The Auto-inhibitory Function of Importin alpha  Is Essential in Vivo*

Michelle T. HarremanDagger §, Mary R. Hodel§, Patrizia Fanara§, Alec E. Hodel§, and Anita H. Corbett§

From the § Department of Biochemistry, School of Medicine and the Dagger  Graduate Program in Biochemistry, Cell and Developmental Biology, Emory University, Atlanta, Georgia 30322

Received for publication, October 25, 2002, and in revised form, December 10, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Proteins that contain a classical nuclear localization signal (NLS) are recognized in the cytoplasm by a heterodimeric import receptor composed of importin/karyopherin alpha  and beta . The importin alpha  subunit recognizes classical NLS sequences, and the importin beta  subunit directs the complex to the nuclear pore. Recent work shows that the N-terminal importin beta  binding (IBB) domain of importin alpha  regulates NLS-cargo binding in the absence of importin beta  in vitro. To analyze the in vivo functions of the IBB domain, we created a series of mutants in the Saccharomyces cerevisiae importin alpha  protein. These mutants dissect the two functions of the N-terminal IBB domain, importin beta  binding and auto-inhibition. One of these importin alpha  mutations, A3, decreases auto-inhibitory function without impacting binding to importin beta  or the importin alpha  export receptor, Cse1p. We used this mutant to show that the auto-inhibitory function is essential in vivo and to provide evidence that this auto-inhibitory-defective importin alpha  remains bound to NLS-cargo within the nucleus. We propose a model where the auto-inhibitory activity of importin alpha  is required for NLS-cargo release and the subsequent Cse1p-dependent recycling of importin alpha  to the cytoplasm.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In eukaryotes, the nuclear envelope provides an essential barrier that separates the nuclear genome from the intermediary metabolism, signaling systems, and translation machinery of the cytoplasm. Selective bi-directional transport of macromolecules across this nuclear envelope regulates critical cellular processes such as gene expression (1, 2). All nucleocytoplasmic transport of macromolecules occurs through large proteinaceous structures, called nuclear pore complexes (NPC),1 that perforate the nuclear envelope (3, 4). These macromolecular cargoes are specifically targeted to and transported through NPCs by a family of soluble nuclear transport receptors (5, 6).

The small GTPase, Ran, governs the interactions between the nuclear transport receptors and macromolecular cargoes (5, 7). Import receptors bind cargo in the absence of RanGTP, whereas export receptors bind cargo in a trimeric complex with RanGTP (5, 8). This mode of regulation requires an asymmetric distribution of RanGTP, with more RanGTP in the nucleus than in the cytoplasm. To achieve this asymmetry, the Ran regulatory proteins are compartmentalized with the GTPase activating protein (RanGAP), which generates RanGDP, in the cytoplasm (9) and the guanine nucleotide exchange factor (RCC1), which generates RanGTP, in the nucleus (10).

The best-characterized nuclear import process occurs via receptor recognition of a classical nuclear localization signal (NLS). This classical NLS is typified by a cluster of basic amino acids (monopartite) or two clusters of basic amino acids separated by a 10-12 amino acid linker (bipartite) (11, 12). A heterodimeric import receptor, composed of importins alpha  and beta  (also known as karyopherin alpha  and beta ), mediates the nuclear import of proteins that contain a classical NLS (13-15). Over the last several years many studies have led to a detailed model for the individual steps in the classic nuclear transport cycle (5, 16): 1) importin alpha  binds to the NLS-cargo to form a trimeric import complex with importin beta ; 2) this NLS-cargo/importin alpha /importin beta  complex is targeted to the NPC by importin beta ; 3) the complex then translocates into the nucleus where it encounters RanGTP; 4) upon binding RanGTP, importin beta  dissociates from NLS-cargo/importin alpha ; 5) NLS-cargo is released from importin alpha ; and 6) once cargo is released, importin alpha  is recycled to the cytoplasm by its export receptor, Cse1p/CAS, in a trimeric complex with RanGTP. Thus, the directionality and efficiency of nuclear import of NLS-cargo is accomplished not only by the Ran GTPase cycle but also by an additional series of protein-protein interactions, occurring in defined locations, that result in changes in affinities of the transport receptor for NLS-cargo.

Dissociation of the NLS-cargo/importin alpha /importin beta  import complex is critical for delivery of NLS-cargo into the nucleus (7). RanGTP-mediated dissociation of importin beta  from the trimeric import complex (17) results in an NLS-cargo/importin alpha  complex. Precisely how NLS-cargo is then released from importin alpha  is unknown; however, a recent study shows that Cse1p and the nucleoporin, Nup2p, can facilitate cargo release in vitro (18). The release of cargo is important in the subsequent functions of both the cargo (7) and importin alpha  (19-21). For example, a recent study identified a critical cargo, TPX2, of the importin alpha /beta complex whose release from importin alpha  is essential for mitotic progression (22). Although this study did not determine the mechanism of cargo release, it highlighted the importance of understanding how cargoes are efficiently dissociated from importin alpha  within the nucleus to mediate their cellular function. Furthermore, Cse1p, the export receptor for importin alpha , can only interact with importin alpha  that is not bound to NLS-cargo (19-21). Thus, for importin alpha  to be recycled to the cytoplasm, it must be dissociated from NLS-cargo within the nucleus. This ensures that importin alpha  is recycled to the cytoplasm only when it has released NLS-cargo; and thereby provides a mechanism for assuring uni-directional transport of NLS-cargo into the nucleus.

The convergence of data from structural analyses and in vitro binding studies provides insight into how importin alpha  binds to and regulates interactions with NLS-cargo (17, 23-26). Domain analysis has shown that importin alpha  has an N-terminal domain that binds importin beta  (the importin beta binding domain or IBB), a central armadillo domain that constitutes the NLS binding pocket, and a C-terminal region that appears to be important for binding to its export receptor Cse1p/CAS (21, 27). Structural studies have extended the knowledge obtained from this domain analysis. Conti et al. (25) solved the structure of truncated Saccharomyces cerevisiae importin alpha  (amino acid residues 89-530) in the presence of an NLS peptide revealing details of how the central armadillo domain of importin alpha  creates specific binding pockets for NLS-cargo. More recently, the structure of full-length mouse importin alpha , solved in the absence of NLS-cargo, showed that the N-terminal IBB domain of importin alpha  can form an intramolecular interaction with the NLS-binding pocket of importin alpha  (23). This observation suggested that the IBB, in addition to mediating binding to importin beta  (28), could have a second role as an auto-inhibitory domain to regulate cargo binding (23). In support of this hypothesis, in vitro binding studies have shown that importin alpha  lacking the proposed auto-inhibitory domain (Delta IBB-alpha ), binds more tightly to NLS-cargo than full-length importin alpha  (26), and that this auto-inhibition of full-length importin alpha  binding to NLS-cargo is relieved in the presence of importin beta  (17, 26). Further analysis of the N-terminal IBB domain of importin alpha  revealed a proposed internal NLS that could serve as an auto-inhibitory sequence to regulate NLS binding through intramolecular competition for the NLS binding site (23, 24). Taken together, these studies suggest a dual role for the N-terminal domain of importin alpha  in 1) binding to importin beta  and 2) auto-inhibition of NLS-cargo binding.

