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J Biol Chem, Vol. 274, Issue 32, 22610-22617, August 6, 1999

Plant Importin  Binds Nuclear Localization Sequences with High Affinity and Can Mediate Nuclear Import Independent of Importin ^*

Stefan Hübner, Harley M. S. Smith^§, Wei Hu, Chee Kai Chan, Hans-Peter Rihs^¶, Bryce M. Paschal, Natasha V. Raikhel^§**, and David A. Jans

From the Nuclear Signalling Laboratory, Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra ACT 2601, Australia, ^§ Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824-1312, ^¶ BGFA, Bochum, Germany D-44789, and the  Center for Cell Signalling, University of Virginia, Charlottesville, Virginia 22908

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Nuclear import of conventional nuclear localization sequence (NLS)-containing proteins initially involves recognition by the importin (IMP) / heterodimer, where IMP binds the NLS and IMP targets the IMP/NLS-containing protein complex to the nuclear pore. Here we examine IMP from the plant Arabidopsis thaliana (At-IMP), which exhibits nuclear envelope localization typical of IMP rather than IMP in other eukaryotic cell systems. We show that At-IMP recognizes conventional NLSs of two different types with high affinity (K_d of 5-10 nM), in contrast to mouse IMP (m-IMP), which exhibits much lower affinity (K_d of 50-70 nM) and only achieves high affinity in the presence of m-IMP. Unlike m-IMP, At-IMP is thus a high affinity NLS receptor in the absence of IMP. Interestingly, At-IMP was also able to bind with high affinity to NLSs recognized specifically by m-IMP and not m-IMP, including that of the maize transcription factor Opaque-2. Reconstitution of nuclear import in vitro indicated that in the absence of exogenous IMP subunit but dependent on RanGDP and NTF2, At-IMP was able to mediate nuclear accumulation to levels comparable with those mediated by m-IMP/. Neither m-IMP nor - was able to mediate nuclear import in the absence of the other subunit. At-IMP's novel NLS recognition and nuclear transport properties imply that plants may possess an IMP-mediated nuclear import pathway independent of IMP in addition to that mediated by IMP/.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The entry of karyophilic proteins into the nucleus through the nuclear pore complex (NPC)¹ is effected by specific targeting signals called nuclear localization sequences (NLSs) (1, 2), and is a receptor-mediated (3, 4), energy-dependent (5, 6) process. The key factors involved are members of the NLS-recognizing importin/karyopherin family (7-11), the monomeric GTPase Ran/TC4 (12, 13), and auxiliary proteins such as NTF2/p10 (14, 15). In the first step, the NLS-containing protein is recognized by the importin (IMP) heterodimer through the NLS-binding IMP subunit (3, 7, 9) and targeted to the NPC through the affinity of the IMP subunit (8, 10, 11, 16) for NPC components (17, 18). In the second step requiring cytoplasmic RanGDP (19, 20), the transport complex is translocated through the NPC (21), and IMP and the NLS-bearing protein are released into the nucleoplasm through the action of Ran GTP (19). Alternative signal-mediated nuclear import pathways have recently been identified, where either IMP itself (22-24) or related homologs (25-27) fulfill the role of both IMP and - in binding NLSs and targeting them to the NPC (25, 26, 28).

