From the Laboratoire de Transport
nucléocytoplasmique, Unité Mixte de Recherche 144 Institut
Curie-CNRS, 26, rue d'Ulm, 75248 Paris Cedex 05, France and the
¶ Institute of Biomolecular Sciences, School of
Biomedical Sciences, University of St. Andrews, The North Haugh,
St. Andrews, KY169TS, Scotland
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ABSTRACT |
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I The Rel/NF- I Different pathways have been described to account for the nuclear
import of various types of karyophilic proteins (33, 34). Proteins
carrying a basic amino acid stretch NLS interact with a heterodimeric
NLS receptor composed of two subunits, importins The goal of the present study was to characterize precisely the
requirements for nuclear localization of I Cells and Culture Conditions--
Adherent or S3 suspension HeLa
cells were maintained in exponential growth in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum.
DNA Transfections--
For transient expression experiments,
HeLa cells were transfected by electroporation as described previously
(20) and cultured subsequently for 24 h before analysis.
Preparation of HeLa Cell Cytosol--
HeLa cell cytosol was
prepared as described by Paschal and Gerace (58). 109
exponentially growing HeLa S3 cells were collected by centrifugation at
300 × g for 10 min. The cells were washed twice with
phosphate-buffered saline and once with lysis buffer (5 mM
Hepes, pH 7.4; 5 mM potassium acetate, pH 7.4; 2 mM magnesium acetate; 1 mM EGTA; 2 mM dithiothreitol; and protease inhibitors: 10 µg/ml each
aprotinin, leupeptin, pepstatin; and 200 µg/ml
4-(2-aminoethyl)benzenesulfonyl fluoride (Uptima)). The cell pellet was
resuspended in 1 volume of lysis buffer and disrupted in a tight
fitting stainless steel homogenizer (as judged by phase-contrast
microscopy). The homogenate was diluted with 0.1 volume of 10 × transport buffer (20 mM Hepes, pH 7.4, 110 mM
potassium acetate, pH 7.4, 2 mM magnesium acetate, 0.5 mM EGTA, 1 mM dithiothreitol, and protease
inhibitors) and centrifuged at 40,000 × g for 30 min
at 4 °C. The supernatant was centrifuged further at 100,000 × g for 1 h. The resulting supernatant (~20 mg/ml as
measured with the protein assay kit (Bio-Rad)) was aliquoted, frozen in
liquid N2, and stored at
To prepare depleted cytosol, 100 µl of HeLa cell cytosol (2 mg of
proteins) was incubated overnight at 4 °C with 10 µg of GST or 20 µg of GST-I Nuclear Import Assay--
Digitonin-permeabilized HeLa cells
were prepared according to Adam et al. (62). Cells grown on
coverslips were permeabilized with 55 µg/ml digitonin (Sigma) in
transport buffer. A standard 50-µl nuclear import assay was performed
in transport buffer containing an energy-regenerating system (1 mM ATP, 0.5 mM GTP, 10 mM creatine phosphate, and 0.4 unit/ml creatine phosphokinase), 15 µg/ml
BSA-NLS-FITC, 30 µl of HeLa cell cytosol (~20 mg/ml), and 40 µg/ml or 10 µg/ml SV5-tagged versions of wt I NLS, SLN Peptides, and Preparation of Transport Substrate
(BSA-NLS-FITC)--
Peptides containing the SV40 large T antigen wt
NLS (cgggPKKKRKVED) or reverse NLS (SLN; cgggDEVKRKVED) were
synthesized with an NH2-terminal Cys for chemical coupling
reactions followed by a (Gly)3 linker. BSA was conjugated
to peptides and FITC according to Görlich et al. (38).
It should be noted that the resulting fusion proteins contain more than
10 NLS or SLN peptides.
Immunofluorescence Microscopy--
For indirect
immunofluorescence analysis, transfected HeLa cells were fixed with 3%
paraformaldehyde and permeabilized with 0.1% Triton X-100 for 10 min.
Digitonin-permeabilized HeLa cells were fixed with 2% paraformaldehyde
and 0.025% glutaraldehyde and permeabilized with 0.1% Triton X-100
for 5 min. Monoclonal antibodies to SV5-Pk-tag (63) or to I Plasmids--
DNA encoding the SV5-tagged version of I
To generate the SV5-tagged GST-I Expression of Recombinant Proteins--
Recombinant SV5-tagged
wt I
Expression vectors for His-tagged hSRP1- Both Endogenous and Overexpressed I
These results show that in HeLa cells, endogenous as well as
overexpressed I Nuclear Import of I
In unstimulated cells, I Nuclear Import of I
The subcellular localization of GFP-wt I
To confirm that the ankyrin repeats were responsible for the
nuclear import of I Nuclear Import of I
Nuclear import of either wt I Nuclear Import of I I
Cytosol was prepared for transport assays as usual except that, prior
to the assay, it was incubated with either GST or GST-I In response to distinct external stimuli, specific transcription
factors are activated which function to regulate the activity of unique
sets of target genes. The regulated nuclear import of transcription
factors provides an efficient mechanism to restrict their access to the
transcriptional machinery. However, in any case of signal-mediated
transcriptional regulation, termination of a regulated transcriptional
event is also required to turn off the cellular response and return to
a "resting" state. In principle, different mechanisms could operate
to turn off a transcriptional response, each of which could have
distinct consequences. Recently, it has been recognized that nuclear
export allows a transcriptional response not only to be terminated but
also to be subsequently reinitiated with minimal delay. The NF- Role of NF- I
The partner of IB
controls the transcriptional activity of
nuclear factor (NF)-
B by retaining it in the cytoplasm; but, when
expressed in the nucleus, it can also inhibit the interaction of
NF-
B with DNA and promote the export of NF-
B from the nucleus to
the cytoplasm. Here, we report that I
B
, when not bound to
NF-
B, is constitutively transported to the nucleus, and we confirm
that the interaction of I
B
with NF-
B retains I
B
in the
cytoplasm. Nuclear import of I
B
does not result from passive
diffusion but from a specific energy-dependent transport
process that requires the ankyrin repeats of I
B
. Nuclear
accumulation of I
B
is dependent on importins
and
as well
as the small GTPase Ran, which are also responsible for the nuclear
import mediated by basic nuclear localization sequences (NLS). However,
these proteins are not sufficient to promote I
B
nuclear
translocation. Factor(s) can be removed selectively from cell extracts
with ankyrin repeats of I
B
which strongly reduce import of
I
B
but not of proteins containing basic NLS. These findings
indicate that I
B
is imported in the nucleus by a piggy-back
mechanism that involves additional protein(s) containing a basic NLS
and able to interact with ankyrin repeats of I
B
.
