Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235
Nuclear protein import requires a nuclear localization signal (NLS) receptor and at least three other
cytoplasmic factors. The subunit of the NLS receptor,
Rag cohort 1 (Rch1), enters the nucleus, probably in a
complex with the
subunit of the receptor, as well as
other import factors and the import substrate. To learn more about which factors and/or events end the import
reaction and how the import factors return to the cytoplasm, we have studied nucleocytoplasmic shuttling of
Rch1 in vivo. Recombinant Rch1 microinjected into
Vero or tsBN2 cells was found primarily in the cytoplasm. Rch1 injected into the nucleus was rapidly exported in a temperature-dependent manner. In contrast, a mutant of Rch1 lacking the first 243 residues
accumulated in the nuclei of Vero cells after cytoplasmic injection. After nuclear injection, the truncated
Rch1 was retained in the nucleus, but either Rch1 residues 207-217 or a heterologous nuclear export signal,
but not a mutant form of residues 207-217, restored nuclear export. Loss of the nuclear transport factor RCC1
(regulator of chromosome condensation) at the nonpermissive temperature in the thermosensitive mutant cell line tsBN2 caused nuclear accumulation of wild-type Rch1 injected into the cytoplasm. However, free
Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported. These results suggested that RCC1 acts at an earlier step in Rch1 recycling, possibly the disassembly of an import complex
that contains Rch1 and the import substrate. Consistent
with this possibility, incubation of purified RanGTP
and RCC1 with NLS receptor and import substrate prevented assembly of receptor/substrate complexes or stimulated their disassembly.
NUCLEAR protein import in eukaryotic cells occurs
through openings in the nuclear envelope that
are spanned by nuclear pore complexes (NPC)1
(Forbes, 1992 It is likely that all of the soluble transport factors mentioned above enter the nucleus in a complex with the cargo
protein, although this has not been demonstrated directly.
While karyopherin The mechanism by which the NLS receptor subunits are
returned to the cytoplasm after completion of the import
reaction is still unknown. Export of karyopherin To study the requirements for nucleocytoplasmic shuttling of the NLS receptor We show here that recycling of Rch1 to the cytoplasm is
dependent on a nuclear export signal within residues 207 and 217 and on temperature. Loss of RCC1 activity leads
to accumulation of the receptor in the nuclei of microinjected tsBN2 cells, although RCC1 is not necessary for the
export reaction itself. We propose that the RCC1-catalyzed
exchange of GDP for GTP on Ran is necessary to destabilize or disassemble the import complex and is therefore required for recycling of karyopherin Plasmids and Proteins
Bacterial expression constructs for human Rch1 were derived by subcloning the Rch1 cDNAs coding for amino acids (aa) 33-529 or aa 244-529
(Cuomo et al., 1994 Maltose binding protein (MBP)-Rch1 fusion constructs were obtained
by PCR amplifying DNA encoding Rch1 residues 197-221 with the primers 5 SV40 T-antigen was purified from insect cells as described by Schneider et al. (1994) Bacterial Expression and Purification of Proteins
To purify nearly full-length (aa 33-529) and truncated (aa 244-529) Rch1
peptides, Ran, RCC1, and karyopherin Rch1185-529, Rch1NES-Rch1244-529, Rch1nes-Rch1244-529, and HIVNES-Rch1244-529 were purified as insoluble proteins. pET19 plasmids containing
the corresponding genes were transformed into BL21 (DE3). Expression
of the proteins, harvesting, and disruption of the cells were performed as
described above. The cell suspension was centrifuged, and proteins were
solubilized from the cell pellet in 8 M urea according to Podust et al.
(1995) MBP fusion proteins were expressed and purified according to a New
England Biolabs protocol (1994). After purification, proteins were dialyzed against 20 mM Hepes/KOH, pH 7.6, 10% glycerol, and 5 mM mercaptoethanol.
Cell Culture
BHK21 (Stoker and MacPherson, 1964 Microinjection
For microinjection experiments, cells were plated for at least 24 h (Vero)
or 36 h (tsBN2 and BHK21) before microinjection on glass coverslips. A
microinjector and a manipulator (models 5246 and 5171, respectively; Eppendorf Scientific, Inc., Madison, WI) mounted on an inverted microscope
(model IM35; Carl Zeiss, Oberkochen, Germany) were used to deliver
samples. All proteins were used at a concentration of 2-4 mg/ml and the
solutions were centrifuged for 30 min at 14,000 g before injection. Microinjection needles were pulled from glass capillaries (Clark Electromedical Instruments, Reading, UK) on an automatic pipette puller (Zeitz Instruments, Augsburg, Germany).
Immunofluorescent Staining
Microinjected cells were washed three times with PBS, fixed in 4% ice-cold paraformaldehyde in PBS for 15 min, permeabilized for 15 min in
0.2% Triton X-100, and blocked for 1 h in 10% FCS in PBS. T7-tagged
Rch1 proteins were visualized by staining for 2 h at room temperature
with a mouse monoclonal anti-T7 antibody (Novagen Corp., Madison,
WI) at a 1:500 dilution and a Cy3-linked secondary anti-mouse antibody
(Jackson ImmunoResearch Laboratories, West Grove, PA) at 1:200 for 1 h
at room temperature. MBP fusion proteins were detected with a polyclonal anti-MBP serum (New England Biolabs) at a 1:100 dilution followed by a Cy3-conjugated anti-rabbit antibody (Jackson ImmunoResearch Laboratories) at 1:200 for 1 h at room temperature. The coverslips
were mounted in 90% glycerol containing 0.1 mg/ml paraphenylenediamine in PBS (Johnson and Araujo, 1981) and viewed on a fluorescence
microscope (model Axiovert 35; Carl Zeiss) using a 63× objective.
Quantification
Quantification was done by capturing images of fluorescent cells with a
digital camera (CCD camera, model C 4880; Hamamatsu Phototonics,
Bridgewater, NJ). The amount of fluorescence in each fixed cell or in the
nucleus of the cell was evaluated using the Image-/MetaMorph Imaging
System (Universal Imaging Corp., West Chester, PA). Background measurements were obtained by evaluating the fluorescence of noninjected
fixed cells or nuclei.
