©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The C Terminus of the Nuclear RAN/TC4 GTPase Stabilizes the GDP-bound State and Mediates Interactions with RCC1, RAN-GAP, and HTF9A/RANBP1 (*)

Stephanie A. Richards (1), Karen M. Lounsbury (§) , Ian G. Macara

From the (1)Department of Pathology, University of Vermont Medical College, Burlington, Vermont 05405-0068

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ran/TC4 is a member of the Ras superfamily of GTPases. It is unusual in being predominantly nuclear and because it possesses an acidic -DEDDDL sequence instead of a consensus prenylation domain at the C terminus. Ran is required for nuclear protein import and cell cycle progression, and has been implicated in mRNA processing and export and DNA replication. The inhibition of cell cycle progression by a dominant gain-of-function mutant of Ran has been shown to be abrogated by removal of the -DEDDDL sequence, suggesting that this domain is essential for Ran function. We demonstrate here that the -DEDDDL sequence stabilizes GDP binding to Ran, and that the domain is required for high affinity interaction with a Ran-binding protein, HTF9A/RanBP1. HTF9A functions as a co-stimulator of Ran-GAP (GTPase activating protein) activity on wild-type Ran, but in the absence of the acidic C terminus of Ran, HTF9A behaves as a Ran-GAP inhibitor. An antibody directed against the C-terminal region preferentially recognizes the GTP-bound form of Ran, suggesting that this domain undergoes a nucleotide-dependent conformational change. The results suggest that the acidic C-terminal domain is important in modulating the interaction of Ran with regulatory factors, and implicate Ran-binding proteins in mediating the effects of Ran on cell cycle progression.


INTRODUCTION

The Ran/TC4 GTPase, referred to here as Ran, is a member of the Ras superfamily of small guanine nucleotide-binding proteins(1) . Ran is unique in this family in that it is largely localized to the cell nucleus and lacks a consensus site for post-translational prenylation (2). It also has an unusually acidic C-terminal tail (-DEDDDL). Ran homologs have been found in all eukaryotic organisms examined to date, including Spi1 in Schizosaccharomyces pombe(3) , RAN1, RAN2A, and RAN2B in the tomato Lycopersicon esculentum(4) , and GSP1 and GSP2 in Saccharomyces cerevisiae(5) . GSP1 is essential for viability in S. cerevisiae, and the GSP1 gene disruption can be complemented by mammalian Ran (5). Amino acid comparisons show a very high level of sequence conservation across species, and all Ran homologs possess an acidic C-terminal domain.

Ran has been implicated in a number of essential cellular processes including nuclear protein import through the nuclear pore complex (6-8), DNA replication and cell cycle control(9, 10, 11) , nuclear growth (9), and mRNA processing and export(12, 13, 14) . It is possible that the multiplicity of effects induced by loss of Ran function, or by expression of dominant mutants of Ran, may reflect a single primary disruption in an essential process such as nuclear protein import, but this hypothesis remains unproven.

The high degree of conservation of Ran sequences through evolution and the essential nature of its functions indicate that the Ran GTPase cycle is likely to be stringently regulated and that Ran may interact with many other factors. Thus far, a nuclear, Ran-specific guanine nucleotide exchange factor, RCC1, and a cytosolic GTPase activating protein (GAP)()have been identified (15, 16). In addition, using a GTP-Ran overlay assay, a number of putative downstream target proteins have been identified that interact with and stabilize the GTP-bound state of Ran(17) . One of these proteins, HTF9A/RanBP1, has been cloned(18, 19) , and is predominantly cytosolic. Information from this sequence and that of several other partial clones has revealed the existence of a novel, highly conserved Ran-binding domain of approximately 150 amino acid residues in length (20). It is likely that a large family of Ran-binding proteins exists, that may contain one or more copies of this conserved Ran-binding domain(20) .

The mechanisms through which Ran interacts with these proteins are not understood, but several pieces of evidence suggest that the acidic C terminus of Ran may be important in the regulation of Ran activity. A mutant Ran protein that lacks this -DEDDDL sequence fails to bind efficiently to several of the RanBPs, including HTF9A, when tested using the GTP-Ran overlay assay(17) . Furthermore, the C terminus has been implicated in the function of Ran in cell cycle progression(10) . Transfection of 293/Tag cells with a dominant gain-of-function mutant of Ran arrests the cells in G or at the G/S boundary, but deletion of the C-terminal -DEDDDL tail of this mutant abrogates the cell cycle arrest(10) . These results suggest that Ran function requires an intact C terminus. Therefore, it was of interest to examine the effects of deleting the -DEDDDL sequence on the interaction of Ran with its known regulatory factors, and to try to elucidate the essential function provided by this C-terminal domain in Ran function.

