1
Biomedical Research Centre, University of Dundee, Level 5, Ninewells Hospital
and Medical School, Dundee, DD1 9SY, UK
2
Department of Cell Biology and Genetics, College of Life Sciences, Peking
University, Beijing 100871, China
*
Present address: ICRF Laboratories, 44 Lincoln's Inn Fields, London, WC2A 3PX,
UK
Author for correspondence (e-mail:
p.clarke{at}icrf.icnet.uk
)
Accepted May 18, 2001
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SUMMARY |
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Key words: Ran, RCC1, Nucleocytoplasmic transport, Xenopus egg extracts
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INTRODUCTION |
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Ran also plays important roles during cell division (Rush et al.,
1996; Sazer,
1996
). In model cell-free
systems made from Xenopus laevis eggs, elevated levels of Ran-GTP
stabilise microtubule asters and promote mitotic spindle formation
(Carazo-Salas et al., 1999
;
Kalab et al., 1999
; Ohba et
al., 1999
; Wilde and Zheng,
1999
; Zhang et al.,
1999
). Ran-GTP is also
required for nuclear envelope assembly in Xenopus egg extracts
(Hetzer et al., 2000
; Zhang
and Clarke, 2000
) and human
somatic cell extracts (Zhang and Clarke,
2001
). Thus, generation of
Ran-GTP is critical for the functions of this protein. In solution, RCC1
catalyses the exchange of GDP to GTP and vice versa with similar efficiency,
whereas Ran has a higher intrinsic affinity for GDP than GTP (Klebe et al.,
1995b
). The ability of RCC1 to
generate Ran-GTP in vivo would therefore depend critically on a high
concentration of free GTP relative to GDP. Alternatively, additional factors
may determine the directionality of the nucleotide exchange reaction catalysed
by RCC1.
Several other proteins have been identified that are involved in
controlling the localisation or nucleotide-bound state of Ran. Ran-binding
protein 1 (RanBP1) is a mainly cytoplasmic protein that contains a nuclear
export signal (Coutavas et al.,
1993; Richards et al.,
1996
). In the dimeric complex,
RanBP1 is highly specific for the GTP-bound form of Ran (Bischoff et al.,
1995
; Coutavas et al.,
1993
; Hayashi et al.,
1995
) and acts as a
co-activator of GTPase activity with RanGAP (Bischoff et al.,
1995
). RanBP1 and related
domains on the nucleoporin RanBP2 (Yokoyama et al.,
1995
) may thereby contribute
to GTP hydrolysis on Ran and dissociation of nuclear export complexes in the
cytoplasm (Bischoff and Görlich,
1997
; Kehlenbach et al.,
1999
). However, RanBP1 is a
highly mobile protein that rapidly shuttles between the cytoplasm and the
nucleus (Plafker and Macara,
2000
). Interestingly, deletion
of the RanBP1 homologous gene sbp1 in the fission yeast,
Schizosaccharomyces pombe, is rescued by mammalian or yeast
Ran-binding domains that are restricted to the nucleus, indicating that RanBP1
does not require cytoplasmic localisation for its primary function in S.
pombe (Novoa et al.,
1999
). Over-production of
human RanBP1 in yeast or a hamster cell line is antagonistic to a function of
RCC1 (Hayashi et al., 1995
).
Similarly, high concentrations of RanBP1 inhibit nuclear assembly
(Nicolás et al.,
1997
; Pu and Dasso,
1997
) and oppose the effects
of RCC1 on microtubule aster and spindle assembly in Xenopus egg
extracts (Carazo-Salas et al.,
1999
; Kalab et al.,
1999
; Zhang et al.,
1999
). In assays using
purified proteins, RanBP1 inhibits the exchange of GTP to GDP on Ran catalysed
by RCC1, with a lesser effect on the exchange of GDP to GTP, and forms a
trimeric complex with Ran and RCC1 in the absence of guanine nucleotides
(Bischoff et al., 1995
).
Nevertheless, the RanBP1 homologue Yrb1p is required for nuclear protein
import and mRNA export in S. cerevisiae (Schlenstedt et al.,
1995
), indicating a role
complementary to the RCC1 homologue Prp20p (Amberg et al.,
1993
) in vivo.
