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Previous Article | Next Article 
Molecular and Cellular Biology, November 2003, p. 8124-8136, Vol. 23, No. 22
0270-7306/03/$08.00+0 DOI: 10.1128/MCB.23.22.8124-8136.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Max-Planck Institut für Molekulare Physiology, 44227 Dortmund, Germany
Received 7 April 2003/ Returned for modification 28 May 2003/ Accepted 4 August 2003
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ABSTRACT |
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INTRODUCTION |
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The presence or absence of Ran-GTP in nucleosol or cytosol, respectively, is the determining factor for Ran-dependent cargo transport (22). Import receptors of the importin-ß family bind to cargo in the absence of Ran-GTP and release it in the nucleus, where they bind with high affinity to Ran-GTP. In contrast, export cargo binding to export receptors (exportins) requires the presence of Ran-GTP. The dissociation of Ran-GTP complexes with importins and exportins on the cytoplasmic side of the nuclear pore complexes (NPCs) requires the action of Ran-binding proteins (RanBPs) (7, 20, 42) and is made irreversible by RanGAP-catalyzed hydrolysis. RanBP1 is a 23-kDa protein and contains a single conserved Ran-binding domain (RanBD) (14). Because of its small size, RanBP1 can diffuse into the nucleus. However, it is also actively transported into the cytoplasm due to an amino-terminal nuclear export sequence (59). RanBP2 is a 3,224-residue large protein attached to the cytosolic side of the NPC and comprises an amino-terminal leucine-rich region, four RanBDs, eight zinc-finger motifs, and a carboxy terminus with homology to cyclophilin (77). Whereas RanBP1 is an essential component of the nuclear transport system in eukaryotes, RanBP2 could not be identified in yeast organisms and is dispensable for transport in metazoans (74). RanBDs fulfill the definition for guanine-nucleotide binding effector proteins because they bind with a higher affinity to the GTP-bound (Kd 
1 nM) than the GDP-bound (Kd 10 µM) form of Ran (40, 72). However, their effect on Ran is decisively different from that of importins and exportins, the true biological effectors. Whereas the latter inhibit both the intrinsic and RanGAP-stimulated hydrolysis of Ran-GTP (19, 23), RanBDs were shown to costimulate RanGAP-catalyzed hydrolysis (10).
RanGAP was first reported as a Saccharomyces cerevisiae mutant (rna1-1) defective in mRNA processing or transport (27, 31). Bischoff et al. purified RanGAP from HeLa cells (8) and found it to be homologous to RNA1 (6, 9). All known RanGAP proteins show a modular structure. They share a 330-to-350-residue large leucine-rich repeat (LRR) domain, followed by an acidic region of 35 to 50 residues. In higher eukaryotes, an additional domain localizes RanGAP to the NPC. In animals, this is achieved via modification with the ubiquitin-like protein SUMO-1 at the carboxy terminus of RanGAP (43, 45). The modified RanGAP then binds to RanBP2 (46). In plant cells, for which no homologue of RanBP2 is known, RanGAPs have an amino-terminal domain with a conserved WPP motif, which also leads to NPC localization (48, 60). RanGAP-stimulated Ran-GTP hydrolysis occurs either in the cytoplasm or on the cytoplasmic fibrils of the NPC but is not directly coupled to the translocation process itself. This explains why up to 1,000 translocations per NPC per s can be observed (57).
Spindle formation in mitotic cells is believed to be induced by a high local concentration of Ran-GTP, which in turn is created by the attachment of RCC1 (and possibly Ran itself) on chromatin. The mechanism of nuclear envelope formation may be due to a similar preferred RCC1 localization. In HeLa cells, RanGAP is localized to kinetochores and mitotic spindles and the localization is at least partially dependent on RanBP2 (33). Much less is known about the targeting of RanGAP in yeast, but it is mandatory for both yeast and mammalian cells that the localization should be different from that of RCC1 in order to establish and maintain the Ran-GTP gradient around chromatin.