Here we report that the -DEDDDL tail of Ran stabilizes GDP binding to the Ran protein. A C-terminal deletion mutant exhibits accelerated loss of GDP both in the presence of EDTA and when release is catalyzed by the exchange factor RCC1. The rate of GTP hydrolysis on this mutant, catalyzed by Ran-GAP, is also accelerated compared to wild-type Ran. Moreover, the C-terminal tail significantly affects the affinity of GTP-Ran for the Ran-binding protein HTF9A/RanBP1. While HTF9A acts as an enhancer of Ran-GAP activity on wild-type Ran, HTF9A decreases the rate of this hydrolysis on the C-terminal deletion mutant protein. We propose a model for the interaction of Ran with RCC1, Ran-GAP and HTF9A, illustrating a possible mechanism for the involvement of the -DEDDDL tail in these protein-protein interactions.


MATERIALS AND METHODS

Construction and Purification of Recombinant Proteins

Deletion of the C terminus of Ran was accomplished using polymerase chain reaction mutagenesis to introduce a stop codon and remove the final 6 amino acid residues of the open reading frame. The template DNA was the TC4 clone in pUC19 (a gift from P. D'Eustachio). The product was subcloned into pGEX-2T (Pharmacia Biotech Inc.) to generate a glutathione S-transferase-Ran fusion protein. Wild-type Ran and HTF9A (a gift from J. Thorner) were also subcloned into pGEX-2T. Proteins were expressed in Escherichia coli DH5 upon induction with 0.4 mM isopropyl--D-thioglactopyranoside. Proteins were purified over glutathione-Sepharose columns (Pharmacia). GST fusion proteins were cleaved to remove the GST by incubating GST fusion proteins on glutathione-Sepharose beads in 50 mM Tris, pH 7.5, 150 mM NaCl, 2.5 mM CaCl with 19.8 units of thrombin for 1 h at 23 °C. The supernatant was treated with p-aminobenzamidine-Sepharose 4B beads (Sigma) for 30 min at 4 °C to remove the thrombin. A pET11a vector containing the RCC1 open reading frame was a gift from T. Nishimoto. RCC1 protein was induced by addition of isopropyl--D-thioglactopyranoside to E. coli BL21(DE3)LysS that contained the pET-RCC1 vector. The soluble fraction of the bacterial lysate was used as a source of RCC1; lysate from cells not containing vector was used as a control. Proteins were quantitated by the Bradford (21) assay, using BSA as a standard.

Nucleotide Release Assays

Recombinant Ran protein was loaded with [-P]GTP or -GDP (20-40 µCi; 3000 Ci/mmol nucleotide; usually in a 50-µl volume) as described previously(17) . To measure EDTA-stimulated nucleotide release, portions of this solution were diluted into a 10-fold larger volume of buffer containing 50 mM MOPS, pH 7.1, 2 mM GTP or GDP, 1 mM dithiothreitol, 0.1 mg/ml BSA plus 10 mM MgCl, or 5 mM EDTA. Loss of [-P]nucleotide from Ran was measured by filter binding samples and the [-P]nucleotide bound was quantitated by scintillation counting(22) . The amount of [-P]nucleotide bound at t = 0 was defined as 100%, which varied between 10,000 and 50,000 dpm in different experiments. RCC1 catalyzed nucleotide release was determined by the same method, with the aforementioned buffer containing 10 mM MgCl, RCC1 or bacterial lysate, and no EDTA. Reactions were performed at 23 °C in duplicate unless otherwise indicated. Off-rates were calculated using a best-fit nonlinear regression, assuming single exponential decay.

