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Molecular and Cellular Biology, March 2000, p. 1571-1582, Vol. 20, No. 5
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Nuclear Import of I
B
Is Accomplished by a
Ran-Independent Transport Pathway
Shrikesh
Sachdev,
Sriparna
Bagchi,
Donna D.
Zhang,
Angela C.
Mings, and
Mark
Hannink*
Biochemistry Department, University of
Missouri
Columbia, Columbia, Missouri 65212
Received 9 June 1999/Returned for modification 27 July
1999/Accepted 10 December 1999
 |
ABSTRACT |
The inhibitor of kappa B alpha (I
B
) protein is able to
shuttle between the cytoplasm and the nucleus. We have utilized a combination of in vivo and in vitro approaches to provide mechanistic insight into nucleocytoplasmic shuttling by I
B
. I
B
contains multiple functional domains that contribute to shuttling of I
B
between the cytoplasm and the nucleus. Nuclear import of I
B
is
mediated by the central ankyrin repeat domain. Similar to previously described nuclear import pathways, nuclear import of I
B
is
temperature and ATP dependent and is blocked by a dominant-negative
mutant of importin
. However, in contrast to classical nuclear
import pathways, nuclear import of I
B
is independent of soluble
cytosolic factors and is not blocked by the dominant-negative RanQ69L
protein. Nuclear export of I
B
is mediated by an N-terminal
nuclear export sequence. Nuclear export of I
B
requires the CRM1
nuclear export receptor and is blocked by the dominant-negative RanQ69L
protein. Our results are consistent with a model in which nuclear
import of I
B
is mediated through direct interactions with
components of the nuclear pore complex, while nuclear export of
I
B
is mediated via a CRM1-dependent pathway.
 |
INTRODUCTION |
The transport of proteins between
the nucleus and the cytoplasm is an essential activity of eukaryotic
cells. Protein transport across the double lipid bilayer of the nuclear
membrane occurs through large macromolecular complexes termed nuclear
pore complexes (NPCs). Transport is generally dependent upon specific
cis-acting signals within the cargo protein and the
corresponding trans-acting factors that mediate the
translocation of the protein cargo through the NPC (for reviews, see
references 8 and 30). The
best-characterized transport pathway is utilized by proteins that
contain a classical nuclear localization signal (NLS). The prototypic
NLS present in the simian virus 40 (SV40) large T protein contains a
short stretch of basic residues that are critically required for
nuclear localization (15, 25). NLS-bearing proteins are
bound by the heterodimeric importin
-
complex (7, 16-18,
31, 36), which mediates docking of the receptor-cargo complex at
the cytoplasmic face of the NPC. Translocation of the receptor-cargo
complex through the NPC is poorly understood, but is thought to involve
direct interactions between the importin
subunit and specific
components of the NPC (37, 38). Nuclear import is terminated
in the nucleus by RanGTP-induced dissociation of the receptor-cargo
complex, releasing the NLS-bearing cargo into the nucleus (21,
38). The importin
and
proteins are subsequently recycled
back to the cytoplasm to participate in a second round of nuclear
import (29).
The export of proteins from the nucleus is accomplished by an analogous
mechanism. Proteins that are destined to be exported from the nucleus
typically contain nuclear export sequences (NESs) comprised of short
clusters of leucine or other hydrophobic residues (12,
47). NES-bearing proteins are recognized by the nuclear export
receptor, CRM1, in a RanGTP-dependent manner (4, 13, 14, 33,
45). Although the details of nuclear export are poorly
understood, the NES-bearing cargo-CRM1 receptor complex is thought to
dock at the nuclear side of the NPC, followed by translocation and
dissociation at the cytoplasmic face of the NPC.
A critical aspect of both nuclear import and nuclear export is the
asymmetric distribution of RanGTP and RanGDP between the nucleus and
the cytoplasm. The GTPase-activating protein for Ran, RanGAP, is
located at the cytoplasmic face of the NPC, while the guanine
nucleotide exchange factor for Ran, RCC1, is tightly associated with
chromatin (19, 34). This differential distribution of RanGAP
and RCC1 between the cytoplasm and the nucleus has led to the
prediction that Ran will be in the GTP-bound form in the nucleus, while
the GDP-bound form will predominate in the cytoplasm. Experimental
perturbation of the RanGTP-RanGDP gradient inhibits both nuclear import
and nuclear export. For example, the GTP-bound form of the
dominant-negative RanQ69L protein inhibits NLS-dependent nuclear import
in digitonin-permeabilized cells, presumably by inhibiting binding of
an NLS-bearing protein to the importin
-
receptor complex
(21, 38). Likewise, depletion of nuclear RanGTP levels
inhibits NES-dependent nuclear export, presumably by preventing a
NES-bearing protein from binding to CRM1 in a manner that is competent
for transport (4, 39).
Transport of proteins between the nucleus and the cytoplasm provides an
effective mechanism for regulation of gene expression. A striking
example of how gene expression can be regulated at the level of protein
transport between the nucleus and the cytoplasm is provided by the
NF-
B/Rel family of transcription factors and their inhibitory I
B
proteins (reviewed in reference 5). For example, the
I
B
protein is able to both inhibit nuclear import of NF-
B/Rel
proteins and direct the export of NF-
B/Rel proteins from the nucleus
(2, 3, 20, 22, 43). The ability of I
B
to act both in
the cytoplasm and in the nucleus requires that I
B
itself travel
through the NPC. The second ankyrin repeat of I
B
is critically
required for nuclear localization of I
B
and is able to
functionally substitute for a classical NLS (42). In this
report, we have utilized a combination of in vitro and in vivo
approaches to provide mechanistic insight into nuclear shuttling of
I
B
. Our results indicate that nuclear import of I
B
is
accomplished by a Ran-independent mechanism, while nuclear export of
I
B
requires the Ran-dependent CRM1 nuclear export receptor.
 |
MATERIALS AND METHODS |
Construction of recombinant DNA molecules.
