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INTRODUCTION |
The entry of karyophilic proteins into the nucleus through the
nuclear pore complex (NPC)1
is effected by specific targeting signals called nuclear localization sequences (NLSs) (1, 2), and is a receptor-mediated (3, 4),
energy-dependent (5, 6) process. The key factors involved
are members of the NLS-binding importin/karyopherin family (7-11), and
the monomeric GTPase Ran/TC4 (12, 13) and auxiliary proteins such as
NTF2/p10 (14, 15). In the first step, the NLS-containing protein is
recognized by the importin heterodimer through the NLS-binding importin
subunit (3, 7, 9), and then targeted to the NPC through the
affinity of the importin
subunit (8, 10, 11, 16) for NPC components
(17, 18). In the second step requiring cytoplasmic RanGDP (19, 20), the
complex is translocated through the NPC (21) and importin
and the
NLS-containing protein released into the nucleus through the action of
RanGTP (19). Alternative signal-mediated nuclear import pathways
independent of the importin
/
heterodimer have recently been
identified where either importin
(22, 23) or importin
-related
homologs (24-26) appear to fulfil the role of both importin
and
in binding NLSs and targeting them to the NPC (24, 25, 27).
Although NLS receptors from different species share structural and
functional homology, the recognition of different types of NLS by
importin subunits and its importance in the nuclear import process has
not been adequately investigated in quantitative terms (28, 29). In
this study, we compare the NLS binding and transport properties of
mouse and yeast importin subunits. We find that although mouse
(m-IMP
) and yeast (y-IMP
) importin
s can bind different types
of NLS, y-IMP
binds with higher affinity. y-IMP
does not
significantly affect the affinity of y-IMP
for the NLS of the simian
virus SV40 large tumor antigen (T-ag) but does enhance binding to the
NLS of the Xenopus laevis phosphoprotein N1N2. In contrast,
m-IMP
requires m-IMP
for high affinity binding. Nuclear import of
a T-ag-NLS containing fusion protein in vitro was found to
be enhanced by NTF2 but did not require it absolutely, whereas import
of an N1N2 NLS-containing fusion protein had an absolute requirement
for NTF2. Surprisingly, high NTF2 concentrations inhibited nuclear
import in all cases. The results indicate that different types of NLS
have different requirements with respect to the factors mediating
nuclear import, and imply a differential, regulatory role for NTF2 in
nuclear transport.
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MATERIALS AND METHODS |
Fusion Proteins--
The amino acid sequences of the NLSs used
are shown in Table I. Constructs to express fusion proteins
T-ag-CcN-
-Gal, and the NLS-defective mutant derivative
T-ag-Cc-
-Gal containing T-ag sequences fused amino-terminal to the
Escherichia coli
-galactosidase sequence (amino acids
9-1023) have been described (30, 31). The plasmid pPR-N1N2-
-Gal
expressing Xenopus laevis N1N2 amino acids 465-581 fused
amino-terminal to
-galactosidase has been described (32). Plasmids
encoding the N1N2 NLS-mutated derivatives N1N2A-
-Gal and
N1N2B-
-Gal were derived through oligonucleotide site-directed
mutagenesis (U.S.E. mutagenesis kit, Amersham Pharmacia Biotech) of
plasmid pPR2-N1N2-
-Gal. N1N2A-
-Gal and N1N2B-
-Gal possess
Asn-Asn-Ser in place of Lys-Lys-Arg537 and Leu-Gln-Asn in
place of the Ala-Lys-Lys552, respectively (see Table
I).
-galactosidase fusion proteins were expressed, purified, and labeled
with 5-iodoacetamidofluorescein as previously (30, 31). m-IMP
(PTAC58), m-IMP
(PTAC97), y-IMP
(Kap60), y-IMP
(Kap95)
subunits, and human Ran were expressed as glutathione S-transferase (GST) fusion proteins and purified as
described (9, 21, 28, 34). GST-free y-IMP
, m-IMP
, and Ran were prepared by thrombin cleavage (21, 22, 28, 34, 35). Ran was loaded with
GDP as described (36). Recombinant human NTF2 was expressed and
purified using S100 HR column chromatography as described (15). Protein
concentrations were determined using the dye binding assay of Bradford
(37), with bovine serum albumin as a standard.