Additional Ran-binding proteins, unrelated in sequence to RanBP1, have been
identified that may regulate the localisation and guanine nucleotide bound
state of Ran. NTF2/p10 (Moore and Blobel,
1994; Paschal and Gerace,
1995
), binds to Ran-GDP
(Paschal et al., 1996
) and
promotes its import into the nucleus (Ribbeck et al.,
1998
; Smith et al.,
1998
). NTF2/p10 also
stabilises Ran-GDP against nucleotide exchange (Yamada et al.,
1998
). Oki and Nishimoto (Oki
and Nishimoto, 1998
) have
shown that, in Saccharomyces cerevisiae, NTF2 and a novel gene
MOG1 suppress the growth defect of temperature-sensitive allelles of
the Ran homologue, gsp1. Deletion of MOG1 made the yeast
temperature sensitive for growth, a defect that was suppressed by
over-expression of NTF2, suggesting a functional interaction between
their products. Although both Ntf2p and Mog1p are required for nuclear protein
import in S. cerevisiae, over-expression of MOG1 does not
rescue ntf2 mutants, indicating that their functions are distinct.
Mog1p, unlike Ntf2p, binds preferentially to Gsp1p-GTP and is localised to the
nucleus when over-expressed (Oki and Nishimoto,
1998
). Stewart and Baker have
recently determined the crystal structure of Mog1p to
1.9
resolution and have provided evidence that
Mog1p interacts with Ran through a site similar to that bound by NTF-2
(Stewart and Baker, 2000
).
S. cerevisiae Mog1p and a related protein from mouse cause the
release of nucleotide from Ran-GTP (Oki and Nishimoto,
2000
; Steggerda and Paschal,
2000
) although this activity
does not appear to be sufficient to explain the biological function of
Mog1-related proteins.
To identify putative regulators and effectors of Ran in a vertebrate
system, we have screened a Xenopus oocyte cDNA library using the
two-hybrid system of interaction in yeast
(Nicolás et al.,
1997). Here, we report the
identification of a Xenopus protein, named XMog1, which has sequence
similarity to S. cerevisiae Mog1p, a related gene product in
Schizosaccharomyces pombe, as well as plant and mammalian homologues.
XMog1 and Mog1p both rescue the growth arrest caused by deletion of the
related gene in S. pombe, showing the conservation of their
functions. XMog1 is localised to the nucleus and interacts preferentially with
Ran-GTP, causing nucleotide release and forming a complex with nucleotide-free
Ran. However, in conjuction with RanBP1, XMog1 promotes the exchange of GDP
for GTP by causing the release of GDP and the selective binding of GTP to Ran.
XMog1 and RanBP1 also promote the selectivity of the guanine nucleotide
exchange reaction on Ran catalysed by RCC1. Thus, XMog1, together with RanBP1,
may facilitate the generation of Ran-GTP in the nucleus.
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MATERIALS AND METHODS |
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Sequencing of Xenopus cDNA clones
Plasmid DNA from positive colonies was amplified by PCR. The sequences were
compared and aligned using the Lasergene program SEQMAN II (DNASTAR Inc,
Madison, WI). DNA from the yeast colony carrying the longest cDNA fragment was
cloned and sequenced in both directions. The 5' end of the XMog1
sequence was extended by PCR amplification from the library to confirm the
presence of upstream stop codons, indicating that the assigned ATG is the
correct initiation codon. The PCR products obtained were cloned in pGEMTeasy
vector (Promega) and all the clones were sequenced in both directions.
S. pombe strains and genetic methods
All S. pombe media were prepared and used as described previously
(Moreno et al., 1991).
mog1 was deleted from the S. pombe strain
h+/h- leu1-32/leu1-32
ade6-M210/ade6-M216 with the use of PCR generated fragments
(Bähler et al.,
1998
), one of the genomic
copies of mog1 being replaced with a KanR
cassette. The strain obtained this way, FJN1
(h+/p- leu1-32/leu1-32
ade6-M210/ade6-M216 mog1/mog1::kanR), was assayed for tetrad
dissection. mog1-related genes from Xenopus, S. pombe and
S. cerevisiae were amplified by PCR and cloned in pREP41X under
control of the nmt1 promoter (Maundrell,
1993
; Maundrell,
1990
).
Production and purification of recombinant proteins
The Xenopus XMog1 cDNA obtained in the two-hybrid screen was
digested with EcoRI and XhoI and cloned into the GST fusion
vector pGEX-4T-1 (Pharmacia). This construct was used to transform E.
coli BL21 and the fusion protein was expressed using the manufacturer's
standard protocols. The fusion protein was purified on glutatione-Sepharose
(Pharmacia), resuspended at approximately 100 µM in 50 mM Tris HCl, pH 8.0,
snap frozen in liquid nitrogen and stored in aliquots at -70°C. Protein
concentrations were determined by the Bradford assay (Biorad) and Lowry method
(Biorad DC protein assay kit).
