(Received for publication, April 16, 1997, and in revised form, May 23, 1997)
From the Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021
We previously showed that RanGTP forms a 1:1
complex with karyopherin that renders RanGTP inaccessible to RanGAP
(Floer, M., and Blobel, G. (1996) J. Biol. Chem. 271, 5313-5316) and karyopherin
functionally inactive (Rexach, M., and
Blobel, G. (1995) Cell 83, 683-692). Recycling of both
factors for another round of function requires dissociation of the
RanGTP-karyopherin
complex. Here we show using BIAcoreTM, a
solution binding assay, and GTP hydrolysis and exchange assays, with
yeast proteins, that karyopherin
and RanGTP are recycled
efficiently in a reaction that involves karyopherin
, RanBP1,
RanGAP, and the C terminus of the nucleoporin Nup1. We find that
karyopherin
first releases RanGTP from karyopherin
in a
reaction that does not require GTP hydrolysis. The released RanGTP is
then sequestered by RanBP1, and the newly formed karyopherin
binds to the C terminus of Nup1. Finally, RanGTP is converted to RanGDP
via nucleotide hydrolysis when RanGAP is present. Conversion of RanGTP
to RanGDP can also occur via nucleotide exchange in the presence of
RanGEF, an excess of GDP, and if RanBP1 is absent. Additional
nucleoporin domains that bind karyopherin
stimulate recycling of
karyopherin
and Ran in a manner similar to the C terminus of Nup1.
Transport of proteins that contain a nuclear localization signal
(NLS)1 into the nucleus of the cell
requires energy, mobile transport factors, and nuclear pore complexes
(NPC) in the nuclear envelope. Karyopherin heterodimer (also
termed importin
, NLS receptor-p97 complex, PTAC, or Kap60/95)
binds proteins that contain an NLS similar to that of the SV40 large
T-antigen or nucleoplasmin (NLS protein) and brings them to the NPC
(3-12). Karyopherin
binds the NLS protein (2, 3, 7, 13-15),
whereas karyopherin
increases the affinity of karyopherin
for
the NLS (2, 16) and docks the karyopherin
-NLS-protein complex to a
subfamily of NPC proteins (nucleoporins) that contain
XXFG-peptide repeats (2, 14, 17-20). The subsequent
translocation across the NPC requires Ran/TC4 (21, 22) and p10/NTF2
(23, 24). p10 is a dimer (24, 25) that binds RanGDP (26, 27) and
karyopherin
(26, 28) and functions to tether RanGDP to karyopherin
heterodimers that are docked to nucleoporins (26). When Ran is
in its GTP-bound form it disrupts the interaction of karyopherin
with karyopherin
and with FXFG regions of nucleoporins
by forming a complex with karyopherin
(2). The repetitive
interaction of transport factors, substrates, and nucleoporins at the
NPC may facilitate the transport of substrates across the NPC (2, 17).
Accessory factors regulate nuclear transport by modulating Ran. The
GTPase-activating protein for Ran, RanGAP (termed RanGAP1, or Rna1 in
yeast) (29-32), and the nucleotide exchange factor for Ran, RanGEF
(termed RCC1, or Prp20 in yeast) (33-35), are required to sustain
efficient transport of substrates across the NPC (36-39). The Ran
binding protein 1, RanBP1 (40), is also involved in nuclear transport
(41, 42). As the RanGTP-karyopherin complex is resistant to
stimulation of GTP hydrolysis by RanGAP (1, 39, 43), RanGAP-stimulated
GTP hydrolysis cannot dissociate the RanGTP-karyopherin
complex.
However, dissociation of RanGTP-karyopherin
is crucial to recycle
both factors for another round of function.
We show here that the RanGTP-karyopherin complex is disassembled in
the presence of karyopherin
, the C terminus of Nup1 (C-Nup1),
RanGAP, and RanBP1. A detailed analysis of the reaction mechanism
revealed that karyopherin
is first released from RanGTP by
karyopherin
, followed by conversion of RanGTP to RanGDP in the
presence of RanGAP and RanBP1. Interaction of RanBP1 with RanGTP not
only stimulates GTP hydrolysis in the presence of RanGAP but also
prevents reformation of the RanGTP-karyopherin
complex in the
presence of C-Nup1 and karyopherin
. C-Nup1 sequesters the released
karyopherin
by forming a C-Nup1-karyopherin
complex.
Formation of this ternary complex makes rebinding of karyopherin
to
RanGTP less favorable. Nup36 and a fragment containing the
FXFG repeat region of Nup1 function in the disassembly
reaction as well, although with lower activity than C-Nup1.
