From the
RCC1 is an abundant, highly conserved, chromatin-associated
protein whose function is necessary for the preservation of a properly
ordered cell cycle. RCC1 is also necessary for numerous nuclear
processes, including nuclear transport and RNA metabolism; and it
functions enzymatically as a guanine nucleotide exchange factor for a
small, ras-related GTPase called Ran. Studies in several
organisms suggest that RCC1 may be part of a large complex containing
multiple proteins. There is also evidence that RCC1 associates with
chromatin through other proteins and that the binding of the complex to
chromatin varies within the cell cycle. In order to characterize this
putative complex, we have identified a number of other proteins as
candidate components of the complex by their association with a
GST-RCC1 fusion protein. Three of these proteins have previously been
identified (Ran, RanBP1, and hsc70). The fourth protein is novel and
has a molecular mass of 340 kDa. In this report, we discuss a
preliminary characterization of the interactions between these
proteins.
The RCC1 protein is important for maintaining the spatial and
temporal order of the eukaryotic nucleus
(1) . This protein is
required for many nuclear functions including RNA transcription and
processing, DNA replication, nuclear transport, and cell cycle control
(1) . RCC1 is a chromatin-bound protein that acts as a guanine
nucleotide exchange factor for Ran
(2, 3) . Ran is a
small, abundant ras-related GTPase primarily localized in the
nucleus. It has recently been shown that Ran is required for a similar
spectrum of nuclear activities and that it has an essential role in
nuclear transport
(4) . It is currently unknown how the activity
of RCC1 is regulated or whether interactions with other nuclear
components facilitate its proper localization or control. We have
therefore been interested in discovering and characterizing proteins
that interact with RCC1 and that may serve to regulate its distribution
or activity.
It was originally thought that RCC1 binds to DNA
directly, since it both binds to chromatin in vivo and to DNA
cellulose in vitro(5) . However, more recent evidence
indicates that RCC1 associates to chromatin through interactions with
other proteins. RCC1 mutants that lack the ability to bind to DNA are
still able to associate with chromatin in mammalian cells
(6) .
Studies on the Saccharomyces cerevisiae homologue of RCC1,
PRP20, suggest that it binds poorly to DNA on its own, and that its
association to chromatin is facilitated through the formation of a high
molecular weight complex, containing at least three GTP-binding
proteins
(7) . Moreover, the binding of this complex to
chromatin appears to vary within the cell cycle
(7) . A limited
set of proteins is already known to interact with RCC1. Bischoff and
Ponstingl
(3) demonstrated that Ran interacts with RCC1
directly. More recently, it has been demonstrated that RCC1, Ran, and
RanBP1 form a heterotrimeric complex. RanBP1 binds to GTP-Ran
(8) , and it is a cofactor for the Ran GTPase-activating
protein, RanGAP1
(9, 10) . The heterotrimeric complex
probably contains Ran in a nucleotide-free state, and it dissociates
with the addition of GTP but not GDP
(9) . However, there is no
evidence to suggest that the formation of this heterotrimeric complex
is regulated, nor that it is responsible for the localization of RCC1
to chromatin.
To address these questions, we sought to identify
members of a putative RCC1-containing complex via their association
with a glutathione S-transferase (GST)
To characterize proteins that might control RCC1's
localization or activity, we sought to identify proteins that associate
with RCC1 in Xenopus egg extracts. This was done through their
association with a GST-RCC1 fusion protein
(15) bound to
glutathione-Sepharose beads or through their immunoprecipitation from
Xenopus extracts with anti-RCC1 antibodies bound to protein
A-Sepharose beads. Among the proteins associated with GST-RCC1 on
glutathione-Sepharose beads, we consist-ently found a 320-360-kDa
protein to be specifically bound to RCC1 (Fig. 1 A). A
large amount of the Ran protein is also retained in association with
GST-RCC1 under the same conditions, as demonstrated by Western blot
analysis (Fig. 1 B). We estimate that the high molecular
mass RCC1-binding protein has an approximate molecular mass of 340 kDa
(Fig. 1 C). This protein will therefore be called p340
throughout this report. A protein with the same mobility on SDS-PAGE
was also often observed in immunoprecipitations using anti-RCC1
antibodies, but the amount of this protein was somewhat variable
between immunoprecipitations (data not shown). Additionally, we
assessed binding to a number of other GST fusion proteins, including
GST-Ran, GST-furin, GST-Grb2, GST-cyclin B, and the GST moiety alone.
