(Received for publication, October 16, 1995; and in revised form, January 16, 1996)
From the
The heterodimeric karyopherin functions in targeting a nuclear
localization sequence (NLS)-containing protein to the nuclear pore
complex followed by Ran-GTP and p10-mediated translocation of the NLS
protein into the nucleoplasm. It was shown recently that Ran-GTP
dissociated the karyopherin heterodimer and, in doing so, associated
with karyopherin (Rexach, M., and Blobel, G.(1995) Cell 83, 683-692). We show here, using all recombinant yeast
proteins expressed in Escherichia coli, that karyopherin
binds to Ran-GTP and inhibits GTP hydrolysis stimulated by RanGAP (the
Ran-specific GTPase activating protein). Inhibition of
RanGAP-stimulated GTP hydrolysis by karyopherin
was dependent on
karyopherin
concentration relative to Ran-GTP. Complete
inhibition of RanGAP was observed at karyopherin
concentrations
that were equimolar to Ran-GTP. In gel filtration experiments, we found
Ran-GTP and karyopherin
to form a stoichiometric complex. Ran-GDP
bound only weakly to karyopherin
. We propose that stoichiometric
complex formation between karyopherin
and Ran-GTP renders Ran-GTP
inaccessible to RanGAP.
Several soluble transport factors are required for import of a
nuclear localization sequence (NLS)()-containing protein
into nuclei of digitonin-permeabilized mammalian cells. Binding of the
NLS protein to the nuclear rim of digitonin-permeabilized cells is
mediated by a heterodimeric complex, termed
karyopherin(1, 2) , or nuclear pore-targeting complex (3) (karyopherin
is synonymous with NLS
receptor(4) , importin(5, 6) , importin
60(7) , or importin
(8) ; karyopherin
(1) is synonymous with p97(4, 9) , importin
90(7) , or importin
(8) ). Transport of the NLS
protein from the nuclear rim into the nucleus requires the Ras-related
GTPase Ran (10, 11) and a Ran-interactive homodimeric
complex of a protein of 15 kDa that migrates in SDS-PAGE as a protein
of 10 kDa and therefore has been termed p10 (12) (p10 is
synonymous with NTF 2(13) ). These four transport factors are
able to substitute for the requirement of cytosol in import of an NLS
protein into nuclei of digitonin-permeabilized cells, even when
prepared from Escherichia coli as recombinant
proteins(14) . Nevertheless, it is likely that additional
soluble factors are required. These factors may remain with the
digitonin-permeabilized cells in sufficient quantities and therefore
not be limiting or they may perform subtle regulatory tasks that have
not yet been detected with this assay. Among these factors might be
several Ran regulatory proteins, such as a guanine nucleotide exchange
factor(15, 16) , or a GTPase activating protein
(GAP)(17) , or soluble Ran-binding proteins(18) .
A
heterodimeric karyopherin has also been isolated from the yeast Saccharomyces cerevisiae, and recombinant karyopherin
and
subunits have been shown to be able to dock NLS protein at
the nuclear rim of digitonin-permeabilized mammalian
cells(19) . Moreover, it was shown that Ran-GTP (but not
Ran-GDP) dissociated the karyopherin heterodimer and associated with
karyopherin
although the stoichiometry of this association was
not determined(20) . Ran-GTP-mediated dissociation of the
karyopherin heterodimer did not require GTP hydrolysis(20) . An
association of Ran-GTP with karyopherin
might affect the
responsiveness of Ran-GTP to Ran regulatory proteins. As a RanGAP has
recently been identified (17) and in yeast is the product of
the RNA1 gene(21, 22) , we have tested
whether association of karyopherin
with Ran-GTP modifies the
response of Ran-GTP to RanGAP. Using all recombinant proteins, we found
that karyopherin
inhibited RanGAP-stimulated Ran-GTP hydrolysis
in a concentration-dependent manner, yielding complete inhibition at
karyopherin
concentrations that are stoichiometric to Ran-GTP. In
gel filtration experiments, we found that karyopherin
was capable
of forming a stoichiometric complex with Ran-GTP but of binding only
weakly to Ran-GDP.
In a control
experiment, Ran was first labeled with either
[H]GDP or [
-
P]GTP.
