaction was repeated twice, the two values were averaged, and the mean
value was plotted in a histogram. Under optimal condition an ~4-fold
stimulation of nuclear import of BSA-NLS was observed with added wild
type importin
(which corresponds to 100% on the y-axis)
as compared with a negative control lacking exogenous importin
. The
level of nuclear import obtained with the Trp
Ala importin
mutants is plotted as a percentage of the wild type importin
import. The level of BSA-NLS accumulation observed under conditions of
ATP depletion was subtracted from each import reaction (~5-10% of
the total).
Trp
Ala Mutants Through the
NPC--
We next examined whether the importin
triple mutants that
were strongly deficient in supporting the import of cNLS-containing cargoes also were impaired in their ability to translocate through the
NPC. Wild type importin
or the triple mutants containing the W864A
mutation were microinjected in the cytoplasm of NRK cells, and at
various times the cells were fixed and importin
localized by
immunofluorescence staining with antibodies to the S-epitope tag (Fig.
4a and b). As shown
for the triple mutant W342A/W430A/W864A, the injected importin
mutant was strongly concentrated in the nucleus after 1 min (Fig. 4) as
well as after longer times (data not shown). Similar results were
obtained for the other two triple mutants that were highly deficient in
nuclear import (W342A/W472A/W864A and W430A/W472A/W864A, data not
shown). In related studies, it has been reported that the C-terminally deleted importin
mutants, which were deficient for binding to the
IBB domain, also efficiently accumulated in the nucleus permeabilized HeLa cell in the absence of exogenous transport factors (22). We
conclude that the binding of importin
to the importin
·cNLS-cargo complex and the ability of the receptor to translocate
through the NPC are not coupled obligatorily. It is unlikely that the efficient nuclear translocation of the importin
Trp
Ala triple mutants observed in the microinjection experiments occurs as a consequence of their binding to importin
-independent cargoes like
the PTHrP (4), because the interaction between the nonclassical-NLS of
the PTHrP and importin
critically involves tryptophans Trp-342, Trp-430, and Trp-472 (4).
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Fig. 4.
Subcellular localization of microinjected
importin . Wild type (w.t.)
importin
(a, right panel) and the
triple mutant W342A/W430A/W864A (342×430×864)
(b, right panel) were microinjected in
the cytoplasm of NRK cells together with a microinjection marker
(a and b, left panels) to
verify the integrity of the plasma membrane and cell nucleus.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Interactions at the Importin
·IBB-binding
Interface--
The mutagenesis of conserved tryptophans at the
importin
·IBB domain-binding interface described above has
revealed a key role of the tryptophans in the molecular recognition of
the IBB domain. We propose that, at least in the case of residues
Trp-430, Trp-472, and Trp-864, the tryptophans engage in cation-
(23-24) interactions with residues of the IBB domain. The cation-
interaction is a type of long-range, non-covalent intermolecular force
between cationic residues and aromatic side chains. The planar ring of the tryptophans provides
electrons, whereas the cationic guanidinum and
-amino-terminal groups of arginines and lysines, respectively, provide the cationic groups. The importance of cation-
interactions in protein structure is becoming increasingly recognized (23-24). In
contrast to hydrophobic interactions, cation-
contacts are electrostatic interactions between dipoles and are completely abolished
by alanine mutagenesis, as alanine lacks the delocalized nucleophilic
charge of an aromatic ring.
Trends Using Realistic Electrostatics) (24), which predicts
and quantifies cation-
interactions based on ab initio
calculations from three-dimensional structures. Interestingly, CaPTURE
predicts that three of the four importin
tryptophans at the binding
interface that we studied engage in energetically significant
intermolecular cation-
interactions with IBB residues. The energetic
contributions were estimated to be approximately
5.41 kcal/mole and
4.61 kcal/mole for the Trp-864·IBB-Arg-51 and Trp-430·IBB-Lys-18
pairs and
2.67 kcal/mole for Trp-472·IBB-Lys-22 pair, respectively
(Table II). Moreover, as predicted by
visual examination of the three-dimensional structure, the potential energetic contribution due to a cation-
interaction for the
Trp-342·IBB-Arg-13 pair was not significant (<2 kcal/mol), as the
ring of the tryptophan points more toward the main chain of the
IBB-Arg-13 than to the cationic guanidinium group. These values
reinforce the concept that Trp-864 is a hot spot mutation but do not
explain the synergy of hot and silent point mutations observed in
second and third generations (see below).
Energetic contribution of cation- interactions
2
kcal/mol.
(25-26), we note that potential cation-
interactions also are found at the binding interface between importin
and the
conserved arginines/lysines of both classical monopartite (25) and
bipartite (26) NLS-peptides. Because both importin
and importin
may have evolved from a common precursor (4, 27), it is possible that
cation-
interactions represent an evolutionary conserved feature of
nucleoplasmic transport factors governing NLS recognition.