For removal of the GST moiety, the GST-XMog1 fusion protein bound to
glutathione-Sepharose was digested with thrombin as described by the
manufacturer (Pharmacia). To produce 6xHis-tagged XMog1, the fragment
EcoRI/XhoI was cloned in vector pET28b (Novagen), digested
with EcoRI and XhoI and used to transform E. coli
BL21(DE3). The protein was expressed and purified on NiTa-Sepharose (Qiagen)
according to the manufacturer's guidelines, dialysed against PBS and snap
frozen in liquid nitrogen and stored in aliquots at -70°C.
Xenopus RanBP1 and human Ran were produced as a GST fusion proteins
as described previously (Hughes et al.,
1998). In some cases, the GST
moiety was removed by thrombin cleavage.
Antibodies
Antiserum against Xenopus XMog1 was raised by inoculation of
rabbits (Eurogentec, Belgium) with the recombinant protein produced by
thrombin digestion of the GST fusion. The serum was purified using an affinity
matrix of 6xHis-XMog1 covalently attached to CNBr-Sepharose (Pharmacia)
as described previously (Harlow and Lane,
1999). Antibodies to
Xenopus RanBP1 were raised and purified in a similar way. Antibodies
to Ran and RCC1 were purchased from Transduction Laboratories and used as
described previously (Zhang and Clarke,
2000
).
Co-precipitation assays
Xenopus egg extracts were prepared as 10,000 g
supernatants supplemented with an ATP-regenerating system and cyclohexamide,
as described previously (Hughes et al.,
1998). Binding assays were
carried out by supplementing 50 µl of Xenopus egg extract with
GST-XMog1 or GST-Ran fusion proteins to a concentration of 2 µM (except
where stated). Where specified, nucleotides were added at 2 mM. Binding was
allowed to proceed at 21°C for 30 minutes before diluting to 250 µl
with Mg2+-buffer (20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 2.5 mM
MgCl2, 0.1% Triton X-100, 10% (v/v) glycerol) or EDTA-buffer (20 mM
Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 10% (v/v)
glycerol). GST-tagged proteins were precipitated by addition of 20 µl of a
50% slurry of glutathione-Sepharose beads (Pharmacia) in either
Mg2+-buffer or EDTA-buffer. Samples were then incubated at 4°C
for 1 hour with gentle continuous agitation. The beads were recovered by low
speed centrifugation, washed three times with the appropriate buffer and
eluted by resuspending the beads in SDS-PAGE loading buffer. Proteins were
examined on SDS-PAGE by silver staining or immunoblotting. Binding assays
using purified proteins were carried out in a similar fashion in
Mg2+-buffer.
Cell culture, nuclear assembly and fluorescence microscopy
Xenopus XTC cells were cultured on coverslips in L-15 medium
Leibovitz (Sigma) plus 10% FCS and 25% H2O at room temperature. The
cells were fixed with 4% paraformaldehyde in Tris-buffered saline (TBS) for 20
minutes on ice and permeabilized with 0.5% Triton X-100 in TBS for 5 minutes.
Indirect immunofluorescence labelling was carried out by incubating with the
first antibody overnight at 4°C followed by incubation with an
FITC-conjugated secondary antibody for 45 minutes at room temperature.
For expression of XMog1 fused to C-terminus of the Aequorea green fluorescent protein (GFP), XMog1 was cloned into pGFP-C2 (Clontech). Cos-1, HeLa and 3T3 cells were cultured in DMEM (Gibco) plus 10% FCS, transfected with pGFP-XMog1 using Lipofectamine (Gibco) and allowed to express GFP-XMog1 for 24 hours. Cells were fixed in acetone for 1 minute, washed twice in PBS and then mounted. Nuclei were stained with DAPI. Images were captured using a cooled CCD camera (Hamamatsu) mounted on a Zeiss Axiovert microscope and processed on an Apple Macintosh computer using Improvision Openlab and Adobe PhotoShop software.
