* Department of Genetics and Howard Hughes Medical Institute, Duke University Medical Center, Durham,
North Carolina 27710
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Abstract |
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Although importin (Imp
) has been
shown to act as the receptor for basic nuclear localization signals (NLSs) and to mediate their recruitment to
the importin
nuclear import factor, little is known
about the functional domains present in Imp
, with the exception that importin
binding is known to map
close to the Imp
NH2 terminus. Here, we demonstrate that sequences essential for binding to the CAS
nuclear export factor are located near the Imp
COOH terminus and include a critical acidic motif.
Although point mutations introduced into this acidic
motif inactivated both CAS binding and Imp
nuclear
export, a putative leucine-rich nuclear export signal
proved to be neither necessary nor sufficient for Imp
nuclear export. Analysis of sequences within Imp
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
. However, these two NLS-binding sites showed only minimal overlap, thus suggesting that the degeneracy of the Arm repeat region
of Imp
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
with CAS in vitro, thus explaining why the SV-40
T NLS is unable to also function as a nuclear export signal.
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Introduction |
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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 (Imp
) or karyopherin
and importin
(Imp
) or karyopherin
1 (for review see Izaurralde and Adam, 1998
; Mattaj and
Englmeier, 1998
; Weis, 1998
). Imp
functions as the NLS
receptor (Görlich et al., 1994
; Weis et al., 1995
) and also
directly binds to Imp
via a specific domain, located between residues 10 and 55, termed the Imp
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
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
directly
interacts with GTP-bound Ran, a form of Ran that is
largely confined to the nucleus, resulting in the release of
Imp
and the NLS protein cargo (Rexach and Blobel, 1995
; Görlich et al., 1996b
; Izaurralde et al., 1997
). Both
Imp
and Imp
are then separately recycled back to the
cytoplasm, where they can once again support nuclear import. Although Imp
is a critical participant in basic NLS-dependent protein import, it actually serves only as an
adapter to recruit NLS-containing proteins to Imp
,
which is the true mediator of nuclear transport. This is
demonstrated by the finding that the Imp
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
dependent but Imp
independent for nuclear import (Truant et al., 1998a
).
Although Imp 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
is believed to participate in Imp
-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
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
family members are here
termed Imp
1 (also termed hSRP1 or NPI-1), Imp
2
(also termed Rch1, hSRP1
, or pendulin), Imp
3 (also
termed Qip1), Imp
4 (also termed hSRP1
), and Imp
6.
These five variants, which vary in size between 521 and 538 amino acids (aa) in length, can be grouped into three Imp
subfamilies consisting, respectively, of
2, of
1 and
6,
and finally of
3 and
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
to be proposed (see Fig. 1). In brief, the core of all five human Imp
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
, i.e., the
residues flanking the IBB and the acidic motif, tend to be poorly conserved in the different Imp
variants and have
therefore been termed variable regions (Tsuji et al., 1997
).
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In addition to sequences that interact with Imp , Imp
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
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
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
(Sekimoto et al., 1997
). The issue of the Imp
NES is
more complex, in that Imp
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
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 2 form of Imp
and, in
particular, to map Imp
2 sequences involved in NLS
binding and nuclear export. We demonstrate that Imp
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
COOH terminus. Mutation
of this acidic domain is shown to block the ability of Imp
2 to exit the nucleus whereas mutation of the putative
Imp
2 leucine-rich NES has no effect. We also demonstrate that NLS binding by Imp
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
2. Finally, we demonstrate that Imp
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.
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Materials and Methods |
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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
was made by amplifying a full-length importin Imp
1 cDNA, using primers that introduce BamHI sites on both ends, with plasmid pGST/Imp
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
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 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
2
(Rch1) sequence (Cuomo et al., 1994
) except that residue 455 was lysine
instead of glutamic acid. The Imp
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
2 were generated similarly by inserting PCR products (template pVP16/Imp
2) encoding the
indicated amino acids (see Fig. 1) into the EcoRI and XhoI restriction
sites of pVP16. The sequences encoding VP16/
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
2
clones was confirmed by sequencing, using an Abi Prism 377 DNA Sequencer (Perkin Elmer Corp., Foster City, CA).