Yeast Ran and RanGAP
were expressed and purified as described (1). Yeast karyopherin (Kap60) and karyopherin
(Kap95), the Nup1 fragment containing a
FXFG repeat region (AA 432-816) and the Nup2 fragment
containing a FXFG repeat region (AA 186-561), were
expressed as glutathione S-transferase (GST) fusion proteins as described (2, 12). Proteins were purified, and the GST moiety was
cleaved with thrombin as described for GST-fusion proteins (1, 2).
RanGEF, the C terminus of Nup1 (AA 963-1076) (C-Nup1), and Nup36 were
expressed as GST-fusion proteins. The genes or gene fragments encoding
these proteins were amplified by polymerase chain reaction from
Saccharomyces cerevisiae genomic DNA (Promega). The RanGEF
polymerase chain reaction product was inserted into vector pGEX-2TK
(Pharmacia Biotech Inc.) as a BglII-EcoRI
fragment and amplified C-Nup1 and Nup36 were inserted into pGEX-2TK as BamHI-EcoRI fragments. The proteins were
expressed in Escherichia coli strain BLR (Novagen). The
GST-fusion proteins of RanGEF and Nup36 were purified from bacterial
lysates on glutathione-Sepharose beads (Pharmacia), and the GST moiety
was cleaved with thrombin as described for GST-fusion proteins (1, 2).
The GST-fusion protein of C-Nup1 was purified on glutathione-Sepharose
beads and eluted with 10 mM glutathione, as described
previously for GST-fusion proteins (12). The purified proteins were
stored in frozen aliquots at 80 °C.
Yeast RanBP1 (Yrb1) was amplified from S. cerevisiae genomic
DNA (Promega) by polymerase chain reaction and inserted as a NcoI-BamHI fragment into pET-21d vector
(Novagen). Protein was expressed in E. coli strain BLR(DE3)
(Novagen) at 37 °C for 4 h. Cells were harvested by
centrifugation at 2,000 × g, and the cell pellet was
resuspended in ice-cold Tris buffer (10 mM Tris-HCl, pH
6.8, 1 mM MgCl2, and 1 mM
dithiothreitol). After cell lysis using a French pressure cell ammonium
sulfate was added at a final concentration of 55%; RanBP1 was found in
the soluble fraction. The dialyzed 10,000 × g
supernatant was loaded onto a MonoQ fast protein liquid chromatography
column (Pharmacia), and proteins were eluted using a linear gradient
(0-500 mM) of NaCl in Tris buffer. RanBP1 eluted between
50 and 200 mM NaCl. Fractions containing RanBP1 were
pooled, concentrated with a Centricon 10 unit (Amicon), and
fractionated on a Superdex 75 fast protein liquid chromatography column
(Pharmacia) which was equilibrated with buffer A (150 mM KOAc, 20 mM Hepes, pH 7.3, 2 mM
Mg(OAc)2, 1 mM dithiothreitol). RanBP1 eluted
as a dimer with a mobility equal to that of a 70-kDa globular protein.
Fractions containing RanBP1 were pooled and aliquots were stored at
80 °C.
For each experiment, an E. coli lysate containing GST-C-Nup1 was incubated for 20 min at 4 °C with glutathione-agarose beads (Sigma) (2 µg of C-Nup1 per 10 µl of beads) in 0.5 ml of binding buffer (20 mM Hepes, pH 6.8, 150 mM KOAc, 2 mM Mg(OAc)2, 1 mM dithiothreitol, 0.1% Tween 20). Beads were washed 6 times by centrifugation (2,000 × g for 30 s) and resuspension in 0.5 ml of chilled binding buffer. One-step assay: 20 µl of bead slurry was added to siliconized 0.5-ml tubes (Sigma) that contained protein additions as indicated in the figure legends, in a total volume of 40 µl. Tubes were tumbled end over end for 1 h at 4 or 21 °C as indicated in the figure legends. Two-step assay: the bead slurry was incubated for 40 min at 4 °C with Kap95 (0.6 µg for every 10 µl of beads) in 0.5 ml of binding buffer. After washing 3 times with 0.5 ml of binding buffer, beads were resuspended as a 50% slurry and incubated with protein additions as in the one-step assay. Beads were subjected to centrifugation at 2,000 × g for 30 s, and unbound proteins were collected by removing 30 µl from the meniscus; this constitutes the unbound fraction. Beads were washed twice with 0.5 ml of chilled binding buffer as before, and 28 µl of binding buffer was added; this constitutes the bound fraction. 12 or 10 µl of 6 × Laemmli sample buffer with 2-mercaptoethanol was added to bound and unbound fractions, respectively. After incubation at 95 °C for 15 min, proteins in 18 µl of each sample were resolved by SDS-PAGE and stained with Coomassie Blue.