GAP Assays

GAP assays were performed as described previously(23) , with the following modifications. Ran was loaded with [-P]GTP as described above (17) and diluted into a 10-fold larger volume of GAP buffer (40 mM Tris, pH 8.0, 8 mM MgCl, 80 µM BSA, 2 mM GTP). An S100 lysate was prepared from BHK-21 as described (18) and used as a source of GAP activity at a final concentration of 0.067 mg/ml. Reactions were carried out at 23 °C and terminated by filter binding. Nucleotides were released from the filters by addition of nucleotide releasing buffer (1% SDS, 25 mM EDTA, 5 mM ATP, plus 200 µM GDP and GTP, pH 6.0), separated by thin layer chromatography(23) , and quantitated on a Bio-Rad GS-250 Molecular Imager.

Immunoprecipitation of Ran

A peptide corresponding to residues 196-207 of the Ran/TC4 sequence (Multiple Peptide Systems) was conjugated to keyhole limpet hemocyanin by glutaraldehyde(24) ; this sequence is adjacent to the acidic -DEDDDL domain. Anti-Ran serum was generated by the injection of this conjugate into rabbits (Cocalico, Inc.). The resulting antiserum, AR-12, detected a single 25-kDa band by immunoblotting either total extracts of BHK-21 cells or purified recombinant Ran protein. The 25-kDa band was not detected by preimmune serum (data not shown). For immunoprecipitations, recombinant Ran purified from E. coli expressing a pET-Ran vector (17) was loaded with [-P]GTP and/or with [-P]GDP, then incubated for 30 min at 4 °C with a 1:10 dilution of AR-12 antiserum in buffer A (20 mM HEPES, pH 7.3, 20 mM MgCl, 150 mM NaCl, and 0.75% Triton X-100). Following a further incubation for 30 min with protein A-Sepharose, the immunoprecipitates were washed three times with buffer A, resuspended in nucleotide releasing buffer, and analyzed by thin layer chromatography for GDP and GTP as described above(23) .


RESULTS

Deletion of the C-terminal DEDDDL Sequence Alters the Off-rate of Guanine Nucleotides from Ran

It was important initially to determine whether the removal of the C-terminal acidic residues of Ran would alter the basal kinetics of binding and release of GDP and GTP. Consistent with previous findings(14, 17) , the off-rate for [-P]GDP (Fig. 1A) and [-P]GTP (Fig. 1B) from both wild-type Ran and the C-terminal deletion mutant Ran was extremely slow in the presence of Mg (k 0.013 min). When excess EDTA was added the rates of nucleotide release were accelerated. For [-P]GDP (Fig. 1A) the rate of nucleotide release from wild-type Ran was 0.069 min. The deletion of the -DEDDDL sequence further accelerated EDTA-stimulated [-P]GDP release (k 0.15 min). The presence of EDTA also accelerated GTP release from these proteins, but the rates were significantly different (Fig. 1B). First, the release of GTP from wild-type Ran (k 0.99 min) was more rapid than that from DEDDDL Ran (k 0.40 min), which is the opposite of the release pattern observed for GDP. Second, the rate of GTP release from the wild-type protein was far more rapid than the rate of GDP release (Fig. 1A). These results were surprising because the removal of the C-terminal amino acids from other small GTPases such as Ras and Rab3 has no detectable effect on the rates of guanine nucleotide release(25, 26) .


Figure 1: Guanine nucleotide release from wild-type and DEDDDL-Ran-GST fusion proteins. Ran proteins (80 pmol) were loaded with either (A) [-P]GDP or (B) [-P]GTP (20 µCi; 3000 Ci/mmol) as described under ``Materials and Methods,'' and portions were diluted 10-fold into buffer containing either 10 mM MgCl or 5 mM EDTA (total volume of 50 µl). Nucleotide release was measured by nitrocellulose filter binding of 5-µl samples at intervals. Values are means ± 1 S.D. (n = 2-6). , wild-type + MgCl; , wild-type + EDTA; , DEDDDL + MgCl; , DEDDDL + EDTA. C, RCC1-catalyzed GDP release from wild-type or DEDDDL-Ran. Forty pmol of [-P]GDP-loaded wild-type or -DEDDDL-Ran-GST was added to buffer (+10 mM MgCl) with or without recombinant human RCC1. Samples were filter bound over time. Values are means ± range (n = 2). , wild-type - RCC1; , wild-type + RCC1; , DEDDDL - RCC1; , DEDDDL + RCC1.