ELISA-based Binding Assay--
An ELISA-based binding assay (28,
34, 35, 38) was used to examine the affinity of binding between
importin subunits (with and without GST moieties) and NLS-containing
proteins. The latter were coated onto 96-well microtiter plates,
incubated with increasing concentrations of importin subunits, and
bound importin-GST detected using a goat anti-GST primary antibody, an
alkaline phosphatase-coupled rabbit anti-goat secondary antibody, and
the substrate p-nitrophenyl phosphate (34). In experiments
where importin
/
combinations were tested, importin
-GST and
thrombin-cleaved GST-free importin
s were used. Absorbance
measurements at A405 nm were performed over a
90-min period using a plate reader (Molecular Devices), with values
corrected by subtracting absorbance both at 0 min and in wells
incubated without importin (34). Quantitation was also performed in
identical fashion for
-galactosidase, and these values subtracted
from those for the T-ag and N1N2-
galactosidase fusion proteins (28,
34, 35). All fusion proteins were also subjected to a parallel
-galactosidase ELISA (34) to correct for any differences in coating
efficiencies and enable a true estimate of bound importin (28, 34, 35,
38).
In Vitro Nuclear Transport--
Nuclear import kinetics were
measured at the single cell level using mechanically perforated HTC rat
hepatoma cells in conjunction with confocal laser scanning microscopy
as previously (31, 34, 35, 38). Experiments were performed for 40 min
at room temperature in a 5 µl volume containing 30 mg/ml bovine serum
albumin, 2 mM GTP, an ATP regenerating system (0.125 mg/ml
creatine kinase, 30 mM creatine phosphate, 2 mM
ATP), and transport substrate (0.2 mg/ml
5-iodoacetamidofluorescein-labeled fusion protein), or a control
(70-kDa fluorescein isothiocyanate-labeled dextran; Sigma) to assess
nuclear integrity. Where indicated, 4 µM RanGDP, 1 µM m-IMP
or y-IMP
, 1 µM m-IMP
or
y-IMP
, and NTF2 (0.04-6 µM) were added. Image
analysis and curve-fitting were performed as described (34, 35, 38);
the level of accumulation at the nuclear envelope, relative to medium
fluorescence, was measured using NIH Image 1.60 in the line plot mode,
as previously (32, 39).
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RESULTS |
y-IMP
Has Higher NLS Binding Affinity Than m-
IMP
--
We have used an ELISA-based assay to determine the binding
affinities of mouse importin subunits for different NLSs (28, 34, 35,
40), with the results revealing relatively low NLS-binding affinity for
m-IMP
compared with that for the m-IMP
/
heterodimer (28, 34).
We set out to compare the NLS binding affinity of m-IMP
, y-IMP
,
m-IMP
/
, and y-IMP
/
for proteins containing either the T-ag
NLS, or the bipartite NLS of the X. laevis nuclear factor
N1N2 (see Table I). y-IMP
bound these
fusion proteins with significantly (p < 0.05) higher
affinity (about 2-fold) than m-IMP
(Figs.
1 and 2;
see Table II), the latter only attaining high affinity in the presence of m-IMP
. The specificity of binding in all cases was indicated by the fact that binding to NLS mutant derivatives was severely reduced (Table II). The T-ag NLS mutant showed
negligible binding affinity for IMP
/
from either mouse or yeast,
whereas the two N1N2 NLS mutant derivatives showed different importin
binding affinity (Fig. 2). N1N2A-
-Gal, mutated in the proximal arm
of the bipartite NLS, showed apparent dissociation constants
(Kd values) of 48 and 35 nM for
y-IMP
/
and m-IMP
/
, respectively, whereas N1N2A-
-Gal,
mutated in the distal arm, exhibited negligible binding. Clearly, mouse
and yeast IMP
/
are able to bind both types of NLS specifically,
with y-IMP
binding with higher affinity than m-IMP
(Figs. 1 and
2; Table II).
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Table I
NLS sequences contained in the proteins used in this study
Fusion proteins contain the indicated amino acid sequences fused
amino-terminal to E. coli -galactosidase (amino acid 9 1023) (see "Materials and Methods" and Refs. 30 32). The single
letter amino acid code is used with small letters indicating variation
from the wild type sequences (33).
