From the Department of Cell Biology and Neuroscience,
Graduate School of Medicine, Osaka University, 2-2 Yamada-oka and
¶ Institute for Molecular and Cellular Biology, Osaka University,
1-3 Yamada-oka, Suita, Osaka 565-0871, Japan
Received for publication, September 11, 2000, and in revised form, January 16, 2001
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ABSTRACT |
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U1A is a component of the uracil-rich small
nuclear ribonucleoprotein. The molecular mechanism of nuclear import of
U1A was investigated in vivo and in vitro. When
recombinant deletion mutants of U1A are injected into the BHK21 cell
cytoplasm, the nuclear localization signal (NLS) of U1A is found in the
N-terminal half of the central domain (residues 100-144 in
mouse U1A). In an in vitro assay, it was found that the
U1A-NLS accumulated in only a portion of the nuclei in the absence of
cytosolic extract. In contrast, the addition of importin Cellular activities in eukaryotic cells are coordinated via the
continuous and bi-directional transport of macromolecules between the
nucleus and the cytoplasm, which occurs through the nuclear pore
complexes (NPCs).1 The
translocation of proteins through the NPCs is mediated by an active and
selective mechanism that is controlled by saturable receptors and
signals that are termed nuclear localization signals (NLSs) and nuclear
export signals (reviewed in Refs. 1 and 2).
The best characterized active nuclear protein import is mediated by a
basic type NLS, referred to as "classical NLS," and which contains
one or more clusters of basic amino acids. The import of substrates
containing classical NLS such as SV40 large T antigen is initiated by
the formation of an NLS-dependent complex with importin
It has been shown that there are at least five different forms of
importin A small GTPase Ran and its interacting protein NTF2 (nuclear transport
factor 2) are involved in the subsequent translocation step of the
complex through the NPC. It is also known that Ran is essential for
various active transport pathways including the import of snRNPs, as
well as the export of nuclear export signal-containing proteins and
several mRNAs (reviewed in Refs. 1 and 15). In the case of the
nuclear import of proteins, nuclear Ran GTP functions to terminate the
import reaction. For example, after the translocation of the trimeric
complex, which is composed of importin Nuclear pre-mRNA splicing is dependent upon the activity of a
number of trans-acting splicing factors. Small nuclear
ribonucleoprotein particles (snRNPs) play a central role in the
recognition and alignment of splice sites. Proteins contained in the
snRNP particles can generally be divided into the following two
classes: those that are common to all snRNP particles and others that
are associated with a specific snRNP particle (reviewed in Ref. 19).
With the exception of U6, which does not leave the nucleus, the
biogenesis of these U snRNPs requires the bi-directional
transport of the snRNA across the nuclear envelope. Newly synthesized
snRNAs exit to the cytoplasm immediately after transcription in the
nucleus where they undergo RNA processing and assemble with the snRNP common core proteins prior to returning to the nucleus.
Previous studies of the behavior of specific snRNP proteins, using
pulse-chase labeling techniques and cultured somatic cell fractionations, showed that U1 snRNP proteins, U1A and U1C, move to the
nucleus independently of de novo snRNA synthesis,
i.e. they are transported separately from the rest of the
snRNP (20). The study in Xenopus laevis oocytes confirmed
that U1A is targeted to the nucleus independently of its association
with snRNA (21). U1A contains two RNA binding domains (RNP motifs or
RNA recognition motifs). The N-terminal RNA binding domain, along with
a small number of flanking amino acids, is required for binding to U1 snRNA. The remaining central domain, which contains 110 amino acids
residues, is responsible for the nuclear import of U1A (21). It has
been recently reported that the central 110 amino acids can enter the
nucleus in an ATP-dependent and cytosol-independent manner
in an in vitro transport assay system (22). However, the
precise molecular mechanism of the U1A nuclear import and the in
vivo behavior remain unclear.
In this study, we show that the central 45-amino acid region of U1A,
which exhibits considerable sequence similarity with the corresponding
region of U2B", is sufficient for the active nuclear accumulation of
U1A in vivo, which results in a narrowing of the NLS domain
of U1A. The nuclear import mechanism was also investigated using a
recombinant U1A-NLS containing protein and full-length U1A in an
in vitro transport assay system and in living cells. The
results suggest that at least two distinct import pathways, an importin
Expression and Purification of Recombinant
Proteins--
The full length of the mouse U1A gene was
amplified from the mouse Ehrlich ascites tumor cells cDNA
library by PCR using the synthetic oligonucleotide primers
5'-CTTCTGGATCCATGGCCACCATAGCCACCATGCCAG-3' and
5'-ACCGTGGATCCGAATTCTACTTCTTGGCAAAAGAGATCTTC-3'. The PCR product was
inserted into the BamHI and EcoRI site of a
pGEX-6p-2-hGFP vector that has been described previously (23).
Recombinant GST-GFP-U1A fusion protein was expressed by 1 mM isopropyl- Preparation of Ehrlich Ascites Tumor Cells, Cytosolic and Nuclear
Extracts--
A total cytosolic extract of Ehrlich ascites tumor cells
was prepared as described previously (30). The nuclei of Ehrlich ascites tumor cells were resuspended in cold buffer A (50 mM Tris, pH 7.4, 5 mM MgCl2, 0.25 M sucrose, 1 mM dithiothreitol, and protease inhibitors) containing 200 µg/ml RNase to a final concentration of
100 units/ml (1 unit = 3 × 106 nuclei). The
suspension was allowed to stand for 1 h at room temperature and
centrifuged at 15,000 × g for 20 min at 4 °C. The
supernatant was used as nuclear extract in this study.
Microinjection and in Vivo Import Assay--
BHK21 and tsBN2
cells were grown on coverslips at the temperatures indicated in the
figure legends. The microinjection of proteins was performed as
described previously (31). Purified GFP-fused U1A and deletion mutants
of U1A were used at a concentration of 2 mg/ml in capillary for
microinjection experiments. Wild type Ran-GTP and G19VRan-GTP were used
at a concentration of 5 mg/ml. After incubation for the times indicated
in each figure legend, the cells were fixed with 3.7% formaldehyde in
phosphate-buffered saline. GST-tagged U1A-(1-108) was probed with
anti-GST antibodies and fluorescein isothiocyanate-labeled anti-rabbit
IgG. The injected GFP-tagged protein and fluorescent labeled 2nd
antibodies were detected by fluorescent microscopy (Axiophot II; Carl
Zeiss, Inc.). The other details are given in the figure legends.
