©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
RNA1 Encodes a GTPase-activating Protein Specific for Gsp1p, the Ran/TC4 Homologue of Saccharomyces cerevisiae(*)

Jörg Becker (1)(§), Frauke Melchior (3), Volker Gerke (2), F. Ralf Bischoff (4), Herwig Ponstingl (4), Alfred Wittinghofer (1)

From the (1) Max-Planck Institut für Molekulare Physiologie, Abteilung Strukturelle Biologie, 44139 Dortmund, Germany, the (2) Universität Münster Zentrum für Dermatologie, 48149 Münster, Germany, the (3) Departments of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, and the (4) Project Molecular Biology of Mitosis, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ran/TC4 is a ras-related GTP-binding protein predominantly located in the nucleus. Ran/TC4 is essential for nuclear transport and is involved in mitotic control. In Saccharomyces cerevisiae a gene highly homologous to Ran/TC4 has been identified and named GSP1. Like all ras-related GTP-binding proteins, Gsp1p undergoes cycles of GTP hydrolysis and GDP/GTP exchange. The switching between the two different nucleotide bound states regulates the function of these GTP-binding proteins. Here we identify the product of the yeast RNA1 gene as the GTPase-activating protein (GAP) of Gsp1p. RNA1 belongs to a group of genes which are conserved in a variety of different organisms. We have expressed and purified recombinant Gsp1p and Rna1p from Escherichia coli. The GTPase activity of Gsp1p is stimulated 10-fold by Rna1p. In addition, we find that the previously identified human RanGAP1 and rna1p from Schizosaccharomyces pombe are also able to induce GTPase activity of Gsp1p. The GTP hydrolysis of Ran is induced by RanGAP1 and rna1p but not by Rna1p. Implications for the suggested functions of Ran/TC4/Gsp1p in nuclear transport and mitotic control are discussed.


INTRODUCTION

Ran/TC4 (further referred to as Ran) was first cloned from a human teratocarcinoma cDNA library (1) . It is a GTP-binding protein representing a distinct subfamily within the superfamily of ras-related GTP-binding proteins. Ran is a very abundant protein constituting about 0.36% of the total protein content in HeLa cells (2) and it is localized mainly in the nucleus. Like all ras-related GTP-binding proteins, Ran cycles between a GTP and a GDP bound state. The conversion between these two states is very slow as Ran binds guanine nucleotides very tightly and hydrolyses GTP very slowly (3) . The dissociation of Ran-bound GDP is increased 10-fold by the gene product of RCC1() which represents the nucleotide exchange factor of Ran (4) . Mutants of RCC1 enter mitosis prematurely before S-phase has been completed (5, 6, 7) . As RCC1 is involved in the checkpoint control of entry into mitosis, Ran is also thought to be involved in cell cycle control. Ran has been reported to regulate initiation of S-phase (8) , entry into mitosis (8-10), and exit from mitosis (11) . Other groups have shown that Ran is also an essential component of the nuclear transport machinery (12, 13).

Genes homologous to Ran and RCC1 have been identified in the fission yeast Schizosaccharomyces pombe as spi1 and pim1(5) and in the budding yeast Saccharomyces cerevisiae as GSP1/GSP2(14) (also isolated as CNR1/2(15) and PRP20(16) , respectively). A comparison of the deduced amino acid sequences revealed a similarity of about 88% between Ran, spi1, and Gsp1/2p (14) and functional homology was shown between RCC1 and Prp20p (17, 18) , suggesting that the functions of these proteins have been conserved during evolution. In S. cerevisiae the loss of PRP20 causes defects in RNA processing and RNA export from the nucleus (17, 19) . While the exact function of Ran and RCC1 related proteins remains to be shown, another class of proteins, known as GTPase-activating proteins (GAPs), is certain to play an essential role in the regulation of Ran, spi1, and Gsp1p. Recently the purification and characterization of a GTPase-activating protein specific for Ran, named RanGAP1, has been reported (20) . Here we identify RNA1 as a gene encoding GAP specific for the S. cerevisiae Ran homologue Gsp1p. RNA1 has been previously identified as an important component of the RNA export machinery from the nucleus (21, 22, 23) and is highly conserved in different species. We have expressed and purified recombinant Rna1p and Gsp1p and show that Rna1p strongly increases the GTPase activity of Gsp1p. In addition, we show that Gsp1p also interacts with human and S. pombe homologues of Rna1p, indicating that this interaction has been conserved during evolution. While this study was in progress RanGAP1 had been cloned and found to be homologous to RNA1(24) .