RanGAP-mediated GTP hydrolysis has many features that are distinct from the canonical RasGAP and RhoGAP reactions. While the maximal 105-fold stimulation of GTP hydrolysis is similar (2, 4, 16, 25, 30, 35), RanGAP does not employ an arginine finger to stabilize the transition state of the reaction (66). Instead, the crystal structure of Ran in complex with RanBP1 and RanGAP (Fig. 1) shows that Ran itself encodes the basic machinery for GTP hydrolysis and that the positioning of the catalytic glutamine residue appears to be essential for the reaction. Furthermore, while RanBDs were shown to costimulate RanGAP-catalyzed hydrolysis (10, 72), the structure of the ternary complex and multiple turnover kinetics suggested that the catalytic machinery was entirely located on Ran. Previously, it was suggested that the conserved C-terminal highly acidic region of RanGAP is required for Ran binding and for stimulation of GTP hydrolysis (26). Although this acidic region was not visible in the structure, it did not appear to participate in catalysis. We thus wanted to reinvestigate the contribution of this region, using the structure of the LRR domain as a guideline for a deletion construct missing the C terminus of RNA1. Previously, the interaction between RanGAP and Ran was investigated by multiple turnover kinetics using radioactive nucleotides to determine kcat and Km values (6, 35). In order to investigate individual steps of the RanGAP catalyzed reaction, we decided to employ pre-steady-state kinetics by using stopped-flow and fluorescence spectroscopy methods. Although the fluorescent 2'(3')-O-(N-methylanthraniloyl) (mant) analogues of GDP and GTP have been used successfully for the Ras-RasGAP system (2-4, 15) and for Ran-nucleotide and Ran-effector interactions (36, 40, 72), they did not show fluorescence changes in the Ran-RanGAP reaction. In analogy to a previously developed assay to characterize interactions of Ras and Rap with their respective GAPs (38), a highly sensitive, fluorescence-based test system was established with 5-((((2-iodoacetyl) amino) ethyl) amino) naphthalene-1-sulfonic acid (1,5-IAEDANS)-labeled Ran. The modified proteins could be used to characterize the dynamics of the Ran-RanGAP interaction, to analyze equilibrium binding of Ran to RanGAP, and to follow the time course of Ran-GTP hydrolysis. We found that RanBP has an effect on the dynamics of the interaction, whereas the acidic region of RanGAP does not affect catalysis. Instead, deletion of the acidic region on RNA1 in Saccharomyces cerevisiae via homologous recombination shows a pleiotropic phenotype, with major defects in the spindle apparatus.
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MATERIALS AND METHODS |
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Fluorescence measurements and data analysis. All fluorescence measurements were performed at 20°C in a buffer containing 100 mM Tris-HCl, pH 7.5, 5% (vol/vol) glycerol, 5 mM MgCl2, and 5 mM dithioerythritol. Equilibrium measurements were carried out in a FluoroMax II spectrofluorimeter (SPEX Instruments, Edison, N.J.) at an excitation wavelength of 350 nm and a band pass of 1.5 nm. For emission spectra, fluorescence emission was monitored with a band pass of 3 nm between 380 and 600 nm. For titrations, emission was followed at a fixed wavelength of 450 nm with a band pass of 3 nm. Stopped-flow experiments were performed on an SX18 MV Applied Photophysics apparatus (Leatherhead, Surrey, United Kingdom). The fluorescent probes were excited at 350 nm with a band pass of 6.4 nm, and emission at wavelengths of >408 or 470 nm was monitored with cutoff filters. Binding curves, titration experiments, and dissociation experiments were analyzed with Perl scripts and GRAFIT 5.0 (Erithacus Software, Surrey, United Kingdom).
Genetic techniques. For functional analysis, the strain Saccharomyces cerevisiae FY1679 (76) was used. PCR-based gene insertion was performed as described previously (41) using plasmid pFA6a-kanMX4 (73). Primers were designed to allow for amplification of the Geneticin cassette of the plasmid and for subsequent homologous recombination. Transformation was carried out by the LiAc/PEG method (1). Specific insertion was confirmed by PCR.
Fluorescence microscopy.
