©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The RCC1 Protein Interacts with Ran, RanBP1, hsc70, and a 340-kDa Protein in Xenopus Extracts (*)

Hisato Saitoh , Mary Dasso (§)

From the (1) Laboratory of Molecular Embryology, NICHD, National Institutes of Health, Bethesda, Maryland 20892-5430

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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)-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.


MATERIALS AND METHODS

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) .


RESULTS AND DISCUSSION

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.


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 GTPS, 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 GTPS 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 GTPS. 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 GTPS. 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 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.() 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) .

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.


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 GTPS 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 GTPS. 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 GTPS 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 GTPS ( 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 GTPS 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.

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 GTPS (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 GTPS, 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 GTPS (Fig. 2) allows nucleotide exchange and releases complexes that are poised to dissociate. p340GSTRCC1 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 GTPS, RCC1 should not re-associate with p340 after p340 has bound GTPS-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) .

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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Laboratory of Molecular Embryology, NICHD, NIH, Bldg. 18, Rm. 101, Bethesda, MD 20892-5430. Tel.: 301-402-1555; Fax: 301-402-0078.

The abbreviations used are: GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; GTPS, guanosine 5`-3- O-(thio)triphosphate.

W. Burgess and H. Saitoh, unpublished results.

M. Dasso and J. W. Newport, unpublished observations.


ACKNOWLEDGEMENTS

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.


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