Determination of the Functional Domain Organization of the Importin alpha  Nuclear Import Factor

Andrea Herold,* Ray Truant,*Dagger Heather Wiegand,Dagger and Bryan R. Cullen*Dagger

* Department of Genetics and Dagger  Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710

    Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Although importin alpha  (Imp alpha ) has been shown to act as the receptor for basic nuclear localization signals (NLSs) and to mediate their recruitment to the importin beta  nuclear import factor, little is known about the functional domains present in Imp alpha , with the exception that importin beta  binding is known to map close to the Imp alpha  NH2 terminus. Here, we demonstrate that sequences essential for binding to the CAS nuclear export factor are located near the Imp alpha  COOH terminus and include a critical acidic motif. Although point mutations introduced into this acidic motif inactivated both CAS binding and Imp alpha  nuclear export, a putative leucine-rich nuclear export signal proved to be neither necessary nor sufficient for Imp alpha  nuclear export. Analysis of sequences within Imp alpha  that bind to the SV-40 T antigen NLS or to the similar LEF-1 NLS revealed that both NLSs interact with a subset of the eight degenerate armadillo (Arm) repeats that form the central part of Imp alpha . However, these two NLS-binding sites showed only minimal overlap, thus suggesting that the degeneracy of the Arm repeat region of Imp alpha  may serve to facilitate binding to similar but nonidentical basic NLSs. Importantly, the SV-40 T NLS proved able to specifically inhibit the interaction of Imp alpha  with CAS in vitro, thus explaining why the SV-40 T NLS is unable to also function as a nuclear export signal.

Key words: importin alpha nuclear exportnuclear importnuclear localization signal
    Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

NUCLEAR import of proteins bearing a nuclear localization signal (NLS)1 of the basic type first defined in SV-40 T antigen and nucleoplasmin is dependent on two cellular factors termed importin alpha  (Imp alpha ) or karyopherin alpha  and importin beta  (Imp beta ) or karyopherin beta 1 (for review see Izaurralde and Adam, 1998; Mattaj and Englmeier, 1998; Weis, 1998). Imp alpha  functions as the NLS receptor (Görlich et al., 1994; Weis et al., 1995) and also directly binds to Imp beta via a specific domain, located between residues 10 and 55, termed the Imp beta  binding (IBB) domain (Görlich et al., 1996a; Weis et al., 1996). The resultant heterotrimeric complex is targeted to the nuclear pore complex due to the direct interaction of Imp beta  with specific nucleoporins (Görlich et al., 1995; Moroianu et al., 1995). The heterotrimer is then translocated into the nucleus via a poorly understood, energy-dependent process that also requires the activity of the Ran GTPase and a Ran cofactor termed NTF2 or p10 (Moore and Blobel, 1993; Nehrbass and Blobel, 1996; Paschal et al., 1996). Once the heterotrimer reaches the nucleus, Imp beta  directly interacts with GTP-bound Ran, a form of Ran that is largely confined to the nucleus, resulting in the release of Imp alpha  and the NLS protein cargo (Rexach and Blobel, 1995; Görlich et al., 1996b; Izaurralde et al., 1997). Both Imp alpha  and Imp beta  are then separately recycled back to the cytoplasm, where they can once again support nuclear import. Although Imp alpha  is a critical participant in basic NLS-dependent protein import, it actually serves only as an adapter to recruit NLS-containing proteins to Imp beta , which is the true mediator of nuclear transport. This is demonstrated by the finding that the Imp alpha  IBB domain is itself a potent NLS when attached to a carrier protein (Görlich et al., 1996a; Weis et al., 1996) and also by the identification of at least one other NLS that is Imp beta  dependent but Imp alpha  independent for nuclear import (Truant et al., 1998a).

Although Imp beta  is a member of a family of related proteins, including the distinct nuclear import factor transportin and the nuclear export factors Crm1 and CAS, only Imp beta  is believed to participate in Imp alpha -dependent nuclear import processes (Izaurralde and Adam, 1998; Mattaj and Englmeier, 1998; Weis, 1998). On the other hand, there are at least five different human forms of Imp alpha  that display significantly different tissue expression patterns (Cortes et al., 1994; Cuomo et al., 1994; Görlich et al., 1994; O'Neill and Palese, 1995; Weis et al., 1995; Köhler et al., 1997; Tsuji et al., 1997; Nachury et al., 1998) and that, in some cases, have been shown to interact differentially with specific basic NLS sequences (Miyamoto et al., 1997; Nadler et al., 1997; Sekimoto et al., 1997). As suggested by Köhler et al. (1997), these Imp alpha  family members are here termed Imp alpha 1 (also termed hSRP1 or NPI-1), Imp alpha 2 (also termed Rch1, hSRP1alpha , or pendulin), Imp alpha 3 (also termed Qip1), Imp alpha 4 (also termed hSRP1gamma ), and Imp alpha 6. These five variants, which vary in size between 521 and 538 amino acids (aa) in length, can be grouped into three Imp alpha  subfamilies consisting, respectively, of alpha 2, of alpha 1 and alpha 6, and finally of alpha 3 and alpha 4, where proteins within one grouping are ~80% identical and proteins in different groups are ~50% identical (Köhler et al., 1997; Tsuji et al., 1997). Alignment of these five proteins has permitted a rough structural domain organization of Imp alpha  to be proposed (see Fig. 1). In brief, the core of all five human Imp alpha  proteins consists of eight degenerate repeats of an ~42 aa, relatively hydrophobic sequence termed the armadillo (Arm) motif. Two additional conserved domains can also be identified. The first of these coincides with the IBB domain whereas the second conserved sequence, which is rich in acidic residues, extends from approximately residue 463- 509 (Köhler et al., 1997). Other regions of Imp alpha , i.e., the residues flanking the IBB and the acidic motif, tend to be poorly conserved in the different Imp alpha  variants and have therefore been termed variable regions (Tsuji et al., 1997).


