(Received for publication, October 5, 1995; and in revised form, December 4, 1995)
From the
Nuclear protein import is accomplished by two sequential events:
docking at the nuclear pore complex followed by ATP-dependent
translocation across the nuclear envelope. Docking of nuclear targeted
proteins requires a 56-kDa nuclear localization signal receptor
(-karyopherin, importin-
, SRP1
) and a 97-kDa protein
(
-karyopherin, importin-
). Components necessary for
translocation include the Ran/TC4 GTPase and NTF2/B-2. The functions of
these factors at a molecular level remain unclear. We have now found
that a complex of Ran, in the GTP-bound state, with either the Ran
binding protein, RanBP1, or an isolated Ran binding domain binds with
high affinity and specificity to
-karyopherin to form a ternary
complex. We find that a C-terminal truncation mutant of Ran,
-DE
Ran, also binds to
-karyopherin and that
-DE Ran can
associate with a cytosolic, multiprotein complex that contains
-karyopherin and another
-DE Ran binding protein of 115/120
kDa. These data suggest a physical link between docking and
translocation mediated by a Ran GTPase-Ran binding protein complex.
The mediated nuclear transport of proteins that bear nuclear
localization signals (NLS) ()proceeds through two distinct
steps. Docking at the nuclear envelope is followed by energy-dependent
translocation of the proteins through the nuclear pore
complex(1, 2, 3) . Recognition and docking of
NLS substrates require a 9 S cytosolic complex that includes
p56/
-karyopherin and
p97/
-karyopherin(4, 5) . The
-karyopherin is
the rat homolog of human SRP1
, Xenopus importin-
,
and yeast SRP1 and functions as a receptor for NLS-containing proteins (5, 6, 7, 8) . The 97-kDa protein,
-karyopherin, also called importin-
, is required for docking
of the NLS substrate, but the exact function of this protein is not
known(5) . It has been suggested that
-karyopherin acts as
an adapter that links the
-karyopherin-NLS substrate complex with
the nuclear pore complex through nucleoporin peptide
repeats(5, 9, 10) . The docking complex
disassembles as a late event in translocation leaving
-karyopherin
associated with the nuclear pore complex while
-karyopherin enters
the nucleus with the NLS substrate(10) .
ATP-dependent
translocation of NLS-containing proteins through the nuclear pore
complex is mediated by the Ran GTPase and a 10-kDa protein,
NTF2/B-2(11, 12, 13) . While these components
are known to be necessary for the import of NLS-containing proteins,
their mechanism of action has not been elucidated. Ran is a 25-kDa
Ras-related GTPase that is predominantly localized to the
nucleus(14, 15) . Expression of a GTPase-deficient
mutant of Ran, or depletion of the Ran GTPase-activating protein,
Rna1p, blocks nuclear import both in vitro and in intact
cells(16, 18, 19) , ()demonstrating that GTP hydrolysis is necessary for nuclear
transport. After GTP hydrolysis, Ran-GDP may enter the nucleus where
guanine nucleotide exchange can be stimulated by the
chromatin-associated factor, RCC1(19) . Ran-GTP would then exit
the nucleus to complete the cycle (for review see (20) ).
Two potential downstream effectors that bind to Ran-GTP have been identified, RanBP1/HTF9A, a 29-kDa cytosolic protein, and RanBP2/NUP358, a 360-kDa nuclear pore protein(21, 22, 23) . These proteins associate with Ran through one or more conserved Ran binding domains (RanBDs). These domains stabilize Ran in the GTP-bound state and co-activate the GTPase-activating protein, Rna1p(24, 25, 26) . Additionally, antibodies raised against RanBP2 inhibit nuclear translocation, which suggests that RanBP2 may be a necessary constituent in the nuclear import mechanism(22) . No requirement for RanBP1 in nuclear protein import has yet been established.
Removal of the acidic C terminus of
Ran reduces binding to RanBPs but enhances binding to two unidentified
proteins of 90 and 115/120 kDa (24) . In this report we
identify the 90-kDa protein as -karyopherin. Direct interaction of
-karyopherin to wild-type Ran is promoted by association of Ran
with RanBP1 or with an isolated Ran binding domain. These results
suggest that
-karyopherin links the docking and translocation
steps of nuclear import by forming a ternary complex with the Ran
GTPase and a RanBP.
