3017
Journal of Cell Science 111, 3017-3026 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
JCS3833
The role of the Ran GTPase in nuclear assembly and DNA replication:
characterisation of the effects of Ran mutants
Mike Hughes1,*, Chuanmao Zhang1,2,*, Johanna M. Avis1,‡, Christopher J. Hutchison3 and Paul R. Clarke1,§
1School of Biological Sciences, University of Manchester, G38 Stopford Building, Oxford Road, Manchester M13
2Department of Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing 100871, China
3Department of Biological Sciences, University of Dundee, Dundee DD1 4HN, UK
9PT, UK
*The first two authors contributed equally to this work
‡Present address: Department of Biochemistry and Applied Molecular Biology, UMIST, PO Box 88, Manchester M60 1QD, UK
§Author for correspondence (e-mail: [email protected])
Accepted 14 August; published on WWW 23 September 1998
SUMMARY
The Ran GTPase plays a critical role in nucleocytoplasmic
transport and has been implicated in the maintenance of
nuclear structure and cell cycle control. Here, we have
investigated its role in nuclear assembly and DNA
replication using recombinant wild-type and mutant Ran
proteins added to a cell-free system of Xenopus egg
extracts. RanQ69L and RanT24N prevent lamina
assembly, PCNA accumulation and DNA replication. These
effects may be due to the disruption of nucleocytoplasmic
transport, since both mutants inhibit nuclear import of a
protein carrying a nuclear localisation signal (NLS).
RanQ69L, which is deficient in GTPase activity, sequesters
importins in stable complexes that are unable to support
the docking of NLS-proteins at the nuclear pore complex
INTRODUCTION
Ran, a member of the Ras small GTPase superfamily, is highly
conserved in eukaryotes from yeast to humans and is found
largely in the nucleus (Bischoff and Ponstingl, 1991b; Drivas
et al., 1990). It plays a central role in directing the active
transport of macromolecules through nuclear pore complexes
(NPCs) (Görlich and Mattaj, 1996). Like other GTPases, Ran
exists in GTP and GDP bound states that interact differently
with regulators and effectors. The major nucleotide exchange
factor for Ran, RCC1, is localised in the nucleus where it is
thought to generate Ran-GTP (Bischoff and Ponstingl, 1991a;
Klebe et al., 1995b; Ohtsubo et al., 1987). The intrinsic GTPase
activity of Ran is very low, but is stimulated by the interaction
of a GTPase-activating protein (RanGAP) located on the
cytoplasmic face of the nuclear pore (Matunis et al., 1996;
Bischoff et al., 1994; Mahajan et al., 1997; Saitoh et al., 1997).
Ran binding protein 1 (RanBP1) is also cytoplasmic (Richards
et al., 1996) and interacts specifically with the GTP-bound
form of Ran (Coutavas et al., 1993), stimulating GTPase
activation by RanGAP1 10-fold (Bischoff et al., 1995). The
compartmentalised localisation of these regulators has been
(NPC). RanT24N, in contrast to wild-type Ran-GDP,
interacts only weakly with importin α and nucleoporins,
and not at all with the import factor p10, consistent with
its poor activity in nuclear import. However, RanT24N does
interact stably with importin β, Ran binding protein 1 and
RCC1, an exchange factor for Ran. We show that Ran-GDP
is essential for proper nuclear assembly and DNA
replication, the requirement being primarily before the
initiation of DNA replication. Ran-GDP therefore mediates
the active transport of necessary factors or otherwise
controls the onset of S-phase in this system.
Key words: Ran, Ran-binding protein, Nucleocytoplasmic transport,
Lamin, DNA replication
proposed to generate a high concentration of Ran GTP in the
nucleus, and a low or very localised concentration of Ran-GTP
in the cytoplasm. This asymmetric distribution is thought to be
crucial for the directionality of nucleocytoplasmic transport
(Görlich et al., 1996b; Izaurralde et al., 1997).
