From the National Institute of Agrobiological
Resources, Tsukuba, Ibaraki 305-8602, Japan, the ¶ Central
Research Institute of Electric Power Industry, Chiba 270-1194, Japan,
the
Department of Biology, Faculty of Science, Niigata
University, Niigata 950-2181, Japan, the ** National Institute of
Genetics, Structural Biology Center, Mishima, Shizuoka 411-8540, Japan,
the
Department of Cell Biology and
Neuroscience, Graduate School of Medicine, Osaka University, Suita,
Osaka 565-0871, Japan, the §§ Department of
Molecular, Cellular and Developmental Biology, Yale University, New
Haven, Connecticut 06520-8104, and the ¶¶ Department of
Biology, Faculty of Science, Ochanomizu University, Bunkyo-ku,
Tokyo 112-8610, Japan
Received for publication, July 19, 2000, and in revised form, December 19, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Nuclear import of proteins that contain classical
nuclear localization signals (NLS) is initiated by importin The most characteristic feature of an eukaryotic cell is the
presence of a nuclear envelope, which separates the cell into two major
compartments, the nucleus and the cytoplasm. Communication between
these two compartments takes place through the nuclear pore complex
(NPC)1 (for review, see Refs.
1-3). The NPC allows molecules smaller than 40-60 kDa to diffuse
across, while larger proteins and RNA-protein complexes must be
actively transported through the NPC in a signal-mediated and
energy-dependent manner. Nuclear proteins involved in
nuclear activities, such as DNA replication, transcriptional RNA
synthesis, and RNA splicing, must enter into the nucleus. Conversely,
RNA, such as mRNA synthesized in the nucleus, must be transported
into the cytoplasm where it is translated to protein. In plants,
nucleocytoplasmic transport has been implicated in functional
regulation of a number of plant photomorphogenesis related protein
factors (4). For example, constitutive photomorphogenic 1 (COP1), a
repressor of photomorphogenesis, has been shown to shuttle between the
nucleus and cytoplasm in response to a change of light environment.
COP1 exists predominantly in the cytoplasm in the light while it
accumulates in the nucleus in the dark, suggesting that nuclear protein
transport is an underlying mechanism for the regulation of COP1
activity (5).
Multiple pathways of nucleocytoplasmic transport have been
identified, each likely to be involved in carrying a distinct group of
proteins (for review, see Refs. 3 and 6). Among them, the best
characterized is the import of proteins containing a classical
nuclear localization signal (NLS) that consists of either a short
stretch of 3-5 basic amino acids or two basic domains separated by a
spacer, referred to as monopartite and bipartite NLS, respectively (7).
Yeast mating factor (Mat Both importin As in many vertebrates, multiple isoforms of importin In this paper we report the cloning and functional analysis of a novel
rice importin cDNA Cloning
A rice expressed sequence tag (EST) clone C3059 (accession
number D23592) with high homology to rice importin DNA Constructions
GST-rice Importin GST-COP1 NLS-GFPs--
Chimeric DNA of GST-COP1 NLS-GFPs were
generated essentially as described previously (36). 5'-End
oligonucleotide primers corresponding to each putative COP1 NLS and
their missense mutants (see Fig. 5A) used for PCR are
as follows: 1) COP1 bWW,
5'-ACGGAATTCAACCAGAGCACCGTGAGCATCGCGCGCAAAAAACGCATCCATGCGCAGTTCAACGATCTGCAGGAATGCTATCTGCAGAAACGCCGCCAGCTGGCGGATCAGCCGAACAGCATGAGTAAAGGAGAAGAACTTTTCACTGGAGTT-3'; 2) COP1 bXW,
5'-ACGGAATTCAACCAGAGCACCGTGAGCATCGCGAACACCACCAACATCCATGCGCAGTTCAACGATCTGCAGGAATGCTATCTGCAGAAACGCCGCCAGCTGGCGGATCAGCCGAACAGCATGAGTAAAGGAGAAGAACTTTTCACTGGAGTT-3'; 3) COP1 bWX,
5'-ACGGAATTCAACCAGAGCACCGTGAGCATCGCGCGCAAAAAACGCATCCATGCGCAGTTCAACGATCTGCAGGAATGCTATCTGCAGACCAACAACCAGCTGGCGGATCAGCCGAACAGCATGAGTAAAGGAGAAGAACTTTTCACTGGAGTT-3'; 4) COP1 bXX,
5'-ACGGAATTCAACCAGAGCACCGTGAGCATCGCGAACACCACCAACATCCATGCGCAGTTCAACGATCTGCAGGAATGCTATCTGCAGACCAACAACCAGCTGGCGGATCAGCCGAACAGCATGAGTAAAGGAGAAGAACTTTTCACTGGAGTT-3'; 5) COP1 mW,
ACGGAATTCAACCTGCTGACCCTGCTGGCGGAACGCAAACGCAAAATGGAACAGGAAGAAGCGGAACGCATGAGTAAAGGAGAAGAACTTTTCACTGGAGTT-3'. The NLS-encoding sequences are indicated by italic and the endonuclease restriction sites are indicated by boldface. Underlined are basic amino
acid cluster regions. The 3'-end primer used was
5'-ACGCTCGAGTTATTTGTAGAGCTCATCCATGCCATGTGT-3'.
