From the
Ran/TC4 is a member of the Ras superfamily of GTPases. It is
unusual in being predominantly nuclear and because it possesses an
acidic -DEDDDL sequence instead of a consensus prenylation domain at
the C terminus. Ran is required for nuclear protein import and cell
cycle progression, and has been implicated in mRNA processing and
export and DNA replication. The inhibition of cell cycle progression by
a dominant gain-of-function mutant of Ran has been shown to be
abrogated by removal of the -DEDDDL sequence, suggesting that this
domain is essential for Ran function. We demonstrate here that the
-DEDDDL sequence stabilizes GDP binding to Ran, and that the domain is
required for high affinity interaction with a Ran-binding protein,
HTF9A/RanBP1. HTF9A functions as a co-stimulator of Ran-GAP (GTPase
activating protein) activity on wild-type Ran, but in the absence of
the acidic C terminus of Ran, HTF9A behaves as a Ran-GAP inhibitor. An
antibody directed against the C-terminal region preferentially
recognizes the GTP-bound form of Ran, suggesting that this domain
undergoes a nucleotide-dependent conformational change. The results
suggest that the acidic C-terminal domain is important in modulating
the interaction of Ran with regulatory factors, and implicate
Ran-binding proteins in mediating the effects of Ran on cell cycle
progression.
The Ran/TC4 GTPase, referred to here as Ran, is a member of the
Ras superfamily of small guanine nucleotide-binding
proteins(1) . Ran is unique in this family in that it is largely
localized to the cell nucleus and lacks a consensus site for
post-translational prenylation (2). It also has an unusually acidic
C-terminal tail (-DEDDDL). Ran homologs have been found in all
eukaryotic organisms examined to date, including Spi1 in Schizosaccharomyces pombe(3) , RAN1, RAN2A, and RAN2B
in the tomato Lycopersicon esculentum(4) , and GSP1 and
GSP2 in Saccharomyces cerevisiae(5) . GSP1 is essential
for viability in S. cerevisiae, and the GSP1 gene disruption can be complemented by mammalian Ran (5). Amino
acid comparisons show a very high level of sequence conservation across
species, and all Ran homologs possess an acidic C-terminal domain.
Ran has been implicated in a number of essential cellular processes
including nuclear protein import through the nuclear pore complex
(6-8), DNA replication and cell cycle
control(9, 10, 11) , nuclear growth (9), and
mRNA processing and export(12, 13, 14) . It is
possible that the multiplicity of effects induced by loss of Ran
function, or by expression of dominant mutants of Ran, may reflect a
single primary disruption in an essential process such as nuclear
protein import, but this hypothesis remains unproven.
The high
degree of conservation of Ran sequences through evolution and the
essential nature of its functions indicate that the Ran GTPase cycle is
likely to be stringently regulated and that Ran may interact with many
other factors. Thus far, a nuclear, Ran-specific guanine nucleotide
exchange factor, RCC1, and a cytosolic GTPase activating protein
(GAP)
The mechanisms
through which Ran interacts with these proteins are not understood, but
several pieces of evidence suggest that the acidic C terminus of Ran
may be important in the regulation of Ran activity. A mutant Ran
protein that lacks this -DEDDDL sequence fails to bind efficiently to
several of the RanBPs, including HTF9A, when tested using the GTP-Ran
overlay assay(17) . Furthermore, the C terminus has been
implicated in the function of Ran in cell cycle
progression(10) . Transfection of 293/Tag cells with a dominant
gain-of-function mutant of Ran arrests the cells in G
Here we report that the
-DEDDDL tail of Ran stabilizes GDP binding to the Ran protein. A
C-terminal deletion mutant exhibits accelerated loss of GDP both in the
presence of EDTA and when release is catalyzed by the exchange factor
RCC1. The rate of GTP hydrolysis on this mutant, catalyzed by Ran-GAP,
is also accelerated compared to wild-type Ran. Moreover, the C-terminal
tail significantly affects the affinity of GTP-Ran for the Ran-binding
protein HTF9A/RanBP1. While HTF9A acts as an enhancer of Ran-GAP
activity on wild-type Ran, HTF9A decreases the rate of this hydrolysis
on the C-terminal deletion mutant protein. We propose a model for the
interaction of Ran with RCC1, Ran-GAP and HTF9A, illustrating a
possible mechanism for the involvement of the -DEDDDL tail in these
protein-protein interactions.