EXPERIMENTAL PROCEDURES

Cloning of Gsp1 and RNA1

GSP1 and RNA1 were cloned by polymerase chain reaction using genomic yeast DNA as a template. To isolate GSP1 the following primers were used: GSP1-3, 5`-CTCATTTATATTATCCATGGCTGCCCCAGC-3` (underlined is the restriction site NcoI); GSP1-2, 5` GAAAGCTTGTTCTCGTTTGTCCC (underlined is the restriction site HindIII). The resulting DNA fragment contained a 5` NcoI and a 3` HindIII restriction site which facilitated the cloning into pET3d-Ran (25) . The protein coding region of Ran in pET3d was removed as an NcoI/HindIII fragment and GSP1 was inserted to yield pET3d-GSP1. To isolate RNA1 the following primers were used: RNA1-1, 5`-GGGCCATGGCTACCTTGCACTTCGTTCC-3` (underlined is the restriction site NcoI) and RNA1-2; 5`-GGATCCTAGCTATTTGATTTCAGTTTCAGCTAAACG-3` (underlined is the restriction site BamHI). The polymerase chain reaction generated a NcoI restriction site at the 5` end and a BamHI restriction site at the 3` end of the resulting DNA fragment. The polymerase chain reaction fragment was cloned into pET3d as an NcoI/BamHI fragment to yield pET3d-RNA1. Both expression plasmids were transformed into Escherichia coli strain BL21(DE3).

Expression of Gsp1p

BL21(DE3) cells containing pET3d-GSP1 were diluted and spread on LB plates containing 200 µg/ml ampicillin to a density of 1000 colonies per plate and incubated overnight. For 2 liters of LB medium containing 200 µg/ml ampicillin, one plate of cells was washed off and incubated for about 2 h at 37 °C to yield an A of 0.5-0.9. 10 µM Isopropyl-1-thio--D-galactopyranoside was added and cells were further incubated until they ceased to grow after about 3 h. Cells from a total of 20 liters of medium were harvested by centrifugation to yield about 80 g (wet weight). Cells were resuspended at 3 ml/g cell paste in buffer A (20 mM potassium phosphate, pH 6.0, 100 µM GDP, 10 mM DTE, 2 mM MgCl, 1 mM phenylmethylsulfonyl fluoride). Cells were lysed by adding 1 mg/ml lysozyme and incubated 1 h at 4 °C while stirring. To decrease the viscosity of the lysate, DNA was digested by adding DNase I (10 µg/ml). In order to uniformly load Gsp1p with GDP, 5 mM EDTA was added and the lysate incubated at 30 °C. After 30 min, 10 mM MgCl was added to stabilize the Gsp1pGDP complex. The lysate was sonicated 5 2 min on ice at 50% (Branson) and cleared at 40,000 g for 1 h. The resulting supernatant was diluted 1:10 with buffer A to reduce the ionic strength and applied onto a SP-Sepharose column (600 ml). A 3-liter gradient from 0 to 500 mM KCl was applied and Gsp1p eluted between 150 and 200 mM KCl. Pooled fractions were concentrated by ultrafiltration and applied on a Superdex 200 (26/60) FPLC column equilibrated with buffer B containing 20 mM potassium phosphate (pH 6.0), 2 mM DTE, 2 mM MgCl. Peak fractions were concentrated by ultrafiltration (Amicon), dialyzed against buffer B, shock frozen in liquid nitrogen, and kept at -80 °C.