Transient Expression Vector and sGFP-COP1 NLS-sGFP--
An
oligonucleotide encoding an extra 5 amino acids (Leu-Ile-Gly-Gly-Gly)
and incorporating three restriction enzyme sites (ApaI,
XmaI, and SpeI) was inserted at the 3'-end of the
original sGFP (S65T) gene (38). The resulting plasmid was
designated as psGFPcs. On the other hand, a PCR product of sGFP with
XbaI and XmaI restriction sites at the 5'-end,
and a SacI site at the 3'-end was produced and subcloned
into pBI221 (CLONTECH) using the XbaI
and SacI sites. Then a fragment encoding sGFP and the nos terminator was excised with XmaI and
EcoRI digestion, and inserted between the XmaI
and EcoRI sites of the psGFPcs plasmid. The resulting
plasmid was designated pdsGFP. A PCR fragment corresponding to each
COP1 NLS sequence with ApaI and XbaI site at 5'-
and 3'-end, respectively, was inserted between two adjacent sGFPs of
pdsGFP to generate sGFP-COP1 NLS-sGFP fusions and used for transient expression assays. The sGFP-COP1 NLS-sGFP DNAs were also digested with
HindIII and SacI, and the resulting fragments
containing the CaM 35S-promoter and the nos terminator
flanking the sGFP-COP1 NLS-sGFP were cloned into the pIG121-Hm (39)
transformation vector. The sequences of forward and reverse
primers used for the PCR are as follows: 1) COP1 bWW,
5'-GGGGGGCCCAATCAGTCAACTGTCTCAATT-3'/5'-CCCCCCGGGACTATTTGGTTGGTCTGCCAA-3'; 2) COP1 bXW,
5'-GGGGGGCCCAATCAGTCAACTGTCTCAATTGCTAACACCACCAACATTCATGCTCAGTTC-3'/5'-CCCCCCGGGACTATTTGGTTGGTCTGCCAA-3'; 3) COP1 bWX,
5'-GGGGGGCCCAATCAGTCAACTGTCTCAATT-3'/5'-CCCCCCGGGACTATTTGGTTGGTCTGCCAACTGGTTGTTGGTTTGGAGGTAACATTC-3'; 4) COP1 bXW,
5'-GGGGGGCCCAATCAGTCAACTGTCTCAATTGCTAACACCACCAACATTCATGCTCAGTTC-3'/5'-CCCCCCGGGACTATTTGGTTGGTCTGCCAACTGGTTGTTGGTTTGGAGGTAACATTC-3'; 5) COP1 mW,
5'-GGGGGGCCCAATCTTCTGACACTTCTTGCG-3'/5'-CCCCCCGGGCCTCTCAGCTTCTTCCTGTTC-3'.
Analysis of Transcript Levels of Rice Importin Transient Expression Assays
Onion epidermis was peeled off and placed inside up on plates
containing MS medium (4.2 g/liter MS plant salt mixture, 30 g/liter
sucrose, 4% Gelrite, pH 5.8) and 2.5 mg/liter amphotericin B
antifungal agent (Sigma-Aldrich Co.). Plasmid DNAs (6.75 µg) harboring the fusion genes were precipitated onto 1.6-µm gold particles (0.75 µg) and the particles were resuspended in 60 µl of
100% ethanol. A 10-µl aliquot of the suspension was loaded onto a
particle delivery disc and the segments of onion epidermis were
bombarded with the particles (PDS-1000/He; Bio-Rad). Bombardment conditions were as recommended by the manufacturer.
Plant Growth Conditions and Arabidopsis Transformation
Arabidopsis thaliana was grown at 22 °C under
constant fluorescent illumination of 85 µmol m Fluorescence Microscopy
sGFP fusion proteins in the transient expression assays were
observed with a microscope (Olympus AX70, Tokyo, Japan) with Nomarski
optics or epifluorescence optics. For subcellular localization of sGFP
fusion proteins of the transgenic Arabidopsis, whole roots were viewed under a stereo fluorescence microscope (Leica MZFL III).
Photomicrographs were taken using 35 mm film and the figures were
assembled using Adobe Photoshop software (Adobe Systems Inc., San Jose, CA).
Protein Purification
Expression and purification of recombinant proteins and in
vitro binding and nuclear import assays were carried out as
described previously (36). Recombinant GST-NLS-GFPs, rice importin
Cloning of Rice Importin NLS Binding and PTAC Formation by Rice Importin
As shown in Fig. 2B, rice importin
We next examined PTAC formation by rice importin Activity of Rice Importin
As shown in Fig. 3A, rice
importin
Upon addition of mouse Ran-GDP and energy-regenerating mixture, T-GFP
was efficiently translocated into the nucleus (Fig. 3B, panel
a). The translocation of the substrate was rice importin Differential Gene Expression of the Three Rice Importin
Transcripts of the three rice importin
We previously reported that transcription of rice importin Identification of a Bipartite Type NLS in COP1 Protein--
To
further investigate any functional differences between the rice
importin
Analysis of the amino acid sequence of the COP1 protein using PSORT, a
computer program for the prediction of protein sorting signals and
localization sites in amino acid sequences, on the internet
predicted two putative NLS sequences, a monopartite-(mW-COP1 NLS) and a
bipartite-type (bWW-COP1 NLS), in the COP1 protein (Fig.
5A). To examine their
functional NLS activities, we inserted oligonucleotides corresponding
to each of these putative NLSs and their missense mutants (Fig.
5A) between two adjacent GFPs in a CaMV 35S-promoter-driven
plasmid and bombarded into onion epidermis. For convenience herein, we
have designated these fusion proteins bWW-, bXW-, bWX-, bXX-, and
mW-dsGFP, respectively. As shown in Fig. 5B, bWW-dsGFP was
localized exclusively in the nucleus (Fig. 5B, panels a and
f). In contrast, neither mW-dsGFP (Fig. 5B, panels
e and j) nor the missense mutants of bWW-dsGFP (Fig. 5B, panels b-d, and g-i) were nuclear localized.
Some GFP signal around the nucleus in Fig. 5B, panels b-e,
is likely to be due to the fact that the cytoplasm is compacted beneath
the plasma membrane and around the nucleus because of the large vacuole
that occupies most of the cell volume in onion epidermal cells. These results suggest that only the bipartite-type NLS, but not the monopartite-type one, in the COP1 protein is functional as a NLS and
mutations in either the basic stretch of amino acids of bWW abolish its
NLS activity. These findings were further supported by the observation
that the subcellular distribution of bWW- and bXW-dsGFP in transgenic
Arabidopsis root cells bWW-dsGFP was found to be localized
predominantly in the nucleus (Fig. 5C, panel a) while its
missense mutant bXW-dsGFP was found to be distributed throughout the
cytoplasm (Fig. 5C, panel b).
This subcellular localization (Fig. 5) and following in
vitro binding (Fig. 6) and nuclear
import (Fig. 7) analyses demonstrate that
COP1 protein bears a functional bipartite-type NLS consisting of two
stretches of basic amino acids separated by 14 residues. Although the
PSORT program also predicted a monopartite-type NLS in COP1 protein, it
did not show any NLS activity in our assays. These results are in
consistent with the observations of Stacey et al. (48), in
which they identify a core domain between residues 293 and 392 that is
capable of mediating nuclear localization of N-terminal fused GUS or
GFP.