In Vitro Assembly of NLS Receptor-Import
Substrate Complexes
Assembly of complexes was performed according to an ELISA protocol
(Goding, 1983 Subcellular Localization of Rch133-529 and Rch1244-529
To determine the subcellular localization of Rch133-529 and
Rch1244-529, each protein was microinjected into the cytosol of Vero cells. The cells were either fixed immediately after injection or after a 2.5-h incubation at 37°C. The exogenous Rch1 was visualized by staining its T7-tag with
anti-T7 antibodies. As shown in Fig. 1 A, a and b, Rch133-529
was localized predominantly in the cytoplasm at both time
points. Although cells injected with Rch1244-529 showed cytoplasmic staining immediately after injection (Fig. 1 A, c),
strong nuclear staining was observed 2.5 h later (Fig. 1 A,
d). The time course of nuclear uptake of the two proteins
was followed by injecting cells with either Rch133-529 or
Rch1244-529, fixing them at various time points as indicated (Fig. 1 B), and determining the amount of nuclear fluorescence as a percentage of the total cell fluorescence in the
injected cells. The graph shows that the fraction of nuclear
Rch133-529 remained at ~20% over a period of 2.5 h, consistent with the predominantly cytoplasmic importin
Export of Rch133-529 and Rch1244-529
To address the question of whether nuclear accumulation
of Rch1244-529 could be due to a defect in recycling of this protein to the cytoplasm, export of Rch133-529 and Rch1244-529 was examined more closely. The purified proteins were
microinjected into the nuclei of Vero cells, and the cells
were fixed either immediately after injection or after a 30-min
incubation at 37°C. Fig. 2 A shows that cells microinjected
with Rch133-529 showed almost exclusively nuclear staining
initially (Fig. 2 A, a) and strong cytoplasmic staining after
a 30-min incubation (Fig. 2 A, b). In sharp contrast, the
staining pattern of cells injected with Rch1244-529 was nuclear at both time points (Fig. 2 A, c and d). Analysis of
the Rch1 export kinetics (Fig. 2 B) demonstrated a rapid
decrease of nuclear fluorescence in cells injected with Rch133-529. However, the amount of nuclear Rch1244-529 fluorescence remained constant over the entire time period
(Fig. 2 B). These observations indicate that Rch133-529 injected into the nuclei of Vero cells was rapidly exported,
whereas Rch1244-529 was not. The results also suggest that a
polypeptide sequence between aa 1 and 244 of Rch1 may
be necessary to allow nuclear export of Rch1.
Another Rch1 deletion mutant, Rch1185-529, was used to
narrow down the region that might contain the information necessary for nuclear export of the NLS receptor subunit. Rch1185-529 was injected into the nuclei of Vero cells,
and the cells were fixed and stained either immediately after injection or after a 30-min incubation at 37°C. Fig. 3 A
shows that shortly after injection, Rch1185-529 was still in
the nucleus of injected cells (Fig. 3 A, a), whereas 30 min
later, translocation to the cytoplasm had occurred (Fig. 3
A, b). Comparison of the export kinetics of Rch1185-529 and
Rch133-529 (Fig. 3 B) confirmed that the rate of export of
Rch1185-529 was comparable to that of Rch133-529. These results support the idea that the sequence between aa 185 and 244 of Rch1 is necessary to direct nuclear export of
Rch1.
Rch1 Contains a Nuclear Export Signal
To examine the possibility that a nuclear export signal
could reside in the region between amino acids 185-244,
the cDNA sequences of the mouse, human, Xenopus, and
yeast NLS receptor To determine which sequence in the region between aa
185-244 was necessary to direct nuclear export of Rch1,
two DNA fragments from this region were chosen, one including the putative NES (coding for aa 197-217) and a
second, downstream from the putative NES (coding for aa
218-244). The fragments were obtained by PCR and cloned into the pMal-vector to allow expression of a fusion
construct between the MBP, a 42-kD bacterial protein,
and the Rch1 cDNA fragment.
The proteins MBP197-217 and MBP218-244 were then injected into the nuclei of BHK21 cells, and the cells were
fixed at different time points after injection to monitor the
export reaction. As a control, MBP was also injected. Immunofluorescence was performed using a polyclonal anti-MBP serum and a secondary antibody coupled to Cy3.
The amount of nuclear fluorescence was measured using
quantitative fluorescent microscopy.
The fluorescent micrographs (Fig. 4 A) show that both
MBP and MBP218-244 were nuclear at the 0-min (Fig. 4 A, a
and c) and the 10-min (Fig. 4 A, b and d) time points. In
contrast, cells injected with MBP197-217 showed nuclear
staining only at the 0-min time point (Fig. 4 A, e) and cytoplasmic staining after a 10-min incubation (Fig. 4 A, f).
The export kinetics shown in Fig. 4 B confirm that neither
MBP nor MBP218-244 were exported over the time period
monitored. However, MBP197-217 was very rapidly exported
from the nucleus to the cytoplasm. We therefore conclude
that the amino acids 197-217, encompassing the putative
NES, were sufficient to direct nuclear export of MBP.
In another set of experiments, the putative nuclear export
signal was directly investigated. A cDNA fragment coding
for amino acids 207-217 was cloned into the Rch1244-529
pET19-vector, fusing residues 207-217 to the amino terminus of Rch1244-529 and resulting in Rch1NES-Rch1244-529.