Solution Binding Assay--
The binding ability of purified
recombinant U1A to recombinant importin Cell-free Transport Assay--
In vitro transport
assays were performed essentially as described previously (32) with
some modifications. In order to decrease the remaining factors in the
cytoplasm after digitonin permeabilization, cells were incubated at
25 °C for 10 min after digitonin permeabilization and then washed
three times with TB. The assay solution (10 µl) in TB containing 1%
bovine serum albumin was incubated with permeabilized cells for 15 min
with or without 1 mM ATP, 5 mM creatine
phosphate, and 20 units/ml creatine phosphokinase at 30 °C, or on
ice, as described in the respective figure legends. The assay solution contained import factors, substrate proteins, and a Ran mixture (4 µM Ran-GDP, 0.4 µM RanBP1, 0.4 µM RanGAP1, and 0.4 µM NTF2) or a cytosolic
extract as described in each figure legend. After the import reaction,
the cells were fixed with 3.7% formaldehyde. T-NLS was prepared as a
control transport substrate by chemical cross-linking of
allophycocyanin (Calbiochem) with SV40 T antigen NLS-containing
peptides as described previously (30).
The Central 45-Amino Acid Domain of U1A Is Sufficient for the
Nuclear Localization in Living Cells--
In order to investigate the
nuclear import mechanism of U1A in living somatic cells, recombinant
GFP-tagged mouse U1A (Fig. 2A, construct a) was injected
into the cytoplasm of cultured BHK21 cells. As shown in Fig.
1, U1A efficiently accumulated in the nucleus after only 5 min of incubation at 37 °C, whereas no nuclear import of U1A was observed when the cells were incubated on ice. In
order to clarify the region that is responsible for nuclear accumulation in living somatic cells, we next constructed four deletion
mutants (Fig. 2A, constructs
b-e). U1A protein can be divided into three domains, the
N-terminal and C-terminal RNA binding domains and the central 110-amino
acid domain, that was reported to be necessary and sufficient for
nuclear transport in Xenopus oocytes (21). Twenty min after
the injection, the subcellular localization of these mutant proteins
was examined. As shown in Fig. 2B, both the N-terminal and
C-terminal RNA binding domains were located in the cytoplasm, whereas
the two mutants that contain the central domains accumulated in the
nucleus. Since these two mutants contain both GFP and GST tags, they
are too large to diffuse passively through the nuclear pores. Moreover, the accumulation of these deletion mutants was not observed when the
cells were incubated on ice (Fig. 2B, d, and data not shown for construct e). Therefore, we conclude that the N-terminal
half of the central domain (residues 100-144 in mouse U1A) is
sufficient for the active nuclear import of U1A in living somatic
cells.
Nuclear Accumulation of U1A-NLS in an in Vitro Transport Assay
System--
In order to investigate the molecular mechanism of U1A
import, the central 110-amino acid region, fused to GST and GFP
(U1A-NLS), was used as a transport substrate (Fig. 2A, construct
d) in the following experiments. When U1A-NLS was incubated with
digitonin-permeabilized cells (32) in the presence of an ATP
regeneration system, a portion (~10-20%) of the cells showed
nuclear accumulation of U1A-NLS in the absence of exogenous cytosolic
extracts (Fig. 3A, a). In
contrast, when the U1A-NLS was incubated with crude cytosolic extracts
in digitonin-permeabilized cells, an obvious and uniform nuclear
accumulation in all cells was observed (Fig. 3A, b). When the cells were incubated on ice or in the absence of ATP, no nuclear import was observed (Fig. 3A, c-f). Furthermore, wheat germ
agglutinin treatment completely inhibited the nuclear import of U1A-NLS
(data not shown). These results were highly reproducible, strongly
suggesting that the nuclear import of U1A-NLS can occur via two
distinct pathways, cytosolic extract-dependent and
-independent ones. Moreover, it should be noted that when
GST-U1A-(100-144)-GFP was used as an import substrate, the same
results were obtained in the in vitro transport assay (data
not shown).
Importin U1A-NLS Forms a Complex with Rch1/Importin U1A-NLS Forms a Complex with All of Three Importin U1A Binds to a Domain That Is Distinct from SV40 T-NLS-binding Site
of Importin
We next examined the effect of classical NLS on the trimeric complex
formation of U1A-NLS/importin Cytosol-independent Nuclear Import of U1A-NLS--
In order to
determine the exact ratio of cells that import the U1-NLS in the
absence of cytosol, we counted the nuclei in which the U1A-NLS
accumulated in a cytosol-independent manner. As a result, it was found
that the U1A-NLS accumulated in the nuclei of 19.1 ± 8.5% of the
digitonin-permeabilized cells (total ~740 cells/12 independent
experiments) in the absence of cytosolic extracts. We next attempted to
exclude the possibility that this is due to the impairment of nuclear
membrane integrity. First, SV40 T antigen NLS substrates did not enter
the nuclei, in which the U1A-NLS accumulated in the absence of cytosol
(Fig. 3B). Second, cytosol-independent nuclear accumulation
was completely inhibited when the cells were incubated on ice or in the
absence of ATP (Fig. 3A), which is consistent with the
previous report (22). Third, cells in which the U1A-NLS accumulated in
the nucleus in a cytosol-independent manner were not stained with
anti-lamin monoclonal antibodies without permeabilization with Triton
X-100 (data not shown).
In order to exclude the possibility that the permeabilized cells still
contained a significant amount of transport factors, i.e.
importin Full-length U1A Does Not Accumulate in the Nucleus in a
Cytosol-independent Manner--
The above data, which were obtained
using the U1A-NLS, suggest that U1A is able to accumulate in the
nucleus by two distinct import pathways, importin
Small GTPase Ran Is Involved in the Nuclear Import of U1A in Living
Cells--
In order to determine whether the cytosol-independent
import of U1A is actually suppressed in living cells, the issue of
whether or not the nuclear import of U1A is dependent on the small
GTPase Ran in living cells was examined, since the importin
To confirm further the involvement of Ran in the nuclear import of U1A
in living cells, tsBN2, which is a temperature-sensitive mutant derived
from the BHK21 cell line (34) and has a point mutation in
rcc1 gene, was used (35). We showed previously that the loss
of RCC1 leads to the suppression of nuclear import of classical
NLS-containing proteins in living cells (36). The tsBN2 cells were
incubated at the permissive or non-permissive temperature, and GFP-U1A
was injected into the cytoplasm. The nuclear import of U1A was clearly
inhibited in the cells that had been incubated at the non-permissive
temperature (Fig. 8), whereas U1A migrated into the nucleus of the
cells cultured at the permissive temperature (data not shown). Based on
these data, we conclude that Ran is involved in the nuclear import of
U1A, and the Ran-independent pathway for U1A is negatively regulated in
living mammalian cultured cells.