INTRODUCTION
Top
Abstract
Introduction
References
B1
transcription factors are critical regulators of genes involved in
diverse cellular processes such as immune and inflammatory responses as
well as cell proliferation and apoptosis (1, 2). These factors share a
Rel homology domain responsible for the dimerization, nuclear
localization, and DNA binding functions (3, 4). In most unstimulated
cells, NF-
B dimers are held in an inactive state in the cytoplasm by
I
B inhibitory proteins that mask their nuclear localization sequence
(NLS) (5-8). In response to various stimuli, recently identified
protein kinase cascades are activated, resulting in the phosphorylation
of I
B proteins on two serine residues at their amino-terminal domain (9-14). This modification triggers polyubiquitination of I
B
proteins, which then undergo rapid degradation via the 26 S proteasome
(1). As a consequence, the NLS of NF-
B is exposed, and the
transcription factor translocates to the nucleus where it activates
responsive genes (15). In particular, NF-
B induces efficient
resynthesis of I
B
through the activation of I
B
mRNA
transcription (16-18). Newly synthesized I
B
accumulates in the
nucleus where it negatively regulates NF-
B-dependent
transcription by inhibiting the NF-
B/DNA interaction and by
transporting NF-
B back to the cytoplasm (19, 20). This latter
function of I
B
is conferred by a leucine-rich nuclear export
sequence, located in its COOH-terminal region, which is homologous to
the nuclear export signal found in HIV-1 Rev and PKI (the inhibitor of
the catalytic subunit of protein kinase A) (20-23). Such nuclear
export sequences are specifically recognized by the nuclear protein
CRM1 (exportin-1) (24-27), which promotes the transport of nuclear
export sequence-containing proteins and in particular
NF-
B·I
B
complexes from the nucleus to the cytoplasm.
B
is composed of a surface-exposed NH2-terminal
region, not essential for binding to RelA (p65), followed by a central, protease-resistant domain containing five ankyrin repeats and a
compact, highly acidic COOH-terminal region connected to the core by a
flexible linker (28). Both the central ankyrin domain and the linker
region are essential for the interaction of I
B
with Rel factors
(28-31). I
B
lacks an SV40 large T antigen- or nucleoplasmin-like
NLS (basic NLS) or any other motif described to serve as NLS (8).
Besides the ability of newly synthesized I
B
to localize in the
nucleus, it has been reported that I
B
overexpressed from a
transfected plasmid or microinjected into the cytoplasm also
distributes to both the cytoplasmic and nuclear compartments (8, 20).
Its molecular mass (37 kDa), which is below the theoretical cutoff of
the nuclear pore complex, led to the hypothesis that I
B
might
enter the nucleus by diffusion (8, 32).
and
or
karyopherins
and
1 (35-44); for alternative nomenclatures, see
Ref. 34. hnRNP A1 import depends on another motif called M9, which is
recognized by transportin or karyopherin
2 (45-47), whereas other
members of the karyopherin
family, karyopherins
3 and
4, are
responsible for the nuclear import of ribosomal proteins (48-51).
Recognition of karyophilic substrates by these karyopherins
leads
to the targeting of karyophilic proteins to the nuclear pore complex, a
specialized and elaborated structure of the nuclear envelope through
which the exchange of macromolecules between the nucleus and cytoplasm
occurs (34). Karyopherin
family members share a common
NH2-terminal binding motif for RanGTP (52), a small GTPase
essential for most nucleocytoplasmic transport pathways (53-56). Ran
is thought to be distributed asymmetrically between the nucleus and
cytoplasm with the GTP-bound form in the nucleus and the GDP-bound form
in the cytoplasm. Both GTPase Ran and the factor p10 or NTF2 (57-59)
mediate the translocation of karyopherin-karyophilic protein complexes
in the nucleus. Interaction of these complexes with RanGTP in the
nucleus promotes the release of karyophilic proteins in this
compartment and subsequent recycling of the NLS receptors (42, 60,
61).
B
as well as the molecular mechanisms underlying this transport process. We found that
NF-
B-free I
B
localizes constitutively in the cytoplasm and in
the nucleus, and we confirm that the interaction of I
B
with
NF-
B retains I
B
in the cytoplasm. Moreover we demonstrate that
the nuclear import of I
B
does not result from passive diffusion but rather from an energy-dependent process that is
mediated by the ankyrin repeats of I
B
. Nuclear import of I
B
requires importins
and
(karyopherins
and
1) as well as
the GTPase Ran. However, these proteins are not sufficient to promote
I
B
nuclear translocation. Factor(s) can be removed selectively
from cell extracts with ankyrin repeats of I
B
which strongly
reduce import of I
B
but not of proteins containing a basic amino
acid stretch NLS. These findings indicate that I
B
is imported in
the nucleus by a piggy-back mechanism that involves additional
protein(s) containing a basic NLS and able to interact with ankyrin
repeats of I
B
.
