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Human Rangtpase-Activating Protein Rangapi Is a Homologue of Yeast Rnalp Involved in Mrna Processing and Transport (TC4/Rccl/Fi*Gl/Nucleocytoplasmic Transport) F

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Human Rangtpase-Activating Protein Rangapi Is a Homologue of Yeast Rnalp Involved in Mrna Processing and Transport (TC4/Rccl/Fi*Gl/Nucleocytoplasmic Transport) F Proc. Natl. Acad. Sci. USA Vol. 92, pp. 1749-1753, February 1995 Biochemistry Human RanGTPase-activating protein RanGAPi is a homologue of yeast Rnalp involved in mRNA processing and transport (TC4/RCCl/fi*gl/nucleocytoplasmic transport) F. RALF BISCHOFF, HEIKE KREBBER, TORE KEMPF, INGRID HERMES, AND HERWIG PONSTINGL Division for Molecular Biology of Mitosis, German Cancer Research Center, POB 101949, D-69009 Heidelberg, Germany Communicated by Hans Neurath, University of Washington, Seattle, WA, November 22, 1994 ABSTRACT RanGAPI is the GTPase activator for the parallel to our work on human RanGAP1 we have purified the nuclear Ras-related regulatory protein Ran, converting it to major RanGAP activity from Sc. pombe cells and identified it the putatively inactive GDP-bound state. Here, we report the to be Rnalp. amino acid sequence of RanGAPI, derived from cDNA and peptide sequences. We found it to be homologous to murine Fugl, implicated in early embryonic development, and to MATERIALS AND METHODS Rnalp from Saccharomyces cerevisiae and Schizosaccharomyces Purification of Rnalp from Sc. pombe. The Sc. pombe strain pombe. Mutations of budding yeast RNA] are known to result 972h-, provided by Jorg D. Hoheisel (German Cancer Re- in defects in RNA processing and nucleocytoplasmic mRNA search Center, Heidelberg), was grown to a density of OD6wi transport. Concurrently, we have isolated Rnalp as the major = 1 in M9 minimal medium (18). The cells were collected by RanGAP activity from Sc. pombe. Both this protein and re- 10-min centrifugation at 5000 x g, and spheroblasts were combinant Rnalp were found to stimulate RanGTPase activ- prepared as described by Melchior et al. (19). Spheroblasts ity to an extent almost identical to that of human RanGAP1, from 10 ml of packed cells were lysed by Dounce homogeni- indicating the functional significance of the sequence homol- zation in 50 ml of lysis buffer (20 mM Tris HCl, pH 7.5/1 mM ogy. The Ran-specific guanine nucleotide exchange factor EDTA/1 mM 2-mercaptoethanol) containing protease inhib- RCC1 and its yeast homologues are restricted to the nucleus, itors (1). After centrifugation for 1 hr at 70,000 x g, ammo- while Rnalp is reported to be localized to the cytoplasm. We nium sulfate to 60% saturation was added to the supernatant. suggest a model in which both activities, nuclear GDP-to-GTP The precipitate was removed by centrifugation for 20 min at exchange on Ran and cytoplasmic hydrolysis of Ran-bound 70,000 x g, and the supernatant was directly chromatographed GTP, are essential for shuttling of Ran between the two on a Fractogel EMD AFTA 650/S column (10 x 50 mm; cellular compartments. Thus, a defect in either of the two Merck) at a flow rate of 2 ml/min, applying a linear gradient antagonistic regulators of Ran would result in a shutdown of from 60% saturated ammonium sulfate in lysis buffer to lysis Ran-dependent transport processes, in agreement with the buffer containing 100 mM NaCl. The buffer of fractions con- almost identical phenotypes described for such defects in taining RanGAP activity was changed on a Nap 25 column budding yeast. (Pharmacia) to 100 mM NaCl in lysis buffer, and the sample was applied to a Mono Q column (HR 5/5; Pharmacia) equil- Ran is a Ras-related, mainly nuclear protein (1, 2) which in its ibrated in the same buffer. Pure RanGAP/Rnalp was eluted GTP-bound form is thought to represent the ON state of a at "400 mM NaCl, using a linear gradient to 1 M salt in lysis regulatory pathway involved in the onset of mitosis (3, 4), buffer. RanGAP assays using Ran [y-32P]GTP have been done initiation of S-phase (4), exit from mitosis (5), import of as described (16). proteins with nuclear localization signals into the nucleus (6, Generation of Fragments and Amino Acid Sequence Deter- 7), maintenance of nuclear structure, and pre-mRNA process- mination. Purified human RanGAP1 was cleaved with trypsin ing and export into the cytoplasm (8, 9). Homologues of Ran (sequencing grade; Boehringer Mannheim) in 100 mM am- have been identified in many different species (for review, see monium bicarbonate, pH 8.3/1 M guanidinium chloride for 16 ref. 10). Activation of Ran requires the exchange of bound hr at 37°C. Rnalp isolated from Sc. pombe was incubated for GDP for GTP, which is stimulated by its specific nucleotide 16 hr at 37°C with endoproteinase Lys-C (sequencing grade; exchange factor (11), designated RCC1 in mammals (12), Boehringer Mannheim) in 50 mM Tris-HCl, pH 8.5/10% SRM1/PRP20/MTR1 in Saccharomyces cerevisiae (9, 13, 