Similarly, a mutant form of the putative NES with the conserved leucines 110 and 112 exchanged for alanine and glycine (L110A, L112G) was fused to the amino terminus of
Rch1244-529, resulting in Rch1nes-Rch1244-529. As a positive
control for this experiment, sequences encoding the NES
of the HIV Rev protein were cloned into the same site of
Rch1244-529. It was shown earlier that the Rev NES, when coupled to BSA, promoted its export after injection of
NES-BSA into Xenopus oocytes or mammalian cell nuclei
(Fischer et al., 1995 Nuclear export of Rch1NES-Rch1244-529, Rch1nes-Rch1244-529,
and HIVNES-Rch1244-529 was then assayed after injection
into nuclei of Vero cells. Cells were fixed either at a 0-min
time point or 30 min later, followed by immunofluorescent staining of the exogenous protein. The fluorescent micrographs in Fig. 5 A demonstrate that cells injected with either Rch1NES-Rch1244-529 or HIVNES-Rch1244-529 showed
nuclear staining immediately after injection (Fig. 5 A, a
and c) and cytoplasmic staining after a 30-min incubation
(Fig. 5 A, b and d), but cells injected with Rch1nes-Rch1244-529 showed nuclear staining at both time points
(Fig. 5 A, e and f). To monitor the time course of the export reaction, cells were fixed at different time points after injection, and immunofluorescence was performed using
the anti-T7 antibody. The amount of nuclear fluorescence
was determined and plotted against time after microinjection. As a control, Rch1244-529 was also injected. The export
kinetics in Fig. 5 B show that although Rch1244-529 and
Rch1nes-Rch1244-529 were not exported, Rch1NES-Rch1244-529
was exported at a rate very similar to that of HIVNES-Rch1244-529. These results demonstrate that export of the
deletion mutant was restored not only by the established
nuclear export signal of HIV-Rev (Fischer et al., 1995
Export of Rch1 Is Temperature Dependent
If export of Rch1 from the nucleus depends on the nuclear
export signal that is deleted in Rch1244-529, one might also expect the export process, like the nuclear import process,
to be temperature dependent. To address this question, we
tested export of the NLS receptor
Role of RCC1 in Receptor Recycling
Addition of RanGTP to the heterodimeric complex of
karyopherin
The import kinetics of Rch1 in the presence of RCC1
demonstrated that Rch133-529 was mostly cytoplasmic in
tsBN2 cells incubated at 33.5°C (Fig. 7 B). The nuclear accumulation of Rch1 in the absence of RCC1 (Fig. 7 B)
demonstrates that RCC1 is required directly or indirectly
for recycling of Rch1 to the cytoplasm. In a control experiment using the parental cell line BHK21, Rch133-529 was mostly cytoplasmic at both temperatures (Fig. 7 B), demonstrating that the altered distribution of Rch1 at the restrictive temperature is caused by the RCC1 mutation
rather than by the elevated temperature.
RCC1 Is Not Required for Export of Rch1
Two simple alternatives can be proposed for the role of
RCC1 in receptor recycling. In one model, transport of
Rch1 back into the cytoplasm might require RanGTP generated by RCC1. Loss of RCC1 would lead to depletion of
RanGTP in the nucleus, and subsequently to a defect in
receptor recycling. If this idea were correct, export of
Rch1 should be inhibited when Rch1 was injected into nuclei of tsBN2 cells that had been incubated at 39.5°C before microinjection and therefore lacked active RCC1. In
the other model, RCC1 would act at a step before export
of Rch1, for example in the disassembly of the import
complex. Rch1 accumulated in tsBN2 nuclei at the restrictive temperature after cytoplasmic injection would then be
part of a nondisassembled import complex. If this possibility were correct, free Rch1 injected directly into the nucleus should be exported in a fashion independent of RCC1.
To differentiate between these two possibilities, we injected Rch133-529 into the nuclei of tsBN2 cells that had
been incubated at the permissive and restrictive temperatures. The cells were fixed either immediately after injection (Fig. 8 A, a and c) or 30 min later (Fig. 8 A, b and d),
and Rch133-529 was visualized by immunofluorescence. At
both temperatures, Rch133-529 injected into the nuclei was
found in the cytoplasm after a 30-min incubation. The time
course (Fig. 8 B) shows clearly that nuclear export kinetics of Rch1 were identical in the presence or absence of
RCC1. In a control experiment in BHK21 cells, Rch1 export kinetics were also identical at 33.5 and 39.5°C (Fig. 8
B). No export of Rch1 to the cytoplasm was observed at
0°C (data not shown), consistent with the temperature dependence of the Rch1 export reaction (Fig. 6). These results rule out a requirement for RCC1 in the export reaction itself and support the idea that it is required in an earlier step of receptor recycling.
RCC1 Destabilizes the NLS Receptor/Substrate
Complex In Vitro
The results presented in Fig. 8 suggested that RCC1 was
not required for nuclear export of free Rch133-529 in vivo, but rather for a step before export. One step that must occur before Rch1 recycling is the disassembly of the NLS
receptor To develop a method to test this possibility, we first assembled NLS receptor/substrate complexes using purified
proteins in a solid phase assay. Karyopherin
The influence of RanGDP and RanGTP on the assembly or stability of the NLS receptor/substrate complexes
was then tested. RanGDP in the presence of GDP and
Mg2+ did not affect the assembly of NLS receptor/substrate complexes (Fig. 9 C, compare column 1 with column
3). When RanGTP was added, NLS receptor/T-antigen
complex formation was slightly reduced in the presence of
Mg2+ and GTP (Fig. 9 C, column 5). If Ran was loaded
with GTP These results confirm that RanGTP prevents formation
of the NLS receptor complex or induces its disruption as
reported (Rexach and Blobel, 1995 Karyopherin In this report, we have investigated the requirements in
vivo for recycling of the The importin The IBB domain is completely absent in the Rch1244-529
deletion mutant, precluding its activity in nuclear protein
import. Nevertheless, nuclear entry of this protein was still
observed. One possible explanation for this apparent contradiction is that Rch1244-529 might enter the nucleus piggyback on a protein with more than one NLS. Rch1244-529 is
capable of binding to NLSs in a transport substrate (Cuomo
et al., 1994 In our experiments, Rch1244-529 was found to accumulate
in the nuclei over a 2.5-h period (Fig. 1), while Gallay et al.
(1996) Compared with the nuclear export of pyruvate kinase,
nuclear export of Rch133-529 was very rapid (Fig. 2). The
high concentration of NLS receptor The pathway of karyopherin A Role for RCC1 in Rch1 Recycling to the Cytoplasm
RCC1 was also found to be a requirement for the recycling
of the NLS receptor On the other hand, export of free receptor from the nucleus to the cytoplasm was not dependent on RCC1 (Fig.