GTP Hydrolysis and Nucleotide Exchange AssaysGTP
hydrolysis assays were conducted as described previously (1). 15 nM Ran-[-32P]GTP and 25 nM
karyopherin
were incubated for 10 min at 21 °C, and then RanGAP,
karyopherin
, C-Nup1, and RanBP1 were added as indicated in the
figure legends. Reactions were incubated for 20 min at 21 °C.
Nucleotide exchange assays were performed essentially as GTP hydrolysis
assays, except that Ran was labeled with [
-32P]GTP,
and 200 µM GTP and 200 µM GDP were added to
the exchange reaction. 15 nM Ran-[
-32P]GTP
was incubated with 25 nM karyopherin
for 10 min at
21 °C, and then RanGEF, karyopherin
, C-Nup1, and RanBP1 were
added. Reactions were incubated for 20 min at 21 °C.
The BIAcoreTM methodology is
described elsewhere (44). Experiments were conducted on a BIAcoreTM
(upgrade) instrument (BIAcore Inc.). Ran was immobilized on a CM5
sensor chip (BIAcore Inc.; research grade) by amine coupling. The
surface was activated with 5 µl of a 1:1 mixture of 0.05 M N-hydroxysuccinimide and 0.2 M N-ethyl-N-(3-dimethylaminopropyl)carbodiimide
hydrochloride at a flow rate of 5 µl/min. Ran was coupled by
injecting 30 µl of a solution containing 10 µg/ml Ran in 10 mM NaOAc, pH 5.0, 2 mM Mg(OAc)2.
The surface was blocked by injecting 30 µl of 300 mM Tris-HCl, pH 8.0, 0.1 mM GTP, 2 mM
Mg(OAc)2. Typically, this resulted in immobilization of
300-400 resonance units (RU) of Ran on the sensor chip surface. We
determined using a method described in Ref. 1 that only ~40% of the
Ran was GTP-bound and ~60% was GDP-bound; therefore, we estimate
that 120-160 RU of RanGTP were immobilized. All experiments were
performed in buffer A containing 1 mM EGTA, 0.005% Tween
20, and 0.1 mM GTP at a flow rate of 10 µl/min at
25 °C. The Ran surface was regenerated by injecting 5 µl of 0.5%
Triton X-100 and 0.01% SDS in water. To obtain RanGMP-PCP, 30 µM Ran was incubated for 1 h at 21 °C in 0.5 ml
of buffer A with 5 mM EDTA, 2 mM GMP-PCP, and
no Mg(OAc)2. The exchange reaction was quenched by adding
20 mM MgCl2, and unbound nucleotide was removed
on a Nap5 spin column (Pharmacia). This resulted in Ran containing
~39% GMP-PCP, ~60% GDP, and less than 1% GTP, determined as
described (1). A RanGMP-PCP surface was generated as described for
Ran.
We previously showed that RanGTP forms a complex with karyopherin
(1, 2). Estimates from the inhibition of RanGAP by karyopherin
indicate an affinity below 1 nM for the RanGTP-karyopherin
interaction2 (0.3 nM for
mammalian proteins (39)). Here we investigated the disassembly of the
yeast RanGTP-karyopherin
complex and its regulation by transport
factors and nucleoporins. As RanGAP is synthetically lethal with the C
terminus of the nucleoporin Nup1 (45) and with karyopherin
(46), we
investigated whether the C terminus of Nup1 and RanGAP are involved in
the disassembly of the RanGTP-karyopherin
complex.
Karyopherin binds to the C terminus of Nup1 (C-Nup1) (AA 963-1076)
(Fig. 1, lane 1) and is released in the
presence of RanGTP (lanes 2 and 3) but not RanGDP
(lanes 4 and 5). This indicates that complex
formation of RanGTP with karyopherin
abolishes the interaction of
karyopherin
with C-Nup1. Neither RanGDP nor RanGTP bound to C-Nup1
(not shown). Preincubation of karyopherin
with RanGTP for 15 min at
4 °C also abolished binding of karyopherin
to C-Nup1 (Fig.
2A, compare lane 1 to lane
2). We used this observation as an assay to detect disassembly of
the RanGTP-karyopherin
complex in the presence of different
transport factors. Addition of RanGAP to karyopherin
and RanGTP
that had been preincubated did not stimulate disassembly of the
RanGTP-karyopherin
complex as judged by the inability of
karyopherin
to bind to C-Nup1 (lane 3). However,
addition of karyopherin
led to binding of some karyopherin
to
C-Nup1 (lane 4). Karyopherin
bound directly to C-Nup1 in
the absence of karyopherin
(not shown). The binding of karyopherin
to C-Nup1 will be discussed elsewhere.3
Most importantly, when karyopherin
and RanGAP were added together, karyopherin
binding to C-Nup1 was restored (lane 5).
