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INTRODUCTION |
Nuclear import of proteins bearing a classical nuclear
localization sequence (NLS)1
has been recently reviewed in Refs. 1-3. In digitonin-permeabilized cells, nuclear import of NLS-bearing proteins is mediated by a karyopherin 
·karyopherin 
1 (Kap·Kap1) heterodimer.
Kap and Kap1 are also called importins and , respectively.
Kap binds to the NLS, whereas Kap1 interacts with the nuclear
pore complex (NPC), resulting in docking of the cargo at the NPC. Other
soluble factors including the small GTPase Ran, p10 (also called NTF2), and the small Ran-binding protein RanBP1 help to translocate the cargo-carrier complex through the NPC and release the cargo inside the
nucleus in the presence of free GTP. The NPC is a large complex of
proteins (nucleoporins) that spans the nuclear membrane and has
filamentous projections into both the nucleus and cytoplasm (3-6).
Although several nucleoporins interact with Kap1 in
vitro, the exact sequence of interactions between Kap1,
nucleoporins, and other soluble factors that result in translocation
from the cytoplasmic to the nuclear side of the NPC and back is not
well understood.
Ran binds to guanine nucleotides and cycles between a GDP-bound form
(RanGDP) and a GTP-bound form (RanGTP). Nuclear transport is thought to
require interconversion between these two forms (7-9). The
interconversion of RanGTP and RanGDP is regulated by two factors. Ran
GTPase activating protein 1 (RanGAP1), located predominantly in the
cytosol and the cytoplasmic surface of the NPC, activates Ran GTPase
activity and thus converts RanGTP to RanGDP. RanGAP1 is targeted to the
NPC by covalent modification with a small ubiquitin-like molecule
(SUMO-1) (10, 11). SUMO-RanGAP1 is attached to the C terminus of
Nup358 and is the only species of RanGAP1 retained in
digitonin-permeabilized cells (10, 12, 13). RCC1, a predominantly
intranuclear guanine nucleotide exchange factor for Ran, allows
the release of bound nucleotide and reloading of Ran (7, 8, 14-16).
The cellular concentration of GTP is higher than that of GDP;
therefore, RCC1 is thought to favor the formation of RanGTP. The
cellular distribution of these two factors suggests that nuclear Ran is
predominantly in the GTP-bound form, whereas cytoplasmic Ran is mostly
GDP-bound.
RanGTP binds to Kap1 leading to the release of Kap along with the
bound cargo into the nucleus (17-19). The Kap1·RanGTP complex
then travels back through the NPC (20) presumably to initiate another
cycle of import. Binding of Kap1 to RanGTP inhibits RanGAP1-mediated
GTP hydrolysis (21). Yet nuclear import is favored when Ran is provided
in its GDP bound form and is inhibited by nonhydrolyzable analogs of
GTP and mutants of Ran that do not hydrolyze GTP (19, 22-28). The site
and mechanism by which GTP hydrolysis occurs after formation of the
Kap1·RanGTP complex are not well understood. RanBP1 binds strongly
to RanGTP (29-31) and weakly to RanGDP (32, 33) and forms trimeric
complexes with Kap1 and either RanGDP or RanGTP (34, 35). In
vitro studies with yeast proteins have shown that RanBP1 in
concert with Kap and a C-terminal portion of the nucleoporin Nup1p
can reverse the inhibition of RanGAP activity imposed by binding of RanGTP to Kap1 (36). Similar results have been reported with mammalian factors (37). However, it is not clear whether and where
these reactions occur in cells. Recent studies provide evidence that
GTP hydrolysis is not required for translocation of cargo through the
NPC (38-40). This leaves open the possibility that GTP hydrolysis is
required for recycling of the Kap1·RanGTP complex for repeated
rounds of import.
Nup358 (also termed RanBP2) is a 358-kDa nucleoporin located at the
tips of the cytoplasmic fibrils of the NPC (41, 42). Its domains
include a leucine-rich region, four RanBP1-homologous (RBH) domains, a
zinc finger region, scattered FG and FXFG repeats characteristic of a subset of nucleoporins, and a
cyclophilin-homologous domain (41, 42). RanGTP binds to Nup358 in
overlay and microtiter plate assays (25, 41, 42). Electron microscopy
of rat liver nuclear envelope preparations shows that Ran loaded with
GTP or its nonhydrolyzable analog GMPPNP interacts with the cytoplasmic side of the NPC (25, 41). These findings are consistent with an
interaction between RanGTP and Nup358 at the NPC. In addition, Nup358
appears to form a trimeric complex with RanGTP and Kap1 in
vitro (43). The binding properties of Nup358 are reminiscent of
those of RanBP1, suggesting that Nup358 may be involved in GTP
hydrolysis at the NPC. On the other hand, the location of Nup358 at the
tips of the cytoplasmic fibrils of the NPC suggests that it may be
involved in the initial docking of substrate prior to import.
Many of the recent advances in characterizing nuclear import factors
were accomplished by using fluorescent import substrates in a
digitonin-permeabilized cell assay (44). One of the major tasks that
currently face the field is dissecting the events that occur at the NPC
during import (3), a challenge that cannot be met at the resolution
level of this assay. Ultrastructural studies of import to date (19,
45-52) have focused mainly on microinjection experiments in intact
cells, in which identifying the functions of individual factors is difficult.