During nuclear import, proteins containing lysine-rich
nuclear localisation signals (NLS) are recognised by a
receptor protein, importin β (karyopherin β) via an adaptor
protein, importin α (karyopherin α) (Görlich and Mattaj,
1996). In a binding assay using purified recombinant proteins,
Rexach and Blobel (1995) showed that Ran-GTP releases an
NLS/importin α/importin β complex from an immobilised
nucleoporin and disassociates NLS/importin α from importin
β. Ran-GTP binds directly to importin β, an interaction that
is stabilised by RanBP1 until it interacts with RanGAP1,
when RanBP1 stimulates the hydrolysis of GTP to GDP
(Bischoff and Görlich, 1997; Bischoff et al., 1995; Floer and
Blobel, 1996; Lounsbury and Macara, 1997; Lounsbury et al.,
1996; Rexach and Blobel, 1995). Another import factor, p10
(NTF2) (Moore and Blobel, 1994; Paschal and Gerace, 1995)
binds to Ran-GDP and importin β, stabilising their interaction
with a nucleoporin (Nehrbass and Blobel, 1996). These
3018 M. Hughes and others
results are consistent with a model in which the interaction
of the NLS-protein/receptor complex with the NPC is
determined by the GDP/GTP cycle of Ran, with Ran-GDP
promoting association with nucleoporins through FXFG
repeat sequences while Ran-GTP causes dissociation of the
docked complex. However, the site of action of Ran has been
controversial: Görlich et al. (1996a,b) have proposed that
Ran-GTP dissociates the importin/NLS-protein complex at
the nucleoplasmic face of the NPC, while other studies
(Delphin et al., 1997; Melchior et al., 1995) have suggested
that Ran-GTP is required for docking of import complexes at
the cytoplasmic face of the nuclear pore complex via
RanBP1-like domains on the nucleoporin RanBP2 (Wu et al.,
1995; Yokoyama et al., 1995). Nevertheless, Ran-GDP and
free GTP are required for nuclear protein import in a
reconstituted cell-free assay, whereas Ran-GTP is much less
effective (Görlich et al., 1996b; Weis et al., 1995). The
interaction of nuclear export factors (‘exportins’) such as
CAS (Kutay et al., 1997) and Crm1 (Fornerod et al., 1997;
Fukuda et al., 1997; Neville et al., 1997; Ossareh-Nazari et
al., 1997; Stade et al., 1997) with their substrates is regulated
by Ran in the opposite way to the importins, being stimulated
by the GTP-bound form present in the nucleus (Fornerod et
al., 1997; Kutay et al., 1997, 1998). The RanGTP/exportin/substrate complexes may be dissociated by
GTP hydrolysis after translocation through the NPC and
interaction with RanGAP1 and RanBP1 at the cytoplasmic
side (Bischoff and Görlich, 1997). In addition, it remains
possible that GTP hydrolysis on Ran plays a role during the
translocation of complexes through the NPC.
Ran and its interacting proteins may also play roles during
the cell division cycle, being implicated in changes in nuclear
architecture, DNA replication and mitosis (reviewed by Avis
and Clarke, 1996; Rush et al., 1996; Sazer, 1996). Such roles
may represent requirements for nucleocytoplasmic transport
during cell cycle transitions, although there has been some
evidence that Ran has distinct functions (Sazer, 1996). A useful
model system to study nuclear assembly and DNA replication,
as well as nucleocytoplasmic transport, is provided by
concentrated Xenopus egg extracts. In this cell-free system, the
structural integrity of the nucleus is crucial for the assembly of
replication complexes and the establishment of S-phase
(Hutchison, 1994). Although it is likely that active import of
proteins plays an important role in these events (Cox, 1992),
the precise role has remained unclear due to the lack of specific
reagents to perturb the process.
In this study, we have investigated the role of Ran in
nuclear assembly and DNA replication in Xenopus egg
extracts. We have compared two mutants of Ran that are
defective in the GTP/GDP cycle and have dominant effects,
presumably because they form stable complexes with other
factors. RanQ69L (where glutamine 69 is changed to
leucine), is defective in its GTPase activity, even when
stimulated by RanGAP1 (Klebe et al., 1995a). It is therefore
locked in the GTP-bound state. RanQ69L inhibits nuclear
protein import when added to a system of permeabilised
human cell nuclei complemented with transport factors
(Palacios et al., 1996; Weis et al., 1996) and when expressed
in cells (Carey et al., 1996; Schlenstedt et al., 1995).
However, its effects on nuclear assembly and DNA
replication have not been determined prior to this study.
RanT24N (where threonine 24 is changed to asparagine) does
not bind GTP and has a reduced affinity for GDP (Klebe et
al., 1995a). This mutant causes aberrant chromatin
decondensation and blocks cell cycle progression in Xenopus
egg extracts (Clarke et al., 1995; Dasso et al., 1994;
Kornbluth et al., 1994). RanT24N inhibits nuclear export of
proteins when injected into nuclei (Richards et al., 1997), but
reports of its effects on nuclear protein import have been
somewhat contradictory (Carey et al., 1996; Dasso et al.,
1994; Kornbluth et al., 1994; Palacios et al., 1996; Richards
et al., 1997; Weis et al., 1996).
Here, we show that both mutants inhibit nuclear protein
import when added early during nuclear assembly, disrupt the
accumulation of lamins and PCNA within the nucleus, and
inhibit the initiation of DNA replication. RanQ69L completely
blocks the interaction of an NLS-protein with the NPC,
whereas Ran T24N permits only weak import into preassembled nuclei. By determining the stable complexes formed
by these proteins we show that the Ran-GTP/importin/NLSprotein complex is unable to interact stably with the NPC
without hydrolysis of GTP on Ran. We also demonstrate that
Ran-GDP is essential for proper nuclear assembly and the
initiation of S-phase, most likely because it is necessary for the
import of structural or regulatory factors required prior to the
initiation of DNA replication.
MATERIALS AND METHODS
Construction of Ran fusion expression vectors
Glutathione-S-transferase (GST) fusions of human Ran and its
mutants T24N and Q69L were constructed by PCR mutagenesis to
introduce specific cloning sites into the cDNA. PCR was carried out
using the following primers: 5′ CGG GAT CCG CCA TAT GGC TGC
GCA GGG AGA GCC CCA GG 3′ and 5′ GAT CCG CTC GAG TCT
TAT CAC AGG TCA TCA TCC TCA TCC GGG 3′. The PCR
products were cloned into the pCRII vector (Invitrogen) and then
subcloned into pGEX-5X-2 (Pharmacia) to yield ORFs that were in
frame for fusion protein expression.