These results indicate that the RanGTP-karyopherin
complex is
disrupted in the presence of karyopherin
, C-Nup1, and RanGAP and
that RanGTP is converted to RanGDP through GTP hydrolysis stimulated by
RanGAP.
To test whether conversion of RanGTP to RanGDP occurred, we measured
GTP hydrolysis. RanGAP-stimulated GTP hydrolysis by Ran was completely
inhibited in the presence of karyopherin (not shown) (1). However,
addition of 1 µM karyopherin
and 10 nM
RanGAP to a mixture of 15 nM RanGTP and 25 nM
karyopherin
that had been preincubated resulted in 25% GTP
hydrolysis (Fig. 2B). If 200 nM C-Nup1 was added
to this reaction, 70% of the Ran-bound GTP was hydrolyzed, and if 1 µM C-Nup1 was added, 80% of the Ran-bound GTP was
hydrolyzed. These results demonstrate that in the presence of
karyopherin
RanGAP stimulates GTP hydrolysis by Ran that was bound
to karyopherin
and that this reaction is stimulated by C-Nup1.
As the C-Nup1-stimulated conversion of RanGTP to RanGDP was only 80%,
we investigated whether RanBP1 could complete the reaction, as RanBP1
binds to RanGTP (40, 47) and enhances RanGAP-stimulated GTP hydrolysis
by Ran (41, 43, 47) (Fig. 9). Addition of RanBP1 and RanGAP to a
mixture of RanGTP and karyopherin that had been preincubated did
not promote GTP hydrolysis (not shown). However, when RanBP1 and RanGAP
were added together with karyopherin
and C-Nup1, GTP hydrolysis was
greatly stimulated (Fig. 3). In a control reaction that
contained 15 nM RanGTP and 25 nM karyopherin
that had been preincubated, 10 nM RanGAP, 100 nM karyopherin
, and 100 nM C-Nup1, 22% of
the Ran-bound GTP was hydrolyzed (Fig. 3, closed symbols).
When 50 nM RanBP1 was added, 80% of the Ran-bound GTP was
hydrolyzed, and when 100 nM RanBP1 was added, 95% of the
Ran-bound GTP was hydrolyzed. Noticeably, the concentration of RanGAP
required was lower in the presence of RanBP1 (open
symbols); this is consistent with previous findings on
RanBP1 function in the absence of karyopherin
(41, 47) (Fig. 9).
These results demonstrate that RanGTP-karyopherin
complex is fully
disassembled in a reaction that requires karyopherin
and is
stimulated by RanGAP, RanBP1, and the C terminus of Nup1.
To determine if RanGAP is essential for the disassembly reaction, we
tested whether the nucleotide exchange factor for Ran, RanGEF, could
replace RanGAP. RanGEF stimulates exchange of GDP and GTP on Ran with
equal efficiency (48). Due to the higher concentration of GTP in the
cell (49), it is generally assumed that RanGEF exchanges Ran-bound GDP
for GTP in vivo (48). Nevertheless, RanGEF can exchange
Ran-bound GTP for GDP in vitro if an excess of GDP is
provided (48). Although recombinant RanGEF stimulated nucleotide
exchange on Ran (not shown), it was incapable of stimulating exchange
when RanGTP was bound to karyopherin (Fig.
4A) (39). However, exchange of Ran-bound GTP
occurred when 10 nM RanGEF, 200 µM GDP, 200 µM GTP, and increasing amounts of karyopherin
and
C-Nup1 were added to 15 nM RanGTP and 25 nM
karyopherin
that had been preincubated (Fig. 4A). GTP
exchange reached 80% in the presence of 1 µM karyopherin
and 1 µM C-Nup1. Addition of C-Nup1 in the absence of
karyopherin
did not result in GTP exchange, whereas addition of 1 µM karyopherin
in the absence of C-Nup1 promoted
exchange of 25% of the Ran-bound GTP (not shown). We also tested the
effect of RanBP1 on this reaction. 50 nM RanBP1 inhibited
exchange of Ran-bound GTP when added to a reaction that contained 10 nM RanGEF, 200 µM GDP, 200 µM
GTP, 1 µM karyopherin
, 1 µM C-Nup1, and
15 nM RanGTP and 25 nM karyopherin
that had
been preincubated (Fig. 4B). This result is consistent with previous reports on the inhibitory effect of RanBP1 on the exchange of
Ran-bound GTP when karyopherin
is absent (43, 47).