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Fig. 1.   Structure of Imp alpha 2 mutants. The conserved structural domains present in all Imp alpha  family members are shown at the top of the figure whereas the structure of the Imp alpha 2 deletion and missense mutants used in this work are given below. The names of the deletion mutants indicate which NH2 residues are missing in the particular mutant protein. The location of the M1, M2, and M3 missense mutations within Imp alpha 2, and the identity of the introduced mutation, are given at the bottom.

In addition to sequences that interact with Imp beta , Imp alpha  is predicted to contain a binding site(s) for basic NLS sequences and also a nuclear export signal (NES) to permit its rapid return to the cytoplasm. Only in one case has the binding site for a basic NLS been mapped, i.e., for the RAG 1 NLS on Imp alpha 1, where binding was shown to require the Arm motifs four through seven or five through eight (Cortes et al., 1994). Based on this result, and also on the finding that a form of Imp alpha 1 lacking the first 77 aa and the last 63 aa retained the ability to bind the SV-40 T antigen NLS (T NLS), it has been suggested that basic NLS binding maps to the Arm repeat region of Imp alpha  (Sekimoto et al., 1997). The issue of the Imp alpha  NES is more complex, in that Imp alpha  has, on one hand, been proposed to exit the nucleus via a leucine-rich NES located between residues 207 and 217 in Arm repeat 3 (Boche and Fanning, 1997), that would be predicted to be dependent on the Crm1 nuclear export factor (Fornerod et al., 1997; Stade et al., 1997), whereas on the other hand Imp alpha  has also been proposed to exit the nucleus via an unmapped NES that is dependent on the distinct nuclear export factor CAS (Kutay et al., 1997a).

In this article, we have attempted to more fully define the domain organization of the alpha 2 form of Imp alpha  and, in particular, to map Imp alpha 2 sequences involved in NLS binding and nuclear export. We demonstrate that Imp alpha 2 binds to CAS, but not to Crm1, in vivo and show that CAS binding is critically dependent on the conserved acidic domain located near the Imp alpha  COOH terminus. Mutation of this acidic domain is shown to block the ability of Imp alpha 2 to exit the nucleus whereas mutation of the putative Imp alpha 2 leucine-rich NES has no effect. We also demonstrate that NLS binding by Imp alpha 2 involves not only the Arm repeats but also the flanking variable regions and show that different NLS sequences bind to distinct regions within Imp alpha 2. Finally, we demonstrate that Imp alpha  is unable to simultaneously bind a basic NLS and the CAS nuclear export factor, thus providing an explanation for why such basic NLS sequences are unable to also function as an NES.

    Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Construction of Molecular Clones

The human CAS cDNA sequence was amplified by PCR using primers containing unique 5'-BamHI and 3'-SalI restriction sites using pCAD/ CAS as a template (Brinkmann et al., 1995) and inserted into the polylinker sites BamHI and SalI present in pGBT9 (Clontech Laboratories, Palo Alto, CA). The resultant pGAL4/CAS plasmid is predicted to express a fusion protein consisting of the DNA-binding domain of GAL4 (aa 1-147) fused to the NH2 terminus of full-length CAS. pGAL4/Imp beta  was made by amplifying a full-length importin Imp beta 1 cDNA, using primers that introduce BamHI sites on both ends, with plasmid pGST/Imp beta  as a template (Truant et al., 1998a). The PCR product was then cut with BamHI and ligated into pGBT9 digested with BamHI. The truncated form of Imp beta  was made similarly, using a 5' primer that starts at aa 262. pGAL4/TNLS was made by inserting annealed oligos encoding the amino acids NH2-GGTPPKKKRKVEDP-COOH (boldfaced letters represent the T NLS) into the restriction sites EcoRI and SalI present in pGBT9. Plasmid pGAL4/LEF-1 encodes the GAL4 DNA-binding domain fused to aa 297-399 of LEF-1, which includes the LEF-1 NLS, and has been described (Prieve et al., 1996).

A cDNA encoding full-length human Imp alpha 2 (Rch1) was isolated from a human T cell cDNA library by PCR amplification with primers introducing unique EcoRI (5') and XhoI (3') restriction sites. This cDNA was found to encode a predicted protein identical to the published Imp alpha 2 (Rch1) sequence (Cuomo et al., 1994) except that residue 455 was lysine instead of glutamic acid. The Imp alpha 2 cDNA was attached 3' to, and in frame with, sequences encoding the VP16 transcription activation domain by insertion into the EcoRI and XhoI restriction sites present in pVP16 (Bogerd et al., 1995). Deletion mutants of Imp alpha 2 were generated similarly by inserting PCR products (template pVP16/Imp alpha 2) encoding the indicated amino acids (see Fig. 1) into the EcoRI and XhoI restriction sites of pVP16. The sequences encoding VP16/alpha 2M1, M2, and M3 were generated using recombinant PCR with overlapping primers that introduced the missense mutations indicated in Fig. 1. The outside primers used in the second round of amplification were designed to allow efficient in-frame insertion into pVP16. The integrity of the resultant Imp alpha 2 clones was confirmed by sequencing, using an Abi Prism 377 DNA Sequencer (Perkin Elmer Corp., Foster City, CA).

The pVP16/Imp alpha 4 expression plasmid contains a cDNA encoding full-length human Imp alpha 4 (hSRP1gamma ) (Köhler et al., 1997) isolated from a human HeLa cDNA library (Clontech Laboratories) by PCR amplification using primers that introduced flanking 5' EcoRI and 3' BglII restriction sites. The resulting PCR product was inserted into pVP16 digested with EcoRI and BamHI. The pVP16/Imp alpha 1 expression plasmid contains a cDNA encoding full-length Imp alpha 1 (hSRP1) (Weis et al., 1995) obtained by PCR amplification using the same HeLa cDNA library. The primers used introduced unique 5' EcoRI and 3' BamHI restriction sites that were then used to insert the open reading frame into the EcoRI and BamHI restriction sites present in the pVP16 polylinker.