8). Since inactivation of the RCC1 homologue prp20 in
yeast also resulted in nuclear accumulation of the yeast
Rch1 homologue (Koepp at al., 1996), the authors speculated that RanGTP supplied by prp20 might be necessary
for the exit of the yeast NLS receptor RanGTP, but not RanGDP, was reported to destabilize
the karyopherin ; Rout and Wente, 1994
) and is characterized
by an energy requirement, signal dependence, and receptor involvement. In vitro assays using semipermeabilized
cells have allowed the identification of the soluble factors
essential for nuclear protein import (Adam et al., 1990
;
Newmeyer and Forbes, 1990
; Moore and Blobel, 1992
),
and studies of their interactions with each other and with
nuclear pore proteins have led to models of the protein import pathway that suggest several distinct steps. In the
cytoplasm, the
subunit of the karyopherin heterodimer,
also called importin
, recognizes the nuclear localization
signal (NLS) (for review see Dingwall and Laskey, 1991
;
Garcia-Bustos et al., 1991
) of a karyophilic protein in a
temperature- and hence energy-independent manner. Subsequently the karyopherin
subunit mediates docking of
the complex to the cytoplasmic periphery of the nuclear
pore (Adam and Adam, 1994
; Görlich et al., 1994
, 1995a
;
Imamoto et al., 1995a
,b; Moroianu et al., 1995a
,b; Radu et
al., 1995
; Weis et al., 1995
). Ran, a member of the Ras superfamily of guanine nucleotide binding proteins, binds in
its GTP form to RanBP2 close to this initial docking site
(Melchior et al., 1995
). GTP hydrolysis of RanGTP, which
has been shown to be necessary for translocation of the
import substrate (Melchior et al., 1993
; Moore and Blobel, 1993
; Schlenstedt et al., 1995
), occurs at the same site and
probably allows the undocking of the karyopherin/substrate complex. This GTP hydrolysis is stimulated by
RanGAP1 at the nuclear envelope (Bischoff et al., 1994
,
1995a
,b; Corbett et al., 1995
; Matunis et al., 1996
; Mahajan
et al., 1997
). Possible links between Ran and the karyopherin/substrate complex are pp15 (Grundmann et al., 1988
; Moore and Blobel, 1994
; Paschal and Gerace, 1995
;
Nehrbass and Blobel, 1996
; Paschal et al., 1996
) and
RanBP1 (Chi et al., 1996
). The import complex is proposed
to move to the immediate vicinity of the nuclear pore before
translocation into the nucleus via several binding sites for
pp15 and karyopherin
(Moroianu et al., 1995b
; Paschal
and Gerace, 1995
; Radu et al., 1995
; Nehrbass and Blobel,
1996
).
remains bound to the nuclear side of
the NPC and is very rapidly reexported (Görlich et al.,
1995b
; Moroianu et al., 1995b
), the transport substrate,
karyopherin
, RanGDP, and pp15 appear to enter the
nucleoplasm. However, it is not known how or when the dissociation of the import complex is induced. Considering
that directionality must be maintained during transport,
dissociation may occur solely as the termination step of
the import reaction in the nucleus (Melchior and Gerace,
1995
; Görlich and Mattaj, 1996
). Alternatively, reiterated
dissociation and association reactions may occur during
translocation through the pore (Rexach and Blobel, 1995
;
Görlich et al., 1996b
). Recent data show that RanGTP destabilizes the
-heterodimer of karyopherin (Rexach and
Blobel, 1995
). Exchange of GDP for GTP on Ran in the
nucleus could therefore induce the dissociation of the import complex. The now free import substrate would be retained in the nucleus by association with, for example,
DNA or chromatin, while the import factors could be recycled. The GDP/GTP exchange factor for Ran in the nucleus is RCC1 (regulator of chromosome condensation) (Bischoff and Ponstingl, 1991a
,b; for review see Dasso,
1993
), first identified as the mutant gene in tsBN2 cells, a
thermosensitive mutant line derived from BHK21 cells
(Nishimoto and Basilico, 1978
; Nishimoto et al., 1978
).
is faster
and therefore apparently distinct from that of karyopherin
(Görlich et al., 1995b
; Moroianu et al., 1995b
). Both subunits are too large to return to the cytoplasm by simple diffusion and must migrate against a concentration gradient
since the NLS receptor resides mostly in the cytoplasm.
These observations suggest that they probably require active reexport. It is also clear that the mechanism of nuclear
exit of karyopherin
must be different from its entry, as it
returns to the cytoplasm without the NLS-bearing protein. Consistent with this, the importin
-binding domain (IBB
domain) of karyopherin
is responsible for nuclear entry
of the NLS receptor
subunit but does not mediate its export (Görlich et al., 1996a
; Weis et al., 1996
).
subunit in vivo, we used two
forms of this protein, Rch133-529 and Rch1244-529. Both were
originally identified through their interaction with the
RAG-1 recombination-activating protein (Cuomo et al.,
1994
) and have been shown to interact directly and specifically with NLSs (Cuomo et al., 1994
; Gallay et al., 1996
).
Rch133-529 was reported to substitute for karyopherin
1 in
an in vitro nuclear import assay (Moroianu et al., 1995b
), whereas Rch1244-529 inhibited nuclear transport of HIV-1 matrix protein when coexpressed in human 293 cells with
HIV-1 matrix protein cDNA (Gallay et al., 1996
) and also
inhibited RAG-1-mediated recombination in vivo (Cuomo
et al., 1994
). The mechanism of this inhibition is not known.
to the cytoplasm.
Materials and Methods
) from a pET21a vector into pET19a using NdeI and
XhoI sites from the pET19a multiple cloning site. The resulting fusion
constructs contained NH2-terminal T7- and His-tags. The Rch1 cDNA encoding for aa 185-529 (Cuomo et al., 1994
) was cloned from a yeast expression vector (Cuomo et al., 1994
) into the pET21a vector using EcoRI
and XhoI restriction sites and subsequently into the pET19a vector using
NdeI and XhoI restriction, resulting in a construct analogous to that for aa 33-529 or aa 244-529. For nuclera export signal (NES)-Rch1 constructs, the following oligonucleotides were used: 5
-TAT GCT CCC TCC TCT AGA ACG ACT CAC GCT ACA and 5
-TAT GTA GCG TGA GCT
GTT CTA GAG GAG GGA GCA for HIVNES-Rch1244-529; 5
-TAT
GGC AGT GGA TCC ACT GTT GGC TCT CCT TGC AGT TCC TCA
and 5
-TAT GAG GAA CTG CAA GGA GAG CCA ACA GTG GAT
CCA CTG CCA for Rch1NES-Rch1244-529; 5
-TAT GGC AGT GGA
TCC ACT GGC AGC TGG ACT TGC AGT TCC TCA and 5
-TAT
GAG GAA CTG CAA GTC CAG CTG CCA GTG GAT CCA CTG
CCA for Rch1nes-Rch1244-529. They were constructed by annealing the
two complementary oligonucleotides and cloning them into the NdeI site
of pET19a containing the Rch1244-529 cDNA. Correct orientation of the inserted fragment was verified by DNA sequencing. The karyopherin
cDNA in pET30a (Chi et al., 1995
) was a generous gift from S. Adam
(Northwestern University, Chicago, IL). Ran wt cDNA (Ren et al., 1993
)
was cloned from pET9c into pET15a using NdeI and BamHI restriction.
The RCC1 cDNA (Ohtsubo et al., 1987
) was cloned from pET3b (T. Dobner, University of Regensburg, Regensburg, Germany) into the pET15a
vector via NdeI and BamHI restriction sites.