The I
B
clones used in this study were derived from the avian I
B
cDNA
clone isolated by Davis et al. (9) and constructed by
standard techniques (44). To construct the GST (glutathione S-transferase)-I
B
expression vector, an
EcoRI fragment containing 69 bp of 5' nontranslated
sequence, the entire 954 bp of the I
B
open reading frame, and 762 bp of 3' nontranslated sequence was cloned into the EcoRI
site of pGEX-2T in the proper orientation for expression. The
GST-I
B
-
N69 expression vector contains an I
B
insert that
lacks the first 69 codons of the I
B
open reading frame. The
GST-I
B
-
C51 expression vector contains an I
B
insert that
contains a termination codon at codon 268 (41). The
GST-I
B
-
Ank2 expression vector contains a deletion which
removes codons 98 to 142 (42). The GST-I
B
-ARD (ankyrin
repeat domain) expression vector contains codons 70 to 254 of I
B
cloned into the SmaI site of pGEX3X. The GFP (green
fluorescent protein)-I
B
expression vectors were constructed by
inserting the various I
B
fragments from the GST-based vectors
into the GFP-C3 eukaryote expression vector (Clontech). The GST-NLS
expression vector contains an oligonucleotide which encodes an NLS
derived from the SV40 large T protein inserted into the SmaI
site of pGEX1. The GST-M9 expression vector contains an oligonucleotide
which encodes the M9 nuclear import sequence from the hnRNP A1 protein
cloned into the SmaI site of pGEX1 (35). The
pQE32-derived expression vectors for wild-type Ran and the RanQ69L
protein and the pQE60-derived expression vector for the importin
(45-462) protein were obtained from Dirk Gorlich (University of
Heidelberg). A pET-based expression vector for the importin
-binding
domain of importin
(IBB) was obtained from Steve Adam (Northwestern
University). A pQE32-derived expression vector for RanBP1 was obtained
from Iain Mattaj's laboratory (EMBL, Heidelberg, Germany).
Expression and purification of recombinant proteins.
The
recombinant GST fusion proteins were expressed in Escherichia
coli strain BL21(DE3). Cultures were grown to an optical density
at 600 nm of 0.4 and induced with 0.2 mM isopropyl thiogalactoside (IPTG) (Sigma) for 4 h at 20°C. The bacterial cell pellets were resuspended in ice-cold phosphate-buffered saline (PBS [pH 7.4]) containing 0.1% Triton X-100; 0.2 mM dithiothreitol (DTT); 1 mM phenylmethylsulfonyl fluoride; and 1 µg (each) of antipain,
aprotinin, leupeptin, pepstatin, and soybean trypsin-chymotrypsin
inhibitor per ml. Lysis of the cell pellets was conducted by brief
sonication on ice. The lysates were cleared by centrifugation at
14,000 × g for 15 min at 4°C and the soluble GST
fusion proteins were bound to glutathione-agarose beads (Sigma) for 30 min at room temperature. The glutathione-agarose beads were extensively
washed with ice-cold PBS (pH 7.4), and the recombinant GST fusion
proteins were eluted with 10 mM reduced glutathione in 50 mM Tris-Cl
(pH 8.0) and dialyzed against transport buffer (20 mM HEPES (pH 7.3),
110 mM potassium acetate, 5 mM sodium acetate, 2 mM magnesium acetate,
1 mM EGTA).
To label the proteins with fluorescein, the recombinant GST fusion
proteins were first dialyzed against labeling buffer (20 mM sodium
phosphate buffer [pH 7.2], 150 mM sodium chloride) at 4°C
overnight. Fluorescein 5'-maleimide (Pierce Chemical Co.) was added at
an equimolar ratio, and the mixtures were incubated for 2 h on
ice. The reactions were quenched by the addition of 50 mM
-mercaptoethanol. The labeled proteins were equilibrated against
transport buffer by using Centricon-3 (Amicon) columns.
The Ran proteins, RanBP1, the importin
(45-462) protein, and the
IBB protein were expressed as His-tagged proteins and purified by
metal-chelate affinity chromatography (Invitrogen). RanBP1, importin
(45-462), and the IBB protein were dialyzed against transport
buffer prior to use. The purified Ran proteins were dialyzed against
Ran loading buffer (10 mM HEPES [pH 7.3], 160 mM potassium acetate, 5 mM magnesium acetate, 1 mM DTT) at 4°C overnight. The dialyzed Ran
proteins were incubated with 1 mM GTP in the presence of 15 mM EDTA for
60 min at room temperature. Magnesium chloride was added to a final
concentration of 30 mM. The loading reactions were performed
immediately prior to the import reactions, and the Ran proteins were
placed on ice during the permeabilization step. A mock loading sample
in which the respective Ran protein was left out of the loading
reaction was included to ensure that the nucleotide loading conditions
did not affect the nuclear import reactions.
Each of the recombinant proteins was examined by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All proteins
were greater than 90% pure, as determined by Coomassie blue staining
of the respective SDS-PAGE gels. Protein concentrations were determined
by Bradford assays (Bio-Rad) in accordance with the instructions
provided by the manufacturer.
In vitro nuclear import assay.