To
further investigate this interaction, GST-HTF9A and GST were loaded
onto glutathione-Sepharose beads and mixed with equal amounts of either
wild-type or
From these data we can conclude that the C-terminal -DEDDDL
sequence is not necessary for the binding of HTF9A to Ran, but does
increase the affinity of Ran for HTF9A. The inhibition of GTP release
suggests that the HTF9A acts to significantly stabilize the GTP binding
to Ran, and/or HTF9A inhibits the interaction between RCC1 and the
GTP-Ran, perhaps by competing for a common binding site on the Ran
protein.
A number of studies suggest that the guanine nucleotide-bound
state of Ran affects a variety of essential cellular functions. For
example, Kornbluth et al.(9) showed that the addition
of T24N Ran, a putative, dominant loss-of-function mutant, to cycling
cell-free Xenopus oocyte extracts blocks the dephosphorylation
of p34
In tsBN2 cells, a mammalian cell line carrying a
temperature-sensitive mutation in the RCC1 gene, incubation at the
nonpermissive temperature leads to the rapid loss of the RCC1
protein(28, 29, 30) . Because RCC1 is an
exchange factor for Ran, it is expected that depletion of RCC1 would
lead to the accumulation of Ran predominantly in the GDP-bound state.
If the temperature shift occurs during S phase of the cell cycle, the
cells undergo premature chromosome condensation and enter mitosis
aberrantly; the resulting cells accumulate micronuclei and
die(28) . If the cells are shifted in G
Surprisingly, the
overexpression of G19V Ran, a dominant gain-of-function mutant which is
constitutively GTP-bound,
We propose that the Ran
C-terminal DEDDDL tail stabilizes the GDP-bound state, possibly by
folding into the guanine nucleotide binding pocket and mimicking the
negative charge on the
When Ran is in the GTP-bound state, it is
able to interact with the Ran-GAP and a number of Ran-binding proteins,
including HTF9A/RanBP1. The Ran-GAP is capable of binding to Ran and
stimulating GTP hydrolysis, but the -DEDDDL tail may inhibit more
efficient binding of the Ran-GAP to GTP-Ran (Fig. 3). When
GTP-Ran binds to HTF9A/RanBP1, the orientation of the C-terminal tail
could change again, resulting in stabilization of Ran in its GTP-bound
state. The binding of the HTF9A/RanBP1 also decreases the functional
interaction of GTP-Ran with RCC1 (Fig. 5A), possibly
through competition for overlapping binding sites on the Ran protein.
The binding of HTF9A/RanBP1, movement of the C-terminal tail, and
stabilization of the GTP-bound Ran could enhance the binding of the
Ran-GAP to GTP-Ran, which would lead to the accelerated rate of GTP
hydrolysis that is seen with wild-type Ran in the presence of
HTF9A/RanBP1 (Fig. 6A). Upon the hydrolysis of GTP to
GDP, the C-terminal tail returns to its initial orientation,
stabilizing the GDP on Ran. The proposed role of the C terminus of Ran
is unlike that of the C terminus in other small GTPases such as Ras and
Rab3A. The C terminus of these proteins is not known to have a
significant effect of the kinetics of nucleotide release and
hydrolysis(25, 26) , and there is no evidence that the C
terminus of Ras undergoes a nucleotide- dependent conformational shift.
Why does the deletion of the -DEDDDL sequence abrogate the cell
cycle arrest induced by a dominant gain-of-function mutant of Ran? The
We thank Jeremy Thorner (University of California,
Berkeley) for the HTF9A clone, Takeharu Nishimoto (Kyushu University,
Japan) for the RCC1 clone, and Peter D'Eustachio (New York
University) for the TC4 clone. We also thank Colleen McKiernan for
helpful discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)have been identified (15, 16). In
addition, using a GTP-Ran overlay assay, a number of putative
downstream target proteins have been identified that interact with and
stabilize the GTP-bound state of Ran(17) . One of these
proteins, HTF9A/RanBP1, has been cloned(18, 19) , and is
predominantly cytosolic. Information from this sequence and that of
several other partial clones has revealed the existence of a novel,
highly conserved Ran-binding domain of approximately 150 amino acid
residues in length (20). It is likely that a large family of
Ran-binding proteins exists, that may contain one or more copies of
this conserved Ran-binding domain(20) .
or at
the G
/S boundary, but deletion of the C-terminal -DEDDDL
tail of this mutant abrogates the cell cycle arrest(10) . These
results suggest that Ran function requires an intact C terminus.