Expression and purification of Ran fusion proteins
Ran proteins were expressed and purified by a modification of the
method of Klebe et al. (1993). GST-fusion proteins were purified from
bacterial cell lysate on glutathione-Sepharose (Pharmacia) and loaded
with the appropriate nucleotide as described by Bischoff and
Ponstingl (1995). To remove glutathione, EDTA and excess
nucleotide, protein solutions were spun on Centricon-30 spin
concentrators (Amicon) at 4°C and the buffer changed for PBS + 20
µM of the appropriate nucleotide. The proteins were snap frozen in
liquid nitrogen and stored in aliquots at −70°C.
Binding assays
Proteins associating with Ran proteins were determined using a
modification of the method of Saitoh et al. (1996). 50 µl aliquots of
Xenopus egg extracts (10,000 g supernatants; Clarke et al., 1995)
supplemented with a GST-Ran fusion protein (2 µM, except were
stated) were incubated at 20°C for 20 minutes before diluting 10-fold
with ice-cold dilution buffer (20 mM Tris-HCl, pH 8.0, 50 mM NaCl,
0.1% (v/v) Triton X-100, 10% (v/v) glycerol), supplemented with 1
mM EDTA or 2.5 mM MgCl2 where stated. Complexes were bound
to glutathione-Sepharose beads (20 µl of a 50% slurry) at 4°C for 1
hour with continuous gentle agitation. The beads were recovered by
low speed centrifugation and washed three times in the appropriate
dilution buffer. Proteins were eluted by resuspending the beads in
Role of Ran in nuclear assembly 3019
SDS-PAGE loading buffer and analysed by silver staining of SDSPAGE gels or immunoblotting. Antibodies used were raised against
Ran BP1 (Coutavas et al., 1993; Nicolás et al., 1997), p10 (Moore and
Blobel, 1994), importin α, importin β (Görlich et al., 1995), RanBP2
(Yokoyama et al., 1995) and nuclear pore complex proteins (mAb414;
Finlay and Forbes, 1990).
Nuclear assembly and immunofluorescence microscopy
Nuclear assembly in Xenopus egg extracts (Hutchison et al., 1988)
was initiated by the addition of demembranated Xenopus sperm
heads (1,000/µl) followed by incubation at 23°C. To examine nuclear
morphology, samples removed at various times during the incubation
were fixed and stained on a slide with DAPI (Hutchison et al., 1988).
For immunofluorescence labelling, samples were fixed with the EGS
(Hutchison et al., 1988) for 30 minutes at 37°C. The fixed nuclei were
then recovered by centrifuging onto coverslips through a cushion of
30% glycerol. Lamin B3 and PCNA were detected using anti-lamin
antibody L6 5D5 and anti-PCNA antibody PC10 followed by an
FITC-conjugated secondary antibody (Zhang et al., 1996). To
monitor DNA replication, 4 µM biotin-16-dUTP was added to the
extracts 20 minutes prior to fixation and incorporated biotin was
visualized with strepavidin conjugated to Texas Red. Images were
captured on a Zeiss Axioskop microscope using a cooled CCD
camera and processed using Improvision Openlab and Adobe
Photoshop software.
Nuclear import assays
Import of the NLS-carrying protein, nucleoplasmin (20 µg/µl)
(Philpott et al., 1991) labelled with 5(6)-carboxyfluorescein-Nhydroxysuccinimide ester (FLUOS, Boehringer Mannheim), was
assessed 60 minutes after addition of the substrate. The reactions were
carried out in 20 µl of Xenopus egg extract supplemented with
nucleoplasmin (20 µg/µl) and sperm chromatin (1,000/µl). Samples
were fixed with 4% formaldehyde (Hutchison et al., 1998). Fixed
nuclei were recovered by centrifugation through a glycerol cushion.
Images were captured and processed as above.
appeared slightly after lamina formation and increased in
intensity at 60 minutes when DNA replication was initiated
(data not shown). We examined the role of Ran in this process
by comparing the T24N and Q69L mutants of Ran expressed
as recombinant proteins and added to the extracts prior to
sperm heads. As shown in Fig. 1, both mutants had a dramatic
effect on nuclear assembly. In the case of RanT24N (preloaded with GDP and added at 10 µM), the nuclei underwent
the transition from elongated sperm head to a more compact
shape after 20 minutes. The nuclei subsequently became
rounded, but with highly condensed, homogeneously staining
chromatin, as described previously (Dasso et al., 1994;
Kornbluth et al., 1994). 10 µM RanQ69L (pre-loaded with
GTP) also produced small nuclei, but were more elongated
with less highly condensed chromatin. In contrast, addition of
wild-type Ran (pre-loaded with GDP) had no detrimental
effect on nuclear assembly (Fig. 1).