The plasmid pSG424 (Sadowski and Ptashne, 1989) was used for construction of plasmids that allow expression of GAL4(1-147) fusion proteins in mammalian cells. To generate pGAL4/CAS, a cDNA sequence encoding full-length CAS was obtained by digesting the yeast expression plasmid pGAL4/CAS with BamHI and SalI and inserting the resultant fragment into pSG424 cut with the same restriction enzymes. Plasmid pGAL4/Impbeta Delta 1/261 was made by inserting a BamHI fragment derived from the corresponding yeast expression plasmid into the BamHI restriction site present in pSG424.

The VP16 fusion proteins used in the mammalian system were generated by inserting PCR products encoding Imp alpha 2 or its mutant forms into pBC12/cytomegalovirus (CMV)/VP16 (Bogerd et al., 1995) using the restriction sites NcoI and BglII. Templates for amplification were the corresponding cDNA sequences present in the yeast expression plasmids. The resulting plasmids allow the expression of Imp alpha 2 fusion proteins that contain a COOH-terminal VP16 domain. The mammalian indicator plasmid pG6(-31)HIVLTRDelta TARCAT, referred to here as pG6/CAT, and the internal control plasmid pBC12/CMV/beta -Gal have been described (Southgate and Green, 1991; Fridell et al., 1997), as have the mammalian two-hybrid expression constructs pGAL4/Crm1, pRev/VP16, and pRevM10/VP16 (Bogerd et al., 1995).

The pGST/alpha 2/HIS expression plasmid, encoding wild-type Imp alpha 2 fused to glutathione-S-transferase (GST) (NH2 terminal) and a 6× His tag (COOH terminal), was made by insertion of cDNA sequences encoding wild-type Imp alpha 2 into pGEX 4T-1 (Pharmacia Biotech, Piscataway, NJ) using the restriction sites EcoRI and XhoI. A plasmid encoding a GST/ alpha 2M1/HIS fusion protein was constructed using the identical approach. In both cases, the COOH-terminal 6× His tag was added by cutting the plasmid with the restriction enzymes PpuMI and XhoI. This digestion removes a fragment encoding the last 5 aa of Imp alpha 2 including the stop codon. Then annealed oligos encoding the missing 5 aa, and a 6× His tag followed by a stop codon were inserted. The inserted sequence also contained an EcoRI and a SalI restriction site between the last aa of Imp alpha 2 and the His tag. The pGST/alpha 2 M2/HIS plasmid, containing the M2 missense mutation of Imp alpha 2, was generated by inserting a NgoM1/PpuM1 fragment from the yeast expression plasmid pVP16/M2 into the same restriction sites present in pGST/alpha 2/HIS. Plasmid pGST/alpha 2Delta 491/529/HIS was made by PCR amplification of a cDNA sequence encoding Imp alpha 2 aa 364-490, using primers that contained a 5' NgoMI restriction site as well as 3' EcoRI and SalI restriction sites followed by a 6× HIS tag, a stop codon, and an XhoI site. This PCR product was then inserted into GST/ alpha 2/HIS digested with NgoMI and XhoI.

Plasmid pHT-CAS was constructed by BamHI and SalI digesting plasmid pGAL4/CAS and ligating the resulting fragment into BamHI-SalI- digested pQE32 vector (QIAGEN, Santa Clarita, CA). The resultant construct encodes the CAS protein with an NH2-terminal 6-His tag. Plasmids pGSTalpha 2 and pGSTalpha 2M2 were constructed similarly by digesting plasmids pVP16/Impalpha 2 and pVP16/Impalpha 2M2 with EcoRI and XhoI and ligating the resulting fragments into EcoRI-XhoI-digested pGEX 4T-1 (Pharmacia Biotech). The resulting plasmids encode the Impalpha 2wt or Impalpha 2M2 protein fused to GST separated by a thrombin protease cleavage site. Plasmid pGSTRanQ69L has been described (Truant et al., 1998a).

Yeast Two-hybrid Analysis

The interaction between Impalpha 1, Impalpha 4, and wild-type and mutant forms of Imp alpha 2, on the one hand and CAS, wild-type and Delta 1/261-deleted Imp beta , the LEF-1 NLS and the T NLS on the other, was assayed using the yeast two-hybrid protein interaction system (Fields and Song, 1989). Plasmids encoding the appropriate GAL4(1-147) DNA-binding domain and VP16 fusion proteins were transformed into the yeast indicator strain Y190 (Harper et al., 1993) by standard techniques. After 3 d of growth at 30°C on selective culture plates, double transformants were transferred to selective medium. The following day, cultures were assayed for beta -galactosidase (beta -gal) activity (Bogerd et al., 1995). However, due to the slower growth of yeast cells containing pGAL4/LEF-1(297-399), these transformants were incubated for 5 d on plates and liquid cultures were incubated for ~36 h at 30°C before beta -gal activity was measured.

Mammalian Two-hybrid Assay

Human 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, gentamycin, fungizone, and glutamine. On the day before transfection, cells were seeded in 6-well dishes at a density of 3 × 105 cells per 35-mm well. The cells were transfected with 0.5 µg of the reporter plasmid pG6/CAT, 2 µg of the plasmids encoding the appropriate VP16 and GAL4 fusion proteins, and 100 ng of pBC12/CMV/beta -gal using the calcium phosphate method. At ~48 h posttransfection, cell lysates were prepared and CAT and beta -gal levels quantified as described (Bogerd et al., 1995).

Recombinant Protein Expression

GST fusion proteins were expressed in the Escherichia coli strains BL21 (Novagen, Madison, WI) (GST/alpha 2/HIS, GST/alpha 2 M2/HIS) or TOPP1 (Stratagene, La Jolla, CA) (GST/alpha 2 M1/HIS, GST/alpha 2Delta 491/529/HIS). Overnight cultures were diluted 1:5 and then grown for 4.5 h without induction. Cells were collected by centrifugation, resuspended in GST buffer (20 mM Hepes, pH 7.4, 0.5 M NaCl, 10% glycerol) and lysed by sonication. The lysate was centrifuged and the GST fusion protein present in the supernatant absorbed to glutathione-Sepharose 4B (Pharmacia Biotech.), washed with GST buffer, and then eluted with elution buffer (1× PBS, 10 mM glutathione, pH 7.4). The proteins were then concentrated in a centricon-10 concentrator (Amicon, Beverly, MA).