EXPERIMENTAL PROCEDURES
80 °C.
B
(68-243) immobilized on 20 µl of
glutathione-Sepharose beads (Amersham Pharmacia Biotech). Mixtures were
subsequently centrifuged, and the postcentrifugation supernatant is
defined as depleted cytosol.
B
or
GST-I
B
(68-243), respectively. The reaction was allowed to
proceed for 45 min at 30 °C. Similar results were obtained with an
untagged version of I
B
(data not shown).
B
(10B
(28)) and polyclonal antibodies to NF-
B p65 (C-20, Santa Cruz) or to
GST (64) were applied for 30 min followed by a 30-min incubation with
Texas red or FITC-conjugated donkey anti-mouse or anti-rabbit IgG
(Jackson). Coverslips were mounted in phosphate-buffered saline
containing 50% glycerol. Confocal laser scanning microscopy and
immunofluorescence analysis were performed with a TCS4D confocal
microscope based on a DM microscope interfaced with a mixed gas
argon-krypton laser (Leica Laser Technik). Fluorescence acquisitions
were performed with the 488 nm and 568 nm laser lines to excite FITC
and Texas red dyes, respectively, with a × 100 oil immersion PL
APO objective. Data presented on a same figure were registered at the
same laser and multiplier settings. To quantify fluorescence intensity,
optical slices of 20 different cells/condition were recorded as
512 × 512-pixel images with the same preset parameters. A region
of interest corresponding to the nuclear area was then created in each
cell, and the mean density was calculated within this area using NIH
image software. For measurement of I
B
or GST-I
B
(68-243) nuclear content, values obtained with nuclei incubated in the absence
of I
B
or GST-I
B
(68-243) and stained with both primary and
secondary antibodies were considered as negative controls and
subtracted from the values obtained for nuclei incubated with the
import substrate. For measurement of BSA-NLS-FITC nuclear content,
areas registered outside the cells were considered as background. All
data were saved in different series, and statistical analysis (mean
intensities and standard deviation) was performed.
B
sequences for amino acids 1-317 or 68-317 were amplified by
polymerase chain reaction using the pcDNA3-I
B
ctag vector
(20) as template and cloned into the BamHI/XbaI
restriction sites of the eukaryotic expression vector pEGFP-C1
(CLONTECH) generating pEGFP-wt I
B
ctag or
pEGFP-I
B
(68-317) ctag, respectively. Sequences encoding amino
acids 68-265 or 68-243 of I
B
were amplified using
pcDNA3-I
B
ctag vector as a template and cloned into the
BamHI/EcoRI restriction sites of pcDNA3 ctag (pcDNA3-I
B
ctag without the
BamHI/EcoRI-wt I
B
fragment) generating pcDNA3-I
B
(68-265) ctag or pcDNA3-I
B
(68-243) ctag,
respectively. The corresponding BamHI/XbaI
restriction fragments were inserted into
BamHI/XbaI-cleaved pEGFP-C1 vector generating
pEGFP-I
B
(68-265) ctag or pEGFP-I
B
(68-243) ctag,
respectively. The plasmid encoding 4N
C has been described previously
(65).
B
(68-243) in bacteria, a
BamHI/EcoRI restriction fragment encoding amino
acids 68-243 of I
B
was generated using
pcDNA3-I
B
(68-243) ctag and inserted into
BamHI/EcoRI-cleaved pGEX ctag vector (63)
generating pGEX-I
B
(68-243) ctag.
B
was produced and purified as described previously (66).
pGEX-2T (Amersham Pharmacia Biotech) or pGEX-I
B
(68-243) ctag
plasmids were transformed into Escherichia coli DH5
or
BL21(DE3) respectively. Cells were grown to an
A600 of 0.4 and induced with 1 mM
isopropyl-1-thio-
-D-galactopyranoside for 3 h at
37 °C. Recombinant GST or SV5-tagged GST-I
B
(68-243) were
purified on glutathione-Sepharose beads (Amersham Pharmacia Biotech),
eluted with glutathione, and dialyzed at 4 °C against transport
buffer. The protein concentration was determined with the protein assay
kit (Bio-Rad), and the sample was aliquoted, quick frozen in liquid
N2, and stored at
80 °C. NF-
B p65 (amino acids
12-317) was expressed in bacteria and purified as described (67).
, His-tagged IBB (pKW312)
and wild-type untagged importin
/p97 (pKW291) were provided by K. Weis (University of California, San Francisco), and proteins were
expressed and purified as described (44, 68). Plasmid for GST-Ran
expression was a gift from M. Dasso, and Ran wt was purified as
described (NIH, Bethesda; 69). Expression and purification of RanQ69L
were performed essentially as described (70) using the expression
vector provided by C. Dingwall (Stony Brook, NY). His-tagged p10
expression and purification were done as described (41) using the p10
expression vector provided by G. Blobel (The Rockefeller Institute, New York).