Therefore, it was of interest to examine the effects of deleting the
-DEDDDL sequence on the interaction of Ran with its known regulatory
factors, and to try to elucidate the essential function provided by
this C-terminal domain in Ran function.
Construction and Purification of Recombinant
Proteins
Deletion of the C terminus of Ran was accomplished
using polymerase chain reaction mutagenesis to introduce a stop codon
and remove the final 6 amino acid residues of the open reading frame.
The template DNA was the TC4 clone in pUC19 (a gift from P.
D'Eustachio). The product was subcloned into pGEX-2T (Pharmacia
Biotech Inc.) to generate a glutathione S-transferase-Ran
fusion protein. Wild-type Ran and HTF9A (a gift from J. Thorner) were
also subcloned into pGEX-2T. Proteins were expressed in Escherichia
coli DH5 upon induction with 0.4 mM
isopropyl-
-D-thioglactopyranoside. Proteins were purified
over glutathione-Sepharose columns (Pharmacia). GST fusion proteins
were cleaved to remove the GST by incubating GST fusion proteins on
glutathione-Sepharose beads in 50 mM Tris, pH 7.5, 150 mM NaCl, 2.5 mM CaCl
with 19.8 units of thrombin
for 1 h at 23 °C. The supernatant was treated with p-aminobenzamidine-Sepharose 4B beads (Sigma) for 30 min at 4
°C to remove the thrombin. A pET11a vector containing the RCC1 open
reading frame was a gift from T. Nishimoto. RCC1 protein was induced by
addition of isopropyl-
-D-thioglactopyranoside to E.
coli BL21(DE3)LysS that contained the pET-RCC1 vector. The soluble
fraction of the bacterial lysate was used as a source of RCC1; lysate
from cells not containing vector was used as a control. Proteins were
quantitated by the Bradford (21) assay, using BSA as a standard.
Nucleotide Release Assays
Recombinant Ran protein
was loaded with [-
P]GTP or -GDP
(20-40 µCi; 3000 Ci/mmol nucleotide; usually in a 50-µl
volume) as described previously(17) . To measure EDTA-stimulated
nucleotide release, portions of this solution were diluted into a
10-fold larger volume of buffer containing 50 mM MOPS, pH 7.1,
2 mM GTP or GDP, 1 mM dithiothreitol, 0.1 mg/ml BSA
plus 10 mM MgCl
, or 5 mM EDTA. Loss of
[
-
P]nucleotide from Ran was measured by
filter binding samples and the
[
-
P]nucleotide bound was quantitated by
scintillation counting(22) . The amount of
[
-
P]nucleotide bound at t =
0 was defined as 100%, which varied between 10,000 and 50,000 dpm in
different experiments. RCC1 catalyzed nucleotide release was determined
by the same method, with the aforementioned buffer containing 10 mM MgCl
, RCC1 or bacterial lysate, and no EDTA. Reactions
were performed at 23 °C in duplicate unless otherwise indicated.
Off-rates were calculated using a best-fit nonlinear regression,
assuming single exponential decay.
GAP Assays
GAP assays were performed as described
previously(23) , with the following modifications. Ran was
loaded with [-
P]GTP as described above (17) and diluted into a 10-fold larger volume of GAP buffer (40
mM Tris, pH 8.0, 8 mM MgCl
, 80 µM BSA, 2 mM GTP). An S100 lysate was prepared from BHK-21
as described (18) and used as a source of GAP activity at a
final concentration of 0.067 mg/ml. Reactions were carried out at 23
°C and terminated by filter binding. Nucleotides were released from
the filters by addition of nucleotide releasing buffer (1% SDS, 25
mM EDTA, 5 mM ATP, plus 200 µM GDP and
GTP, pH 6.0), separated by thin layer chromatography(23) , and
quantitated on a Bio-Rad GS-250 Molecular Imager.