Expression of Rna1p

A 200-ml overnight culture of BL21 cells containing pET3d-RNA1 was diluted into 20 liters of LB medium containing 100 µg/ml ampicillin and grown at 37 °C to a A of 0.1. Isopropyl-1-thio--D-galactopyranoside was added to a concentration of 10 µM and the culture was further incubated for 5 h. Cells were harvested by centrifugation and stored frozen at -20 °C. Cells were resuspended in buffer A lacking GDP (3 ml/g cell paste) and lysed as described before. The cleared lysate was applied directly onto a DEAE Sephadex column (600 ml) and eluted with a 3-liter 0-1.5 M KCl gradient. Rna1p eluted at about 600 mM salt. Fractions were treated as described above including the purification on a Superdex 200 (26/60) FPLC column (Pharmacia). The final protein solution was dialyzed against standard buffer C (64 mM Tris Cl, pH 7.4, 2 mM DTE, 5 mM MgCl).

Protein Determination

Protein concentrations were determined according to Bradford (26) using bovine serum albumin as a standard. The concentration of Gsp1p was defined as the amount of purified Gsp1p that is able to bind GDP as measured by the nitrocellulose filter binding test (27) . 1 µM Gsp1p was incubated for 30 min at 30 °C in the presence of 5 mM EDTA, 20 mM potassium phosphate (pH 6.0), 2 mM DTE, and 5 µM GDP and 43 nM [H]GDP (11.5 Ci/mmol, Amersham). The Gsp1p-nucleotide complex was stabilized by adding 10 mM MgCl. The amount of protein bound GDP was measured by determining the filter bound radioactivity (BA85, Schleicher & Schüll, Dassel).

GTPase Activity

To measure the GTPase reaction, Gsp1p was first loaded with [-P]GTP. 30 µM Gsp1p was incubated in the presence of 5 mM EDTA, 20 mM potassium phosphate (pH 6.0), 2 mM DTE, 150 µM GTP, and 5 pM [-P]GTP (6000 Ci/mmol) (ICN). The Gsp1p-nucleotide complex was stabilized by adding 10 mM MgCl after 30 min. Unbound nucleotide was removed by gel filtration (NAP-5, Pharmacia LKB Biotech). The rate of GTP hydrolysis was measured at 30 °C by determining the filter bound radioactivity after filtering aliquots of the GTPase reaction through nitrocellulose filters. Data were fitted to a single-exponential function using the computer program GraFit (Erithacus Software). Alternatively, the GTPase reaction was followed by measuring the formation of inorganic phosphate using a modified charcoal method (28) . 50-µl aliquots of the GTPase reaction were thoroughly mixed with 200 µl of 20 mM phosphoric acid containing 5% (w/v) charcoal. This mixture was centrifuged in a microcentrifuge at full speed for 10 min and the formation of -P was determined by measuring 100 µl of the supernatant in a scintillation counter. The GTPase activity of Ran, Rab7, and H-Ras was measured essentially as described above except that standard buffer C was used as a reaction buffer.

Cell Fractionation

A wild type S. cerevisiae strain was grown in rich medium to late logarithmic growth phase (A = 10), and harvested by centrifugation at 1000 g for 5 min. The cells were washed in standard buffer C containing 0.1 mM phenylmethylsulfonyl fluoride, resuspended in 10 ml of lysis buffer, and lysed by vortexing for 5 1 min with an equal volume of glass beads (diameter 0.5 mm). The crude extract was first centrifuged at 4 °C, 10,000 g for 30 min, and then at 4 °C, 100,000 g for 1 h. 30 mg of soluble protein was applied onto a 1-ml Resource Q (Pharmacia) chromatography column and eluted with an linear gradient, ranging from 0 to 1500 mM KCl. 1-ml fractions were collected, and GTPase activity was measured using filter binding assays. 1 µM Gsp1p was incubated for 15 min at 30 °C with column fractions diluted 1:100.