In this study, we bring ultrastructural analysis and Ran GTPase
activity measurements to the digitonin-permeabilized cell system. This
approach is combined with in vitro binding and GTPase assays, as well as standard import assays, to examine where and how the
Kap1·RanGTP complex is recycled at the NPC. We show that Nup358 is
a site at which the first (docking of Kap·NLS) and the last
(recycling of Kap1·RanGTP) steps of nuclear import are linked and
that the resulting GTP hydrolysis by Ran is required for subsequent
import. Using ultrastructural and in vitro solution binding
assays, we show that the RBH domains of Nup358 serve as RanGDP- or
RanGTP-dependent docking sites for Kap1 on the
cytoplasmic fibers of the NPC. The Kap1·RanGTP·RBH complex, a
likely intermediate in the recycling of Kap1·RanGTP, is not
dissociated by RanBP1, suggesting that GTP hydrolysis by Ran is a
prerequisite for the dissociation of Kap1 from Nup358 during
transport. Kap and NLS peptide bound to the Kap1·RanGTP·RBH
complex resulting in GTP hydrolysis by free and NPC-associated RanGAP1.
This GTP hydrolysis was necessary for the initiation of further cycles
of nuclear import. These findings are integrated into a model for the
termination of one cycle of import and the initiation of the next.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Permeabilization--
HeLa cells were cultured
in Dulbecco's modified Eagle's medium supplemented by 10% fetal calf
serum, 2 mM glutamine, and 100 units/ml each of penicillin
and streptomycin. For electron microscopy and GTPase assays, 50-80%
confluent cells were digitonin-permeabilized and stored essentially as
described (53). Cells were nonenzymatically dissociated in dissociation
solution (Sigma C-5914) for 15 min at 37 °C and washed three times
by low speed centrifugation in cold transport buffer (TB) (20 mM HEPES, pH 7.4, 110 mM potassium acetate, 2 mM magnesium acetate, 1 mM EGTA). They were
then suspended in TB with 35 µg/ml digitonin and incubated for 5 min
on ice followed by three washes in cold TB. They were resuspended in TB
with 10 mg/ml bovine serum albumin (Sigma A-7030) and 5%
Me2SO at 107 cells/ml and stored in aliquots at
80 °C.
Recombinant Proteins--
Human Ran was expressed in
Escherichia coli BL21(DE3) from a pET-9C construct (29) and
purified as described (21). Recombinant human Ran was loaded with
either GDP, GTP, or GMPPNP as described (21) prior to use for gold
conjugation, binding assays, or import assays. The murine RanGAP1 open
reading frame was amplified by polymerase chain reaction from a
construct kindly provided by Mark Rush (54) and subcloned into pGEX 4T3
(Amersham Pharmacia Biotech), from which it was expressed in E. coli as a GST fusion protein, purified by binding to
glutathione-Sepharose, and eluted by thrombin cleavage according to the
manufacturer's instructions. The Kap1 open reading frame was
amplified from a human cDNA library (CLONTECH),
subcloned into pGEX4T3, expressed in E. coli as a GST fusion
protein, and purified by binding to glutathione-Sepharose. For electron
microscopic and binding studies, Kap1 was eluted with thrombin; for
GTPase assays, it was eluted with glutathione according to the
manufacturer's instructions as a GST fusion protein. Murine Kap2
(55), expressed with a C-terminal His tag from a pET21A construct, was
a kind gift from Michael Matunis. The Nup358-4 protein (12) was a kind
gift from Jian Wu. The Nup358-1 construct was produced by amplifying
the region of the Nup358 cDNA that encodes amino acids 996-1963
from a plasmid containing the 7-4 fragment of Nup358 (41) provided by
Jian Wu. It was subcloned into pGEX4T3 and expressed as a GST fusion
protein in E. coli. Recombinant RanBP1, expressed from a
pET11 construct (kindly provided by Elias Coutavas), was a gift from
Yuh-Min Chook and Tschong-Uk Park.
Conjugation of Recombinant Proteins to Colloidal Gold
Particles--
The optimum amount of recombinant protein needed to
stabilize colloidal gold beads (BBI International) was determined by
titration according to the manufacturer's instructions. Colloidal gold
beads, 10 nm in diameter, were incubated with the recombinant protein for 5 min at room temperature and brought up to pH 9 with 0.2 M potassium carbonate. Bovine serum albumin was added to 10 mg/ml and the coated beads were then concentrated 50-fold by
centrifugation according to the manufacturer's instructions and stored
at 4 °C for up to 1 month. Gold conjugates were checked by electron
microscopy to ensure that the preparation is monodisperse with no
clumping of gold particles.
Electron Microscopy--
Approximately 5 × 104
digitonin-permeabilized HeLa cells were incubated with 1 µl of 10-nm
gold conjugate for 40 min at 22 °C in 30 µl of TB followed by
overnight fixation in 5% glutaraldehyde at 4 °C. Samples were
processed for transmission electron microscopy and viewed with a Jeol
100CX microscope (Jeol USA, Peabody, MA). Two photographs per cell were
taken from ultrathin sections at × 26,000 magnification from 5-6
cells for each experimental point. For quantitation of Kap1 binding
to the NPC, the gold beads associated with all the morphologically
preserved NPCs visible in the photograph (approximately 40 NPCs per
sample) were counted and divided by the number of NPCs. All electron
micrographs were scanned and saved using Adobe Photoshop 4.0 software.
Quantitative analysis of the images was carried out with NIH Image 1.61 software.
In Vitro Binding Assays--
Binding reactions contained 10 µl
of glutathione-Sepharose beads (Amersham Pharmacia Biotech) in a total
volume of 46 µl in transport buffer with Tween 20 (20 mM
HEPES, pH 7.4, 110 mM potassium acetate, 2 mM
magnesium chloride, and 0.1% Tween 20) and 0.5 mg/ml Pefabloc SC
(Roche Molecular Biochemicals). Nup358-1, Nup358-4, and Kap1 were
present at 0.25 µM each; Ran was at 0.8 µM.