RESULTS
B
Can Localize to the
Nucleus in the Absence of Cell Stimulation--
To investigate the
mechanisms responsible for the nuclear accumulation of I
B
, we
first addressed the question of whether the ability of I
B
to be
imported into the nucleus depends on cell activation. In unstimulated
HeLa cells, I
B
was distributed throughout both the nucleus and
the cytoplasm, whereas NF-
B p65 was localized predominantly in the
cytoplasmic compartment (Fig. 1A). This result suggests that
even in the absence of cell activation, a fraction of I
B
which
does not interact with p65 is expressed in the nucleus. To confirm this
result, HeLa cells were transfected with a plasmid encoding a tagged
version of I
B
which could be detected with an anti-tag antibody
(Fig. 1B, panel a). Overexpressed I
B
displayed a cytoplasmic as well as a nuclear localization in HeLa
cells. This distribution has also been observed recently in fibroblasts
lacking Rel proteins (66), indicating that the ability of I
B
to
be expressed in the nucleus is not dependent on the presence of
endogenous NF-
B family members. To establish whether the nuclear
localization of I
B
was caused by serum activation of the cells,
transfected HeLa cells were treated with 100 µg/ml cycloheximide for
1 h, leading to the complete disappearance of the protein in both
nuclear and cytoplasmic compartments (Fig. 1B, panel
b). Cycloheximide was then removed, and cells were incubated for 2 additional h without serum. Under this unstimulated condition, newly
synthesized I
B
accumulated in both the nucleus and the cytoplasm
(Fig. 1B, panel c), indicating that nuclear
localization of overexpressed I
B
could occur in the absence of
stimulation of HeLa cells. Cells were then incubated with tumor
necrosis factor-
for 30 min leading to degradation of I
B
(Fig.
1B, panel d). Tumor necrosis factor-
was then
removed, and newly synthesized I
B
again accumulated in both
cytoplasmic and nuclear compartments (Fig. 1B, panel
e).
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Fig. 1.
Constitutive localization of
I B
in both nuclear
and cytoplasmic compartments. Panel A, endogenous
I
B
or NF-
B p65 was localized in HeLa cells by indirect
immunofluorescence using I
B
mouse monoclonal antibodies and
NF-
B p65 rabbit polyclonal antibodies. Panel B,
transfected epitope-tagged wt I
B
was immunolocalized using an
anti-tag monoclonal antibody followed by a FITC-conjugated anti-mouse
antibody. Transfected HeLa cells cultured in the presence of serum
(a) were washed and treated for 1 h with 100 µg/ml
cycloheximide (CHX; b). This drug was then
removed, and the cells were incubated in the absence of serum for
2 h (c) followed by a 30-min stimulation with tumor
necrosis factor (TNF)-
(d) and a 30-min chase
(e). Cells were visualized with a confocal laser scanning
microscope, and photographs correspond to the accumulation of four
optical sections in one projection. Data were recorded at the same
laser and multiplier settings.
B
can localize to the nucleus in the absence of
cell stimulation or after treatment with tumor necrosis factor-
.
B
Is an Active Process Inhibited by
NF-
B p65--
To establish whether nuclear localization of I
B
was caused by passive diffusion or by a specific nuclear import
mechanism, properties of I
B
nuclear transport were analyzed using
semipermeabilized cells (62). Digitonin-permeabilized HeLa cells were
incubated for 45 min at 30 °C with recombinant wt I
B
in the
presence of HeLa cytosol, an energy source (ATP, GTP, and an
ATP-regenerating system), and BSA-NLS-FITC as a control for import of a
karyophilic protein (Fig. 2). Under this
experimental condition both BSA-NLS-FITC and I
B
accumulated in
the nucleus. Accumulation of either BSA-NLS-FITC or wt I
B
did not
occur when the cytosol was replaced by BSA or when apyrase was added to
cytosol (Fig. 2 and Table I), indicating that the nuclear import of wt I
B
as BSA-NLS-FITC was not the result of diffusion into the nucleus but was rather a specific cytosol-
and energy-dependent nuclear import process. Replacement of
HeLa cytosol by BSA (or other inhibitory conditions, see Fig. 4) led to
the interaction of I
B
with cytoplasmic structures including the
nuclear envelope. This effect was probably caused by the intrinsic
ability of the NH2-terminal region of I
B
to promote
retention on similar structures under certain experimental conditions
but was not relevant for nuclear import activity (data not shown).
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Fig. 2.
Nuclear import of
I B
in
semipermeabilized HeLa cells requires both cytosol and energy and is
inhibited by p65. In the left panel,
digitonin-permeabilized HeLa cells were incubated at 30 °C for 45 min with BSA-NLS-FITC and recombinant wt I
B
in the presence of
HeLa cytosol (12 mg/ml) and an energy-regenerating system (ATP), BSA
(12 mg/ml), and ATP, HeLa cytosol, and apyrase (Sigma, 24 units/ml) or
HeLa cytosol, ATP, and recombinant p65(12-317) (20 µg/ml) as
indicated. In the right panel, digitonin-permeabilized HeLa
cells were incubated at 30 °C for 45 min with BSA-NLS-FITC only, in
the presence of HeLa cytosol (12 mg/ml) and an energy-regenerating
system (ATP). After incubation, cells were processed for indirect
immunofluorescence with a tag-specific monoclonal antibody followed by
a Texas red-conjugated donkey anti-mouse antibody. Cells were
visualized with a confocal laser scanning microscope, and photographs
correspond to the accumulation of four optical sections in one
projection.