To analyze the in vivo requirement for of the auto-inhibitory activity of importin alpha , we created a series of importin alpha  mutants. These mutants dissect the two functions of the N-terminal domain, importin beta  binding and auto-inhibition. One of these mutants specifically decreases auto-inhibitory function without impacting RanGTP-regulated binding to importin beta . Despite normal binding to both importin beta  and Cse1p, this importin alpha  mutant is unable to function in vivo. These experiments demonstrate that the auto-inhibitory function of importin alpha  is essential in vivo. Our data support the hypothesis that the auto-inhibitory mutant is deficient in NLS-cargo release from importin alpha  and the subsequent recycling of importin alpha  to the cytoplasm. Thus, we propose that the auto-inhibitory function of importin alpha  is necessary for efficient release of NLS-cargo into the nucleus via intramolecular competition of the N-terminal IBB domain at the NLS-binding site.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Strains, Plasmids, and Chemicals-- All chemicals were obtained from Sigma or USBiological unless otherwise noted. All DNA manipulations were performed according to standard methods (29), and all media was prepared by standard procedures (30). All yeast strains and plasmids used in this study are described in Table I. A complete deletion of the SRP1 open reading frame was created using a standard PCR-based strategy (31) in the wild-type diploid ACY247 to create the haploid Delta SRP1 strain (ACY324) maintained by an SRP1 URA3 plasmid (pAC876). Importin alpha -GFP and A3-importin alpha -GFP were integrated at the endogenous importin alpha  locus of wild-type (ACY192) and cse1-1 (ACY671) cells using a standard integration strategy. This integration strategy creates a duplication at the endogenous importin alpha  locus such that both endogenous importin alpha  and importin alpha -GFP are expressed from an SRP1 promoter.

                              
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Table I
Strains and plasmids used in this study

Generation of Importin alpha  Mutants-- Amino acid substitutions were introduced in the S. cerevisiae importin alpha  (SRP1) coding region using PCR. For most mutants, mutagenesis was carried out on importin alpha  in the bacterial expression vector pProEX-HTb (PerkinElmer Life Sciences). Mutations were subcloned from the bacterial expression vector into the yeast expression plasmid for in vivo studies. An 88-amino acid N-terminal deletion of importin alpha , Delta IBB-alpha (pAC959), was created using the following PCR-based strategy. An importin alpha  plasmid (pAC876) was used as a template to amplify the importin alpha  promoter and Delta IBB-alpha open reading frame starting at a PCR-introduced ATG start codon preceding leucine residue 89. These products were further amplified by PCR to form Delta IBB-alpha (amino acid residues 89-542) expressed from the endogenous importin alpha  promoter. The resulting PCR product was cloned into the yeast centromeric (CEN) plasmid pRS315 (32). For all constructs generated, the presence of each desired mutation and the absence of any other mutations were confirmed by DNA sequencing.

In Vivo Functional Analysis-- The in vivo function of each of the importin alpha  mutants was tested using a plasmid shuffle technique (33). Plasmids encoding each of the importin alpha  mutant proteins were individually transformed into SRP1 deletion cells (ACY324) containing the URA3 SRP1 maintenance plasmid, pAC876. Single transformants were grown in liquid culture to saturation, serially diluted (1:10), and spotted on minimal media plates lacking leucine as a control or on fluoro-orotic acid (5-FOA) plates. The drug 5-FOA eliminates the URA3 plasmid-encoded wild-type importin alpha  (pAC876) (33). Plates were incubated at 30 °C for 3 days.

Immunoblot Analysis-- Immunoblot analysis was performed essentially as described previously (34). Briefly, cultures were grown to log phase in appropriate media at 30 °C. Cells were harvested by centrifugation and washed twice in water and once in PBSMT (100 mM KH2PO4, pH 7.0, 15 mM (NH4)2SO4, 75 mM KOH, 5 mM MgCl2, 0.5% Triton X-100). Cells were subsequently lysed in PBSMT with protease inhibitors (0.5 mM phenylmethylsulfonyl fluoride, 3 µg/ml each of aprotinin, leupeptin, chymostatin, and pepstatin) by glass bead lysis. Equal amounts of total protein (generally 10 µg) were resolved by SDS-PAGE and immunoblotted with monoclonal anti-myc antibody (1:2000 dilution; Oncogene), polyclonal anti-GFP antibody (1:10,000 dilution) (35), anti-GST (1:5000; Santa Cruz Biotechnology), anti-His (1:5000; Santa Cruz Biotechnology), polyclonal anti-Cse1p (1:5000) (36), or polyclonal anti-importin beta  antibody (1:5000 dilution) (37).

Expression and Purification of Recombinant Proteins-- Assays were performed with purified recombinant S. cerevisiae proteins Srp1p (importin alpha ), Kap95p (importin beta ), Cse1p, and Gsp1p (Ran). Full-length His6-importin alpha  (residues 1-542), His6-Delta IBB-importin alpha  (residues 89-530), His6-IBB-GFP (importin alpha  residues 1-88), His6-SV40 (SPKKKRKVEAS)-NLS-GFP, His6-Cse1p, and His6-Ran were expressed in the Escherichia coli strain BL21(DE3) and purified by nickel affinity chromatography essentially as described previously (26, 38, 39). Importin beta  was expressed and purified as described previously (26). GST-importin alpha  and GST-A3-importin alpha  were expressed as described elsewhere (40).

Fluorescence Depolarization Assay-- Fluorescence anisotropy measurements were carried out using an ISS PC1 fluorometer fitted with polarization filters. The dissociation constants for the binding of SV40 NLS-GFP and IBB-GFP to importin alpha  were measured essentially as described previously (26, 38). Briefly, SV40 NLS-GFP or IBB-GFP was diluted in PBS to the desired concentration (~20 nM) in a total volume of 1 ml in a 1-cm quartz cuvette. Changes in the anisotropy of the GFP fluorophore were monitored as aliquots of the full-length wild-type or mutant importin alpha  proteins were successively added to the assay volume. Changes in anisotropy were used to calculate the fraction of the GFP fluorophore bound, yielding a binding isotherm for the reaction. The binding isotherm was then fit through nonlinear regression to a simple binding equation to obtain dissociation constants. All Kd values were calculated as detailed at http://www.biochem.emory.edu/Hodel/Research/BindingCurves/fitting_curves.htm. The dissociation constant for importin alpha  binding to importin beta  was measured using a competition assay (41) with IBB-GFP. The assay was carried out as described above except changes in the anisotropy of IBB-GFP were monitored in the presence of increasing amounts of importin beta . This yielded a dissociation constant for IBB-GFP binding to importin beta . To measure the binding of importin alpha  to importin beta , the binding of IBB-GFP was examined in the presence of three different concentrations of full-length wild-type or mutant importin alpha . The Kd values for wild-type and mutant importin alpha  proteins binding to importin beta  were determined by fitting the resulting binding curves to an equation for the fraction of IBB-GFP bound as a function of Kd for IBB, Ki for the full-length importin alpha  protein, the total IBB-GFP concentration, the total importin beta  concentration, and the total concentration of the full-length importin alpha  protein. Binding energies were calculated using Delta G = RT ln Kd, where RT = -0.59 kcal/mol.

Solid Phase Binding Assay-- As previously described (26), importin beta  was covalently coupled to epoxy-activated Sepharose beads (Amersham Biosciences) at a concentration of 1 mg/ml. Approximately 0.2 mg/ml purified full-length wild-type or full-length mutant importin alpha  or Delta IBB-alpha was incubated with 0.5 ml of importin beta -coupled beads in binding buffer (50 mM Tris-HCl, pH 7.8, 100 mM NaCl, 20 mM dithiothreitol) for 3 h at 4 °C. The beads were then washed five times with 1 ml of binding buffer, and beads were either eluted with elution buffer (50 mM Tris-HCl, pH 7.8, 1 M NaCl) or incubated with a RanGTP-RS as described previously (39). Briefly, recombinant Ran was incubated with a RanGTP-RS (20 units of acetate kinase, 10 mM GTP, and 10 mM sodium acetyl phosphate) for 15 min on ice before addition to the beads at 4 °C for 2 h. The supernatants from each reaction were subsequently saved as the eluant fractions. The beads were washed twice with binding buffer before elution with elution buffer. The unbound (2% of total unbound), bound (2% of total bound), and eluant (2% of total eluant) fractions were resolved on a 10% SDS-PAGE gel and visualized by Coomassie Brilliant Blue staining (42).

