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

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 β was made by amplifying a full-length importin Imp β1 cDNA, using primers that introduce BamHI sites on both ends, with plasmid pGST/Imp β 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 β 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 α2 (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 α2 (Rch1) sequence (Cuomo et al., 1994) except that residue 455 was lysine instead of glutamic acid. The Imp α2 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 α2 were generated similarly by inserting PCR products (template pVP16/Imp α2) encoding the indicated amino acids (see Fig. 1) into the EcoRI and XhoI restriction sites of pVP16. The sequences encoding VP16/α2M1, 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 α2 clones was confirmed by sequencing, using an Abi Prism 377 DNA Sequencer (Perkin Elmer Corp., Foster City, CA).

The pVP16/Imp α4 expression plasmid contains a cDNA encoding full-length human Imp α4 (hSRP1γ) (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 α1 expression plasmid contains a cDNA encoding full-length Imp α1 (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/ImpβΔ1/261 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 α2 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 α2 fusion proteins that contain a COOH-terminal VP16 domain. The mammalian indicator plasmid pG6(−31)HIVLTRΔTARCAT, referred to here as pG6/CAT, and the internal control plasmid pBC12/CMV/β-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/α2/HIS expression plasmid, encoding wild-type Imp α2 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 α2 into pGEX 4T-1 (Pharmacia Biotech, Piscataway, NJ) using the restriction sites EcoRI and XhoI. A plasmid encoding a GST/ α2M1/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 α2 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 α2 and the His tag. The pGST/α2 M2/HIS plasmid, containing the M2 missense mutation of Imp α2, was generated by inserting a NgoM1/PpuM1 fragment from the yeast expression plasmid pVP16/M2 into the same restriction sites present in pGST/α2/HIS. Plasmid pGST/α2Δ491/529/HIS was made by PCR amplification of a cDNA sequence encoding Imp α2 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/ α2/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 pGSTα2 and pGSTα2M2 were constructed similarly by digesting plasmids pVP16/Impα2 and pVP16/Impα2M2 with EcoRI and XhoI and ligating the resulting fragments into EcoRI-XhoI–digested pGEX 4T-1 (Pharmacia Biotech). The resulting plasmids encode the Impα2wt or Impα2M2 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 Impα1, Impα4, and wild-type and mutant forms of Imp α2, on the one hand and CAS, wild-type and Δ1/261-deleted Imp β, 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 β-galactosidase (β-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 β-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/β-gal using the calcium phosphate method. At ∼48 h posttransfection, cell lysates were prepared and CAT and β-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/α2/HIS, GST/α2 M2/HIS) or TOPP1 (Stratagene, La Jolla, CA) (GST/α2 M1/HIS, GST/α2Δ491/529/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 Impα2, 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 DH5α 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-β-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 α2 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 α2. 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 Impα2 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.

Protein Interaction Domains of Imp α2

We next constructed a series of deletion mutants of Imp α2 in the context of the Vp16/Imp α2 fusion protein and determined whether these would retain the ability to bind to ImpβΔ1/261, 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 α (Prieve et al., 1996; Sekimoto et al., 1997).

The structure of the 19 deletion mutants of Imp α2 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.

As expected, deletion of the first 53 or 80 aa of Imp α2, which includes the IBB domain (Görlich et al., 1996a ; Weis et al., 1996), blocked binding to Imp β 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 α2, 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 α2 and further demonstrate that the sequences essential for T NLS binding extend NH₂ terminal to the first Arm motif of Imp α2.

Progressive deletion from the COOH terminus of Imp α2 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 β and to both the LEF-1 and T NLS (Table III). Further deletion of Imp α2 residues between positions 449 and 471 resulted in the additional loss of LEF-1 NLS binding but did not affect binding to Imp β 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 β 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 β, binding by Imp α2 (Table III). These data therefore further confirm the distinction between the Imp α2 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/ α2Δ136/249, were negative for T NLS binding (Table III). However, these mutants did permit the NH₂-terminal border of the CAS-binding domain of Imp α2 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 α2, in mediating binding to CAS. These data also map the NH₂ terminus of the LEF-1 NLS binding sequence in Imp α2 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 α2 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 α2 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 α2 acidic domain to generate Imp α2 mutants M2 (residues 469–474) and M3 (residues 476 –481) (Fig. 1). These missense mutants were then tested for binding to Imp β, 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 α2 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 β, T NLS, or LEF-1 NLS. These data therefore confirm that the conserved Imp α2 acidic domain is critical for specific CAS binding in vivo.

Imp α2 Binds to CAS but Not to Crm1

We next asked whether Imp α2 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 Δ1/261 deletion mutant of Impβ, 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 Impα2 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 β. In contrast, both the wild-type and M1 mutant form of Imp α2 interacted strongly with both GAL4/CAS and the GAL4 ImpβΔ1/261 fusion protein but failed to interact detectably with Crm1. These data therefore suggest that Imp α2 contains a CAS-dependent, but not a Crm1-dependent, NES sequence.

In addition to the mammalian two-hybrid data presented in Fig. 2, we also assessed the ability of a subset of the α2 mutations described in Fig. 1 to interact with either CAS or ImpβΔ1/261 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 α2 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 α2 are severely inhibited for CAS, but not for Imp β, binding in the mammalian cell nucleus.

CAS-binding Domain of Imp α2 Mediates Nuclear Export

We next wished to ask whether the ability to interact with CAS was indeed critical for the nuclear export of Imp α2 from the mammalian cell nucleus. As described in more detail elsewhere (Truant et al., 1998b ), we have observed that recombinant Imp α2 accumulates in the nucleus of microinjected human cells. To demonstrate nuclear export, we therefore mixed recombinant wild-type full-length Imp α2 fused to GST with a rabbit IgG tracer protein and microinjected it into a single nucleus located in multinucleate cells. If Imp α2 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 α2 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 α2 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 Impα2 is not required for nuclear export. In contrast, GST–Imp α2 fusion proteins containing either the Δ491/529 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 α2 in human cells.

Imp α 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 α2 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 α2 molecule, these binding domains do not obviously overlap. However, if the T NLS and the CAS nuclear export factor could bind Imp α simultaneously, then one might predict that proteins containing the T NLS would be exported from the nucleus in a ternary complex with Imp α and CAS just as they are imported into the nucleus as a ternary complex with Imp α and Imp β.

To test whether the T NLS and CAS can indeed bind Imp α2 simultaneously, we performed an in vitro protein– protein interaction assay using recombinant wild-type and M2 mutant Imp α2 and CAS as well as Ran protein (Fig. 5). As reported previously by Kutay et al. (1997a), we observed that efficient binding of Imp α 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 α2 in vitro (Fig. 5, B and C), thus also confirming the in vivo data demonstrating that the Imp α2 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 α2 (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 α2 binding.

To examine whether the T NLS would interfere with CAS binding to Imp α2, 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 α2 whereas the mutant peptide had no effect. We therefore conclude that Imp α2 is unable to simultaneously bind to both CAS and the T NLS.