The data suggest that the C-terminal acidic tail can function to stabilize the GDP-bound state of Ran, and may destabilize the GTP-bound state. This hypothesis predicts that loss of the C-terminal domain will accelerate the rate of GDP release catalyzed by the Ran exchange factor, RCC1. To test this hypothesis we performed GDP release assays on wild-type and DEDDDL Ran-GST fusion proteins, in the presence or absence of recombinant human RCC1 (Fig. 1C). As predicted by the previous experiments, RCC1-catalyzed GDP release is significantly faster from the DEDDDL mutant (k 1.7 min) than from the wild-type Ran (k 0.21 min). However, these experiments were performed at subsaturating concentrations of RCC1. Therefore, an alternative explanation for the more rapid GDP release from the DEDDDL Ran would be that the C-terminal deletion mutant has a higher affinity for RCC1, which would result in a higher turnover rate for GDP. To test this possibility we performed a competition binding assay for RCC1 using [-P]GDP-loaded wild-type Ran. We measured the rate of RCC1-catalyzed [-P]GDP release from this Ran in the presence of increasing concentrations of nonradiolabeled, GDP-loaded wild-type or DEDDDL-Ran-GST fusion proteins. (To avoid complications that might arise from homotypic interactions of the GST domains, the [-P]GDP wild-type Ran was prepared by thrombin-cleavage of a GST fusion protein.) The DEDDDL-Ran-GST competes for RCC1-catalyzed GDP release from [-P]GDP wild-type Ran with a similar affinity to the competition by wild-type Ran-GST; the apparent K is about 0.36 µM (Fig. 2). Therefore, these results indicate that the accelerated release of GDP from the DEDDDL Ran is not due to more efficient interaction with RCC1, and support the hypothesis that the acidic -DEDDDL sequence stabilizes GDP binding to Ran.


Figure 2: Wild-type and DEDDDL-Ran-GST fusion proteins have similar affinities for RCC1. To measure the relative affinities for RCC1, a competition exchange assay was performed at 23 °C as described previously (26). Increasing concentrations of GDP-wild-type-GST-Ran or DEDDDL-GST-Ran were added to 80 fmol of wild-type thrombin-cleaved Ran that had been loaded with [-P]GDP (final assay volume = 200 µl) as described under ``Materials and Methods'' and in the legend to Fig. 1. In the presence of RCC1, nucleotide release was measured over 3 min by nitrocellulose filter binding. Values are means ± range. , wild-type; , DEDDDL.



The Rate of Ran-GAP-catalyzed GTP Hydrolysis Is Accelerated in the Absence of the C-terminal DEDDDL Sequence

To examine the interaction of both wild-type and DEDDDL Ran with Ran-GAP, we performed GAP assays using a BHK-21 cell S100 lysate as a source of Ran-GAP activity. This lysate had no detectable Ran exchange factor activity and the total radioactivity did not decrease significantly over time (data not shown). In the absence of lysate, the basal rate of GTP hydrolysis on these fusion proteins was similar over a 30-min period, as shown in Fig. 3. The Ran-GAP activity in the lysate stimulated GTP hydrolysis on both the wild-type and DEDDDL Ran. Interestingly, the GAP-catalyzed rate of hydrolysis by the DEDDDL mutant was significantly greater than that by wild-type Ran, as quantitated by phosphoimager analysis. These results suggest that the C-terminal DEDDDL sequence may impede Ran-GAP stimulation of the intrinsic Ran GTPase activity.


Figure 3: Ran-GAP stimulates GTP hydrolysis on wild-type and DEDDDL-Ran-GST. Twenty-three pmol of wild-type or DEDDDL-Ran-GST was loaded with [-P]GTP (see Fig. 1 and ``Materials and Methods'') and added to a buffer with a BHK-21 S100 lysate containing Ran-GAP activity, or BSA. The assay was executed as described under ``Materials and Methods,'' with samples taken over 30 min. The lysate contained no detectable nucleotide exchange activity. Total recovery of radioactivity was equal (± 5%) at all time points. %GTP bound was calculated as (dpm GTP/(dpm GDP + dpm GTP)) 100. , wild-type - lysate; , wild-type + lysate; , DEDDDL - lysate; , DEDDDL + lysate.