Reactions were allowed to proceed for 1 h at 4 °C with
rotation. After pelleting the beads, the supernatant was collected, and
the beads were washed twice in cold TB-0.1% Tween 20. For the
experiment shown in Fig. 4, a further 1 h incubation was carried
out in the presence or absence of RanBP1 at 4 °C as indicated. The
bound and unbound fractions were subjected to SDS-PAGE on 5-20%
gradient gels. Proteins were visualized by Coomassie Blue staining of
gels, Amido Black staining of blots, or Western blotting as indicated
in the text and figure legends. In Western blots, Kap, by virtue of
its C-terminal His tag, was detected using a nickel-nitrilotriacetic
acid-horseradish peroxidase (Ni-NTA horseradish peroxidase) conjugate
(Qiagen) according to the manufacturer's instructions (see Fig. 7).
Detection was by chemiluminescence using SuperSignal reagents (Pierce). Quantitation of Kap binding was performed by densitometric analysis of blots using Scan Analysis version 2.56 software (Biosoft).
GTPase Assays--
Ran was loaded with
[
-32P]GTP as described (21) and used at 0.4 µM in all reactions. When Kap1 was used, it was
preincubated at 0.25 µM for 30 min at 4 °C with the
[-32P]RanGTP before the other components were added,
in both in vitro and permeabilized cell assays. In
vitro assays were done in the presence or absence of the following
as indicated in Fig. 8A: 0.25 µM GST-Nup358-1,
0.6 µM Kap, 1.2 µM NLS peptide (56), and
20 nM RanGAP1. For permeabilized cell assays using
digitonin-permeabilized cells, 5 × 104 cells were
used per assay ± 10 nM RanGAP1 in the presence or absence of 0.6 µM Kap and 1.2 µM NLS
peptide, as indicated in Fig. 8B. All reactions were carried
out in 45 µl of hydrolysis buffer (20 mM HEPES, pH 7.4, 100 mM potassium acetate, 20 mM magnesium acetate, 1 mM dithiothreitol, and 0.5 mg/ml bovine serum
albumin) (21). Hydrolysis was allowed to proceed for 30 min at 22 °C and stopped by adding 300 µl of hydrolysis buffer; half of the reaction was then passed through a nitrocellulose filter. The filter
was washed with 5 ml of cold hydrolysis buffer, and the radioactivity
retained on it was counted in a scintillation counter.
Import Assays--
GST-Kap1 fusion protein was immobilized on
glutathione-Sepharose beads, and an excess of either RanGTP or
RanGMPPNP was added. After a 1-h incubation at 4 °C, the beads were
washed twice, and the Kap1·RanGTP and Kap1·RanGMPPNP
complexes were eluted with glutathione according to the manufacturer's
instructions. The buffer was changed to TB by centrifugation through
preequilibrated MicroSpin columns (Amersham Pharmacia Biotech) prior to
use in import reactions. HeLa cells were grown on glass coverslips for 24 h, permeabilized with 30 µg/ml digitonin in TB for 5 min at room temperature, and washed three times with transport buffer. Import
reactions included 0.1 µM Kap1·RanGTP or
Kap1·RanGMPPNP complexes, 0.6 µM Kap, 5 µg/ml
tetramethylrhodamine B isothiocyanate-NLS-bovine serum albumin (kindly
provided by Mary S. Moore) (38), 0.3 µM p10, 10 nM RanGAP1, 1 µM RanBP1, and 2 mM
GTP or GMPPNP in TB. The mixtures, except for RanGAP1, were
preincubated for 15 min on ice. RanGAP1 was added immediately before
the mixtures were added to permeabilized cells. Import was allowed to
proceed for 15 min at room temperature, and cells were washed twice in
TB, fixed in 2% formaldehyde in TB, and visualized using a Zeiss
Axiophot microscope using a × 63 objective. Images were captured
with Adobe Photoshop 4.0 software.
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RESULTS |
Karyopherin 1 Docks at the RBH Domains of Nup358 by Forming
Trimeric Complexes with Either RanGDP or RanGTP--
Karyopherin 1
(Kap1) binds to several nucleoporins in vitro (17,
57-62). Of these, the nucleoporin Nup358 is located at the tips of the
cytoplasmic fibrils of the NPC, where the initial docking of import
substrates is believed to occur prior to import (46, 49). It contains a
leucine-rich region, four domains that are homologous to RanBP1 (RBH
domains), eight zinc fingers, a cyclophilin-homologous domain, and
scattered FG repeats that are characteristic of a subset of
nucleoporins (41, 42) (Fig. 1). Trimeric
complexes between RanBP1, Kap1, and either RanGTP or RanGDP have
been demonstrated in solution binding assays (34, 35). This would
suggest that trimeric complexes could be formed between Kap1, RBH
domains, and either RanGDP or RanGTP. However, in overlay and
microtiter plate assays, binding of Kap1 to Nup358 was found to be
enhanced by RanGTP but not RanGDP (43).
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Fig. 1.
Schematic diagrams of Nup358 and the Nup358-1
and Nup358-4 constructs. R1-4, RBH domains 1-4;
CycH, cyclophilin-homologous domain.
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Ran-dependent binding of Kap1 to Nup358 would be of
potential significance for both import of NLS-bearing cargo and
recycling of Kap1 after a round of import. RanGDP is required for
import of Kap and NLS-bearing cargo (19, 28) in addition to Kap1; therefore, complexes involving Kap1, RanGDP, and Nup358 may act as
intermediates in nuclear import. On the other hand, after a round of
import, Kap1 is bound to RanGTP and probably exported as a
Kap1·RanGTP complex. This complex may bind to Nup358 as its last
station on the NPC before recycling. With these considerations in mind,
we carried out solution binding assays to determine whether Kap1 can
bind to Nup358 in the presence of RanGDP or RanGTP, and which domains
of Nup358 are involved in such binding.