Immunoprecipitation of Ran
A peptide corresponding
to residues 196-207 of the Ran/TC4 sequence (Multiple Peptide
Systems) was conjugated to keyhole limpet hemocyanin by
glutaraldehyde(24) ; this sequence is adjacent to the acidic
-DEDDDL domain. Anti-Ran serum was generated by the injection of this
conjugate into rabbits (Cocalico, Inc.). The resulting antiserum,
AR-12, detected a single 25-kDa band by immunoblotting either total
extracts of BHK-21 cells or purified recombinant Ran protein. The
25-kDa band was not detected by preimmune serum (data not shown). For
immunoprecipitations, recombinant Ran purified from E. coli expressing a pET-Ran vector (17) was loaded with
[-
P]GTP and/or with
[
-
P]GDP, then incubated for 30 min at 4
°C with a 1:10 dilution of AR-12 antiserum in buffer A (20 mM HEPES, pH 7.3, 20 mM MgCl
, 150 mM NaCl, and 0.75% Triton X-100). Following a further incubation for
30 min with protein A-Sepharose, the immunoprecipitates were washed
three times with buffer A, resuspended in nucleotide releasing buffer,
and analyzed by thin layer chromatography for GDP and GTP as described
above(23) .
Deletion of the C-terminal DEDDDL Sequence Alters the
Off-rate of Guanine Nucleotides from Ran
It was important
initially to determine whether the removal of the C-terminal acidic
residues of Ran would alter the basal kinetics of binding and release
of GDP and GTP. Consistent with previous
findings(14, 17) , the off-rate for
[-
P]GDP (Fig. 1A) and
[
-
P]GTP (Fig. 1B) from both
wild-type Ran and the C-terminal deletion mutant Ran was extremely slow
in the presence of Mg
(k
0.013 min
). When excess EDTA was added the rates of
nucleotide release were accelerated. For
[
-
P]GDP (Fig. 1A) the rate
of nucleotide release from wild-type Ran was
0.069
min
. The deletion of the -DEDDDL sequence further
accelerated EDTA-stimulated [
-
P]GDP release (k
0.15 min
). The
presence of EDTA also accelerated GTP release from these proteins, but
the rates were significantly different (Fig. 1B). First,
the release of GTP from wild-type Ran (k
0.99 min
) was more rapid than that from
DEDDDL
Ran (k
0.40 min
), which
is the opposite of the release pattern observed for GDP. Second, the
rate of GTP release from the wild-type protein was far more rapid than
the rate of GDP release (Fig. 1A). These results were
surprising because the removal of the C-terminal amino acids from other
small GTPases such as Ras and Rab3 has no detectable effect on the
rates of guanine nucleotide release(25, 26) .
Figure 1:
Guanine nucleotide release from
wild-type and DEDDDL-Ran-GST fusion proteins. Ran proteins (80
pmol) were loaded with either (A)
[
-
P]GDP or (B)
[
-
P]GTP (20 µCi; 3000 Ci/mmol) as
described under ``Materials and Methods,'' and portions were
diluted 10-fold into buffer containing either 10 mM MgCl
or 5 mM EDTA (total volume of 50 µl). Nucleotide
release was measured by nitrocellulose filter binding of 5-µl
samples at intervals. Values are means ± 1 S.D. (n = 2-6).
, wild-type + MgCl
;
, wild-type + EDTA;
,
DEDDDL +
MgCl
;
,
DEDDDL + EDTA. C,
RCC1-catalyzed GDP release from wild-type or
DEDDDL-Ran. Forty
pmol of [
-
P]GDP-loaded wild-type or
-
DEDDDL-Ran-GST was added to buffer (+10 mM MgCl
) with or without recombinant human RCC1. Samples
were filter bound over time. Values are means ± range (n = 2).
, wild-type - RCC1;
, wild-type
+ RCC1;
,
DEDDDL - RCC1;
,
DEDDDL +
RCC1.
The
data suggest that the C-terminal acidic tail can function to stabilize
the GDP-bound state of Ran, and may destabilize the GTP-bound state.