Nuclear import of BSA-NLS-FITC, IB
, and GST-I
B
(68-243) in
semipermeabilized HeLa cells
B
has been shown to prevent nuclear
import of NF-
B by masking its NLS (5, 6, 8). Moreover, nuclear
localization of I
B
is inhibited in vivo by
overexpressed NF-
B members p65 or p50 (8). To investigate whether
NF-
B can control the nuclear accumulation of I
B
, nuclear
import of I
B
was analyzed in semipermeabilized cells in the
presence of extracts, energy-regenerating system, and recombinant Rel
domain of p65 (amino acids 12-317; p65(12-317)). The Rel domain is
necessary and sufficient for association with I
B
, dimerization,
DNA binding, and nuclear localization (28). As shown in Fig. 2,
p65(12-317) inhibited the nuclear import of I
B
without affecting
that of BSA-NLS-FITC. This result indicates that the domains involved in the nuclear import of I
B
and p65 are mutually masked when these proteins interact. Therefore, nuclear accumulation of I
B
is
an active process that is inhibited by NF-
B p65.
B
Is Mediated by Its Ankyrin
Repeats--
None of the previously described nuclear localization
signals can be recognized in the sequence of I
B
. To define the
motif responsible for its nuclear import, nucleocytoplasmic
distribution of fusion proteins between the green fluorescent protein
(GFP) and different domains of I
B
was analyzed. These fusion
proteins had predicted molecular masses greater than 50 kDa and
therefore were not able to diffuse into the nucleus. Plasmids encoding
GFP fused to tagged version of wild-type I
B
(GFP-wt I
B
),
I
B
lacking the NH2-terminal domain
(GFP-I
B
(68-317)) or both NH2- and COOH-terminal
domains (GFP-I
B
(68-265)), or I
B
ankyrin repeats
(GFP-I
B
(68-243)) (Fig.
3A) were generated and
transiently transfected in HeLa cells. Overexpressed proteins were
detected both with GFP fluorescence and with a specific anti-tag
antibody.
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Fig. 3.
Nuclear import of
I B
is mediated by its
ankyrin repeats. Panel A, schematic drawing of I
B
and SV5-tagged GFP or
-galactosidase-I
B
fusion proteins. The
positions of the amino acids that delimit the different domains of
I
B
(NH2-terminal domain, residues 1-68; ankyrin
repeats domain, residues 68-243; linker domain, residues 243-265; and
COOH-terminal domain, residues 265-317) are indicated in
bold, and the names of the overexpressed proteins are
indicated on the left. Panel B, epitope-tagged
GFP or
-galactosidase-I
B
fusion proteins were transiently
expressed in HeLa cells, stained using a tag-specific antibody
(upper) or visualized directly with GFP fluorescence
(lower). For the GFP-wt I
B
fusion protein, cells
expressing high levels of the exogenous proteins are also shown (see
inset). Cells were visualized with a confocal laser scanning
microscope, and photographs correspond to the accumulation of four
optical sections in one projection. Panel C,
upper, digitonin-permeabilized HeLa cells were incubated at
30 °C for 45 min with BSA-NLS-FITC and either 40 µg/ml recombinant
GST-I
B
(68-243) or 20 µg/ml GST in the presence of HeLa cytosol
(12 mg/ml) and an ATP-regenerating system. After incubation, cells were
processed for indirect immunofluorescence with an anti-GST polyclonal
antibody or a monoclonal anti-tag antibody followed by a Texas
red-conjugated donkey anti-rabbit antibody or a Texas red-conjugated
donkey anti-mouse antibody respectively. Lower,
digitonin-permeabilized HeLa cells were incubated at 30 °C for 45 min with BSA-NLS-FITC and recombinant GST-I
B
(68-243) in the
presence of HeLa cytosol (12 mg/ml) and ATP, BSA (12 mg/ml), and ATP or
HeLa cytosol and apyrase (Sigma, 24 units/ml) as indicated. After
incubation, cells were processed for indirect immunofluorescence with
an anti-GST polyclonal antibody followed by a Texas red-conjugated
donkey anti-rabbit antibody. Cells were visualized with a confocal
laser scanning microscope, and photographs correspond to the
accumulation of four optical sections in one projection.
B
depended on its level
of expression (Fig. 3B). In cells expressing low amounts of
GFP-wt I
B
, this protein was essentially cytoplasmic, but in cells
expressing higher amounts, this protein partitioned equally between
nucleus and cytoplasm (Fig. 3B, compare the inset
with the rest of the panel). However when the
NH2-terminal domain of I
B
(GFP-I
B
(68-317)) or
both NH2- and COOH-terminal domains (GFP-I
B
(68-265)
and GFP-I
B
(68-243)) were deleted, the resulting fusion proteins
were localized in both the nucleus and the cytoplasm whatever their
expression level. It should be noted that the immunofluorescence signal
obtained with these proteins was more intense in the nucleus than in
the cytoplasm. Because both GFP-wt I
B
and GFP-I
B
(68-317) are able to interact with NF-
B (data not shown), the different distribution of these two protein suggests that the
NH2-terminal domain of I
B
prevents the nuclear
accumulation of GFP-wt I
B
. Both NH2- and
COOH-terminal domains of I
B
were also fused to
-galactosidase
(Fig. 3A; 43) but none of these regions was able to direct
-galactosidase to the nucleus (Fig. 3B). These results indicate that the ankyrin repeats of I
B
are necessary and
sufficient to target this protein in the nucleus of intact cells and
confirm a recent study showing that the nuclear localization of
I
B
requires the integrity of hydrophobic residues within the
second ankyrin repeat (66).