The Ran-binding Protein HTF9A/RanBP1 Binds to the DEDDDL Ran, but Has Reduced Ability to Stabilize the GTP-bound State

A 28-kDa Ran-binding protein that specifically interacts with Ran in the GTP-bound state has been cloned and called RanBP1(18) . It is identical to a putative open reading frame (203 amino acid residues in length) sequenced previously and named HTF9A(19) . Our previous data using the Ran overlay assay on whole cell extracts detected a 28-kDa Ran-binding protein that possesses properties similar to recombinant HTF9A and which did not appear to interact with the DEDDDL mutant(17) .

To further investigate this interaction, GST-HTF9A and GST were loaded onto glutathione-Sepharose beads and mixed with equal amounts of either wild-type or DEDDDL Ran, which had been loaded with [-P]GTP. Other experiments showed that the binding of GST-HTF9A was specific for GTP-Ran; there was no detectable binding of GDP-Ran to GST-HTF9A (data not shown). Surprisingly, we found that both the wild-type and DEDDDL Ran proteins bound to HTF9A specifically and to a similar extent (approximately 60% of the total added [-P]GTP Ran bound to the GST-HTF9A) (Fig. 4A), which is unlike the result of the Ran overlay assay(17) . Therefore, the acidic C-terminal tail is not essential for the binding of GTP-Ran to HTF9A, but likely increases the affinity of the interaction between these two proteins.


Figure 4: HTF9A/RanBP1 binds to wild-type and DEDDDL-Ran but stabilization of GTP-DEDDDL-Ran is reduced. A, binding of GTP-Ran to GST-HTF9A. Forty five pmol of GST or 22.5 pmol of GST-HTF9A were bound to 20 µl of glutathione-Sepharose beads. Four pmol of wild-type or -DEDDDL thrombin-cleaved Ran proteins which had been loaded with [-P]GTP (0.12 µCi of bound [-P]GTP) were mixed with the beads in 50 mM MOPS, pH 7.1, 10 mM MgCl, 0.1 mg/ml BSA, and were then washed 3 times in the same buffer (1.0 ml per wash). Assays were performed in duplicate, and nucleotides associated with the GSH beads were quantitated by scintillation counting. DPM indicates the amount of [-P]GTP-wild type or -DEDDDL-Ran (62,500 dpm/pmol of protein) associated with the GST or GST-HTF9A on glutathione-Sepharose beads. B, EDTA-induced GTP release from the wild type- or DEDDDL-RanGST-HTF9A complex. Equal amounts (1.6 pmol) of wild-type or -DEDDDL-Ran were loaded with [-P]GTP and incubated with 5.5 pmol of GST-HTF9A or GST as described under ``Results.'' Nucleotide release in EDTA was measured over as described under ``Materials and Methods.'' Values are means ± range. , wild-type + GST; , DEDDDL + GST; , wild-type + GST-HTF9A; , DEDDDL + GST-HTF9A.



Using an overlay assay on cell extracts, we demonstrated previously that Ran-binding proteins stabilize the GTP-bound state of Ran(17) . To determine if the binding of recombinant HTF9A to GTP-Ran could inhibit EDTA-stimulated release, nucleotide release assays were performed as described for Fig. 1. After loading wild-type or DEDDDL Ran with [-P]GTP, GST or GST-HTF9A was added at 23 °C for 10 min to allow complex formation. The mixtures were then added to EDTA-containing buffer. Fig. 4B shows that the preincubation of wild-type Ran with GST-HTF9A can dramatically reduce the EDTA-stimulated nucleotide release from the wild-type protein (k with HTF9A 0.028 min compared to k without HTF9A 0.46 min). However, the inhibitory effect of HTF9A on release from the DEDDDL mutant was significantly smaller (k with HTF9A 0.10 min compared to k without HTF9A 0.23 min). This result suggests that either the affinity of the DEDDDL mutant for HTF9A is lower than that of wild-type Ran, or that the deletion of the C terminus reduces the inhibition efficiency. At a 10-fold higher concentration of HTF9A, GTP release was almost completely inhibited from both wild-type and DEDDDL Ran (data not shown). Therefore, the efficiency of inhibition is not reduced by loss of the C-terminal domain, and the most likely explanation of the data is that HTF9A exhibits a lower affinity for DEDDDL Ran than for wild-type Ran.