GST-importin alpha  proteins were bound to glutathione-Sepharose (Amersham Biosciences) and tested for interaction with Cse1p essentially as described previously (40), with the following modifications. The GST-importin alpha -conjugated beads were washed five times with PBSMT before incubation with purified Cse1p (10 µg) and Gsp1p (20 µg) in the presence or absence of the RanGTP-RS in a total volume of 500 µl. The bound (50% of total bound) fractions were resolved on a 8% SDS-PAGE gel and immunoblotted as described. For quantification, immunoblots were analyzed using a fluorescence imager and Quantity One Software (Bio-Rad).

Microscopy-- Indirect immunofluorescence microscopy was performed as described elsewhere (37) with the following conditions. The myc antibody was used at 1:100 dilution, and the GFP antibody was used at 1:2000 for incubation with cells overnight at 4 °C. The Texas Red-labeled anti-mouse secondary antibody (Jackson ImmunoResearch, 1:1000 dilution) was incubated with cells for 2 h at room temperature. DNA was stained with DAPI (1 µg/ml). Samples were viewed through a Texas Red-optimized filter (Chroma Technology) using an Olympus BX60 epifluorescence microscope equipped with a Photometrics Quantix digital camera.

Direct fluorescence microscopy was used to localize GFP fusion proteins in live cells. For all experiments, cells were stained with DAPI to visualize the DNA and confirm the location of the nucleus. The localization of the fusion proteins was monitored by directly viewing the GFP signal in living cells through a GFP-optimized filter as described for indirect immunofluorescence microscopy.

Immunoprecipitation of Importin alpha -myc-- Agarose-conjugated anti-myc antibody beads (9E-10 Santa Cruz Biotechnology) were used to immunoprecipitate importin alpha -myc proteins. Wild-type cells (ACY192) expressing importin alpha -myc (pAC891) or A3-importin alpha -myc (pAC894) from the endogenous SRP1 promoter were transformed with galactose-inducible plasmids encoding GFP (pAC1042) alone or a bipartite NLS (KRTADGSEFESPKKKRKVE)-GFP (pAC1045) (38). Cells were grown to early-log phase in synthetic media containing 2% raffinose, induced with 2% galactose, and incubated at 30 °C for 3 h. Lysates were prepared as described for immunoblot analysis. Six milligrams of total protein lysate was incubated with myc antibody-coupled agarose beads (10 µl of volume of packed beads) for 5 h at 4 °C in the absence or presence of a RanGTP-RS. Beads were washed twice in PBSMT and once in PBSM before elution with 30 µl of sample buffer (125 mM Tris-HCl, pH 6.8, 250 mM dithiothreitol, 5% SDS, 0.25% bromphenol blue, 25% glycerol). The bound (50% of total bound) fractions were resolved on a 10% SDS-PAGE gel and immunoblotted as described. For quantification, immunoblots were analyzed using a fluorescence imager and Quantity One Software (Bio-Rad).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Generation of Mutant Importin alpha  Proteins-- Previous structural and in vitro analyses identified an auto-inhibitory sequence within the N-terminal IBB domain of importin alpha  (23, 24, 26). The structural studies suggested that this auto-inhibitory domain interacts with the NLS binding pocket by mimicking an NLS sequence (23, 24). Alignment of the N-terminal domains of importin alpha  proteins from yeast, mouse, and human reveals three clusters of conserved basic amino acids that could serve as auto-inhibitory NLS sequences (Fig. 1). To identify the amino acid residues that are required for the auto-inhibitory function of importin alpha , we carried out an alanine scan by individually substituting each of these basic clusters in yeast importin alpha  (Srp1p) to generate importin alpha  mutants referred to as A1 (33RRRR36 right-arrow AAAA), A2 (44RKAKR48 right-arrow AAAAA), and A3 (54KRR56 right-arrow AAA) (Fig. 1). The full-length mouse importin alpha  crystal structure showed that residues 44-54 (corresponding to yeast importin alpha  residues 49-59) interact with the larger NLS binding pocket (24). Although this suggests that our A3 mutant should target this intramolecular interaction, we completed a broader, unbiased alanine scan and analyzed the function of each of the mutant importin alpha  proteins, A1, A2, and A3.


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Fig. 1.   Alignment of the N-terminal domains of importin alpha  proteins. Amino acid sequence alignment of yeast (sc) (residues 30-60), mouse (mm), and human (hs) importin alpha  proteins is shown. The conserved clusters of basic residues that were mutated to alanine to generate the A1, A2, and A3 mutants in S. cerevisiae importin alpha  are indicated by the boxes.

In Vivo Function of Importin alpha  Mutants-- To test the function of the mutant importin alpha  proteins in vivo, each mutant was transformed into yeast cells deleted for the endogenous importin alpha  gene (SRP1) and plasmid shuffle (see "Experimental Procedures") was used to replace the functional wild-type copy of SRP1 (Fig. 2A). This results in Delta SRP1 cells that express each of the mutant importin alpha  proteins from their own promoter on a low copy centromeric plasmid as the only copy of importin alpha . Controls demonstrate that a wild-type importin alpha  plasmid can rescue Delta SRP1 cells, whereas neither a vector alone nor the N-terminally truncated importin alpha  (Delta IBB-alpha ) can functionally replace SRP1. Results shown in Fig. 2A indicate that none of the importin alpha  mutant proteins, A1, A2, or A3, can function in vivo.


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Fig. 2.   Functional analysis of the importin alpha  mutants in vivo. A, the SRP1 deletion strain (ACY324) maintained by a plasmid encoding wild-type importin alpha  and expressing either wild-type or mutant importin alpha  proteins was spotted onto minimal media (control) or 5-FOA plates as described under "Experimental Procedures." All plates were incubated at 30 °C for 3 days. B, levels of importin alpha  expressed in wild-type cells (ACY192) were examined by immunoblotting with an anti-myc antibody. Ten micrograms of total yeast protein was loaded in each lane. The positions of full-length importin alpha  and Delta IBB-alpha are indicated by the arrows.

To confirm that each mutant protein is expressed at a similar level to wild-type importin alpha , we analyzed their expression using a C-terminal triple myc tag. Wild-type importin alpha -myc can functionally replace endogenous importin alpha  (data not shown). Immunoblotting of the myc-tagged importin alpha  proteins demonstrates that each of the mutant proteins is expressed at approximately the same level as wild-type importin alpha  (Fig. 2B, compare lanes 3-6 with lane 2). This suggests that none of the mutations in the N-terminal IBB domain of importin alpha  significantly affect the level of the protein within the cell, but rather that these mutations perturb the function of the importin alpha  proteins. Importin alpha  undergoes a series of defined protein-protein interactions during a nuclear transport cycle (16). Thus, to determine the basis for the loss of function of each of the importin alpha  mutants, we investigated the interaction of the mutant importin alpha  proteins with both NLS-cargo and nuclear transport factors essential for importin alpha  function.

Identification of an Importin alpha  Mutant with Decreased Auto-inhibitory Function-- To examine both NLS binding and auto-inhibitory function for each importin alpha  protein, we used a quantitative, fluorescence anisotropy, solution binding assay (26, 38). Full-length wild-type importin alpha  binds weakly to a classical SV40-NLS due to the N-terminal auto-inhibitory IBB domain. However, when the same experiment was carried out in the presence of a stoichiometric amount of importin beta , the N-terminal auto-inhibition of full-length importin alpha  was relieved and much tighter binding to the SV40-NLS was observed. Thus, this assay actually examines three aspects of importin alpha  function: binding to NLS-cargo (directly), auto-inhibitory function (directly, measured by the ability of full-length importin alpha  to bind NLS-cargo in the absence of importin beta ), and binding to importin beta  (indirectly, based on the relief of auto-inhibition).