For use in affinity chromatography assays, wild-type and M2 mutant forms of Impalpha 2, as well as the Q69L mutant of Ran, which does not support hydrolysis of bound GTP (Bischoff et al., 1994), were expressed, purified, and cleaved as described (Truant et al., 1998a). CAS protein was expressed in the E. coli DH5alpha strain. After 2 h of growth of a 1:10 dilution from an overnight culture in 2XYT medium at 37°C, the culture was induced with 0.5 mM isopropylthio-beta -D-galactoside (IPTG) for 4 h at 37°C. After harvesting by centrifugation, the cells were lysed by sonication, centrifuged, and then the supernatant was saved. The pellet was detergent extracted (B-Per; Pierce Chemical Co., Rockford, IL), recentrifuged, and the second supernatant was incubated along with the original supernatant with nickel-agarose beads (QIAGEN). The His-tagged CAS protein was eluted from the nickel-agarose beads on an imidazole gradient and then further purified on a Q-Sepharose ion exchange column (Pharmacia Biotech). SV-40 T NLS peptides were made synthetically. The peptide sequences used were: T NLS wild type, 125-YPKKKRKVEDP-135 and T NLS mutant(K128T), 125-YPKtKRKVEDP-135.

Protein Affinity Chromatography

Purified wild-type and M2 mutant Imp alpha 2 proteins were covalently coupled at a 6 mg/ml concentration to active ester-agarose beads (Affi-10; Bio-Rad Laboratories, Hercules, CA). The protein-coupled beads were then used to make 10-µl columns in 100-µl borosilicate glass micropipets. Approximately 4 µg of CAS protein was preincubated on ice with 6 µg RanQ69L and 1 mM GTP in ACB buffer (10 mM Hepes, pH 7.4, 10% glycerol, 50 mM NaCl, 1 mM MgCl2) in a 75-µl final volume. This entire mix was then loaded onto microcolumns containing either wild-type or M2 mutant Imp alpha 2. The columns were then washed with 6-column vol of 100 mM NaCl ACB buffer and finally eluted with 5-column vol 500 mM MgCl2. The entire bound fraction was analyzed by 10% SDS-PAGE (Ready-gels; Bio-Rad Laboratories) and visualized by Coomassie blue staining (R-250, Life Technologies, Inc.). For SV-40 T NLS peptide competition experiments, 100 µg (~100 fold molar excess) of SV-40 T NLS wild-type or mutant peptides were mixed with the CAS/RanQ69L/GTP mixture before loading onto the Impalpha 2 columns.

Mammalian Cell Microinjection

HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, gentamycin, fungizone, and glutamine. 2 d before microinjection, HeLa cells were seeded onto CELLocate microgrid coverslips (Eppendorf Scientific, Hamburg, Germany) at a density of 2 × 105 cells per 35-mm dish. To increase the percentage of multinucleated cells, cultures were serum starved overnight. The next day, cells were replenished with serum containing media 4-6 h before microinjection. The proteins (final concentration in PBS ~2 µg/µl) were coinjected with a rhodamine-conjugated goat IgG tracer (final concentration 1.5 µg/µl; Cappel Laboratories, Malvern, PA) to verify the site of injection. After injection, cells were incubated at 37°C for 35 min and then fixed with 3% paraformaldehyde in PBS (Fridell et al., 1997). The GST fusion protein was visualized by indirect immunofluorescence using a polyclonal affinity-purified rabbit anti-GST antibody and fluorescein-conjugated donkey anti-rabbit antiserum. Cellular localization of the injected proteins was determined using a Leica DMRB fluorescence microscope (Leica USA, Deerfield, IL) at a 100× magnification.

    Results
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

As noted above, Imp alpha  is predicted to interact with Imp beta , with a range of different basic NLS sequences and with at least one nuclear export factor. To test whether this prediction is correct, we asked whether the alpha 2 (RchI) form of Imp alpha  would be able to interact with Imp beta , with CAS, and with two different NLS sequences, derived from the T NLS (Kalderon et al., 1984) or the transcription factor LEF-1 (Prieve et al., 1996), in the yeast two-hybrid protein-protein interaction assay.

As shown in Table I, full-length Imp alpha 2 proved able to specifically interact with full-length human CAS and with both the T NLS and the LEF-1 NLS, as shown by the readily detectable activation of beta -gal expression in cells expressing a VP16/Imp alpha 2 fusion protein and the appropriate GAL4 fusions. However, Imp alpha 2 failed to interact with the GAL4 DNA-binding domain itself or with full-length human Imp beta . This latter result is expected, as the interaction of Imp beta  with Ran-GTP in the nucleus is predicted to block Imp alpha  binding (Rexach and Blobel, 1995; Görlich et al., 1996b). We therefore removed most of the Ran-GTP-binding domain of Imp beta , which is known to be located at the Imp beta  NH2 terminus (Chi and Adam, 1997; Kutay et al., 1997b), by deletion of Imp beta  residues 1-261. The resultant Imp beta Delta 1/261 protein proved able to effectively interact with Imp alpha 2 in the two-hybrid assay (Table I).

                              
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Table I
Interaction of Imp alpha 2 with Various Cellular Targets Measured by Yeast Two-hybrid Analysis

As noted above, there are five known forms of Imp alpha  that can be subdivided into three families consisting of alpha 1 and alpha 6, alpha 3 and alpha 4, and finally alpha 2. To test whether the ability of alpha 2 to interact with CAS and Imp beta  was a general property of these family members, we asked whether alpha 1 and alpha 4 would also be able to interact with these two proteins in the yeast two-hybrid assay. As shown in Table II, both Imp alpha 1 and Imp alpha 4 interacted strongly with the GAL4/Imp-beta Delta 1/261 fusion protein and less strongly, but still detectably, with CAS. We therefore conclude that both Imp beta  and CAS binding are conserved properties of Imp alpha  family members and would therefore be predicted to map to sequences conserved among Imp alpha  family members.