For this purpose, two recombinant fragments of Nup358 were expressed in
E. coli as fusion proteins with an N-terminal GST tag (Fig.
1). The first fragment, called Nup358-1, contains the RBH1 domain, the
eight zinc fingers, and several FG repeats. The second fragment, called
Nup358-4, contains the RBH4 domain, the cyclophilin-homologous domain,
and several FG repeats (Fig. 1). The constructs were intended to
include as much representative sequence from Nup358 as possible within
the constraints imposed by the bacterial expression system. We wanted
to express the RBH domains in their natural context within Nup358
sequences and to include most of the FG repeats, which have been
implicated in the binding of Kap1 to other nucleoporins (17, 57, 58, 60, 63, 64).
We first incubated immobilized Nup358-1 and Nup358-4 with Kap1 and
either RanGDP or RanGTP. Bound and unbound material was analyzed by
SDS-PAGE (Fig. 2). Very little Kap1
bound to either of the Nup358 fragments, despite the presence of FG
repeats in both fragments; close inspection of the original gels
reveals a faint band (see Fig. 7A, lane 2). However, in the
presence of either RanGTP or RanGDP, there was strong binding of
Kap1 to both Nup358-1 and Nup358-4 (Fig. 2). These data indicate
that each of the two Nup358 fragments is capable of forming trimeric complexes with Kap1 and either RanGTP or RanGDP. When incubations were carried out in the presence of increasing concentrations of
RanBP1, there was complete inhibition of Ran-mediated binding of
karyopherin 1 to Nup358-1 and Nup358-4, indicating that karyopherin 1 and Ran form trimeric complexes with the RBH domains of Nup358 (Fig. 3).
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Fig. 2.
Kap1 binds to
Nup358-1 and Nup358-4 in the presence of either RanGDP or RanGTP.
Nup358-1 (left) or Nup358-4 (right) immobilized
on glutathione-Sepharose beads at 0.25 µM were incubated
with 0.25 µM Kap1 in the presence or absence of 0.8 µM RanGDP or RanGTP. Bound and unbound fractions were
resolved by SDS-PAGE and visualized by Coomassie Blue staining.
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Fig. 3.
RanBP1, when added concomitantly, competes
with Nup358 for the formation of trimeric complexes with Ran and
Kap1. Binding reactions were carried out
as in Fig. 2 but in the presence or absence of 1, 2, or 4 µM RanBP1, as indicated.
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The Preformed Kap1·RanGTP·RBH Complex Resists Dissociation
by RanBP1--
RanBP1 has been implicated in the recycling of
Kap1·RanGTP complexes (36, 37). However, these complexes may
encounter and bind to Nup358 on their way out of the NPC before they
encounter RanBP1, because the latter is a cytosolic protein (65). It
was therefore important to determine whether RanBP1 would be able to
dissociate a Kap1·Ran·RBH complex after it is formed. Kap1 and either RanGDP or RanGTP were incubated with Nup358-4 immobilized on
beads in order to form Kap1·RanGDP·RBH and
Kap1·RanGTP·RBH complexes, respectively, as before. The
beads were then washed and subjected to a second incubation with
increasing concentrations of RanBP1 followed by SDS-PAGE of bound and
unbound fractions (Fig. 4). The preformed
Kap1·RanGTP·RBH trimeric complex was resistant to
dissociation by RanBP1. In contrast, the preformed trimeric complex of
Kap1·RanGDP·RBH was readily dissociated by RanBP1
(Fig. 4). Identical results were obtained with Nup358-1 (data not
shown). Hence, once a trimeric RBH·Kap1·RanGTP complex is
formed, RanBP1 is unable to disassemble the complex, whereas a trimeric
complex of RBH·Kap1·RanGDP can be disassembled by RanBP1 (or
presumably another RBH domain of Nup358). These results suggest that
GTP hydrolysis is needed before Kap1 can move from one RBH to the
next or be released by RanBP1.
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Fig. 4.
The trimeric
Nup358·RanGTP·Kap1 complex resists
dissociation by RanBP1. Binding reactions with 0.25 µM Nup358-4 on glutathione-Sepharose beads, 0.25 µM Kap1, and 0.8 µM either RanGDP or
RanGTP were allowed to proceed for 1 h, followed by two washes and
another 1-h incubation in the presence or absence of 1 or 2 µM RanBP1. Bound and unbound fractions were visualized by
SDS-PAGE and Coomassie Blue staining.
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Ultrastructural Analysis of Kap1 Docking at the NPC--
To
assess the significance of these in vitro binding data in
the context of intact NPCs, Kap1 was coupled to 10-nm colloidal gold
and incubated with digitonin-permeabilized cells, either alone or in
the presence of RanGDP or RanGTP. Electron microscopic inspection of
sections of these cells confirmed the in vitro binding data
(Fig. 5). There was very little binding
to NPCs of Kap1-gold alone (Fig. 5, top panel), whereas
significant gold decoration of NPCs occurred when either RanGDP (Fig.
5, middle panel) or RanGTP (Fig. 5, bottom panel)
was present during the incubation. Localization of the gold particles
to the cytoplasmic fibrils of the NPC and competition by RanBP1 (see
below) indicate that they are indeed decorating Nup358.
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Fig. 5.
Kap1 docks at the
NPC through either RanGDP or RanGTP. Recombinant Kap1 was
conjugated to 10-nm gold beads and incubated with
digitonin-permeabilized HeLa cells in the presence or absence of 0.8 µM RanGDP or RanGTP. Photographs were taken from
ultrathin sections at × 26,000 original magnification.
Scale bars represent 100 µm.
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Electron microscopic analysis also confirmed the in vitro
competition data with concomitantly added RanBP1 (data not shown) and
with RanBP1 added after docking (Fig. 6).
Kap1-gold was first allowed to dock in digitonin-permeabilized
cells, either alone or with RanGDP or RanGTP. This was followed by a
second incubation in the presence or absence of RanBP1, and the average
number of Kap1-gold particles per NPC under each condition was then
determined. There was considerable reduction of NPC-bound gold
particles when a preformed Kap1-gold·RanGDP·Nup358 complex was
exposed to RanBP1, whereas the preformed Kap1-gold·RanGTP·Nup358
complex remained stable (Fig. 6). Interestingly, Kap1 binding that
occurred in the absence of exogenously added Ran was also diminished by
RanBP1 treatment, suggesting that this binding is mediated in part by the formation of a trimeric complex with residual endogenous RanGDP and
Nup358. In fact, residual Ran can be seen at the nuclear envelope in
digitonin-permeabilized cells by immunofluorescence (66).