14), (vol/vol) acetonitrile. Cleavage with CNBr, separation of pep- and piml in Schizosaccharomyces pombe (5, 15). Suppression tides by reversed-phase chromatography, and sequence deter- of the phenotypes that result from temperature-sensitive mu- mination was done as described (16). tations in the RCC1 homologues of Sc. pombe and Sa. cerevi- Cloning of Human RanGAP1. Based on the peptide se- siae, by overexpressing the corresponding Ran homologues (5, quences of purified RanGAP1 indicated in Fig. 1, degenerate 8, 9, 15), indicates that RCC1 and Ran participate in the same oligonucleotides were synthesized containing inosine in the regulatory pathway. wobble position at a degeneracy of three or four. PCR ampli- Ran has a very low intrinsic GTPase activity. It is stimulated fication with a HeLa Agtll cDNA library (Clontech) resulted 105-fold by the Ran GTPase activator RanGAP1 (16, 17), in a 800-bp fragment that was cloned into pBluescript KS' which we have purified from HeLa cells. This protein is an after digestion with EcoRI and BamHI. The 800-bp EcoRI- antagonistic regulator to RCC1, converting Ran into its GDP- BamHI fragment that was found to code for RanGAP1, as bound putative OFF form. Here we report the determination of verified by the sequences of additional RanGAP1 peptides, its amino acid sequence, based on data from peptides and was used to generate a hybridization probe labeled with cDNA.t The completed sequence revealed homologies to mu- [a-32P]ATP using the DECAprimeII-DNA-labeling kit (Am- rine Fugl and to Sa. cerevisiae and Sc. pombe Rnalp. In bion, Austin, TX). A HeLa Agtll cDNA library was screened as in ref. 20. Cross-hybridizing clones were purified and used The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in tThe sequence reported in this paper has been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. EMBL/GenBank data base (accession no. X82260). 1749 Downloaded by guest on September 25, 2021 1750 Biochemistry: Bischoff et aL Proc. Natl. Acad Sci. USA 92 (1995) for DNA isolation. Inserts cut with EcoRI were isolated from shaker. After the time intervals indicated in Fig. 5, 10 ,lI of agarose gels using the JetSorb-DNA-elution kit (Genomed, tsBN2 lysate and 10 ,ul of a 1:4 dilution of BHK21 lysate in lysis Bad Oeynhausen, Germany) and subcloned into pBluescript buffer, respectively, were added to 70 ,ul of 1 ,uM Ran- KS'. Both strands of the inserts were sequenced by using the [3H]GDP in incubation buffer containing 250 ,tM unlabeled T7Sequencing kit (Pharmacia). GDP. Ran-bound radioactivity was determined after 10-min Cloning and Expression of Sc. pombe RNA1. Oligonucleo- incubation at 25°C on a rotation shaker by filtering the sample tides representing the N terminus (5'-CGGAATTCATAT- through nitrocellulose as described (1). GTCGCGTTTTTCAATA-3') and C terminus of Rnalp (5'- CGGGATCCCTAAATATGAGCTTTTGA-3') were used to RESULTS AND DISCUSSION amplify the coding sequence from Sc. pombe genomic DNA, which was provided by Jorg D. Hoheisel. The resulting 1161-bp Human RanGAPi Is Structurally Homologous to Murine Nde I-BamHI fragment was ligated into the pET3a expression Fugl and to Yeast Rnalp. RanGAP1 was purified from HeLa vector. After transformation into Escherichia coli BL21(DE3), cells as a homodimeric 65-kDa protein (16) and cleaved into protein expression was induced for 2 hr with 0.5 mM isopropyl peptides that were sequenced. The partial amino acid se- /3-D-thiogalactoside after bacteria were grown at 37°C to an quences (Fig. 1) were used to design degenerate primers, and OD6wo of 0.4 in a 4-liter culture. Cells were harvested and lysed a 800-bp DNA fragment was obtained from a HeLa kgtll as described (16). Recombinant Rnalp was purified from the cDNA library by PCR. By screening the same library with the soluble fraction as described above for endogenous Rnalp. amplification product, a cDNA of 2.9 kb could be isolated. The However, a larger proportion of recombinant Rnalp already nucleotide sequence comprises an open reading frame coding precipitated at <60% ammonium sulfate saturation. for a protein of 587 amino acid residues and a calculated Mr Determination of Ran-Specific Guanine Nucleotide Ex- of 63,577. The primary structure suggests an N-terminal do- change Activity in tsBN2 and BHK21 Cells. Ran-[3H]GDP was main of 358 residues, separated by an extremely acidic string prepared by incubating 2 ,tM bacterially expressed Ran-GDP of some 40 residues from a C-terminal region of 190 residues. (16) with 10 ,tM [3H]GDP (10 Ci/mmol; NEN; 1 Ci = 37 GBq) The sequence appears to be unrelated to that of GTPase in 25 mM Mes, pH 6.5/500 mM NaCl/1 mM 2-mercaptoetha- activators for other Ras-related proteins, but it is highly ho- nol/10% (vol/vol) glycerol/10 mM EDTA for 30 min on ice. mologous (88% identical residues) to that of Fugl (21), the The buffer was changed to 20 mM Hepes-NaOH, pH 7.4/5 mM murine homologue ofyeast Rnalp (Fig.
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