This hypothesis predicts that loss of the C-terminal domain will
accelerate the rate of GDP release catalyzed by the Ran exchange
factor, RCC1. To test this hypothesis we performed GDP release assays
on wild-type and DEDDDL Ran-GST fusion proteins, in the presence
or absence of recombinant human RCC1 (Fig. 1C). As
predicted by the previous experiments, RCC1-catalyzed GDP release is
significantly faster from the
DEDDDL mutant (k
1.7 min
) than from the wild-type Ran (k
0.21 min
). However,
these experiments were performed at subsaturating concentrations of
RCC1. Therefore, an alternative explanation for the more rapid GDP
release from the
DEDDDL Ran would be that the C-terminal deletion
mutant has a higher affinity for RCC1, which would result in a higher
turnover rate for GDP. To test this possibility we performed a
competition binding assay for RCC1 using
[
-
P]GDP-loaded wild-type Ran. We measured
the rate of RCC1-catalyzed [
-
P]GDP release
from this Ran in the presence of increasing concentrations of
nonradiolabeled, GDP-loaded wild-type or
DEDDDL-Ran-GST fusion
proteins. (To avoid complications that might arise from homotypic
interactions of the GST domains, the
[
-
P]GDP wild-type Ran was prepared by
thrombin-cleavage of a GST fusion protein.) The
DEDDDL-Ran-GST
competes for RCC1-catalyzed GDP release from
[
-
P]GDP wild-type Ran with a similar
affinity to the competition by wild-type Ran-GST; the apparent K
is about 0.36 µM (Fig. 2). Therefore, these results indicate that the
accelerated release of GDP from the
DEDDDL Ran is not due to more
efficient interaction with RCC1, and support the hypothesis that the
acidic -DEDDDL sequence stabilizes GDP binding to Ran.
Figure 2:
Wild-type and DEDDDL-Ran-GST fusion
proteins have similar affinities for RCC1. To measure the relative
affinities for RCC1, a competition exchange assay was performed at 23
°C as described previously (26). Increasing concentrations of
GDP-wild-type-GST-Ran or
DEDDDL-GST-Ran were added to 80 fmol of
wild-type thrombin-cleaved Ran that had been loaded with
[
-
P]GDP (final assay volume = 200
µl) as described under ``Materials and Methods'' and in
the legend to Fig. 1. In the presence of RCC1, nucleotide release was
measured over 3 min by nitrocellulose filter binding. Values are means
± range.
, wild-type;
,
DEDDDL.
The Rate of Ran-GAP-catalyzed GTP Hydrolysis Is
Accelerated in the Absence of the C-terminal DEDDDL Sequence
To
examine the interaction of both wild-type and DEDDDL Ran with
Ran-GAP, we performed GAP assays using a BHK-21 cell S100 lysate as a
source of Ran-GAP activity. This lysate had no detectable Ran exchange
factor activity and the total radioactivity did not decrease
significantly over time (data not shown). In the absence of lysate, the
basal rate of GTP hydrolysis on these fusion proteins was similar over
a 30-min period, as shown in Fig. 3. The Ran-GAP activity in the
lysate stimulated GTP hydrolysis on both the wild-type and
DEDDDL
Ran. Interestingly, the GAP-catalyzed rate of hydrolysis by the
DEDDDL mutant was significantly greater than that by wild-type
Ran, as quantitated by phosphoimager analysis. These results suggest
that the C-terminal DEDDDL sequence may impede Ran-GAP stimulation of
the intrinsic Ran GTPase activity.
Figure 3:
Ran-GAP stimulates GTP hydrolysis on
wild-type and DEDDDL-Ran-GST. Twenty-three pmol of wild-type or
DEDDDL-Ran-GST was loaded with [
-
P]GTP
(see Fig. 1 and ``Materials and Methods'') and added to a
buffer with a BHK-21 S100 lysate containing Ran-GAP activity, or BSA.
The assay was executed as described under ``Materials and
Methods,'' with samples taken over 30 min. The lysate contained no
detectable nucleotide exchange activity. Total recovery of
radioactivity was equal (± 5%) at all time points. %GTP bound
was calculated as (dpm GTP/(dpm GDP + dpm GTP))
100.
, wild-type - lysate;
, wild-type + lysate;
,
DEDDDL - lysate;
,
DEDDDL +
lysate.
The Ran-binding Protein HTF9A/RanBP1 Binds to the
A 28-kDa Ran-binding protein that specifically interacts
with Ran in the GTP-bound state has been cloned and called
RanBP1(18) . It is identical to a putative open reading frame
(203 amino acid residues in length) sequenced previously and named
HTF9A(19) . Our previous data using the Ran overlay assay on
whole cell extracts detected a 28-kDa Ran-binding protein that
possesses properties similar to recombinant HTF9A and which did not
appear to interact with the DEDDDL Ran, but Has Reduced Ability to Stabilize the GTP-bound
State
DEDDDL mutant(17) .