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Fig. 6.
Kap1 docked at the
NPC through RanGTP resists displacement by RanBP1.
Kap1-conjugated 10-nm gold beads were allowed to dock at the NPC in
digitonin-permeabilized HeLa cells alone, through RanGDP, or through
RanGTP. After docking, a further incubation was carried out in the
absence or presence of 1 µM RanBP1. The average number of
beads per NPC was derived by counting the number of beads associated
with approximately 40 NPCs for each experimental condition.
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Thus, the binding of Kap1·RanGTP to Nup358 is stable both in
vitro and in permeabilized cells, whereas Kap1 docked through RanGDP can be readily displaced. These results suggest that once a
Kap1·RanGTP complex resulting from one round of import binds to
Nup358, another round of NLS import may not be possible before GTP
hydrolysis occurs.
Induction of GTP Hydrolysis by Kap and NLS--
The
Kap1·RanGTP complex is resistant to GTP hydrolysis by RanGAP (21).
RanBP1 can form a trimeric complex with Kap1 and RanGTP in
vitro (34, 35), and in this context, the addition of Kap allows
GTP hydrolysis in the presence of RanGAP (36, 37). However, with the
high concentration of Nup358 RBH domains at the NPC (67) and the
cytosolic location of RanBP1 (65), the Kap1·RanGTP complex is
likely to encounter a Nup358 RBH domain and bind to it before it
encounters RanBP1. Because the Kap1·RanGTP·Nup358 complex thus
formed cannot be dissociated by RanBP1 (see above), GTP hydrolysis
would have to occur on Nup358 at the cytoplasmic fibers of the NPC. It
was therefore important to determine whether Kap would interact with
the Kap1·RanGTP·Nup358 complex and stimulate GTP hydrolysis in
the presence of RanGAP1. To assess the relevance of these processes to
the further import of NLS-bearing substrate, it was also of interest to
see whether they are affected by the presence of NLS cargo.
As shown in Fig. 7A, when
Kap and NLS peptide were added to immobilized Nup358-4 alone, they
did not bind to a significant extent (Fig. 7A, lane 1).
However, binding occurred when both Kap1 and Ran were present,
indicating the formation of NLS·Kap·Kap1·RanGTP·Nup358 complexes (Fig. 7A, lanes 2-4). Surprisingly, RanGTP caused
even stronger binding of Kap than RanGDP (Fig. 7A, lanes
3-4). GTP remained unhydrolyzed throughout the assay (data not
shown). Similar results were seen when Nup358-1 was used instead of
Nup358-4 (data not shown). Binding of Kap was diminished in the
absence of NLS peptide (Fig. 7B), indicating cooperative
binding of Kap and NLS to the rest of the complex. These results
were quantitated by scanning densitometry (Fig. 7C).
Ultrastructural studies (46, 49) have suggested that the initial
docking site in nuclear import is at the tips of the cytoplasmic fibers
of the NPC. Because this is where Nup358 is localized as well (41), the
NLS·Kap·Kap1·RanGTP·Nup358 complexes may represent
initial docking events in classical NLS-mediated nuclear import.
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Fig. 7.
Kap and NLS can form
a pentameric complex with Nup358, Kap1, and
either RanGDP or RanGTP. A, Kap and NLS bind to
Nup358 in a Kap1- and Ran-dependent fashion.
Glutathione-Sepharose beads with immobilized Nup358-4 at 0.25 µM were incubated with 0.25 µM Kap1, 0.6 µM Kap, 1.2 µM NLS peptide, and 0.8 µM RanGDP or RanGTP as indicated. Bound fractions were
visualized by SDS-PAGE, Western blotting, and Amido Black staining.
Kap was also visualized by Ni-NTA horseradish peroxidase treatment
followed by chemiluminescence detection (see under "Experimental
Procedures"). Unbound fractions were visualized by SDS-PAGE and
Coomassie Blue staining. B, Kap and NLS bind
cooperatively in the formation of pentameric
NLS·Kap·Kap1·Ran·Nup358 complexes.
Glutathione-Sepharose beads with immobilized Nup358-4 at
0.25 µM were incubated with 0.25 µM Kap1
and 0.6 µM Kap in the presence or absence of 1.2 µM NLS peptide, 0.8 µM RanGDP or RanGTP as
indicated. Bound Kap was visualized by Ni-NTA horseradish peroxidase
treatment followed by chemiluminescence detection (see under
"Experimental Procedures"). C, the blot shown in
B was subjected to densitometric analysis with Scan Analysis
version 2.56 software. The amount of Kap is expressed in arbitrary
units.
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To test whether GTP hydrolysis could be induced within the trimeric and
higher order RanGTP-containing complexes described above, we
preincubated Kap1 and Ran[-32P]GTP at 4 °C to
allow binary complex formation and then incubated the Kap1·RanGTP
complex at 22 °C with RanGAP1 and various combinations of Kap,
NLS peptide, and Nup358-1. The reactions were stopped and passed
through a nitrocellulose filter, and the amount of unhydrolyzed
Ran-bound GTP was measured by counting radioactivity that remained
bound to the filter after washing (21). The Nup358-1·RanGTP·Kap1 trimeric complexes were resistant to RanGAP1 activity (Fig.
8A). Addition of Kap
resulted in partial GTP hydrolysis, and when NLS peptide was included
in the reaction, complete hydrolysis occurred (Fig. 8A).