                              
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Table II
Both CAS and Imp beta  Binding Are Conserved among Different Imp alpha  Family Members

Protein Interaction Domains of Imp alpha 2

We next constructed a series of deletion mutants of Imp alpha 2 in the context of the Vp16/Imp alpha 2 fusion protein and determined whether these would retain the ability to bind to Impbeta Delta 1/261, CAS, and to the T NLS and LEF-1 NLS. It should be noted that the T NLS (NH2-PKKKRKVE-COOH) and the LEF-1 NLS (NH2-KKKKRKREK-COOH) are both lysine-rich NLS sequences that have been previously shown to specifically interact with full-length Imp alpha  (Prieve et al., 1996; Sekimoto et al., 1997).

The structure of the 19 deletion mutants of Imp alpha 2 used in these experiments is shown in Fig. 1. These consist of four mutants progressively deleted from the NH2 terminus, eight mutants deleted progressively from the COOH terminus, and seven mutants deleted internally, starting from residue 137. It is important to note that every one of these mutants gave rise to a readily detectable interaction with at least one of the four protein targets described above (Table III), thus demonstrating that each mutant fusion protein was expressed at functional levels in the yeast cell nucleus.

                              
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Table III
Protein Interaction Profiles of a Panel of Imp alpha 2 Mutants

As expected, deletion of the first 53 or 80 aa of Imp alpha 2, which includes the IBB domain (Görlich et al., 1996a; Weis et al., 1996), blocked binding to Imp beta  but had at most a modest effect on binding to CAS, T NLS, and LEF-1 NLS (Table III). Surprisingly, further NH2-terminal deletion, to either residue 104 or residue 138, also entirely blocked T NLS binding by Imp alpha 2, although LEF-1 NLS and CAS binding remained readily detectable. Therefore, these data demonstrate that the LEF-1 and T NLSs bind to distinguishable domains in Imp alpha 2 and further demonstrate that the sequences essential for T NLS binding extend NH2 terminal to the first Arm motif of Imp alpha 2.

Progressive deletion from the COOH terminus of Imp alpha 2 demonstrated that residues located between 496 and 501, i.e., within the conserved acidic domain, were critical for CAS binding but dispensable for binding to Imp beta  and to both the LEF-1 and T NLS (Table III). Further deletion of Imp alpha 2 residues between positions 449 and 471 resulted in the additional loss of LEF-1 NLS binding but did not affect binding to Imp beta  or the T NLS. In fact, deletion of additional residues up to position 363, i.e., deletion of Arm repeats 7 and 8, failed to inhibit either T NLS or Imp beta  binding, although both LEF-1 NLS binding and CAS binding remained undetectable. However, deletion of residues 321-363, which form Arm repeat 6, did block T NLS, but not Imp beta , binding by Imp alpha 2 (Table III). These data therefore further confirm the distinction between the Imp alpha 2 sequences required to bind the T NLS and LEF-1 NLS and demonstrate that the sequences required for LEF-1 binding extend COOH terminal to the eighth and last Arm motif.

All internal deletion mutations, starting with VP16/ alpha 2Delta 136/249, were negative for T NLS binding (Table III). However, these mutants did permit the NH2-terminal border of the CAS-binding domain of Imp alpha 2 to be mapped to between 382 and 394 for weak CAS binding or between 365 and 382 for strong binding. Importantly, this mapping therefore excludes any role for the putative leucine-rich NES sequence, located between residues 207 and 217 in Imp alpha 2, in mediating binding to CAS. These data also map the NH2 terminus of the LEF-1 NLS binding sequence in Imp alpha 2 to between residues 249 and 278, i.e., to at least 145 residues COOH terminal to the border of the sequences required for binding the functionally equivalent T NLS (Table III).

The data derived from the deletion mutants presented in Fig. 1 demonstrate that CAS binding is dependent on Imp alpha 2 sequences located in the conserved COOH-terminal acidic domain but independent of the putative leucine-rich NES located between 207 and 216 (Boche and Fanning, 1997). To more fully confirm this hypothesis, we mutated the leucine-rich sequence present in Imp alpha 2 by substitution of alanine residues for leucines to generate mutant M1 (Fig. 1). Comparable mutations have previously been shown to entirely block NES function for several leucine-rich NESs (Malim et al., 1991; Wen et al., 1995). Similarly, we also substituted alanine in place of conserved residues located in the Imp alpha 2 acidic domain to generate Imp alpha 2 mutants M2 (residues 469-474) and M3 (residues 476 -481) (Fig. 1). These missense mutants were then tested for binding to Imp beta , CAS, the T NLS and the LEF-1 NLS by two-hybrid analysis. As shown in Table III, the M1 mutation had no effect on Imp alpha 2 binding to any of these four protein targets. In contrast, the M2 mutation eliminated, and the M3 mutation strongly inhibited, CAS binding without affecting binding to Imp beta , T NLS, or LEF-1 NLS. These data therefore confirm that the conserved Imp alpha 2 acidic domain is critical for specific CAS binding in vivo.

Imp alpha 2 Binds to CAS but Not to Crm1

We next asked whether Imp alpha 2 would be able to bind to CAS, or to the leucine-rich NES-specific nuclear export factor Crm1 (Fornerod et al., 1997; Stade et al., 1997), in mammalian cells using a previously described mammalian version of the two-hybrid assay (Bogerd et al., 1995). In this assay, fusions of the GAL4 DNA-binding domain to full-length human Crm1 or CAS, or to the Delta 1/261 deletion mutant of Impbeta , were coexpressed in the mammalian cell nucleus together with fusion proteins consisting of the VP16 transcription activation domain linked to wild-type or M1 mutant forms of Impalpha 2 or to wild-type or M10 mutant forms of the human immunodeficiency virus type 1 Rev protein. The Rev protein is known to contain an active leucine-rich NES whereas the M10 Rev mutant encodes a defective form of this NES (Malim et al., 1989; Fischer et al., 1995; Wen et al., 1995).