In Vitro Binding of COP1 NLSs to Rice Importin Preferential Nuclear Import of bWW-GFP by Rice Importin
In the present work, we have isolated a novel cDNA for an
importin In addition to the primary structural data, our in vitro
assays using recombinant rice importin Both rice importin Although both rice importin , a
protein that recognizes and binds to the NLS in the cytoplasm. In this paper, we have cloned a cDNA for a novel importin
homologue from rice which is in addition to our previously isolated rice importin
1a and
2, and we have named it rice importin
1b. In vitro binding and nuclear import assays using recombinant
importin
1b protein demonstrate that rice importin
1b functions
as a component of the NLS-receptor in plant cells. Analysis of the transcript levels for all three rice importin
genes revealed that
the genes were not only differentially expressed but that they also
responded to dark-adaptation in green leaves. Furthermore, we also show
that the COP1 protein bears a bipartite-type NLS and its nuclear import
is mediated preferentially by the rice importin
1b. These data
suggest that each of the different rice importin
proteins carry
distinct groups of nuclear proteins, such that multiple isoforms of
importin
contribute to the regulation of plant nuclear protein transport.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-2) contains a NLS consisting of basic and
hydrophobic amino acid residues and has also been shown to be
functional in plants (8). The NLS-containing proteins are initially
recognized and bound in the cytoplasm by NLS-receptor, a heterodimer
consisting of importin
and importin
subunits. Importin
binds the NLS specifically, forming a stable pore targeting complex
(PTAC) (9-11), whereas importin
mediates the docking of the PTAC
to the cytoplasmic face of the NPC (12-14). Translocation of the
docked PTAC into the nucleus is mediated by the small GTPase, Ran (15,
16).
and
have been identified in a wide range of
species, including vertebrates, yeast, and plants. However, while only
a single isoform of importin
involved in classical NLS-directed
nuclear transport has been isolated in most species, multiple isoforms
of importin
have been identified in various species (2, 3). Recent
studies suggest that such multiplicity of importin
isoforms might
contribute to the tissue-specific or temporal regulation of nuclear
protein import. For example, at least six different human isoforms of
importin
have been identified, and they show significantly
different tissue-dependent expression patterns (11,
17-24). They have also been shown to differentially interact with
specific NLS sequences (22, 25-28).
have been
identified from Arabidopsis (aIMP
(29), AtKAP
(30), ATHKAP2 (31), and AtIMP
1-4 (32)) and rice (rice importin
1 (33)
and rice importin
2 (34)). Intriguingly, in vitro binding
assays using recombinant plant importin
s have revealed differential
recognition of different types of NLS. The aIMP
can bind all three
of the typical classes of plant NLSs, namely, the NLS of SV40 large
T-antigen (T-NLS, a monopartite type), that of maize transcription
factor Opaque 2 (O2-NLS, a bipartite type), and that of
maize transcription factor R (R-NLS, a yeast Mat
2 type) (35). In
contrast, rice importin
1 bound to the T- and O2-NLS, but not to the
R-NLS (36). Rice importin
1 also mediated nuclear import of fusion
proteins containing either T-NLS or O2-NLS, but not R-NLS, in
digitonin-permeabilized HeLa cells (36). All these data suggest that
multiple isoforms of importin
may serve as a control point for the
regulation of nuclear protein import. It is also noteworthy that two
closely related importin
s have been identified in rice (37). This
is the only report thus far of multiple importin
isoforms
identified in a single species. Whether such multiplicity is a common
characteristic among plants is not yet clear, as no importin
s have
yet been isolated from other plants. At least in rice, however, this
may provide an additional point for regulation of nuclear protein import.
homologue which is in addition to our previously
isolated rice importin
1 (33) and
2 (34). Because it shares very
high homology (82.8% identity) with the rice importin
1, we have
named this novel homologue rice importin
1b and have renamed the
rice importin
1 as rice importin
1a. We show that rice importin
1b selectively binds to different types of plant NLSs and mediates
the nuclear import of NLS substrates in digitonin-permeabilized HeLa
cells. Comparison of the transcript levels of the three isoforms of
rice importin
reveals some differential expression, not only in
different tissues, but also in response to light. Furthermore, we also
demonstrate that the COP1 protein bears a bipartite-type NLS and its
nuclear translocation is mediated preferentially by the rice importin
1b.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1 (33) was
obtained from the Rice Genome Project team in the National Institute of
Agrobiological Resources, Ministry of Agriculture, Forestry & Fisheries, Japan. We used this EST clone as a probe to screen a
cDNA library constructed in
gt11 using poly(A) RNA from the
leaves of rice seedlings grown in the light for 14 days, and obtained a
positive clone of about 2.4 kilobases in length. However, since
this clone did not cover the full-length coding sequence, we carried
out the 5' rapid amplification of cDNA ends using a Marathon
cDNA Amplification Kit (CLONTECH, Palo Alto, CA). The 5'-end of cDNA was amplified by PCR using a gene-specific primer, 5'-GGGGATAATCCTCACGTTGGAGGAAGGC-3', according to the
manufacturer's instructions. The products were cloned into pT-Adv
vector using the AdvanTAgeTM PCR Cloning Kit
(CLONTECH) and sequenced. The full-length importin
1b cDNA was used to generated a GST-rice importin
1b
construct, as described below.