To examine NLS binding and the auto-inhibitory function for each importin alpha  variant, we performed the fluorescence anisotropy assay using a monopartite SV40-NLS-cargo (26, 38). The assay was carried out with each importin alpha  protein in the presence and absence of importin beta  (Fig. 3). Typical curves for binding of SV40-NLS-GFP to wild-type importin alpha  in the absence (open circle ) and presence (black-triangle) of importin beta  are shown in Fig. 3A. As described under "Experimental Procedures," these curves were used to calculate Kd values for the interaction between the NLS-cargo and importin alpha  (Table II). The change in free energy (Delta G) for the binding of each importin alpha  protein to NLS-cargo in the absence () and presence (black-square) of importin beta  is shown in Fig. 3B. As previously demonstrated (24, 26), wild-type importin alpha  binds to SV40-NLS-GFP weakly (Kd ~ 500 nM), but the affinity increases ~30-fold (Kd ~ 18 nM) in the presence of importin beta . The A1-importin alpha  and A2-importin alpha  mutants show weak binding to SV40-NLS-GFP, comparable to wild-type importin alpha , suggesting that the N-terminal auto-inhibitory function is intact in these mutants. However, the binding to NLS is not enhanced in the presence of importin beta  showing that there is no relief of auto-inhibition. This observation implies that the A1-importin alpha  and A2-importin alpha  mutants have decreased binding to importin beta .


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Fig. 3.   Analysis of the auto-inhibitory function of importin alpha  mutants. A, binding of an NLS-GFP cargo to full-length importin alpha  protein was measured by anisotropy in the absence (open circle ) or presence (black-triangle) of a stoichiometric amount of importin beta . The anisotropy is plotted versus the concentration of importin alpha  on a logarithmic scale. B, binding of an NLS-GFP cargo to each full-length importin alpha  protein indicated was measured by fluorescence anisotropy in the absence () or presence (black-square) of a stoichiometric amount of importin beta . For each importin alpha  protein, the data were fit to an ideal binding curve to yield a value for Kd. The calculated binding energy (Delta G, kcal/mol) is shown on the left axis. Standard deviations are indicated by error bars. C, the N-terminal IBB domain of importin alpha  (WT-IBB (triangle )) and A3-importin alpha  (A3-IBB ()) were fused to GFP, and their binding to Delta IBB-alpha was measured by anisotropy. The anisotropy is plotted versus the concentration of importin alpha  on a linear scale.

                              
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Table II
Binding of importin alpha  proteins to NLS-cargo

In contrast to the A1 and A2 mutants, the A3-importin alpha  mutant binds to SV40-NLS-GFP more tightly (Kd ~ 73 nM) than full-length wild-type importin alpha  (Kd ~ 500 nM), an ~7-fold increase in affinity. This suggests that A3-importin alpha  has decreased auto-inhibitory function. When the assay is performed in the presence of importin beta , the affinity of SV40-NLS-GFP for A3-importin alpha  increases to approximately the same binding affinity (Kd ~ 20 nM) as measured for wild-type importin alpha  (Kd ~ 18 nM), which shows that the residual auto-inhibition can be relieved by importin beta  and suggests that the A3-importin alpha  mutant retains normal binding to importin beta . In addition, these data show that the A3-importin alpha /beta complex is able to bind NLS-cargo with approximately the same affinity as the wild-type importin alpha /beta complex. Similar results were obtained when two different cargoes containing distinct NLS sequences were used (data not shown).

Our analysis of the auto-inhibition of A3-importin alpha  suggests that this mutant IBB domain has a decreased affinity for the NLS binding pocket compared with a wild-type IBB domain. To directly examine this interaction, we analyzed binding of the IBB domain to the NLS binding pocket in trans. We compared the binding of wild-type and A3 IBB-GFP to Delta IBB-alpha using fluorescence anisotropy (Fig. 3C). Wild-type IBB-GFP binds to Delta IBB-alpha with a Kd of ~14 ± 5 µM. Binding of A3 IBB-GFP to Delta IBB-alpha was significantly less and was too weak to accurately measure in this assay.

Interaction of Importin alpha  Proteins with Importin beta -- The interaction of importin alpha  with importin beta  is essential for targeting the import complex to nuclear pores (5). Therefore, to evaluate the in vivo role of the auto-inhibitory function of importin alpha , it is critical to demonstrate that we have separated the two functions of the N-terminal IBB domain of importin alpha  (importin beta  binding and auto-inhibition). Although the auto-inhibitory binding assay suggests that A3-importin alpha  retains wild-type binding to importin beta , it does not measure this binding directly. To quantitatively measure the binding of importin alpha  to importin beta  we utilized two assays. First, we used a competition assay where we examined the ability of each full-length importin alpha  protein to compete with the IBB-GFP for binding to importin beta . Second, we performed a direct binding assay to measure binding of wild-type and A3 IBB-GFP to importin beta .

For the competition binding assay, we first used fluorescence anisotropy to measure the binding of wild-type IBB-GFP to importin beta . This yielded a Kd of ~17 ± 5 nM (Fig. 4). The competition experiment was then carried out in the presence of three concentrations of each importin alpha  protein (wild-type, A1, A2, and A3) in competition with IBB-GFP. This analysis yields equilibrium binding constants for the interaction of wild-type and each of the mutant importin alpha  proteins with importin beta  (Table III). Full-length importin alpha  binds to importin beta  with a Kd of ~0.6 µM. Both A1-importin alpha  and A2-importin alpha  show decreased affinity for importin beta  with decreases of ~15-fold and ~28-fold, respectively. In contrast, as suggested by the NLS binding experiments, A3-importin alpha  binds to importin beta  with approximately the same affinity (Kd ~ 0.4 µM) as wild-type importin alpha  (Kd ~ 0.6 µM). To further confirm that the A3 mutation within the IBB domain does not affect the interaction between importin alpha  and importin beta , we performed the direct binding assay. This assay measures binding of wild-type and A3 IBB-GFP to importin beta  using fluorescence anisotropy. Binding curves for IBB-GFP and A3 IBB-GFP are shown in Fig. 4. A3 IBB-GFP binds to importin beta  with the same affinity (Kd ~ 20 ± 5 nM) as wild-type IBB-GFP (Kd ~ 17 ± 5 nM). These results indicate that the A3 mutant of importin alpha  has compromised auto-inhibitory function but binds to importin beta  with wild-type affinity. Because this mutant dissects the two functions of the IBB domain, we focused our subsequent experiments on the A3 mutant to analyze the in vivo contribution of the auto-inhibitory activity of importin alpha .


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Fig. 4.   Quantitative analysis of the interaction between importin beta  and A3-importin alpha . The N-terminal IBB domains of importin alpha  (WT-IBB (open circle )) and A3-importin alpha  (A3-IBB (black-square)) were fused to GFP, and their binding to importin beta  was measured by anisotropy. Data are plotted as anisotropy versus the concentration of importin beta  on a logarithmic scale.

                              
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Table III
Binding of importin alpha  proteins to importin beta

Dissociation of the Importin alpha /Importin beta  Complex by RanGTP-- To determine whether the A3-importin alpha  can be dissociated from importin beta  by RanGTP, we performed a bead dissociation assay. We covalently attached purified recombinant importin beta  to activated epoxy beads and then incubated the beads with purified importin alpha  to pre-form the importin alpha /beta complex. We have previously shown that full-length importin alpha  specifically binds to the importin beta -coupled beads (26). Both wild-type and A3-importin alpha  can bind to the importin beta  beads (Fig. 5A, lanes 2 and 4). Fig. 5B shows that both wild-type and A3-importin alpha  are released from importin beta  upon addition of an RanGTP-regenerating system (RanGTP-RS) (39). Similar results were obtained when the experiment was carried out using Ran loaded with 100 µM GTPgamma S in place of the RanGTP-RS to modulate the nucleotide bound state of Ran (data not shown). RanGDP, which does not bind with high affinity to importin beta  (43), did not dissociate any of the pre-formed complexes (data not shown).