IBB Domain Inhibits the Nuclear Accumulation of U1A in Living
Cells--
Finally, to determine whether the nuclear import of
full-length U1A is dependent upon importin Is U1A-NLS One of the Conventional NLSs?--
In this study, we
showed that the 100-144-amino acid region in mouse U1A is sufficient
for targeting to the nucleus in living mammalian cells. Furthermore, it
was found that this domain forms a complex with importin
In addition, it was demonstrated that U1A-NLS binds to a unique site of
importin
U1A and U2B" are closely related and components of U1 snRNP and U2
snRNP, respectively. Both proteins consist of two RNA binding domains
at the N- and C-terminal ends that are highly conserved, whereas the
central domains show relatively poor conservation. The NLSs of the two
snRNP proteins, which are located in the poorly conserved central
domain, showed efficient cross-competition and behaved similarly in the
in vitro nuclear transport assay (22). The comparison of
U1A-NLS (45 amino acids as demonstrated in this study) with U2B"-NLS
(56 amino acids as reported previously (22)) revealed characteristic
amino acid sequence similarity, which has a basic charged cluster
divided by acidic amino acids and is surrounded by polar amino acids
(Fig. 2A). Although the issue of whether U2B"-NLS is
transported into the nucleus in an importin A Possible Involvement of Two Distinct Nuclear Import Pathways for
U1A--
It was found that in digitonin-permeabilized cells,
GST/GFP-tagged U1A-NLS accumulated in the nuclei in a portion of the
cells in the absence of a cytosolic extract. This accumulation was not inhibited by the IBB domain of importin Why Should Cells Provide Two Distinct Import Pathways for
U1A?--
Human hnRNP K protein contains two different NLSs, a
conventional basic type NLS (39) and KNS (hnRNP K nuclear
shuttling domain), that mediates nuclear entry in a saturable but
cytosol-independent and an ongoing RNA polymerase II
transcription-dependent manner (40). However, in the wild
type K protein, the classical NLS overrides KNS, and the nuclear
import of the wild type K protein requires importin
Thus, although RCC1 and the K protein show two NLS activities and
appear to be mediated by two distinct pathways in vitro, both of them as well as U1A preferentially utilize one of the two
pathways. Why do cells utilize and regulate two distinct import pathways for one karyophile? Michael et al. (40) suggested
that the K protein has more than one function in the nucleus and that each function may be achieved by being targeted to different subnuclear domains by distinct pathways. In fact, RCC1 has two distinct biological functions. One is to generate a Ran-GTP gradient across the nuclear pores (42), and the other is to create a Ran-GTP gradient near the
chromatin surface, which is required for mitotic spindle formation (43-45). O'Connor et al. (46) found a unique form of U1A
that is not associated with the U1 snRNP (termed snRNP-free U1A or SF-A) and that is complexed with a novel set of non-snRNP proteins. This non-snRNP complex, which contains the U1A, has been suggested to
perform an important function in both splicing and polyadenylation of
pre-mRNA (47). These findings show that the U1A has two distinct functions in the nucleus, suggesting a localization in the distinct subdomains for each to function properly, which supports the suggestion by Michael et al. (40). Alternatively, the fact that U1A, K protein, and RCC1 are important for cell function and extremely abundant in cells may explain the existence of the two import pathways,
that is the backup system may be required for the nuclear import of
certain karyophiles. Consistent with this notion, it has been
demonstrated that various importins overlap in their ability to import
ribosomal proteins. In mammals, ribosomal proteins L23a, S7, and L5 can
be imported alternatively by any of the four receptors, importin /
and
Ran induced the uniform nuclear accumulation of U1A-NLS in all cells.
Furthermore, U1A was found to bind the C-terminal portion of importin
. In addition, the in vitro nuclear migration of
full-length U1A was found to be exclusively dependent on importin
/
and Ran. Moreover, in living cells, the full-length U1A
accumulated in the nucleus in a Ran-dependent manner, and
nuclear accumulation was inhibited by the importin
binding domain
of importin
. These results suggest that the nuclear import of U1A
is mediated by at least two distinct pathways, an importin
/
and
Ran-dependent and an -independent pathway in permeabilized
cells, and that the latter pathway may be suppressed in intact cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
in the cytoplasm, which is referred to as the nuclear
pore-targeting complex. Importin
recognizes the NLS and binds to
importin
via its N-terminal sequences, which are rich in basic
amino acids, and is referred to as the importin
binding (IBB)
domain (3). Importin
accounts for the targeting of the complex to
the NPC. In addition to this classical import pathway, several
different types of pathways have been identified, which involve
importin
or importin
family members that bind directly to their
cognate cargoes without importin
(4-6).
in the human and mouse that display significantly different tissue expression patterns (7, 8). It has been also shown
that these importin
isoforms interact differentially with specific
NLS (9-11). As evidenced by the primary sequences, these molecules can
be grouped into three subfamilies, which are referred to Rch1, NPI-1,
and Qip1, and which show ~50% amino acid identity with one another.
Individuals within the same subfamily show more than an 80% amino acid
identity. The alignment of these importin
isoforms indicates a
rough structural domain organization of importin
(12). There are
three conserved domains as follows: the N-terminal IBB domain, the
hydrophilic C-terminal regions, and a large central domain that
consists of tandemly repeated relatively hydrophobic modules known as
armadillo (Arm) motifs. It has been proposed that the Arm repeat domain
of importin
harbors the binding site for NLSs (10, 12-14).
/
and a karyophile with
the classical NLS, through the nuclear pores, the direct binding of
nuclear Ran GTP to importin
causes the dissociation of the trimeric
complex, thus releasing the cargo (16-18).