p340 bound to GST-Ran under the same conditions that allowed its
binding to GST-RCC1 (see Fig. 5). However, none of the other GST
proteins bound detectable amounts of p340 (Fig. 1 A, data
not shown). These controls demonstrated that p340 does not bind
nonspecifically to the GST moiety of the fusion proteins, nor does it
bind promiscuously to all bacterially expressed GST fusion proteins.
The association of hsc70 with GST-RCC1 is intriguing,
but its functional significance is not yet clear. Although the GST-RCC1
protein appears to be both completely soluble and functional, it is
clearly possible that hsc70 could be recognizing improperly folded
regions of the bacterially expressed GST-RCC1 and binding to those
regions as a molecular chaperone. However, there are two observations
that may suggest that this interaction has functional significance.
First, we had previously found that hsc70 was the major egg extract
protein that bound to RCC1 covalently coupled to Sepharose
beads.
We wished to determine whether the
endogenous RCC1 in the egg extract might be part of a complex whose
properties are consistent with our observations on GST-RCC1. We
therefore examined the endogenous protein's behavior by gel
filtration chromatography (Fig. 3). We found that Xenopus RCC1 chromatographed as a high molecular mass complex
(400-600 kDa) on a Sephacryl S200 column when the experiment was
performed in the presence of EDTA. In the presence of 2.5 mM
magnesium, RCC1 was found in at least two peaks, one of a similar
molecular mass to that seen in the presence of EDTA, the other being
somewhat smaller (approximately 150 kDa). Bacterially expressed RCC1
protein behaved as a monomer when chromatographed on the same gel
filtration column in the absence of Xenopus extracts (data not
shown). These results strongly indicated that other proteins associate
with RCC1 in Xenopus egg extracts. Furthermore, RCC1
sedimented as a high molecular mass peak on 5-20% sucrose
gradients (data not shown). Some Ran and hsc70 also chromatographed in
the high molecular mass fractions, in amounts consistent with them
being a component of the complex containing RCC1 (data not shown).
However, the distribution of Ran and hsc70 throughout many fractions
from the gel filtration column made it difficult to identify discrete
complexes containing them. We could not determine the distribution of
p340, because we do not yet have antibodies that recognize it. The
behavior of the endogenous RCC1, Ran and hsc70 is therefore consistent
with our observations on GST-RCC1 and its associated proteins.
We have noted that
p340 behaves differently from the other two major proteins that we find
associated with GST-RCC1, hsc70 and Ran. Ran and hsc70 were only
partially inhibited in their binding by the addition of GTP
In summary, we have found that
the RCC1 protein interacts with a number of other proteins in
Xenopus egg extracts, possibly as a large, multi-protein
complex. These interactions appear to be regulated by the guanine
nucleotide binding state of Ran or some other GTP-binding protein. We
have identified a number of proteins as candidate members of this
complex: Ran, hsc70, and p340. RanBP1 may also be a member of the
complex. The p340 protein is particularly intriguing, since it
associates with Ran in an RCC1-independent fashion, and may also
associate with RCC1 in a Ran-independent manner. p340 is therefore an
excellent candidate for a molecule that could modulate the activity of
the RCC1/Ran system in response to DNA or to the state of the cell
cycle. We are currently in the process of investigating the
interactions between p340 and RCC1 further.