-TTT GAA TTC GAC TTG GTT ATT AAG TAC GGT G and
5
-TTT TCT AGA TAA AGA TGA CAT ATC AGG AAC TG and residues 218-244 using the primers 5
-TTT GAA TTC ATG TCA TCT TTA
GCA TGT GGC and 5
-TTT TCT AGA GGT GCA GGA TTC TTG
TTG C. The cDNA encoding Rch133-529 in pET19 served as the template.
The PCR fragments were cloned into the pMal-Vector (New England Biolabs, Beverly, MA) using EcoRI and XbaI restriction sites. The DNA
fragments were sequenced and plasmids containing the correct sequence
were transformed into BL21 bacteria for expression.
.
, the corresponding pET plasmids were transformed into Escherichia coli BL21 (DE3) containing the
pLys plasmid. Bacteria were grown in 1000 ml Luria-Bertani broth to an
OD600 of 0.7 at 30°C (37°C for Ran wt and RCC1) and expression was induced by addition of 2 mM IPTG for 3 h. Cells were harvested by centrifugation and frozen in 0.2 M Tris-HCl, pH 8.0, 0.5 M NaCl, 20 µg/ml leupeptin, 2 mM PMSF, and 1 mg/ml aprotinin. Cells were thawed at 37°C
and disrupted by ultrasonification after a 15-min incubation at 4°C. To the
cleared supernatant of the cell lysates, imidazole, pH 7.6, was added to a
final concentration of 30 mM and the solution was loaded on 0.5 ml Ni-NTA-Sepharose (Qiagen, Chatsworth, CA) equilibrated in 0.5 M NaCl
and 30 mM imidazole, pH 7.6. After extensive washing with 30 and 60 mM
imidazole, pH 7.6, and 0.5 M NaCl, proteins were eluted by step elution
with 0.5 M imidazole, pH 7.0, and 0.5 M NaCl, and pooled fractions were
dialyzed overnight against 20 mM Hepes/KOH, pH 7.6 (Rch1 derivatives,
Ran, karyopherin
), or 20 mM Tris-HCl, pH 7.6 (RCC1), 10% glycerol,
and 5 mM mercaptoethanol.
. Solubilized proteins were dialyzed to remove the urea, and the protein-containing solution was applied to Ni-NTA-agarose as described above. The eluate was adjusted to 5% glycerol and then dialyzed against
20 mM Hepes/KOH, pH 7.6, 10% glycerol, and 5 mM mercaptoethanol.
), tsBN2 cells, a temperature-sensitive mutant line derived from BHK21 cells (Nishimoto and Basilico,
1978
), and Vero cells (Earley and Johnson, 1988
) were grown in DME
(GIBCO BRL, Gaithersburg, MD) supplemented with antibiotics, 10%
FCS (5% for BHK21 cells) (GIBCO BRL), and (for Vero cells) nonessential amino acids (GIBCO BRL) in a humidified incubator at 37°C (Vero)
or 33.5°C (tsBN2 and BHK21) under a 10% CO2 atmosphere.
). ELISA immunoplates (Nunc, Rochester, NY) were
coated with 2 µg of karyopherin
in 50 µl of PBS for 2 h at room temperature, followed by saturation of residual nonspecific binding sites with 3%
BSA in PBS for 1 h at room temperature. The soluble proteins were
added in the following amounts: 1 µg of Rch133-529, 1 µg SV-40 T-antigen,
2 µg of Ran (RanGDP, RanGTP, or RanGTP-
S), and 2 µg of RCC1, as
indicated in the figure legend. Additionally, the corresponding nucleotides were present at 2 mM. The binding reaction was performed for 2 h
at room temperature in binding buffer (Bischoff and Ponstingl, 1991b
) (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM DTT, and 5 mM MgCl2). The
plates were washed three times briefly and three times for 10 min each,
and either anti-T-antigen antibody Pab101 (Gurney et al., 1980
) (1 µg/
well in 50 µl of PBS) or anti-T7 antibody (0.1 µl/well in 50 µl of PBS) was
added and incubated overnight, followed by washing of the plates as
above and incubation with 1 µl/well peroxidase-coupled anti-mouse secondary antibody (Jackson ImmunoResearch) in 100 µl of PBS for 1 h at
room temperature. Plates were washed again and the ELISA was developed
by adding 100 µl of peroxidase substrate (50 mM potassium phosphate,
pH 5.7, 0.03% H2O2, 2 mM 2,2
-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid; Sigma Chemical Co., St. Louis, MO) for 5 min. The absorbance of the
reaction product was measured at 405 nm using a reference filter at 595 nm
in an ELISA reader (model MR5000; DYNEX Technologies, Chantilly, VA).
Results
observed in Xenopus cells (Görlich et al., 1996a
). However,
nuclear staining of Rch1244-529 increased slowly from 12 to
38% over the same period of time. Since nuclear protein
import took place over the entire 2.5-h period, these results suggest that Rch133-529 was rapidly recycled to the cytoplasm, but Rch1244-529 was not.
Fig. 1.
(A) Rch1244-529 accumulates in the nuclei of Vero cells in vivo. Vero cells were injected into the cytoplasm with either Rch133-529
(a and b) or Rch1244-529 (c and d) and fixed immediately (a and c) and 2.5 h (b and d) after injection. Immunofluorescence was performed using an antibody against the T7-tag of the extracellular Rch1 proteins and a Cy3-coupled secondary antibody. Micrographs
were taken with a digital camera mounted on a fluorescence microscope. (B) Time course of nuclear accumulation of Rch1244-529. Vero
cells were injected either with Rch133-529 or Rch1244-529 and fixed at various time points as indicated on the x-axis. Cells were immunostained, and the amount of Rch1 fluorescence was measured as described in Materials and Methods. The staining of nuclear Rch1 is expressed as the percentage of total cell fluorescence residing in the nucleus and plotted against time after microinjection. For each time
point, the average value obtained from 10-20 cells in two to three independent experiments is shown; the standard deviation of the
mean is indicated by error bars. Bar, 25 µm.
[View Larger Versions of these Images (83 + 23K GIF file)]
Fig. 2.
(A) Rch1244-529 is defective in nuclear export. Rch133-529 (a and b) and Rch1244-529 (c and d) proteins were injected into nuclei of
Vero cells. Fluorescence micrographs show cells fixed and stained 0 min (a and c) or 30 min (b and d) after injection. (B) Export kinetics
of Rch1. Vero cells were injected into the nucleus with either Rch133-529 or Rch1244-529 and incubated for the indicated time periods.