1b--
A fragment corresponding to the
amino-terminal portion (about 300 base pairs) of rice importin
1b
with an artificial EcoRI site at the 5'-end was generated by
PCR using the 5'-rapid amplification of cDNA ends fragment as
template DNA. The forward and reverse primers used were
5'-GGGAATTCATGTCGCTGCGGCCGAGCGAGCGG-3' and
5'-GGCAACCCCTCCAACTTCTGCTGGAGCG-3', respectively. The PCR fragment
contains a NotI site near the 3'-end. After double digestion
with EcoRI and NotI, the fragment was inserted between the EcoRI and NotI sites of pGEX-6p-1
(Amersham Pharmacia Biotech). The rest of the rice importin
1b
sequence was digested from the cDNA clone obtained from the rice
seedling cDNA library and then cloned into the NotI site
at the 3'-end of the amino-terminal portion. Both nucleotide sequence
and orientation were checked by DNA sequencing.
s--
Plant
materials and total RNA samples were prepared as previously described
(33). The RNA samples were treated with DNase (RT-grade, Nippon Gene,
Tokyo) to remove genomic DNA contaminants. Transcript levels of
importin
s in the RNA samples were examined with the ABI
PRISMSTM 7700 Sequence Detection System essentially
according to the manufacturer's instruction (PE Applied Biosystems,
Foster City, CA) and were normalized with transcript levels of 18 S
rRNA. Reverse transcription and amplification of transcripts were
carried out using TaqManTM EZ RT-PCR Kit (PE Applied
Biosystems) in the presence of one of the specific primer-probe sets
shown below. The sequences of forward and reverse primers and TaqMan
probes used for the analyses were as follows: 1) rice importin
1a, 5'-CGATAAGAAGCTCGAAAGCCTT-3'/5'-AAAGCAACTTGCGGAACTGTGT-3' and
5'-CTGCTATGATTGGTGGAGTTTATTCGGACG-3'; 2) rice importin
1b, 5'-AATCAGGAGTGTTCCCAAGGC-3'/5'-ATCCCCAGTGACGATGTTACC-3' and
5'-TGGAACTTCTCATGCATCCTTCGGC-3'; 3) rice importin
2,
5'-AGCTCCAATTTCTGGCAGTGAT-3'/5'-CAACAGCATGCGCAGTCTTTGC-3' and
5'-TGGTCGATGAGGAGAAAGCATGTCTTG-3'; 4) 18 S rRNA,
5'-GAGAAACGGCTACCACATCCAA-3'/5'-CTAAAGCGCCCGGTATTGTTAT-3' and
5'-AAGGCAGCAGGCGCGCAAATTA-3'.
2
s
1, using Supermix (Sakata Seeds Co., Ltd., Yokohama,
Japan) and vermiculite (mixed in 1:1). Seedlings were germinated
aseptically on half-concentrated MS medium supplemented with 2%
sucrose, 0.3 mg/ml thiamine (HCl), 0.5 mg/ml nicotinic acid, 0.05 mg/ml
pyridoxine (HCl) and as required with 20 µg/ml hygromycin.
Arabidopsis ecotype Columbia was transformed by
Agrobacterium tumefaciens (EHA 101)-mediated T-DNA transfer
using the floral dip procedure (40). Hygromycin-resistant seedlings
were selected and allowed to self seed for amplification.
1a,
2 (36), rice importin
1 (41), and Ran (42, 43) were prepared as described previously.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1b--
By searching the rice EST data
base, we identified an EST clone (accession number D23592) showing high
homology with rice importin
1 previously reported in Ref. 33. Using
this clone as a probe, we screened a rice seedling cDNA library and
obtained a positive clone. Since DNA sequence analysis indicated that
this clone lacked a 5'-segment of the open reading frame, we employed the 5'-rapid amplification of cDNA ends method to isolate a
corresponding full-length cDNA clone. The full-length cDNA of
the clone is predicted to encode a protein of 534 amino acids with a
calculated molecular mass of 58.5 kDa (Fig.
1). The deduced amino acid sequence of the clone showed 82.8% identity with rice importin
1. Therefore, we
designated this novel cDNA as rice importin
1b and renamed the
rice importin
1 as rice importin
1a. Rice importin
1b also shares significant amino acid homology with other previously identified importin
s, such as rice importin
2 (27.5%), aIMP
(59%
(29)), AtKAP
(68% (30)), and yeast SRP1 (54% (44)) (Fig. 1). Like the other importin
homologues, rice importin
1b contains the main characteristics of importin
: the importin
-binding domain (IBB domain) at the amino terminus (45, 46), the eight tandem armadillo
(arm) repeats of 42 amino acids (47), a COOH-terminal acidic region,
and two variable regions flanking the arm repeats region (Fig. 1).
View larger version (82K):
[in a new window]
Fig. 1.
Multiple sequence alignment of rice
importin s. Protein sequences were
aligned using GENETYX genetic information processing software (Software
Development Co., LTD, Tokyo, Japan). Identical amino acid residues
across either all three or two of the clones are indicated by
black and gray boxes, respectively. IBB domain,
arm repeats (arm), variable region (V), and
acidic region are indicated in the upper line. DDBJ
accession numbers of rice importin
1b,
1a, and
2 are
AB034311, AB004660, and AB006788 respectively.
1b--
To
address the ability of rice importin
1b to bind NLSs, we performed
an in vitro binding assay using native gel electrophoresis. On a native gel, complex formation between two proteins gives a new
band with a mobility different from that of either of the proteins
alone. NLS-GFP fusion proteins, T-, O2-, R-, and Tm-GFP, were used as
NLS substrates (36) throughout the functional analysis of rice importin
1b (Fig. 2A). Tm-GFP, a
point mutant of T-GFP in which the sixth residue, a lysine, of the
T-NLS was replaced by a threonine, was used as a negative control.
View larger version (48K):
[in a new window]
Fig. 2.
In vitro protein binding
analyses. A, amino acid sequences of SV40 large
T-antigen NLS (T-NLS), mutant T-NLS (Tm-NLS),
maize transcription factor opaque 2 NLS (O2-NLS), and maize
transcription factor R NLS (R-NLS) are shown in a
single-letter code. Basic amino acid clusters are indicated in
bold face and amino acid replacement in Tm-NLS is
underlined. These NLSs were inserted between GST and GFP to
generate recombinant GST-NLS-GFP fusion proteins designated T-, Tm-,
O2-, and R-GFP, respectively. B and C, in
vitro protein binding assays were analyzed by native gel
electrophoresis. 20 pmol of each protein was mixed in 15 µl of
transport buffer (TB) (20 mM Hepes, pH 7.3, 110 mM potassium acetate, 2 mM magnesium acetate, 5 mM sodium acetate, 0.5 mM EGTA, 2 mM dithiothreitol, 1 µg/ml each of aprotinin, leupeptin,
and pepstatin A) supplemented with 250 mM sucrose and
incubated for 1 h at room temperature. 7.5% polyacrylamide gels
were run in the presence of 1 mM dithiothreitol and 1 mM EGTA in both the gels and the running buffer and stained
with Coomassie Blue.