HeLa cells were grown in
Dulbecco's modified Eagle's medium (low glucose) supplemented with
10% fetal bovine serum (FBS) in a 37°C, 5% CO2
incubator. Approximately 16 h prior to the transport assays,
6.5 × 105 HeLa cells were plated onto 35 mm-diameter
plates containing glass coverslips. The in vitro nuclear transport
assays were conducted essentially as described in reference
1. In brief, cells on coverslips were permeabilized
with 50 µg of digitonin per ml (Calbiochem) in transport buffer for 5 min on ice. The transport reactions were typically conducted for 30 min
at 30°C, except where noted. A standard 50-µl transport reaction
contained an energy-regenerating system (1 mM ATP, 0.1 mM GTP, 5 mM
creatine phosphate, 20 U of creatine phosphokinase per ml), protease
inhibitor mix, 2 mM DTT, and 15 µl of rabbit reticulocyte lysate (50 mg of total protein per ml). The unlabeled import substrates were added
to a final concentration of 100 µg/ml, while the fluorescein-labeled
import substrates were added to a final concentration of 50 µg/ml.
For some samples, HeLa cells growing on coverslips were pretreated with
10 nM leptomycin B for 30 min prior to digitonin permeabilization, and
10 nM leptomycin B was included throughout the 30-min time course of
the transport reactions. For the energy-dependence experiments, the
reticulocyte lysates were pretreated with 25 U of apyrase per ml for 30 min prior to the import reactions, which were performed in the absence
of ATP, creatine phosphate, and creatine phosphokinase.
The competition experiments were carried out at room temperature with a
100-fold molar excess of unlabeled protein relative to the
fluorescein-labeled protein, as indicated in the figures and legends.
For the importin
(45-462) blocking experiments, the
digitonin-permeabilized cells were preincubated for 10 min with 5 µg
of the recombinant importin
(45-462) protein at room temperature in
transport buffer before initiation of the import reaction. The
Ran-dependence experiments were performed in the presence of 5 µg of
the respective Ran protein preloaded with GTP. The IBB competition
experiment was performed in the presence of 15 µg of the IBB
polypeptide. The RanBP1 experiments were carried out in the presence of
15 µg of RanBP1.
Indirect immunofluorescence assays were conducted on coverslips as
previously described (42). The primary antibody was an anti-GST monoclonal antibody (Santa Cruz Biotechnology) used at a
concentration of 1:100 in PBS (pH 7.4) containing 10% FBS. Anti-mouse fluorescein isothiocyanate (FITC)-conjugated secondary antibody was
used at a concentration of 1:100 in PBS (pH 7.4) containing 10% FBS.
The coverslips were mounted onto glass slides with Mowiol containing
2.5% DABCO (Sigma). Pictures shown in the figures were taken with a
×40 oil immersion lens on a Nikon Optiphot-2 equipped with a Nikon
UFX-IIA 35-mm camera. Unless otherwise noted, equivalent exposure time
periods were used for all panels shown in the same figure. The
negatives were scanned into Photoshop 3.0 (Adobe) and compiled into the
figures shown. All panels shown in the same figure were treated
identically during developing and compilation.
Confocal laser scanning microscopy was performed with a ×60
oil-immersion lens on a Nikon Diaphot microscope equipped with a
Bio-Rad MRC-600 laser. The z-sections were captured as TIFF files by
using CoSMOS software. The images were compiled with Photoshop 3.0 and
treated identically during figure construction.
HeLa cell transfections.
HeLa cells were purchased from
American Type Culture Collection and cultured in Dulbecco's modified
Eagle's medium containing 10% FBS. HeLa cells were transfected with
the GFP-I
B
expression plasmids by using Fugene 6 according to the
manufacturor's instructions (Boehringer Mannheim). Expression of the
GFP-I
B
fusion proteins was confirmed by immunoblot analysis.
Localization of the GFP-I
B
fusion proteins was determined by
indirect immunofluorescence with an anti-GFP antibody (Chemicon).
 |
RESULTS |
Nuclear shuttling of I
B
in digitonin-permeabilized HeLa
cells.
A digitonin-permeabilized cell assay was used to
characterize nuclear import of I
B
. I
B
was expressed as a
GST fusion protein in E. coli, and the ability of the
GST-I
B
fusion protein to accumulate in the nuclei of
digitonin-permeabilized HeLa cells was monitored by indirect
immunofluorescence with a monoclonal antibody directed against GST. The
NLS from the SV40 large T protein was fused to GST (GST-NLS) to serve
as a positive control for nuclear import. Nuclear staining was not
observed in the absence of an import substrate (Fig.
1A). In the presence of reticulocyte lysate and an energy-regenerating system, GST-NLS was efficiently imported into the nucleus (Fig. 1C). Under these conditions, neither GST nor a GST fusion protein containing a mutant NLS was imported into
the nucleus (data not shown).

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FIG. 1.
Nuclear accumulation of I B in
digitonin-permeabilized cells is leptomycin B sensitive. HeLa cells
were either left untreated (A to C) or were treated with 10 nM
leptomycin B (LMB [D to F]) for 30 min prior to permeabilization with
digitonin. Digitonin-permeabilized HeLa cells were incubated for 30 min
at 30°C in the presence of reticulocyte lysate and an
energy-regenerating system. Leptomycin B (10 nM) was included in the
import reaction for the samples shown in panels D to F. The cells were
incubated in the absence of import substrate (A and D) and in the
presence of either GST-I B (B and E) or GST-NLS (C and F). The
cellular localization of the import substrates was visualized by
indirect immunofluorescence with a monoclonal antibody directed against
GST.
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|
In contrast to GST-NLS, the GST-I
B
protein did not efficiently
accumulate in the nucleus in a standard nuclear import reaction (Fig.