Quantification was performed as in Fig. 1 B. Bar, 25 µm.
[View Larger Versions of these Images (20 + 74K GIF file)]
Fig. 3.
(A) Rch1185-529 is efficiently exported from the nucleus.
Rch1185-529 was injected into the nuclei of Vero cells, which were
fixed and immunostained 0 min (a) or 30 min (b) later. (B) Cells
were injected into the nucleus with either Rch1185-529 or Rch133-529
to compare export kinetics of the two proteins. After injection,
cells were fixed and stained. Nuclear fluorescence was determined and plotted against time after microinjection as in Fig. 1 B.
Bar, 25 µm.
[View Larger Versions of these Images (38 + 19K GIF file)]
subunit were first compared and
screened for conserved leucines in a hydrophobic environment, a motif common to several nuclear export sequences (Gerace, 1995
; Nigg, 1997
). The region between aa 207 and 217 met these characteristics: DPLPLLALLAVPD.
The amino acids shown in bold are conserved and amino
acids shown in italics are similar among the species mentioned above.
Fig. 4.
(A) Export of MBP-Rch1 fusion constructs. BHK21
cells were injected into the nucleus with purified proteins MBP (a
and b), MBP fused to Rchl residues 218-244 (c and d), or MBP
fused to Rchl residues 197-217 (e and f), fixed 0 min (a, c, and e)
or 10 min (b, d, and f) later, and examined by immunofluorescence. (B) Export kinetics of MBP-Rch1 fusion constructs. After
nuclear injection of MBP, MBP218-244, and MBP197-217, the cells
were fixed and immunostained using an anti-MBP polyclonal serum. Nuclear fluorescence at different times after injection was
determined and plotted against time as in Fig. 1 B. Bar, 25 µm.
[View Larger Versions of these Images (21 + 84K GIF file)]
).
),
but also by amino acids 207-217 of Rch1, indicating that they function as a nuclear export signal. Leucines 110 and
112 of Rch1 appear to be necessary for this function.
Fig. 5.
(A) Export of Rch1244-529 is restored by a nuclear export signal. HIVNES-Rch1244-529 (a and b), Rch1NES-Rch1244-529 (c and d), and Rch1nes-Rch1244-529 (e and f) were injected into nuclei of Vero cells. Cells were fixed either at a 0-min time point (a,
c, and e) or 30 min later (b, d, and f). Immunofluorescence was performed using an anti-T7 antibody. (B) Export kinetics
of Rch1244-529, HIVNES-Rch1244-529, Rch1NES-Rch1244-529, and
Rch1nes-Rch1244-529. Nuclear fluorescence at different time points
after injection was determined as described (Fig. 1 B). Bar, 25 µm.
[View Larger Versions of these Images (76 + 27K GIF file)]
subunit at 0°C. Vero
cells were shifted to 0°C immediately before nuclear microinjection of Rch133-529, and Rch1244-529 and then incubated on ice for various time periods. Cells were fixed and
the exogenous tagged Rch1 was visualized by immunofluorescence. Cells injected with both forms of Rch1 showed
nuclear staining not only immediately after injection (Fig. 6 A, a and c), but also after a 30-min incubation on ice
(Fig. 6 A, b and d). Quantitative evaluation of nuclear fluorescence in cells injected with Rch133-529 and Rch1244-529
confirmed that the intensity of nuclear fluorescence was
constant over the 30-min time period (Fig. 6 B). These results support the idea that recycling of Rch1 is temperature dependent and show that neither Rch133-529 nor
Rch1244-529 can exit the nucleus by simple diffusion.
Fig. 6.
(A) Export of Rch1 is temperature dependent. Rch133-529 and Rch1244-529 were injected into the nuclei of Vero cells at 0°C. The
cells were either fixed (a and c) or incubated on ice for 30 min and then fixed (b and d). Cells were immunostained and fluorescent micrographs were taken. (B) Export of Rch1 is impaired at 0°C. The graph shows the quantification of the nuclear fluorescence of cells injected into the nucleus with Rch133-529 or Rch1244-529 and incubated on ice as indicated on the x-axis before fixing and immunostaining.
[View Larger Versions of these Images (19 + 74K GIF file)]
and
leads to the dissociation of the heterodimer in vitro (Rexach and Blobel, 1995
; Görlich et al.,
1996b
). The fact that the nuclear protein RCC1 is the only
known Ran guanine exchange factor, and plays a role in
nuclear protein import in vivo (Tachibana et al., 1994
;
Dickmanns et al., 1996
), suggested that RCC1 could be involved in the release of Rch1 from the import complex by providing RanGTP. Since the disassembly of the import
complex must be a prerequisite for the recycling of the import factors, RCC1 would then indirectly affect the shuttling of the NLS receptor
subunit. To investigate the possible effect of RCC1 on receptor recycling, we used tsBN2
cells, mutant derivatives of BHK21 cells that express thermolabile RCC1 (for review see Dasso, 1993
). To compare
shuttling of Rch1 in the presence and absence of RCC1, Rch133-529 was injected into the cytoplasm of tsBN2 cells. The cells were then fixed immediately or incubated for 4 h
at either the restrictive or permissive temperature. The
subcellular distribution of Rch133-529 was then determined
by immunofluorescent staining. Fig. 7 A shows that in the
presence of RCC1, cells displayed the predominantly cytoplasmic staining typical of injected Rch133-529 (Fig. 7 A, a
and b). In contrast, Rch1 was predominantly nuclear in the
absence of RCC1 (Fig. 6 A, compare b [33.5°C] and d
[39.5°C]).
Fig. 7.
(A) Effect of RCC1 depletion on Rch1 recycling. tsBN2
cells were injected with Rch133-529 into the cytoplasm followed by
incubation at 33.5°C (a and b) or 39.5°C (c and d). Fluorescence micrographs show the localization of Rch1 in cells that were fixed after 0 min (a and c) or 4 h (b and d) of incubation under the conditions indicated. (B) Time course of nuclear accumulation of cytoplasmically injected Rch133-529 in the absence and presence of
RCC1. tsBN2 and BHK21 cells were injected into the cytoplasm
with Rch133-529 and incubated at 39.5°C or, as a control, at 33.5°C
for various time periods, fixed and stained. Quantification was
done as in Fig. 1 B and the percentage of Rch1 in the nucleus was
expressed as a function of time after injection. Bar, 25 µm.