We thank Dr. Mark Rush for his kind gift of the
anti-RanBP1 antibodies and for useful comments on the manuscript. We
thank Dr. Lawrence Samelson and Dr. Juan Bonafacio for the kind gifts
of the GST-Grb2 and GST-furin expression plasmids, respectively.
Finally, we also thank Drs. Julie Donaldson, Richard Klausner, Kathleen
Steinmann, and Alan Wolffe for useful discussions and comments on this
manuscript.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-RCC1
(
)
fusion protein (GST-RCC1) in Xenopus laevis egg
extracts. In this study, we show that at least four proteins bound
specifically to GST-RCC1 in extracts. Consistent with the behavior of
endogenous RCC1-containing complexes, magnesium and guanine nucleotides
released these proteins from their association to GST-RCC1 protein.
Three of these proteins have previously been identified: Ran, RanBP1,
hsc70. The fourth protein has an apparent molecular mass of 340 kDa and
has not been previously described in its association with RCC1. This
protein is an attractive candidate for a regulator of the RCC1/Ran
system in Xenopus. We have made a preliminary characterization
of the interactions between these proteins. We report evidence that Ran
can interact with p340 in a manner that is RCC1-independent and
demonstrate that different interactions show differential sensitivities
to guanine nucleotide analogs.
Buffers and Reagents
Sephacryl S200 and
glutathione-Sepharose resins were obtained from Pharmacia Biotech Inc.
Glutathione-Sepharose beads were prewashed with Buffer C before all
experiments. In all figures, proteins were analyzed on 4-20%
SDS-polyacrylamide gradient gels (Novex) unless otherwise stated.
Buffer A is 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.5
mM dithiothreitol, 2 mM EDTA. Buffer B is 20
mM Tris-HCl, pH 8.0, 50 mM NaCl, 0.5 mM
dithiothreitol, 2.5 mM MgCl. Buffer C is 20
mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA,
0.1% Triton X-100, 10% glycerol. Buffer D is 20 mM Tris-HCl,
pH 8.0, 50 mM NaCl, 2.5 mM MgCl
, 0.1%
Triton X-100, 10% glycerol.
Preparation of Xenopus Egg Extracts
Fractionated
interphase egg extracts were prepared according to the methods
described by Smythe and Newport
(11) . These experiments were
conducted exclusively using the membrane-free soluble portion of
fractionated extracts, produced after a high speed centrifugation of
the crude egg lysate. The RCC1-depleted and mock-depleted extracts used
in Fig. 5were prepared as described previously
(12) .
Figure 5:
p340 associates with GST-Ran in an
RCC1-independent manner. A, mock-treated ( lane 1) and
RCC1-depleted ( lane 2) extracts were made by incubation of
clarified egg cytosol with either nonspecific IgG or affinity-purified
anti-RCC1 antibodies bound to protein A-Sepharose beads, respectively
(12). 150 µl of each extract were incubated with 200 µg of the
GST fusion proteins for 10 min at room temperature. 1.2 ml of ice-cold
Buffer C and 20 µl of glutathione-Sepharose beads were added to the
reaction mixture. Incubation was continued for 2 h at 4 °C with
gentle shaking. The glutathione-Sepharose beads and proteins associated
with them were pelleted by low speed centrifugation. The beads were
washed three times with Buffer C, and the proteins bound to the beads
were eluted with 20 µl of SDS-sample buffer, electrophoresed on an
8% SDS-PAGE gel, and visualized by Coomassie Brilliant Blue staining.
The region corresponding to p340 is shown. Lane 3 ( Control) shows the p340 bound to GST-RCC1 instead of
GST-Ran in a parallel incubation using a mock-depleted extract.
B, mock-depleted ( lane 1) and RCC1-depleted ( lane
2) extracts used in A were subjected to Western blot
analysis using anti-RCC1 antibodies as a probe. The position of the
RCC1 protein is indicated on the right side of the
panel.