Nuclei assembled in extracts containing 10 µM of either
mutant were defective in lamina formation (Fig. 2A) and
PCNA accumulation (Fig. 2B). DNA replication was
completely absent. At lower concentrations (1-5 µM), the
mutants produced less pronounced effects: with RanT24N
there was some nuclear growth and weak lamina staining but
no DNA replication. At similar concentrations of RanQ69L,
DNA replication assays
DNA synthesis was assayed by a modification of the method of Blow
and Laskey (1986). Demembranated Xenopus sperm heads (1,000/µl)
were added to 20 µl of egg extract, and the reactions were then
supplemented with Ran proteins where stated and 0.5 µl of [α32P]dCTP (3,000 Ci/mmol, 10 µCi/µl). After incubation at 23°C for
40 minutes, the reactions were stopped by addition of 180 µl 0.5%
SDS, 20 mM EDTA, 20 mM Tris-HCl, pH 8.0, and 0.5 mg proteinase
K. The mixture was incubated at 37°C for at least one hour, then
extract with phenol, phenol/chloroform and chloroform. 50 µl of the
reaction was spotted on to Whatman GF/C paper and the filters were
washed in 5% TCA for 4× 10 minutes. Incorporation of radioactivity
was determined by liquid scintillation counting.
RESULTS
Effects of Ran mutants on nuclear assembly and
DNA replication
Nuclear assembly from demembranated sperm heads added to
Xenopus egg extracts was monitored by fixing samples during
the course of the assembly reaction and staining DNA with
DAPI. Immunofluorescence detection of lamin B3 and the
DNA polymerase δ auxilliary factor PCNA were used to
follow the formation of the nuclear lamina and DNA
replication
complexes,
respectively.
Chromatin
decondensation occured at 20-40 minutes (Fig. 1), with the
appearance of a nuclear lamina at 40 minutes. PCNA first
Fig. 1. Effects of Ran mutants on nuclear assembly and DNA
replication. 10 µM Ran wild-type, T24N or Q69L proteins were
added to aliquots of Xenopus egg extracts immediately prior to
addition of demembranated sperm heads at 1,000/µl. A control
incubation had buffer only added. Samples were removed at the
times shown for fixing and staining with DAPI to reveal DNA. Bar,
10 µM.
3020 M. Hughes and others
the growth of nuclei was heterogeneous, with some large nuclei
showing lamina staining, while others remained small and
without a lamina (data not shown).
Effects of RanT24N and RanQ69L on nuclear protein
import
To assess the relationship between the effects of Ran Q69L and
T24N mutants on nuclear assembly and nuclear protein import
in Xenopus egg extracts, we assessed the uptake of
fluorescently labelled nucleoplasmin, a substrate which
contains an NLS and is imported by a Ran-dependent
mechanism. When Ran mutants (10 µM) were added prior to
sperm heads, the resulting nuclei were completely deficient in
nucleoplasmin import (Fig. 3A). In contrast, wild-type Ran had
no inhibitory effect.
A.
B.
Fig. 2. Effect of Ran mutants on lamina formation and PCNA
accumulation. Nuclei were assembled in Xenopus egg extracts
containing 10 µM Ran wild-type, T24N or Q69L proteins, as in Fig.
1. Biotin-16-dUTP was added at the start of the incubation. After 100
minutes, nuclei were fixed. The distribution of lamin BIII (A) and
PCNA (B) detected by imunofluoresence was compared to DNA
staining by DAPI. Biotin incorporation indicating DNA synthesis
was detected with strepavidin conjugated to Texas Red. Bar, 10 µM.
To distinguish direct effects on nuclear import from
possible secondary effects due to disruption of nuclear
assembly, extracts were supplemented with Ran proteins
after the assembly of nuclei for 90 minutes (Fig. 3B). Under
these conditions, wild-type Ran also had no detrimental
effect on nucleoplasmin import. However, RanQ69L
inhibited the import of nucleoplasmin at this stage. In
addition, no staining of the nuclear envelope was apparent,
indicating that RanQ69L also prevented docking of
nucleoplasmin at the nuclear envelope (NE). At the same
concentration, RanT24N decreased the extent of
nucleoplasmin import, but some nuclear accumulation was
apparent, with a distinct labelling of the nuclear periphery,
which may indicate docking at the NE.
These results show that both Ran mutants disrupt nuclear
protein import. However, RanT24N seems to act in part by
inhibiting the establishment of nucleocytoplasmic transport
during nuclear assembly, whereas RanQ69L has a powerful
dominant effect even in preformed nuclei.
Molecular interactions of RanQ69L and RanT24N:
Ran BP1
Presumably, the dominant effects of RanQ69L and RanT24N
proteins are due to the sequestering of factors involved in
nucleocytoplasmic transport and nuclear assembly. We
therefore examined interacting proteins that co-precipitate from
egg extracts to examine the molecular basis for these effects. In
these experiments, we used Ran proteins produced as fusions
with glutathione-S-transferase (GST) and co-precipitating
proteins were detected with antibodies. GST alone was used as
a control. The GST-tagged Ran proteins produced effects on
nuclear assembly and nucleoplasmin import identical to the
untagged forms (data not shown), demonstrating that the GST
fusion does not affect their function.