To analyze further the effect of RanGEF and RanBP1 on the disassembly
of RanGTP-karyopherin complex, we carried out solution binding
assays similar to those described in Fig. 2A. As expected, RanGEF could replace RanGAP (Fig. 5A) when an
excess of GDP (lane 4), but not GTP (lane 5), was
present in addition to karyopherin
and C-Nup1. Karyopherin
was
required, as RanGEF and GDP alone did not promote karyopherin
binding to C-Nup1 (lane 6). This result suggests that RanGTP
is accessible to RanGAP, or RanGEF, after the RanGTP-karyopherin
complex is disassembled by the combined action of karyopherin
and
C-Nup1. Surprisingly, RanGEF bound to the C terminus of Nup1
(lanes 4-7); we are currently investigating the
significance of this interaction. We then tested whether inhibition of
RanGEF function by RanBP1 had an effect on RanGTP-karyopherin
disassembly. Unexpectedly, disassembly occurred even in the presence of
RanBP1 (compare lane 7 to lane 4). This
surprising result suggests that RanBP1 can directly stimulate disassembly of the RanGTP-karyopherin
complex in the presence of
karyopherin
and C-Nup1. Indeed RanBP1 stimulated disassembly in the
presence of karyopherin
and C-Nup1 (Fig. 5B, lane
4) but not in the absence of karyopherin
(lane 5).
Thus karyopherin
is required to disrupt the RanGTP-karyopherin
complex in the presence of RanBP1 and C-Nup1. Neither RanBP1 nor RanGTP
bound to the karyopherin
-C-Nup1 complex (not shown). These
results suggest that RanBP1 stimulates disassembly of the
RanGTP-karyopherin
complex by preventing reformation of the complex
after it has been disassembled in the presence of karyopherin
and
C-Nup1. RanBP1 may accomplish this task by sequestering RanGTP that had been released from karyopherin
, since RanBP1 binds RanGTP (not shown) (40, 43, 47).
To analyze in detail the mechanism of RanGTP-karyopherin disassembly we used BIAcoreTM (44) which allows a direct measurement of
on/off rates of protein-protein interactions. Ran containing ~40%
GTP and ~60% GDP was immobilized on a CM5 sensor chip by amine
coupling as described under "Experimental Procedures." 100 µg/ml
karyopherin
was injected for 3 min at a flow rate of 10 µl/min
over the Ran surface which typically resulted in binding of 500 resonance units (RU) of karyopherin
(Fig.
6A). This was followed by a wash-out phase of
3 min to monitor karyopherin
dissociation. We then examined whether
the dissociation rate of the RanGTP-karyopherin
complex increases
when other proteins are injected during the wash-out phase. For these
experiments 100-200 RU of karyopherin
were bound to the Ran
surface, dissociation was allowed to proceed for 1 min, and solutions
containing different factors were injected for 1 min. Strikingly,
injection of karyopherin
caused the release of all karyopherin
from RanGTP (Fig. 6B). The amount released was dependent on
the concentration of karyopherin
injected with a linear relation
between released karyopherin
and the concentration of karyopherin
(0-2 µg/ml karyopherin
) (Fig. 6B,
inset). This result indicates that karyopherin
disassembles the RanGTP-karyopherin
complex and that this
disassembly is a first order reaction with respect to karyopherin
.
The apparent rate of RanGTP-karyopherin
dissociation in the
presence of saturating concentrations of karyopherin
was faster
than the detection limit of BIAcoreTM (>0.1 s
1); hence,
stimulation of RanGTP-karyopherin
dissociation by karyopherin
could not be measured directly. The rate of RanGTP-karyopherin
dissociation in the absence of karyopherin
was calculated to be
4.5 × 10
4 s
1 based on the conditions
of these experiments. We therefore estimate that karyopherin
stimulates dissociation of the RanGTP-karyopherin
complex by at
least 3 orders of magnitude. RanGAP did not increase the amount of
karyopherin
released in the presence of limiting amounts of
karyopherin
(not shown). This result is in agreement with the
notion that RanGAP interacts with RanGTP only after its release from
karyopherin
.
To test whether intrinsic GTP hydrolysis by Ran is a prerequisite for
karyopherin -induced dissociation of karyopherin
, we used
RanGMP-PCP instead of RanGTP; GMP-PCP is a non-hydrolyzable analog of
GTP. Ran was incubated with GMP-PCP as described under "Experimental
Procedures" to obtain Ran that was ~39% GMP-PCP, ~60% GDP, and
less than 1% GTP-bound. RanGMP-PCP was immobilized on a CM5 sensor
chip as described for Ran. Karyopherin
bound to RanGMP-PCP with the
same apparent kinetics as to RanGTP under these conditions (compare
Fig. 6, C to A). When karyopherin
was
injected during the wash-out phase, all the karyopherin
was
released (Fig. 6C). The release of karyopherin
from
RanGMP-PCP showed the same dependence on the concentration of
karyopherin
as release of karyopherin
from RanGTP (not shown).
This result demonstrates that GTP hydrolysis is not required for the
karyopherin
-dependent dissociation of RanGTP from
karyopherin
.