B
, a recombinant fusion protein between GST
and the ankyrin repeats of I
B
(GST-I
B
(68-243)) was
produced in bacteria and tested for its ability to be imported to the
nucleus in semipermeabilized HeLa cells. GST-I
B
(68-243)
visualized using either anti-GST or anti-SV5 tag antibodies was
imported efficiently into nuclei in a cytosol- and
energy-dependent manner (Fig. 3C, upper and lower panels). In contrast, GST is not
accumulated in the nucleus in the presence of cytosol and energy (Table
I and Fig. 3C, upper panel). This result
confirms, by the permeabilized cells assay, that the nuclear import of
I
B
is mediated by its ankyrin repeats.
B
Is a GTPase Ran-dependent
Process--
To gain insight into the molecular mechanism allowing
I
B
to enter the nucleus, the involvement of the small GTPase Ran
was investigated. Indeed, Ran is involved not only in the nuclear import of basic NLS-containing proteins but also in the nuclear import
of snRNPs, hnRNPs as well as in nuclear export of both RNAs and
proteins (53, 54, 71, 72).
B
or GST-I
B
(68-243) and
BSA-NLS-FITC in semipermeabilized cells was analyzed in the presence of
HeLa extracts, an energy-regenerating system, and GTP
S, a nonhydrolyzable analog of GTP (Table I and Fig.
4, A and B). GTP
S at a 5 mM concentration strongly inhibited nuclear
accumulation of these proteins, indicating that GTP hydrolysis is
required for wt I
B
nuclear import. To test the involvement of Ran
in this process, a nuclear import assay was performed in the presence of either a recombinant GTPase-deficient Ran mutant (RanQ69L) or
recombinant wild-type Ran (Ran wt). Nuclear import of BSA-NLS-FITC, wt
I
B
and GST-I
B
(68-243) was blocked by RanQ69L, whereas
transport of these proteins was only slightly impaired in the presence
of wt Ran (Table I and Fig. 4, A and B). In
contrast, neither RanQ69L nor Ran wt affected diffusion into the
nucleus (data not shown). These results indicate that the GTPase Ran is
necessary for the ankyrin repeat-mediated nuclear import of
I
B
.
View larger version (108K):
[in a new window]
Fig. 4.
Nuclear import of
I B
involves the
GTPase Ran. Digitonin-permeabilized HeLa cells were incubated at
30 °C for 45 min with BSA-NLS-FITC and either recombinant wt
I
B
(panel A) or recombinant GST-I
B
(68-243)
(panel B) in the presence of HeLa cytosol and an
ATP-regenerating system in the absence (control) or in the presence of
5 mM GTP
S, 3 µM RanQ69L, or 3 µM Ran wt as indicated. After incubation, cells were
processed for indirect immunofluorescence with either a tag-specific
monoclonal antibody followed by a Texas red-conjugated donkey
anti-mouse antibody for detection of wt I
B
or an anti-GST rabbit
polyclonal antibody followed by a Texas red-conjugated donkey
anti-rabbit antibody for detection of GST-I
B
(68-243). Cells were
visualized with a confocal laser scanning microscope, and photographs
correspond to the accumulation of four optical sections in one
projection.
B
Requires Both Importins
and
--
Different hypotheses could account for the
Ran-dependent nuclear transport of I
B
. I
B
might
enter the nucleus by interacting with a NLS-containing protein
(piggy-back), through a direct interaction with one of the component of
the NLS import machinery or by using another Ran-dependent
import pathway. To distinguish between these mechanisms, the ability of
a peptide corresponding to the SV40 large T antigen NLS to compete with
the nuclear import of wt I
B
or GST-I
B
(68-243) and
BSA-NLS-FITC was analyzed. As shown in Fig.
5, A and B, NLS but
not SLN peptide (reversed NLS sequence), completely blocked transport
of the analyzed karyophilic proteins in the nucleus of permeabilized
cells, indicating that nuclear import of I
B
required the basic
NLS receptor, importin
(See also Table I). Because importin
binds importin
to mediate basic NLS-dependent import,
the involvement of the importin
in the nuclear uptake of I
B
was then analyzed. For this purpose, a recombinant protein
corresponding to the importin
-binding site of importin
(IBB)
reported previously to act as a competitive inhibitor of the basic NLS
nuclear import machinery (68, 73), was added in the nuclear import
assay. This recombinant protein blocked nuclear import of BSA-NLS-FITC
as well as nuclear accumulation of wt I
B
or GST-I
B
(68-243)
(Table I and Fig. 5, A and B) but did not affect
diffusion into the nucleus (data not shown). Taken together, these
results indicate that importins
and
are required for the
ankyrin repeat-mediated nuclear import of I
B
. Therefore the
involvement of another Ran-dependent pathway would be
minimal in such a system.
View larger version (111K):
[in a new window]
Fig. 5.
Nuclear import of
I B
requires the basic
NLS receptor (importins
/
).
Digitonin-permeabilized HeLa cells were incubated at 30 °C for 45 min with BSA-NLS-FITC and either recombinant wt I
B
(panel
A) or recombinant GST-I
B
(68-243) (panel B) in
the presence of HeLa cytosol and an ATP-regenerating system in the
absence (control) or in the presence of 1 mM NLS peptide, 1 mM SLN peptide, or IBB (5 µM in panel
A and 20 µM in panel B), as indicated.
After incubation, cells were processed for indirect immunofluorescence
with either a tag-specific monoclonal antibody followed by a Texas
red-conjugated donkey anti-mouse antibody for detection of wt I
B
or an anti-GST polyclonal antibody followed by a Texas red-conjugated
donkey anti-rabbit antibody for detection of GST-I
B
(68-243).
Cells were visualized with a confocal laser scanning microscope, and
photographs correspond to the accumulation of four optical sections in
one projection.