Wild-type Ran pre-loaded with GTP or GDP associated with
low levels of Ran-binding protein 1 (RanBP1). When Ran was
preloaded with GTPγS, which is resistant to hydrolysis,
additional RanBP1 was recovered, while the Q69L mutant of
Ran, locked in a GTP-bound state, bound large amounts of
RanBP1 (Fig. 4A). This is consistent with a preference of
Xenopus RanBP1 for the GTP-bound conformation of Ran
(Nicolás et al., 1997; Pu and Dasso, 1997). It is likely that wildtype Ran-GTP fails to co-precipitate RanBP1 effectively
because of RanGAP-assisted GTP hydrolysis on Ran, which is
stimulated by RanBP1 (Bischoff et al., 1995). RanT24N was
able to coprecipitate RanBP1 from Xenopus egg extracts nearly
as efficiently as RanQ69L (Fig. 4A), although it does not bind
GTP (Klebe et al., 1995a). Previously, RanT24N has been
shown to form a stable complex with RCC1 under similar
conditions (Dasso et al., 1994). Using purified proteins, wildtype Ran forms a trimeric complex with RCC1 and RanBP1
that may be nucleotide-free (Bischoff et al., 1995). Therefore,
one explanation for the stable interaction between RanT24N
and RanBP1 in the extracts could be as part of a trimeric
complex with RCC1.
To examine further the interactions between Ran, RanBP1 and
RCC1, we carried out a binding assay using purified proteins.
RanBP1 binding to GST-Ran was detected by western blotting
as before (Fig. 4B). We found that the antibody against RCC1
that we had available also cross-reacted with GST-Ran (data not
shown), making identification of RCC1 difficult by this method.
Role of Ran in nuclear assembly 3021
Nucleoplasmin
Nucleoplasmin
Fig. 3. RanQ69L and RanT24N
inhibit nucleoplasmin import. Nuclei
were assembled in Xenopus egg
extracts with the addition of Ran
proteins or buffer only (control) at
the start of the incubation (A) or after
90 minutes (B). Fluorescein-labelled
nucleoplasmin was added at the start
of the incubation or after 110
minutes, respectively. Samples were
fixed and stained with DAPI after a
further 60 minutes incubation. Bar,
10 µM.
presence or absence of Mg2+. Addition of RCC1 markedly
increased the interaction of RanBP1 with RanT24N or wild-type
Ran, even in the presence of Mg2+. Since RanBP1 does not
interact with RCC1 in the absence of Ran (Bischoff et al., 1995),
these results strongly suggest that the association of RanBP1
with RanT24N in egg extracts is stabilised by the co-operative
binding of RCC1 in a trimeric complex.
Interactions with p10
p10 (NTF-2) interacts with Ran-GDP and stabilises the
γ
However, RCC1 could be visualised on silver-stained protein
gels as a polypeptide migrating slightly faster than GST-Ran
(Fig. 4C). In the absence of RCC1 and with Mg2+ ions present,
RanBP1 interacted in a dimeric complex with Ran-GTP but not
Ran-GDP (Fig. 4B,C). In the presence of EDTA, which chelates
Mg2+ ions and destabilises nucleotide binding to Ran, a weak
association of RanBP1 with Ran-GDP was detected. This may
be due to the partial formation of a nucleotide-free complex
(Bischoff et al., 1995). RanT24N also formed a weak complex
with RanBP1 in the absence of RCC1, in this case both in the
Fig. 4. Interactions of Ran mutants with RanBP1 and
RCC1. (A) RanBP1 coprecipitated from Xenopus egg
extracts with GST-Ran proteins and detected on a western
blot using a specific antibody. (B,C) RCC1 stabilises the
association of Ran wild-type and T24N proteins in a
purified protein binding assay. Incubations containing
GST-Ran proteins and RanBP1 (both at 2 µM) were
carried out in buffer containing Mg2+ or EDTA with or
without 2 µM RCC1. GST-Ran proteins were precipitated
on glutathione beads, and analysed on a 10% acrylamide
SDS-PAGE gels for blotting with anti-RanBP1 antibodies
after transfer to a nitocellulose membrane (B) or silver
staining of the gel (C).
γ
3022 M. Hughes and others
Fig. 5. Interactions of Ran mutants with p10/NTF-2. (A) p10
coprecipitated from Xenopus egg extracts with 2 µM GST-Ran
proteins or GST alone as a control were separated on a 15%
polyacrylamide gel, transferred to nitro-cellulose and probed with an
antibody against Xenopus p10 (1:1,000 dilution). (B) In a separate
experiment, precipitations using 10 µM GST-Ran proteins were
carried out with or without the addition of 10 µM RCC1.
assembly of the importin/NLS-protein complex with
nucleoporins in assays using purified proteins (Clarkson et
al., 1996; Moore and Blobel, 1994; Nehrbass and Blobel,
1996; Paschal et al., 1996). We found that Ran-GDP does
indeed bind p10 strongly in Xenopus egg extracts (Fig. 5A).