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Fig. 5.   Dissociation of the importin alpha /importin beta  complex by RanGTP. A, recombinant importin beta  was bound to epoxy beads and incubated with recombinant importin alpha  to pre-form the importin alpha /importin beta  complex as described under "Experimental Procedures." Samples were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. For each protein, U (unbound) (lanes 1 and 3) and B (bound) (lanes 2 and 4) fractions are shown. B, the pre-formed importin alpha /importin beta  complexes were incubated with a RanGTP-RS. The bound (B) (lanes 1 and 3) and eluant (E) (lanes 2 and 4) fractions were analyzed as described for A. The positions of importin alpha , Ran, and importin beta  (which minimally leaches off the beads) are indicated by the arrows.

Localization of A3-importin alpha -- Our in vitro experiments demonstrated that the A3-importin alpha  protein interacts with importin beta  in a RanGTP-dependent manner that is comparable to wild-type importin alpha . One prediction from these experiments is that the A3 protein should be efficiently targeted to the nucleus in vivo through a productive interaction with importin beta . Wild-type S. cerevisiae importin alpha  protein is localized throughout the cell with some accumulation at the nuclear rim and/or within the nucleus depending on the tag (37, 44). This steady-state localization reflects a dynamic state where importin alpha  enters the nucleus and is then exported by Cse1p (19, 20, 44). To analyze the intracellular localization of importin alpha , we first utilized C-terminal triple myc-tagged fusion proteins. These tagged proteins were expressed from the importin alpha  promoter on a centromeric plasmid and localized by indirect immunofluorescence (Fig. 6A). Wild-type importin alpha -myc is localized throughout the cell but accumulates within the nucleus (Fig. 6A, panel A). The A3-importin alpha -myc protein also accumulates within the nucleus (Fig. 6A, panel D). For comparison, the localization of A2-importin alpha , which has compromised binding to importin beta , is also shown (Fig. 6A, panel G). It is clear that, in contrast to wild-type and A3-importin alpha , the A2 protein does not accumulate within the nucleus. Although the localization of wild-type and A3-importin alpha  was very similar, we consistently noticed less cytoplasmic signal for the A3 protein. This raised the possibility that the A3 protein could have a more nuclear steady-state localization than wild-type importin alpha .


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Fig. 6.   Localization of importin alpha  proteins. A, wild-type cells (ACY192) expressing myc-tagged importin alpha  plasmids were grown at 30 °C to log phase. Cells were prepared for indirect immunofluorescence and stained with anti-myc antibody to visualize importin alpha  (panels A, D, and G) and with DAPI to visualize DNA (panels B, E, and H) as described under "Experimental Procedures." Corresponding DIC images are shown (panels C, F, and I). B, integrated importin alpha -GFP or A3-importin alpha -GFP was viewed in wild-type (ACY 192) (panels A and C) or cse1-1 (panels E and G) cells by direct fluorescence. All cultures were grown at 30 °C. Corresponding DIC images are shown (panels B, D, F, and H). C, integrated A3-importin alpha -GFP cells were grown at 30 °C to log phase. Cells were prepared for indirect immunofluorescence and stained with anti-GFP antibody to visualize importin alpha  (panel A) and with DAPI to visualize DNA (panel B). The corresponding DIC image is shown (panel C).

To address this possibility, we re-examined the localization using an assay that can more readily distinguish between importin alpha  at the nuclear rim and importin alpha  within the nuclear interior (44). This assay relies on visualization of an importin alpha -GFP fusion protein that is expressed from the endogenous importin alpha  promoter. We integrated both wild-type and A3-importin alpha -GFP fusion proteins at the endogenous importin alpha  locus as described under "Experimental Procedures." As previously reported (44), wild-type importin alpha  accumulates at the nuclear rim in wild-type cells when visualized in this manner (Fig. 6B, panel A). However, A3-importin alpha -GFP is localized within the nucleus (Fig. 6, B (panel C) and C) suggesting that this mutant protein is not efficiently recycled to the cytoplasm. The localization of A3-importin alpha -GFP is similar to that of importin alpha -GFP in cse1-1 mutant cells where importin alpha  is not efficiently recycled to the cytoplasm (Fig. 6B, panel E) (20, 36). A3-importin alpha -GFP also accumulates within the nucleus of cse1-1 cells (Fig. 6B, panel G).

Co-immunoprecipitation of Importin alpha  and NLS-cargo-- Because A3-importin alpha  has decreased auto-inhibitory activity and a more steady-state nuclear localization than wild-type importin alpha , it seems likely that this mutant protein accumulates within the nucleus bound to NLS-cargo. Thus, A3-importin alpha  should bind to more NLS-cargo than wild-type importin alpha  in cell lysates. To test this prediction, we performed a co-immunoprecipitation with cells expressing either myc-tagged wild-type importin alpha  or myc-tagged A3-importin alpha  together with a bipartite NLS-GFP cargo. The bipartite NLS-GFP protein is localized to the nucleus in vivo (data not shown).

Presumably, at least two major NLS-cargo/importin alpha  complexes exist: 1) the trimeric import complex of NLS-cargo/importin alpha /importin beta ; and 2) NLS-cargo/importin alpha , which may exist transiently after importin beta  is released. Immunoprecipitation of importin alpha  could isolate both complexes; however, addition of RanGTP should dissociate the trimeric complex, because RanGTP causes a conformational change in importin beta  that results in importin beta  dissociation from importin alpha  (17, 45). If A3-importin alpha  is defective in NLS-cargo release from importin alpha , it should bind more NLS-cargo in cell lysates than wild-type importin alpha  under the same conditions.

Agarose-conjugated anti-myc antibody beads were used to immunoprecipitate importin alpha -myc from lysates (see "Experimental Procedures"). The bound fractions were analyzed for co-immunoprecipitation of the bipartite NLS-GFP reporter protein and importin beta . As a control, GFP alone was not co-immunoprecipitated with either of the importin alpha  proteins (data not shown). The experiment was carried out in the presence of a RanGTP-RS to isolate NLS-cargo/importin alpha  complexes in the absence of importin beta . The RanGTP-RS dissociates importin beta  from the complex and decreases the amount of NLS-GFP co-immunoprecipitated (compare lanes 1 and 2). This confirms that in the presence of RanGTP the primary co-immunoprecipitated complex isolated is not the trimeric import complex of NLS-cargo/importin alpha /importin beta  but a complex of NLS-cargo/importin alpha . Thus, we compared the amount of NLS-GFP co-immunoprecipitated with wild-type and A3-importin alpha  under the same conditions. Results of the experiment indicate that, in the presence of RanGTP, ~3-fold more NLS-GFP co-immunoprecipitated with A3-importin alpha -myc than wild-type importin alpha -myc (Fig. 7A, compare lanes 2 and 3). Expression of the importin alpha -myc proteins and the NLS-cargo protein was similar in each lysate (data not shown). Protein bands were compared by fluorescence imaging under conditions where none of the bands were saturated. For each sample the amount of NLS-cargo present was calculated relative to the amount of importin alpha  precipitated in that experiment.


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Fig. 7.   A3-importin alpha  is defective in NLS-cargo release. A, co-immunoprecipitation of importin alpha  and NLS-cargo. Wild-type cells (ACY192) expressing the bipartite NLS-GFP cargo protein (pAC1045) were transformed with wild-type importin alpha -myc (pAC891) or A3-importin alpha -myc (pAC894). Cells were grown to early-log phase, and expression of NLS-GFP was induced with galactose. Binding assays were carried out as described under "Experimental Procedures." Wild-type importin alpha  (lanes 1 and 2) and A3-importin alpha  (lane 3) fractions are shown probed with anti-importin beta  antibody to detect importin beta , anti-myc antibody to detect importin alpha , and anti-GFP antibody to detect the bound NLS-GFP reporter cargo. Lanes are designated "-" or "+" to indicate whether binding was carried out in the absence or presence of a RanGTP-RS. B, in vitro interaction of importin alpha  and Cse1p. Wild-type and A3-importin alpha  were expressed in E. coli as GST fusion proteins. The importin alpha  GST fusion proteins (~30 µg) were bound to glutathione beads and incubated with purified His-tagged Cse1p (10 µg) and His-tagged Gsp1p (20 µg). Lanes are designated "-" or "+" to indicate whether binding was carried out in the absence or presence of a RanGTP-RS. C, in vivo interaction of importin alpha  and Cse1p. Wild-type cells expressing importin alpha -GFP (panels A and C), A3-importin alpha -GFP (panels E and G), or A3-ED-importin alpha -GFP (panels I and K) were transformed with either a 2µ plasmid expressing Cse1p (pAC640) or a vector control (pAC8). Cells were grown to early-log phase, and the GFP signal was viewed directly in living cells. Corresponding DIC images are shown.