Radiolabelled nucleotide assays
Assays were adapted from Dasso et al. (Dasso et al.,
1994). To make up a stock of
protein loaded with radioactive nucleotide sufficient for several assays,
GST-Ran (1 nmol) was incubated with the appropriate nucleotide in 50 µl
loading buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% (v/v) lubrol, 1 mM
MgCl2) containing 6 mM EDTA for 30 minutes at room temperature. The
protein was diluted with 450 µl of cold loading buffer and the bound
nucleotide stabilised with 20 mM MgCl2. In exchange assays, free
nucleotide was added to the loaded Ran. Similar amounts of nucleotides were
used for loading on Ran or added free for exchange: either 0.1 µmol of
unlabelled GDP/GTP (Sigma) or 0.185 MBq [3H]GDP/[3H]GTP
(Amersham) were used for exchange assays, or 0.037 MBq
[
-32P]GTP (Amersham) for GTPase assays. For each assay, 25
µl of loaded Ran was added to an equal volume of reaction mixture to start
the reaction. Reaction mixtures contained RCC1, RanBP1 or XMog1 in exchange
buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1% (v/v) lubrol) to give final
protein concentrations indicated in the figure legends and 10 mM
MgCl2. Reactions were performed at room temperature (21°C). To
stop the reaction, samples were added to 5 ml of stop buffer (20 mM Tris-HCl,
pH 7.5, 25 mM MgCl2, 100 mM NaCl) and immediately filtered through
nitrocellulose (Hybond ECL, Amersham). The filters were washed with a further
20 ml of stop buffer, dried and radioactivity bound to the filters was
determined by scintillation counting. Where experiments were performed in
triplicate, results are shown as mean values with error bars indicating the
standard error of the means.
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RESULTS |
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To assess the Ran-binding properties of XMog1, further two-hybrid analysis
was carried out using wild-type Ran, RanQ69L and RanT24N as baits. The latter
mutant is defective in GTP binding and has a reduced affinity for GDP (Klebe
et al., 1995a). In each case,
the yeast grew on selective (His-) media, indicating that XMog1 was
able to interact with all of the Ran proteins
(Fig. 2). Since both wild-type
and RanQ69L are likely to be predominantly GTP-bound when expressed in yeast
nuclei, whereas RanT24N may be nucleotide-free, these data suggest an
interaction between XMog1 and GTP-bound or nucleotide-free Ran. However, they
do not exclude the possibility of an interaction with Ran-GDP.
|
To test the specificity of the interaction between XMog1 and Ran, we also used several related small GTPases as baits in the two-hybrid system. When Cdc42, Rac and Rho (including mutants locked in the GTP-bound state like RanQ69L or equivalent to RanT24N), were co-transformed with XMog1, all failed to produce strong growth of colonies when plated on selective media (His-). When colonies grown on non-selective media (His+) were tested for ß-galactosidase activity, in all cases the activity was negligible, in contrast to the activity given by the XMog1/Ran interaction, showing that the interaction of XMog1 with Ran is specific (data not shown).
The related gene mog1 is essential for viability in S.
pombe
To compare the function of XMog1 with the product of S. cerevisiae
MOG1 and the related gene in S. pombe, we carried out a
complementation analysis in S. pombe. A diploid S. pombe
strain was created in which one of the genomic copies of the mog1
gene was replaced by insertion of a KanR cassette. By
tetrad analysis, in which haploid spores are germinated following dissection
of asci, we consistently obtained germination and growth of two colonies from
four spores (Fig. 3A). This is
consistent with the failure of haploid cells lacking mog1
(mog1) to grow. Microscopic examination of the dissected
spores after germination revealed that the
mog1 spores did
germinate to produce a few cells that then failed to divide further (data not
shown). Together, these results indicate that mog1 is not required
for germination but is essential for continued cell viability in S.
pombe. In S. cerevisiae, deletion of MOG1 produces a
temperature-sensitive growth defect, whereby cells grow at 26°C but not at
34°C (Oki and Nishimoto,
1998
). However, growth of
S. pombe
mog1 cells was not rescued at any
temperature in the range of 18°C to 36°C (data not shown).