RESULTS

Analogous to mammalian cells where Ran is regulated by RCC1, Gsp1p of S. cerevisiae is regulated by the gene product of PRP20(14, 15) . Mutants of PRP20 are defective in processing and exporting mRNA from the nucleus (17, 19) . A similar phenotype was observed in yeasts carrying mutations in the RNA1 gene. Mutations in the RNA1 gene disrupted the mRNA 3`-end formation and prevented the transport of newly synthesized mRNA to the cytoplasm (19, 21, 22, 23) . In addition, in yeast cells where the GSP1 gene was under control of a GAL1 promotor, mRNA began to accumulate inside the nucleus 15 h after a switch to a glucose medium (15) . The observation that mutants of PRP20, RNA1, and GSP1 have similar phenotypes suggested that their gene products might function in a common pathway. Furthermore, immunofluorescence microscopy with antiserum against rna1p, the Rna1p homologous protein of S. pombe, showed a preferential staining of the nuclear periphery (29). Rna1p is therefore positioned to play a potential role in nuclear transport. Through these observations Rna1p, for which no biochemical function had yet been identified, became a prime candidate as a protein that could potentially interact with Gsp1p and that could possibly act as a GTPase-activating protein for Gsp1p. To investigate the interaction between Gsp1p and Rna1p we expressed and purified recombinant Gsp1p and Rna1p. GSP1 and RNA1 genes were cloned into pET3d and expressed in E. coli strain BL21(DE3). After induction with isopropyl-1-thio--D-galactopyranoside, large amounts of expressed proteins were visible in a Coomassie-stained SDS gel (Fig. 1, A and B, lanes 2).


Figure 1: Expression of Gsp1p (A) and Rna1p (B) in E. coli. SDS-PAGE of total bacterial lysates of cells containing pET3d (A and B, lane 1), pET3d-GSP1 (A, lane 2), or pET3d-RNA1 (B, lane 2). SDS-PAGE of pooled fractions after a SP-Sepharose chromatography (A, lane 3) followed by Superdex 200 FPLC gel filtration (A, lane 4) or after DEAE-Sephadex chromatography (B, lane 3) followed by Superdex 200 FPLC gel filtration (B, lane 4).



Purification of Gsp1p

Bacterially expressed Gsp1p had an apparent molecular mass of 24 kDa which is in good keeping with the calculated molecular mass of 24,810 Da. It was purified in a two-step purification procedure. First the soluble bacterial protein extract was fractionated by gradient elution (0-500 mM KCl) from a SP-Sepharose column at pH 6.0. Fractions containing Gsp1p were identified by SDS-PAGE, pooled (Fig. 1A, lane 3), and further purified on a Superdex 200 FPLC chromatography column at pH 6.0. A typical purification from 80 g of cells produced about 80 mg of >90% pure protein as judged by SDS-PAGE (Fig. 1A, lane 4). The purified protein showed a minor second band when the SDS gel was overloaded. Most likely the lower band is a degradation product. A similar degradation band has been observed during the purification of other small GTPases (30) . Any attempts to purify Gsp1p at a higher pH failed, as the protein did not bind to anion or to cation exchange columns under these conditions. Attempts to neutralize the buffer after the first purification step also failed since even the most careful titration rendered the protein inactive, as measured by its ability to bind GDP. Therefore all subsequent measurements involving Gsp1p were performed at pH 6.0.