We also used BIAcoreTM to investigate the role of C-Nup1 in the
disassembly of the RanGTP-karyopherin complex. C-Nup1 did not
release karyopherin
from RanGTP when injected during the wash-out
phase (not shown). Also, coinjection of C-Nup1 with karyopherin
did
not stimulate release over the levels seen with karyopherin
alone
(not shown). These results were surprising as C-Nup1 greatly stimulates
the disruption of the RanGTP-karyopherin
complex by karyopherin
, as judged by the GTP hydrolysis assay (Fig. 2B), yet
does not stimulate RanGAP activity directly (not shown). To understand
the role of C-Nup1 in the RanGTP-karyopherin
disassembly reaction,
we compared the GTP hydrolysis and BIAcoreTM experiments. In the
BIAcoreTM experiments the disassembly reaction is monitored in real
time and not at equilibrium as in the GTP hydrolysis assay. In the GTP
hydrolysis assay the released karyopherin
may rebind to RanGTP
before RanGAP stimulates GTP hydrolysis, whereas in the BIAcoreTM
experiment the released karyopherin
is removed by constant flow and
cannot rebind to RanGTP. In BIAcoreTM rebinding during the wash-out
phase is significant only when high density surfaces and low flow rates
are used (50). As there was only ~160 RU of RanGTP immobilized on the
surface, and there was no change in the RanGTP-karyopherin
dissociation rate at flow rates of up to 30 µl/min, we assume that
rebinding of karyopherin
to RanGTP did not occur. To test whether
C-Nup1 affects rebinding of karyopherin
to RanGTP, we coinjected
karyopherin
during the wash-out phase; this retards the diffusion
of dissociated karyopherin
from the Ran surface and may promote
rebinding of karyopherin
before it is removed by the wash. When 5 nM karyopherin
was coinjected with 35 nM
karyopherin
(2 µg/ml), release of karyopherin
was completely
inhibited (Fig. 7A). Strikingly, release of
karyopherin
from the RanGTP surface was restored when 35 nM C-Nup1 was coinjected with 35 nM karyopherin
and 5 nM karyopherin
(Fig. 7B). This
result indicates that C-Nup1 sequesters karyopherin
and prevents
reformation of the RanGTP-karyopherin
complex.
As yeast karyopherin also binds to the FXFG repeat
region of Nup1 (2), Nup2 (2), and Nup36 (26), we tested whether these
nucleoporins or fragments thereof could replace C-Nup1 in the
disassembly of the RanGTP-karyopherin
complex using the GTP
hydrolysis assay. As a control, addition of 0.75 µM
karyopherin
and 10 nM RanGAP to 15 nM
RanGTP and 25 nM karyopherin
that had been preincubated
resulted in 11% hydrolysis of the Ran-bound GTP (Fig.
8). Addition of 1 µM C-Nup1 to this
mixture resulted in 70% hydrolysis. When 1 µM FXFG-Nup1
was added instead of C-Nup1, 34% of the Ran-bound GTP was hydrolyzed.
Likewise, addition of 1 µM FXFG-Nup2 resulted
in 15% hydrolysis, and addition of 1 µM full-length
Nup36 resulted in 38% hydrolysis. These findings demonstrate that the
FXFG repeat region of Nup1 and full-length Nup36 can efficiently stimulate the RanGTP-karyopherin
disassembly
reaction.
Nup36, in addition to binding karyopherin (26), also binds
RanGTP4 (51) presumably through a
Ran-binding domain in its C terminus (26, 51-53). We find that Nup36
can stimulate GAP activity in a manner similar to RanBP1 (Fig.
9) (51). Addition of 1 µM Nup36 (open squares) resulted in greater stimulation of GAP
activity than addition of 1 µM RanBP1 (closed
circles). This result suggests that Nup36 functions in the
RanGTP-karyopherin
disassembly reaction by stimulating conversion
of RanGTP to RanGDP via GTP hydrolysis in the presence of RanGAP, as
well as by sequestering karyopherin
.
Our results suggest a model for RanGTP-karyopherin disassembly
(Fig. 10). First, karyopherin
is released from
RanGTP by karyopherin
in a reaction that does not require GTP
hydrolysis (Fig. 10, step 1; Fig. 6). RanGTP is then bound
by RanBP1 which prevents reformation of the RanGTP-karyopherin
complex in the presence of C-Nup1 and karyopherin
(Fig. 10,
step 2; Fig. 5B). RanGTP bound to RanBP1 has a
higher affinity for RanGAP than unbound RanGTP, so that conversion of
RanGTP to RanGDP is enhanced (Fig. 10, step 3; Figs. 3 and
9) (41, 43, 47). RanBP1 binding to RanGTP also prevents interaction of
RanGTP with RanGEF (Fig. 10, step 4; Fig. 4B)
(43, 47). The newly formed karyopherin
binds to C-Nup1 and makes
rebinding of karyopherin
to RanGTP less favorable (Fig. 10,
step 5; Figs. 2A, 5, and 7). Nup36 and the
FXFG repeat region of Nup1 can also function in this step of
the reaction (Fig. 8). Additional factors may modulate the disassembly
reaction (e.g. p10 (26) or Dis3 (54)).