Rat brain
cytosol proteins were prepared as described previously(3) .
BHK21 cell proteins were prepared by addition of hot SDS sample buffer
to 100-mm plates of confluent BHK21 cells that had been rinsed twice
with phosphate-buffered saline. Cell lysate was passed through a
26-gauge syringe needle, and insoluble material was removed by
centrifugation at 12,000 g for 5 min. Protein samples
(150 µg) were separated by 8% SDS-PAGE and transferred to
nitrocellulose for analysis by the Ran overlay assay, as described
previously(24) . Equal amounts of Ran-bound
[
-
P]GTP (approximately 300,000 cpm) were
diluted into 10 ml of binding buffer for the overlay. After 30 min of
incubation, overlays were washed and exposed to film(24) .
The -DE Ran affinity matrix was prepared by applying lysate
from 1 liter of E. coli expressing GST-
-DE Ran
(approximately 3 mg) onto 1 ml of glutathione-Sepharose beads. After 30
min, the beads were washed twice with phosphate-buffered saline and
once with 25 mM MOPS, pH 7.1, plus 1 mM EDTA. The
beads were resuspended in the same buffer plus 2 mM GTP and
incubated for 30 min. Magnesium acetate (20 mM) was then
added, and the
-DE Ran-Sepharose was centrifuged for 2 min at 2000
g and washed with binding buffer(24) .
Cleared supernatant was incubated with the -DE Ran-Sepharose
for 1-2 h. The mixture was then washed three times with binding
buffer plus 0.05% Tween 20 and once with binding buffer and then loaded
onto a small column. Purified proteins were eluted with 4 ml of 10
mM glutathione in binding buffer.
We showed previously that deletion of the C terminus of Ran
reduces the affinity of Ran-GTP for the Ran-binding proteins RanBP1 and
RanBP2(17) . In addition, the C-terminal deletion mutant of
Ran, -DE Ran, binds avidly to unidentified proteins of 90 and
115/120 kDa(24) . These proteins (
-DE RanBPs) associate
specifically with
-DE Ran that is in the GTP-bound state; they
inhibit release of GTP and are located primarily in the soluble
fraction of cell extracts (not shown). The affinity of the proteins for
-DE Ran appeared to be higher than that to wild-type Ran, possibly
as a consequence of the unmasking of a region on Ran normally blocked
by the C terminus. We reasoned that the formation of a RanBP-Ran
heterodimer may present the Ran in a conformation able to bind more
efficiently to the 90- and 115/120-kDa proteins.
To test this
hypothesis, BHK21 cell extract was separated by SDS-PAGE, transferred
to nitrocellulose, and incubated with a complex of
[P]GTP-Ran and RanBP1. As predicted, the
formation of a Ran-RanBP1 complex dramatically increased the apparent
affinity of Ran for the 90- and 115/120-kDa
-DE RanBPs (Fig. 1). The RanBP1 also effectively competed out binding on
the nitrocellulose to endogenous RanBP1 and RanBP2 from BHK21 cells.
The association of Ran with the 90- and 115/120-kDa proteins was
further intensified when Ran was complexed with an isolated RanBD of
RanBP1 (Fig. 1) or of RanBP2 (not shown). Removal of the C
terminus from RanBP1 also enhanced the binding of Ran to the
-DE
RanBPs (Fig. 1). These results show that the behavior of the
-DE Ran mutant can be mimicked by the heteromeric association of
full-length Ran with a Ran binding domain. The data suggest that this
association causes a change in the conformation of the C terminus of
the Ran GTPase that exposes a binding site for the 90- and p115/120-kDa
proteins.
Figure 1:
A Ran-RanBP1 complex is able to bind to
-DE Ran binding proteins. Six lanes of BHK21 cell extract (150
µg of protein) were separated by 8% SDS-PAGE and transferred to
nitrocellulose. Lanes were then incubated separately for 30 min with
300,000 cpm of [
-
P]GTP-GST-
-DE Ran
alone (lane 5) or with [
P]GTP-Ran
(wild type) plus the indicated constructs of GST-RanBP1 fusion proteins (lanes 1-4) or plus GST as a negative control (lane
6), each in 10 ml of binding buffer. (Specific activity of the
[
P]GTP was 3000 Ci/mmol.) RanBD consists of
residues 27-160 of RanBP1, RanBP1
-C of residues
1-160, and RanBP1
-N of residues 27-203.
