Molecular Cloning of a Novel Importin alpha  Homologue from Rice, by Which Constitutive Photomorphogenic 1 (COP1) Nuclear Localization Signal (NLS)-Protein Is Preferentially Nuclear Imported*

Chang-Jie JiangDagger §, Kazuhiro Shoji, Rikyu Matsuki, Akiko BabaDagger , Noritoshi InagakiDagger , Hiroshi Ban||, Toshisuke Iwasaki||, Naoko Imamoto**, Yoshihiro YonedaDagger Dagger , Xing-Wang Deng§§, and Naoki Yamamoto¶¶

From the Dagger  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 Dagger Dagger  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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nuclear import of proteins that contain classical nuclear localization signals (NLS) is initiated by importin alpha , a protein that recognizes and binds to the NLS in the cytoplasm. In this paper, we have cloned a cDNA for a novel importin alpha  homologue from rice which is in addition to our previously isolated rice importin alpha 1a and alpha 2, and we have named it rice importin alpha 1b. In vitro binding and nuclear import assays using recombinant importin alpha 1b protein demonstrate that rice importin alpha 1b functions as a component of the NLS-receptor in plant cells. Analysis of the transcript levels for all three rice importin alpha  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 alpha 1b. These data suggest that each of the different rice importin alpha  proteins carry distinct groups of nuclear proteins, such that multiple isoforms of importin alpha  contribute to the regulation of plant nuclear protein transport.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (Matalpha -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 alpha  and importin beta  subunits. Importin alpha  binds the NLS specifically, forming a stable pore targeting complex (PTAC) (9-11), whereas importin beta  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).

Both importin alpha  and beta  have been identified in a wide range of species, including vertebrates, yeast, and plants. However, while only a single isoform of importin beta  involved in classical NLS-directed nuclear transport has been isolated in most species, multiple isoforms of importin alpha  have been identified in various species (2, 3). Recent studies suggest that such multiplicity of importin alpha  isoforms might contribute to the tissue-specific or temporal regulation of nuclear protein import. For example, at least six different human isoforms of importin alpha  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).

As in many vertebrates, multiple isoforms of importin alpha  have been identified from Arabidopsis (aIMPalpha (29), AtKAPalpha (30), ATHKAP2 (31), and AtIMPalpha 1-4 (32)) and rice (rice importin alpha 1 (33) and rice importin alpha 2 (34)). Intriguingly, in vitro binding assays using recombinant plant importin alpha s have revealed differential recognition of different types of NLS. The aIMPalpha 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 Matalpha -2 type) (35). In contrast, rice importin alpha 1 bound to the T- and O2-NLS, but not to the R-NLS (36). Rice importin alpha 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 alpha  may serve as a control point for the regulation of nuclear protein import. It is also noteworthy that two closely related importin beta s have been identified in rice (37). This is the only report thus far of multiple importin beta  isoforms identified in a single species. Whether such multiplicity is a common characteristic among plants is not yet clear, as no importin beta 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.

In this paper we report the cloning and functional analysis of a novel rice importin alpha  homologue which is in addition to our previously isolated rice importin alpha 1 (33) and alpha 2 (34). Because it shares very high homology (82.8% identity) with the rice importin alpha 1, we have named this novel homologue rice importin alpha 1b and have renamed the rice importin alpha 1 as rice importin alpha 1a. We show that rice importin alpha 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 alpha  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 alpha 1b.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

cDNA Cloning

A rice expressed sequence tag (EST) clone C3059 (accession number D23592) with high homology to rice importin alpha 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 lambda 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 alpha 1b cDNA was used to generated a GST-rice importin alpha 1b construct, as described below.

DNA Constructions

GST-rice Importin alpha 1b-- A fragment corresponding to the amino-terminal portion (about 300 base pairs) of rice importin alpha 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 alpha 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.

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 alpha 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 alpha 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 alpha 1a, 5'-CGATAAGAAGCTCGAAAGCCTT-3'/5'-AAAGCAACTTGCGGAACTGTGT-3' and 5'-CTGCTATGATTGGTGGAGTTTATTCGGACG-3'; 2) rice importin alpha 1b, 5'-AATCAGGAGTGTTCCCAAGGC-3'/5'-ATCCCCAGTGACGATGTTACC-3' and 5'-TGGAACTTCTCATGCATCCTTCGGC-3'; 3) rice importin alpha 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'.

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-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.