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Fig. 1.
y-IMP binds the T-ag
NLS with higher affinity than m-IMP as
quantitated using an ELISA-based binding assay. Microtiter plates
were coated with T-ag-CcN- -Gal or the NLS-deficient T-ag-Cc- -Gal,
and incubated with increasing concentrations of m-IMP (left
panel) or y-IMP (right panel) in the absence or
presence of m-IMP or y-IMP as indicated. Curves were fitted for
the function B(x) = Bmax
(1 e kB), where x is
the concentration of importin, and B is the level of
importin bound, with the apparent dissociation constants
(Kd values, representing the importin concentration
yielding half-maximal binding) indicated. The results are from a single
typical experiment performed in triplicate (S.D. not greater than 9%
the value of the mean), with pooled data shown in Table II.
UD, Kd unable to be determined due to low
binding.
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Fig. 2.
y-IMP binds the N1N2
NLS with higher affinity than m-IMP .
Measurements were performed as described in the legend to Fig. 1.
Curves for wild type N1N2- -Gal (top panels), mutant
derivatives N1N2A- -Gal (middle panels) and N1N2B- -Gal
(bottom panels) are shown for m-IMP (left
panels) or y-IMP (right panels) in the absence or
presence of m-IMP or y-IMP as indicated. The results are from a
single typical experiment performed in triplicate (S.D. not greater
then 9% the value of the mean), with pooled data shown in Table
II.
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Table II
Binding parameters of NLS-containing -gatactosidase fusion proteins
for yeast and mouse importins as measured using an ELISA-based assay
Sequence details are presented in Table I. Data represent the mean ± S.E. (number in parentheses) for the apparent dissociation constant
(Kd) determined as outlined under "Materials and
Methods" (28, 34).
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Interestingly, the affinity of y-IMP
after precomplexation to
y-IMP
for the T-ag NLS was not significantly different from that in
its absence (Fig. 1 and Table II), but was enhanced 2-fold (p < 0.02) in the case of the N1N2 NLS (Fig. 2; Table
II). This was in contrast to the significantly larger effect of
m-IMP
on NLS recognition by m-IMP
(p < 0.03),
where the presence of m-IMP
increased the binding affinity 2- and
4-fold for the T-ag and N1N2 NLSs respectively (Table II). Clearly,
y-IMP
is a higher affinity NLS receptor than m-IMP
, which
requires m-IMP
to achieve high affinity. That y-IMP
increased the
binding affinity of m-IMP
for the bipartite N1N2 NLS but not for the
T-ag NLS implies that different NLSs have different requirements to
achieve high affinity recognition by importin.
Nuclear Import Conferred by the T-ag NLS Does not Require
NTF2--
The ability of m-IMP
/
and y-IMP
/
to mediate
nuclear import in a reconstituted in vitro system was
compared (Fig. 3). In the presence of
RanGDP, y- or m-IMP
or
alone could not mediate nuclear import of
T-ag-CcN-
-Gal (data not shown) with the levels of nuclear
accumulation similar to those in the absence of importin subunits (Fig.
3, A-C and Table
III). This was in contrast to the combination of m-IMP
/
where maximal nuclear accumulation relative to that in the cytoplasm (Fn/cmax)
was about 5-fold, half-maximal levels (t1/2)
being achieved within 5.3 min. In the presence of NTF2, the nuclear
import rate was enhanced significantly (t1/2 = 2.6 min; p < 0.05), whereas
Fn/cmax remained unaffected
(Fn/cmax of 5.0; see Table III).

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Fig. 3.