Binding to Ran-GTP and Ran-GTPγS was detected at lower
levels, although it is possible that this association was due
to residual Ran-GDP, either present in the preparations of
recombinant proteins or generated upon addition to the
extracts. RanQ69L, locked in the GTP bound state, failed to
coprecipitate any p10. Similarly, RanT24N also failed to
interact with any detectable p10. One possibility might be
that p10 fails to interact with a nucleotide-free form of Ran
that is stabilised by the T24N mutation (Bischoff et al.,
1995). We found that addition of RCC1 to extracts markedly
reduces the binding of p10 to wild-type Ran-GDP (and does
not stabilise the interaction of p10 with RanT24N or
RanQ69L; Fig. 5B). This may be the result of stabilising the
nucleotide-free form of wild-type Ran, or the physical
exclusion of p10 from the complex formed with both
RanBP1 and RCC1 under these conditions (Nicolás et al,
1997). Together, these results indicate that p10 interacts only
with wild-type Ran-GDP in the extracts under conditions
similar to those found in intact cells and does not interact
with either of the Ran mutants.
T24N and Q69L mutations of Ran perturb
interactions with importins
Importin β co-precipitated from Xenopus egg extracts with
wild-type Ran preloaded with GDP or GTP (Fig. 6A), as
previously reported (Saitoh et al., 1996). Wild-type RanGTPγS recovered an increased amount of importin β, as did
the Q69L mutant of Ran, consistent with an increased affinity
of the GTP-bound form of Ran for direct binding to importin
Fig. 6. Interactions of Ran mutants with importins. Detection of
importin α and importin β co-precipitating with 2 µM GST-Ran
proteins or GST alone as a control. Western blots were probed with
specific antibodies against Xenopus importins (both at 1:5,000
dilution). (B) The experiment was performed using buffers
containing EDTA or Mg2+. The minor band that migrates slightly
more slowly than importin α is a contaminating polypeptide present
in the GST-Ran preparations that reacts with the antibody against
importin α.
β. Nevertheless, significant association with Ran preloaded
GDP was also found (Fig. 6A), even though the lack of
association with RanBP1 (Fig. 4A) and the strong binding of
p10 (Fig. 5A) under the same conditions indicated that Ran
remained predominantly GDP bound in the extract. RanT24N
also showed a strong association with importin β (Fig. 6A).
Importin α associated with Ran preloaded with either GDP
or GTP, although the amount co-precipitating with Ran
preloaded with GTP was lower (Fig. 6A). Importin α does
not interact directly with Ran, but forms a complex with
importin β, p10 and nucleoporins when Ran is in the GDPbound form (Görlich et al., 1996b; Rexach and Blobel, 1995).
It is likely, therefore, that a similar complex accounts for the
interaction between Ran-GDP and both importins together
with p10 in Xenopus egg extracts. However, RanQ69L
showed an even stronger association with importin α (Fig.
6A) than Ran-GDP, as did wild-type Ran preloaded with
GTPγS (data not shown).
To investigate the stability of the interaction with importins
further, Ran proteins were recovered from egg extracts in the
presence of Mg2+ or EDTA (Fig. 6B), conditions shown
previously to alter the binding of some proteins to wild-type
Ran (Saitoh et al., 1996). Ran wild-type and Q69L proteins
bound significant amounts of importin β under both conditions,
whereas the binding of importin β to RanT24N was abolished
in the presence of EDTA. This was in contrast to the
association of RCC1 with RanT24N which is stable in the
presence of EDTA, either in solution assays (Fig. 4C) or in egg
extracts (Dasso et al., 1994). The association of importin α
with wild-type Ran was also blocked by precipitation in EDTA
(Fig. 6B), showing that the interaction of Ran-GDP with
Role of Ran in nuclear assembly 3023
importin α requires bound nucleotide or is otherwise Mg2+dependent.
Interactions with nucleoporins
We also compared the interactions of RanQ69L, RanT24N and
wild-type Ran with components of the NPC, using antibodies
that recognise RanBP2 (Nup358/p340) or other nucleoporins
(Fig. 7A). Although RanBP2 has four domains thought to
interact specifically with Ran-GTP, we found that Ran-GDP
co-precipitated higher levels of RanBP2 than Ran-GTP.
Furthermore, RanQ69L failed to bind detectable RanBP2 in the
extracts whereas RanT24N precipitated low levels. Nup214
bound to both GDP and GTP-loaded wild-type Ran, and to a
lesser extent with RanQ69L and RanT24N. Nup153 appeared
to bind Ran-GDP more strongly than Ran-GTP, and did not
associate with RanQ69L or RanT24N (Fig. 7B). Nup62, also
recognised by the antibody, failed to coprecipitate with any of
the Ran proteins (data not shown).
Ran-GDP is required for assembly of a replication
competant nucleus
To examine further the role of Ran during nuclear assembly and
the initiation of DNA replication, we disrupted the function of
the endogenous protein present in the extracts by adding a
specific antibody directed against Ran followed by incubation at
4°C for 1 hour. When sperm heads were added after this
treatment the result was small, elongated nuclei with only weak
lamin and PCNA staining and no DNA replication (Fig. 8).