DEDDDL Ran, which had been loaded with
[
-
P]GTP. Other experiments showed that the
binding of GST-HTF9A was specific for GTP-Ran; there was no detectable
binding of GDP-Ran to GST-HTF9A (data not shown). Surprisingly, we
found that both the wild-type and
DEDDDL Ran proteins bound to
HTF9A specifically and to a similar extent (approximately 60% of the
total added [
-
P]GTP Ran bound to the
GST-HTF9A) (Fig. 4A), which is unlike the result of the
Ran overlay assay(17) . Therefore, the acidic C-terminal tail is
not essential for the binding of GTP-Ran to HTF9A, but likely increases
the affinity of the interaction between these two proteins.
Figure 4:
HTF9A/RanBP1 binds to wild-type and
DEDDDL-Ran but stabilization of GTP-
DEDDDL-Ran is reduced. A, binding of GTP-Ran to GST-HTF9A. Forty five pmol of GST or
22.5 pmol of GST-HTF9A were bound to 20 µl of glutathione-Sepharose
beads. Four pmol of wild-type or -
DEDDDL thrombin-cleaved Ran
proteins which had been loaded with [
-
P]GTP
(0.12 µCi of bound [
-
P]GTP) were mixed
with the beads in 50 mM MOPS, pH 7.1, 10 mM
MgCl
, 0.1 mg/ml BSA, and were then washed 3 times in the
same buffer (1.0 ml per wash). Assays were performed in duplicate, and
nucleotides associated with the GSH beads were quantitated by
scintillation counting. DPM indicates the amount of
[
-
P]GTP-wild type or -
DEDDDL-Ran
(62,500 dpm/pmol of protein) associated with the GST or GST-HTF9A on
glutathione-Sepharose beads. B, EDTA-induced GTP release from
the wild type- or
DEDDDL-Ran
GST-HTF9A complex. Equal amounts
(1.6 pmol) of wild-type or -
DEDDDL-Ran were loaded with
[
-
P]GTP and incubated with 5.5 pmol of
GST-HTF9A or GST as described under ``Results.'' Nucleotide
release in EDTA was measured over as described under ``Materials
and Methods.'' Values are means ± range.
,
wild-type + GST;
,
DEDDDL + GST;
, wild-type
+ GST-HTF9A;
,
DEDDDL +
GST-HTF9A.
Using an
overlay assay on cell extracts, we demonstrated previously that
Ran-binding proteins stabilize the GTP-bound state of Ran(17) .
To determine if the binding of recombinant HTF9A to GTP-Ran could
inhibit EDTA-stimulated release, nucleotide release assays were
performed as described for Fig. 1. After loading wild-type or
DEDDDL Ran with [
-
P]GTP, GST or
GST-HTF9A was added at 23 °C for 10 min to allow complex formation.
The mixtures were then added to EDTA-containing buffer. Fig. 4B shows that the preincubation of wild-type Ran
with GST-HTF9A can dramatically reduce the EDTA-stimulated nucleotide
release from the wild-type protein (k
with HTF9A
0.028 min
compared to k
without HTF9A
0.46 min
). However, the
inhibitory effect of HTF9A on release from the
DEDDDL mutant was
significantly smaller (k
with HTF9A
0.10
min
compared to k
without
HTF9A
0.23 min
). This result suggests that
either the affinity of the
DEDDDL mutant for HTF9A is lower than
that of wild-type Ran, or that the deletion of the C terminus reduces
the inhibition efficiency. At a 10-fold higher concentration of HTF9A,
GTP release was almost completely inhibited from both wild-type and
DEDDDL Ran (data not shown). Therefore, the efficiency of
inhibition is not reduced by loss of the C-terminal domain, and the
most likely explanation of the data is that HTF9A exhibits a lower
affinity for
DEDDDL Ran than for wild-type Ran.
HTF9A/RanBP1 Inhibits RCC1-mediated Nucleotide Release on
Ran
To measure the effect of HTF9A on RCC1 activity toward Ran,
the wild-type and DEDDDL mutant proteins were loaded with
[
-
P]GTP and preincubated with a molar
excess of GST or GST-HTF9A, as described above, before addition of
recombinant RCC1. Under these conditions HTF9A completely blocked the
RCC1-catalyzed release of nucleotide from wild-type Ran, with the k
without HTF9A
0.56 min
and the k
with HTF9A
0
min
(Fig. 5A). However, Fig. 5B shows that the rate of GTP release from the
DEDDDL mutant in the presence of HTF9A was higher than that from
wild-type Ran under the same conditions (k
without HTF9A
1.6 min
versusk
with HTF9A
0.076
min
). In addition, we observed a more rapid
RCC1-catalyzed release of GTP from this protein (k
1.6 min
) than from wild-type Ran (k
0.56 min
), as was
the case for GDP release catalyzed by RCC1 (see Fig. 2).