Although NLS receptors from different species share structural and functional homology, experimental evidence suggests that nuclear import in plant cells has unique features compared with that in other eukaryotes. In contrast to the latter, in vitro transport in plant cells appears not to be inhibited by the nucleoporin-binding lectin wheat germ agglutinin and to occur at low temperature and in the absence of exogenously added cytosol (see Refs. 29 and 30). In addition, the NLS-binding IMP subunit from Arabidopsis thaliana (At-IMP) shows nuclear envelope association (30, 31) in similar fashion to IMP in mammalian and other cell systems (8, 10, 11); IMP in mammalian and yeast systems shows predominantly nucleoplasmic location as well as cell cycle-dependent localization in either cytoplasm or nucleus in Drosophila (32). A linkage of At-IMP with the cytoskeleton has also recently been demonstrated, with a mechanistic role in nuclear import surmised (33). Because of these novel properties, we set out to quantitate the NLS binding properties of At-IMP for the first time using an ELISA-based assay (34, 35). We find that At-IMP binds NLSs of different types with high affinity independent of an IMP subunit, in contrast to the IMP subunits from mouse and yeast, which require their respective IMP subunits to achieve high affinity binding (21, 34-36). At-IMP, together with Ran/TC4 and NTF2 and in the absence of IMP, was able to mediate nuclear import in vitro to levels comparable with those mediated by mouse IMP/ (m-IMP/). m-IMP was unable to mediate nuclear import in the absence of m-IMP. At-IMP thus shows unique properties, being able to fulfill both NLS recognition and nuclear import in the absence of IMP.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Fusion Proteins and Peptides-- The amino acid sequences of the NLSs contained within the fusion proteins and peptides used are shown in Table I. The plasmid expressing Xenopus laevis N1N2 amino acids 465-581 fused amino-terminal to -galactosidase was derived by inserting a 351-base pair EcoRV/NcoI-filled in fragment from the N1N2 cDNA (39) into the unique SmaI site of the plasmid vector pPR2 (37). The T-ag peptides pep101Lys and pep101Thr (40) and maize transcription factor (TF) Opaque-2 (O2) bipartite NLS-containing pepO2 (31, 41) have all been described previously (see Table I).

-Galactosidase fusion proteins were expressed and purified as previously (37, 38). HisGAL4 was purified from bacterial extracts using nickel-nitrilotriacetic acid-agarose (Qiagen) (23), and His-tagged At-IMP (ACC bankit 209141) was similarly expressed and purified (30). The m-IMP (PTAC58; ACC D55720) and m-IMP (PTAC97; ACC D45836) and yeast IMP (Kap95; ACC U19028) subunits were expressed as glutathione S-transferase (GST) fusion proteins and purified as previously (9, 21, 34, 35, 42), with GST-free m-IMP prepared by thrombin cleavage (9, 34, 35). Human Ran was similarly expressed as a GST fusion protein, GST-free Ran being prepared by thrombin cleavage and loaded with GDP as described (43, 44). Recombinant human NTF2 was expressed and purified using S100 HR column chromatography (15).

Protein concentrations were determined using the dye binding assay of Bradford (45), with bovine serum albumin as a standard.

ELISA-based Binding Assay-- An ELISA-based binding assay (34, 35, 42, 46) was used to examine the binding affinity between importin subunits (with and without GST moieties) and NLS-containing proteins or peptides. The latter were coated onto 96-well microtiter plates and incubated with increasing concentrations of IMP subunits, and detection of bound IMP-GST was performed using a goat anti-GST primary antibody, an alkaline phosphatase-coupled rabbit anti-goat secondary antibody, and the substrate p-nitrophenyl phosphate (34). In some experiments, including all those with At-IMP, affinity-purified antibody specific to At-IMP (30, 31), which cross-reacts with mouse and yeast IMP but not  (Ref. 30 and data not shown), was used instead of anti-GST antibody. Absorbance measurements were performed over 90 min using a plate reader (Molecular Devices), with values corrected by subtracting absorbance both at 0 min and in wells incubated without IMP (34). To examine importin binding to the NLSs in the case of the T-ag- and N1N2--galactosidase fusion proteins, quantitation was performed in parallel for -galactosidase, and these values were subtracted from those for the respective fusion proteins (34, 35, 42). Fusion proteins were also subjected to a parallel -galactosidase ELISA (see Ref. 34) to correct for any differences in coating efficiencies and enable a true estimate of bound importin (34-36, 42, 46). Experiments were also performed in which IMPs (GST-free) were coated onto microtiter plates, and m-IMP-GST and y-IMP-GST binding was assessed in similar fashion to the NLS-binding ELISAs above.