[View Larger Versions of these Images (61 + 23K GIF file)]
Fig. 8.
(A) Export of free Rch1 is not dependent on RCC1.
Rch133-529 was injected into the nuclei of tsBN2 cells preincubated for 4 h at 33.5 or 39.5°C. Cells were fixed and stained immediately after injection (a and c) or incubated for 30 min before fixing and
immunostaining (b and d). (B) Export of free Rch1 is independent of RCC1. tsBN2 and, as a control, BHK21 cells, were incubated for 4 h at 33.5 or 39.5°C before nuclear injection with
Rch133-529. For injections at 0°C, cells were first incubated at
33.5°C, but shifted to 0°C immediately before injection. After incubation at the indicated temperatures for the indicated time periods, cells were fixed and stained. Quantification was performed
as described in Fig. 1 B. Bar, 25 µm.
[View Larger Versions of these Images (26 + 45K GIF file)]
complex with the imported cargo protein.
RanGTP was reported to disassemble the NLS receptor
complex in vitro (Rexach and Blobel, 1995
) by binding
to karyopherin
at a binding site that overlaps with the
karyopherin
binding site (Moroianu et al., 1996b
; Görlich et al., 1996b
). These results supported the idea that the
role of RCC1 might be to destabilize an NLS receptor/substrate complex in the nucleus.
and, as a
control, BSA were immobilized on ELISA plates and soluble Rch133-529 was added. After washing, bound Rch133-529
was detected using the anti-T7 antibody and a peroxidase-coupled secondary antibody. Fig. 9 A shows that Rch133-529
was bound in the presence of karyopherin
, but not in the
presence of BSA, indicating that Rch133-529 bound specifically to karyopherin
. To test whether the immobilized
Rch133-529/karyopherin
complexes were able to bind
T-antigen as an NLS-bearing protein, we immobilized karyopherin
in the ELISA plate and then added either
T-antigen alone or T-antigen together with Rch133-529.
Bound T-antigen was detected using a specific monoclonal
antibody and a peroxidase-coupled second antibody. Fig. 9
B shows that bound T-antigen was detectable in the presence of karyopherin
and Rch133-529 but not with karyopherin
alone, indicating the assembly of an
NLS receptor complex with the transport substrate T-antigen.
Fig. 9.
RCC1 inhibits formation of the NLS receptor/substrate
complex. (A) Karyopherin was immobilized on an ELISA plate
before addition of different amounts of Rch133-529, as indicated on the x-axis of the graph. Plates were washed and Rch1 bound to
the solid phase was detected in a colorimetric reaction. (B) Karyopherin
was immobilized on ELISA plates. T-antigen was
added in the amounts indicated on the x-axis of the graph, either
alone or together with Rch133-529. Plates were washed, and the amount of T-antigen bound to the solid phase was determined in a colorimetric reaction. (C) Karyopherin
was immobilized on ELISA immunoplates before addition of either Rch133-529 and T-antigen (columns 1 and 3-7) or T-antigen alone (column 2).
The binding reactions were then supplemented with RanGDP
and GDP (column 3); RanGDP, GDP, and RCC1 (column 4);
RanGTP and GTP (column 5); RanGTP-
S and GTP-
S (column 6); or RanGTP, GTP, and RCC1 (column 7). MgCl2 was
present in all reactions at 5 mM. The T-antigen bound to each
solid phase was determined in a colorimetric reaction. The columns
depict the mean values from at least two separate experiments;
the standard deviation of the mean is indicated by error bars.
[View Larger Versions of these Images (9 + 40K GIF file)]
-S, a nonhydrolyzable GTP analogue, complex
formation was strongly inhibited in the presence of Mg2+
and GTP
-S (Fig. 9 C, compare columns 5 and 6). However, if RCC1 and GTP were present, NLS receptor complex formation was inhibited by ~60% (Fig. 9 C, column
7), while RCC1 in the presence of GDP had no effect (Fig.
9 C, column 4). These results indicate that when RanGTP
was added without RCC1, GTP hydrolysis on Ran occurred, but GDP bound to Ran was not exchanged for
GTP in the presence of Mg2+ (Bischoff and Ponstingl,
1991b
), and therefore did not destabilize or impair formation of the NLS receptor/T-antigen complex. On the other
hand, when RanGTP, RCC1, and GTP were added, GDP
bound to Ran after GTP hydrolysis was exchanged for GTP.
; Moroianu et al., 1996b
)
and demonstrate that RCC1-catalyzed exchange of GDP
for GTP on Ran inhibited formation of the NLS receptor/
substrate complex or dissociated the existing complexes.
Discussion
Sequences Required for Recycling to
the Cytoplasm
subunit of the NLS receptor
(Rch1) from the nucleus to the cytoplasm. Rch1 was found
predominantly in the cytoplasm with only ~20% of the exogenous
subunit localized to the nucleus in Vero cells
and ~30% in tsBN2 cells. These results are in accordance
with the subcellular distribution of the endogenous
subunit, which was found in higher concentrations in the cytoplasm than in the nucleus in Xenopus X1177 cells (Görlich
et al., 1996a
). This similarity indicates that Rch133-529 behaves in these assays much like the endogenous
subunit.
-binding domain of karyopherin
(Görlich et al., 1996a
; Weis et al., 1996
) was mapped to residues
1-65 of importin
and required aa 21-65 or 1-52. In contrast, Rch133-529 was reported to support nuclear import in
vitro as efficiently as full-length karyopherin
1 (Moroianu et al., 1995b
), and even a karyopherin
1 mutant
lacking the first 49 amino acids was reported to bind to
karyopherin
in vitro, albeit with reduced efficiency (Moroianu et al., 1996a
). Another group observed nuclear
entry of Rch133-529 in vivo (Gallay et al., 1996
), consistent with nuclear import activity. The experimental variables
that give rise to the discrepancies between these reports
have not been resolved. However, our experiments showed
nuclear entry and exit of Rch133-529 in vivo (Figs. 1, 2, and
7), as expected for an
subunit active in nuclear protein
import, and our in vitro binding assays confirmed interaction of Rch133-529 with both karyopherin
and an NLS-bearing cargo protein as expected for an NLS receptor
complex (Fig. 9).