Figure 5:
HTF9A/RanBP1 reduces RCC1-catalyzed GTP
release from Ran, and has a lower affinity for DEDDDL-Ran. Twenty
pmol of wild-type (A) or
DEDDDL (B) Ran were
loaded with [
-
P]GTP as described in the
legend to Fig. 1 and under ``Materials and Methods,'' and
incubated with 190 pmol of GST or GST-HTF9A. RCC1 nucleotide release
assays were performed as described under ``Materials and
Methods.'' Values are the means ± range.
, GST
- RCC1;
, GST + RCC1;
, GST-HTF9A - RCC1;
, GST-HTF9A + RCC1. C, HTF9A has a lower affinity
for
DEDDDL Ran than for wild-type Ran. Increasing concentrations
of GST-HTF9A were incubated with constant amounts of
[
-
P]GTP-wild type or -
DEDDDL-Ran. The
complex was added to an RCC1-containing buffer for 5 min before
termination by filter binding. Values are means ± range.
, wild-type;
,
DEDDDL.
A
likely interpretation of these results is that they all reflect a
reduced affinity of HTF9A for the DEDDDL mutant. Further evidence
to support this interpretation is shown in Fig. 5C.
Here, different concentrations of GST-HTF9A were bound to equal amounts
of thrombin-cleaved [
-
P]GTP-loaded
wild-type or
DEDDDL Ran, and the complexes were added to
RCC1-containing buffers for 5 min at 23 °C. The results show that
saturating amounts of HTF9A were able to inhibit RCC1-mediated GTP
release from either protein, but that the affinity of the mutant for
HTF9A is lower than that of wild-type. For wild-type Ran the apparent K
is about 1.5 nM, and for the
DEDDDL mutant the apparent K
is
about 60 nM. Because only a portion of the total Ran protein
binds nucleotide (27) and the proportion of active HTF9A is
unknown, these affinities are relative to each other and not absolute
values.
HTF9A/RanBP1 Is a Co-activator of Ran-GAP for Wild-type
Ran But Decreases Ran-GAP Activity on the
To
investigate the effect of HTF9A on Ran-GAP, we performed GAP assays on
Ran that had been preincubated with either GST or GST-HTF9A. The basal
rate of GTP hydrolysis by wild-type Ran was very low (kDEDDDL Mutant
0.002 min
), and
HTF9A did not act as a Ran-GAP (Fig. 6A). In the absence
of HTF9A and presence of Ran-GAP activity, the rate of hydrolysis is
increased by about 10-fold (k
0.018
min
). However, in the presence of both HTF9A and
Ran-GAP activity, the rate of GTP hydrolysis was further increased to
approximately 20-fold over the basal rate (k
0.039 min
), demonstrating that the
HTF9A/RanBP1 can function as a co-activator of Ran-GAP. These results
confirm an earlier report of this phenomenon by Bischoff et
al.(16) , and the observation by Beddow et al.(20) that an isolated Ran-binding domain can function as a
co-activator of Ran-GAP. As described above (Fig. 3) and seen
here (Fig. 6B), Ran-GAP acted on the
DEDDDL mutant
more efficiently than on wild-type Ran (
DEDDDL k
0.058 min
). Surprisingly, no
co-activation was observed when the
DEDDDL mutant was preincubated
with HTF9A and tested in the GAP assay (Fig. 6B).
Rather, the addition of HTF9A significantly decreased this
activity (k
0.023 min
).
These results suggest that together the C-terminal DEDDDL tail and
HTF9A play an important role in the interaction of the Ran GTPase with
Ran-GAP.
Figure 6:
HTF9A/RanBP1 enhances Ran-GAP activity for
wild-type Ran but decreases Ran-GAP activity for DEDDDL-Ran.
Wild-type- (A) or
DEDDDL-Ran (B) (16 pmol) was
loaded with [
-
P]GTP (see Fig. 1) and
incubated with 115 pmol of GST or GST-HTF9A as described under
``Results.'' Samples were filter bound over 30 min and
analyzed by thin layer chromatography and PhosphoImager analysis.