Interaction of A3-importin alpha  with Cse1p-- For importin alpha  to be functional in vivo, it must be efficiently recycled to the cytoplasm by its export receptor, Cse1p/CAS. Formation of this export complex requires both the direct interaction between importin alpha  and Cse1p and the dissociation of NLS-cargo (19-21). To examine the physical interaction of A3-importin alpha  with Cse1p, we used a bead binding assay. Both wild-type and A3-importin alpha  were expressed in E. coli as GST fusion proteins as described under "Experimental Procedures." These proteins were bound to glutathione beads and incubated with purified His6-tagged Cse1p in the absence or presence of a RanGTP-RS. As shown in Fig. 7B, A3-importin alpha  binds to Cse1p in a RanGTP-dependent manner. Furthermore, in the presence of RanGTP, Cse1p binding to A3-importin alpha  is comparable to binding to wild-type importin alpha  (Fig. 7B, compare lanes 2 and 3). Protein bands were compared by immunoblotting and fluorescence imaging. Results shown are typical of three independent experiments. In addition, both wild-type importin alpha  and A3-importin alpha  interacted with Cse1p to the same extent in a two-hybrid assay (data not shown).

As a complement to the in vitro binding experiments, we examined importin alpha  recycling by Cse1p in vivo. We took advantage of the C-terminal GFP fusion protein, importin alpha -GFP, expressed from the importin alpha  promoter on a centromeric plasmid. Previous work demonstrates that fusion of GFP to the C terminus of wild-type importin alpha  results in a protein that can function in vivo when expressed as the only copy of importin alpha  (44). However, plasmid expression results in increased amounts of importin alpha -GFP and thus a more nuclear localization than observed for integrated importin alpha -GFP protein (as shown in Fig. 6B). When expressed on a centromeric plasmid, both wild-type importin alpha -GFP and A3-importin alpha -GFP show primarily nuclear localization (Fig. 7C, panels A and E). Overexpression of Cse1p decreases the nuclear localization of wild-type importin alpha -GFP presumably by facilitating export (44). Thus, cells that overexpress Cse1p show a more diffuse (throughout the cell) localization pattern for importin alpha -GFP. As previously reported, we find that wild-type importin alpha -GFP is recycled to the cytoplasm and diffusely localized throughout the cell when Cse1p is overexpressed on a high copy plasmid (Fig. 7C, compare panels A and C). This diffuse localization is observed in ~80% of cells examined. In contrast, A3-importin alpha -GFP remains in the nucleus even when Cse1p is overexpressed (Fig. 7C, compare panels E and G). There are two possible explanations for why A3-importin alpha -GFP is not recycled to the cytoplasm in cells that overexpress Cse1p. First, A3-importin alpha -GFP may be bound to NLS-cargo and therefore unable to efficiently interact with Cse1p. Several lines of experimentation have shown that importin alpha  cannot interact simultaneously with NLS-cargo and Cse1p (19-21), which presumably prevents recycling of importin alpha  with NLS-cargo still bound. Thus, overexpression of Cse1p should not cause redistribution of a mutant importin alpha -GFP protein if it is still bound to NLS-cargo. Second, the A3 mutation in importin alpha  could directly interfere with the physical interaction between importin alpha  and Cse1p. However, our results shown in Fig. 7B demonstrate that A3-importin alpha  is still able to bind to Cse1p. This strongly suggests that the lack of recycling of A3-importin alpha  to the cytoplasm by Cse1p is due to a tighter interaction between A3-importin alpha  and NLS-cargo in the nucleus.

To examine whether the lack of A3-importin alpha  recycling was in fact due to bound NLS-cargo, we utilized a known importin alpha  mutant, ED-importin alpha  (D203K/E402R), that has significantly reduced binding to NLS-cargo (22). The interaction between S. cerevisiae ED-Delta IBB-alpha and SV40-NLS is too weak to detect in the anisotropy assay implying that the binding constant is at least in the millimolar range (data not shown). Because this mutant protein cannot bind to NLS-cargo, it can be combined with the A3 mutation, to create an A3-importin alpha  mutant protein (A3-ED-importin alpha ) that cannot bind NLS-cargo. If this protein can be efficiently recycled to the cytoplasm, it confirms that bound cargo prevents recycling of A3-importin alpha . Alternatively, if the reason that A3-importin alpha  is not recycled to the cytoplasm is independent of binding to NLS-cargo, then regardless of the ED mutation, A3-ED-importin alpha  would not be recycled and would remain in the nucleus.

When the ED mutations are combined with A3-importin alpha  to create A3-ED-importin alpha -GFP, the steady localization of the double-mutant protein is within the nucleus (Fig. 7C, panel I) indistinguishable from the localization of wild-type or A3-importin alpha -GFP. However, in contrast to the result obtained with A3-importin alpha -GFP, overexpression of Cse1p causes relocalization of A3-ED-importin alpha -GFP to the cytoplasm in ~60% of cells (Fig. 7C, panel K). Immunoblotting demonstrates that each of the importin alpha -GFP proteins is expressed at approximately the same level as wild-type importin alpha -GFP and that cells transformed with the CSE1 plasmid all have a similar increase in expression of Cse1p (data not shown). Results of these experiments suggest that A3-importin alpha  accumulates in the nucleus bound to NLS-cargo and thereby support our hypothesis that the auto-inhibitory function of importin alpha  is required for efficient NLS-cargo release in the nucleus. In addition, these results show that A3-importin alpha  can interact with Cse1p in vivo when its ability to bind NLS-cargo is abrogated.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study demonstrates that the N-terminal IBB domain of importin alpha  has two essential functions in vivo. This domain is known to bind importin beta  for targeting of the import complex to the nuclear pore (14, 15, 46). In addition, previous structural studies demonstrated that the IBB domain contained an NLS-like sequence that could compete directly with NLS binding to importin alpha  through an intramolecular interaction (23, 24). Investigations of the in vitro behavior of importin alpha  confirmed that the N-terminal domain inhibited binding of NLS-cargo to importin alpha  (24, 26). This auto-inhibitory function was relieved when importin beta  bound to sequences of the N-terminal domain of importin alpha  just upstream of the NLS-like sequence (47). Here we create mutants of importin alpha  that dissect the two functions of the N-terminal domain, importin beta  binding and auto-inhibition. We demonstrate that mutations within the NLS-like sequence in importin alpha  (mutant A3) specifically decrease the auto-inhibitory function of the N terminus without affecting binding to importin beta  and provide evidence that this auto-inhibitory function is required for efficient NLS-cargo release in the nucleus. Yeast cells expressing this mutant of importin alpha  as the sole copy of importin alpha  are not viable, demonstrating that the NLS-like sequence mediates an essential function of importin alpha  in vivo.