Antibodies
Affinity purified anti- Xenopus RCC1 antibodies were prepared as described previously
(12) . The anti-Ran/TC4 rabbit polyclonal serum was as described
in Kornbluth et al.(13) . Monoclonal anti-heat shock
protein 70 antibodies (clone 3a3) were purchased from Affinity
BioReagents. These monoclonal antibodies recognize both hsp70 and
hsc70, the cellular heat shock cognate protein. The anti-RanBP1 rabbit
polyclonal antibodies were the kind gift of Dr. Mark Rush. Western
blotting was carried out using standard procedures
(14) . All
Western blots were visualized using an LumiGLO chemiluminescence kit,
exactly according to the manufacturer's suggestions (Kirkegaard
& Perry Laboratories).
Fractionation of the RCC1 Complex
50 µl of egg
extract (about 2 mg of protein) were mixed with an equal volume of
Buffer A or Buffer B. This mixture was centrifuged for 10 min in a
refrigerated (4 °C) Eppendorf microcentrifuge at maximum speed. The
supernatant was loaded onto a Sephacryl S200 gel filtration column (1.0
30 cm) which had previously been equilibrated with Buffer A or
B. Column chromatography was carried out at 4 °C at a flow rate of
200 µl/min. 10-µl aliquots of each fraction were separated on
12.5% SDS-PAGE, and transferred to the poly(vinylidene fluoride)
membranes. Detection of RCC1 was carried out by Western blot analysis
by using affinity-purified anti-RCC1 antibodies.
Preparations of RCC1-binding Proteins
The GST-RCC1
and GST-Ran constructs used were as described previously
(15) .
The GST moiety alone was expressed from a pGEX-KG vector without any
insert
(16) . The GST-cyclin B, GST-furin, and GST-Grb2
expression plasmids were constructed as described elsewhere
(17, 18, 19) . GST and all of the GST fusion
proteins were expressed and prepared as described previously
(15) .
Figure 1:
The association of p340
and Ran with GST-RCC1. A, 150 µl of egg extracts were
incubated for 10 min at room temperature with 200 µg of either GST
or GST-RCC1. 1.2 ml of ice-cold Buffer C and 20 µl of
glutathione-Sepharose beads were added into the reaction mixture.
Incubation was continued for 2 h at 4 °C with gentle shaking. The
glutathione-Sepharose beads and proteins associated with them were
pelleted by low speed centrifugation. The beads were washed three times
with Buffer C, and the bound proteins were eluted with 20 µl of SDS
sample buffer. The eluted proteins were analyzed by SDS-PAGE and
visualized by silver staining. Lane 1, GST without extract;
lane 2, GST plus extract; lane 3, GST-RCC1 without
extract; lane 4, GST-RCC1 plus extract; and lane 5, 1
µl of total extract. - and + indicate the absence and
the presence of extract, respectively. Molecular mass standards are
indicated on the left. The positions of p340 and Ran (p25) are
shown on the right. B, samples as shown in A were
subjected to Western analysis using anti-Ran antibodies. Lane
numbers are as in A. C, extract proteins associated with
GST-RCC1 were separated on a 6% SDS-polyacrylamide gel and stained with
Coomassie Blue ( lane 1). -Macroglobulin was
electrophoresed on the same polyacrylamide gel under nonreducing
conditions ( lane 2) or reducing conditions ( lane 3).
Additional protein markers are shown in lane
4.