, GST - lysate;
, GST + lysate;
,
GST-HTF9A - lysate;
, GST-HTF9A +
lysate.
The C Terminus of Ran Undergoes a Guanine
Nucleotide-dependent Change in Conformation
All of the above
data indicate that the acidic C-terminal domain is involved in the
regulation of guanine nucleotide binding to Ran and GTP hydrolysis by
the Ran GTPase. They also suggest that this domain undergoes a guanine
nucleotide-dependent change in conformation. To test this hypothesis
more directly, we analyzed the ability of an anti-Ran antiserum, AR-12,
to distinguish between the GDP- and GTP-bound states of Ran. This
antiserum was generated against a C-terminal, Ran-specific peptide
directly adjacent to the -DEDDDL sequence. Recombinant Ran was loaded
with a mixture of [-
P]GTP and
[
-
P]GDP, then immunoprecipitated with
AR-12, and the Ran-bound nucleotides were separated by thin layer
chromatography and quantitated. As can be seen from Fig. 7(lane 2), the antiserum specifically
immunoprecipitated the GTP-Ran about 20-fold more efficiently than it
did the GDP-bound Ran. In a separate experiment, no immunoprecipitation
of [
-
P]GDP-loaded Ran could be detected
over background (using preimmune serum). Together these results suggest
that in the GDP-bound state, the Ran C-terminal domain is folded in
such a way as to be inaccessible to the anti-Ran antibodies, and that
binding of GTP triggers a conformational change that exposes the AR-12
epitopes.
Figure 7:
Immunoprecipitation of GTP- or GDP-bound
Ran with anti-Ran antiserum AR-12. Two µg of recombinant Ran was
loaded with either a mixture of [-
P]GTP and
[
-
P]GDP (lanes 1, 2, 5, and 6) or [
-
P]GDP alone (lanes 3,
4, 7, and 8) as in Fig. 1, then immunoprecipitated with
anti-Ran antiserum AR-12 or preimmune antiserum (PreAR-12).
Eluted precipitates and 1% of the supernatants were separated by thin
layer chromatography and analyzed by autoradiography. Supernatants
represent the total mixture including unbound
nucleotide.
and prevents entry into mitosis.
Furthermore, the presence of this mutant protein in cell-free extracts
prevents DNA replication, and suppresses nuclear growth but not nuclear
assembly.
, they arrest
at the G
/S boundary(28) .
(
)also arrests
mammalian cells in G
or at the G
/S boundary
(10). However, the addition of this protein to cell-free Xenopus egg extracts has no effect on nuclear formation or entry into
mitosis, but does inhibit DNA replication slightly(9) . In S. cerevisiae, the analogous gain-of-function mutation in the
Ran homolog GSP1 is lethal(5) . These reports suggest that the
regulated guanine nucleotide exchange and hydrolysis on Ran is an
essential cellular process. The data presented here implicate the
C-terminal tail of Ran in this regulation.
-phosphate of GTP; in this conformation,
the -DEDDDL tail is not available for interaction with Ran-binding
proteins. The Ran exchange factor, RCC1, catalyzes the exchange of GDP
for GTP. When bound to Ran, the
-phosphate of GTP could displace
the -DEDDDL tail, which would undergo a conformational change to expose
the C terminus to the environment. The destabilization of GTP binding
on Ran may result from repulsion forces exerted between the negatively
charged
-phosphate on GTP and the extremely negative charge of the
DEDDDL tail. This destabilization would account for the more rapid loss
of GTP from wild-type Ran than from the
DEDDDL mutant in EDTA (Fig. 1B).
DEDDDL mutant can still interact efficiently with RCC1 and
Ran-GAP; therefore, we can speculate that the loss of function is most
likely related to the reduced efficiency of interaction with
HTF9A/RanBP1 and other similar proteins. In the absence of this
interaction, the signal transmitted by the ``on''
conformation of Ran is interrupted and subsequent cellular events are
not initiated. Therefore, we hypothesize that the C-terminal -DEDDDL
tail of Ran plays a dual role in Ran-mediated signal transduction. Not
only is this domain involved in the stabilization of GDP on the Ran
protein, but it is of critical importance for interaction of GTP-Ran
with the Ran-binding proteins that function as downstream effectors.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.