This is consistent with the cooperative binding of Kap and NLS
peptide in the formation of pentameric complexes (see above). The
results were identical when Kap with or without NLS peptide was
added to a preformed Kap1·RanGTP·Nup358 complex (data not
shown). In addition, identical results were obtained when RanBP1 was
substituted for Nup358-1 (data not shown). Thus, the presence of
transport cargo favors the hydrolysis of GTP, consistent with a role
for GTP hydrolysis by Ran during nuclear import.
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Fig. 8.
GTP hydrolysis catalyzed by free RanGAP1 and
NPC-associated SUMO-RanGAP1 is triggered by Kap and NLS. A, maximal stimulation of GTP hydrolysis
in vitro by free RanGAP1 within trimeric complexes requires
both Kap and NLS peptide. -32P-Labeled RanGTP was
preincubated with or without Kap1 prior to adding the other
indicated components. The amount of radioactivity retained on a
nitrocellulose filter was expressed as a percentage of
control (radioactivity retained in the Nup358-RanGTP-Kap1
complex was set at 100%). B, NPC-associated SUMO-RanGAP1
catalyzes partial GTP hydrolysis. -32P-Labeled RanGTP
was preincubated with or without Kap1 and GTPase assays were carried
out in the presence of 5 × 104
digitonin-permeabilized HeLa cells per reaction; other components were
added as indicated. The amount of radioactivity retained on the filter
in the reaction containing RanGTP, cells, and Kap1 was set at
100%.
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To study GTP hydrolysis by Ran within intact NPCs in a context closer
to the in vivo situation, we used digitonin-permeabilized HeLa cells (Fig. 8B). RanGAP1 exists in cells in two forms,
a free cytosolic form and an NPC-bound form that is covalently modified with a small ubiquitin-like peptide (SUMO-1) (10, 11). The latter form
(SUMO-RanGAP1) is attached to the C terminus of Nup358 and is the only
species retained in digitonin-permeabilized cells (10, 13). Assays were
carried out in a manner similar to the in vitro assays with
preincubation of [-32P]RanGTP with or without Kap1.
GTP hydrolysis assays were then started by adding
digitonin-permeabilized cells with or without Kap and NLS peptide
(Fig. 8B). The digitonin-permeabilized cells served as a
source of both Nup358 and NPC-associated SUMO-RanGAP1. When RanGTP was
added to digitonin-permeabilized cells in the absence of Kap1, GTP
was readily hydrolyzed (Fig. 8B). When RanGTP was
preincubated with Kap1, there was maximal inhibition of GAP activity. Thus SUMO modification and NPC association of RanGAP1 do not
override the inhibitory effect of Kap1 on GTP hydrolysis. As in the
in vitro experiment, addition of Kap stimulated partial GTP hydrolysis. The addition of NLS did not further stimulate GTP
hydrolysis by NPC-associated SUMO-RanGAP1 (Fig. 8B). The
presence of residual unhydrolyzed GTP under these conditions suggests
that some Kap1·RanGTP associated with the four RBH domains of
Nup358 might not be accessible to SUMO-RanGAP1, which is attached to the C terminus of Nup358. Indeed, this residual GTP was completely hydrolyzed in a Kap and NLS-dependent fashion when
recombinant free RanGAP1 was included in addition to the permeabilized
cells (data not shown). Thus, the two forms of RanGAP
soluble and
NPC-associatedmay play complementary roles in GTP hydrolysis at the
NPC.
GTP Hydrolysis Is Required for Recycling of the
Kap1·RanGTP Complex for Further Rounds of Import--
Several
previous studies have shown that import of classical NLS bearing
substrates requires GTP hydrolysis by Ran (22-28). On the other hand,
a recent study indicated that GTP hydrolysis might not be required for
the translocation step of the nuclear import of classical NLS-bearing
proteins (38). In that study, Ran was supplied in its GDP-bound form in
the import reactions. However, as discussed previously, Ran is likely
to be in the GTP bound form and in complex with Kap1 at the end of a
round of import; and GTP hydrolysis may then be required for a
recycling step before this Kap1·RanGTP complex can be utilized for
another round of import. To address this question, RanGTP or Ran loaded with the nonhydrolyzable GTP analog GMPPNP was added in excess to
GST-Kap1 immobilized on glutathione-Sepharose beads. After washing
to remove unbound Ran, the Kap1·RanGTP and Kap1·RanGMPPNP complexes thus formed were eluted from the beads with glutathione (Fig.
9A) and used in standard
import reactions in permeabilized cells in the presence of free GTP and
GMPPNP, respectively (Fig. 9B). As shown in Fig.
9B, the Kap1·RanGTP complex supported import, whereas
the Kap1·RanGMPPNP complex did not. These data show that GTP
hydrolysis is required for recycling of a Kap1·RanGTP complex and
reinitiation of nuclear import of a classical NLS-bearing substrate.
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Fig. 9.
GTP hydrolysis is required for reinitiation
of nuclear import. A, preparation of Kap1·RanGTP
and Kap1·RanGMPPNP complexes. RanGTP or RanGMPPNP was added in
excess to GST-Kap1 immobilized on glutathione-Sepharose beads. After
washing, the Kap1·RanGTP and Kap1·RanGMPPNP complexes were
eluted with glutathione, exchanged into transport buffer, and checked
by SDS-PAGE and Coomassie Blue staining. B, recycling of the
Kap1·RanGTP complex for nuclear import requires GTP hydrolysis.
Import reactions were carried out in permeabilized cells as described
under "Experimental Procedures." In the left panel,
Kap1·RanGTP complex and GTP were added, whereas the right
panel contained Kap1·RanGMPPNP complex and GMPPNP. In
addition, both reactions contained NLS-bovine serum albumin-rhodamine,
Kap, p10, RanBP1, and RanGAP1.