The pVP16/Imp 4 expression plasmid contains a cDNA encoding full-length human Imp
4 (hSRP1
) (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
1 expression plasmid contains a
cDNA encoding full-length Imp
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/Imp
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 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
2 fusion proteins that contain a COOH-terminal VP16 domain. The mammalian indicator plasmid
pG6(
31)HIVLTR
TARCAT, referred to here as pG6/CAT, and the internal control plasmid pBC12/CMV/
-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/2/HIS expression plasmid, encoding wild-type Imp
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
2 into pGEX 4T-1 (Pharmacia Biotech, Piscataway, NJ)
using the restriction sites EcoRI and XhoI. A plasmid encoding a GST/
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
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
2
and the His tag. The pGST/
2 M2/HIS plasmid, containing the M2 missense mutation of Imp
2, was generated by inserting a NgoM1/PpuM1
fragment from the yeast expression plasmid pVP16/M2 into the same restriction sites present in pGST/
2/HIS. Plasmid pGST/
2
491/529/HIS
was made by PCR amplification of a cDNA sequence encoding Imp
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/
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
pGST2 and pGST
2M2 were constructed similarly by digesting plasmids
pVP16/Imp
2 and pVP16/Imp
2M2 with EcoRI and XhoI and ligating
the resulting fragments into EcoRI-XhoI-digested pGEX 4T-1 (Pharmacia Biotech). The resulting plasmids encode the Imp
2wt or Imp
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 Imp1, Imp
4, and wild-type and mutant forms
of Imp
2, on the one hand and CAS, wild-type and
1/261-deleted Imp
, 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
-galactosidase (
-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
-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/-gal using the calcium phosphate method. At ~48 h posttransfection, cell lysates were prepared and CAT and
-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/2/HIS, GST/
2 M2/HIS) or TOPP1
(Stratagene, La Jolla, CA) (GST/
2 M1/HIS, GST/
2
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 Imp2, 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 DH5
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-
-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 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
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 Imp
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.
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Results |
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As noted above, Imp is predicted to interact with Imp
,
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
2 (RchI) form of
Imp
would be able to interact with Imp
, 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 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
-gal expression in cells
expressing a VP16/Imp
2 fusion protein and the appropriate GAL4 fusions. However, Imp
2 failed to interact with the GAL4 DNA-binding domain itself or with full-length human Imp
. This latter result is expected, as the
interaction of Imp
with Ran-GTP in the nucleus is predicted to block Imp
binding (Rexach and Blobel, 1995
;
Görlich et al., 1996b
). We therefore removed most of the
Ran-GTP-binding domain of Imp
, which is known to be
located at the Imp
NH2 terminus (Chi and Adam, 1997
; Kutay et al., 1997b
), by deletion of Imp
residues 1-261.
The resultant Imp
1/261 protein proved able to effectively interact with Imp
2 in the two-hybrid assay (Table I).
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As noted above, there are five known forms of Imp that can be subdivided into three families consisting of
1
and
6,
3 and
4, and finally
2. To test whether the ability of
2 to interact with CAS and Imp
was a general
property of these family members, we asked whether
1
and
4 would also be able to interact with these two proteins in the yeast two-hybrid assay. As shown in Table II,
both Imp
1 and Imp
4 interacted strongly with the GAL4/Imp-
1/261 fusion protein and less strongly, but
still detectably, with CAS. We therefore conclude that
both Imp
and CAS binding are conserved properties of
Imp
family members and would therefore be predicted
to map to sequences conserved among Imp
family members.
|
Protein Interaction Domains of Imp 2
We next constructed a series of deletion mutants of Imp
2 in the context of the Vp16/Imp
2 fusion protein and
determined whether these would retain the ability to bind
to Imp
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
(Prieve et al., 1996
; Sekimoto et al., 1997
).
The structure of the 19 deletion mutants of Imp 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.
|
As expected, deletion of the first 53 or 80 aa of Imp 2,
which includes the IBB domain (Görlich et al., 1996a
;
Weis et al., 1996
), blocked binding to Imp
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
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
2 and further demonstrate that the sequences essential for T NLS binding extend NH2 terminal to the first Arm motif of Imp
2.
Progressive deletion from the COOH terminus of Imp
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
and
to both the LEF-1 and T NLS (Table III). Further deletion
of Imp
2 residues between positions 449 and 471 resulted
in the additional loss of LEF-1 NLS binding but did not affect binding to Imp
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
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
, binding by Imp
2 (Table III). These data
therefore further confirm the distinction between the Imp
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/
2
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
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
2, in mediating binding to CAS. These data also map
the NH2 terminus of the LEF-1 NLS binding sequence in
Imp
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 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
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
2 acidic domain to
generate Imp
2 mutants M2 (residues 469-474) and M3
(residues 476 -481) (Fig. 1). These missense mutants were
then tested for binding to Imp
, 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
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
, T NLS, or
LEF-1 NLS. These data therefore confirm that the conserved Imp
2 acidic domain is critical for specific CAS
binding in vivo.