/
and Ran-dependent pathway and an -independent pathway, are involved in the mediation of the nuclear import of U1A-NLS
in vitro. However, it was demonstrated that the nuclear import of full-length U1A is exclusively dependent on importin
/
and Ran in living cells, which poses a new question as to how these two
pathways are regulated in the mediation of the import of U1A
appropriately in cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside for
20 h at 20 °C in Escherichia coli strain BL21 (DE3)
and purified with glutathione-Sepharose (Amersham Pharmacia Biotech)
following the manufacturer's recommendations. The GST portion was
cleaved with PreScission protease (Amersham Pharmacia Biotech)
overnight in transport buffer (TB, 20 mM HEPES, pH 7.3, 110 mM CH3COOK, 5 mM
CH3COONa, 2 mM (CH3COO)2Mg, 0.5 mM EGTA, 2 mM dithiothreitol, 1 µg/ml each of
leupeptin, pepstatin, and aprotinin) at 4 °C. The GST portion and
PreScission protease were then removed by centrifugation with
glutathione-Sepharose. Four U1A deletion mutants were prepared by the
PCR with appropriate oligonucleotide primers. For construction of an
expression vector of GST-U1A-(1-108) (Fig. 2A, construct
b), the PCR product was inserted into the
BamHI/EcoRI site of pGEX-6P2 vector (Amersham
Pharmacia Biotech). For construction of expression vectors of
GST-U1A-(208-287)-GFP, GST-U1A-(100-209)-GFP, and
GST-U1A-(100-144)-GFP (Fig. 2A, constructs c-e), the PCR
products were inserted into the BamHI and SmaI
site of a pGEX-2T-GFP vector. The above expression vectors were
verified by DNA sequencing and expressed by 1 mM
isopropyl-
-D-thiogalactopyranoside for 20 h at
20 °C in E. coli strain BL21 (DE3). These recombinant
proteins were purified using glutathione-Sepharose following the
manufacturer's recommendations and were then dialyzed against TB.
Recombinant mouse Rch1/PTAC58 (24), mouse importin
(25), Ran,
RanBP1 (23), and NPI-1 deletion mutants (26) proteins were prepared from E. coli as previously described. GFP-Rch1, GFP-Qip1,
and GST-RanGAP1 were expressed and purified as described previously (27). Preparation of recombinant human NTF2 and GST-NLS-GFP was
performed as described previously (28). G19V Ran was prepared as
described previously (29).
and importin
was
examined using solution binding assay. All assays were performed in TB
(total volume of 300 µl). 100 pmol of GST-U1A-NLS was immobilized on
10 µl of a packed volume of glutathione-Sepharose beads by batchwise
incubation for 1 h at 4 °C. 100 pmol of recombinant importin
and/or importin
were added to the immobilized GST-U1A-NLS, and
the resulting suspension was incubated for 1 h at 4 °C in 300 µl of TB containing 1% of bovine serum albumin and 0.05% of Nonidet
P-40 in the presence or absence of 200 pmol of Ran-GTP or Ran-GDP. The
beads were then washed with TB extensively and eluted with 10 mM glutathione. The eluted materials were analyzed by
SDS-PAGE. For the pull-down experiments using a nuclear extract, 100 pmol of GST-tagged NPI-1 deletion mutants were immobilized on
glutathione-Sepharose, and 300 µl of the nuclear extract of mouse
Ehrlich ascetics tumor cells was added in the presence or absence of
100 pmol of importin
. After a 1-h incubation at 4 °C, the bound
fractions were analyzed by immunoblotting with anti-U1A monoclonal antibody.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nuclear accumulation of U1A in living
cells. Purified GFP-tagged mouse U1A (2 mg/ml) was injected into
the cytoplasm of BHK21 cells. After incubation for the indicated times
in each panel at 37 (a and b) or 4 °C
(c), the cells were fixed with 3.7% formaldehyde in
phosphate-buffered saline.
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Fig. 2.
The central 45-amino acid domain is
responsible for the nuclear localization of U1A. A,
schematic presentation of mouse U1A and its deletion mutants for the
analysis of nuclear accumulation activity, expressed in E. coli as recombinant GFP and/or GST fusion proteins. The
numbers below the proteins refer to amino acid positions.
The lower part shows an amino acid comparison between
U1A-NLS and U2B"-NLS. Bold letters show basic amino acids.
Asterisks indicate acidic amino acids. B, each
fragment (construct b-e in A) was injected into
the cytoplasm of cultured BHK21 cells. After a 20-min incubation at the
indicated temperature, cells were fixed, and the subcellular
localization of the injected proteins was detected by GFP or polyclonal
anti-GST antibodies and fluorescein isothiocyanate-conjugated
anti-rabbit IgG.
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Fig. 3.
Nuclear import of U1A-NLS in an in
vitro transport assay. A,
GST-U1A-(100-209)-GFP (Fig. 2A, construct d) was used as a
substrate for the permeabilized Mandin Darbey bovine kidney
cells. The import reaction was performed for 15 min in the absence
(a, c, and e) or presence (b, d, and
f) of an Ehrlich ascites tumor cell cytosolic extract at
30 °C (a, b, e, and f) or on ice (c
and d). An energy-regenerating system was added in the
upper panel. The reactions were terminated by fixation, and
import was analyzed by fluorescence microscopy. B,
GST-U1A-(100-209)-GFP or T-NLS (SV40 T antigen NLS-conjugated
allophycocyanin, see "Experimental Procedures") was added to
digitonin-permeabilized cells and incubated with an energy-regenerating
system in the absence (upper panels) or presence
(lower panels) of the cytosolic extract.
GST-U1A(100-209)-GFP but not T-NLS accumulated in a portion of the
nuclei even in the absence of the cytosol.
/
and Ran Mediate the Cytosol-dependent
Import Pathway of U1A-NLS--
We next attempted to analyze the
cytosolic extract-dependent pathway and to understand
better the transport factors for U1A-NLS. The central domain of U1A
contains some basic amino acid residues (see Fig. 2), although 100-125
in mouse U1A, which corresponds to 94-119 in human, has been shown not
to be sufficient for the nuclear accumulation of U1A in
Xenopus oocytes and in vitro (21, 22). To
determine whether importin
/
is able to mediate the nuclear
import of U1A-NLS, we examined the effect of the IBB domain, which is
the importin
binding domain of importin
and which is well known
to inhibit competitively the importin
/
-dependent import pathway. The IBB domain was added to the in vitro
import assay in the presence of a cytosolic extract. As shown in Fig. 4a, the IBB domain strongly
inhibited the nuclear accumulation of U1A-NLS except for 10-20% of
the cells. In addition, the issue of whether the nuclear import was
reconstituted by recombinant importin
/
and small GTPase Ran was
tested. After a 15-min incubation at 30 °C with Rch1 (one of the
importin
family molecules), importin
, and a Ran mixture (see
"Experimental Procedures"), the U1A-NLS accumulated in the nucleus
efficiently (Fig. 4c). When Rch1 or importin
alone was
added in the presence of a Ran mixture, or when Rch1/importin
were
added in the absence of the Ran mixture, the U1A-NLS only
accumulated in 10-20% of the nuclei, and no stimulation of nuclear
uptake of U1A-NLS was observed (data not shown). These results indicate
that Rch1/importin
, in conjunction with Ran, is able to mediate the
import of U1A-NLS into the nucleus. It should be noted that the nuclear
import of the U1A-(100-144) region was reconstituted with importin
/
and Ran to a similar extent to that of U1A-(100-209) in the
in vitro transport assay system (data not shown).