For this, 10 µM Ran was incubated in the presence of
either 10 nM [
H]GDP (34 Ci/mmol, DuPont
NEN) or 10 nM [
-
P]GTP (6000
Ci/mmol, DuPont NEN) in exchange buffer for 30 min at 21 °C. This
resulted in labeled Ran that contained 16% GTP and 20% GDP, as
determined on the reversed-phase column. After addition of 20 mM Mg(OAc)
, 5 µM Ran-[
H]GDP or 5 µM Ran-[
-
P]GTP was incubated with 2.5
µM Kap95 for 10 min at 21 °C. The reaction mixture was
subjected to gel filtration as described, and the elution of
radioactivity was monitored by measuring 10-µl aliquots of each
fraction in a scintillation counter.
Ran (Gsp1), RanGAP (Rna1), and karyopherin (Kap95), all
from S. cerevisiae, were expressed as recombinant proteins in E. coli (see ``Experimental Procedures''). Each of
the purified recombinant proteins yielded a single band of the expected
electrophoretic mobility upon SDS-PAGE analysis (see Fig. 1for
purified RanGAP and Fig. 4for purified Ran and karyopherin
). The purified recombinant Ran was determined to contain 40% GDP
and 25% GTP, whereas 35% is nucleotide-free (data not shown). To assay
for GTP hydrolysis, the endogenous Ran-bound GDP or GTP were in part
exchanged with [
-
P]GTP (see
``Experimental Procedures''). Under the conditions used for
the exchange reaction (no exogenously added cold GTP), we found that
only about 13% of the Ran was complexed with GTP, as determined by
measuring the bound nucleotide on a reversed-phase column. Hence, of
the 300 nM Ran used in the incubation reaction with RanGAP,
only about 40 nM was present as Ran-GTP. After incubation with
RanGAP, GTP hydrolysis was measured using a nitrocellulose filter
binding assay in which hydrolyzed
PO
is not
retained by the filter, whereas Ran-bound nonhydrolyzed
[
-
P]GTP is retained. The recombinant GAP
was indeed active. Increasing concentrations yielded increasing rates
of GTP hydrolysis, with as little as 1.0 nM GAP yielding more
than 70% GTP hydrolysis and 2.0 nM GAP resulting in near 100%
GTP hydrolysis within the 10-min reaction time (Fig. 2).
Strikingly, in the presence of 40 nM karyopherin
, the
GAP-stimulated GTP hydrolysis was completely inhibited (Fig. 2).
Figure 1: Purified yeast RanGAP (Rna1). RanGAP was purified as a GST-fusion protein as described under ``Experimental Procedures.'' The thrombin-cleaved RanGAP is >90% pure as judged by SDS-PAGE.
Figure 4:
Ran
forms a complex with karyopherin (Kap95) that can be detected by
gel filtration. Complex formation between Ran and Kap95 was analyzed on
a Superdex 200 FPLC column as described under ``Experimental
Procedures.'' 13 of 24 fractions were analyzed by SDS-PAGE. A, fractions were analyzed by electrophoresis on a 12%
polyacrylamide gel and stained with Coomassie Blue. Upper
panel, 2.5 µM Kap95 was incubated in buffer A. Middle panel, 2.5 µM Kap95 was incubated with 5
µM Ran-GTP. Lower panel, 2.5 µM Kap95 was incubated with 5 µM Ran-GDP. B,
2.5 µM Kap95 was incubated with 5 µM Ran-[
H]GDP (open circles) or 5
µM Ran-[
-
P]GTP (closed
circles). Elution of radioactivity was monitored by counting
10-µl aliquots of each of the 24 fractions in a scintillation
counter.
Figure 2:
GAP activity is inhibited by karyopherin
(Kap95). GAP activity was assayed as described under
``Experimental Procedures'' by incubating 40 nM Ran-[
-
P]GTP in the presence of
increasing amounts of RanGAP with (
) or without (
) 40 nM Kap95.
To determine whether karyopherin inhibits GAP-stimulated GTP
hydrolysis in a concentration-dependent manner, 40 nM Ran-[
-
P]GTP (see Fig. 2) was
incubated with 1 nM RanGAP and increasing amounts of
karyopherin
. Inhibition of GAP activity was maximal at 30 to 40
nM karyopherin
(Fig. 3). Increasing the
concentration of RanGAP did not overcome the inhibition of karyopherin
(Fig. 2). These data suggested that karyopherin
did
not inhibit GAP activity by interacting with RanGAP directly, but
rather by forming a stoichiometric complex with Ran-GTP.
Figure 3:
Inhibition of GAP activity is dependent on
karyopherin (Kap95) concentration. GAP activity was determined as
described under ``Experimental Procedures'' after incubating
40 nM Ran[
-
P]GTP in the presence
of 1 nM RanGAP and increasing amounts of
Kap95.