HTF9A/RanBP1 Inhibits RCC1-mediated Nucleotide Release on Ran

To measure the effect of HTF9A on RCC1 activity toward Ran, the wild-type and DEDDDL mutant proteins were loaded with [-P]GTP and preincubated with a molar excess of GST or GST-HTF9A, as described above, before addition of recombinant RCC1. Under these conditions HTF9A completely blocked the RCC1-catalyzed release of nucleotide from wild-type Ran, with the k without HTF9A 0.56 min and the k with HTF9A 0 min (Fig. 5A). However, Fig. 5B shows that the rate of GTP release from the DEDDDL mutant in the presence of HTF9A was higher than that from wild-type Ran under the same conditions (k without HTF9A 1.6 minversusk with HTF9A 0.076 min). In addition, we observed a more rapid RCC1-catalyzed release of GTP from this protein (k 1.6 min) than from wild-type Ran (k 0.56 min), as was the case for GDP release catalyzed by RCC1 (see Fig. 2).


Figure 5: HTF9A/RanBP1 reduces RCC1-catalyzed GTP release from Ran, and has a lower affinity for DEDDDL-Ran. Twenty pmol of wild-type (A) or DEDDDL (B) Ran were loaded with [-P]GTP as described in the legend to Fig. 1 and under ``Materials and Methods,'' and incubated with 190 pmol of GST or GST-HTF9A. RCC1 nucleotide release assays were performed as described under ``Materials and Methods.'' Values are the means ± range. , GST - RCC1; , GST + RCC1; , GST-HTF9A - RCC1; , GST-HTF9A + RCC1. C, HTF9A has a lower affinity for DEDDDL Ran than for wild-type Ran. Increasing concentrations of GST-HTF9A were incubated with constant amounts of [-P]GTP-wild type or -DEDDDL-Ran. The complex was added to an RCC1-containing buffer for 5 min before termination by filter binding. Values are means ± range. , wild-type; , DEDDDL.



A likely interpretation of these results is that they all reflect a reduced affinity of HTF9A for the DEDDDL mutant. Further evidence to support this interpretation is shown in Fig. 5C. Here, different concentrations of GST-HTF9A were bound to equal amounts of thrombin-cleaved [-P]GTP-loaded wild-type or DEDDDL Ran, and the complexes were added to RCC1-containing buffers for 5 min at 23 °C. The results show that saturating amounts of HTF9A were able to inhibit RCC1-mediated GTP release from either protein, but that the affinity of the mutant for HTF9A is lower than that of wild-type. For wild-type Ran the apparent Kis about 1.5 nM, and for the DEDDDL mutant the apparent K is about 60 nM. Because only a portion of the total Ran protein binds nucleotide (27) and the proportion of active HTF9A is unknown, these affinities are relative to each other and not absolute values.

From these data we can conclude that the C-terminal -DEDDDL sequence is not necessary for the binding of HTF9A to Ran, but does increase the affinity of Ran for HTF9A. The inhibition of GTP release suggests that the HTF9A acts to significantly stabilize the GTP binding to Ran, and/or HTF9A inhibits the interaction between RCC1 and the GTP-Ran, perhaps by competing for a common binding site on the Ran protein.

HTF9A/RanBP1 Is a Co-activator of Ran-GAP for Wild-type Ran But Decreases Ran-GAP Activity on the DEDDDL Mutant

To investigate the effect of HTF9A on Ran-GAP, we performed GAP assays on Ran that had been preincubated with either GST or GST-HTF9A. The basal rate of GTP hydrolysis by wild-type Ran was very low (k 0.002 min), and HTF9A did not act as a Ran-GAP (Fig. 6A). In the absence of HTF9A and presence of Ran-GAP activity, the rate of hydrolysis is increased by about 10-fold (k 0.018 min). However, in the presence of both HTF9A and Ran-GAP activity, the rate of GTP hydrolysis was further increased to approximately 20-fold over the basal rate (k 0.039 min), demonstrating that the HTF9A/RanBP1 can function as a co-activator of Ran-GAP. These results confirm an earlier report of this phenomenon by Bischoff et al.(16) , and the observation by Beddow et al.(20) that an isolated Ran-binding domain can function as a co-activator of Ran-GAP. As described above (Fig. 3) and seen here (Fig. 6B), Ran-GAP acted on the DEDDDL mutant more efficiently than on wild-type Ran (DEDDDL k 0.058 min). Surprisingly, no co-activation was observed when the DEDDDL mutant was preincubated with HTF9A and tested in the GAP assay (Fig. 6B). Rather, the addition of HTF9A significantly decreased this activity (k 0.023 min). These results suggest that together the C-terminal DEDDDL tail and HTF9A play an important role in the interaction of the Ran GTPase with Ran-GAP.