1b and each of the
NLS-GFPs migrated as single bands (lanes 1, 3, 5, 7, and
9). A mixture of rice importin
1b with T-GFP gave a new
major band of retarded mobility, with a little of the unbound protein
running as in the control (Fig. 2B, lane 2). As was the case
with rice importin
1a (36), the complex of O2-GFP and rice importin
1b showed almost indistinguishable mobility relative to O2-GFP alone
on the gel (Fig. 2B, lane 6). However, complex formation
between the two proteins is apparent as all of the rice importin
1b
shifted upward, giving a much darker band (Fig. 2B, lane 6).
In contrast, a mixture of rice importin
1b with either R-GFP or
Tm-GFP gave no new visible bands, with migration of each protein as in
the control (Fig. 2B, lanes 4 and 8). These
results suggest that the rice importin
1b selectively binds to T-
and O2-NLS, but not to R-NLS. The binding was NLS-specific as the rice
importin
1b did not bind to the Tm-GFP that has been shown to be
nonfunctional (Fig. 2B, lane 4).
1b with rice
importin
1 and NLS-GFPs (Fig. 2C). Each protein migrated
as a single band on the gel (Fig. 2C, lanes 1, 2, 6, 9, and
12). A mixture of rice importin
1b with rice importin
1 gave a new major band of retarded mobility, with some unbound
proteins migrating at the position of the controls (Fig. 2C, lane
5), indicating a direct binding of the two proteins. Addition of
either T-GFP (Fig. 2C, lane 4) or O2-GFP (Fig. 2C,
lane 11), but not Tm-GFP (Fig. 2C, lane 8), to the
mixture resulted in formation of a large complex with lower mobility on
the gel relative to the complex formed either between rice importin
1b and
1 (Fig. 2C, lane 5) or between rice importin
1b and either T- (Fig. 2C, lane 3) or O2-GFP (Fig.
2C, lane 10). SDS-polyacrylamide gel electrophoresis of
these bands confirmed that the complex contained rice importin
1b
and
1 and either T- or O2-GFP (data not shown).
1b in the in Vitro Import Assay using
Digitonin-permeabilized HeLa Cells--
To assess the functional
activity of rice importin
1b in the process of nuclear import of
proteins, we performed an in vitro nuclear import assay
using digitonin-permeabilized HeLa cells. T-GFP and Tm-GFP were used as
transport substrates, as positive and negative controls, respectively.
The T-NLS containing proteins have been most commonly used as the
transport substrate in nuclear import assays.
1b, in conjunction with rice importin
1, efficiently
accumulated T-GFP at the nuclear rim (Fig. 3A, panel
a). In contrast, rice importin
1 alone was not sufficient to
direct the substrate to the nuclear rim (Fig. 3A, panel c).
Such accumulation did not occur when Tm-GFP was used as substrate (Fig.
3A, panel e).
View larger version (45K):
[in a new window]
Fig. 3.
The effect of rice importin
1b on the nuclear binding and import of GST-NLS-GFP
assayed in vitro using digitonin-permeabilized HeLa
cells. Part A, for nuclear binding, a 10-µl assay
sample was incubated on ice for 20 min. GST-NLS-GFP was 0.2 µg; rice
importin
1 was 6 pmol; rice importin
1b was 6 pmol. Part
B: for nuclear import, a 10-µl assay sample was incubated at
25 °C for 20 min in the presence of mouse Ran-GDP (42 pmol), 1 mM ATP, 20 units/ml creatine phosphokinase, 5 mM creatine phosphate, and 1 mM GTP.
GST-NLS-GFP was 0.2 µg; rice importin
1 was 3 pmol; and rice
importin
1b was 12 pmol. In both parts A and
B: a and b, T-GFP + importin
1 + importin
1b; c and d, T-GFP + importin
1;
and e and f, Tm-GFP + importin
1 + importin
1b. After incubation, cells were fixed with 3.7% formaldehyde in
TF. Panels a, c, and e, fluorescence images; panels b,
d, and f; Nomarski images.
1b-dependent (Fig. 3B, panel c) and
NLS-specific (Fig. 3B, panel e). Omission of
Ran-GDP or depletion of ATP by hexokinase from the transport solution
abolished translocation of the substrate into the nucleus (data not shown).
s--
To investigate the expression of the three rice importin
s in different tissues and to examine the effect of light on their expression in green leaves, we performed quantitative analysis of RNA
levels using the ABI PRISMSTM 7700 Sequence Detector (PE
Applied Biosystems).
s were detected in all the
tissues tested, with minimum levels in green leaves and relatively
higher levels in nongreen tissues including roots, etiolated leaves,
and calli (Fig. 4). However, the tissue
expression patterns for the different importin
s varied
significantly. Rice importin
1b and
2 showed maximal transcript
levels in roots (Fig. 4, B and C). In contrast,
the highest levels of rice importin
1a were detected in calli (Fig.
4A).
View larger version (18K):
[in a new window]
Fig. 4.
Transcript analyses of rice importin
s. Total RNA samples were prepared from
tissues of either dark-(etiolated leaves) or light-grown (green leaves
and roots) rice plants (14-days-old). For dark adaptation, the
light-grown plants were further grown in complete darkness for the
indicated hours (6, 12, and 24 days, respectively). In each
panel, the highest transcript levels were regarded as 100% of relative
expression.
1a is
down-regulated by light (33). Herein, we examined whether this is also
the case with the other two rice importin
s. As shown in Fig. 4, the
three rice importin
s displayed varied transcript patterns during
dark-adaptation in green leaves. Dark treatment of rice seedlings for
24 h significantly increased the transcript levels of rice
importin
1a by about 10-fold (Fig. 4A). Rice importin
2 also showed a modest, 5-fold increase in transcript levels after
24 h in the dark (Fig. 4C). In comparison, rice
importin
1b showed only a minor change in transcript levels during
dark-adaptation (Fig. 4B). Thus it appears that the rice
importin
s express differentially not only in different tissues but
also in response to light.
s in nuclear import processes, we identified the NLS in the
COP1 protein and examined its interaction with rice importin
s.