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 alpha 1a, alpha 2 (36), rice importin beta 1 (41), and Ran (42, 43) were prepared as described previously.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cloning of Rice Importin alpha 1b-- By searching the rice EST data base, we identified an EST clone (accession number D23592) showing high homology with rice importin alpha 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 alpha 1. Therefore, we designated this novel cDNA as rice importin alpha 1b and renamed the rice importin alpha 1 as rice importin alpha 1a. Rice importin alpha 1b also shares significant amino acid homology with other previously identified importin alpha s, such as rice importin alpha 2 (27.5%), aIMPalpha (59% (29)), AtKAPalpha (68% (30)), and yeast SRP1 (54% (44)) (Fig. 1). Like the other importin alpha  homologues, rice importin alpha 1b contains the main characteristics of importin alpha : the importin beta -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 this window]
[in a new window]
 
Fig. 1.   Multiple sequence alignment of rice importin alpha 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 alpha 1b, alpha 1a, and alpha 2 are AB034311, AB004660, and AB006788 respectively.

NLS Binding and PTAC Formation by Rice Importin alpha 1b-- To address the ability of rice importin alpha 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 alpha 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 this window]
[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.

As shown in Fig. 2B, rice importin alpha 1b and each of the NLS-GFPs migrated as single bands (lanes 1, 3, 5, 7, and 9). A mixture of rice importin alpha 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 alpha 1a (36), the complex of O2-GFP and rice importin alpha 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 alpha 1b shifted upward, giving a much darker band (Fig. 2B, lane 6). In contrast, a mixture of rice importin alpha 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 alpha 1b selectively binds to T- and O2-NLS, but not to R-NLS. The binding was NLS-specific as the rice importin alpha 1b did not bind to the Tm-GFP that has been shown to be nonfunctional (Fig. 2B, lane 4).

We next examined PTAC formation by rice importin alpha 1b with rice importin beta 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 alpha 1b with rice importin beta 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 alpha 1b and beta 1 (Fig. 2C, lane 5) or between rice importin alpha 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 alpha 1b and beta 1 and either T- or O2-GFP (data not shown).

Activity of Rice Importin alpha 1b in the in Vitro Import Assay using Digitonin-permeabilized HeLa Cells-- To assess the functional activity of rice importin alpha 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.

As shown in Fig. 3A, rice importin alpha 1b, in conjunction with rice importin beta 1, efficiently accumulated T-GFP at the nuclear rim (Fig. 3A, panel a). In contrast, rice importin beta 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 this window]
[in a new window]
 
Fig. 3.   The effect of rice importin alpha 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 beta 1 was 6 pmol; rice importin alpha 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 beta 1 was 3 pmol; and rice importin alpha 1b was 12 pmol. In both parts A and B: a and b, T-GFP + importin beta 1 + importin alpha 1b; c and d, T-GFP + importin beta 1; and e and f, Tm-GFP + importin beta 1 + importin alpha 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.

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 alpha 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).

Differential Gene Expression of the Three Rice Importin alpha s-- To investigate the expression of the three rice importin alpha 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).

Transcripts of the three rice importin alpha 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 alpha s varied significantly. Rice importin alpha 1b and alpha 2 showed maximal transcript levels in roots (Fig. 4, B and C). In contrast, the highest levels of rice importin alpha 1a were detected in calli (Fig. 4A).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4.   Transcript analyses of rice importin alpha 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.

We previously reported that transcription of rice importin alpha 1a is down-regulated by light (33). Herein, we examined whether this is also the case with the other two rice importin alpha s. As shown in Fig. 4, the three rice importin alpha 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 alpha 1a by about 10-fold (Fig. 4A). Rice importin alpha 2 also showed a modest, 5-fold increase in transcript levels after 24 h in the dark (Fig. 4C). In comparison, rice importin alpha 1b showed only a minor change in transcript levels during dark-adaptation (Fig. 4B). Thus it appears that the rice importin alpha s express differentially not only in different tissues but also in response to light.

Identification of a Bipartite Type NLS in COP1 Protein-- To further investigate any functional differences between the rice importin alpha s in nuclear import processes, we identified the NLS in the COP1 protein and examined its interaction with rice importin alpha s.

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).


View larger version (41K):
[in this window]
[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.

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.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 6.   In vitro interactions between rice importin alpha s and COP1 NLSs. Native gel electrophoresis was carried out as described in the legend to Fig. 2.


View larger version (93K):
[in this window]
[in a new window]
 
Fig. 7.   Preferential nuclear translocation of bWW-GFP by rice importin alpha 1b. Nuclear binding and import assays were carried out as described in the legend to Fig. 3.

In Vitro Binding of COP1 NLSs to Rice Importin alpha s-- In vitro interaction between the COP1 NLS and each of the three rice importin alpha 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 alpha 1a (Fig. 6A, lane 2) and alpha 1b (Fig. 6B, lane 2), but not by alpha 2 (Fig. 6C, lane 2). All the missense mutants of bWW- and mW-GFP were not recognized by any rice importin alpha s. Addition of rice importin beta 1 to the mixtures gave rise to the formation of PTAC consisting of bWW-GFP, rice importin beta 1, and either rice importin alpha 1a (Fig. 6D, lane 2) or alpha 1b (Fig. 6D, lane 5). No such PTAC formation was observed with rice importin alpha 2 (data not shown).