The activity of the purified protein was determined by its ability to bind GDP. Comparing the measured protein concentration with the amount of protein able to bind GDP in a nitrocellulose filter assay, we consistently found that only 50-70% of purified Gsp1p was active. This phenomenon has been observed with other small GTP-binding proteins assayed in filter binding assays. In the case of Ran, fluorescence spectroscopy showed that Ran forms a 1:1 complex with the guanine nucleotide although filter binding tests indicated less nucleotide binding (25) . Testing the nucleotide-bound state of purified Gsp1p on high performance liquid chromatography (31) we found that >90% of the protein was complexed with guanine nucleotide. Thus we conclude that Gsp1p forms a tight equimolar complex with the nucleotide as has been observed for all other ras-related GTP-binding proteins. Insufficient recovery of radioactive nucleotide in the nitrocellulose filter assay could either be attributed to insufficient binding of Gsp1p itself or to limited protein denaturation which leads to nucleotide release. In general we noticed that the nucleotide binding activity of Gsp1p is very sensitive to changes in pH or ionic strength of the solvent and to freezing/thawing procedures.

Purification of Rna1p

Bacterially expressed Rna1p migrated with an apparent molecular mass of 46 kDa (Fig. 1B) which correlates the calculated molecular mass of 46,103 Da. Rna1p was purified in a two-step purification procedure involving a gradient elution (0-1500 mM KCl) on a DEAE Sephadex column at pH 6.0 and a Superdex 200 FPLC chromatography step (Fig. 1B, lanes 3 and 4). A typical preparation produced 50-60 mg of >95% pure protein per 80 g of cell paste as judged by SDS-PAGE (Fig. 1B, lane 4). Unlike Gsp1p, Rna1p was very stable tolerating changes in pH as well as changes in the ionic strength of the solvent.

Interaction between Gsp1p and Rna1p

To measure the effect of Rna1p on the GTPase activity of Gsp1p, 0.5 µM Gsp1p was incubated with 0.1 nM Rna1p. The charcoal assay demonstrated that 0.1 nM Rna1p increases GTP hydrolysis 30-fold over the intrinsic rate when initial rates were compared (Fig. 2A). The rate of GTP hydrolysis is dependent on the concentration of Rna1p in the concentration range of 0.05 to 10 µM Gsp1p (Fig. 2B). Treating the Rna1p induced GTP hydrolysis as an enzymatic reaction, we attempted to determine the Kand k given that Ran and Gsp1p are 88% identical, we therefore expected a Kvalue similar to that for RanGAP1 stimulated GTP-hydrolysis of Ran which is 0.43 µM(3) . Unexpectedly the Kof the Rna1p-induced GTP hydrolysis appears to be much higher than that of RanGAP1-induced GTP hydrolysis of Ran. Due to protein instability of Gsp1p at higher protein concentrations we were unable to follow the rate of GTP hydrolysis at substrate concentrations higher than 10 µM. However, our estimates indicate that Kand V must be at least 30 µM and 300 s, respectively (Fig. 2B). Both values are almost 100-fold higher than the Kand V of the RanGAP1-induced GTP hydrolysis of Ran. As the intrinsic GTP hydrolysis of Gsp1p is 5 10 s, we calculated that the GTP hydrolysis of Gsp1p is stimulated at least 0.5-1 10-fold. This increase in GTP hydrolysis is significantly higher than that of other GTPase-activating proteins, like RanGAP1 (10-fold) (3) and that of RasGAP (2, 5 10-fold) (32, 33) .


Figure 2: Rna1p activates the GTPase activity of Gsp1p. 0.5 µM Gsp1p complexed with [-P]GTP was incubated with () or without () 0.1 nM Rna1p for increasing amounts of time. Liberated [-P] was determined in a charcoal assay (27) (A). The rate increase of GTP hydrolysis is dependent on the substrate (Gsp1p) concentration. 0.1 nM Rna1p was incubated with increasing amounts of Gsp1p as indicated and the initial rates of GTP hydrolysis are represented as a function of Gsp1p concentration (B). Data were fitted non-linearly to a hyperbolic function (the Michaelis-Menten equation) using the computer program GraFit (Erithacus Software).



The ability of Rna1p to accelerate the GTP hydrolysis appears to be specific for Gsp1p, as Rna1p is unable to increase the GTPase activity of H-Ras or Rab7 even when incubated with 100-fold more Rna1p (10 nM) than used with Gsp1p (Fig. 3). Therefore, Rna1p appears to be a GTPase-activating protein specific for Gsp1p.