View larger version (41K):
[in a new window]
Fig. 5.
Identification of COP1 NLS.
A, PSORT program predicted two putative NLS sequences, a
monopartite- (mW) and a bipartite-type (bWW), in the COP1 protein. The
position and nature of mutations within bWW were indicated (bXW, bWX,
and bXX). B, DNA constructs encoding COP1 NLS-dsGFPs under
CaMV 35S-promoter were bombarded into onion epidermis and their
subcellular localization were visualized for sGFP. Only the bWW-dsGFP
(a and f), but not its mutants (b-d
and g-i) and mW-dsGFP (e and j), was
nuclear localized. Panels a-e, fluorescence images;
panels f-j, Nomarski images. C,
stereofluorescence microscopic images of nuclear localization of
bWW-dsGFP (a), but not bXW-dsGFP (b), in root
cells of transgenic Arabidopsis plants.
View larger version (39K):
[in a new window]
Fig. 6.
In vitro interactions between rice
importin s and COP1 NLSs. Native gel
electrophoresis was carried out as described in the legend to Fig.
2.
View larger version (93K):
[in a new window]
Fig. 7.
Preferential nuclear translocation of bWW-GFP
by rice importin 1b. Nuclear binding and
import assays were carried out as described in the legend to Fig.
3.
s--
In
vitro interaction between the COP1 NLS and each of the three rice
importin
s was examined using the native gel electrophoresis method.
Oligonucleotides corresponding to each of these putative NLSs and their
missense mutants (Fig. 5A) were inserted between GST and GFP
to generate recombinant GST-COP1 NLS-GFP fusion proteins in
Escherichia coli as described previously (36). For
convenience herein, we have designated these fusion proteins bWW-,
bXW-, bWX-, bXX-, and mW-GFP, respectively. As shown in Fig. 6, bWW-GFP
was specifically bound by rice importin
1a (Fig. 6A, lane
2) and
1b (Fig. 6B, lane 2), but not by
2 (Fig.
6C, lane 2). All the missense mutants of bWW- and mW-GFP
were not recognized by any rice importin
s. Addition of rice
importin
1 to the mixtures gave rise to the formation of PTAC
consisting of bWW-GFP, rice importin
1, and either rice importin
1a (Fig. 6D, lane 2) or
1b (Fig. 6D, lane
5). No such PTAC formation was observed with rice importin
2
(data not shown).
1b--
Because bWW-GFP was recognized in vitro by both
rice importin
1a (Fig. 6A) and
1b (Fig. 6B)
and further formed PTAC in the presence of rice importin
1 (Fig.
6D), we next carried out an in vitro nuclear
protein import assay. As shown in Fig. 7, both rice importin
1a
(Fig. 7B) and
1b (Fig. 7A), in conjunction with rice importin
1, docked bWW-GFP (Fig. 7A, panels a
and b, B, panels a and b), but not its
missense mutants and mW-GFP (Fig. 7A, panels c-j), to the
nuclear envelope. Surprisingly, however, further translocation of the
bWW-GFP into the nucleus occurred preferentially in the presence of
rice importin
1b (Fig. 7A, panels k and l).
Although nuclear docking occurred with rice importin
1a, almost no
(if any) further nuclear translocation proceeded (Fig. 7B, panels
c and d). These results together with those shown in
Fig. 6 suggest that a functional difference exists between the multiple
importin
isoforms in the plant nuclear protein import system.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
homologue in rice and named it rice importin
1b.
Analysis of its primary structure reveals that rice importin
1b
contains the main characteristics of other importin
s, namely an IBB
domain, arm repeats, a COOH-terminal acidic region, and variable
regions flanking the arm repeats (Fig. 1). The IBB domain is a highly conserved stretch of basic amino acids and is well known to be responsible for the interaction with importin
(20, 49). The
putative IBB domain of rice importin
1b displays maximum homology
with those of other importin
s. Such high homology of IBB domains
among different importin
s may explain why different isoforms of
importin
can interact with the same importin
. For instance, all
three rice importin
s can bind to rice importin
1 (Fig.
2B; Refs. 36 and 41). On the other hand, the region of arm
repeats is another well conserved domain and is responsible for NLS
binding. Mapping of the NLS-binding sites within importin
molecules
has suggested that different NLS types interact with different subsets
of arm repeats (17, 50, 51). However, recent studies indicate that NLS
binding by importin
also requires the variable regions (51). In the
case of the human importin
2 (also termed Rch1, hSRP1
, or
pendulin), the first variable region is required for T-NLS-binding,
while the second one is required for binding to LEF-1 NLS (51). The
variable regions of rice importin
1b show only minimum identities to
those of the other importin
s (Fig. 1), and such variability may
confer an importin
with its NLS specificity (26). The COOH-terminal acidic region has been demonstrated to be the site for interaction with
CAS (51), a nuclear transport factor that exports importin
from the
nucleus (52).
1b protein demonstrated that rice importin
1b can bind functional plant NLSs and rice importin
1, forming a stable PTAC (Fig. 2), and mediate nuclear import of
NLS-proteins in digitonin-permeabilized HeLa cells (Fig. 3). These data
strongly suggest that rice importin
1b functions as a component of
the NLS receptor in plant cells.
1a and
1b proteins showed selective binding to
T-NLS and O2-NLS, but not to R-NLS (36, Fig.
2A). This is in contrast to the Arabidopsis
aIMP
, which binds all the typical plant NLSs (35). Moreover, a
different activity was also seen between rice importin
1a and
1b
proteins in mediation of nuclear import of COP1 NLS-GFP in the present
work (Fig. 7). In fact, preferential affinities between distinct
importin
s and different NLS-containing proteins have also been
reported in human importin
s. Human DNA helicase Q1 (25) and RCC1
(22) are most efficiently imported into the nucleus by human importin
3, and transcription factor Stat1 is imported into the nucleus by
NP1 (human importin
1), but not by Rch1 (human importin
2), in
response to interferon-r (27). RanBP3 interacts preferentially with
both human importin
3 and
4, but its nuclear import was most
efficient in the presence of importin
3 (28). All these data suggest
a diverse specificity among importin
s with respect to NLS
recognition, such that each importin
preferentially imports a
distinct group of proteins into the nucleus. In addition, the
differential expression patterns of rice importin
s (Fig. 4) support
the hypothesis that multiple isoforms of importin
s might contribute
to spatial and temporal regulation of nuclear protein import.