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Deletion of Mog1 in S. pombe is complemented by
XMog1 or S. cerevisiae MOG1
The S. pombe strain diploid heterozygous for mog1 was
transformed with the empty vector or plasmids that expressed XMog1, S.
pombe Mog1p or S. cerevisiae Mog1p. One colony from each
transformation was allowed to sporulate and the resulting spores were
germinated. Approximately 50 colonies from each transformation were
transferred to media with or without thiamine (50 µg/ml). Thiamine
represses the nmt1 promoter, blocking the production of
plasmid-encoded Mog1 protein. None of the colonies derived from the
transformation with the empty vector showed retardation when plated on media
with thiamine nor the KanR phenotype. However,
transformation with the other plasmids resulted in approximately 50% of the
colonies showing growth retardation when plated on media with thiamine. After
a few rounds of plate selection, growth retardation became evident
(Fig. 3B). Colonies that still
showed growth retardation in media with thiamine had a
KanR phenotype, indicating they were mog1
(this was confirmed by PCR analysis; data not shown). Thus, expression of
S. pombe Mog1p, S. cerevisiae Mog1p or Xenopus
XMog1 rescues the lethal phenotype of
mog1 cells
(Fig. 3B). Together, these data
demonstrate a remarkable conservation of the function of Mog1-related proteins
between yeasts and vertebrates.
Nuclear localisation of XMog1
A polyclonal antibody raised against XMog1 and affinity purified against
the recombinant protein recognised a single major polypeptide in
Xenopus egg and somatic Xenopus XTC cell extracts that
migrated on SDS-PAGE with an apparent molecular mass of 34 kDa, identical to
the migration of recombinant XMog1 protein
(Fig. 4A). XMog1, which has a
calculated molecular mass of 20.4 kDa, therefore migrates aberrantly on
SDS-PAGE in a similar fashion to S. cerevisiae Mog1p (Stewart and
Baker, 2000). Indirect
immunofluorescence of Xenopus somatic XTC cells using this antibody
revealed a predominantly nuclear localisation of XMog1, with exclusion from
nucleoli (Fig. 4B). Consistent
with this finding, XMog1 expressed as a fusion with green fluorescent protein
(GFP) showed a nuclear localisation in three different mammalian cell lines
(Fig. 4C).
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XMog1 is a Ran-binding protein
In solution binding assays, XMog1 interacted directly with Ran, binding
strongly to wild-type Ran loaded with GTP and more weakly with Ran-GDP
(Fig. 5A). In the presence of
EDTA, which chelates Mg2+ ions required for nucleotide binding to
Ran, a strong association was also seen, indicating that XMog1 also forms a
complex with nucleotide-free Ran. XMog1 interacted strongly with both
RanQ69L-GTP, which carries a mutation in the switch II region, and RanT24N,
which is deficient in nucleotide binding
(Fig. 5B). Similar interactions
were seen using either GST-tagged Ran and untagged XMog1
(Fig. 5A,B) or untagged Ran and
GST-tagged XMog1 (Fig. 5C; data
not shown), confirming that the GST moiety does not affect the binding
properties of XMog1 in this assay. Consistent with these results and the
two-hybrid analysis in yeast, XMog1 present in Xenopus egg extracts
interacted more strongly with Ran loaded with GTP than with the GDP-bound
form, but also interacted strongly with Ran under conditions that promote the
formation of nucleotide-free Ran (Fig.
5B,C).
|
In the absence of other proteins, XMog1 selectively releases GTP from
Ran
To investigate the biochemical function of XMog1 further, we examined the
effect of XMog1 on the binding of guanine nucleotides to Ran. Initially, we
examined the effect on nucleotide exchange by observing the loss of Ran-bound
[3H]GTP when incubated with free GDP
(Fig. 6A). Under these
conditions, XMog1 promoted loss of radioactivity in a concentration-dependent
manner when Ran was preloaded with [3H]GTP, but not
[3H]GDP (Fig. 6A).
Significant release of radioactivity was observed when the amount of XMog1 in
the assay was similar or greater than that of Ran (50 pmol). However, XMog1
also caused release of [3H]GTP from Ran in a similar experiment
performed without free nucleotide, which is essential for the nucleotide
exchange reactions catalysed by RCC1 or EDTA
(Fig. 6B). Thus, in the absence
of other proteins, XMog1, like S. cerevisiae Mog1p (Oki and
Nishimoto, 2000) and mouse
Mog1 (Steggerda and Paschal,
2000
), is a GTP release factor
for Ran rather than a guanine nucleotide exchange factor.
|
The concentration dependence of the effect of XMog1 on the loss of
[3H]GTP was similar using wild-type Ran or the Q69L mutant, which
is deficient in GTPase activity (Fig.
6B). The rate of loss of radioactivity was also identical for Ran
preloaded with [-32P]GTP, rather than [3H]GTP
(data not shown). These results indicated that XMog1 causes loss of GTP from
Ran without stimulation of GTP hydrolysis. They also showed that the
interaction of XMog1 with Ran is not disrupted by the Q69L mutation,
consistent with the analysis of their interactions by two-hybrid analysis
(Fig. 6A) and co-precipitation
experiments (Fig. 6B). The
release of [3H]-GTP from Ran by XMog1 was stimulated by chelation
of Mg2+ by EDTA (Fig.