), and a substrate with multiple NLSs could still
interact with endogenous karyopherin
after binding one
molecule of Rch1244-529. The endogenous NLS receptor
subunit could thus mediate the transport reaction. However, our data do not rule out the possibility that Rch1244-529 could enter the nucleus via diffusion.
reported that Rch1244-529 was exclusively cytoplasmic at ~48 h after the corresponding cDNA was transfected into 293S cells. This discrepancy might be explained
by the very different times at which localization was assayed. When cells were microinjected with cDNA encoding Rch1244-529 and incubated for extended time periods,
nuclear accumulation of the protein was initially observed
for ~4-5 h, but after that nuclear staining was reduced (data not shown). Slow leaking from the nucleus has been
observed with other proteins. For example, pyruvate kinase injected into the nuclei of Xenopus oocytes was gradually transported out of the nucleus (Schmidt-Zachmann
et al., 1993
).
subunit in the cytoplasm, which probably depends on the fast export reaction
from the nucleus, could be very important in maintaining
directionality in the import reaction. Rch1244-529, however,
proved to be deficient in this fast nuclear export reaction
(Fig. 2). The fast nuclear exit of Rch1 is mediated by a sequence in the region between aa 207 and 217 (Figs. 4 and
5) that probably carries an NES since the HIV Rev NES
fused to Rch1244-529 restored nuclear export of the deletion
mutant to a very similar extent as aa 207-217 (Fig. 5). Typically, NESs contain several Leu-residues and are rich in
hydrophobic amino acids (for review see Gerace, 1995
; Nigg, 1997
). The region 207-217 in Rch1 shows similar
characteristics: Rch1, DPLPLLALLAVPD; PKI NES,
LALKLAGLDI (Wen et al., 1995
); and Rev NES, LPPLERLTL (Fischer et al., 1995
; Wen et al., 1995
). Interestingly, the leucine residues indicated in bold are conserved
among human Rch1, mouse importin, Xenopus importin 1, and yeast SRP1p
subunits, and residues indicated in italics are similar (Yano et al., 1992
; Cuomo et al., 1994
; Görlich et al., 1994
; Imamoto et al., 1995a
), consistent with
their probable importance in Rch1 function. We have
shown here that the conserved leucines are necessary for
the NES activity of amino acids 207-217.
recycling must be different from that for its entry into the nucleus to achieve accumulation of nuclear proteins in the nucleus. One mechanistic difference probably lies in the proteins that accompany
karyopherin
during the transport through the pore in the
import and export reactions. Import of karyopherin
is
mediated by karyopherin
, whereas export is not, since
the IBB domain of karyopherin
supports only nuclear
entry, but not exit of karyopherin
(Görlich et al., 1996a
;
Weis et al., 1996
). Our data indicate that Rch1 export depends on an NES and on energy, suggesting a receptor-mediated export reaction. However, the putative export
receptor and other proteins required for recycling remain
to be identified. Interestingly, Aitchinson et al. (1995)
reported that a yeast mutant that lacked Nup120p and displayed defects in mRNA export also accumulated importin
in the nucleus, pointing to a possible role for NPC proteins in receptor recycling.
subunit. Loss of RCC1 in tsBN2
cells led to nuclear accumulation of cytoplasmically injected Rch133-529 (Fig. 7). The time course of nuclear accumulation of Rchl33-529 depicted in Fig. 7 B shows that when
tsBN2 cells were shifted to the restrictive temperature directly after the injection, the Rch1 distribution was predominantly cytoplasmic for about the first hour. By 2 h after the shift-up, however, the intensity of nuclear staining
of Rch133-529 was markedly increased and continued to increase over the time period monitored. We speculate that
during the first hour after shift-up, active RCC1 was still present. This time course is consistent with the observation that inhibition of nuclear protein import in vivo in
tsBN2 cells was first detectable after 2 h at the restrictive
temperature (Dickmanns et al., 1996
). Trapping of Rch1 in
the nucleus, as observed here, may well be responsible for
the nuclear protein import defect of tsBN2 cells at the restrictive temperature.
subunit from the
nucleus. However, we demonstrate here that at least for
tsBN2 cells, the exit event itself was not affected by the
loss of RCC1 (Fig. 8). While this manuscript was under revision, Richards et al. (1997)
independently demonstrated
that RCC1 was not necessary for nuclear export of an
NES-bearing protein. Nevertheless, RanGTP did appear
to be necessary for export. These findings are consistent with our proposal that RCC1 plays a role in an earlier step
of NLS receptor recycling.
heterodimer in vitro (Rexach and Blobel, 1995
; Moroianu et al., 1996b
). We confirm this result
here using a solid phase assay and show that Ran, GTP,
and RCC1 release an NLS-bearing protein from an NLS
receptor/substrate complex or prevent formation of these
complexes (Fig. 9 C). These results, taken together with our in vivo data, suggest that RCC1 could induce the disassembly of the import complex by providing RanGTP,
which dissociates karyopherin
from the import complex,
thereby lowering the affinity of karyopherin
to NLSs
(Rexach and Blobel, 1995
). RanGTP will therefore facilitate the release of the imported protein from the NLS receptor
subunit, which in turn would be a prerequisite for
the recycling of the
subunit. Interaction of the imported
protein with other nuclear proteins or DNA, and/or association of karyopherin
with other proteins involved in
the recycling process, could then promote the dissociation
of import substrate from the
subunit.
Received for publication 21 May 1997 and in revised form 8 July 1997.
A DAAD fellowship to Irene Boche under the Hochschulsonderprogramm II program, the support of the Deutsche Forschungsgemeinschaft (Fa 138/4-3), Vanderbilt University, and the National Science Foundation Shared Instrumentation grant BIR-9419667 are gratefully acknowledged.We thank Melanie Hauser (Universtiy of Munich, Munich, Germany) for help in microinjection experiments, Achim Dickmanns (The Scripps Research Institute, La Jolla, CA) for sharing reagents and helpful discussions, Andreas Zeitvogel (Vanderbilt University) for purified SV-40 T-antigen, Chuck Cole (Dartmouth Medical School, Hanover, NH) for tsBN2 cells, and James Patton (Vanderbilt University) for helpful criticism. We also thank Steve Adam (Northwestern University Medical School, Evanston, IL), Marjorie Oettinger (Massachusetts General Hospital, Boston, MA), and Thomas Dobner (University of Regensburg, Regensburg, Germany) for plasmids.
aa, amino acid(s);
IBB domain, importin--binding domain;
MBP, maltose binding protein;
NES, nuclear export signal;
NLS, nuclear localization signal;
NPC, nuclear pore complex;
RCC1. regulator of chromosome condensation 1; Rch1, Rag cohort 1.
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