1a and
1b, together with rice
importin
1, complexed with COP1 NLS-GFP and docked to the nuclear envelope, translocation of the fusion protein into the nucleus occurred
preferentially in the presence of rice importin
1b (Fig. 7). This
suggests that the nuclear import of COP1 protein in vivo is
mediated, at least most efficiently, by the importin
1b and that
in vitro binding between an importin
and an NLS does not necessarily mean that the nuclear import of the NLS protein is performed by that importin
. Indeed, similar phenomena have been reported in human importin
s. Human RanBP3 NLS shows a binding activity to both human importin
3 and
4, however, its nuclear import was mediated most efficiently by human importin
3 (28). Nachury et al. (24) observed a consistently lower nuclear
import activity of hSRP1r (human importin
3) for bovine serum
albumin-SV40 large T-antigen NLS when compared with that of hSRP1
(human importin
2) or NP1. Taken together, all these data seem to
suggest that the nature of the interaction between a distinct importin
and a specific cognate NLS protein is also a critical factor for
nuclear protein import process. This idea is also supported by the
evidence that
-catenin, that contains arm repeats, alone can dock
onto the nuclear envelope and further translocate into the nucleus in a
NLS-independent manner without assistance of importin and Ran-GTPase
(53, 54). Thus it is conceivable that arm repeats in importin
may
interact with NPC and such interaction could be affected by binding of
a specific NLS-protein to this domain.
![]() |
ACKNOWLEDGEMENT |
---|
We are grateful to Dr. Patrick J. Hussey (School of Biological Science, Royal Holloway, University of Lodon, Egham, Surrey, United Kingdom) for critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by the Special Coordination Fund for Promoting Science and Technology, Enhancement of Center-of-Excellence, to the National Institute of Agrobiological Resources from the Science and Technology Agency of Japan, the Ministry of Agriculture, Forestry and Fisheries of Japan (BDP-00-I-1), and Scientific Research C Grant-in-Aid 0868076 from the Japanese Ministry of Education, Science, Sports and Culture (to N. I. and Y. Y.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB03431l.
§ To whom correspondence should be addressed: Dept. of Plant Physiology, National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan. Tel.: 0298-38-8383; Fax: 0298-38-8383; E-mail: cjjiang@abr.affrc.go.jp.
Published, JBC Papers in Press, December 20, 2000, DOI 10.1074/jbc.M006430200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
NPC, nuclear
pore complex;
COP1, constitutive photomorphogenic 1;
NLS, nuclear
localization signal;
PTAR, pore targeting complex;
PCR, polymerase
chain reaction;
GST, glutathione S-transferase;
IBB domain, importin -binding domain.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Imamoto, N., Kamei, Y., and Yoneda, Y. (1998) Eur. J. Histochem. 42, 9-20[Medline] [Order article via Infotrieve] |
2. | Mattaj, I. W., and Englmeier, L. (1998) Annu. Rev. Biochem. 67, 265-306[CrossRef][Medline] [Order article via Infotrieve] |
3. | Pemberton, L. F., Blobel, G., and Rosenblum, J. S. (1998) Curr. Opin. Cell Biol. 10, 392-399[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Nagy, F.,
and Schafer, E.
(2000)
EMBO J.
19,
157-163 |
5. | von Arnim, V. G., and Deng, X.-W. (1994) Cell 79, 1035-1045[Medline] [Order article via Infotrieve] |
6. | Wozniak, R. W., Rout, M. P., and Aitchison, J. D. (1998) Trends Cell Biol. 8, 184-188[CrossRef][Medline] [Order article via Infotrieve] |
7. | Dingwall, C., and Laskey, R. A. (1991) Trends Biochem. Sci. 16, 478-481[CrossRef][Medline] [Order article via Infotrieve] |
8. | Raikhel, N. (1992) Plant Physiol. 100, 1627-1632 |
9. | Imamoto, N., Shimamoto, T., Kose, T., Takao, T., Tachibana, T., Matsubae, M., Sekimoto, T., Shimamoto, T., and Yoneda, Y. (1995) FEBS Lett. 368, 415-419[CrossRef][Medline] [Order article via Infotrieve] |
10. | Imamoto, N., Shimamoto, T., Takao, T., Tachibana, T., Kose, T., Matsubae, M., Sekimoto, T., Shimonishi, Y., and Yoneda, Y. (1995) EMBO J. 14, 3617-3626[Abstract] |
11. | Görlich, D., Prehn, S., Laskey, R. A., and Hartmann, E. (1994) Cell 79, 767-778[Medline] [Order article via Infotrieve] |
12. | Iovine, M. K., Watkins, J. L., and Wente, S. R. (1995) J. Cell Biol. 131, 1699-1713[Abstract] |
13. |
Kraemer, D. M.,
Strambio-de-Castillia, C.,
Blobel, G.,
and Rout, M. P.
(1995)
J. Biol. Chem.
270,
19017-19021 |
14. | Radu, A., Moore, M. S., and Blobel, G. (1995) Cell 81, 215-222[Medline] [Order article via Infotrieve] |
15. | Melchior, F. B., Paschal, J., Evans, J., and Gerace, L. (1993) J. Cell Biol. 123, 1649-1659[Abstract] |
16. | Moore, M. S., and Blobel, G. (1993) Nature 365, 661-663[CrossRef][Medline] [Order article via Infotrieve] |
17. | Cortes, P., Ye, Z.-S., and Baltimore, D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7633-7637[Abstract] |
18. | Cuomo, C. A., Kirch, S. A., Gyuris, J., Brent, R., and Oettinger, M. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6156-6160[Abstract] |
19. | O'Neill, R. E., and Palese, P. (1995) Virology 206, 116-125[Medline] [Order article via Infotrieve] |
20. | Weis, K., Mattaj, I. W., and Lamond, A. I. (1995) Science 269, 1049-1053 |
21. | Kohler, M., Ansieau, S., Prehn, S., Leutz, A., Haller, H., and Hartmann, E. (1997) FEBS Lett. 417, 104-108[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Kohler, M.,
Speck, C.,
Christiansen, M.,
Bischoff, F. R.,
Prehn, S.,
Haller, H.,
Gorloch, D.,
and Hartmann, E.