In Vitro Nuclear Transport-- Nuclear import kinetics were analyzed at the single cell level using mechanically perforated HTC rat hepatoma cells, cultured as previously (37, 38), in conjunction with confocal laser scanning microscopy (35, 42, 46). Experiments were performed as described (36) for 40 min at room temperature in a 5-µl volume containing 30 mg/ml bovine serum albumin, 2 µM GTP, and an ATP regenerating system (0.125 mg/ml creatine kinase, 30 mM creatine-phosphate, 2 mM ATP), transport substrate (0.2 mg/ml IAF-labeled fusion protein), and where indicated, 4 µM RanGDP, 0.15 µM NTF2, 1 µM m-IMP, 1 µM m-IMP, or 0.6 µM At-IMP. In inhibition experiments, specific antibody to either IMP (30, 31) (final concentration of 20 µg/ml) or IMP (10) (mAb3E9; final concentration of ~200 µg/ml from purified ascites fluid) was included in the assay. Image analysis and curve-fitting was performed as described (35, 42, 46); the level of accumulation at the nuclear envelope, relative to medium fluorescence, was determined using NIH Image 1.60 in line plot mode as previously (47).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

At-IMP Displays High Binding Affinity to Different Types of NLSs-- Our previous results using an ELISA-based assay to determine the binding affinities of IMP subunits for different NLSs indicated relatively low affinity on the part of m-IMP compared with that for the m-IMP/ heterodimer (34-36, 42). We set out to quantitate the NLS binding affinity of At-IMP using this assay, where proteins and peptides were used that contained the NLS of T-ag, the bipartite NLS of the X. laevis nuclear factor N1N2 (36, 39), or the less well defined NLSs of the yeast TF GAL4 (48) and the plant TF O2 (49) (see Table I). We found that At-IMP bound all of these NLSs, present in fusion proteins and/or as peptides, with 2-5-fold higher affinity than m-IMP (Figs. 1 and 2, Table II, and data not shown), the latter only attaining binding affinity comparable with that of At-IMP in the presence of m-IMP (Table II). The specificity of binding in all cases was indicated by the fact that binding to NLS-mutant derivatives was severely reduced (less than 8% maximal binding compared with the respective wild type derivative; Table II). The results indicate that At-IMP is able to recognize different types of NLS with high affinity independent of an IMP subunit (see below).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   At-IMP binds to conventional NLSs with high affinity in the absence of an IMP subunit as quantitated using an ELISA-based binding assay. Microtiter plates were coated with T-ag-CcN--Gal or the NLS-deficient T-ag-Cc--Gal (top panels) or with N1N2--Gal (bottom panels) and incubated with increasing concentrations of m-IMP (right panels) or At-IMP (left panels) in the absence or presence of m-IMP as indicated. Curves were fitted for the function B(x) = Bmax (1  ekB), where x is the concentration of importin, and B is the level of importin bound (34, 35, 42). The apparent Kd values representing the IMP concentration yielding half-maximal binding for T-ag-CcN--Gal were 50 and 3 nM, respectively, for m-IMP without or with precomplexed m-IMP, while those for At-IMP were 8.3 and 7.2 nM in the absence or presence of m-IMP, respectively. Binding to T-ag-Cc--Gal was negligible. The KD values for N1N2--Gal were 12.2 and 1 nM, respectively, for m-IMP without or with precomplexed m-IMP, while those for At-IMP were 7.1 and 6 nM in the absence or presence of m-IMP, respectively. The results are from a single typical experiment performed in triplicate (S.D. value not greater then 9% the value of the mean), with pooled data shown in Table II.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   At-IMP binds to the GAL4 NLS with high affinity as quantitated using an ELISA-based binding assay. Microtiter plates coated with HisGAL4 were incubated with increasing concentrations of mouse importins (left panel) or At-IMP (right panel) in the absence or presence of m-IMP as indicated. The apparent Kd values for m-IMP and m-IMP/ were 23 and 20 nM, respectively, while those for At-IMP were 6.1 and 10.8 nM in the absence or presence of m-IMP, respectively. The results are from a single typical experiment performed in triplicate (S.D. not greater then 11% the value of the mean), with pooled data shown in Table II.