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DISCUSSION |
Nuclear import of classical NLS-bearing proteins is mediated by a
Kap·1 heterodimer. Other soluble factors involved in the process include Ran, p10, RanBP1, RanGAP1, and GTP. Binding of RanGTP
to Kap1 dissociates Kap from Kap1, resulting in the release of
Kap and its NLS-bearing cargo into the nucleus. In this study, we
analyze how and where the Kap1·RanGTP complex is recycled at the
NPC to initiate another round of import. Data from in vitro
binding and GTPase activity assays are correlated with findings from
ultrastructural and functional assays in permeabilized cells to provide
a clearer picture of the events that actually occur on intact NPCs.
With this approach, we show that the RBH domains are a site where
docking of NLS cargo is linked to GTP hydrolysis by Ran and that this
hydrolysis is a prerequisite for repeated cycles of import.
Ran-dependent Docking of Kap1 on RBH Domains at the
NPC--
Kap1 is involved in the docking of NLS-bearing proteins at
the NPC. In vitro and in cell lysates, Kap1 has been
shown to bind to several nucleoporins (17, 34, 36, 43, 57-62, 64). In
several cases, binding of Kap1 has been localized to the FG repeat
regions of nucleoporins (17, 57, 58, 60, 63, 64), and in another case,
it has been localized to the -helical coiled-coil rod domain of the
nucleoporin p62 (61).
After translocation through the NPC, RanGTP binds to Kap1, leading
to the release of the Kap·NLS complex in the nucleus. The
mechanism of reexport of Kap1 after a round of nuclear import is not
well understood. A leucine-rich nuclear export sequence in yeast
Kap1 has been reported to mediate its export to the cytoplasm and
its interaction with GLFG-containing yeast nucleoporins (63). It is not
known whether other soluble export factors are involved in Kap1
export, but it is probably reexported in a complex with RanGTP (20).
The Kap1·RanGTP complex is then presumably recycled to initiate
further rounds of import. In vitro, binding of RanGTP to
Kap1 releases it from some nucleoporins (17, 43, 61), but it
enhances its binding to Nup358 (Fig. 2) (43). Nup358 is the outermost
known nucleoporin on the cytoplasmic side of the NPC and is likely to
be the first station for nuclear import of NLS-bearing proteins as well
as the last station for the reexported Kap1·RanGTP complex on the
NPC.
Our data (Figs. 4 and 5) show that either RanGDP or RanGTP can target
Kap1 to Nup358 by forming trimeric complexes and that the bulk of
Kap1 docking on Nup358 is Ran-dependent. Furthermore, although RanGDP can bind either to the zinc fingers or the RBH domains
of Nup358 (67), Ran-dependent docking of Kap1 occurs through the RBH sites only, as evidenced by the fact that this binding
can be prevented by concomitantly added excess RanBP1 (Fig. 3).
Ultrastructural studies with Kap1-gold conjugates in digitonin
permeabilized cells confirm RanGDP- and RanGTP-dependent docking of Kap1 at the cytoplasmic fibrils (Fig. 5) and competition by RanBP1 (Fig. 6). It should be noted that, although RanBP1 inhibits Kap1 binding to Nup358, it enhances docking and import of
NLS-bearing proteins (34). The mechanism of this enhancement is not
clear; it is possible that RanBP1 stabilizes NLS docking at NPC sites other than the RBH domains of Nup358.
It is of interest in this context to compare our data to those of
Delphin et al. (43). Their overlay and microtiter plate assays (in agreement with our solution binding assays) show that RanGTP
enhances the binding of Kap1 to Nup358. However, when they added
recombinant Kap1 to isolated nuclear envelopes and detected it by
immunoelectron microscopy, the substantial amount of Kap1 docking on
the cytoplasmic side of the NPC was reduced by RanGTP instead of the
increase one might have expected. This is probably due to the presence
of other nucleoporins on the cytoplasmic side of the NPC and the fact
that RanGTP dissociates Kap1 from some nucleoporins (see above). Our
data, on the other hand, were obtained by directly coupling recombinant
Kap1 to colloidal gold and applying it to digitonin-permeabilized
cells with or without Ran (Fig. 5). We saw little docking in the
absence of Ran, but docking was markedly enhanced in the presence of
either RanGDP or RanGTP (Figs. 5 and 6). It thus appears that gold
conjugation of Kap1 has led to a reduction in docking on other
nucleoporins while leaving Ran-dependent docking on RBH
domains relatively intact. This may be due to selective masking of
certain domains on Kap1 or to steric interference by the bulk of the
gold particle. In any case, this has allowed us to examine docking on
Nup358 in relative isolation from the background of other nucleoporins. We have thus been able to confirm the in vitro binding and
competition data and to show that Ran-dependent docking of
Kap1 occurs on intact NPCs.
As shown diagrammatically in Fig. 10,
formation of a Kap1·RanGTP·Nup358 complex is likely to be the
last step in the reexport of the Kap1·RanGTP complex through the
NPC. An important feature of the Kap1·RanGTP·Nup358 complex is
that once it is formed, it resists dissociation by subsequently added
RanBP1 both in vitro and on intact NPCs, indicating that it
is relatively stable (Figs. 4 and 6). The RanGDP-containing complex, on
the other hand, is readily dissociated by subsequently added RanBP1.
These data imply that GTP hydrolysis by Ran is needed in order to allow
movement of Kap1.
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Fig. 10.
Model for recycling of the
Kap1·RanGTP complex and initiation of a
second round of import. See text for details.
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Docking of Kap·NLS, GTP Hydrolysis, and Import--
The
stability of the Kap1·RanGTP·Nup358 complex in comparison to the
Kap1·RanGDP·Nup358 complex (see above) suggested that GTP
hydrolysis is required before translocation and import can occur.