Our data on RanGTP-karyopherin disassembly in vitro
explains previously reported genetic interactions in S. cerevisiae between RanGAP, karyopherin
, and the C terminus of
Nup1 (45, 46). A mutant form of RanGAP is synthetically lethal with a
mutant form of Nup1 that lacks the C terminus (45). Our data can
explain this synergistic effect as RanGAP and C-Nup1 cooperate to
dissociate the RanGTP-karyopherin
complex (Fig. 2) and recycle each
factor for a new round of function. A mutant form of RanGAP is also
synthetically lethal with a mutant form of karyopherin
(46). This
genetic interaction can be explained as well, since our data show that RanGAP promotes recycling of karyopherin
by stimulating
RanGTP-karyopherin
disassembly (Fig. 2).
RanGTP-karyopherin complex is disassembled by karyopherin
through active release as karyopherin
increases the rate of RanGTP-karyopherin
dissociation (Fig. 6B). Release of
karyopherin
from RanGTP is linearly dependent on the concentration
of karyopherin
(Fig. 6B, inset); this
indicates that RanGTP-karyopherin
disassembly is a first order
reaction with respect to karyopherin
. We estimate that karyopherin
stimulates RanGTP-karyopherin
dissociation by at least 3 orders
of magnitude. Release of RanGTP from karyopherin
presumably occurs
through formation of an intermediate RanGTP-karyopherin
-karyopherin
complex, followed by dissociation of RanGTP. We did not detect this
intermediate complex possibly because the displacement reaction is too
fast to be resolved using BIAcoreTM. The proposed displacement
mechanism is supported by data that demonstrate that RanGTP and
karyopherin
have partially overlapping binding sites on karyopherin
(55-57). Thus karyopherin
could interact with the
RanGTP-karyopherin
complex through that part of its binding site on
karyopherin
that is not occupied by RanGTP; this interaction might
then displace RanGTP from the overlapping site. The displacement
reaction is reversible as RanGTP dissociates karyopherin
from
karyopherin
(2, 28), presumably by forming the same intermediate
ternary complex. However, karyopherin
preferentially binds to
RanGTP over karyopherin
(2, 55). This difference in affinity would
force the RanGTP-karyopherin
disassembly reaction in the direction
of RanGTP-karyopherin
complex formation (Fig. 10, step
1). However, in the presence of C-Nup1, RanGAP, and RanBP1, the
equilibrium is shifted toward formation of the karyopherin
complex (Fig. 2, 3, and 5B). The presence of a GST-NLS
fusion protein did not affect the disassembly reaction when tested in
the GTP hydrolysis or BIAcoreTM experiments.2
GTP hydrolysis is not required for the release of karyopherin from
RanGTP by karyopherin
; this is evidenced by the fact that release
of karyopherin
from RanGMP-PCP occurs with the same efficiency as
release from RanGTP (Fig. 6C). This finding offers new
insight into the function of small Ras-like GTP-binding proteins.
GTP-binding proteins switch between an active GTP-bound form and an
inactive GDP-bound form (58). The GTP-bound protein often forms a
complex with a downstream effector molecule. This interaction is
thought to be terminated by GTP hydrolysis (see for example interaction
of Ras with Raf-kinase (59)) as the GDP-bound form generally has a
lower affinity for the effector than the GTP-bound form. As GTP-binding
proteins are often resistant to GTPase-activating proteins when bound
to an effector (60), the intrinsic GTP hydrolysis is thought to trigger
complex disassembly (59). Our results on RanGTP-karyopherin
disassembly suggest that instead a "release factor" terminates the
interaction between the GTP-binding protein and the effector. In our
case karyopherin
is the release factor. Release factors analogous
to karyopherin
may exist for other GTP-binding proteins. The
karyopherin
-dependent release of RanGTP from
karyopherin
occurs much faster than the intrinsic hydrolysis of
Ran-bound GTP (krelease > 0.1 s
1
compared with kcat = 5 × 10
5
s
1 (32)). Our results also suggest that
RanGTP-karyopherin
dissociation in the absence of karyopherin
does not require GTP hydrolysis, as dissociation of the
RanGMP-PCP-karyopherin
complex occurred with the same apparent
kinetics as the dissociation of the RanGTP-karyopherin
complex
under the conditions described2 (Fig. 6). We are currently
investigating the exact rates of association and dissociation. We
propose that GTP hydrolysis during NLS-protein transport across the NPC
is required to recycle Ran and karyopherin
so that each can perform
multiple rounds of function.