Figure 3: Rna1p is specific for Gsp1p. 1 µM Gsp1p (, ), H-Ras (, ), and rab7 protein (, ) complexed with [-P]GTP was incubated at 30 °C with (open symbols) or without (closed symbols) 0.1 nM Rna1p (Gsp1p) or 10 nM Rna1p (H-Ras, Rab7). GTPase activity was determined as the decrease of [-P]GTP bound to nitrocellulose filters. Data are presented as percent of the radioactivity bound to Gsp1p 30 s after addition of Rna1p.



Interaction of Ran/Gsp1p with Rna1p Homologues

Genes homologous to RNA1 have been found in different organisms. Comparison of the deduced amino acid sequence of RNA1 (S. cerevisiae) (23) , rna1 (S. pombe) (29), CEC29E4 (C. elegans) (34) , fug1 (mouse) (35), and RanGAP1 (human) (24) reveals that these proteins are highly conserved (24) . The conserved primary structure of the RNA1 related genes was also suggestive of a conserved function. We therefore tested whether Rna1p homologues from different species could activate GTP hydrolysis of Gsp1p. As shown in Fig. 4A, 0.1 nM RanGAP1 purified from HeLa cells and 0.1 nM bacterially expressed rna1p were able to increase the GTPase activity of Gsp1p. The observed rates of the induced GTPase activities are very similar, suggesting that these GTPase-activating proteins are functionally and structurally closely related.


Figure 4: The GTPase activity of Gsp1p is activated by S. pombe rna1p and human RanGAP1 while the GTPase activity of Ran is induced by RanGAP1 and rna1p but not by Rna1p. 1 µM Gsp1p (A) or Ran (B) complexed with [-P]GTP was incubated at 30 °C with 0.1 nMS. cerevisiae Rna1p () or 0.1 nMS. pombe rna1p (), or 0.1 nM human-RanGAP1 (▾) or with buffer (). GTPase activity was determined as the decrease of [-P]GTP bound to nitrocellulose filters. Data are presented as the percentage of the amount of radioactivity bound to Ran/Gsp1p 30 s after addition of the respective GAP (``zero time point'').



Testing whether the GTPase activity of human Ran could also be enhanced by S. cerevisiae Rna1p and S. pombe rna1p, we found that Rna1p was unable to increase the GTP hydrolysis of Ran while rna1p could (Fig. 4B). The observation that Gsp1p is able to interact with all three GAPs investigated while Ran is not indicates that the specificity of the interaction rests mainly with the nucleotide-binding protein rather than with the GTPase-activating protein.

It was suggested that in order for Ran to facilitate import and export of proteins and small nuclear RNP into and out of the nucleus, Ran would have to shuttle from the cytoplasm into the nucleus and back. For this mechanism it was suggested that two sets of GEFs and GAPs specific for Ran would have to exist (36) , each regulating one cycle of GTP binding, GTP hydrolysis, and GDP dissociation specific for one direction of this bidirectional transport. To investigate whether more than one Gsp1p-specific GAP activity exists in yeast, we fractionated a 100,000 g supernatant of a protein extract from S. cerevisiae on a Resource Q column (Fig. 5). We detected only one discrete peak of GAP activity specific for Gsp1p eluting at about 550 mM salt. Purified Rna1p eluted at the same salt concentration under these conditions. Therefore, we did not find any evidence for more than one GAP activity specific for Gsp1p, although we cannot exclude the possibility of overlapping activities.


Figure 5: GTPase activating activity specific for Gsp1p in a cell extract from S. cerevisiae. A 100,000 g supernatant of a crude cell lysate was eluted from a Resource Q FPLC column using a 0-1500 mM KCl gradient. 1-ml fractions were analyzed for protein concentration () and GTPase activity of Gsp1p (hatched bars). GTPase activity was determined in filter binding assays. The hatched bar on the left represents 100% GTP hydrolysis and the solid bar represents the intrinsic amount of GTP hydrolysis by Gsp1p.