Indeed, nonhydrolyzable analogs of GTP and mutants of Ran that do not
hydrolyze GTP have been reported to inhibit nuclear import (22-28). On
the other hand, a recent study showed that the nonhydrolyzable GTP
analog GMPPNP can support classical NLS-mediated import in the presence
of RanGDP (38). However, a round of nuclear import results in a
Kap1·RanGTP complex, and it is this complex that needs to be
recycled and used in a subsequent round of import. A hydrolysis
requirement for recycling would be bypassed if RanGDP were used as a
starting point. Indeed, as shown in Fig. 9, when cells were presented
with a Kap1·RanGMPPNP complex, import failed to occur, whereas the
Kap1·RanGTP complex was able to support import under similar conditions.
Our studies of GTP hydrolysis in vitro and in permeabilized
cells (Fig. 8) revealed a requirement for Kap and NLS peptide for
maximal GTP hydrolysis at the NPC. The trimeric
Kap1·RanGTP·Nup358 complex was resistant to the action of
RanGAP1. The addition of Kap and NLS peptide cooperatively induced
complete GTP hydrolysis in the presence of RanGAP1. This did not result
from a dissociation of Kap1 from Nup358 and RanGTP, but rather from
cooperative binding of Kap and NLS to the Kap1·RanGTP·Nup358
complex (Fig. 7). Ultrastructural studies indicate that the initial
docking site of NLS bearing cargo is at the tips of the cytoplasmic
fibrils (46, 49), where Nup358 resides. Thus, the pentameric
NLS·Kap·Kap1·Ran·Nup358 complexes may represent early
intermediates in the nuclear import pathway for classical NLS-bearing
proteins (Fig. 10). The small amount of Kap·NLS binding in the
absence of Ran could be mediated by interaction of Kap1 with the
repeat regions present in our Nup358 constructs (Fig. 1), but the
significant increase seen in the presence of Ran, particularly RanGTP,
occurs in all probability through the RBH domains. However, even in the
presence of RanGTP, the amount of Kap·NLS bound was small compared
with the input, suggesting that the pentameric complexes represent
unstable transient intermediates in nuclear import, the function of
which is to induce GTP hydrolysis. In summary, the RBH domains of
Nup358 appear to be a site where docking of Kap·NLS results in GTP
hydrolysis and hence recycling of the Kap1·RanGTP complex, thus
linking the termination of one cycle of import and the initiation of
the next (Fig. 10).
Maximal GTP hydrolysis in vitro required both Kap and NLS
(i.e. the formation of the pentameric complex). In
permeabilized cells, under the same conditions, NPC-associated
SUMO-RanGAP1 catalyzed only about 60% hydrolysis of GTP (Fig.
8B). This is unlikely to be due to inadequate quantity of
NPC-associated SUMO-RanGAP1, because near-complete GTP hydrolysis
occurred in the absence of Kap1(Fig. 8B). It thus appears
that the RanGTP-Kap1 complex bound to a subset of the RBH domains on
Nup358 may not be accessible to SUMO-RanGAP1 that is tethered to the
C-terminal portion of Nup358. The addition of free recombinant RanGAP1
to the cells resulted in complete hydrolysis (not shown). These data
suggest that the two forms of RanGAP1 cooperate in GTP hydrolysis at
the NPC. Indeed, free RanGAP has been shown to enhance nuclear import in permeabilized cells and becomes essential if Ran is provided in its
GTP-bound form (19). Furthermore, yeast RanGAP (Rna1p), which is
neither SUMO-modified nor targeted to the NPC, has been shown to be
essential for import (68).
Another difference between NPC-associated SUMO-RanGAP1 and free
unmodified RanGAP1 is that NLS peptide did not enhance GTP hydrolysis
by the former (Fig. 8B). A comparison with yeast may be
relevant to this observation. Hydrolysis of GTP after the formation of
a yeast Kap1·RanGTP complex can be accomplished in a reaction involving RanBP1, Kap, and the C terminus of the yeast nucleoporin Nup1 (36). It is interesting to note that in the same study, direct
binding of the C terminus of Nup1 to Kap was observed. It may well
be that the C terminus of Nup1, by binding to Kap, played a role
similar to that of the NLS peptide in the present study. By analogy, it
is possible that NLS-like sequences in other components of the NPC in
permeabilized cells played a similar role with regard to NPC-associated
SUMO-RanGAP1, thus obviating the need for NLS peptide (Fig.
8B). In any case, the functional significance of this
difference between free and NPC-associated RanGAP1 remains to be
determined. One possibility is that free, unmodified RanGAP1 may link
GTP hydrolysis to NLS availability, whereas NPC-associated RanGAP1 may
be involved in the recycling of Kap1 back to the cytosol in the
presence of "empty" Kap.
Two types of models have been proposed for the initiation and
termination of nuclear import. One assumes that the
NLS·Kap·Kap1 complex forms in the cytosol and then docks at
the NPC (69). In this case, it is proposed that GTP hydrolysis
(recycling) is mediated by RanBP1 and occurs independently from the
docking step of import. The other model postulates that the
Kap1·RanGTP complex binds to Nup358, that GTP hydrolysis occurs
there as Kap and NLS release Kap1 from RanGTP, and that this is
followed by the formation of a more stable RanGDP-containing complex
(70). Our data agree with the second model in that GTP hydrolysis
occurs on Nup358. However, we show that Kap and NLS bind to the
Kap1·RanGTP·Nup358 complex rather than destabilize it, that
RanGTP is more favorable for this binding than RanGDP, and that
hydrolysis occurs within a pentameric
NLS·Kap·Kap1·RanGTP·Nup358 complex.
To summarize, we have shown that Nup358 at the cytoplasmic fibers of
the NPC provides a site where GTP hydrolysis by Ran terminates one
cycle of import as Kap and NLS dock to initiate the next cycle. This
may be the first and last of several steps at the NPC that are required
to complete a cycle of nuclear import.
§¶ and
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Laboratory of Cell
Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327-8096; Fax:
212-327-7880; E-mail: blobel@rockvax.rockefeller.edu.