1B). The inability of I
B
to localize to the nucleus in vitro was
surprising, because either endogenous or ectopically expressed I
B
can readily be detected in the nucleus in vivo (2, 3, 42).
Furthermore, Turpin et al. have recently reported that I
B
is
actively imported into the nuclei of digitonin-permeabilized HeLa cells
(46). Because I
B
contains a canonical NES and is able
to direct the nuclear export of NF-
B/Rel proteins (3, 42), we asked if inhibition of nuclear export would enable
nuclear accumulation of I
B
in vitro. HeLa cells were pretreated
with 10 nM leptomycin B, a specific inhibitor of the nuclear export receptor CRM1 (13, 14, 33, 45), for 30 min prior to
permeabilization, and 10 nM leptomycin was included throughout the
30-min time course of the import reaction. This regimen of leptomycin B
treatment markedly increased the accumulation of GST-I
B
in the
nuclei of digitonin-permeabilized HeLa cells (compare panels B and E of
Fig. 1). Leptomycin B treatment had no effect on nuclear accumulation of GST-NLS (Fig. 1F). Nuclear accumulation of an I
B
protein containing a short N-terminal hexahistidine tag was also markedly increased by leptomycin B treatment (data not shown).
To further characterize nuclear accumulation of I
B
in
digitonin-permeabilized cells, increasing amounts of FITC-labeled GST-I
B
protein were added to in vitro import reactions in the absence or presence of leptomycin B. Nuclear accumulation of I
B
was markedly enhanced by leptomycin B at 2.5 and 5.0 µg of input I
B
protein. However, nuclear accumulation of I
B
was
leptomycin B independent at 10.0 and 20.0 µg of input I
B
protein (data not shown). This concentration range at which nuclear
accumulation of GST-I
B
became leptomycin B independent was highly
reproducible in multiple experiments (data not shown). It is likely
that the addition of excess I
B
protein titrates out one or more
factors that are limiting for nuclear export of I
B
.
I
B
contains multiple functional domains that contribute to
rapid shuttling between the cytoplasm and the nucleus.
To define
regions within I
B
that specify nuclear import and nuclear export,
we constructed a series of mutant GST-I
B
fusion proteins (Fig.
2). These mutant proteins were expressed
in E. coli, purified with glutathione-agarose, and assayed
for their ability to accumulate in the nucleus of
digitonin-permeabilized HeLa cells in the absence and presence of
leptomycin B (Fig. 3). To ensure that
nuclear export of I
B
was not saturated by excess input protein,
the ability of leptomycin B to increase nuclear accumulation of the
mutant I
B
proteins was measured at 2.5 µg of input protein.

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FIG. 2.
Domain organization of I B . I B contains an
N-terminal signal response domain, a central ARD comprised of six
ankyrin repeats, and a C-terminal PEST domain. The location of two
canonical leucine-rich NESs (amino acids 45 to 55 and 273 to 283) in
I B is indicated (LLLL). The structure of the mutant I B
proteins used in these experiments is diagrammed.
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FIG. 3.
Definition of nuclear import and NESs in I B .
Nuclear import reactions were performed with the indicated protein
substrates in either the absence (A, C, E, G, I, and K) or presence of
10 nM leptomycin B (B, D, F, H, J, and L). The cellular localization of
the import substrates was determined by indirect immunofluorescence
with either a monoclonal antibody directed against GST or by direct
fluorescence with FITC-labeled proteins. Parallel experiments with both
detection methods were done for all protein samples, with the exception
of the GST-I B - C251 protein, which was only detected by direct
fluorescence.
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|
Consistent with our previous demonstration that the second ankyrin
repeat of I
B
is critically required for nuclear localization of
I
B
in transfected cells, a mutant I
B
protein that contained a deletion of the second ankyrin repeat (GST-I
B
-
Ank2; Fig. 3C
and D) did not accumulate in the nucleus of digitonin-permeabilized HeLa cells, in either the absence or presence of leptomycin B. Likewise, a mutant I
B
protein that contained alanine residues in
place of four hydrophobic residues within the second ankyrin repeat was
defective for nuclear import (GST-I
B
-114A4 [data not shown]).
These mutant I
B
proteins were unable to accumulate in the nucleus
of digitonin-permeabilized cells even when 10 µg of input I
B
protein was added to the import reactions (data not shown).
I
B
contains a canonical leucine-rich NES located in its C
terminus (amino acids 273 to 283). Several reports have suggested that
this NES is required for I
B
-mediated nuclear export of NF-kB/Rel
proteins (3, 42). However, a C-terminal-truncated GST-I
B
fusion protein, which lacked the C-terminal 51 amino acids
of I
B
(GST-I
B
-
C51), remained leptomycin B dependent for
nuclear accumulation (Fig. 3G and H). Likewise, a mutant full-length GST-I
B
protein containing four leucine-to-alanine substitutions within the canonical leucine-rich NES of I
B
was also leptomycin B
dependent for nuclear accumulation (GST-I
B
-273A4; Fig. 3K and L).
That these I
B
mutants remained leptomycin B dependent for nuclear
accumulation despite deletion or mutation of the C-terminal NES
indicates the presence of additional functional NES(s) in I
B
.
A mutant I
B
protein that lacked the N-terminal 69 amino acids of
I
B
(GST-I
B
-
N69) was able to accumulate in the nucleus in
a leptomycin B-independent manner (Fig. 3E and F). The ability of the
GST-I
B
-
N69 protein to accumulate in the nucleus in a leptomycin B-independent manner was not due to saturation of I
B
export by excess protein, because significant leptomycin B-independent nuclear accumulation was observed at 1 µg of input FITC-labeled GST-I
B
-
N69 protein (data not shown). That removal of the
N-terminal 69 amino acids of I
B
enabled leptomycin B-independent
nuclear accumulation of I
B
suggests the presence of a functional
nuclear export sequence within the N-terminal domain of I
B
.