To assay
directly for an association of Ran with karyopherin , we carried
out gel filtration experiments. For these experiments, the endogenous
Ran-bound GDP or GTP was exchanged in the presence of a 10-fold molar
excess of either GTP or GDP to ensure that most of the Ran would be
bound either to GTP or GDP. To allow for complex formation, 2.5
µM karyopherin
was incubated either with 5.0
µM Ran-GTP or Ran-GDP. In a control reaction, 2.5
µM karyopherin
was incubated without Ran-GTP or
Ran-GDP. Each of the reaction mixtures was then subjected to gel
filtration on a Superdex 200 FPLC column, and fractions were collected,
trichloroacetic acid-precipitated, and analyzed by SDS-PAGE and
Coomassie blue staining of the gel. The bulk of karyopherin
eluted at fractions 12 and 13 (Fig. 4A, upper
panel). When preincubated with a 2-fold molar excess of Ran-GTP,
there was a dramatic shift of the karyopherin
peak to fractions
10 and 11. Moreover, karyopherin
coeluted with about half of the
Ran-GTP, whereas the other half of Ran-GTP peaked at fraction 18 (Fig. 4A, middle panel). These data indicated
that karyopherin
and Ran-GTP formed a stoichiometric complex.
Some binding to karyopherin
could also be detected when Ran was
loaded with GDP prior to incubation (Fig. 4A, lower
panel). However, we found that under our exchange conditions there
was still about 10% GTP bound to Ran, as determined on the
reversed-phase column. To determine whether there was indeed binding of
Ran-GDP to karyopherin
, Ran was labeled with
[
H]GDP prior to incubation with karyopherin
. Gel filtration on the Superdex 200 column resulted in
cofractionation of some of the radioactivity with the karyopherin
-Ran complex (Fig. 4B, open circles).
When Ran was labeled with [
-
P]GTP prior to
incubation with karyopherin
, all of the labeled Ran
cofractionated with the karyopherin
-Ran complex (Fig. 4B, closed circles). These results
indicated that Ran-GDP also binds to karyopherin
, but with much
lower affinity than does Ran-GTP.
Our data here show that the RanGAP-stimulated GTP hydrolysis
of Ran-GTP is inhibited by karyopherin in a
concentration-dependent manner. Complete inhibition was observed when
the amount of karyopherin
was equimolar to Ran-GTP. In gel
filtration experiments, Ran-GTP and karyopherin
were found to
form a stoichiometric complex. We suggest that binding of Ran-GTP to
karyopherin
renders Ran-GTP inaccessible to RanGAP.
Ran-GTP
binding to karyopherin does not appear to affect the slow
intrinsic GTPase activity of Ran, as no differences in the rates of GTP
hydrolysis could be detected during a 1-h incubation of Ran-GTP either
in the absence or presence of karyopherin
(data not shown).
Unlike Ras, which has an affinity for GTP that is about 1 order of
magnitude higher than that for GDP(26) , Ran has a 10-fold
higher affinity for GDP than it has for GTP(27) . Up to 80% of
the cellular Ran is thought to be located in the nucleus(28) ,
whereas RanGAP is thought to be located in the cytoplasm(29) .
Hence, the cytoplasmic RanGAP is likely to keep the cytoplasmic
concentration of Ran-GTP very low. This seems logical as cytoplasmic
Ran-GTP would be detrimental for nuclear import. It would dissociate
the karyopherin heterodimer in the cytoplasm, associate with
karyopherin , and thereby prevent targeting of NLS protein to the
nuclear pore complex(20) .
The formation of a complex
between Ran-GTP and karyopherin that renders Ran-GTP inaccessible
to RanGAP is reminiscent of the formation of a complex of Ras-GTP with
its downstream effector Raf kinase that has been proposed to render
Ras-GTP inaccessible to RasGAP(30, 31) . Ras-Raf
interaction is thought to be terminated through intrinsic GTP
hydrolysis of Ras-GTP, resulting in the release of Raf
kinase(32) . Similarly, the intrinsic GTPase activity of Ran
could result in a dissociation of Ran from karyopherin
as the
affinity of the latter for Ran-GDP is lower than for Ran-GTP (Fig. 4).
It remains to be determined whether
(Ran-GTPkaryopherin
) complex formation might serve to
down-regulate protein import into the nucleus in vivo.