DISCUSSION

We have demonstrated here that Rna1p from S. cerevisiae is a GTPase-activating protein for Gsp1p. Sequence analysis reveals that RNA1 belongs to a family of conserved genes, including human (RanGAP1), mouse (fug1), S. pombe (rna1), S. cerevisiae (RNA1), and C. elegans(24) . When compared to members of the RasGAP family, Rna1p homologues are relatively small and significantly stronger conserved. Furthermore, the conserved regions extend over the entire protein, while in RasGAPs only a small region which possesses catalytic activity is conserved.

In Caenorhabditis elegans the open reading frame coding for a gene homologous to RNA1 was identified during the genome sequencing project (34) . This DNA sequence exists as a tandem repeat, however, the functional significance of this tandem repeat is unclear. Our biochemical analysis suggests that Rna1p homologous proteins from different species are also functionally conserved as they react with Ran homologous proteins from different species. This finding is corroborated by the observation that rna1 from S. pombe can functionally replace the RNA1 gene from S. cerevisiae(29) .

The most prominent features of Rna1p homologous proteins are the so called leucine-rich repeats (LRR) constituting the major part of the protein sequence. LRRs have been found in proteins with different functions and intracellular localizations and participate in protein-protein interaction (37) . The three-dimensional crystal structure of one of the LRR containing proteins, the porcine RNase inhibitor, has been determined recently (38) and shown to have a nonglobular horseshoe-like structure where the LRR's form repeating - structural units. The -strands form a curved -sheet constituting the inner circumference, while the -helices align on the outer circumference (38) . In addition to the conserved leucines the LRR-containing proteins contain a conserved Asn, Cys, or Thr at a defined position of the repeat, but the functional and evolutionary significance of the various consensus residues is as yet unknown. Rna1p homologous proteins have a conserved asparagine at this position. Furthermore, the RNase inhibitor contains 14 repeats while Rna1p contains only 8, two of which appear to be separated by intervening sequences (29) . Although it is likely that the LRRs of Rna1p homologues form similar characteristic - structural units it will be interesting to see the structural and functional difference between these proteins.

Comparing the primary structures of GAPs for Ran with those for Ras, Rap, Rho, and Rab, these proteins show no apparent similarity (39) . It is striking that although GAPs specific for different GTP-binding proteins share no apparent sequence homology, at least Ran-GAP and Ras-GAP stimulate GTP hydrolysis at least 10-fold (this paper, Refs. 3 and 32). In the case of the GTPase activity of Ras it has been argued that residues of Ras-GAP contribute to the active site to stabilize the reaction intermediate and/or transition state of the reaction (40) . Potential residues for such stabilization, i.e. arginines, have been identified as totally conserved residues in Ras-GAP and their mutagenesis reduces k approximately 50-fold.() It is interesting that similar residues are also totally conserved in Ran-GAP's.

In addition to the yeast proteins, mammalian Rna1p homologues contain an additional 260 amino acid residues at the C-terminal end. As rna1p from S. pombe is able to induce the GTPase activity of Ran, we conclude that these C-terminal extensions are not involved in inducing GTP hydrolysis or providing protein specificity. This observation that the GTP hydrolysis of human Ran is not induced by Rna1p from S. cerevisiae is reminiscent of the cross-reactivity of human H-Ras and yeast Ras1p with human RasGAP and Ira1p from yeast. While the GTPase activity of Ras1p was stimulated by Ira1p and RasGAP, the GTP hydrolysis of H-Ras was only catalyzed by RasGAP but not by Ira1p (41) .