B
Is Imported into the Nucleus by a Piggy-back
Mechanism--
To distinguish a basic NLS-dependent
piggy-back mechanism and a direct interaction of I
B
with the
basic NLS nuclear import machinery, we analyzed the nuclear import
properties of BSA-NLS-FITC and either wt I
B
or
GST-I
B
(68-243) in the presence of an energy-regenerating system
and the recombinant purified nuclear import machinery consisting of the
importins
and
, Ran, and p10. Although the addition of these
recombinant proteins was sufficient to promote nuclear accumulation of
BSA-NLS-FITC, neither wt I
B
nor GST-I
B
(68-243) was
imported in this experimental condition (Fig.
6A).
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[in a new window]
Fig. 6.
I B
is imported
in the nucleus by a piggy-back mechanism. Panel A,
digitonin-permeabilized HeLa cells were incubated at 30 °C for 45 min with BSA-NLS-FITC and either recombinant wt I
B
or recombinant
GST-I
B
(68-243) in the presence of an ATP-regenerating system and
the recombinant basic NLS nuclear import machinery consisting of 1 µM importin
, 400 nM importin
, 1.4 µM Ran wt, and 1.5 µM p10. Panel
B, digitonin-permeabilized HeLa cells were incubated at 30 °C
for 30 min with BSA-NLS-FITC and recombinant wt I
B
in the
presence of an ATP-regenerating system and HeLa cytosol previously
incubated with either GST or GST-I
B
(68-243) beads (depleted
cytosol). After incubation, cells were processed for indirect
immunofluorescence with a tag-specific monoclonal antibody followed by
a Texas red-conjugated donkey anti-mouse antibody. Cells were
visualized with a confocal laser scanning microscope, and photographs
correspond to the accumulation of four optical sections in one
projection.
B
(68-243) immobilized on glutathione-Sepharose beads. Depletion of cytosol with
GST had no effect on the ability of the cytosol to support nuclear
import of either BSA-NLS-FITC or wt I
B
. However, cytosol depleted
with GST-I
B
(68-243), although still able to support nuclear
import of BSA-NLS-FITC, was strongly affected in its I
B
nuclear
import activity (Fig. 6B). This result shows that cytosol contains cellular factor(s) able to bind ankyrin repeats of I
B
and essential for the nuclear import of I
B
but not of proteins containing a basic amino acid stretch NLS. I
B
is thus likely imported in the nucleus by a piggy-back mechanism that involves additional protein(s) containing a basic NLS and able to interact with
ankyrin repeats of I
B
.
DISCUSSION
B
transcription factor constitutes a well documented example of this
phenomenon. The transcriptional activity of NF-
B is regulated mainly
by its subcellular localization, which is determined by the level of
expression as well as by the nucleocytoplasmic distribution of the
I
B proteins. In particular, I
B
retains NF-
B in an inactive
form in the cytoplasm (5, 6, 8), but it can also enter the nucleus
where it inhibits NF-
B/DNA interaction and transports NF-
B back
to the cytoplasm (19, 20). To define more clearly the physiological conditions that lead to the nuclear expression of I
B
and
subsequent termination of the NF-
B-dependent
transcription, it was necessary to characterize the requirements and
mechanisms accounting for I
B
nuclear import.
B in the Cytoplasmic Retention of I
B
--
In
the present report, we show that when I
B
is not bound to NF-
B,
it is constitutively imported into the nucleus both in vivo
and in vitro. Cytoplasmic NF-
B-free I
B
exists when
the I
B
expression level exceeds the amount of NF-
B or when
these proteins are distributed differentially between the nucleus and cytoplasm. Overexpression of I
B
by transient transfection with plasmid encoding I
B
(8, 20) and physiological situations such as
NF-
B-induced de novo synthesis of I
B
(19) or
dissociation of the NF-
B·I
B
complex by phosphorylation of
I
B
tyrosine 42 (74) have been reported to produce NF-
B-free
I
B
. Subcellular distribution of I
B
has been investigated in
some of these conditions and found to be both cytoplasmic and nuclear
(75). In contrast, when I
B
is overexpressed as an NF-
B-bound
form, it localizes exclusively in the cytoplasm (8). These data suggest
that interaction of NF-
B with I
B
in the cytoplasm could either
prevent diffusion of I
B
into the nucleus by forming a complex
that is unable to translocate freely through the nuclear pore or mask a
region of I
B
involved in its nuclear import. Using
semipermeabilized cells, we show here that I
B
does not pass to
the nucleus by diffusion but is transported there by an extract- and
energy-dependent pathway. Both in vitro and
in vivo, the ankyrin repeats appear sufficient to promote
the nuclear import of I
B
. This result confirms a recent study
showing that the nuclear localization of I
B
requires the
integrity of hydrophobic residues within the second ankyrin repeat
(66). It has been well documented that the five ankyrin repeats of
I
B
bind the NF-
B Rel homology domain, and the sixth degenerated ankyrin repeat, also called the "linker" region, also participates in the interaction with NF-
B (28, 30, 76). The Rel
homology domain of p65 prevents not only the transport of I
B
but
also the nuclear accumulation of GST-I
B
(68-243) in the nucleus
of semipermeabilized cells (a higher concentration of p65 is necessary
to inhibit nuclear import of GST-I
B
(68-243); data not shown).
Taken together, these results demonstrate that NF-
B retains
I
B
in the cytoplasm by masking its ankyrin repeats, which are
essential for nuclear import. In unstimulated cells, cytoplasmic
retention of NF-
B·I
B
complexes is therefore caused by a
mutual masking of the sequences responsible for the nuclear import of
both proteins.