Ability of
y-IMP / and
m-IMP / to mediate
nuclear import of a T-ag NLS-containing protein or mutant variant
in vitro using purified components in the absence or
presence of NTF2. Nuclear import was reconstituted in mechanically
perforated HTC cells in the presence of an ATP-regenerating system
containing GTP/GDP and using GDP-loaded Ran as described under
"Materials and Methods." A, visualization of binding at
the nuclear envelope (top panels) or nuclear accumulation
(middle panels) of the T-ag-NLS-containing fusion protein
T-ag-CcN- -Gal, or lack of nuclear accumulation of the NLS-mutant
derivative T-ag-Cc- -Gal (bottom panels) mediated by
importin subunits in the absence or presence of Ran and NTF2 (0.08 µM) after 10 and 30 min, respectively. B,
visualization at early time points of nuclear accumulation as indicated
of T-ag-CcN- -Gal mediated by m-IMP / (left panels)
and y-IMP / (right panels) in the presence of RanGDP in
the absence (top panels) and presence (bottom
panels) of NTF2. C, nuclear import kinetics of
T-ag-CcN- -Gal and T-ag-Cc- -Gal mediated by m-IMP /
(left panel) or y-IMP / (right panel) in the
presence of Ran and NTF2 (0.08 µM). Results shown are
from a single typical experiment where each data point represents at
least five separate measurements for each of Fn,
Fc, and background fluorescence (see "Materials and
Methods"). Data is fitted for the function
Fn/c = Fn/cmax(1 e kt), where
Fn/cmax is the maximal level of
nuclear accumulation, k is the rate constant, and
t is time in minutes. Pooled data are presented in Table
III.
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Table III
Kinetics of nuclear import reconstituted in vitro using purified
proteins
Results represent the mean ± S.E. from data (see Figs.
3C and 4C) fitted to the function
Fn/c(t) = Fn/cmax*(1 e kt),
where Fn/cmax is the maximal level of
nuclear accumulation, k is the rate constant, and
t is time in minutes. NTF2 was used at the optimal
concentration for nuclear accumulation (see Fig. 5).
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In the absence of exogenously added NTF2, y-IMP
/
mediated nuclear
import to levels (Fn/cmax of 5.8;
t1/2 = 4.9 min) only slightly higher than
m-IMP
/
, while in the presence of NTF2, both the import rate and
the level of accumulation (Fn/cmax of 9.4; t1/2 = 1.8 min) were increased further
(p < 0.05; see Table III). The specificity of
transport in all cases was demonstrated by the fact that the
T-ag-Cc-
-Gal protein, containing a nonfunctional NLS, did not
accumulate to any significant extent (Fig. 3, A and C, and Table III). The results thus demonstrate that both
m-IMP
/
and y-IMP
/
can mediate nuclear import of an T-ag
NLS-containing transport substrate independent of NTF2 (Fig. 3 and
Table III).
We also examined the ability of m-IMP
/
and y-IMP
/
to
mediate binding to the nuclear envelope in the absence of RanGDP/NTF2 (Fig. 3A, top panel). No targeting of
T-ag-CcN-
-Gal to the nuclear envelope could be observed in the
absence of exogenously added importin subunits (results not shown).
Although m-IMP
or
alone could not mediate binding, as expected,
the combination of the two was able to do so with high efficiency (Fig.
3A). The maximal level of accumulation mediated by
y-IMP
/
at the nuclear envelope, as determined by image analysis
using the line plot mode (39), was 4.6 ± 0.6 times higher than
that in the cytoplasm, significantly higher (p < 0.008) than that effected by m-IMP
/
of 2.4 ± 0.1 (n = 4). This activity, together with its higher
affinity compared with m-IMP
/
for the T-ag-NLS (Table II), is
presumably the basis of the ability of y-IMP
/
to mediate nuclear
import more efficiently than m-IMP
/
.
Nuclear Import Conferred by the N1N2 NLS Requires
NTF2--
Nuclear import kinetic measurements were also performed
using the bipartite NLS-containing substrate N1N2-
-Gal (Fig.
4 and Table III). As for
T-ag-CcN-
-Gal, nuclear accumulation was negligible in the absence of
either RanGDP or m-IMP
/
. In the presence of m-IMP
/
and
RanGDP but in the absence of NTF2, a lack of nuclear accumulation was
also evident, although nuclear accumulation was clearly observed in the
presence of NTF2, with an Fn/cmax of
3.3 and t1/2 of 7.8 min.

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Fig. 4.