In the experiment shown in Fig. 1, GST-RCC1 was allowed to
form complexes with extract proteins for 10 min under standard
conditions ( i.e. in the presence of magnesium and nucleotides)
prior to dilution in the EDTA-containing buffer used for the
purification of GST-RCC1 and its associated proteins. Bischoff and
Ponstingl
(9) have demonstrated that EDTA causes Ran's
rate of nucleotide dissociation to increase, and that it stabilizes
complexes between RCC1 and nucleotide-free Ran. These complexes rapidly
dissociate with the addition of magnesium and guanine nucleotides. We
therefore wished to determined whether any proteins bound to GST-RCC1
could be released by the addition of magnesium and guanine nucleotides,
in a manner consistent with nucleotide exchange allowing complex
dissociation. In this experiment, the samples were prepared as in
Fig. 1
, except that after a final wash in magnesium-containing
buffer, we treated the beads with a buffer containing both GTPS
and magnesium. Proteins eluted in this way were visualized by silver
staining after SDS-PAGE (Fig. 2 A). We found that
approximately half of the p340 was released under these conditions, as
well as two other prominent bands with apparent molecular masses of 70
and 25 kDa. None of these proteins was released when nucleotides were
added in the presence of EDTA (data not shown). GTP and GDP also
facilitated the full release of p70 and p25, as judged on Western blots
(data not shown). These results suggest that, although guanine
nucleotide exchange appears to be required for the elution of p70 and
p25, nucleotide hydrolysis is probably not necessary. However, the
pattern of release for p340 was more complex; p340 was also eluted with
buffer containing GTP rather than GTP
S, although the elution was
less complete. GDP did not cause extensive release of bound p340
protein (data not shown). We estimate that GDP was less than 10% as
effective as GTP
S for p340 release. These observations suggest
that nucleotide exchange must occur to allow release of p340. Further,
p340 release is only facilitated by exchange with guanine
trinucleotides, although hydrolysis does not appear to be required for
dissociation of p340 from RCC1.
Figure 2:
Three
proteins dissociate from GST-RCC1 in a nucleotide-dependent manner.
A, 150 µl of egg extract extracts were incubated with 200
µg of either GST ( lanes 2, 3, and 4) or
GST-RCC1 ( lanes 5, 6, and 7) for 10 min at
room temperature. 1.2 ml of ice-cold Buffer C (1 mM EDTA) and
20 µl of glutathione-Sepharose beads were added to this mixture.
Incubation was continued for 2 h at 4 °C with gentle shaking. The
glutathione-Sepharose beads and proteins associated with them were
pelleted by low speed centrifugation. The beads were washed three times
with Buffer C, followed by 300 µl of Buffer D (2.5 mM
MgCl). The beads were then washed with 300 µl of Buffer
D plus 100 µM GTP
S. The eluted proteins from the
final two washes were precipitated by adding 1.2 ml of ice-cold
acetone. The precipitated proteins were resuspended in 20 µl of SDS
sample buffer. Proteins remaining on the beads after the final wash
were eluted with 20 µl of SDS sample buffer. The proteins were
analyzed by SDS-PAGE, loading equal final volumes of the proteins which
were eluted from the beads with magnesium only ( lanes 2 and
5), magnesium and nucleotides ( lanes 3 and
6), and sample buffer ( lanes 4 and 7). The
proteins were visualized by silver staining. B, in order to
determine whether p70 is distinct from GST-RCC1, a sample prepared as
in lane 6 of A was electrophoresed on a SDS-gel of a
different composition (15% separating gel) (20). The proteins were
visualized by silver staining, and the region around 70 kDa is shown.
C, samples were prepared as in A and subjected to
Western blot analysis with anti-hsc70/hsp70 ( upper panel),
anti-RanBP1 ( middle panel), and anti-Ran ( lower
panel) antibodies. In each case, lane numbers correspond
to those in A.