·IBB domain binding interface cannot be understood without
considering the structural plasticity of the ligand, the IBB domain.
Previous studies have shown that the IBB domain adopts different
conformations in different structural contexts. Whereas it is mostly
-helical when bound to importin
(9), it is essentially
unstructured in solution (28). Furthermore, a portion of the domain
adopts an extended conformation when bound to the cNLS binding groove
of importin
, suggesting an auto-inhibitory mechanism for binding to
the NLS cargo (29). Such conformational flexibility suggests that, in
solution, the IBB domain exists in a range of conformations with low
energy barriers between them. Similar structural plasticity was
reported previously for human immunodeficiency virus type 1 (HIV-1) Rev
(30), another small, basic importin
-binding protein (1-3), and
probably reflects an intrinsic propensity of arginine-rich polypeptides
(30).
may follow a population selection model
(31, 32). Accordingly, the receptor would select the helical
conformation of the IBB domain, which complements most favorably the
importin
-binding cavity (Fig. 5),
because of the electrostatic complementarity of basic IBB side chains
projecting along the helix peptide and acidic residues of importin
.
The binding of the helical IBB-conformer would be energetically driven by the large enthalphic nature of the
binding,2 which is
accompanied by the formation of over 40 specific contacts between the
protein and the peptide (9). Moreover, the high affinity binding
of a distinct helical IBB conformer must be dependent on the degree of
structural stabilization of the helix imposed by importin
. We
propose that the tryptophans of importin
control this step of the
binding reaction by stabilizing the helical conformation of the IBB
domain primarily via cation-
interactions with IBB-Arg-51 and
Lys-18/22.
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Fig. 5.
Model for involvement of tryptophans in
selection of the helical IBB domain by importin
. Tryptophans 430, 472, and 864 (represented
as space-filled objects) located at the N and C termini of the
IBB-helix, respectively, are suggested to stabilize the interaction of
the C-terminal domain of importin
with the IBB domain helical form.
The IBB-helix is mostly unfolded when not bound to importin
, but is
in equilibrium with a population of the helically folded form.
provides a rational explanation for the existence of weak and hot spots
mutations. Whereas a single residue Trp-864 would stabilize the C
terminus of the IBB helix, three separated residues near the N-terminal
end of the helix could play overlapping roles. Residues Trp-430 and
Trp-472 are located in immediate proximity to the N terminus of the IBB
helix (Fig. 5), and Trp-342 is next to the 310 helix
(IBB-residues 15-18) (Fig. 1a). The hot spot can be
interpreted as a destabilization of the C terminus of the IBB helix due
to the loss of the single tryptophan located in that area (33).
Conversely, it is possible that mutations W342A, W430A, and W472A do
not individually play a critical role, as they can complement each
other. The synergy of hot spot and silent mutants would derive from the
fact that each of the three silent mutations on the N terminus of the
IBB helix is enhanced by a mutation destabilizing the C terminus of the
IBB helix, which leads to the inability of importin
to select the
helical form of the IBB domain.
Ala mutants the small methyl group
of alanines is incompatible with cation-
interactions with the IBB
side chains. This reduces the constraints on the helix stabilization
and, in turn, prevents the high affinity binding of the helical IBB
domain by importin
.
·IBB domain binding interface is not a static structural state
wherein a protein and the ligand fit together as lock and key, as
originally proposed by Chothia and Janin (36). Rather the molecular
recognition of the IBB domain by importin
may be dissected into the
following two distinct events: (i) the selection of a helical IBB
domain sub-population inside the acidic groove of importin
, which
stabilizes the IBB-helix (residues 22-51) via tryptophan residues; and
(ii) the stable binding of the IBB-side chains arranged on the surface of the IBB-helix to the acidic binding cavity of importin
. The concept that the specificity of a NLS is encoded in its primary as well
as secondary structure may be crucial for understanding the flexible
yet highly specific recognition of a NLS by nuclear transport receptors.
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ACKNOWLEDGEMENTS |
---|
We thank Janna Bednenko for help with the flow cytometry and Sarmad Al-Bassam for technical assistance.
![]() |
FOOTNOTES |
---|
* The work was supported by National Institutes of Health Grant GM41955 (to L. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Strukturelle Biologie, Max-Planck-Institut fuer
Molekulare Physiologie, Dortmund, Germany 44227.
§ To whom correspondence should be addressed: Dept. of Cell and Molecular Biology, The Scripps Research Inst., 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-8514; Fax: 858-784-9132; E-mail: lgerace@scripps.edu.
¶ Supported by a Human Frontier Science Program post-doctoral fellowship.
Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M301137200
2 G. Cingolani, C. Koerner, T. Guan, and L. Gerace, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
NLS, nuclear
localization signal;
cNLS, classical nuclear localization signal;
PTHrP, parathyroid hormone-related protein;
IBB, importin binding;
NPC, nuclear pore complex;
GST, glutathione S-transferase;
BSA, bovine serum albumin.
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REFERENCES |
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