-DE Ran
consists of residues 1-197 of Ran. Following incubation, the
overlays were washed and exposed to x-ray film overnight. The positions
of rainbow molecular weight standards (Amersham Corp.) are
noted.
These proteins are potentially important effectors of Ran
function. We therefore pursued the identification of the 90-kDa protein
by affinity purification using a GST fusion of -DE Ran. Fusion to
GST did not affect the binding of
-DE Ran to the 90- and
115/120-kDa proteins or increase binding to nonspecific proteins in
overlay assays (Fig. 1, lane 5). Using the rat brain
cytosol fractions defined by Moore and Blobel(3) , we observed
that Ran is found exclusively in fraction B, and the RanBPs and
-DE RanBPs were both present in fraction A, the component required
for docking of NLS substrates(3, 11) . We therefore
prepared a modified fraction A from rat brain cytosol as an initial
step in the purification of the 90-kDa protein. This partially purified
material was then affinity-purified using a glutathione-Sepharose
matrix coupled to GST-
-DE Ran. The affinity step yielded the 90-
and 115/120-kDa proteins, purified nearly to homogeneity (Fig. 2, lane 2). Peptide sequences of two separate
trypsin fragments of the 90-kDa protein were identical to sequences
within p97/
-karyopherin, an essential component of the 9 S nuclear
docking complex (Table 1) (4) .
Figure 2:
Purification of the 90-kDa protein
-DE Ran binding protein by
-DE Ran affinity chromatography. A, rat brain cytosol was applied to 100 ml of a DE52 anion
exchange cellulose slurry, washed with 150 mM sodium acetate,
and eluted with 500 mM sodium acetate (as described under
``Experimental Procedures''). The eluate was mixed with
GST-
-DE Ran-GTP coupled to glutathione-Sepharose column beads and
eluted with 10 mM glutathione. Shown is a silver-stained gel
of 50 µg of DE52 eluate (lane 1),
-DE Ran
affinity-purified protein (10% of preparation) (lane 2), and
proteins present in 100 µl of the GST-
-DE Ran matrix before
addition of the partially purified
-DE RanBP preparation (lane
3). B, autoradiogram of the
[
-
P]GTP-GST-
-DE Ran overlay of
fractions from the p90 purification. Portions of rat brain cytosol (lane 1), DE52 eluate (lane 2),
-DE Ran
affinity-purified material (lane 3), and GST-
-DE Ran
affinity matrix material (lane 4) were subjected to SDS-PAGE
and
-DE Ran overlay as described in Fig. 1.
To confirm that
-karyopherin is a Ran binding protein, recombinant
-karyopherin carrying a His
-tag (5) was tested
for its ability to interact with
[
-
P]GTP-Ran in an overlay assay. The
conditions that enhance binding of Ran to
-karyopherin (Fig. 3, lower panel) paralleled those that promote
binding of Ran to the 90-kDa protein from brain cytosol (upper
panel). Wild-type Ran in the presence of GST alone did not
detectably bind recombinant
-karyopherin (lane 1). As
predicted, however, both
-DE Ran (lower panel, lane 5)
and a complex of wild-type Ran with the isolated Ran binding domain of
RanBP1 (lane 2) did associate with recombinant
-karyopherin. The addition of
-karyopherin to the Ran-RanBP1
complex also abolished binding of the complex to immobilized
-karyopherin and to the 90- and 115/120-kDa brain proteins (lane 4). It did not reduce binding to RanBP2, however (lane 3), confirming that
-karyopherin interacts with a
region on Ran distinct from the RanBD binding site. Note that the
recombinant
-karyopherin was less efficient in competition for
binding to the
-DE Ran (lane 6). We do not currently
understand the basis for this difference.
Figure 3:
Interaction of
[-
P]GTP-
-DE Ran with
-karyopherin. 150 µg of rat brain cytosol proteins (upper
panel) or 0.5 µg of recombinant
His
-
-karyopherin (lower panel) were separated
by 8% SDS-PAGE and transferred to nitrocellulose.