As shown in Fig. 2, we observed a readily detectable interaction between wild-type Rev and Crm1 that was blocked by the M10 NES mutation, but not between Rev and either CAS or Imp beta . In contrast, both the wild-type and M1 mutant form of Imp alpha 2 interacted strongly with both GAL4/CAS and the GAL4 Impbeta Delta 1/261 fusion protein but failed to interact detectably with Crm1. These data therefore suggest that Imp alpha 2 contains a CAS-dependent, but not a Crm1-dependent, NES sequence.


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Fig. 2.   Interaction of Imp alpha 2 and HIV-1 Rev with human nucleocytoplasmic transport factors measured by mammalian two-hybrid assay. Human 293T cells were transfected with one of the indicated GAL4 DNA-binding domain fusion protein expression plasmids, one of the indicated VP16 transcription activation domain fusion protein expression plasmids, the two-hybrid interaction indicator construct pG6/CAT and finally the internal control plasmid pBC12/CMV/beta -gal. Induced CAT activities were measured at 48 h after transfection, as previously described (Bogerd et al., 1995), and were corrected for small differences in transfection efficiency revealed by analysis of beta -gal expression levels.

In addition to the mammalian two-hybrid data presented in Fig. 2, we also assessed the ability of a subset of the alpha 2 mutations described in Fig. 1 to interact with either CAS or Impbeta Delta 1/261 in the mammalian nucleus. In brief, these data (Table IV) confirmed the yeast data (Table III) showing that the NH2-terminal border of the CAS-binding domain in Imp alpha 2 was located near to residue 365 and also located the COOH-terminal border of this domain between residues 491 and 501. In addition, these data (Table IV) revealed that both the M2 and the M3 mutant of Imp alpha 2 are severely inhibited for CAS, but not for Imp beta , binding in the mammalian cell nucleus.

                              
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Table IV
Interaction of Imp a2 Mutants with CAS and Imp beta  Measured by Mammalian Two-hybrid analysis


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Fig. 3.   CAS mediates the nuclear export of Imp alpha 2. Recombinant fusion proteins consisting of GST linked to full-length, wild-type Imp alpha 2 and to the M1, Delta 491/529 or M2 mutants of Imp alpha 2, were purified from bacteria, mixed with a rhodamine-conjugated rabbit IgG tracer, and then microinjected into a single nucleus in a multinucleate HeLa cell. Subcellular localization of the injected proteins was determined at 35 min after injection by double-label immunofluorescence microscopy, as previously described (Fridell et al., 1997). GST fusion proteins able to undergo nucleocytoplasmic shuttling to the uninjected nuclei present in the same cell (B and E) must contain a functional NES whereas mutants lacking nuclear export capacity (H and K), as well as the IgG tracer (A, D, G, and J), remain confined to the single injected nucleus.

CAS-binding Domain of Imp alpha 2 Mediates Nuclear Export

We next wished to ask whether the ability to interact with CAS was indeed critical for the nuclear export of Imp alpha 2 from the mammalian cell nucleus. As described in more detail elsewhere (Truant et al., 1998b), we have observed that recombinant Imp alpha 2 accumulates in the nucleus of microinjected human cells. To demonstrate nuclear export, we therefore mixed recombinant wild-type full-length Imp alpha 2 fused to GST with a rabbit IgG tracer protein and microinjected it into a single nucleus located in multinucleate cells. If Imp alpha 2 contains a functional NES, to go with the NLS activity known to reside in the IBB motif, it should export from the injected nucleus and then import into the uninjected nuclei present in the same cell. This is indeed precisely the activity that was seen, in that the injected GST/Imp alpha 2 fusion protein is redistributed to all the nuclei present in an injected cell (Fig. 3 B) whereas the IgG tracer remains in the single injected nucleus (Fig. 3 A). The same result was obtained upon injection of the M1 mutant form of full-length GST/Imp alpha 2 into one nucleus in a multinucleate cell (Fig. 3, D and E), thus demonstrating that a functional form of the putative leucine-rich NES present between residues 207 and 217 in Impalpha 2 is not required for nuclear export. In contrast, GST-Imp alpha 2 fusion proteins containing either the Delta 491/529 deletion mutation or the M2 missense mutation that knocks out CAS binding (Tables III and IV failed to demonstrate nucleocytoplasmic shuttling (Fig. 3, H and K) even though both proteins are efficiently imported into the nucleus when injected into the cell cytoplasm (data not shown). We therefore conclude that CAS binding is a critical step in the nuclear export of Imp alpha 2 in human cells.

Imp alpha  Is Unable to Bind CAS and the T NLS Simultaneously

The mapping data presented in Table III, and summarized in Fig. 4, demonstrate that the CAS-binding domain in Imp alpha 2 is located between residues 383 and 497 whereas the SV-40 T NLS binding sequences are located between residues 81 and 362. Therefore, at least in this linear representation of the Imp alpha 2 molecule, these binding domains do not obviously overlap. However, if the T NLS and the CAS nuclear export factor could bind Imp alpha  simultaneously, then one might predict that proteins containing the T NLS would be exported from the nucleus in a ternary complex with Imp alpha  and CAS just as they are imported into the nucleus as a ternary complex with Imp alpha  and Imp beta .


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Fig. 4.   Functional domain organization of Imp alpha 2. The figure correlates the location of the conserved structural domains of Imp alpha  with the approximate location of sequences in Imp alpha 2 required for binding to Imp beta , to CAS, to the LEF-1 NLS and the T NLS. The speckled areas are essential for binding to each target while the flanking clear boxes define the ambiguity as to the precise border of the critical Imp alpha 2 sequences. IBB, Imp beta  binding motif; V, variable regions.