Overnight cultures of Saccharomyces cerevisiae FY1679 were diluted to an A600 of 0.2 and grown in yeast-peptone-dextrose medium to early log phase (A600 = 0.6). Ten milliliters of the culture was then fixed by the addition of 4% (vol/vol) formaldehyde (5 min at room temperature [RT]), subjected to centrifugation (800 x g, 3 min, RT), and resuspended in phosphate-buffered saline with 4% (vol/vol) formaldehyde. After 1 h of incubation at RT, cells were washed three times in phosphate-buffered saline, and the resultant pellet was resuspended in buffer KP (50 mM potassium phosphate [pH 7], 1.2 M sorbitol, 1 mM MgCl2). Cell walls were digested with 40 µl of Zymolyase 100T and 5 µl of glucuronidase (15 min, 30°C) and washed two times in buffer KP. Twenty microliters of the resultant suspension of fixed cells was treated with monoclonal anti-
-tubulin antibodies or with DAPI (4',6'-diamidino-2-phenylindole; Sigma-Aldrich) as described previously (17). Microscopy was carried out on an Axiophot microscope equipped with a x100 Neofluar lens (Carl Zeiss Inc., Jena, Germany). Images were recorded with a mounted charge-coupled device camera, and Adobe Photoshop was used to optimize contrast and brightness.
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RESULTS |
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220 µM) (Fig. 4C). To further investigate the dynamic nature of the interaction, the dissociation rate for the Ran-RanGAP complex was determined by competition experiments. This approach was feasible for both the binary Ran-GppNHp-RanGAP and the ternary Ran-GppNHp-RanBP1-RanGAP complexes, because both RanBP1 and nucleotide dissociate slowly from Ran (36, 72), while RanGAP dissociation is fast. A fluorescent complex of Ran94-GppNHp-RanGAP was preformed, and a 50-fold excess of unlabeled Ran-GppNHp was added in the stopped-flow apparatus. The decrease in fluorescence was fitted with a single exponential and reflected the koff value (150 s-1). If RanBP1 is present in the experiment, the dissociation rate constant is about sixfold higher (880 s-1) (Fig. 5).
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Ras-GDP can be induced to bind to RasGAP by adding aluminum fluoride, and this is dependent on the presence of an arginine finger in RasGAP (3, 50). Similar complexes have also been observed for other Ras-like GTP-binding proteins, such as Rho (3) and Rap1, which does not use an arginine for GTPase stimulation (38). In contrast, and for reasons that are unclear to us, Ran94-GDP shows no observable increase in binding affinity for RanGAP in the presence of fluoroaluminate (AlFx). In the presence of RanBP, which already induces tight binding between Ran47-GDP and RanGAP, there is no observable fluorescence change upon binding of AlFx, although we have shown that AlFx binds under these conditions (66).
RanGAP-catalyzed hydrolysis of Ran-GTP. The RanGAP-catalyzed hydrolysis of Ran-GTP was monitored by single-turnover stopped-flow reactions in order to learn more about individual and possibly rate-limiting steps in the overall reaction and whether or not RanBP-type proteins have a modulatory effect on such steps. For the interaction of Ras with its cognate RasGAP proteins, different kinetic mechanisms have been observed. For p120GAP, very fast association of substrates and release of products were found, and the chemical reaction is believed to be rate limiting (2, 38, 65). A completely different situation is observed during neurofibromin (NF1)-catalyzed hydrolysis of Ras-GTP, during which binding is also fast, but the overall reaction is limited by either the phosphate or Ras-GDP release step (5, 38). Here, measurements were done with Ran47 because this mutant showed the largest difference in fluorescence between the GDP- and GTP-bound states.
Mixing 1.5 µM Ran47-GTP-RanBP1 with an excess of 12.5 µM RanGAP showed a monophasic fluorescence increase (Fig. 6A). At lower concentrations of RanGAP, a second, slower phase was observed. For all concentrations, a single- or double-exponential function (Fig. 6B and C) can describe the resultant fluorescence transients. The fast, initial phase is characteristic of saturation kinetics and reaches its maximum rate at about 2.5 µM RanGAP. The corresponding rate of 10 s-1 is similar to the kcat that was observed with simple phosphate detection multiple-turnover assays in the absence of RanBP1 (30, 35) and suggests that this phase reflects the hydrolysis reaction and/or the phosphate release. This also supports the notion that the reporter group and the V47C mutation do not affect the rate-limiting step of the GTPase reaction by Ran.