View this table: [in this window] [in a new window]   Table II NLS binding parameters of At-IMP in the presence and absence of m-IMP as measured using an ELISA-based assay

That At-IMP bound GAL4 so well was somewhat surprising, since its NLS is recognized exclusively by IMP subunits and not by IMP (see Ref. 23). The data in Fig. 2 and Table II (see legend) illustrate this, showing clearly that m-IMP, in contrast to At-IMP and m-IMP, does not recognize the GAL4 NLS. Similarly, the NLS contained within pepO2 is recognized with high affinity by m-IMP (K_d of 10.1 nM) rather than m-IMP (K_d of 80 nM), and pepO2 is bound by At-IMP with high affinity (K_d of 11.7 nM; see Table II). Since At-IMP recognizes the GAL4 and O2 NLSs with high affinity, it clearly possesses some of the NLS binding properties of IMP.

Importin  Subunits Can Recognize At-IMP without Affecting NLS Binding Affinity-- Binding of IMP to IMP has been shown to increase the NLS binding affinity of the latter (21, 34, 35). We decided to investigate whether At-IMP is able to dimerize with the IMP subunits m-IMP and y-IMP employing our ELISA-based assay. We found higher affinity binding of At-IMP to y-IMP (K_d of 10 ± 2.5 nM, n = 5) than to m-IMP (K_d of 20 ± 5.1 nM, n = 13). For comparison, m-IMP was also tested, exhibiting highest affinity for m-IMP (K_d of 2.2 ± 0.9 nM, n = 4), as expected, and a K_d of 21 ± 1.2 nM (n = 3) for y-IMP.

Since At-IMP exhibited high affinity binding to m-IMP, we also tested whether binding of m-IMP influenced NLS recognition by At-IMP, using our ELISA-based assay and antibodies to either At-IMP or m-IMP-GST. The affinity of At-IMP for the NLSs within the various fusion proteins and peptides after precomplexation to m-IMP was not significantly different from that in its absence (Figs. 1 and 2; Table II). This was in stark contrast to the effect of m-IMP on NLS recognition by m-IMP (Table II), which significantly increased the binding affinity for the various NLSs 4-8-fold (see also Refs. 34 and 35).

At-IMP Can Mediate Nuclear Protein Import Independent of IMP-- The NLS-recognition properties of At-IMP implied that it possessed some of the properties of IMP as well as IMP, so that we decided to compare the ability of At-IMP to that of the mouse importins to mediate nuclear import in a reconstituted in vitro system in the presence of purified RanGDP and NTF2. Since plant protoplasts retain sufficient amounts of transport factors, including IMP, to support nuclear import in the absence of exogenously added cytosol (29, 30), we used our well characterized system of mechanically perforated rat hepatoma cells, which requires the addition of cytosol and an ATP-regenerating system for NLS-dependent nuclear accumulation (35-37, 42, 46). We initially examined the ability of At-IMP and the mouse importins to mediate binding to the nuclear envelope in the absence of Ran/NTF2 (Fig. 3A). No significant targeting of T-ag-CcN--Gal to the nuclear envelope was observed in the absence of exogenously added importin subunits (Fig. 3A, bottom right panel, and results not shown); although m-IMP (Fig. 3A, top right panel) or - alone could not mediate binding as expected, the combination of the two was able to do so with high efficiency (Fig. 3A, bottom left panel). Significantly, At-IMP was able to perform the same function in the absence of m-IMP (Fig. 3A, top left panel); the maximal level of accumulation at the nuclear envelope, as determined by image analysis using the line plot mode (47), was 2.6 ± 0.2 (n = 16) times that in the cytoplasm, compared with a level of 2.4 ± 0.1 (n = 15) effected by m-IMP/. This activity was consistent with At-IMP's ability to localize at the nuclear envelope/NPC (30, 31).