Using an alternative SDS-PAGE system
(20) , we determined that the 70-kDa band was in fact a doublet
and that it can be clearly distinguished from the GST-RCC1 protein
(Fig. 2 B). In Western blotting analysis, p70 co-migrated
with a band that was recognized by antibodies specific for the heat
shock protein hsp70 and its heat shock cognate, hsc70
(Fig. 2 C). p25 co-migrated with a band that was
recognized by anti-Ran antibodies (Fig. 2 C). In order to
identify p340, we purified the protein and subjected it to peptide
analysis.(
)
The sequence thus obtained did not
indicate any significant homology to sequences presently in the Swiss
Protein Sequence Data Base. We therefore believe that p340 is a
previously uncharacterized protein. We are currently in the process of
obtaining cDNA clones of p340 using the information we have obtained
from peptide analysis. We also tested whether RanBP1 associates with
GST-RCC1. By Western blotting analysis, we found that some RanBP1 was
retained with GST-RCC1 (Fig. 2 C). The majority of
retained RanBP1 was eluted with GTP
S. We could not detect any
specific bands in the eluted supernatant that co-migrated with RanBP1
on the silver stained gel (Fig. 2 A), although a
nonspecific band could be observed near the region where RanBP1 was
found on Western blots. This failure to observe a RanBP1 band on
silver-stained gels makes us suspect that either RanBP1 was present in
substoichiometric amounts in a complex with Ran, hsc70, and p340, or
that it interacts with RCC1 as a member of a distinct, less abundant
complex. However, we could not quantitate the amount of RanBP1
associated with GST-RCC1, because of its co-migration with the
nonspecific band and because we do not have purified Xenopus RanBP1 as a standard. Clearly, the number of discrete complexes
that are assembled by different subsets of these proteins remains to be
elucidated.
(
)
The binding of hsc70 to this resin
suggested that hsc70 does not bind to the GST moiety of the fusion
protein. Further, hsc70 does not significantly associate with any other
GST fusion protein we examined except GST-Ran. Second, a number of
reports have indicated a role for hsc70 in nuclear transport
(21, 22, 23) . Given the clear demonstrations
that Ran is essential for nuclear transport, it is worth speculating
that the requirement for hsc70 in nuclear transport reflects its direct
physical interaction with a component(s) of the RCC1/Ran system
(24, 25) .
Figure 3:
RCC1 associates with a high molecular mass
complex in Xenopus extracts. A, egg extracts were
fractionated on a Sephacryl S200 column in the presence of EDTA
( upper panel) or in the presence of magnesium ( lower
panel). Aliquots of each fraction were separated by SDS-PAGE and
subjected to Western analysis using anti-RCC1 antibodies. Numbers above
the panels indicate the elution positions of standards. B, the
amount of RCC1 per lane in A was quantitated using a Molecular
Dynamics densitometer. The results from +EDTA samples (⊡)
and +magnesium samples () are as
indicated.
The
data presented above suggested that guanine nucleotides are capable of
disrupting previously established interactions between RCC1 and other
proteins in semipurified complexes. We also wished to know whether the
addition of exogenous nucleotides to extracts would enhance or inhibit
the primary formation of these protein complexes in the presence of the
full complement of extract proteins. We incubated the extract with
nucleotides for 10 or 30 min prior to the addition of
glutathione-Sepharose beads. The association of p340 to GST-RCC1 was
extremely sensitive to the presence of 100 µM GTPS
(Fig. 4 A), and it was abolished with a 10 min GTP
S
preincubation. These results support the specificity of p340 binding to
GST-RCC1, since a nonspecific association would not be expected to
change so dramatically in response to GTP
S. Under the same
conditions, GST-RCC1's association with Ran was reduced
approximately 50%, while its association to hsc70 was not measurably
decreased (Fig. 4 B). Ran binding was not completely
abolished even when the GST-RCC1 and extracts were incubated with
GTP
S for 30 min prior to the addition of the glutathione-Sepharose
beads. The association of p340, Ran, and hsc70 to GST-RCC1 was not
significantly changed by preincubation of extracts with exogenous GDP
or GTP (Fig. 4 A, data not shown).
Figure 4:
GTPS inhibits the association of p340
and GST-RCC1. A, 150 µl of egg extracts and 200 µg of
GST-RCC1 protein were incubated for 10 min at room temperature without
added nucleotides ( lane 1), with 100 µM added GDP
( lane 2), or with 100 µM added GTP
S
( lane 3). 1.2 ml of ice-cold Buffer C and 20 µl of
glutathione-Sepharose beads were added into the reaction mixture.