Control incubations with buffer alone (Fig. 8) or irrelevant
antibodies (data not shown) produced no detrimental effect, the
nuclei forming a lamina and undergoing DNA replication. The
defect produced by the anti-Ran antibody could be fully restored
by addition of Ran-GDP, whereas Ran-GTP was much less
effective, indicating that Ran-GDP is specifically required for the
assembly of nuclei competant for DNA replication (Fig. 8).
Ran-dependent processes are required prior to DNA
replication
We utilised Ran mutant proteins to determine the timing of the
Fig. 7. Interaction of Ran mutants with RanBP2 and other
nucleoporins. Co-precipitation of nucleoporins with GST-Ran
proteins. RanBP2 (A) was identified by a specific antibody (1:1,000
dilution) and Nup214 and Nup153 (B) were detected by monoclonal
antibody mAb414 (1:10,000 dilution) after separation on 6% gels
and transfer to nitrocellulose membranes.
requirement for Ran and NLS-mediated nuclear protein
import during the establishment of DNA replication (Figs 9,
10). At 40 minutes after addition of sperm heads to the
extracts, nuclear lamina formation is just becoming apparent,
and PCNA is present, but incorporation of biotin-dUTP is not
detectable, suggesting that DNA replication is at the initiation
stage. When 10 µM wild-type Ran (or buffer alone) was added
at this point, and the nuclei fixed 40 minutes later, a complete
nuclear lamina was apparent with PCNA staining and strong
incorporation of biotin-dUTP, indicating that S-phase was
proceeding (Fig. 9). Similarly, DNA replication continued
when 10 µM RanQ69L or RanT24N were added at 40
minutes, although they continued to inhibit nuclear protein
import after this time (Fig. 3B).
A similar experiment was performed using the incorporation
of [α-32P]dCTP as a more quantitative measurement of DNA
synthesis (Fig. 10). When added at the start of the assembly
reaction, both RanQ69L and RanT24N completely blocked
DNA replication, whereas wild-type Ran had a small
stimulatory effect, consistent with results shown in Fig. 2.
When RanQ69L or RanT24N were added after 40 minutes of
assembly, subsequent DNA replication proceeded in the
presence of added Ran proteins, although a partial inhibitory
effect of RanT24N and RanQ69L was found. This may be due
to effects on some nuclei initiating replication after this time
(data not shown). Alternatively, it may indicate that disrupting
a function of Ran after initiation slows or partially suppresses
DNA replication, although replication can clearly proceed even
under conditions where no Ran-dependent nuclear protein
import is apparent.
DISCUSSION
In this study, we have investigated the role of Ran in nuclear
assembly and the initiation of DNA replication in a cell-free
sytem of Xenopus egg extracts. We have utilised mutants of
Ran that are blocked at different stages of the GTPase cycle.
RanQ69L (locked in the GTP-bound conformation) and
RanT24N (defective in nucleotide binding) both sequester
components of the import machinery and exert inhibitory
effects on nuclear protein import in this system. Using these
mutants we show that Ran-dependent processes, most likely
nuclear protein import, are required during this process until
the initiation of DNA replication but are not essential during
the subsequent progression of S-phase.
RanT24N has dramatic effects on the architecture of nuclei
assembled in Xenopus egg extracts, producing very small
nuclei with condensed and homogenously staining chromatin
(this study; Dasso et al., 1994; Kornbluth et al., 1994). We
show that these nuclei have little or no detectable lamina, very
similar to nuclei formed in the presence of an excess of
RanBP1 (Nicolás et al., 1997). They also fail to accumulate
PCNA and do not undergo DNA replication. We find that
RanT24N decreases import of an NLS-protein in preformed
nuclei, although it permits some docking at the NE, in general
agreement with Weis et al. (1996). While RanT24N interacts
well with importin β, this complex associates very poorly with
importin α and p10. Therefore Ran T24N is not itself active in
NLS-protein import and partially inhibits import mediated by
endogenous Ran, probably by sequestering importin β.
3024 M. Hughes and others
Fig. 8. Ran-GDP is required
for nuclear assembly and DNA
replication. Anti-Ran antibody
was added to the extracts and
incubated for 60 minutes on ice
before the addition of sperm
heads at 1,000/µl and biotindUTP. Where indicated, 10 µM
Ran-GDP or Ran-GTP were
added at the same time as the
sperm heads. The extracts were
incubated at 23°C for 80
minutes, before fixing and
processing for detection of
biotin incorporated into
replicated DNA and lamin B3
or PCNA. Bar, 10 µM.