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Fig. 4.
IBB domain suppresses the
cytosol-dependent import and importin
/
is able to reconstitute
the import of U1A into the nucleus. GST-U1A-NLS(100-209)-GFP and
IBB domain of Rch1 were added to the permeabilized cells in the
presence (a) or absence (b) of cytosolic extract.
Import reactions were performed with recombinant importin
and
(0.4 µM each) in the absence (c) or presence
(d) of IBB domain. All reactions contain ATP regeneration
system.
in the Absence of
Ran-GTP--
It is well known that the classical basic type NLS
directly binds to importin
, forming a ternary complex with importin
and
in the absence of Ran-GTP and that this complex targets the
nuclear envelope to translocate through the nuclear pores. In order to
confirm that the U1A-NLS binds to importins, a solution binding assay
with recombinant U1A-NLS and importins was performed. As shown in Fig.
5A, although the interaction
of the U1A-NLS with Rch1 or with importin
was not detected under
our assay conditions, it obviously formed a ternary complex with Rch1
and importin
. On the other hand, GST-C-terminal RNA binding
domain-GFP (Fig. 2A, construct c) did not bind to the
importin
and Rch1-importin
complex (data not shown).
Consistent with the previous reports, the GST-SV40 T antigen-NLS-GFP,
which was used as a positive control, bound directly to Rch1 but not to
importin
and formed a ternary complex with Rch1 and importin
.
In the case of U1A-NLS, although high concentrations (up to 1 µM) of recombinant proteins were used, no obvious direct
binding between U1A-NLS and Rch1 was detected (data not shown). It has
been demonstrated that the direct binding of Ran-GTP to importin
causes the dissociation of the ternary complex including importin
/
and the NLS substrates (16, 18). Therefore, to determine
whether Ran-GTP dissociates the Rch1/importin
/U1A-NLS, a solution
binding assay was performed in the presence of Ran-GTP or Ran-GDP. The
results clearly demonstrated that Ran-GTP but not Ran-GDP induced the
dissociation of the Rch1-importin
-U1A-NLS complex (Fig.
5B). These results support the view that the nuclear import
of U1A is mediated by Rch1/importin
and Ran.
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Fig. 5.
NLS domain of U1A forms a trimeric complex
with Rch1 and importin but does not directly
bind to Rch1. A, GST-U1A-NLS-(100-144)-GFP or GST-SV40
T antigen NLS-GFP was immobilized to glutathione-Sepharose and
incubated with GFP-tagged Rch1 and/or importin
for 1 h. Since
Rch1 and GST-U1A-NLS-GFP showed the same mobility on SDS-PAGE,
GFP-tagged Rch1 was used in these experiments. After extensive washing,
the bound proteins were analyzed by SDS-PAGE followed by Coomassie
Brilliant Blue staining. Although the specific reasons are
unclear at this time, the recombinant GFP-Rch1 always showed two bands
on SDS-PAGE even if they lacked the GFP tag, as described previously
(24). B, Ran-GTP causes the dissociation of the U1A-importin
/
complex. The binding assay of U1A-NLS-(100-144)/Rch1/importin
was performed in the presence of Ran-GTP or Ran-GDP. C,
U1A-NLS forms complexes with all three of the importin
subfamilies in the presence of importin
. GST-U1A-NLS-(100-209)-GFP
was incubated with GFP-Rch1, GFP-Qip1, or MBP-NPI-1 in the presence or
absence of importin
. After incubation for 1 h at 4 °C and
extensive washing, the bound proteins were analyzed by SDS-PAGE
followed by Coomassie Brilliant Blue staining. Lanes
7-10 show input proteins. Asterisks, importin
.
Subfamilies--
It has been reported that three types of importin
subfamilies show distinct substrate specificity (8, 9, 26). As a
result, we examined the issue of whether other importin
subfamily molecules, NPI-1 and Qip1, are able to form a complex with U1A-NLS in
the presence or absence of importin
. The results clearly showed
that, whereas the U1A-NLS did not bind to NPI-1 and Qip1 directly, in
the presence of importin
, U1A-NLS formed a complex with
NPI-1/importin
or Qip1/importin
as efficiently as with Rch1/importin
(Fig. 5C). GST-U1A-RBD2-GFP, used as a
negative control, did not form complex with importin
/
(data not
shown). Identical results were obtained by using the U1A-NLS-(100-144) construct (data not shown) suggesting that the nuclear import of U1A
can be mediated by all of three distinct importin
subfamilies.
--
As described above, importin
is divided into
three functional domains as follows: the N-terminal IBB domain, the
central Arm repeat domain, and the C-terminal region. The central
portion lacking the first 77 amino acids and the last 63 amino acids
retains the ability to bind to the monopartite SV 40 T antigen NLS and the bipartite nucleoplasmin NLS, indicating that the conventional basic
type NLS binding domains map to the Arm repeat region of importin
(26). U1A-NLS-(100-144) contains not only positively but negatively
charged amino acids and differs from the conventional NLSs. To
understand more clearly the binding domain of importin
for U1A, we
assessed the binding ability of several NPI-1 deletion mutants (Fig.
6A) to endogenous U1A in a
mouse Ehrlich ascites tumor cell nuclear extract, since U1A is mainly
contained in the nuclear extract but not the cytosol. It has previously
been reported that residues 1-538 (full-length NPI-1), 78-538
(IBB-deleted NPI-1), and 1-475 (C-terminal region-deleted NPI-1) bind
to the conventional NLSs, whereas residues 404-538 (C-terminal
fragment of NPI-1) do not (26). On the other hand, as shown in Fig.