Imp 2 Binds to CAS but Not to Crm1
We next asked whether Imp 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
1/261 deletion
mutant of Imp
, 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 Imp
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 . In contrast, both the wild-type
and M1 mutant form of Imp
2 interacted strongly with
both GAL4/CAS and the GAL4 Imp
1/261 fusion protein but failed to interact detectably with Crm1. These
data therefore suggest that Imp
2 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 2 mutations described in Fig. 1 to interact with either
CAS or Imp
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
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
2 are severely inhibited for CAS, but not for Imp
, binding in the mammalian cell nucleus.
|
|
CAS-binding Domain of Imp 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 2
from the mammalian cell nucleus. As described in more
detail elsewhere (Truant et al., 1998b
), we have observed
that recombinant Imp
2 accumulates in the nucleus of
microinjected human cells. To demonstrate nuclear export,
we therefore mixed recombinant wild-type full-length Imp
2 fused to GST with a rabbit IgG tracer protein and microinjected it into a single nucleus located in multinucleate cells. If Imp
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
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
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 Imp
2 is not required for nuclear export. In contrast, GST-Imp
2 fusion
proteins containing either the
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
2 in human cells.
Imp 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 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
2 molecule, these binding domains
do not obviously overlap. However, if the T NLS and the CAS nuclear export factor could bind Imp
simultaneously, then one might predict that proteins containing
the T NLS would be exported from the nucleus in a ternary complex with Imp
and CAS just as they are imported into the nucleus as a ternary complex with Imp
and Imp
.
|
To test whether the T NLS and CAS can indeed bind
Imp 2 simultaneously, we performed an in vitro protein-
protein interaction assay using recombinant wild-type and
M2 mutant Imp
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
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
2 in vitro (Fig. 5, B and C), thus also confirming the in
vivo data demonstrating that the Imp
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
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
2 binding.
|
To examine whether the T NLS would interfere with
CAS binding to Imp 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
2
whereas the mutant peptide had no effect. We therefore
conclude that Imp
2 is unable to simultaneously bind to
both CAS and the T NLS.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
and if the latter, the identification and delineation of the CAS-dependent NES present
in Imp
. Using the two-hybrid assay for in vivo protein-
protein interactions, we have demonstrated that the distinct Imp
2, Imp
1, and Imp
4 forms of Imp
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
2 with CAS and showed that Imp
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
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
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
2 (refer to Fig.
4). Finally, we demonstrate that nucleocytoplasmic shuttling of Imp
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
2 Arm repeat 3 is neither necessary nor sufficient for the nuclear export of Imp
2 and
suggest that the published identification of this sequence
as the Imp
NES (Boche and Fanning, 1997
) is incorrect.
Rather, our data confirm the proposal by Kutay et al. (1997a)
that Imp
nuclear export is dependent on the interaction of Imp
with the CAS nuclear export factor and
map the Imp
sequences required for this interaction to
the conserved Imp
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 2 that mediate CAS binding, we wished to also determine which Imp
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
1 (Cortes et al., 1994
), little had been reported about the
identity of Imp
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
specifically (Prieve et al., 1996
; Sekimoto et al., 1997
).
Analysis of the sequence requirements for T NLS and
LEF-1 NLS binding by Imp 2 produced two surprising
findings. First, the Imp
2 sequences required for binding
to these two NLS sequences were clearly distinct, with T
NLS binding mapping between Imp
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
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
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
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
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
appear to make a significant contribution to NLS binding, this result is not, in retrospect, surprising.
Although the interaction of Imp with Imp
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
. Although it has been suggested that basic NLS binding to an Imp
/Imp
heterodimer is of higher affinity than binding by Imp
alone
(Rexach and Blobel, 1995
; Efthymiadis et al., 1998
), it is
nevertheless apparent that NLS binding by Imp
can be
readily detected in the nucleus of cells under conditions
where Imp
and Imp
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
/CAS complex in a process
analogous to its import by the Imp
/Imp
complex? One
possibility is that CAS binding and basic NLS binding by
Imp
is mediated by two distinct, mutually incompatible Imp
protein conformations, a mechanism which has also
been invoked to explain why Imp
is unable to simultaneously bind to Imp
and Ran-GTP. Alternately, CAS
binding and NLS binding might simply sterically interfere
with one another. In either case, CAS binding, and hence
Imp
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
preferentially, although the underlying
mechanism for this effect has remained unclear (Kutay et
al., 1997a
). Although the extensive overlap between the
Imp
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
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
2 (refer to Fig. 5). These data demonstrate
that Imp
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
-dependent NES.
After submission of this article, a paper was published
that bears on the issue of the functional domain organization of Imp 2. Specifically, Conti et al. (1998)
have published the crystal structure of a truncated form of yeast
Imp
bound to two molecules of an SV-40 T NLS peptide.
Although yeast Imp
is somewhat different in structure to
human Imp
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
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
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
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;
-gal,
-galactosidase;
CMV, cytomegalovirus;
GST, glutathione-S-transferase;
IBB, importin
binding domain;
Imp
, importin
;
Imp
, importin
;
NES, nuclear export signal;
NLS, nuclear localization signal;
T
NLS, SV-40 T antigen NLS.
![]() |
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