Preferential Nuclear Import of bWW-GFP by Rice Importin alpha 1b-- Because bWW-GFP was recognized in vitro by both rice importin alpha 1a (Fig. 6A) and alpha 1b (Fig. 6B) and further formed PTAC in the presence of rice importin beta 1 (Fig. 6D), we next carried out an in vitro nuclear protein import assay. As shown in Fig. 7, both rice importin alpha 1a (Fig. 7B) and alpha 1b (Fig. 7A), in conjunction with rice importin beta 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 alpha 1b (Fig. 7A, panels k and l). Although nuclear docking occurred with rice importin alpha 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 alpha  isoforms in the plant nuclear protein import system.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present work, we have isolated a novel cDNA for an importin alpha  homologue in rice and named it rice importin alpha 1b. Analysis of its primary structure reveals that rice importin alpha 1b contains the main characteristics of other importin alpha 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 beta  (20, 49). The putative IBB domain of rice importin alpha 1b displays maximum homology with those of other importin alpha s. Such high homology of IBB domains among different importin alpha s may explain why different isoforms of importin alpha  can interact with the same importin beta . For instance, all three rice importin alpha s can bind to rice importin beta 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 alpha  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 alpha  also requires the variable regions (51). In the case of the human importin alpha 2 (also termed Rch1, hSRP1alpha , 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 alpha 1b show only minimum identities to those of the other importin alpha s (Fig. 1), and such variability may confer an importin alpha  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 alpha  from the nucleus (52).

In addition to the primary structural data, our in vitro assays using recombinant rice importin alpha 1b protein demonstrated that rice importin alpha 1b can bind functional plant NLSs and rice importin beta 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 alpha 1b functions as a component of the NLS receptor in plant cells.

Both rice importin alpha 1a and alpha 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 aIMPalpha , which binds all the typical plant NLSs (35). Moreover, a different activity was also seen between rice importin alpha 1a and alpha 1b proteins in mediation of nuclear import of COP1 NLS-GFP in the present work (Fig. 7). In fact, preferential affinities between distinct importin alpha s and different NLS-containing proteins have also been reported in human importin alpha s. Human DNA helicase Q1 (25) and RCC1 (22) are most efficiently imported into the nucleus by human importin alpha 3, and transcription factor Stat1 is imported into the nucleus by NP1 (human importin alpha 1), but not by Rch1 (human importin alpha 2), in response to interferon-r (27). RanBP3 interacts preferentially with both human importin alpha 3 and alpha 4, but its nuclear import was most efficient in the presence of importin alpha 3 (28). All these data suggest a diverse specificity among importin alpha s with respect to NLS recognition, such that each importin alpha  preferentially imports a distinct group of proteins into the nucleus. In addition, the differential expression patterns of rice importin alpha s (Fig. 4) support the hypothesis that multiple isoforms of importin alpha s might contribute to spatial and temporal regulation of nuclear protein import.

Although both rice importin alpha 1a and alpha 1b, together with rice importin beta 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 alpha 1b (Fig. 7). This suggests that the nuclear import of COP1 protein in vivo is mediated, at least most efficiently, by the importin alpha 1b and that in vitro binding between an importin alpha  and an NLS does not necessarily mean that the nuclear import of the NLS protein is performed by that importin alpha . Indeed, similar phenomena have been reported in human importin alpha s. Human RanBP3 NLS shows a binding activity to both human importin alpha 3 and alpha 4, however, its nuclear import was mediated most efficiently by human importin alpha 3 (28). Nachury et al. (24) observed a consistently lower nuclear import activity of hSRP1r (human importin alpha 3) for bovine serum albumin-SV40 large T-antigen NLS when compared with that of hSRP1alpha (human importin alpha 2) or NP1. Taken together, all these data seem to suggest that the nature of the interaction between a distinct importin alpha  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 beta -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 alpha  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 beta -binding domain.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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[Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
28. Welch, K., Franke, J., Kohler, M., and Macara, I. G. (1999) Mol. Cell. Biol. 19, 8400-8411[Abstract/Free Full Text]
29. Hicks, G. R., Smith, H. M. S., Lobreaux, S., and Raikhel, N. V. (1996) Plant Cell 8, 1337-1352[Abstract/Free Full Text]
30. Ballas, N., and Citovsky, V. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10723-10728[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
36. Jiang, C. J., Imamoto, N., Matsuki, R., Yoneda, Y., and Yamamoto, N. (1998) J. Biol. Chem. 273, 24083-24087[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]
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[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.