Nuclear import of
N1N2- -Gal and its mutant derivatives
reconstituted in vitro mediated by m-
IMP / and
y-IMP / in the absence
or presence of NTF2. A, visualization (after 30 min at
room temperature) of nuclear accumulation of N1N2- -Gal and its
mutant derivatives (N1N2A- -Gal and N1N2B- -Gal) mediated by
m-IMP / (left panels) or y-IMP / (right
panels) in the presence of RanGDP and NTF2. B, images
after 30 min of nuclear accumulation of N1N2- -Gal mediated by
m-IMP / (left panels) and y-IMP / (right
panels) in the presence of RanGDP in the absence (top
panels) and presence (bottom panels) of NTF2 (0.15 µM). C, nuclear import kinetics of
N1N2- -Gal (top panels) or mutant derivatives
N1N2A- -Gal (middle panels) and N1N2B- -Gal
(bottom panels) mediated by m-IMP / (left
panels) or y-IMP / (right panels) in the presence
of RanGDP in the absence or presence of NTF2. Results shown are from a
single typical experiment where each data point represents at least
five separate measurements for each of Fn, Fc,
and background fluorescence. Pooled data are presented in Table
III.
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Similar results were observed for y-IMP
/
in the presence of
Ran-GDP, no nuclear accumulation being observed in the absence of NTF2
(Fig. 4; Table III); in its presence y-IMP
/
yielded an Fn/cmax of 4.2 (t1/2 = 4.1 min). Of the two N1N2-NLS mutant
derivatives, N1N2A-
-NLS was excluded from the nucleus, whereas
N1N2B-
-Gal showed reduced nuclear accumulation in the presence of
RanGDP and NTF2 (Fn/cmax of 2.8, t1/2 = 13.9 min and
Fn/cmax of 2.6, t1/2 = 8.4 min for y-IMP
/
and
m-IMP
/
, respectively) compared with wild type N1N2-
-gal.
Clearly, although both arms of the bipartite N1N2 NLS are necessary for
optimal nuclear import, mutation of the proximal arm has a much greater
effect on nuclear import (Fig. 4) through its more severe effect on
importin binding (Fig. 2).
Inhibition of Nuclear Import by High Levels of NTF2--
Previous
studies have shown that NTF2 is necessary for nuclear import in several
vertebrate systems, being able to bind directly to RanGDP, importin
and proteins of the NPC (15, 21, 41-44). Because the T-ag and N1N2
NLSs appeared to have different requirements for NTF2 for nuclear
import, we performed transport measurements in the presence of
increasing concentrations of NTF2 (Fig.
5). Although NTF2 concentrations up to
0.15-0.3 µM enhanced nuclear import of the T-ag
NLS-containing fusion protein in the case of both m-IMP
/
and
y-IMP
/
, higher concentrations inhibited transport strongly (Fig.
5A). Optimal nuclear import efficiency appeared to be
obtained with an NTF2 concentration of about 0.08 µM.

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Fig. 5.
Relationship between NTF2 concentration and
nuclear import efficiency. Maximal nuclear accumulation
(Fn/cmax) and import rate
(t1/2, the time in min at which nuclear
accumulation is half-maximal) is plotted against the NTF2 concentration
for nuclear import of T-ag-CcN- -Gal (A) or N1N2- -Gal
(B) mediated by m-IMP / (left panels) or
y-IMP / (right panels) in the presence
of Ran. Results are for the means ± S.E. from a series
of three separate experiments.
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Similar results were also observed for N1N2 NLS-mediated nuclear import
(Fig. 5B), whereby m-IMP
/
or y-IMP
/
mediated
import was enhanced in the presence of NTF2 concentrations up to 0.3 and 0.6 µM, respectively, with the optimal concentration
of NTF2 for N1N2 NLS-mediated nuclear accumulation being about 0.15 µM. Nuclear accumulation was inhibited essentially
completely by NTF2 concentrations above 0.6 µM. NTF2 thus
appears to play a regulatory role in nuclear import, its concentration
determining whether it has enhancing or inhibitory effects.