Incubation was continued for 2 h at 4 °C with gentle shaking. The
glutathione-Sepharose beads and proteins associated with them were
pelleted by low speed centrifugation. The beads were washed three times
with Buffer C, and the proteins bound to the beads were eluted with 20
µl of SDS sample buffer. Equal volumes of each of the elutions were
separated by SDS-PAGE and visualized by silver staining. The positions
of p340 are indicated. B, samples identical to those in A were Western blotted using anti-Ran, anti-RanBP1, and
anti-hsc70/hsp70 antibodies. Lane numbers are as in
A.
It is noteworthy
that [-
P]GTP-bound Ran protein will bind to
a series of renatured proteins from Chinese hamster ovary cell extracts
that have been electrophoresed on an SDS-gel and blotted to
nitrocellulose
(26) . One of the bands revealed by this
technique was a Ran-binding protein with a molecular mass greater than
200 kDa. We therefore wished to determine whether p340 can bind to Ran
in a manner that is independent of RCC1. To do this, we incubated
GST-Ran fusion protein with extracts lacking RCC1. The association
between GST-Ran and p340 did not require RCC1 (Fig. 5), since
GST-Ran bound p340 even in interphase cytosol that had been over 98%
depleted of RCC1 by incubation with affinity-purified anti-RCC1
antibodies bound to protein A-Sepharose beads
(12) . These
observations suggest either that p340 can bind to both RCC1 and Ran
independently or that RCC1 binds in conjunction with Ran. We believe
that the former possibility is true, because while almost all of the
Ran protein was eluted with GTP
S as shown in Fig. 2, a
substantial fraction of p340 remained associated with GST-RCC1.
Unfortunately, we cannot report conclusively on this question, since we
have not yet been able to achieve a complete depletion of Ran from our
extracts. Since Ran can associate with p340 in an RCC1-independent
fashion, it is possible that it is homologous to the mammalian protein
described by Lounsbury et al.(26) . An interesting
feature of the mammalian protein was that it interacts well with
GTP-bound Ran, but poorly with GDP-bound Ran.
S
(Fig. 4), but both were almost completely eluted from GST-RCC1 by
the addition of magnesium and nucleotides (Fig. 2 C). On
the other hand, p340 was completely inhibited from GST-RCC1 binding by
GTP
S, but it was incompletely eluted by the addition of magnesium
and nucleotides. There are a number of models that could account for
such behavior. We would like to suggest one hypothesis that we are
currently in the process of testing: p340 may interact with GTP-bound
Ran
(26) . After hydrolysis, GDP-bound Ran is released from
p340. When p340 is free of Ran, RCC1 becomes associated with p340. RCC1
subsequently facilitates both nucleotide exchange of Ran and its
transfer in the GTP-bound state to p340. hsc70 may also be involved at
this stage. In the presence of EDTA, the complex would be frozen and
unable to dissociate (Fig. 1 A). The addition of
magnesiumand GTP
S (Fig. 2) allows nucleotide exchange and
releases complexes that are poised to dissociate.
p340
GST
RCC1 complexes without Ran would not dissociate
under the same conditions. This model would also predict that under
conditions where p340 is bound with GTP-Ran and nucleotide hydrolysis
cannot occur, such as in the presence of GTP
S, RCC1 should not
re-associate with p340 after p340 has bound GTP
S-Ran
(Fig. 4). In some ways, this idea is reminiscent of the
interactions proposed by Bischoff et al.(9) for the
association of RCC1, Ran, and RanBP1. They showed that a heterotrimeric
complex of these proteins is formed when Ran is nucleotide free, but
that the complex is dissociated by the addition of magnesium and GTP.
The most notable difference between the behavior of p340 and RanBP1 in
these two scenarios is that our model predicts that p340 may associate
with RCC1 in a Ran-independent manner (Fig. 2 A), while
RanBP1 clearly does not
(9) .
S, guanosine 5`-3- O-(thio)triphosphate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.