In addition to its interactions with components of the import
machinery, RanT24N forms a stable complex with RCC1
blocking its exchange activity (Clarke et al., 1995; Dasso et al.,
1994; Klebe et al., 1995b). Presumably, inhibition of RCC1
prevents the generation of nuclear Ran-GTP required for the
release of NLS-cargo into the nucleoplasm and for nuclear
export (Richards et al, 1997). The complex with RCC1 also
sequesters RanBP1 (this study and Nicolás et al., 1997) and
possibly other factors required for export, including the recycling
of importins. When added during the early stages of nuclear
assembly, RanT24N would have access to RCC1 localised on
the chromatin (C. Zhang, unpublished) and thus prevent the
establishment of nucleocytoplasmic transport following the
completion of the nuclear envelope. This may account for the
abolition of both docking and import by RanT24N under these
conditions. RCC1 may also have a separate function in
Fig. 9. Ran-dependent processes
are required prior to DNA
replication. Nuclei were
assembled in Xenopus egg
extracts as in Figs 1-3, except that
Ran proteins were added after 40
minutes of assembly. At this time,
a nuclear lamina was apparent and
PCNA staining was present, but
no DNA replication was
detectable, as shown by samples
fixed at 40 minutes. Bar, 10 µM.
chromatin decondensation that is disrupted by RanT24N,
accounting for the more severe effect of RanT24N than
RanQ69L on this process, even though RanQ69L completely
inhibits NLS-mediated nuclear protein import.
RanQ69L presumably has a dominant inhibitory effect on the
docking of the NLS-cargo because it sequesters the importins
in complexes that are unable to undergo GTP hydrolysis or
interact with the NPC. These results are consistent with a model
in which the Ran-GTP/importin β/RanBP1 complex is
disrupted by GTP hydrolysis, stimulated by RanGAP1 localised
at the cytoplasmic face of the NPC (Mahajan et al., 1997). GTP
hydrolysis releases RanBP1, promoting the assembly of an
import complex between Ran-GDP, importin β and the importin
α/NLS protein cargo, together with p10. A striking feature of
our results is the exchange of RanBP1 for p10 when Ran is
converted from the GTP to GDP-bound state. This import
Role of Ran in nuclear assembly 3025
Ran-GDP is not generated by GTP hydrolysis to permit nuclear
assembly, but it is possible that Ran-GTP has a dominant
inhibitory effect under these conditions. Ran-dependent import
is not essential for S-phase in this system once nuclear
assembly has occurred and DNA replication has been initiated.
However, nuclear import may be necessary during S-phase in
somatic cells where the process is much more protracted and
may be under additional controls. The requirement for Randependent processes prior to initiation is likely to be due to the
import of lamins and other proteins required because of
structural constraints on DNA replication (Ellis et al., 1997),
as well as components of the replication machinery, such as
PCNA. It remains possible that Ran and interacting factors
such as RCC1 play a more direct role in the assembly of
replication complexes or the control of their activation.
Fig. 10. Quantification of the effects of Ran mutants on DNA
replication. Extracts were supplemented with either wild-type (WT)
Ran, Ran T24N, Ran Q69L or buffer immediately prior to addition of
sperm heads (left hand set of conditions) or after 40 minutes of
nuclei formation. [α-32P]dCTP was added either simultaneously with
the Ran proteins (+40) or 10 minutes after addition of the Ran
proteins (+50). After incubation for a further 40 minutes, reactions
were stopped and processed as described in Materials and Methods.
At each time point, buffer samples were normalised to 100% and the
percentage replication was calculated for each condition.
complex could then interact with the NPC at FXFG repeats on
RanBP2 and other nucleoporins via p10 and importin β. The
role of the RanBP1-like domains in RanBP2 that interact
specifically with Ran-GTP when isolated (Yokoyama et al.,
1995) is still unclear. In contrast to previous studies showing
that isolated Ran-GTP can interact directly with RanBP2
(Yokoyama et al., 1995; Delphin et al., 1997), we show that in
Xenopus egg extracts under similar conditions to those found in
cells, RanQ69L does not interact stably with RanBP2. This is
probably because of the large excess of RanBP1 that competes
for binding to Ran in the GTP-bound conformation.
In addition to the stable association with importin β,
RanQ69L interacts strongly with importin α. Since RanQ69L
disrupts the interaction between importin α and importin β
(Görlich et al., 1996b; Rexach and Blobel, 1995), it is likely
that these interactions represent distinct complexes with RanGTP. Recently, Kutay et al. (1997) have shown that Ran-GTP
and importin α interact via the nuclear export protein, CAS1,
which may account for the interaction that we observe in egg
extracts. Furthermore, although it disrupts the docking of
import complexes, RanQ69L may still interact with the NPC
via specific nucleoporins such as Nup214, perhaps as a
component of other transport complexes.
We have shown that Ran-GDP is required for nuclear
assembly and the initiation of DNA replication, while RanGTP is unable to substitute. This is consistent with an essential
role for nuclear protein import, since Ran-GDP is required for
this process (Görlich et al., 1996b; Weis et al., 1996). It is
perhaps surprising that when Ran-GTP is added, sufficient
We are very grateful to the following for kind gifts of antibodies:
D. Görlich (importins and RanBP7), M. Goldberg (nucleoporins), M.
S. Moore (p10), T. Nishimoto (RanBP2), G. Murphy and M. Rush
(RanBP1) and A. Wittinghofer (RCC1, Ran). We also thank F. R.
Bischoff and A. Wittinghofer for Ran cDNAs, and M. Goldberg and
T. D. Allen for discussion. This work was supported by grants to
P.R.C. from the Royal Society, Cancer Research Campaign (CRC) and
Medical Research Council.
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