RanGAP stimulates the disassembly of the RanGTP-karyopherin complex
(Fig. 2) by converting RanGTP to RanGDP after the karyopherin
-dependent release of RanGTP from karyopherin
thereby preventing reformation of the complex. We found that RanGEF can
replace RanGAP in the disassembly reaction when GDP is present (Fig.
4A). RanGAP, apart from being a cytosolic protein (61), is
also localized at the NPC (38, 62, 63) and in the nucleoplasm (64). In contrast, RanGEF is located mainly in the nucleoplasm bound to chromatin (65, 66), although it may also bind to
nucleoporins2 (Fig. 5A). Disassembly of
RanGTP-karyopherin
in vivo may occur in the presence of
both RanGAP and RanGEF at the NPC. However, due to the higher
concentration of GTP versus GDP in the cell (49), conversion
of RanGTP to RanGDP via RanGEF is probably not efficient. It is
therefore likely that in vivo only RanGAP is responsible for
conversion of RanGTP to RanGDP.
Our results suggest that RanBP1 has three functions. First, RanBP1
binds to RanGTP after its release from karyopherin by karyopherin
and prevents reformation of the RanGTP-karyopherin
complex when
C-Nup1 is present (Fig. 5B). Second, RanBP1 interaction with
RanGTP increases the affinity of RanGTP for RanGAP which results in
stimulation of GAP activity (Figs. 3 and 9) (41, 43, 47). Third, RanBP1
binding to RanGTP prevents interaction of RanGTP with RanGEF (Fig.
4B) (43, 47) which might be advantageous in vivo
to prevent futile exchange of Ran-bound GTP for GTP. RanBP1, RanGTP,
and karyopherin
form a trimeric complex (42, 43, 67, 68) which may
assemble during release of RanGTP from karyopherin
. However, in the
presence of karyopherin
and C-Nup1 this complex is unstable as the
majority of karyopherin
bound to C-Nup1 without associated RanGTP
and RanBP1 (Fig. 5B). We propose that RanBP1 functions to
promote recycling of RanGTP and karyopherin
at the NPC. Our
proposal is consistent with the localization of RanBP1 at the nuclear
envelope (41) and its proposed function in protein import (41,
42).
The C terminus of Nup1 binds karyopherin and stimulates
RanGTP-karyopherin
disassembly (Fig. 2B). We suggest
that C-Nup1 sequesters karyopherin
and inhibits reformation of the
RanGTP-karyopherin
complex (Fig. 7). We also suggest that Nup36 and
the FXFG repeat region of Nup1 may function in a manner
similar to C-Nup1 (Fig. 8) because they also bind karyopherin
(2, 26). The FXFG repeat region of Nup2 binds karyopherin
weakly (not shown) and did not significantly stimulate
RanGTP-karyopherin
disassembly (Fig. 8).
Nup36 was initially identified as a karyopherin binding protein
(26) and was later shown to bind RanGTP4 (51).
Overexpression of a tagged version of Nup36 results in its localization
to the nucleoplasm (51). However, Nup36 localizes to the nuclear
envelope as visualized by immunofluorescence using antibodies against
Nup36.5 In addition to a karyopherin
binding site, Nup36 has a Ran-binding domain similar to the one in
RanBP1 (52, 53). Nup36 stimulates GTP hydrolysis by RanGAP as does
RanBP1 (Fig. 9) (51) and is synthetically lethal with RanGAP (51). This
genetic interaction can be explained by our in vitro data as
both Nup36 and RanGAP cooperate to recycle Ran and karyopherin
for
another round of function. Other nucleoporins, like the mammalian
Nup358 (69, 70) and yeast Nup2 (52, 53), also contain Ran-binding
domains and may function in a manner similar to Nup36.
The involvement of different nucleoporins in the disassembly of the
RanGTP-karyopherin complex has interesting implications for protein
transport into the nucleus. Nucleoporins that bind karyopherin
have different activities in the disassembly of the RanGTP-karyopherin
complex via karyopherin
(Fig. 8). This may be due to different
affinities of the nucleoporins for karyopherin
. If nucleoporins
are localized along the NPC with increasing affinities for karyopherin
(from cytoplasmic to nucleoplasmic sites), disassembly of
RanGTP-karyopherin
complexes and concomitant docking of karyopherin
might occur along an affinity gradient. This affinity gradient
may confer directionality to movement of transport factors and
substrates across the nuclear pore complex.
We thank James Cheetham for initial help with BIAcoreTM and Ulf Nehrbass for providing the Nup36 construct.