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Ability of At-IMP to mediate nuclear protein import reconstituted in vitro in the absence of exogenously added IMP. Nuclear import was reconstituted in mechanically perforated HTC cells in the presence of an ATP-regenerating system containing GTP/GDP and using NTF2 and GDP-loaded Ran as described under "Materials and Methods." A, visualization (after a 10-min incubation at room temperature) of binding at the nuclear envelope of the T-ag-NLS-containing fusion protein T-ag-CcN--Gal mediated by IMP subunits in the absence of Ran and NTF2. B, visualization (after a 25-min incubation at room temperature) of nuclear accumulation mediated by IMP subunits in the presence of Ran and NTF2. Lack of import of the NLS mutant derivative T-ag-Cc--Gal is shown in the bottom panels. C, quantitative results for nuclear import kinetics of the fusion proteins T-ag-CcN--Gal and T-ag-Cc--Gal mediated by mouse IMP subunits (left panel) or At-IMP (right panel) in the presence of Ran and NTF2. Results shown are from a single typical experiment, where each data point represents at least five separate measurements each of Fn, Fc, and background fluorescence (see "Materials and Methods"). Data are fitted for the function Fn/c = Fn/cmax * (1  ekt), where Fn/c is the nuclear/cytoplasmic ratio, k is the rate constant, and t is time (min) (34, 35, 42). Pooled data are presented in Table III.

In the presence of RanGDP and NTF2, m-IMP or  alone could not mediate nuclear import of T-ag-CcN--Gal (Fig. 3B), levels of nuclear accumulation being similar to those in the absence of IMP subunits (Fig. 3C, Table III). This was in contrast to the m-IMP/ combination, where maximal nuclear accumulation relative to that in the cytoplasm (Fn/c_max) was over 5-fold (half-maximal within 2.6 min). Significantly, At-IMP, in the absence of exogenously added IMP subunit, was able to mediate nuclear import to comparable levels (Fn/c_max of 3.7; half-maximal within 8 min). The specificity of transport in all cases was demonstrated by the fact that the T-ag-Cc--Gal protein, containing a nonfunctional NLS, was not accumulated to any significant extent (Fig. 3, B and C; Table III). The results thus demonstrated that At-IMP could mediate nuclear import of an NLS-containing transport substrate independent of an IMP subunit. While nuclear import mediated by At-IMP was enhanced by NTF2, especially in terms of the rate of import, it did not absolutely appear to require it, since an Fn/c_max of over 3 was observed in its absence (Fig. 4; Table III).

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Nuclear import reconstituted in vitro using purified components mediated by At-IMP and m-IMP in the absence and presence of exogenously added m-IMP. Nuclear import kinetics of the fusion protein T-ag-CcN--Gal mediated by IMP subunits in the absence (left) and presence (right) of m-IMP was measured in the presence of an ATP-regenerating system containing GTP/GDP, GDP-loaded Ran, and NTF2 as indicated. Experiments for transport were performed as described in the legend to Fig. 3. Results shown are from a single typical experiment, where each data point represents at least four separate measurements each of Fn, Fc, and background fluorescence. Pooled data are presented in Table III.

Nuclear import mediated by At-IMP was also tested in the presence of the m-IMP subunit (Figs. 3B and 4). Interestingly, nuclear import was reduced by more than 50%, compared with in the absence of m-IMP, in either the absence or presence of NTF2 (Fig. 4; Table III), this inhibition of At-IMP-mediated transport presumably resulting from m-IMP's ability to bind At-IMP (see above). To test whether the nuclear import mediated by At-IMP alone was attributable to small amounts of residual IMP in the assay, inhibition experiments were performed using antibodies to either IMP (29) or IMP (10) (Fig. 5). Clear results were obtained, whereby the antibody to IMP reduced nuclear import mediated by At-IMP together with Ran over 90%, while the antibody to IMP had no inhibitory effect but rather enhanced transport by almost 70%, consistent with the idea that IMP inhibited At-IMP-mediated nuclear import (Fig. 5). In contrast to the results for At-IMP, the antibodies to IMP or IMP both inhibited nuclear import mediated by m-IMP/ in conjunction with Ran, maximal nuclear accumulation being reduced by over 80 and 95%, respectively (Fig. 5). Clearly, the anti-IMP antibody was functional in inhibiting IMP/-mediated nuclear import, the results for At-IMP thus demonstrating that its ability to mediate nuclear import was not attributable to residual contaminating IMP in the assay system; rather, the residual IMP appeared to inhibit its action. An additional control was performed of m-IMP alone together with Ran in the absence or presence of the antibodies, indicating that the background level of accumulation in the absence of exogenously added m-IMP was not increased by the anti-IMP antibody (Fig. 5), further underlining the functional differences between m-IMP and At-IMP.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Inhibition of At-IMP- and m-IMP/-mediated nuclear import by specific antibodies to IMP subunits. Maximal nuclear accumulation of T-ag-CcN--Gal above background (transport in the absence of IMP subunits) mediated by At-IMP, m-IMP, or m-IMP/ in the presence of Ran was measured in the absence or presence of anti-IMP or anti-IMP antibodies (see "Materials and Methods"). The results represent averages from three separate experiments (measurements performed as described in the legend to Fig. 3), with the S.E. indicated. In the case of the low m-IMP-mediated transport, results are expressed relative to those for m-IMP/.