Originally RNA1 was identified in a genetic screen selecting for yeast mutants defective in mRNA processing (22) . Mutants of RNA1 were also defective in exporting mRNA from the nucleus (19, 42, 43). In S. cerevisiae, Rna1p is described as a cytosolic protein apparently excluded from the nucleus (44) . The cytoplasmic localization of Rna1p was hard to reconcile with its apparent functions inside the nucleus, i.e. mRNA processing and export (44) . As Gsp1p is localized mainly inside the nucleus (14) , the regulation of Gsp1p by Rna1p provides the means for Rna1p to exert its function inside the nucleus. This could explain why GSP1 and RNA1 mutants have similar phenotypes (15, 19) . If Rna1p and Gsp1p are localized in different cellular compartments how do they effectively interact with each other? The observation that a small fraction of rna1p in S. pombe is localized to the nuclear envelope (29) suggests that the location of interaction could be the interface between the nuclear matrix and cytoplasm, possibly the nuclear pore. As the nuclear pore complex is crucial for both nuclear import and export it would explain why mutants of Rna1p and Gsp1p effect both processes.

Using in vitro nuclear protein import assays two cytosolic fractions termed A and B have been characterized, each facilitating a specific step of nuclear protein import (45) . Fraction A promotes the first step, the binding of nuclear localization sequence-tagged proteins to the nuclear envelope. Importin was recently found to be involved in this targeting (46) . The second step is ATP-dependent and facilitates the actual translocation of proteins into the nucleus. Ran is the active component of the second step (12, 13) and together with the newly identified importin is the only protein necessary for complete nuclear protein import in vitro(46) . For full import activity, a Ran interacting protein of 10 kDa was found to be required (49) . Importin contains an 8-fold repeat of an hydrophobic motif, the so called arm motif (47) . Among other proteins the arm motif is found in -catenin, plakoglobin, adenomatosis polyposis coli, p120, and smgGDS (47) . The arm motif is believed to function in protein-protein interactions and although it remains to be determined whether Ran interacts directly with importin it is interesting that one of the arm motif containing proteins is a GTPase-regulatory protein.

It is noteworthy that RCC1/Prp20 and RanGAP1/Rna1p have never been implicated in nuclear protein import. Detailed analysis of the interaction between Ran/Gsp1 and their interacting factors like GAPs, GEFs, or the recently cloned Ran-binding proteins (48, 49, 50) will be necessary to show how the cycle of GTP binding, GTP hydrolysis, and GTP/GTP exchange regulates nuclear transport mechanisms or cell cycle events. The multitude of different defects caused by mutations in Ran, RCC1, RNA1, and their relatives from other organisms makes it difficult to assign a unifying function for these proteins. The current data is insufficient to decide whether these proteins have many different functions or whether a defect in one key function is the basis for the pleiotropic defects observed. At this point it is not entirely unlikely that the nucleocytoplasmic transport could be this principal function as the cell cycle regulation depends on the signal transduction from the exterior through the cytoplasm into the nucleus.


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: Max-Planck-Institute für Molekulare Physiologie, Abteilung für Strukturelle Biologie, P. O. Box 10 26 64, 44026 Dortmund, Germany. Tel.: 49-0231-1206-240; Fax: 49-0231-1206-230; E-mail: jbecker@mpi-dortmund.mpg.de.

The abbreviations used are: RCC1, regulator of chromosome condensation; Ran, ras-related nuclear protein; RanGAP1, the human GTPase-activating protein for Ran; PRP20, S. cerevisiae gene functionally homologous to RCC1 involved in precursor RNA processing; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; GSP1, GTP binding suppressor of PRP20, S. cerevisiae gene homologous to Ran/TC4; RNA1, S. cerevisiae gene coding for protein involved in mRNA processing and transport; LRR, leucine-rich repeats; PAGE, polyacrylamide gel electrophoreis; DTE, dithioerythritol.

X. Skinner and L. Wiesmüller, unpublished data.


ACKNOWLEDGEMENTS

We thank C. Koerner for excellent technical assistance, I. Simon for providing purified Rab7 protein, C. Klebe for providing purified Ran protein, and C. Herrmann and R. H. Cool for discussions.


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