[
-
P]GTP-Ran or
[
-
P]GTP-GST-
-DE Ran (2 µg of
protein each; 300,000 cpm; specific activity, 3000 Ci/mmol) were mixed
with the proteins indicated above the top panel, then were
added separately to the nitrocellulose strips in 10 ml of binding
buffer, and incubated for 30 min, as described in Fig. 1. GST
was included in lane 1 as a negative control.
-kar, recombinant
-karyopherin. The nitrocellulose
strips were then washed and exposed to x-ray film overnight. The
positions of the 90-kDa protein, 115/120-kDa protein, and
-karyopherin are indicated by arrows.
Endogenous
-karyopherin is present in the cytosol as part of a multisubunit
complex(4) , and we explored the possibility that Ran interacts
with this complex. Proteins in the modified fraction A from rat brain
cytosol were mixed with GST (Fig. 4A) or GST-
-DE
Ran (Fig. 4B) loaded with GTP. Protein complexes were
then separated by size exclusion chromatography. Fractions were
analyzed by overlay with GST-
-DE Ran to detect the 90- and
115/120-kDa proteins. After exposure to x-ray film, the same
nitrocellulose blot was washed to remove the probe and immunoblotted
with either anti-Ran antiserum (Fig. 4B, bottom
panel) or anti-GST antibody (not shown) to identify fractions
containing GST-
-DE Ran. Two distinct peaks of
-DE RanBPs were
detected in fractions that correspond to sizes of approximately 700 and
200 kDa (Fig. 4A). The 700-kDa peak may represent the 9
S complex(4) . The addition of GST-
-DE Ran-GTP to the
cytosol did not affect the elution profile of the
-DE RanBPs (Fig. 4B), but the GST-
-DE Ran preferentially
associated with the 700-kDa peak. One interpretation of this result is
that other proteins contained in the large complex increase the
affinity of Ran for the
-DE RanBPs.
Figure 4:
Size exclusion chromatography of the 90-
and 115/120-kDa -DE Ran binding proteins. A, 200 µg
of DE52-purified brain cytosol proteins mixed with 3 µg of GST
(negative control) were applied to a Superose 12 FPLC size exclusion
column, and the eluant fractions (0.5 ml each) were separated by 8%
SDS-PAGE and transferred to nitrocellulose. The void volume of the
column was 4.5 ml; total column volume was 20 ml. The nitrocellulose
was then overlaid with [
-
P]GTP-GST-
-DE
Ran. Positions of molecular weight standards are designated by the arrows. B, 3 µg of GST-
-DE Ran was loaded
with GTP and incubated with 200 µg of DE52 eluate proteins for 10
min. This mixture was applied to the Superose 12 column and analyzed as
above (upper panel). After overnight exposure to x-ray film,
the nitrocellulose was washed and immunoblotted with anti-Ran 12
antiserum and horseradish peroxidase-anti-rabbit secondary antibody,
which was detected by chemiluminescence (lower panel).
-kar (recombinant
-karyopherin) indicates the
position of the 90-kDa
-DE Ran binding protein;
-DE indicates the position of GST-
-DE Ran (50
kDa).
Together these data fit
well with the current model for mediated transport of karyophiles
through nuclear pore complexes and, importantly, they link the two
major steps in nuclear import: docking and translocation. We propose a
model in which -karyopherin serves as an adapter between the
-karyopherin-NLS substrate complex and a Ran-RanBP heterodimer.
RanBP1 may stabilize this complex in the cytosol until it is displaced
by RanBP2 at the nuclear pore. Hydrolysis of Ran-GTP, stimulated by the
Ran GTPase-activating protein, Rna1p, then triggers dissociation of the
complex and permits the entry of the NLS substrate-
-karyopherin
heteromer into the nucleus, perhaps with NTF2/B-2. The GDP-Ran released
from the complex also enters the nucleus where RCC1 catalyzes the
exchange of GTP for GDP on Ran such that the cycle can continue. The
tools currently available will now allow for a more detailed
biochemical analysis of the interactions between the growing number of
components that regulate nuclear import.