Indeed, a recent report has suggested that amino acids 45 to 55 of
I
B
comprise a leucine-rich NES (24).
A GST fusion protein containing just the ARD of I
B
(GST-I
B
-ARD) was competent for nuclear import (Fig. 3I and J),
consistent with our previous demonstration that the nuclear
localization of I
B
in transfected cells is mediated by one or
more nuclear import sequences within the ARD (42).
Surprisingly, the GST-I
B
-ARD protein was leptomycin B
dependent for nuclear accumulation despite the absence of both the
N-terminal and C-terminal NESs. The leptomycin B-dependent nuclear
accumulation of the GST-I
B
-ARD protein suggests that, under the
conditions of the in vitro import reaction, the ARD contains an
additional nuclear export sequence.
Nuclear shuttling of I
B
in transfected HeLa cells.
The
failure of the C-terminal NES of I
B
to contribute to nuclear
export of I
B
in digitonin-permeabilized HeLa cells was surprising, because several reports have indicated that this canonical leucine-rich NES is required for I
B
-mediated nuclear export of
NF-
B/Rel proteins (3, 42). To further characterize
nuclear shuttling of I
B
in vivo, we constructed fusion proteins
between the GFP and the various I
B
proteins. These fusion
proteins were expressed in HeLa cells, and the ability of leptomycin B
to alter the nuclear-cytoplasmic distribution of fusion proteins was
measured (Fig. 4 and Table
1).

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FIG. 4.
Nuclear shuttling of GFP-I B chimeric proteins in
transfected HeLa cells. HeLa cells transfected with expression vectors
for the indicated GFP-I B fusion proteins were left untreated or
were treated with leptomycin B for 1 h. The cellular localization
of the fusion proteins was detected by indirect immunofluorescence with
a chicken antibody raised against GFP. More than 200 cells were
examined for each fusion protein (see Table 1 for quantitation), and
representative cells are shown.
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|
The wild-type GFP-I
B
protein was distributed throughout both the
nucleus and the cytoplasm in transfected HeLa cells (Fig. 4A).
Treatment of transfected HeLa cells with leptomycin B for 1 h
resulted in a marked redistribution of the wild-type GFP-I
B
protein to the nucleus (Fig. 4B). In contrast, leptomycin B treatment had no effect on the predominantly cytoplasmic localization of GFP
(Table 1). The increased nuclear localization of GFP-I
B
in the
presence of leptomycin B indicates that the GFP-I
B
protein shuttles between the nucleus and the cytoplasm in transfected HeLa cells.
The ability of the mutant GFP-I
B
proteins to shuttle
between the nucleus and the cytoplasm in a leptomycin B-dependent
manner was also determined. As expected, the GFP-I
B
-
Ank2
protein and the GFP-I
B
-114A4 protein were predominantly
cytoplasmic in the absence of leptomycin B (Fig. 4C and data not
shown). Treatment of the transfected HeLa cells with leptomycin B
resulted in increased nuclear localization of the mutant GFP-I
B
proteins (Fig. 4D and data not shown). That leptomycin B
treatment increased the extent to which these mutant proteins
accumulated in the nucleus suggests that I
B
contains
additional weak nuclear import sequences, likely within one or more of
the remaining intact ankyrin repeats (24, 42).
The GFP-I
B
-
C51 protein and the GFP-I
B
-273A4 protein were
distributed between the nucleus and the cytoplasm in the absence of
leptomycin B, and treatment of the transfected cells with leptomycin B
markedly increased the nuclear localization of these proteins (Fig. 4G,
H, K, and L). These results are consistent with the shuttling phenotype
of these proteins in digitonin-permeabilized cells and demonstrate that
both in vitro and in vivo, the C-terminal NES of I
B
is not
required for nuclear export of I
B
.
The GFP-I
B
-
N69 protein displayed increased nuclear
localization in the absence of leptomycin B, consistent with the
ability of this protein to accumulate in the nucleus of
digitonin-permeabilized cells in the absence of leptomycin B (Fig. 4E).
Leptomycin B treatment increased the percentage of
GFP-I
B
-
N69-positive cells that displayed predominantly nuclear
staining (Table 1).
In contrast to the results obtained in vitro, a GFP fusion protein
containing just the ARD of I
B
also displayed significant nuclear
accumulation in the absence of leptomycin B (Fig. 4I). Leptomycin B
treatment increased the percent of GFP-I
B
-ARD-positive cells that
displayed predominantly nuclear staining (Table 1), suggesting that
this protein, which lacks both of the previously described NESs in
I
B
, is still able to shuttle between the nucleus and the cytoplasm.
Nuclear import of I
B
is energy and temperature dependent and
is blocked by a dominant-negative importin
protein.
To
determine whether nuclear accumulation of I
B
reflects active
nuclear transport or passive diffusion through the NPC, cytosolic
extracts were pretreated with apyrase to deplete the extracts of
high-energy phosphate compounds. The ability of the energy-depleted
extracts to support nuclear import of GST-I
B
or GST-NLS in the
absence of an energy-regenerating system was determined. Nuclear import
of both GST-I
B
(Fig. 5D) and
GST-NLS (Fig. 5H) was inhibited when the cytosolic extracts were
pretreated with apyrase. Similar results were obtained when hexokinase
and glucose were used to deplete the energy pools present in the
cytosolic extracts (data not shown).

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FIG. 5.