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0.3 µM. This value corresponds well to the previously determined Michaelis constant of 0.4 µM in the absence of RanBP1 (30, 35) and is lower than the Kd between Ran-GppNHp-RanBP1 and RanGAP determined by fluorescence equilibrium measurements (Fig. 4), which is approximately sixfold higher. Presumably, the difference arises because GppNHp is not a perfect analog of GTP and leads to a higher dissociation rate. The second phase of the fluorescence transient disappears when the RanGAP concentration is increased (Fig. 6C). Its amplitude curve has the shape of a titration curve and probably reflects the binding equilibrium of Ran-GDP-RanBP1, which is pushed towards a ternary complex at high concentrations of RanGAP and is in the same range as the 2-µM dissociation constant determined independently. While the rate constant for the second step, described as k2, can be determined with reasonable accuracy (for [RanGAP] of <5 µM), it does not change with the RanGAP concentration, but is constant at 0.05 s-1.
The transient obtained in the reaction between Ran-GTP and RanGAP (no RanBP) can be described by a single exponential (Fig. 7). The time scale corresponds to the initial, fast phase observed in the presence of RanBP1. As before, a plot of the rate constants versus [RanGAP] shows saturation kinetics. The maximum rate of 7.9 s-1 is slightly lower than that in the presence of RanBP1. The half-maximal saturation of 1 µM [RanGAP] is again in good agreement with the Kd and is higher than the Km determined previously (30, 35). As with Ran-GppNHp, the affinity of Ran-GTP is about 3.3-fold higher in the presence of RanBP1.
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C330 and RanGAP341 may have destabilized the proteins.
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C344 from here on). Titration of Ran-GppNHp-RanBP1 with RanGAPC344 shows that the deletion mutant has the same affinity as the wild-type RanGAP protein (Fig. 10A) and does not change the dynamics of the interaction (data not shown). Using stopped-flow kinetic experiments as described above (see Fig. 6), we investigated the RanGAP-mediated Ran-GTPase reaction with the deletion construct. Here again, the fluorescent transient and the RanGAP concentration dependence of the first and the second rate are indistinguishable from those for wild-type RanGAP (Fig. 10B and C), indicating that at least the rate-limiting step(s) of the GAP-stimulated hydrolysis reaction is not influenced by the acidic C-terminal tail.
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Heterozygous diploid RanGAPC362 transformants do not show any evident phenotype. The effect of the deletion was then characterized by random spore analysis. Two equally strong populations of haploid cells were identified, and their genotypes were checked by PCR. The fast-growing population represented the wild type, whereas the slow-growing population represented haploid cells with the desired deletion in RanGAP's carboxy-terminal region.
Under the light microscope, mutant cells showed a pleiotropic phenotype (Fig. 11). Many of the cells were larger than wild-type cells and had unusual shapes. A large number of cells was found in various stages of unsuccessful attempts to complete mitosis. Chromatin staining with DAPI yielded further information about the mutant phenotypes (Fig. 12A) and helped to quantify the results (Table 2). Compared to wild-type cells in mitosis, a significant number of the cells (
5%) was enlarged, and some of the cells, predominantly the larger ones, were anucleate (5%). In rare cases, cells with two nuclei were observed (0.2%). Approximately 17% of the cells were found in mitosis, which is significantly reduced compared to wild-type cells during exponential growth (30%). It was not clear whether all of the mitotic cells would have completed mitosis. Rather, it appeared that many cells were arrested in mitosis. For example, cells 3 and 4 (Fig. 12A) show an uneven separation of chromatin. The mother and bud marked "4" were able to separate but are still connected by chromatin. If in these cases septum formation was successful, the resultant cells were probably not provided with a complete set of chromosomes or a nucleus.