To test whether the T NLS and CAS can indeed bind Imp alpha 2 simultaneously, we performed an in vitro protein- protein interaction assay using recombinant wild-type and M2 mutant Imp alpha 2 and CAS as well as Ran protein (Fig. 5). As reported previously by Kutay et al. (1997a), we observed that efficient binding of Imp alpha  by CAS only occurred in the presence of the GTP bound form of Ran (data not shown). This interaction was specific in that CAS bound the wild-type but not the M2 mutant form of Imp alpha 2 in vitro (Fig. 5, B and C), thus also confirming the in vivo data demonstrating that the Imp alpha 2 M2 mutant blocks CAS binding (refer to Tables III and IV. Interestingly, although the NH2 terminally HIS-tagged recombinant CAS protein preparation used in this analysis contained a significant level of breakdown products that presumably represent a nested set of COOH terminally truncated forms of CAS, only the full-length form of CAS was observed to bind to Imp alpha 2 (Fig. 5, compare lane A with B). This further confirms the specificity of this protein-protein interaction and suggests that sequences located towards the CAS COOH terminus are critical for Imp alpha 2 binding.


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Fig. 5.   Analysis of CAS binding to Imp alpha 2 in vitro. Recombinant wild-type or M2 mutant Imp alpha 2 proteins were coupled to agarose beads and used to construct microaffinity columns. Recombinant, NH2 terminally His-tagged CAS protein was preincubated on ice with Ran and GTP before loading onto the columns either directly or after addition of a 100-fold molar excess of a wild-type or defective mutant T NLS peptide. After extensive washing, bound proteins were eluted from the affinity columns and analyzed by SDS-PAGE.

To examine whether the T NLS would interfere with CAS binding to Imp alpha 2, we repeated this binding assay in the presence of an ~100-fold molar excess of synthetic peptides containing either the wild-type SV-40 T NLS or a mutant, nonfunctional form of the T NLS (K128 to T, Kalderon et al., 1984). As shown in Fig. 5, the wild-type T NLS peptide entirely blocked CAS binding by Imp alpha 2 whereas the mutant peptide had no effect. We therefore conclude that Imp alpha 2 is unable to simultaneously bind to both CAS and the T NLS.

    Discussion
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

A major goal of the research described in this article was the determination of whether Crm1 (Boche and Fanning, 1997) or CAS (Kutay et al., 1997a) was required for the nuclear export of Imp alpha  and if the latter, the identification and delineation of the CAS-dependent NES present in Imp alpha . Using the two-hybrid assay for in vivo protein- protein interactions, we have demonstrated that the distinct Imp alpha 2, Imp alpha 1, and Imp alpha 4 forms of Imp alpha  are all able to specifically interact with human CAS in the yeast two-hybrid assay (refer to Tables I and II. Using a mammalian two-hybrid assay, we confirmed the specific interaction of Imp alpha 2 with CAS and showed that Imp alpha 2 failed to interact with Crm1 under conditions where the interaction of Crm1 with the leucine-rich NES present in HIV-1 Rev was readily detectable (refer to Fig. 2). We therefore conclude that Imp alpha  can specifically bind to CAS but not to Crm1 in vivo. By deletion and missense mutation analysis (Tables III and IV, we have further demonstrated that a proposed leucine-rich NES sequence (Boche and Fanning, 1997), located within the third Arm repeat of Imp alpha 2, is entirely dispensable for CAS binding, which instead was shown to largely map to a conserved acidic domain located proximal to the COOH terminus of Imp alpha 2 (refer to Fig. 4). Finally, we demonstrate that nucleocytoplasmic shuttling of Imp alpha 2 is blocked by two distinct mutations within the acidic domain that inhibit CAS binding but is not affected by mutation of the putative leucine-rich NES (refer to Fig. 3). We therefore conclude that the leucine-rich sequence present in Imp alpha 2 Arm repeat 3 is neither necessary nor sufficient for the nuclear export of Imp alpha 2 and suggest that the published identification of this sequence as the Imp alpha  NES (Boche and Fanning, 1997) is incorrect. Rather, our data confirm the proposal by Kutay et al. (1997a) that Imp alpha  nuclear export is dependent on the interaction of Imp alpha  with the CAS nuclear export factor and map the Imp alpha sequences required for this interaction to the conserved Imp alpha acidic domain, together with some adjacent flanking sequences (refer to Fig. 4). Although the CAS-binding domain defined in this analysis is quite large, being minimally ~120 aa in length, it is clearly distinct in sequence from the much smaller leucine-rich NES sequences recognized by the Crm1 nuclear export factor (Fornerod et al., 1997; Stade et al., 1997).

In addition to defining sequences in Imp alpha 2 that mediate CAS binding, we wished to also determine which Imp alpha 2 sequences might be required for basic NLS recognition. Surprisingly, with the exception of a manuscript that mapped RAG1 NLS binding to Arm repeats 4-8 of Imp alpha 1 (Cortes et al., 1994), little had been reported about the identity of Imp alpha  sequences required for this critical step in nuclear import at the time that this work was initiated. The two basic NLS sequences chosen for analysis, the T NLS and the LEF-1 NLS, are both lysine-rich NLS sequences that had been previously shown to bind to Imp alpha  specifically (Prieve et al., 1996; Sekimoto et al., 1997).