6C), although EDTA alone did not cause [3H]GTP release
under these conditions (data not shown). Taken together with the data showing
an affinity of XMog1 for Ran under nucleotide-free conditions
(Fig. 5), these results suggest
that XMog1 interacts with Ran-GTP, causes loss of nucleotide and forms a
complex with nucleotide-free Ran.
Interaction with Ran-binding protein 1 (RanBP1)
Ran interacts strongly with a family of binding proteins related to RanBP1
(Avis and Clarke, 1996).
Although RanBP1 and XMog1 are unrelated in primary sequence, RanBP1 also
interacts strongly with Ran-GTP, including the Q69L mutant
(Nicolás et al.,
1997
). To examine the
relationship between the interactions of RanBP1 and XMog1 with Ran, we
determined the effect of XMog1 on the binding of RanBP1 to Ran. GST-RanBP1
interacted more strongly with Ran preloaded with GTP
S rather than GDP,
as expected. However, addition of XMog1 reduced the interaction between
GST-RanBP1 and Ran-GTP
S. By contrast, XMog1 stabilised the otherwise
weak interaction between RanBP1 and Ran-GDP
(Fig. 7A). Although no direct
interaction between XMog1 and RanBP1 occurred (data not shown), XMog1
precipitated with GST-RanBP1 in the presence of Ran preloaded with GTP
S
or GDP (Fig. 7A). GST-XMog1
also precipitated both RanBP1 and Ran from Xenopus egg extracts (data
not shown). Thus, GST-XMog1, Ran and RanBP1 can form a complex, suggesting
that XMog1 and RanBP1 can interact with different regions of Ran.
|
XMog1 promotes the release of GDP from Ran and the selective binding
of GTP when RanBP1 is present
The ability of XMog1 and RanBP1 to form a complex with Ran simultaneously
(Fig. 7A) prompted us to
examine the combined effects of these proteins on guanine nucleotide binding
to Ran. RanBP1 inhibited the release of [3H]GTP from Ran induced by
XMog1 unless high concentrations of XMog1 were added
(Fig. 7B). XMog1 or RanBP1
alone did not cause the release of [3H]GDP from Ran. However, when
added together, XMog1 and RanBP1 caused a strong stimulation of GDP release,
with >50% of the nucleotide released after 30 minutes
(Fig. 7C), a rate comparable
with the release of GTP by XMog1 alone
(Fig. 6B). These experiments
were performed without the addition of free nucleotides, so the effect of
XMog1 and RanBP1 on Ran-GDP was not due to nucleotide exchange, but rather the
release of nucleotides to form nucleotide-free Ran. RanBP1 has a strong
affinity for Ran-GTP, but also interacts more weakly with nucleotide-free Ran
(Bischoff et al., 1995;
Nicolás et al.,
1997
). Thus, generation of
nucleotide-free Ran by a high concentration of XMog1 may explain why the
binding between RanBP1 and Ran loaded with GTP
S is decreased, whereas
the interaction with Ran loaded with GDP is increased
(Fig. 7A).
When Ran-GDP was incubated in the presence of [3H]GTP (1.2 µM) and a large excess of GDP (700 µM), the combination of XMog1 and RanBP1 promoted the uptake of [3H]GTP by Ran (Fig. 7D). Under these conditions, free exchange catalysed by RCC1 or EDTA did not cause any detectable loading of Ran with [3H]GTP (data not shown). Thus, the combination of XMog1 and RanBP1 loads Ran with GTP preferentially, unlike the nucleotide exchange reaction catalysed by RCC1 or EDTA.
Effects of XMog1 and RanBP1 on the nucleotide exchange activity of
RCC1
In the nucleus, guanine nucleotide exchange on Ran is catalysed by RCC1. We
therefore examined the effects of XMog1 and RanBP1 on the activity of RCC1
assayed by release of [3H]GDP from Ran
(Fig. 8), or by the uptake of
[3H]GDP (Fig. 9A,B)
or [3H]-GTP (Fig.