Figure 6: HTF9A/RanBP1 enhances Ran-GAP activity for wild-type Ran but decreases Ran-GAP activity for DEDDDL-Ran. Wild-type- (A) or DEDDDL-Ran (B) (16 pmol) was loaded with [-P]GTP (see Fig. 1) and incubated with 115 pmol of GST or GST-HTF9A as described under ``Results.'' Samples were filter bound over 30 min and analyzed by thin layer chromatography and PhosphoImager analysis. , GST - lysate; , GST + lysate; , GST-HTF9A - lysate; , GST-HTF9A + lysate.



The C Terminus of Ran Undergoes a Guanine Nucleotide-dependent Change in Conformation

All of the above data indicate that the acidic C-terminal domain is involved in the regulation of guanine nucleotide binding to Ran and GTP hydrolysis by the Ran GTPase. They also suggest that this domain undergoes a guanine nucleotide-dependent change in conformation. To test this hypothesis more directly, we analyzed the ability of an anti-Ran antiserum, AR-12, to distinguish between the GDP- and GTP-bound states of Ran. This antiserum was generated against a C-terminal, Ran-specific peptide directly adjacent to the -DEDDDL sequence. Recombinant Ran was loaded with a mixture of [-P]GTP and [-P]GDP, then immunoprecipitated with AR-12, and the Ran-bound nucleotides were separated by thin layer chromatography and quantitated. As can be seen from Fig. 7(lane 2), the antiserum specifically immunoprecipitated the GTP-Ran about 20-fold more efficiently than it did the GDP-bound Ran. In a separate experiment, no immunoprecipitation of [-P]GDP-loaded Ran could be detected over background (using preimmune serum). Together these results suggest that in the GDP-bound state, the Ran C-terminal domain is folded in such a way as to be inaccessible to the anti-Ran antibodies, and that binding of GTP triggers a conformational change that exposes the AR-12 epitopes.


Figure 7: Immunoprecipitation of GTP- or GDP-bound Ran with anti-Ran antiserum AR-12. Two µg of recombinant Ran was loaded with either a mixture of [-P]GTP and [-P]GDP (lanes 1, 2, 5, and 6) or [-P]GDP alone (lanes 3, 4, 7, and 8) as in Fig. 1, then immunoprecipitated with anti-Ran antiserum AR-12 or preimmune antiserum (PreAR-12). Eluted precipitates and 1% of the supernatants were separated by thin layer chromatography and analyzed by autoradiography. Supernatants represent the total mixture including unbound nucleotide.




DISCUSSION

A number of studies suggest that the guanine nucleotide-bound state of Ran affects a variety of essential cellular functions. For example, Kornbluth et al.(9) showed that the addition of T24N Ran, a putative, dominant loss-of-function mutant, to cycling cell-free Xenopus oocyte extracts blocks the dephosphorylation of p34 and prevents entry into mitosis. Furthermore, the presence of this mutant protein in cell-free extracts prevents DNA replication, and suppresses nuclear growth but not nuclear assembly.

In tsBN2 cells, a mammalian cell line carrying a temperature-sensitive mutation in the RCC1 gene, incubation at the nonpermissive temperature leads to the rapid loss of the RCC1 protein(28, 29, 30) . Because RCC1 is an exchange factor for Ran, it is expected that depletion of RCC1 would lead to the accumulation of Ran predominantly in the GDP-bound state. If the temperature shift occurs during S phase of the cell cycle, the cells undergo premature chromosome condensation and enter mitosis aberrantly; the resulting cells accumulate micronuclei and die(28) . If the cells are shifted in G, they arrest at the G/S boundary(28) .

Surprisingly, the overexpression of G19V Ran, a dominant gain-of-function mutant which is constitutively GTP-bound,()also arrests mammalian cells in G or at the G/S boundary (10). However, the addition of this protein to cell-free Xenopus egg extracts has no effect on nuclear formation or entry into mitosis, but does inhibit DNA replication slightly(9) . In S. cerevisiae, the analogous gain-of-function mutation in the Ran homolog GSP1 is lethal(5) . These reports suggest that the regulated guanine nucleotide exchange and hydrolysis on Ran is an essential cellular process. The data presented here implicate the C-terminal tail of Ran in this regulation.