Analysis of the sequence requirements for T NLS and LEF-1 NLS binding by Imp alpha 2 produced two surprising findings. First, the Imp alpha 2 sequences required for binding to these two NLS sequences were clearly distinct, with T NLS binding mapping between Imp alpha 2 residues 81-362 whereas LEF-1 NLS binding mapped between residues 250 and 470 (refer to Table III and Fig. 4). The second unexpected result was that the Imp alpha 2 sequences required for basic NLS binding extended at least somewhat beyond the Arm repeat sequences into flanking variable regions. Specifically, T NLS binding required Imp alpha 2 sequences located NH2 terminal to residue 105, the first residue of the first Arm repeat, whereas LEF-1 NLS binding clearly required sequences located COOH terminal to residue 447, the last amino acid of the eighth Arm repeat (refer to Table III and Fig. 4). These observations therefore demonstrate that the identity of the Arm repeats required for any given NLS interaction is likely to be quite variable and further reveal that the Imp alpha  Arm repeats, although critical for basic NLS binding, are not necessarily sufficient, in that flanking variable regions may also make an essential contribution. In this regard, it is interesting to note that several papers have reported that certain NLS sequences can be bound by particular members of the Imp alpha  family of NLS receptors but not by others (Miyamoto et al., 1997; Nadler et al., 1997; Sekimoto et al., 1997). As highly variable regions in Imp alpha  appear to make a significant contribution to NLS binding, this result is not, in retrospect, surprising.

Although the interaction of Imp beta  with Imp alpha  in the cell nucleus is blocked by Ran-GTP (Rexach and Blobel, 1995; Görlich et al., 1996b), it has remained unclear why NLS proteins are released by Imp alpha . Although it has been suggested that basic NLS binding to an Imp alpha /Imp beta  heterodimer is of higher affinity than binding by Imp alpha  alone (Rexach and Blobel, 1995; Efthymiadis et al., 1998), it is nevertheless apparent that NLS binding by Imp alpha  can be readily detected in the nucleus of cells under conditions where Imp alpha and Imp beta  do not interact (refer to Tables I and III (Cuomo et al., 1994; Cortes et al., 1995; O'Neill et al., 1995). Why then doesn't the NLS protein return to the cytoplasm along with the Imp alpha /CAS complex in a process analogous to its import by the Imp alpha /Imp beta  complex? One possibility is that CAS binding and basic NLS binding by Imp alpha  is mediated by two distinct, mutually incompatible Imp alpha  protein conformations, a mechanism which has also been invoked to explain why Imp beta  is unable to simultaneously bind to Imp alpha and Ran-GTP. Alternately, CAS binding and NLS binding might simply sterically interfere with one another. In either case, CAS binding, and hence Imp alpha  export from the nucleus, could only occur once the NLS protein was released into the nucleoplasm. It has, in fact, previously been demonstrated that CAS binds to NLS-free Imp alpha  preferentially, although the underlying mechanism for this effect has remained unclear (Kutay et al., 1997a). Although the extensive overlap between the Imp alpha 2 sequences that mediate binding to the LEF-1 NLS and to CAS, respectively (refer to Fig. 4), certainly seems consistent with the hypothesis that these protein-protein interactions might be mutually exclusive, this is less obvious in the case of the SV-40 T NLS, which binds to Imp alpha 2 sequences that do not obviously conflict with the CAS binding site (refer to Fig. 4). Nevertheless, in vitro binding analysis reveals that the SV-40 T NLS, but not a nonfunctional mutant form thereof, can entirely block CAS binding by Imp alpha 2 (refer to Fig. 5). These data demonstrate that Imp alpha 2 is unable to bind to the T NLS and to CAS simultaneously and therefore provide a mechanistic explanation for the finding that the SV-40 T NLS is unable to function as an Imp alpha -dependent NES.

After submission of this article, a paper was published that bears on the issue of the functional domain organization of Imp alpha 2. Specifically, Conti et al. (1998) have published the crystal structure of a truncated form of yeast Imp alpha  bound to two molecules of an SV-40 T NLS peptide. Although yeast Imp alpha  is somewhat different in structure to human Imp alpha 2 (for example, it appears to contain 10 rather than 8 Arm repeats), this report is striking in that the two T NLS peptides were found to bind to Arm repeats one through four and seven through nine, respectively. The authors proposed that the larger, more NH2-terminal NLS binding site was likely to be the functionally relevant site on yeast Imp alpha  for T NLS binding whereas the smaller, more COOH-terminal site might instead play a role in the recognition of bipartite NLSs, such as the one observed in nucleoplasmin. In contrast, the data presented in our article suggest that Imp alpha 2 contains two distinct binding sites for monopartite NLS sequences, with the more NH2-terminal site being specific for NLSs equivalent in sequence to the SV-40 T NLS whereas the more COOH-terminal site may interact specifically with NLSs that are similar to the LEF-1 NLS. From this perspective, the weak binding observed by Conti et al. (1998) between the T NLS peptide and the more COOH-terminal binding site in yeast Imp alpha  may therefore simply reflect the fact that the structure of the T NLS is suboptimal for binding to this latter site, rather than suggesting that this site is not an autonomous binding site for other, more optimal monopartite NLSs.

    Footnotes

Received for publication 9 June 1998 and in revised form 9 September 1998.

   This research was funded by the Howard Hughes Medical Institute. A. Herold was supported by funds from the German Academic Exchange Organization (DAAD).
   Address all correspondence to B.R. Cullen, Department of Genetics, Room 426 CARL Building, Research Drive, Box 3025, Duke University Medical Center, Durham, NC 27710. Tel.: (919) 684-3369. Fax: (919) 681-8979. E-mail: culle002{at}mc.duke.edu

The authors thank G. Grosveld (St. Jude Children's Research Hospital, Memphis, TN) for the human Crm1 cDNA, U. Brinkmann (National Cancer Institute, Bethesda, MD) for the human CAS cDNA clone, M. Waterman (University of California, Irvine, CA) for pGAL4/LEF-1, and M. Malim (University of Pennsylvania, Philadelphia, PA) for wild-type and mutant forms of the SV-40 T NLS peptide.

    Abbreviations used in this paper

aa, amino acid(s); Arm, armadillo motif; beta -gal, beta -galactosidase; CMV, cytomegalovirus; GST, glutathione-S-transferase; IBB, importin beta  binding domain; Imp alpha , importin alpha ; Imp beta , importin beta ; NES, nuclear export signal; NLS, nuclear localization signal; T NLS, SV-40 T antigen NLS.

    References
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
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