An in vivo analysis of the specific defect of A3-importin alpha  shows that this mutant protein accumulates in the nucleus. This nuclear accumulation suggests that A3-importin alpha  is not efficiently recycled from the nucleus to the cytoplasm by the export receptor, Cse1p. Our in vitro data showing that the A3 mutations do not appear to have a direct impact on the binding of Cse1p to importin alpha  suggest that this defect is due to impaired release of cargo in the nucleus. Previous work has shown that the Cse1p/CAS association with importin alpha  does not depend on the N-terminal domain (40, 48). In fact, the Cse1p/CAS binding site has been mapped to the C terminus of human importin alpha  (21). Our in vivo data support these in vitro analyses, because a variant of the A3 mutant of importin alpha , where the NLS-binding function was destroyed (A3-ED), could be efficiently recycled to the cytoplasm by Cse1p. Taken together, these analyses suggest that the accumulation of A3-importin alpha  in the nucleus is due to the persistence of an NLS-cargo/importin alpha  complex in the nucleus, which inhibits the interaction between importin alpha  and Cse1p. This strongly supports the hypothesis that an essential function of the NLS-like sequence in the N-terminal domain of importin alpha  is the efficient release of NLS-cargo from importin alpha  within the nucleus.

The release of NLS-cargo from importin alpha  is necessary for both the function of NLS-cargoes within the nucleus and for recycling of importin alpha  to the cytoplasm. The accumulation of A3-importin alpha  in the nucleus also demonstrates in vivo the intimate link between cargo release and recycling of importin alpha . Because NLS-cargo release is required for binding to Cse1p/CAS (19-21), it has been proposed that Cse1p/CAS may facilitate release of NLS-cargo (7, 18). Unfortunately, the molecular details of the interaction of importin alpha  with Cse1p/CAS have not yet been elucidated. Furthermore, it is not known how Cse1p/CAS distinguishes between the NLS-cargo bound and free importin alpha  proteins. One possibility is that a conformational change in importin alpha  signals cargo release to Cse1p.

Although our data suggest that auto-inhibition is essential for efficient NLS-cargo release from importin alpha , it does not exclude the possibility that additional factors may facilitate this dissociation. Specifically, a recent study demonstrated that in vitro both Cse1p and the nucleoporin Nup2p facilitate dissociation of NLS-cargo from importin alpha  (18). It is not yet clear how Nup2p may function in cargo release, although the observation that Nup2p interacts with both full-length and Delta IBB-alpha (44, 49) leads us to suspect that this function is either independent of, or complementary to, the auto-inhibitory function characterized in this study. Further work will be necessary to determine how importin alpha , Cse1p, and Nup2p interact as well as how Cse1p and Nup2p contribute to NLS-cargo delivery into the nucleus in vivo.

Although our study suggests that the essential role of importin alpha  auto-inhibition is within the nucleus for release of NLS-cargo from importin alpha , we also present quantitative data that suggests a secondary role in recognition/targeting in the cytoplasm. Indeed, a cytoplasmic role would provide a mechanism by which formation of the import complex is cooperative, leading from free importin alpha  to a trimeric import complex consisting of NLS-cargo, importin alpha , and importin beta  (24). We have determined that full-length importin alpha  binds to importin beta  with low affinity (Kd ~ 0.6 µM) in the absence of any NLS-cargo, presumably because the N-terminal domain forms an intramolecular interaction with the NLS-binding pocket. In contrast, the IBB domain alone binds more tightly to importin beta  (Kd ~ 17 nM). This quantitative analysis of protein-protein interactions shows that, in the cytoplasm, NLS-cargo, importin alpha , and importin beta  have a low affinity for each other (micromolar Kd) unless they are coincident, in which case they form a fairly tight trimeric complex (nanomolar Kd). This cooperative complex formation would presumably prevent nonproductive perpetual Ran-dependent cycling of an importin alpha /importin beta  complex without NLS-cargo. Thus importin alpha  is a dynamic adaptor protein that has evolved to couple NLS-cargo import complex formation in the cytoplasm and NLS-cargo import complex dissociation in the nucleus through regulated protein-protein interactions.

We note that the absolute dissociation constants measured in our studies differ from previously reported values (18, 24), particularly in the binding of importin alpha  to importin beta . The values presented in this study are unique in that they are taken from a measurement of binding at equilibrium in solution. Previously reported values were derived from assays that depended on the separation of bound ligand from unbound (i.e. solid phase pull-down (18) or surface plasmon resonance assays (24)). The dependence of these assays on the kinetics of the binding reaction may play a role in the disparity in the published values. Further experimentation will be needed to determine the origin of the variance in measured binding constants.

Although RanGTP binding to importin beta  is the primary signal that initiates import complex disassembly, our data suggest that the IBB is critical for relaying this signal to the NLS binding pocket on importin alpha . Thus, this study incorporates an NLS-cargo release step into the classic nuclear transport pathway. A schematic is shown in Fig. 8: 1) NLS-cargo/importin alpha /importin beta  cooperatively associate to form the trimeric import complex in the cytoplasm; 2) in the nucleus RanGTP mediates dissociation of importin beta  from the import complex to release an NLS-cargo/importin alpha  complex; 3) the N-terminal domain of importin alpha  competes with NLS-cargo to bind the NLS-binding pocket; and 4) NLS-cargo is released into the nucleus, and the auto-inhibitory N-terminal domain is bound to the NLS-binding site. This renders importin alpha  incapable of binding nuclear NLS-containing proteins prior to export to the cytoplasm by Cse1p. This model for protein import requires precise tuning of the thermodynamic interactions between the various species for the reaction to proceed efficiently in a single direction. In addition, it will be interesting to determine what contribution other release factors, such as Cse1p and Nup2p (18, 50), make to cargo release in vivo.


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Fig. 8.   A schematic depicting the proposed role of the N-terminal auto-inhibitory function of importin alpha  in NLS-cargo delivery into the nucleus.

In summary, we present experimental evidence for the auto-inhibition of importin alpha  in vivo and provide a mechanistic understanding of the essential nature of the auto-inhibitory function in NLS-cargo delivery into the nucleus as well as a potential role in NLS-cargo recognition/targeting in the cytoplasm. We propose that changes in the binding affinities of the nuclear transport components underlie and define the mechanism of regulation of nuclear transport. A complete understanding of nuclear transport requires a quantitative model for the nuclear transport process that correlates structural analyses, in vitro interaction energies, and in vivo functionality. Such analyses to determine the energetics of individual mechanisms in the nuclear transport process will be required to fully understand the rapid, selective, and highly regulated processes of nuclear transport.

    ACKNOWLEDGEMENTS

We thank Dr. Maureen Powers for experimental suggestions and D. M. Green, A. Lange, and Dr. S. W. Leung for their critical review of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM-58728 (to A. H. C.) and by National Science Foundation Grant MCB-9874548 (to A. E. H.).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.

To whom correspondence should be addressed: Dept. of Biochemistry, School of Medicine, Emory University, 1510 Clifton Rd., NE, Atlanta, GA 30322. Tel.: 404-727-4546; Fax: 404-727-3954; E-mail: acorbe2@emory.edu.

Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M210951200

    ABBREVIATIONS

The abbreviations used are: NPC, nuclear pore complex; CEN, centromeric; DIC, differential interference contrast; 5-FOA, 5-fluoro-orotic acid; GFP, green fluorescent protein; GST, glutathione S-transferase; IBB, importin beta  binding; NLS, nuclear localization signal; RanGTP-RS, RanGTP regenerating system; PBS, phosphate-buffered saline; DAPI, 4',6-diamidino-2-phenylindole; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Kaffman, A., and O'Shea, E. K. (1999) Annu. Rev. Cell Dev. Biol. 15, 291-339[CrossRef][Medline] [Order article via Infotrieve]
2. Cyert, M. S. (2001) J. Biol. Chem. 276, 20805-20808[Free Full Text]
3. Wente, S. R. (2000) Science 288, 1374-1377[Abstract/Free Full Text]
4. Rout, M. P., and Aitchison, J. D. (2001) J. Biol. Chem. 276, 16593-16596[Free Full Text]
5. Görlich, D., and Kutay, U. (1999) Annu. Rev. Cell Dev. Biol. 15, 607-660[CrossRef][Medline] [Order article via Infotrieve]
6. Macara, I. G. (2001) Microbiol. Mol. Biol. Rev. 65, 570-594[Abstract/Free Full Text]
7. Kuersten, S., Ohno, M., and Mattaj, I. W. (2001) Trends Cell Biol. 11, 497-503[CrossRef][Medline] [Order article via Infotrieve]
8. Izaurralde, E., Kutay, U., vonKobbe, C., Mattaj, I. W., and Görlich, D. (1997) EMBO J. 16, 6535-6547[Abstract/Free Full Text]
9. Hopper, A. K., Traglia, H. M., and Dunst, R. W. (1990) J. Cell Biol. 111, 309-321[Abstract]
10. Ohtsubo, M., Kai, R., Furuno, N., Sekiguchi, T., Sekiguchi, M., Hayashida, H., Kuma, K., Miyata, T., Fukushige, S., Murotsu, T., et al.. (1987) Genes Dev. 1, 585-593[Abstract]
11. Kalderon, D., Roberts, B. L., Richardson, W. D., and Smith, A. E. (1984) Cell 39, 499-509[Medline] [Order article via Infotrieve]
12. Robbins, J., Dilworth, S. M., Laskey, R. A., and Dingwall, C. (1991) Cell 64, 615-623[Medline] [Order article via Infotrieve]
13. Görlich, D., Kostka, S., Kraft, R., Dingwall, C., Laskey, R. A., Hartmann, E., and Prehn, S. (1995) Curr. Biol. 5, 383-392[Medline] [Order article via Infotrieve]
14. Moroianu, J., Blobel, G., and Radu, A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2008-2011[Abstract]
15. Enenkel, C., Blobel, G., and Rexach, M. (1995) J. Biol. Chem. 270, 16499-16502[Abstract/Free Full Text]
16. Conti, E., and Izaurralde, E. (2001) Curr. Opin. Cell Biol. 13, 310-319[CrossRef][Medline] [Order article via Infotrieve]
17. Rexach, M., and Blobel, G. (1995) Cell 83, 683-692[Medline] [Order article via Infotrieve]
18. Gilchrist, D., Mykytka, B., and Rexach, M. (2002) J. Biol. Chem. 277, 18161-18172[Abstract/Free Full Text]
19. Kutay, U., Bischoff, F. R., Kostka, S., Kraft, R., and Görlich, D. (1997) Cell 90, 1061-1071[Medline] [Order article via Infotrieve]
20. Solsbacher, J., Maurer, P., Bischoff, F. R., and Schlenstedt, G. (1998) Mol. Cell. Biol. 18, 6805-6815[Abstract/Free Full Text]
21. Herold, A., Truant, R., Wiegand, H., and Cullen, B. R. (1998) J. Cell Biol. 143, 309-318[Abstract/Free Full Text]
22. Gruss, O. J., Carazo-Salas, R. E., Schatz, C. A., Guarguaglini, G., Kast, J., Wilm, M., Le, Bot, N., Vernos, I., Karsenti, E., and Mattaj, I. W. (2001) Cell 104, 83-93[Medline] [Order article via Infotrieve]
23. Kobe, B. (1999) Nat. Struct. Biol. 6, 301-304[CrossRef][Medline] [Order article via Infotrieve]
24. Catimel, B., Teh, T., Fontes, M. R., Jennings, I. G., Jans, D. A., Howlett, G. J., Nice, E. C., and Kobe, B. (2001) J. Biol. Chem. 276, 34189-34198[Abstract/Free Full Text]
25. Conti, E., Uy, M., Leighton, L., Blobel, G., and Kuriyan, J. (1998) Cell 94, 193-204[Medline] [Order article via Infotrieve]
26. Fanara, P., Hodel, M. R., Corbett, A. H., and Hodel, A. E. (2000) J. Biol. Chem. 275, 21218-21223[Abstract/Free Full Text]
27. Moroianu, J., Blobel, G., and Radu, A. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6572-6576[Abstract/Free Full Text]
28. Görlich, D., Vogel, F., Mills, A. D., Hartmann, E., and Laskey, R. A. (1995) Nature 377, 246-248[CrossRef][Medline] [Order article via Infotrieve]
29. Sambrook, J., and Russell, D. (2001) Molecular Cloning: A Laboratory Manual , 3rd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
30. Adams, A., Gottschling, D. E., Kaiser, C. A., and Stearns, T. (1997) Methods in Yeast Genetics , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
31. Baudin, A., Ozier-Kalogeropoulou, O., Denouel, A., Lacroute, F., and Cullin, C. (1993) Nucleic Acids. Res. 21, 3329-3330[Medline] [Order article via Infotrieve]
32. Sikorski, R. S., and Hieter, P. (1989) Genetics 122, 19-27[Abstract/Free Full Text]
33. Boeke, J. D., Truehart, J., Natsoulis, G., and Fink, G. (1987) Methods Enzymol. 154, 164-175[Medline] [Order article via Infotrieve]
34. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354[Abstract]
35. Seedorf, M., Damelin, M., Kahana, J., Taura, T., and Silver, P. A. (1999) Mol. Cell. Biol. 19, 1547-1557[Abstract/Free Full Text]
36. Hood, J. K., and Silver, P. A. (1998) J. Biol. Chem. 273, 35142-35146[Abstract/Free Full Text]
37. Koepp, D. M., Wong, D. H., Corbett, A. H., and Silver, P. A. (1996) J. Cell Biol. 133, 1163-1176[Abstract]
38. Hodel, M. R., Corbett, A. H., and Hodel, A. E. (2001) J. Biol. Chem. 276, 1317-1325[Abstract/Free Full Text]
39. Marfatia, K. A., Harreman, M. T., Fanara, P., Vertino, P. M., and Corbett, A. H. (2001) Gene (Amst.) 266, 45-56[CrossRef][Medline] [Order article via Infotrieve]
40. Kunzler, M., and Hurt, E. C. (1998) FEBS Lett. 433, 185-190[CrossRef][Medline] [Order article via Infotrieve]
41. Wang, Z. X. (1995) FEBS Lett. 360, 111-114[CrossRef][Medline] [Order article via Infotrieve]
42. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
43. Villa Braslavsky, C. I., Nowak, C., Görlich, D., Wittinghofer, A., and Kuhlmann, J. (2000) Biochemistry 39, 11629-11639[CrossRef][Medline] [Order article via Infotrieve]
44. Booth, J. W., Belanger, K. D., Sannella, M. I., and Davis, L. I. (1999) J. Biol. Chem. 274, 32360-32367[Abstract/Free Full Text]
45. Chook, Y. M., and Blobel, G. (1999) Nature 399, 230-237[CrossRef][Medline] [Order article via Infotrieve]
46. Görlich, D., Henklein, P., Laskey, R. A., and Hartmann, E. (1996) EMBO J. 15, 1810-1817[Abstract]
47. Cingolani, G., Petosa, C., Weis, K., and Müller, C. W. (1999) Nature 399, 221-229[CrossRef][Medline] [Order article via Infotrieve]
48. Dickmanns, A., Bischoff, F. R., Marshallsay, C., Lührmann, R., Ponstingl, H., and Fanning, E. (1996) J. Cell Sci. 109, 1449-1457[Abstract/Free Full Text]
49. Hood, J., Casolari, J. M., and Silver, P. A. (2000) J. Cell Sci. 113, 1471-1480[Abstract/Free Full Text]
50. Solsbacher, J., Maurer, P., Vogel, F., and Schlenstedt, G. (2000) Mol. Cell. Biol. 20, 8468-8479[Abstract/Free Full Text]
51. Winston, F., Dollard, C., and Ricupero-Hovasse, S. L. (1995) Yeast 11, 53-55[Medline] [Order article via Infotrieve]
52. Xiao, Z., McGrew, J. T., Schroeder, A. J., and Fitzgerald-Hayes, M. (1993) Mol. Cell. Biol. 13, 4691-4702[Abstract]


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