9C).
|
|
XMog1 alone had no inhibitory effect on nucleotide exchange on Ran-GDP
catalysed by RCC1 in the presence of free GDP
(Fig. 8A). By contrast, RanBP1
strongly inhibited [3H]GDP release catalysed by RCC1 under these
conditions (i.e. GDP to GDP exchange), as reported previously (Bischoff et
al., 1995). XMog1 partially
overcame the inhibitory effect of RanBP1 on [3H]GDP release
(Fig. 8A); this effect may
represent the release of [3H]GDP by XMog1 and RanBP1 to form
nucleotide free-Ran (Fig. 7C).
In the presence of free GTP, the inhibitory effect of RanBP1 on release of
[3H]GDP by RCC1 (i.e. exchange of GDP to GTP) was much less
pronounced (Fig. 8B; Bischoff
et al., 1995
). XMog1 overcame
the small inhibition by RanBP1, and when added alone, XMog1 stimulated the
rate of [3H]GDP release by RCC1
(Fig. 8B).
In the reverse reaction, XMog1 partially inhibited the exchange of GTP to GDP catalysed by RCC1, assayed by the loading of RanGTP with [3H]GDP (Fig. 9A). RanBP1 also inhibited exchange of GDP for GTP, blocking [3H]GDP uptake; this inhibition by RanBP1 was not competed by XMog1 (Fig. 9B). By contrast, XMog1 and RanBP1 promoted loading of Ran-GDP with [3H]GTP by RCC1, the predominant effect being due to RanBP1 (Fig. 9C). Under these conditions, where [3H]GTP concentration (1.2 µM) was low relative to GDP (700 µM), RCC1 alone failed to load Ran with [3H]GTP, whereas XMog1 and RanBP1 caused a modest loading with [3H]GTP in the absence of RCC1, as before (Figs 7D, 9C). Thus, XMog1 and RanBP1 promote the exchange of GDP to GTP by RCC1 against an unfavourable gradient in nucleotide concentrations.
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DISCUSSION |
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Reduction of Ran-GTP levels by Mog1-related proteins would seem to be
inconsistent with the requirement of Mog1p for nuclear protein import in
S. cerevisiae (Oki and Nishimoto,
1998), because this process
also requires the Ran homologue Gsp1p (Oki et al.,
1998
) and is likely to involve
the maintenance of high levels of Gsp1p-GTP in the nucleus, where Mog1p is
predominantly localised. Furthermore, high levels of Mog1p do not inhibit the
growth of S. cerevisiae (Oki and Nishimoto,
2000
), which might be expected
if nuclear Ran-GTP were depleted. It seems unlikely, therefore, that induction
of GTP release from Ran in the nucleus and formation of a stable
nucleotide-free complex could account for the principal biological activity of
Mog1-related proteins.
In the cellular environment, the interaction of XMog1 with Ran is likely to
be influenced by other proteins that interact with Ran. A major Ran-GTP
binding protein is RanBP1, which forms a trimeric complex with Ran and XMog1
in Xenopus egg extracts, and inhibits GTP release from Ran.
Similarly, RanBP1 homologues inhibit GTP release induced by the mouse
(Steggerda and Paschal, 2000)
and yeast (Oki and Nishimoto,
2000
) Mog1 proteins. However,
in contrast to the effects of XMog1 alone, we find that when RanBP1 is
present, XMog1 causes the release of GDP from Ran and the binding of GTP. In
the presence of RCC1, RanBP1 and XMog1 strongly promote loading of Ran with
GTP while inhibiting the exchange of GTP to GDP. XMog1 and RCC1 are both
localised to the nucleus during interphase, but RanBP1 has been described as a
predominantly cytoplasmic protein. However, it is now clear that RanBP1 in
fact shuttles rapidly between nucleus and cytoplasm in organisms as diverse as
S. cerevisiae (Kunzler et al.,
2000
), mammals (Plafker and
Macara, 2000
) and
Xenopus (F.J.N., C.Z. and P.R.C., unpublished). Therefore, XMog1,
RanBP1 and RCC1 could all interact with Ran in the nucleus.