6B, the full-length NPI-1 and C-terminal region-deleted
NPI-1 interacted with endogenous U1A very weakly. In contrast, the
IBB-deleted NPI-1 and the C-terminal fragment of NPI-1 significantly
bound to U1A (Fig. 6B). Since it was found that the
full-length NPI-1 did not efficiently bind to the recombinant U1A-NLS
in the absence of importin
(Fig. 5C), it was assumed
that the full-length NPI-1 and the C-terminal region-deleted NPI-1
failed to pull down the U1A because of a deficiency of importin
in
the nuclear extract. In fact, the Western blotting experiment with
anti-importin
antibodies showed that the nuclear extract contained
only a small amount of importin
and that no apparent importin
was detected in the samples that were pulled down with the full-length
and the C-terminal region-deleted NPI-1 (data not shown). Therefore, we
added recombinant importin
to the nuclear extract and performed a
pull-down assay. As shown in Fig. 6C, the C-terminal
region-deleted NPI-1 binds to endogenous U1A. Similar results were
obtained using the full-length NPI-1 (data not shown). Moreover, the
C-terminal fragment (residues 404-538) of NPI-1 significantly
bound to U1A (Fig. 6B), whereas it did not bind to the
classical basic type NLS as shown previously (26). From these results,
we conclude that the region 404-475 of NPI-1 is required for the
interaction with U1A. This region is obviously distinct from the
classical basic type NLS-binding site (see "Discussion").
View larger version (29K):
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Fig. 6.
U1A-NLS binds to the C-terminal Armadillo
repeat motif in importin . A,
schematic presentation of NPI-1 deletion mutants. B,
recombinant GST-tagged deletion mutants of NPI-1 were immobilized to
glutathione-Sepharose and incubated with nuclear extract of mouse
Ehrlich ascites tumor cells. The bound fractions were analyzed by
immunoblotting with an anti-U1A monoclonal antibody. C, the
C-terminal deleted fragment of NPI-1 is able to interact with U1A in
the presence of importin
. The same pull-down assay as described in
B was performed with GST-tagged C-terminal deleted fragment
of NPI-1 in the absence (lanes 1-3) or presence
(lanes 4-6) of importin
. The bound U1A was detected
with anti-U1A monoclonal antibody.
/
. When an excess amount of SV40
T-NLS peptide was added to the solution binding assay with U1A-NLS,
importin
and importin
, complex formation of U1A-NLS/importin
/
was not detected (data not shown), indicating that importin
is unable to bind simultaneously to U1A-NLS and T-NLS.
and
, the IBB domain was added to the in
vitro import assay system, which was performed in the absence of
the exogenous cytosol. The nuclear entry of U1A-NLS was still evident in ~20% of the cells, indicating that the cytosol-independent import
of U1A-NLS was not dependent on importin
/
(Fig. 4b). Moreover, when the nuclear import was reconstituted with recombinant importin
/
and Ran, the IBB domain failed to inhibit the nuclear localization of U1A-NLS in some portion of the cells (Fig.
4d). Such cytosol-independent and ATP-dependent
import is compatible with previously reported results (22). However,
they obviously showed that the U1A-NLS accumulated in the nucleus of
nearly all cells in a cytosol-independent manner. The reason for this
discrepancy remains to be elucidated.
/
-dependent and -independent. To determine whether
these pathways actually function in cells, we examined the import of
full-length U1A in the in vitro transport assay system. In
the absence of a cytoplasmic extract, the nuclear accumulation of
GFP-U1A (Fig. 2A, construct a) was not observed at all,
whereas the addition of cytosolic extract induced a remarkable nuclear localization of U1A (Fig. 7A).
We next attempted to reconstitute the nuclear import of U1A with
recombinant importin
/
and Ran. Rch1, NPI-1, and Qip1 were used
in this assay because, as shown in Fig. 5C, U1A-NLS
efficiently formed a complex with all of the importin
subfamily
members in the presence of importin
. The results indicate that all
the subfamilies of importin
mediated the nuclear accumulation of
the full-length U1A in the presence of importin
and Ran (Fig. 7,
d-f). The elimination of the Ran mixture suppressed the
import of U1A (Fig. 7c and data not shown). These results
indicate that the nuclear accumulation of full-length U1A is completely
dependent on importin
/
and Ran and suggest that the
cytosol-independent import pathway may be negatively regulated or
suppressed in cells.
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Fig. 7.
Nuclear accumulation of full-length U1A is
completely dependent on the cytosolic extract. GFP-U1A was added
to the permeabilized cells, followed by incubation for 15 min at
30 °C in the absence (a) or presence (b) of
cytosolic extract. Nuclear import of full-length U1A was reconstituted
by Rch1 (d), NPI-1 (e), or Qip1 (c and
f) with importin in the presence (d-f) or
absence (c) of a Ran mixture.
/
-independent import pathway appeared not to require Ran. GFP-U1A
was co-injected with a dominant-negative mutant G19VRan-GTP,
which is defective with respect to GTP hydrolysis and is known to
inhibit the Ran-dependent nuclear import, into the
cytoplasm of BHK21 cells. As shown in Fig.
8, the G19VRan-GTP completely blocked the
nuclear import of U1A, whereas wild type Ran did not.
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Fig. 8.
Nuclear import of U1A is dependent on Ran and
importin /
.
A, mutant Ran suppresses the nuclear import of U1A. Wild
type Ran or G19VRan was co-injected with GFP-U1A into the cytoplasm of
BHK21 cells (a and b). GFP-U1A was injected into
the cytoplasm in tsBN2 cells, which were cultured at non-permissive
temperature for 6 h (c). After a 20-min incubation at
37 °C, the cells were fixed with 3.7% formaldehyde. B,
GFP-U1A was co-injected with 0.5 mM of MBP-IBB (MBP-tagged
IBB domain of Rch1) or MBP into the cell cytoplasm. After 15 min
incubation at 37 °C, the cells were fixed with 3.7%
formaldehyde.
/
in living cells, we
co-injected the IBB domain with full-length U1A into the cell
cytoplasm. As shown in Fig. 8B, although MBP alone did not
inhibit the nuclear import of U1A, the same concentration of MBP-IBB
strongly suppressed the nuclear accumulation of U1A as well as T-NLS
substrate. From these findings, we conclude that importin
/
mediates the nuclear import of full-length U1A in living cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
in the
absence of Ran-GTP. Although a number of basic type NLSs have been
reported to be recognized by importin
with a high degree of
specificity, these classical NLSs lack a stringent consensus, except
for general features of short stretches of amino acids that contain a
high proportion of positively charged residues (37). Although U1A-NLS
is recognized by all of the importin
subfamilies, it was found that
the U1A-NLS has quite unique features that are different from the known
conventional NLSs such as the SV40 T-NLS. The U1A-NLS was transported
into the nucleus in an importin
/
-dependent and
-independent manner. Moreover, the basic amino acids cluster
(94IAKMKGTFVERDRKREKRKPKSQETP119 in
human U1A-NLS, which corresponds to residues 100-125 in mouse U1A) was
not sufficient for nuclear accumulation in vivo and in vitro (21, 22). Therefore, the NLS activity of U1A required a much
longer domain.
that is distinct from the binding site for the
conventional monopartite and bipartite NLSs, such as those of SV40
large T antigen and Xenopus nucleoplasmin. The crystal structure of yeast importin
revealed that nucleoplasmin NLS and
SV40-T antigen NLS interact with importin
at two sites in the Arm
motifs, namely the second through fourth and seventh and eighth (14,
38). On the other hand, in this study, it was found that the binding
site of importin
for U1A is located in the C-terminal portion of
the Arm repeats (residues 404-475) probably corresponding to Arm 9 and
its flanking regions (Fig. 6). Moreover, we were able to detect the
binding of U1A-NLS to importin
only in conjunction of importin
.