We propose that the Ran C-terminal DEDDDL tail stabilizes the GDP-bound state, possibly by folding into the guanine nucleotide binding pocket and mimicking the negative charge on the -phosphate of GTP; in this conformation, the -DEDDDL tail is not available for interaction with Ran-binding proteins. The Ran exchange factor, RCC1, catalyzes the exchange of GDP for GTP. When bound to Ran, the -phosphate of GTP could displace the -DEDDDL tail, which would undergo a conformational change to expose the C terminus to the environment. The destabilization of GTP binding on Ran may result from repulsion forces exerted between the negatively charged -phosphate on GTP and the extremely negative charge of the DEDDDL tail. This destabilization would account for the more rapid loss of GTP from wild-type Ran than from the DEDDDL mutant in EDTA (Fig. 1B).

When Ran is in the GTP-bound state, it is able to interact with the Ran-GAP and a number of Ran-binding proteins, including HTF9A/RanBP1. The Ran-GAP is capable of binding to Ran and stimulating GTP hydrolysis, but the -DEDDDL tail may inhibit more efficient binding of the Ran-GAP to GTP-Ran (Fig. 3). When GTP-Ran binds to HTF9A/RanBP1, the orientation of the C-terminal tail could change again, resulting in stabilization of Ran in its GTP-bound state. The binding of the HTF9A/RanBP1 also decreases the functional interaction of GTP-Ran with RCC1 (Fig. 5A), possibly through competition for overlapping binding sites on the Ran protein.

The binding of HTF9A/RanBP1, movement of the C-terminal tail, and stabilization of the GTP-bound Ran could enhance the binding of the Ran-GAP to GTP-Ran, which would lead to the accelerated rate of GTP hydrolysis that is seen with wild-type Ran in the presence of HTF9A/RanBP1 (Fig. 6A). Upon the hydrolysis of GTP to GDP, the C-terminal tail returns to its initial orientation, stabilizing the GDP on Ran. The proposed role of the C terminus of Ran is unlike that of the C terminus in other small GTPases such as Ras and Rab3A. The C terminus of these proteins is not known to have a significant effect of the kinetics of nucleotide release and hydrolysis(25, 26) , and there is no evidence that the C terminus of Ras undergoes a nucleotide- dependent conformational shift.

Why does the deletion of the -DEDDDL sequence abrogate the cell cycle arrest induced by a dominant gain-of-function mutant of Ran? The DEDDDL mutant can still interact efficiently with RCC1 and Ran-GAP; therefore, we can speculate that the loss of function is most likely related to the reduced efficiency of interaction with HTF9A/RanBP1 and other similar proteins. In the absence of this interaction, the signal transmitted by the ``on'' conformation of Ran is interrupted and subsequent cellular events are not initiated. Therefore, we hypothesize that the C-terminal -DEDDDL tail of Ran plays a dual role in Ran-mediated signal transduction. Not only is this domain involved in the stabilization of GDP on the Ran protein, but it is of critical importance for interaction of GTP-Ran with the Ran-binding proteins that function as downstream effectors.


FOOTNOTES

*
This work was supported in part by Grant GM50526 from the National Institutes of Health (to I. G. M.). 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.

Supported by Environmental Pathology Training Grant EST3207122. To whom correspondence should be addressed: Dept. of Pathology, Medical Alumni Bldg., Univ. of Vermont, Burlington, VT 05405-0068. Tel.: 802-656-0394; Fax: 802-656-8892; E-mail: srichard@moose.uvm.edu.

§
Supported by National Research Service Award F32CA63801-01.

The abbreviations used are: GAP, GTPase activating protein; RCC1, regulator of chromosome condensation; GST, glutathione S-transferase; BSA, bovine serum albumin; MOPS, 4-morpholinepropanesulfonic acid.

S. A. Richards and I. G. Macara, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Jeremy Thorner (University of California, Berkeley) for the HTF9A clone, Takeharu Nishimoto (Kyushu University, Japan) for the RCC1 clone, and Peter D'Eustachio (New York University) for the TC4 clone. We also thank Colleen McKiernan for helpful discussions.


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