When Ran is imported into the nucleus, it is likely to be initially in the
GDP-bound state. XMog1 may displace the import factor NTF2 from Ran-GDP
(Stewart and Baker, 2000),
although XMog1 does not form a stable complex with Ran-GDP, but rather
increases the affinity for RanBP1 and promotes the loss of nucleotide. Thus,
XMog1 does not behave like a bona fide guanine nucleotide exchange factor
(GEF) such as RCC1, which acts catalytically by increasing the rate at which
equilibrium between GDP- and GTP-bound states is achieved (Klebe et al.,
1995b
), but rather acts
stoichiometrically by destabilising guanine nucleotide binding. A similar
mechanism has been suggested for another GTPase-interacting protein, Mss4,
which forms a complex with nucleotide-free Rab proteins (Geyer and
Wittinghofer, 1997
; Nuoffer et
al., 1997
). RanBP1 favours the
binding of GTP to the nucleotide-free complex between Ran and XMog1 by forming
a strong association with Ran-GTP, thereby effectively removing Ran-GTP from
the equilibrium reaction. Thus, the combination of a nucleotide-destabilising
factor (XMog1) and a Ran-binding protein (RanBP1) that interacts with
nucleotide-free Ran but has a higher affinity for Ran-GTP can produce a
nucleotide-exchange reaction, albeit much less efficiently than RCC1.
RCC1 interacts with Ran-GDP and Ran-GTP with similar affinities, catalysing
the exchange of GDP to GTP and vice versa at similar rates (Klebe et al.,
1995b). Although XMog1 and
RanBP1 inhibit GDP binding catalysed by RCC1, they promote the accumulation of
RanGTP. At higher activities of RCC1, the predominant effect is due to RanBP1,
probably by stabilisation of Ran-GTP and inhibition of the reverse reaction in
which GTP is exchanged to GDP (Bischoff et al.,
1995
). Thus, XMog1 and RanBP1
act as co-factors that ensure that the direction of the exchange reaction
catalysed by RCC1 promotes the generation of Ran-GTP
(Fig. 10). These activities of
XMog1 and RanBP1 may be important in the nucleus, where RCC1 has to work
against an unfavourably high concentration of Ran-GTP relative to that of
Ran-GDP. XMog1 and RanBP1 would also ensure that the generation of Ran-GTP by
RCC1 is not critically dependent on a high concentration of free GTP relative
to GDP.
|
In this way, the generation of Ran-GTP, critical for maintenance of nuclear structure and function, may be regulated by the interaction of Ran with XMog1 and RanBP1. Nucleocytoplasmic shuttling could provide a mechanism to precisely control the level of RanBP1 in the nucleus, perhaps coupling the activity of RCC1 and the generation of Ran-GTP to the rate of nucleocytoplasmic transport. If the nuclear Ran-GTP concentration was too high or RanBP1 levels declined, perhaps owing to over-active nuclear export, XMog1 could also act as a sink for Ran, sequestering the protein in an inactive, nucleotide-free form. These possibilities may be tested by examining the effects on nuclear function of altering the relative levels of these proteins and disrupting their interactions by mutation.
Could the ability of XMog1 and RanBP1 to promote generation of Ran-GTP in
the nucleus explain the phenotype of the deletion of MOG1 in S.
cerevisiae? MOG1 is required for nuclear protein import and
MOG1 deletion makes the yeast temperature-sensitive. Growth at the
restrictive temperature can be rescued by overexpression of the Ran homologue
Gsp1p or Ntf2p (Oki and Nishimoto,
1998). Increased expression of
Gsp1p or promotion of Gsp1p import into the nucleus by expression of Ntf2p may
both compensate for the loss of Mog1p by increasing the total level of Ran,
and therefore Ran-GTP, in the nucleus. To test this possibility, it will be of
interest to examine the genetic interactions between Mog1, RanBP1 and RCC1
homologues in yeast. It also remains possible that Mog1-related proteins have
a more direct function in nuclear import by dissociating import complexes
between Gsp1p/Ran-GDP, importins/karyopherins and their cargoes in
nucleus.
As well as its role during interphase, when maintenance of a high
concentration of Ran-GTP is required to distinguish the environment of the
nucleus from the cytoplasm and thereby determine the direction of
nucleocytoplasmic transport, generation of Ran-GTP plays crucial roles during
cell division. Evidence from Xenopus egg extracts (Carazo-Salas et
al., 1999; Kalab et al.,
1999
; Ohba et al.,
1999
; Wilde and Zheng,
1999
; Zhang et al.,
1999
) indicates that localised
generation of Ran-GTP is likely to play a critical role in centrosomal
nucleation of microtubules and organisation of the mitotic spindle. In
addition, generation of Ran-GTP on the surface of chromatin is critical for
nuclear envelope assembly at the end of mitosis (Hetzer et al.,
2000
; Zhang and Clarke,
2000
; Zhang and Clarke,
2001
). It is possible that
Mog1-related proteins also play roles in these processes.
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ACKNOWLEDGMENTS |
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