It is known that although the interaction of the classical NLS to
importin
is considerably enhanced in the presence of importin
,
the classical NLS is able to bind importin
alone. From these
findings, we propose that the U1A-NLS is a novel basic type NLS, which
interacts with importin
at a site that is distinct from the
conventional NLS.
/
-dependent manner was not confirmed, both of the two
snRNP proteins might be imported into the nucleus in a similar fashion.
but was completely
inhibited by low temperature and ATP depletion, indicating that the
cytosolic extract-independent nuclear accumulation does not involve
passive diffusion. A similar observation was reported by using the
110-amino acid central portion of U1A fused to nucleoplasmin core
region (22). These investigators reported that the central domain of U1A entered the nuclei in a Ran, cytosolic extract-independent but an
ATP-dependent manner. On the other hand, in our in
vitro transport assay, it was found that only when the cytosolic
extract was added did all cells show a nuclear accumulation of U1A-NLS, which was inhibited by the IBB domain of importin
. Moreover, importin
/
formed a complex with U1A-NLS and reconstituted
nuclear entry in digitonin-permeabilized cells. These findings suggest that U1A-NLS is able to accumulate in the nucleus via two distinct active import pathways, importin
/
and Ran-dependent
and -independent. Nonetheless, when the full-length U1A was used as an
import substrate, surprisingly, cytosolic extract-independent nuclear
migration was not observed and the import of the full-length U1A was
exclusively mediated by importin
/
and Ran in the in
vitro transport assay. Furthermore, in living cells, the
dominant-negative form of Ran mutant and the IBB domain of importin
suppressed the nuclear accumulation of full-length U1A. Collectively,
these data point to the fact that importin
/
and Ran mediate the
nuclear import of U1A in vivo and that cytosolic
extract-independent import pathway might be negatively regulated under
ordinary cell conditions.
/
and Ran
in vitro and is independent of polymerase II transcription
in vivo (8). Therefore, the removal of the classical NLS in
the K protein induces KNS activity. Recently, Nemergut and
Macara (41) reported that the import of RCC1 can proceed by at least
two distinct mechanisms. The first is mediated by the N-terminal domain
of RCC1 and is dependent on importin
and
. This pathway
preferentially uses the importin
3 isoform, Qip1. The nuclear
import of wild type RCC1 is, however, also mediated by the second
pathway that is saturable and temperature-sensitive but does not
require soluble transport factors in vitro and the Ran-GTP
gradient in vivo.
,
transportin, RanBP5, and RanBP7 (33). In yeast, both Kap123p and
another related importin
, Pse1p, are able to mediate the import of
ribosomal NLS-bearing substrates into the nucleus in vivo
(40). In summary, our study demonstrates that 1) U1A has the ability to
be transported into the nucleus by at least two distinct pathways and
2) that the import of U1A is regulated to be mediated by an importin
/
-dependent pathway in living mammalian cultured
cells under ordinary conditions. Further studies will be required to
completely understand how these two pathways are regulated in
vivo.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant-in-aid for Scientific Research on Priority Areas (B) (11237202), Grant-in-aid for Scientific Research (B) (12480215), Grants-in-aid for COE Research (07CE2006 and 12CE2007) from the Japanese Ministry of Education, Science, Sports and Culture, the Mitsubishi Foundation, and the Human Frontier Science Program.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.
§ Research fellow of the Japanese Society for the Promotion of Science.
To whom correspondence should be addressed: Dept. of Cell
Biology and Neuroscience, Graduate School of Medicine, Osaka
University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel.:
81-6-6879-3210; Fax: 81-6-6879-3219; E-mail:
yyoneda@anat3.med.osaka-u.ac.jp.
Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.M008299200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NPCs, nuclear pore
complexes;
NTF2, nuclear transport factor 2;
NLS, nuclear localization
signal;
PAGE, polyacrylamide gel electrophoresis;
GST, glutathione
S-transferase;
IBB, importin binding;
PCR, polymerase
chain reaction;
snRNA, small nuclear RNA;
snRNPs, small nuclear
ribonucleoprotein particles;
GFP, green fluorescent protein;
MBP, maltose binding protein;
KNS, hnRNP K nuclear shuttling
domain.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Mattaj, I. W., and Englmeier, L. (1998) Annu. Rev. Biochem. 67, 265-306[CrossRef][Medline] [Order article via Infotrieve] |
2. | Yoneda, Y., Hieda, M., Nagoshi, E., and Miyamoto, Y. (1999) Cell Struct. Funct. 24, 425-433[CrossRef][Medline] [Order article via Infotrieve] |
3. | Görlich, D., Henklein, P., Laskey, R. A., and Hartmann, E. (1996) EMBO J. 15, 1810-1817[Abstract] |
4. | Chan, C. K., Hubner, S., Hu, W., and Jans, D. A. (1998) Gene Ther. 5, 1204-1212[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Nagoshi, E.,
Imamoto, N.,
Sato, R.,
and Yoneda, Y.
(1999)
Mol. Biol. Cell
10,
2221-2223 |
6. |
Yoneda, Y.
(2000)
Genes Cells
5,
777-787 |
7. | Tsuji, L., Takumi, T., Imamoto, N., and Yoneda, Y. (1997) FEBS Lett. 416, 30-34[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Kohler, M.,
Speck, C.,
Christiansen, M.,
Bischoff, F. R.,
Prehn, S.,
Haller, H.,
Görlich, D.,
and Hartmann, E.
(1999)
Mol. Cell. Biol.
19,
7782-7791 |
9. |
Miyamoto, Y.,
Imamoto, N.,
Sekimoto, T.,
Tachibana, T.,
Seki, T.,
Tada, S.,
Enomoto, T.,
and Yoneda, Y.