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DISCUSSION |
This study represents the first quantitative comparison of two
conventional NLS binding receptors and two different NLSs, and their
ability to mediate nuclear protein import in a system using purified
components. Although in vitro systems for nuclear transport
have existed since 1990 (4), surprisingly little kinetic analysis has
been performed. True understanding of the subtleties of
signal-dependent nuclear import requires rigorous quantitation and this study can be seen as a first step in this direction. One of our important findings is that the importin
subunits from mouse and yeast appear to be different in their requirement for importin
to achieve high affinity NLS binding. This
is highlighted by the results for the T-ag NLS, where y-IMP
and
y-IMP
/
have almost identical binding affinities, in stark contrast to m-IMP
, which requires m-IMP
to obtain high affinity binding. In the case of the N1N2 NLS, y-IMP
does enhance y-IMP
binding, but not to such a great extent as the enhancement effected on
m-IMP
binding by m-IMP
. y-IMP
thus appears to resemble
importin
from the plant Arabidopsis thalania to some
extent, the latter being able to bind a variety of NLS types in the
absence of importin
(32). There would thus appear to be a gradation
through evolution in the properties of importin
, the vertebrate
form being much more strongly dependent on the additional presence of
importin
to achieve high affinity NLS binding.
As shown here, both y-IMP
/
and m-IMP
/
can mediate the
nuclear import of proteins carrying the T-ag or N1N2 NLSs in
combination with human Ran and NTF2, but the role of NTF2 in nuclear
import of T-ag is different from that of N1N2. Nuclear import mediated by the T-ag NLS does not require NTF2 absolutely, whereas nuclear accumulation in the case of the N1N2 NLS is completely dependent on
NTF2. Varying the concentration of NTF2 changes nuclear import dramatically, high levels of NTF2 inhibiting nuclear accumulation mediated by both the T-ag and N1N2 NLSs. This is consistent with the
observations of Tachibana et al. (45), who found that
cytoplasmically injected NTF2 strongly inhibited nuclear import
mediated by the T-ag NLS, as well as nuclear export mediated by the
nuclear export sequence of the cAMP-dependent protein
kinase inhibitor PKI, in a dose-dependent manner. The basis
of this may relate to the reported ability of NTF2 to regulate the
functional size of the central transporter element of the NPC (46), as
well as through its ability to associate with RanGDP, importin
, and
nucleoporins including p62 (14, 15, 41, 43, 44, 47), which itself is
located close to the central channel of the NPC (48). The present study
shows that the nuclear import process is finely balanced, with an
individual NLS possessing its own particular properties and
requirements with respect to the different nuclear transport components
to be translocated to the nucleus with optimal efficiency. In this
regard, NTF2 would appear to play a central regulatory role (42,
45-47, 49).
Our mutagenic analysis clearly shows that both arms of the N1N2
bipartite NLS are necessary for high affinity binding by importin
/
as well as efficient nuclear targeting. Mutagenesis of the proximal arm of the NLS reduces importin binding and nuclear import, whereas mutation of the distal arm completely oblates both activities. Essentially similar results have been obtained for the nucleoplasmin NLS (50), our results thus confirming the bipartite nature of the N1N2
NLS (37). From our results here and elsewhere (28, 40), the importin
binding and in vitro nuclear import of bipartite NLS-containing proteins appear to be intrinsically less efficient than
those of proteins containing the T-ag NLS. Although T-ag may be rather
special in terms of being a viral protein that uses the host cell
transport system with unusually high efficiency, it remains unclear as
to whether as yet unidentified transport factors may be involved in
importin-mediated nuclear import of proteins carrying bipartite NLSs
under physiological conditions. Investigation of this possibility is
currently in progress in this laboratory.
The results presented here indicate a close relationship between the
importin binding affinity and rate of nuclear import, where the highest
affinity conformation of NLS and NLS receptor leads to the most
efficient nuclear accumulation. This is illustrated most poignantly by
the two different mutant variants of the N1N2 NLS, which exhibit quite
different importin binding activities; the proximal arm mutant (the
N1N2B-
-Gal protein) is recognized by importins with higher affinity,
and in consequence is able to be transported to the nucleus much more
efficiently than the distal arm mutant (N1N2A-
-Gal). NLS binding by
importins is clearly a crucial step in nuclear import, consistent with
the idea that the affinity of NLS recognition by importins is critical
in determining the rate and maximal extent of NLS-dependent
nuclear import (28, 34, 35, 40).