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DISCUSSION |
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Typical association rates of protein-protein complexes are on the order of 105 to 106 M-1 s-1 (63), and in very rare cases rates of up to 109 M-1 s-1 have been observed (64). For very fast association rates, strong electrostatic complementarity and steering are required. Apparently, this is true for the interaction surface of Ran and RanGAP, which is dominated by large, complementary electrostatic potentials (66). Based on the fluorescence-based test system presented here, we confirmed that Ran and RanGAP indeed interact on a very fast time scale. Ran-GppNHp-RanBP1 and RanGAP bind with an association constant (kon) of 4.5 x 108 M-1 s-1 and dissociate quickly at 900 s-1 (koff). Thus, the interaction is highly dynamic and has a micromolar affinity (Kd) of 2 µM. The increase in affinity due to RanBP1 is 3- to 4-fold and is not simply a decrease in the dissociation and/or an increase in the association rate, as expected, but rather an increase in both the dissociation (6-fold) and association (20-fold) rates, with a net positive effect on affinity. Although the kinetics of the interaction of Ran-GTP with RanGAP have not been determined directly, the similar increase in affinity from 0.3 to 1 µM suggests a corresponding increase in association and dissociation rate constants.
Thus, the major effect of RanBP-type proteins is to increase the dynamics of the interaction between Ran and RanGAP, while the chemical reaction itself is barely affected and increases from 7.9 to 10 s-1, which might not be significant. Although the structural basis for an increase in rates can only be speculated about, we assume that it is due to the localization of the C-terminal end of Ran. This C-terminal extension is tightly bound to the body of the G domain in Ran-GDP (62) and is detached from it and wrapped around RanBP in Ran-GppNHp-RanBD or in the ternary complex shown in Fig. 1. Although the structure of Ran-GTP alone is not available, we can assume from a number of biochemical studies (summarized in reference 71) that the C-terminal end is not stably bound to the G domain and may thus prevent binding of RanGAP (or transport factors). Removal of the C-terminal extension may thus expose more of the polar surface and/or present an additional interaction surface to RanGAP and thereby increase the rate of association and dissociation reactions.
A fast acceleration caused by favorable electrostatic interactions is typically found in cases where speed is crucial for biological function (63). One example is protease inhibitors, for which an accelerated association leads to increased efficiency. Among these inhibitors, very high association rates of up to 5 x 109 M-1 s-1 were observed (64). Highly dynamic interactions have also been found in signal transduction for the binding of Ras to effector molecules. In the case of the interaction between Ras-GppNHp and RafRBD, association and dissociation rate constants of 5 x 107 M-1 s-1 and 7.7 s-1, respectively, have been observed (69), suggesting that a single Ras-GTP molecule is able to activate multiple effector molecules and that even in the presence of an effector(s), Ras can be efficiently downregulated by GAPs.
In the context of nuclear transport, an optimization for speed appears to be favorable for biological function. When Ran-GTP-receptor complexes exit the nucleus, they should be unloaded quickly. This is supported by the fast association between Ran-GTP and RanGAP, the even faster association in the presence of RanBP1 and RanBP2, and the catalytic activity of RanGAP. According to the accepted model, Ran-GTP hydrolysis and translocation through the NPC are not directly coupled (58). While the localization of RanGAP at the NPCs of higher eukaryotes suggests that Ran-GTP hydrolysis occurs in the vicinity of the cytoplasmic filaments, it is clear from measurements (57) and simulations (24, 68) of nuclear transport kinetics that the observed fast translocation rates are only possible if several RanGAP molecules per NPC are acting on Ran-GTP molecules exiting the nucleus and if catalysis by RanGAP is not rate limiting.
Kinetic scheme for Ran inactivation. Binding and association constants determined in this study can be used to derive a kinetic model for the interactions between Ran, RanBP1, and RanGAP (Fig. 13). Based on this model, simulations can be performed that show the Ran-GTP gradient and the flux of Ran and receptor molecules between nucleosol and cytosol (24, 68).
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4 x 10-4 s-1) (40) and the very fast association rate shown above induce ternary Ran-GTP-RanBP1-RanGAP complex formation. After the quasi-irreversible hydrolysis step, phosphate (Pi), RanGAP, and RanBP1 can dissociate from the Ran-GDP product. Rate constants determined in this work suggest that RanGAP dissociates first, and then RanBP1 dissociates. Previous work on different RasGAPs and RapGAP has shown that the chemical step and/or the Pi release step can be fully or partially rate limiting (5, 38, 53). Based on the data presented here, it cannot be decided at which point Pi leaves the complex and whether hydrolysis or Pi release is rate limiting. Time-resolved Fourier-transform infrared spectroscopy experiments are being undertaken to resolve this issue.