(1997)
J. Biol. Chem.
272,
26375-26381 |
10. |
Sekimoto, T.,
Imamoto, N.,
Nakajima, K.,
Hirano, T.,
and Yoneda, Y.
(1997)
EMBO J.
16,
7067-7707 |
11. |
Nadler, S. G.,
Tritschler, D.,
Haffar, O. K.,
Blake, J.,
Bruce, A. G.,
and Cleaveland, J. S.
(1997)
J. Biol. Chem.
272,
4310-4315 |
12. |
Herold, A.,
Truant, R.,
Wiegand, H.,
and Cullen, B. R.
(1998)
J. Cell Biol.
143,
309-318 |
13. | Cortes, P., Ye, Z. S., and Baltimore, D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7633-7637[Abstract] |
14. | Conti, E., Uy, M., Leighton, L., Blobel, G., and Kuriyan, J. (1998) Cell 94, 193-204[Medline] [Order article via Infotrieve] |
15. | Goldfarb, D. S. (1997) Curr. Biol. 7, 13-16 |
16. | Rexach, M., and Blobel, G. (1995) Cell 83, 683-692[Medline] [Order article via Infotrieve] |
17. | Görlich, D., Pante, N., Kutay, U., Aebi, U., and Bischoff, F. R. (1996) EMBO J. 15, 5584-5594[Abstract] |
18. |
Moroianu, J.,
Blobel, G.,
and Radu, A.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
7059-7062 |
19. | Lührmann, R., Kastner, B., and Bach, M. (1990) Biochim. Biophys. Acta 1087, 265-292[Medline] [Order article via Infotrieve] |
20. | Feeney, R. J., and Zieve, G. W. (1990) J. Cell Biol. 110, 871-881[Abstract] |
21. | Kambach, C., and Mattaj, I. W. (1992) J. Cell Biol. 118, 11-21[Abstract] |
22. |
Hetzer, M.,
and Mattaj, I. W.
(2000)
J. Cell Biol.
148,
293-303 |
23. |
Hieda, M.,
Tachibana, T.,
Yokoya, F.,
Kose, S.,
Imamoto, N.,
and Yoneda, Y.
(1999)
J. Cell Biol.
144,
645-655 |
24. | Imamoto, N., Shimamoto, T., Takao, T., Tachibana, T., Kose, S., Matsubae, M., Sekimoto, T., Shimonishi, Y., and Yoneda, Y. (1995) EMBO J. 14, 3617-3626[Abstract] |
25. | Imamoto, N., Shimamoto, T., Kose, S., Takao, T., Tachibana, T., Matsubae, M., Sekimoto, T., Shimonishi, Y., and Yoneda, Y. (1995) FEBS Lett. 368, 415-419[CrossRef][Medline] [Order article via Infotrieve] |
26. |
Sekimoto, T.,
Imamoto, N.,
Nakajima, K.,
Hirano, T.,
and Yoneda, Y.
(1997)
EMBO J.
16,
7067-7077 |
27. | Tachibana, T., Hieda, M., Miyamoto, Y., Kose, S., Imamoto, N., and Yoneda, Y. (2000) Cell Struct. Funct. 25, 115-123[CrossRef][Medline] [Order article via Infotrieve] |
28. | Tachibana, T., Hieda, M., Sekimoto, T., and Yoneda, Y. (1996) FEBS Lett. 397, 177-182[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Sekimoto, T.,
Nakajima, K.,
Tachibana, T.,
Hirano, T.,
and Yoneda, Y.
(1996)
J. Biol. Chem.
271,
31017-31020 |
30. |
Imamoto, N.,
Tachibana, T.,
Matsubae, M.,
and Yoneda, Y.
(1995)
J. Biol. Chem.
270,
8559-8565 |
31. | Yoneda, Y., Imamoto-Sonobe, N., Yamaizumi, M., and Uchida, T. (1987) Exp. Cell Res. 173, 586-595[Medline] [Order article via Infotrieve] |
32. | Adam, S. A., Sterne-Marr, R., and Gerace, L. (1990) J. Cell Biol. 111, 807-816[Abstract] |
33. |
Jakel, S.,
and Görlich, D.
(1998)
EMBO J.
17,
4491-4502 |
34. | Nishimoto, T., Eilen, E., and Basilico, C. (1978) Cell 15, 475-483[Medline] [Order article via Infotrieve] |
35. | Uchida, S., Sekiguchi, T., Nishitani, H., Miyauchi, K., Ohtsubo, M., and Nishimoto, T. (1990) Mol. Cell. Biol. 10, 577-584[Medline] [Order article via Infotrieve] |
36. |
Tachibana, T.,
Imamoto, N.,
Seino, H.,
Nishimoto, T.,
and Yoneda, Y.
(1994)
J. Biol. Chem.
269,
24542-24545 |
37. | Dingwall, C., and Laskey, R. A. (1991) Trends Biochem. Sci. 16, 478-481[CrossRef][Medline] [Order article via Infotrieve] |
38. | Conti, E., and Kuriyan, J. (2000) Structure 8, 329-338[CrossRef][Medline] [Order article via Infotrieve] |
39. | Michael, W. M., Choi, M., and Dreyfuss, G. (1995) Cell 83, 415-422[Medline] [Order article via Infotrieve] |
40. |
Michael, W. M.,
Eder, P. S.,
and Dreyfuss, G.
(1997)
EMBO J.
16,
3587-3598 |
41. |
Nemergut, M. E.,
and Macara, I. G.
(2000)
J. Cell Biol.
149,
835-850 |
42. | Bischoff, F. R., and Ponstingl, H. (1991) Nature 354, 80-82[CrossRef][Medline] [Order article via Infotrieve] |
43. | Carazo-Salas, R. E., Guarguaglini, G., Gruss, O. J., Segref, A., Karsenti, E., and Mattaj, I. W. (1999) Nature 400, 178-181[CrossRef][Medline] [Order article via Infotrieve] |
44. |
Ohba, T.,
Nakamura, M.,
Nishitani, H.,
and Nishimoto, T.
(1999)
Science
284,
1356-1358 |
45. |
Wilde, A.,
and Zheng, Y.
(1999)
Science
284,
1359-1362 |
46. | O'Connor, J. P., Alwine, J. C., and Lutz, C. S. (1997) RNA (New York) 3, 1444-1455[Abstract] |
47. |
Lutz, C. S.,
Cooke, C.,
O'Connor, J. P.,
Kobayashi, R.,
and Alwine, J. C.
(1998)
RNA (New York)
4,
1493-1499 |