Nuclear import of I B is energy and temperature
dependent. Nuclear import reactions into digitonin-permeabilized HeLa
cells were performed with either GST-I B (A to D) or GST-NLS (E to
H). Leptomycin B (10 nM) was included in the nuclear import reactions
with GST-I B (A to C). The import reactions were carried out under
standard conditions (A and E), in the presence of WGA (B and F), on ice
(C and G), or in the presence of 25 U of apyrase per ml (D and H). The
cellular localization of the import substrates was determined by
indirect immunofluorescence with a monoclonal antibody directed against
GST. Panels A and E were from equivalent exposure settings. Panels B to
D and F to H were exposed for 10 to 20 times longer than panels A and
E.
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|
To determine the temperature dependence of I
B
nuclear import, the
import reactions were performed at 4°C instead of at 30°C. Nuclear
import of both GST-I
B
(Fig. 5C) and GST-NLS (Fig. 5G) was
markedly reduced when the import reactions were conducted at 4°C.
Nuclear import of I
B
was restored when the import reactions were
shifted back to 30°C (data not shown).
The effect of wheat germ agglutinin (WGA), a lectin which binds to
N-acetyl-D-glucosamine residues present on many
nucleoporins, was examined. Addition of WGA significantly inhibited
nuclear accumulation of both GST-I
B
and GST-NLS (Fig. 5B and F).
The ability of a dominant-negative importin
protein, importin
(45-462), to perturb nuclear import was also determined. The
importin
(45-462) protein has been demonstrated to block multiple
nuclear transport pathways, presumably by binding irreversibly to
specific components of the NPC (28). Digitonin-permeabilized HeLa cells were preincubated with the mutant importin
(45-462) protein, and leptomycin B-dependent nuclear accumulation of I
B
was examined by confocal laser scanning microscopy. Consistent with
previous results (27), the importin
(45-462) protein
completely blocked nuclear import of GST-NLS (Fig.
6E and F). Similarly, preincubation of
HeLa cells with the importin
(45-462) protein inhibited entry of
both GST-I
B
(Fig. 6A and B) and of the N-terminal-truncated derivative, GST-I
B
-
N69 (Fig. 6C and D). The I
B
proteins, like GST-NLS, accumulated at the nuclear envelope in the presence of
the importin
(45-462) protein (Fig. 6B, D, and F).

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FIG. 6.
Binding of I B to the nuclear membrane in the
presence of a dominant-negative importin protein. Nuclear import
reactions using the indicated proteins were performed in the absence
(A, C, and E) or presence (B, D, and F) of the dominant-negative
importin (45-462) protein. Leptomycin B (10 nM) was included in the
import reactions shown in panels A and B. The images shown are
representative of more than 50 cells that were examined for each import
substrate. The import substrates were visualized by indirect
immunofluorescence by confocal laser scanning microscopy.
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|
Taken together, these results demonstrate that nuclear accumulation of
I
B
does not occur by passive diffusion through open channels of
the NPC. Rather, nuclear import of I
B
is an energy-dependent and
temperature-sensitive process that requires specific components of the NPC.
Rate-limiting factors for nuclear import of I
B
are not lost
during the digitonin-permeabilization procedure.
To investigate
the dependence of I
B
import on soluble protein factors, the
ability of GST-I
B
to accumulate in the nuclei of HeLa cells in
the absence of exogenously added cytosol was determined. Nuclear import
of GST-NLS was strictly dependent on the presence of exogenously added
cytosol (Fig. 7E and F). In contrast,
GST-I
B
readily accumulated in the nuclei of HeLa cells, even when
cytosol was not added to the import reactions (Fig. 7A and B).
Leptomycin B was included in the nuclear import reactions for
GST-I
B
. However, the ability of GST-I
B
to accumulate in the
nucleus in the absence of exogenously added soluble factors was not an
artifact resulting from the inclusion of leptomycin B in the import
reaction, because the GST-I
B
-
N69 protein efficiently accumulated in HeLa cell nuclei in a cytosol-independent manner in the
absence of leptomycin B (Fig. 7C and D).

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FIG. 7.
Nuclear import of I B is independent of exogenous
cytosol. Nuclear import reactions using the indicated proteins were
carried out either in the absence of reticulocyte lysate (A, C, and E)
or in the presence of 100 µg of reticulocyte lysate. Leptomycin B
(LMB) was included in the nuclear import reactions using GST-I B
(A and B). The cellular localization of the import substrates was
determined by indirect immunofluorescence with a monoclonal antibody
directed against GST.
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|
Nuclear import of I
B
is not blocked by saturation of the
NLS-dependent or M9-dependent import pathways.
Nuclear import of
proteins that utilize known nuclear import pathways, such as the
NLS-dependent pathway or the M9-dependent pathway, can be inhibited by
saturation of the respective import receptor (30). As
anticipated, we found that a 100-fold molar excess of GST-NLS (Fig.
8D) or a 7-fold molar excess of IBB (Fig. 8F) markedly reduced nuclear import of FITC-labeled GST-NLS. Likewise, a 100-fold molar excess of GST-M9 significantly inhibited nuclear import of FITC-labeled GST-M9 (Fig. 8J). However, nuclear import of
FITC-labeled GST-I
B
-
N69 was not inhibited by the addition of a
100-fold molar excess of GST (Fig. 8A), GST-NLS (Fig. 8C), a 7-fold
molar excess of IBB (Fig. 8E), or a 100-fold molar excess of GST-M9
(Fig. 8I). Similar results were obtained with the full-length GST-I
B
protein when the nuclear import reactions were carried out
in the presence of leptomycin B (data not shown). Taken together, these
results indicate that nuclear import of GST-I
B
is independent of
either the NLS-dependent or the M9-dependent nuclear import pathway.