(1999)
Mol. Cell. Biol.
19,
7782-7791 |
23. | Tsuji, L., Takumi, T., Imamoto, N., and Yoneda, Y. (1997) FEBS Lett. 416, 30-34[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Nachury, M. V.,
Ryder, U. W.,
Lamond, A. I.,
and Weis, K.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
582-587 |
25. |
Miyamoto, Y.,
Imamoto, N.,
Sekimoto, T.,
Tachibana, T.,
Seki, T.,
Tada, S.,
Enomoto, T.,
and Yoneda, Y.
(1997)
J. Biol. Chem.
272,
26375-26381 |
26. | Nadler, S. G., Tritschler, D., Haffar, O. K., Blake, J., Bruce, A., and Cleaveland, J. S. (1997) J. Biol. Chem. 272, 420-429 |
27. |
Sekimoto, T.,
Imamoto, N.,
Nakajima, K.,
Hirano, T.,
and Yoneda, Y.
(1997)
EMBO J.
16,
7067-7077 |
28. |
Welch, K.,
Franke, J.,
Kohler, M.,
and Macara, I. G.
(1999)
Mol. Cell. Biol.
19,
8400-8411 |
29. |
Hicks, G. R.,
Smith, H. M. S.,
Lobreaux, S.,
and Raikhel, N. V.
(1996)
Plant Cell
8,
1337-1352 |
30. |
Ballas, N.,
and Citovsky, V.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
10723-10728 |
31. |
Németh, K.,
Salchert, K.,
Putnoky, P.,
Bhalerao, R.,
Koncz-Kálman, Z.,
Stankovic,
Stangeland, B.,
Bakó, L.,
Mathur, J.,
Ökrész, L.,
Stabel, S.,
Geigenberger, P.,
Stitt, M.,
Reédei, G. P.,
Schell, J.,
and Koncz, C.
(1998)
Genes Dev.
12,
3059-3073 |
32. | Schledz, M., Leclerc, D., Neuhaus, G., and Merkle, T. (1998) Plant Physiol. 116, 868 |
33. | Shoji, K., Iwasaki, T., Matsuki, R., Uchimiya, H., Miyao, M., and Yamamoto, N. (1998) Gene (Amst.) 212, 279-286[CrossRef][Medline] [Order article via Infotrieve] |
34. | Iwasaki, T., Matsuki, R., Shoji, K., Sanmiya, K., Miyao, M., and Yamamoto, N. (1998) FEBS Lett. 428, 259-262[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Smith, H. M. S.,
Hicks, G. R.,
and Raikhel, N. V.
(1997)
Plant Physiol.
114,
411-417 |
36. |
Jiang, C. J.,
Imamoto, N.,
Matsuki, R.,
Yoneda, Y.,
and Yamamoto, N.
(1998)
J. Biol. Chem.
273,
24083-24087 |
37. | Matsuki, R., Iwasaki, T., Shoji, K., Jiang, C. J., and Yamamoto, Y. (1998) Plant Cell Physiol. 39, 879-884[Medline] [Order article via Infotrieve] |
38. | Chiu, W., Niwa, Y., Zeng, W., Hirano, T., Kobayashi, H., and Sheen, J. (1996) Curr. Biol. 6, 325-330[Medline] [Order article via Infotrieve] |
39. | Akama, K., Shiraishi, H., Ohta, S., Nakamura, K., Okada, K., and Shimura, Y. (1992) Plant Cell Rep. 12, 7-11 |
40. | Clough, S. J., and Ben, A. F. (1998) Plant J. 16, 735-743[CrossRef][Medline] [Order article via Infotrieve] |
41. | Jiang, C. J., Imamoto, N., Matsuki, R., Yoneda, Y., and Yamamoto, N. (1998) FEBS Lett. 437, 127-130[CrossRef][Medline] [Order article via Infotrieve] |
42. | Bischoff, F. R., and Ponstingl, H. (1995) Methods Enzymol. 257, 135-144[Medline] [Order article via Infotrieve] |
43. | Melchior, F., Sweet, D. J., and Gerace, L. (1995) Methods Enzymol. 257, 279-291[Medline] [Order article via Infotrieve] |
44. | Yano, R., Oakes, M., Yamaghishi, M., Dodd, J. A., and Nomura, M. (1992) Mol. Cell. Biol. 12, 5640-5651[Abstract] |
45. | Görlich, D., Henklein, P., Laskey, R. A., and Hartmann, E. (1996) EMBO J. 15, 1810-1817[Abstract] |
46. | Weis, K., Ryder, U., and Lamond, A. I. (1996) EMBO J. 15, 1818-1825[Abstract] |
47. | Peifer, M., Berg, S., and Reynolds, A. B. (1994) Cell 76, 789-791[Medline] [Order article via Infotrieve] |
48. |
Stacey, M. G.,
Hicks, S. N.,
and von Arnim, A. G.
(1999)
Plant Cell
11,
349-363 |
49. | Görlich, D., N., Pante, Kutay, U., Aebi, U., and Bischoff, F. R. (1996) EMBO J. 15, 5584-5594[Abstract] |
50. | Conti, E., Uy, M., Leighton, L., Blobel, G., and Kuriyan, J. (1998) Cell 94, 193-204[Medline] [Order article via Infotrieve] |
51. |
Herold, A.,
Truant, R.,
Wiegand, H.,
and Cullen, B. R.
(1998)
J. Cell Biol.
143,
309-318 |
52. | Kutay, U., Bischoff, F. R., Kostka, S., Kraft, R., and Görlich, D. (1997) Cell 90, 1061-1071[Medline] [Order article via Infotrieve] |
53. | Fagotto, F., Gluck, U., and Gumbiner, B. M. (1998) Curr. Biol. 8, 181-190[Medline] [Order article via Infotrieve] |
54. |
Yokoya, F.,
Imamoto, N.,
Tachibana, T.,
and Yoneda, Y.
(1999)
Mol. Biol. Cell
10,
1119-1131 |