Nuclear import kinetic measurements were also performed in vitro for the first time using the bipartite NLS-containing substrate N1N2--Gal (Table III and data not shown), producing results similar to those for T-ag-CcN--Gal. Maximal levels of At-IMP-mediated import (Fn/c_max of about 3.4; half-maximal within 15.5 min) were obtained in the presence of RanGDP; the rate of import was almost twice as fast in the presence of NTF2 (t_1/2 of ~9 min; Table III). In the presence of m-IMP, accumulation was reduced by ~40 and 60% in the presence and absence of NTF2, respectively. Clearly, independent of IMP, At-IMP is able to mediate nuclear import not only of a T-ag-NLS-containing protein (above) but also of a bipartite NLS-containing protein.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

It has become obvious over the last 2 years that there are multiple signal-dependent nuclear import pathways additional to that mediated by the IMP/ heterodimer. These include those mediated exclusively by IMP-related homologs such as transportin, where no IMP subunit homolog is involved (24-27), and those where soluble cytosolic receptors are not required at all such as nuclear import conferred by the heterogenous nuclear ribonucleoprotein K KNS shuttle sequence (50) and the human immunodeficiency virus type 1 Tat NLS (46). It has also recently been shown that, in the absence of IMP, IMP can mediate NLS recognition and nuclear import of U small nuclear ribonucleoproteins and proteins such as human immunodeficiency virus type 1 Rev (51, 52), TCPTP (22), and parathyroid hormone-related protein (24), implying that IMP itself can function independently as a nuclear import receptor. The present study's findings that IMP from plant is able both to bind NLSs with high affinity and to mediate nuclear import independent of IMP represent evidence for a further variant of a signal-dependent nuclear import pathway. The results show that At-IMP can fulfill the NLS binding and NPC docking roles of the IMP/ heterodimer and thereby may function in analogous fashion to IMP/IMP-related homologs in the pathways mentioned above. This study thus implies that nuclear import pathways mediated exclusively by IMP subunits may exist in plants and possibly other eukaryotes (see Ref. 53). Rice IMP has recently been shown to be able to interact with rice IMP (54) to mediate nuclear import (55), so that it seems likely that the novel IMP-mediated pathway described here may exist in addition to conventional pathways in which IMP acts in conjunction with IMP.