RanGAP has a function during mitosis.
Contrary to previous findings of Haberland et al. (26), we find that RanGAP's acidic carboxy-terminal region does not play a measurable role in the Ran-RanGAP reaction, since the deletion mutant RanGAPC344 is fully functional with respect to Ran-GTP binding and stimulation of Ran-GTP hydrolysis. Presumably, the acidic motif does not play any role in interphase cells, where the main function of Ran is in the transport of cargo in and out of the nucleus. We can also show that the deletion of the acidic region in RanGAP does not affect its cytoplasmic localization in yeast cells (Fig. 14). However, the phenotype of deletion of RanGAP's acidic region suggests that, apart from regulating nuclear transport, RanGAP may play an important role in mitosis and mitotic spindle formation, at least in Saccharomyces cerevisiae.
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C362. Overproduction of Rna1 (Saccharomyces cerevisiae RanGAP) from the strong GAL1 promoter causes chromosome instability (55), which is consistent with uneven chromatin separation of the RanGAPC mutant. Previously, it was shown in Schizosaccharomyces pombe that disturbing the appropriate balance of Ran-GDP and Ran-GTP by inactivation or overexpression of RanGEF, RanGAP, and RanBP1 has severe effects on the passage of cells through mitosis (47, 61). It has been argued that it is difficult to decide whether these effects are directly related to a role of Ran in mitosis or caused indirectly due to a defect in nuclear transport. Here we show that deletion of the C-terminal end of Rna1 from Saccharomyces cerevisiae seems to uncouple the transport effect from the mitotic effects of RanGAP.
In metaphase of HeLa cells, RanGAP localizes at the kinetochores and in the vicinity of the spindle poles (33). Chromosome segregation appears to be guided by a gradient of Ran-GTP that possibly spans between RanGEF and RanGAP. In dividing tobacco cells, RanGAP colocalizes with tubulin and consequently must be close to the spindle apparatus during all steps of mitosis (56). It is also known that hydrolysis of Ran-GTP is required for nuclear envelope formation (12, 28). This implies that RanGAP is involved in the process, since the intrinsic hydrolysis of Ran is very slow. Because of the differences between open mitosis in higher eukaryotes and closed mitosis in lower eukaryotes, it is not clear how these findings can be applied to yeast organisms, which do not have an open mitosis. It has, however, been shown that Ran acts on mitotic microtubules in Schizosaccharomyces pombe and that the imbalance of Ran-nucleotide has severe effects on passage through mitosis (18, 47, 61).
It is generally agreed that a high local concentration of Ran-GTP is required for the mitotic effects of Ran and that attachment of the RanGEF RCC1 to chromatin supplies the positional information for localized Ran activation in higher eukaryotic cells. However, an equal distribution of RanGAP in the mitotic fluid would oppose the formation of the Ran-GTP gradient. This implies that an active process of keeping RanGAP away from RanGEF should be very helpful for the establishment and maintenance of such a gradient. We imagine that the conserved acidic region of RanGAP could be involved in an as yet uncharacterized localization mechanism which involves the acidic region. While our findings on RanGAP having a role beyond regulating nuclear transport strictly apply only to yeast organisms, it will be necessary to investigate whether the conserved acidic region of RanGAP is important for the regulation of the mitotic effects of Ran in higher organisms also.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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) or presence (•) of 1 µM RanBP1 with Schizosaccharomyces pombe RanGAP (Kd, 7 or 2 µM, respectively). (B) Ran94-GDP (0.75 µM) was titrated in the absence (
) (Kd, 220 µM), or 500 mM NaCl (
) (Kd, 
1 mM). Fluorescence spectra were recorded, and calculation of Kd values was done as described in Materials and Methods. Note that the affinities (Kd) of RanBP1 for Ran-GppNHp and Ran-GDP are 1 nM and 10 µM, respectively (