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FIG. 8.
Nuclear import of I B is independent of NLS- and
M9-mediated nuclear import pathways. Nuclear import reactions with
fluorescein-labeled GST-I B - N69 (A, C, E, G, and I),
fluorescein-labeled GST-NLS (B, D, and F), or fluorescein-labeled
GST-M9 (H and J) were performed. The import reactions were performed in
the presence of a 100-fold molar excess of unlabeled GST (A, B, G, and
H), GST-NLS (C and D), or GST-M9 (I and J). For the import reactions
shown in panels E and F, the IBB was included at a final concentration
of 30 µM. The cellular localization of the import substrates was
determined by direct fluorescence.
|
|
I
B
utilizes a high-throughput nuclear import pathway.
The saturability of both nuclear import and nuclear export of I
B
was determined by the ability of FITC-labeled GST-I
B
to
accumulate in the nuclei of digitonin-permeabilized HeLa cells following addition of unlabeled GST-I
B
. These experiments were performed with limiting amounts of FITC-GST-I
B
protein to ensure that nuclear export was not titrated out simply by an excess of the
FITC-labeled protein. Although FITC-labeled GST-I
B
does not
efficiently accumulate in the nucleus in the absence of leptomycin B
(Fig. 9A), addition of a 100-fold molar
excess of unlabeled GST-I
B
resulted in a significant nuclear
accumulation of FITC-labeled GST-I
B
(Fig. 9B), consistent with
the notion that nuclear export of I
B
requires one or more
rate-limiting factors that can be titrated out with an excess of
I
B
protein.

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FIG. 9.
High capacity of the I B nuclear import pathway.
Nuclear import reactions with FITC-GST-I B were performed in the
absence (A and C) or presence (B and D) of 10 nM leptomycin B (LMB).
The import reactions were performed in the absence (A and B) or
presence (C and D) of a 100-fold molar excess of unlabeled
GST-I B . The cellular localization of the import substrates was
determined by direct fluorescence.
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|
Surprisingly, a 100-fold molar excess of unlabeled GST-I
B
did not
inhibit the nuclear accumulation of FITC-labeled GST-I
B
in the
presence of leptomycin B (compare Fig. 9C and D). The inability of an
excess of GST-I
B
to inhibit the nuclear import of FITC-labeled GST-I
B
is not simply a consequence of the N-terminal GST tag, because addition of a 100-fold molar excess of a His-tagged I
B
protein was also unable to inhibit the nuclear accumulation of FITC-labeled GST-I
B
(data not shown). Furthermore, addition of a
100-fold molar excess of unlabeled GST-I
B
-
N69 was also unable
to inhibit nuclear import of FITC-labeled GST-I
B
-
N69 (data not
shown). Taken together, these results indicate that nuclear import of
I
B
is not blocked by a 100-fold molar excess of specific competitor.
Nuclear import of I
B
is independent of GTP hydrolysis by
Ran.
The directionality of classical nuclear import and nuclear
export pathways is determined by the differential distribution of the
GTP-bound and GDP-bound forms of Ran between the nucleus and the
cytoplasm (21). To determine if nuclear shuttling of I
B
is sensitive to perturbation of the asymmetric Ran-nucleotide gradient, nuclear accumulation of GST-I
B
was determined in the presence of the dominant-negative mutant RanQ69L protein bound to GTP.
RanQ69L-GTP blocked nuclear import of GST-NLS (compare Fig. 10G and H)
and GST-M9 (data not shown). In contrast, RanQ69L-GTP did not inhibit
nuclear import of GST-I
B
in the presence of leptomycin B (compare
Fig. 10C and D) or of
GST-I
B
-
N69 in the absence of leptomycin B (compare Fig. 10E
and F). Consistent with the results obtained with the RanQ69L protein,
addition of the wild-type Ran protein preloaded with GTP
S inhibited
nuclear import of GST-NLS but did not inhibit nuclear import of either
GST-I
B
or GST-I
B
-
N69 (data not shown). Furthermore,
while addition of GTP
S or 5'-guanylylimdodiphosphate (GMP-PNP)
completely inhibited nuclear import of GST-NLS and GST-M9 (data not
shown), neither GTP
S nor GMP-PNP blocked nuclear import of
GST-I
B
or GST-I
B
-
N69 (data not shown).

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FIG. 10.
Nuclear import of I B is not inhibited by a
dominant-negative Ran protein. Nuclear import reactions with the
indicated substrate proteins were performed in the absence (A, B, and E
to H) or presence (C and D) of 10 nM leptomycin B (LMB). Parallel
import reactions were performed in the absence (A, C, E, and G) or
presence (B, D, F, and H) of 5 µg of the RanQ69L protein preloaded
with GTP. Localization of the import substrates was determined by
indirect immunofluorescence with a monoclonal antibody directed against
GST.
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|
The ability of the RanQ69L-GTP mutant protein to perturb nuclear export
of I
B
was also determined. Nuclear import reactions were
performed in the presence of the RanQ69L-GTP mutant protein, but in the
absence of leptomycin B. Analysis by confocal laser scanning microscopy
demonstrated that the inclusion of RanQ69L-GTP in the import reaction
allowed leptomycin B-independent nuclear accumulation of GST-I
B
(compare Fig. 11A and B). Nuclear rim accumulation of I
B
was observed in 30 to 50% of the cells (Fig. 11C), consistent with the notion that RanQ69L-GTP interfered with release of I
B
from CRM1 at nuclear pore sites involved in the termination of nuclear export. We hypothesized that the mutant RanQ69L-GTP protein interfered with termination of the nuclear export
reaction by titrating out a specific factor(s) necessary for
disassembly of the CRM1-cargo complex. The Ran-binding protein, RanBP1,
has been suggested to play a critical role in the disassembly of the
receptor-Ran-GTP complex (6). Addition of RanBP1 restored nuclear export of I
B
in the presence of RanQ69L-GTP (Fig. 11D). Thus, the ability of the RanQ69L-GTP mutant protein to inhibit nuclear
export of I
B
may be due to sequestration of endogenous RanBP1.