We and others have shown that the NLS binding affinity of IMP from yeast or mouse increases when it is bound to IMP (21, 34-36). Furthermore, nuclear import of NLS-containing proteins recognized by IMP is dependent on IMP for the NPC docking and translocation steps in these systems (Refs. 8, 11, 18, and 36; see Ref. 56). In this study, we found that in the absence of IMP, At-IMP binds NLSs with high affinity similar to that possessed by yeast and mammalian IMP/ heterodimers. Further, in the absence of exogenously added IMP, At-IMP is able to mediate the nuclear import of proteins containing two different types of NLS; although as elsewhere (54, 55, 57) the NLSs used in the transport studies here are not plant-derived, the possibility that this is the basis of At-IMP's apparent novel properties seems unlikely, since plant NLSs resemble those from other eukaryotes in all respects, e.g. in terms of NLS-type (T-ag-NLS-like, bipartite, and Mat2-like) (29, 30). That residual IMP in our nuclear import assay system is not the basis of the observed results is demonstrated by the lack of inhibition of At-IMP-mediated nuclear import by anti-IMP antibodies (Fig. 5); rather, IMP appears to inhibit At-IMP-mediated nuclear import (Figs. 4 and 5). Based on the results here and elsewhere, At-IMP, in contrast to mouse and yeast IMP subunits (21, 35, 36), thus appears to possess a number of IMP properties/characteristics: 1) At-IMP localizes at the nuclear envelope/NPC in tobacco protoplasts (30) in analogous fashion to IMP subunits in mammalian and yeast systems (e.g. Ref. 8; see, however, Refs. 53 and 58); 2) like mouse and yeast IMP subunits (23), At-IMP recognizes the O2 and GAL4 NLSs; and 3) At-IMP can mediate targeting to the nuclear envelope of NLS-containing transport substrates, as well as their nuclear accumulation in the presence of Ran and NTF2, independent of IMP. These properties appear to be sufficient to enable At-IMP to bind to a variety of NLSs with high affinity and mediate nuclear import in the absence of IMP. In this sense, At-IMP represents a composite of conventional IMP and  subunit activities, although as stated above, this is not meant to imply that it does not have a role in conventional IMP/-mediated transport (55). The specific mechanism by which IMP may mediate IMP-independent targeting to the NPC and subsequent nuclear import is unclear at this stage but may relate to the recent intriguing demonstration that At-IMP associates with the cytoskeleton (33).

The structure and function of the NPC is conserved in all eukaryotes (see Refs. 56, 58, and 59), and the signals determining passage through it are functional in a variety of cell types; e.g. the T-ag NLS functions in yeast and plant cells (60, 61; see Ref. 29), the NLSs of the yeast TF SWI5 (62), and Drosophila morphogen Dorsal (63) can mediate nuclear import in mammalian cells, and the plant-cell functioning NLS of Agrobacterium tumefaciens VirD2 protein is active in yeast cells (64). That At-IMP can bind IMP from both mouse and yeast as shown here implies functional conservation across eukaryotes in terms of the proteins mediating nuclear transport. This is reflected in the relatively high sequence conservation (69 and 77% similarity, respectively) within the IMP binding domains of At-IMP and the mouse and yeast IMP subunits (30). Other regions of IMP, such as the region C-terminal to the IMP binding domain including the first armadillo repeat (At-IMP amino acids 50-118), do not exhibit high homology, showing only 32 and 25% similarity with the mouse and yeast IMP subunits, respectively. A priority of future work will be to determine whether the differences in this domain of At-IMP, and in particular the absence of a series of negatively charged amino acid residues present in the IMP-subunits from other species (see Ref. 30), may constitute the basis of At-IMP's ability to target nuclear import substrates to the NPC in the absence of IMP.

The fact that At-IMP can reconstitute nuclear import in rat nuclei in combination with human Ran and NTF2, as shown here, indicates that transport factors from different species are able to function together to mediate signal-dependent nuclear accumulation. Consistent with this, plant cell cytoplasmic extract is able to support NLS-dependent nuclear import in mammalian cells (65), At-IMP (AtKap) is able to rescue a yeast Srp1 (IMP) mutant defective for transport (66), and importins from rice have recently been shown to be functional in nuclear transport by digitonin-permeabilized HeLa cells (55, 57). The study here can thus be seen as further demonstration of the conservation of the eukaryotic cell nuclear transport apparatus, as well as providing further evidence for its multifaceted nature.

	ACKNOWLEDGEMENTS

We are indebted to Glenn Hicks for critical discussion, to Mary Dasso for providing the Ran-GST-expressing plasmid construct, to Michael Rexach for making the y-IMP/y-IMP expression constructs available, to Steve Adam for providing the anti-IMP  antibody, and to Lyndall Briggs for skilled technical assistance.

	FOOTNOTES

^* This work was supported by Department of Energy Grant DE-FGO2-91ER-20021 (to N. R.).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.