B
Nuclear Import Pathway--
Ankyrin repeats could allow
NF-
B-free I
B
to enter the nucleus via a novel nuclear import
pathway or, alternatively, interact directly or indirectly (piggy-back)
with known nuclear import receptors. The present report shows that
nuclear import of I
B
requires GTP hydrolysis by Ran as well as
the basic NLS receptors, importins
and
. The whole purified
recombinant nuclear import machinery (importins
/
, Ran, and p10)
was however not sufficient to target I
B
or GST-I
B
(68-243)
into the nucleus. When HeLa cytosol was submitted to a GST affinity
column, the flow-through was able to promote nuclear import of both
BSA-NLS-FITC and I
B
. In contrast, the flow-through resulting from
a GST-I
B
(68-243) affinity column was not affected in its ability
to induce BSA-NLS-FITC nuclear import but was unable to promote nuclear
import of I
B
. We thus propose that ankyrin repeats of I
B
probably interact with additional component(s) containing a basic NLS
that is recognized by the basic NLS receptor (piggy-back mechanism).
Similar piggy-back mechanisms accounting for protein nuclear import
have been reported already. For example, the 46-kDa subunit of the
mouse DNA primase does not have an NLS but enters nuclei upon
interaction with the 54-kDa subunit, which carries a basic NLS (77).
Although NF-
B itself contains an NLS and binds I
B
ankyrin
repeats, the Rel homology domain of p65 was unable to target I
B
or GST-I
B
(68-243) into the nucleus of semipermeabilized cells in
the presence of the recombinant basic NLS nuclear import machinery
(data not shown). Moreover, overexpressed wt I
B
displays
identical subcellular distribution in fibroblasts expressing or lacking
p50, p52, p65, or c-Rel (66), indicating that NF-
B is therefore
unlikely to be responsible for the nuclear import of I
B
.
B
required for import may specifically recognize
ankyrin repeats of I
B
or, alternatively, structures shared by
other ankyrin repeat-containing proteins. The presence of ankyrin repeats is a common characteristic of I
B proteins, and,
interestingly, some of them have been shown to localize in the nucleus.
I
B members display an expression pattern depending on cell types and
different affinities for the NF-
B members (for review, see Refs. 3
and 4). I
B
has no NLS and interacts with the same subset of
NF-
B proteins as I
B
. Upon certain stimuli, I
B
is
degraded and subsequently resynthesized, but it accumulates as a
hypophosphorylated protein. Interaction of this newly synthesized
I
B
with NF-
B fails to mask both the NLS and DNA binding domain
of NF-
B and therefore leads to a complex able to enter the nucleus
by piggy-back and activate transcription (78, 79). Bcl-3, an I
B
protein containing two basic NLS in its NH2-terminal
domain, is expressed predominantly in the nucleus of lymphoid cells and
binds NF-
B p50 and p52 homodimers. This interaction does not mask
NF-
B NLS and results in the nuclear import of
(p50)2·Bcl-3 complexes. It has been reported that this transport can be ensured either by p50 NLS or by Bcl-3 NLS (80). From
these data, it appears clearly that the ankyrin repeats of Bcl-3 or
I
B
are not involved in the physiological nuclear import of these
proteins, although the intrinsic ability of these repeats to localize
in the nucleus has been reported recently (66). In particular, when
I
B
is overexpressed from a transfected vector in HeLa cells, it
localizes both in the cytoplasm and in the nucleus (data not shown). On
the other hand, some ankyrin repeat-containing proteins other than from
the I
B family have also been shown to be expressed in the nucleus.
For example, oncogenic intracellular forms of NOTCH can still be
detected in the nucleus when their two putative NLS have been deleted
(81, 82). In addition, a 37-kDa fragment composed of the ankyrin
repeats of the recently identified 104-kDa diacylglycerol kinase
DGK-IV/DGK-
and lacking a recognizable NLS accumulates in the
nucleus (83). It has been reported recently that ankyrin repeats of
53BP2 and GABP
but not Notch1 are able, when fused to a reporter, to
target the resulting fusion protein to the nucleus (66). Whether a
common protein or protein family is responsible for the nuclear import
of ankyrin repeat-containing proteins remains to be elucidated.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Drs. R. Goldsteyn, C. Maison, and J. Salamero for a critical reading of the manuscript. We
thank Drs. K. Weis, M. Dasso, C. Dingwall, G. Blobel, M. Rodriguez, and
T. Galli for the generous gift of expression vectors coding for
importins and
as well as IBB, GST-Ran, RanQ69L, p10, 4N
C,
and anti-GST antibodies, respectively. We thank Ellis Jaffray for
technical support in protein production and purification.
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FOOTNOTES |
---|
* This work was supported in part by grants from the Association de Recherche contre le Cancer and the European Communities Concerted Action Project Rocio II.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.
§ Supported by a fellowship from the Ministere de l'Education Nationale.
Supported by the Medical Research Council.
** To whom correspondence should be addressed. Tel.: 33-1-4234-6366; Fax: 33-1-4234-6367; E-mail: dargemon{at}curie.fr.
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ABBREVIATIONS |
---|
The abbreviations used are:
NF-B, nuclear
factor
B;
NLS, nuclear localization sequence(s);
HIV-1, human
immunodeficiency virus type 1;
GST, glutathione
S-transferase;
BSA, bovine serum albumin;
FITC, fluorescein
isothiocyanate;
wt, wild-type;
GFP, green fluorescent protein;
hnRNP, heterogenous nuclear ribonucleoprotein;
IBB, importin
binding site;
GTP
S, guanosine 5'-3- O-(thio)triphosphate.
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REFERENCES |
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