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FIG. 11.
RanQ69L-GTP inhibits nuclear export of I B through
sequestration of RanBP1. Nuclear import reactions were performed with
2.5 µg of FITC-GST-I B in the absence of leptomycin B (LMB).
RanQ69L-GTP (5 µg) was added to the import reactions shown in panels
B to D, and RanBP1 (15 µg) was added to the import reaction shown in
panel D. Localization of FITC-GST-I B was determined by direct
fluorescence with confocal laser scanning microscopy. A single
z-section of representative cells is shown in each panel.
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 |
DISCUSSION |
In the present study, we have utilized both in vitro and in vivo
approaches to understand how I
B
travels between the cytoplasm and
the nucleus. The results of our experiments lead to three important
conclusions: first, that I
B
rapidly shuttles between the
cytoplasm and the nucleus; second, that I
B
contains multiple domains that specify nuclear import and nuclear export; and third, that
nuclear import of I
B
is mediated by an import pathway that is
mechanistically distinct from classical nuclear import pathways.
Nuclear shuttling of I
B
.
We find that leptomycin B
markedly increased nuclear accumulation of I
B
in both
digitonin-permeabilized cells and in transiently transfected HeLa
cells. Our finding that nuclear accumulation of I
B
was increased
by leptomycin B in digitonin-permeabilized cells is in apparent
contrast to a recent report in which nuclear accumulation of I
B
in digitonin-permeabilized cells was observed in the absence of
leptomycin B treatment (46). However, a careful titration of
input FITC-GST-I
B
protein revealed that leptomycin B-independent
nuclear accumulation of I
B
was observed at high levels of input
protein, suggesting that one or more limiting export factors were
titrated out by an excess of I
B
. It has been established that
rate-limiting nuclear export factors can be lost during
permeabilization with digitonin (10, 26). It is likely that
subtle differences in the conditions used for permeabilization resulted
in more complete loss of CRM1 in the experiments reported by Turpin et
al. (46), such that their digitonin-permeabilized cells were
no longer competent for nuclear export of I
B
.
The ability of leptomycin B to increase nuclear accumulation of
I
B
in both digitonin-permeabilized cells and transiently transfected cells is consistent with several recent reports that leptomycin B treatment markedly increases nuclear accumulation of
either ectopically expressed or endogenous I
B
(24,
40). Taken together, the available experimental evidence strongly
supports a model in which I
B
rapidly shuttles between the nucleus
and the cytoplasm.
I
B
contains multiple cis-acting sequences for
nuclear import and export.
The distribution of I
B
between
the nucleus and the cytoplasm can be altered by specific mutations with
I
B
(24, 42). For example, we have previously
demonstrated that mutations within the second ankyrin repeat of
I
B
resulted in a marked relocalization of ectopically expressed
I
B
from the nucleus to the cytoplasm (42). Consistent
with this result, we find that the integrity of the second ankyrin
repeat is required for nuclear import of I
B
in
digitonin-permeabilized cells. However, mutation or deletion of the
second ankyrin repeat does not completely abolish nuclear import of
I
B
, because leptomycin B increased nuclear accumulation of the
GFP-I
B
-
Ank2 protein in transfected HeLa cells. Likewise, Hope
and coworkers found that GFP-I
B
constructs lacking the second
ankyrin repeat were able to accumulate in the nucleus in a leptomycin
B-dependent manner (24). In our previous work, we found that
the second ankyrin repeat was able to direct nuclear localization of a
heterologous cytoplasmic protein (42). The other ankyrin
repeats of I
B
also possess this nuclear import function
(42). Taken together, the data support a model in which I
B
contains multiple cis-acting nuclear import
sequences within its ARD. Because the steady-state distribution of
I
B
between the nucleus and the cytoplasm is the consequence of
rapid shuttling between the nucleus and the cytoplasm, mutation of any
individual nuclear import sequence will decrease the rate of nuclear
import of I
B
, resulting in a net increase in cytoplasmic
I
B
.
I
B
contains several leucine-rich sequences that resemble
canonical NESs, one located in the N-terminal domain, amino acids 45 to
55, and a second located in the C-terminal domain, amino acids 273 to
283. We find that deletion of the N-terminal 69 amino acids of I
B
enables leptomycin B-independent nuclear accumulation of the
GST-I
B
-
N69 fusion protein in digitonin-permeabilized cells and
of the GFP-I
B
-
N69 fusion protein in transiently transfected HeLa cells. Likewise, Hope and coworkers have demonstrated that mutation of two hydrophobic residues in this N-terminal domain abolished nuclear shuttling of a GST-I
B
fusion protein following microinjection into multinucleated 3T3 cells (24). In these experimental situations, the mutant I
B
proteins are present in a
large excess relative to the endogenous NF-
B proteins. Hence, it is
likely that these assays only measure nuclear export of free I
B
(i.e., I
B
proteins that are not present in a complex with
endogenous NF-
B/Rel proteins). Taken together, these results indicate that the N-terminal NES of I
B
is a major determinant for
nuclear export of free I
B
.
I
B
also contains a leucine-rich NES-like sequence in its C
terminus. Deletion or mutation of this C-terminal NES-like sequence has
no effect on nuclear export of the I
B
proteins