Present address: Julius Maximilians Universität Würzburg, Institut für Anatomie, 97070 Würzburg, Germany.

^** To whom correspondence may be addressed: Nuclear Signalling Laboratory, Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia. Tel.: 00612-62494188; Fax: 00612-62490415; E-mail: David.Jans@anu.edu.au.

To whom correspondence may be addressed: Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824-1312. Tel.: 517-353-2270; Fax: 517-353-9168; E-mail: nraikhel@pilot.msu.edu.

	ABBREVIATIONS

The abbreviations used are: NPC, nuclear pore complex; NLS, nuclear localization sequence; IMP, importin; At-IMP, A. thaliana IMP; m-IMP, mouse IMP; y-IMP, yeast IMP; GST, glutathione S-transferase; T-ag, SV40 large tumor-antigen; ELISA, enzyme-linked immunosorbent assay; TF, transcription factor; O2, Opaque-2.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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				B. L. Conway-Campbell, J. W. Wooh, A. J. Brooks, D. Gordon, R. J. Brown, A. M. Lichanska, H. S. Chin, C. L. Barton, G. M. Boyle, P. G. Parsons, et al. Nuclear targeting of the growth hormone receptor results in dysregulation of cell proliferation and tumorigenesis PNAS, August 14, 2007; 104(33): 13331 - 13336. [Abstract] [Full Text] [PDF]

				I. Meier Composition of the plant nuclear envelope: theme and variations J. Exp. Bot., January 1, 2007; 58(1): 27 - 34. [Abstract] [Full Text] [PDF]

				G. Kass, G. Arad, J. Rosenbluh, Y. Gafni, A. Graessmann, M. R. Rojas, R. L. Gilbertson, and A. Loyter Permeabilized mammalian cells as an experimental system for nuclear import of geminiviral karyophilic proteins and of synthetic peptides derived from their nuclear localization signal regions J. Gen. Virol., September 1, 2006; 87(9): 2709 - 2720. [Abstract] [Full Text] [PDF]

				M. M. Bhatti and W. J. Sullivan Jr. Histone Acetylase GCN5 Enters the Nucleus via Importin-{alpha} in Protozoan Parasite Toxoplasma gondii J. Biol. Chem., February 18, 2005; 280(7): 5902 - 5908. [Abstract] [Full Text] [PDF]

				M. R. M. Fontes, T. Teh, D. Jans, R. I. Brinkworth, and B. Kobe Structural Basis for the Specificity of Bipartite Nuclear Localization Sequence Binding by Importin-{alpha} J. Biol. Chem., July 18, 2003; 278(30): 27981 - 27987. [Abstract] [Full Text] [PDF]

				K. M. Bollman, M. J. Aukerman, M.-Y. Park, C. Hunter, T. Z. Berardini, and R. S. Poethig HASTY, the Arabidopsis ortholog of exportin 5/MSN5, regulates phase change and morphogenesis Development, April 15, 2003; 130(8): 1493 - 1504. [Abstract] [Full Text] [PDF]

				J. K. Forwood, V. Harley, and D. A. Jans The C-terminal Nuclear Localization Signal of the Sex-determining Region Y (SRY) High Mobility Group Domain Mediates Nuclear Import through Importin beta 1 J. Biol. Chem., November 30, 2001; 276(49): 46575 - 46582. [Abstract] [Full Text] [PDF]

				L. J. Schedlich, S. L. Le Page, S. M. Firth, L. J. Briggs, D. A. Jans, and R. C. Baxter Nuclear Import of Insulin-like Growth Factor-binding Protein-3 and -5 Is Mediated by the Importin beta Subunit J. Biol. Chem., July 28, 2000; 275(31): 23462 - 23470. [Abstract] [Full Text] [PDF]

				B. Catimel, T. Teh, M. R. M. Fontes, I. G. Jennings, D. A. Jans, G. J. Howlett, E. C. Nice, and B. Kobe Biophysical Characterization of Interactions Involving Importin-alpha during Nuclear Import J. Biol. Chem., August 31, 2001; 276(36): 34189 - 34198. [Abstract] [Full Text] [PDF]

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