The Ran / TC 4 GTPase - binding domain : Identification by expression cloning and characterization of a conserved sequence motif ( GTP - binding protein / nucleoporin / cell

Ran/TC4 is an essential, nuclear GTPase implicated in the initiation of DNA replication, entry into and exit from mitosis, and in nuclear RNA and protein transport through the nuclear pore complex. This diversity of functions suggests that Ran interacts with a large number of downstream targets. Using an overlay assay, we detected a family of putative target proteins that associate with GTP-bound Ran. The sequence of only one such protein, HTF9a/RanBPI, is known. We have now cloned two additional Ran-binding proteins, allowing identification of a distinctive, highly conserved sequence motif of 150 residues. This motif represents a minimal Ran-binding domain that stabilizes the GTP-bound state of Ran. The isolated domain also functions as a coactivator of Ran-GTPase-activating protein. Mutation of a conserved residue within the Ran-binding domain of HTF9a protein drastically reduced Ran binding. Ran-binding proteins coimwunoprecipitated with epitope-tagged Ran from cell lysates, suggesting that these proteins may associate in vivo. A previously uncharacterized Caenorhabditis elegans gene could encode a protein (96 kDa) possessing two Ran-binding domains. This open reading frame also contains similarities to nucleoporins, suggesting a functional link between Ran and nuclear pore complexes. The Ran/TC4 GTPase is a highly conserved nuclear protein expressed in all eukaryotic cells examined to date (1-3). Disruption of Ran function, by either the introduction into cells of dominant gain-of-function mutants or removal of a regulatory factor, results in a pleiotropic phenotype characterized by cell-cycle arrest (4-8), premature chromosome condensation (2, 8) or exit from mitosis (9), and the accumulation of incorrectly processed nuclear RNA (10-12). In Xenopus oocyte preparations a dominant loss-of-function mutant of Ran blocks nuclear growth and the entry into S phase and inhibits dephosphorylation of the cdc2 p34 mitotic protein kinase (5), preventing the initiation of mitosis. In vitro studies also indicate that Ran may be an essential cytosolic component for the transport of karyophilic proteins into the nucleus through the nuclear-pore complex (13, 14). This extraordinary diversity of functions suggests that Ran likely interacts with a large number of downstream target proteins. Using an overlay assay, we have previously identified a number of proteins that bind to and stabilize the GTP-bound state of Ran (15). The smallest of these proteins, Ran-binding protein 1 (RanBP1), has been cloned and is identical to a previously sequenced open reading frame called HTF9a (16, 17) that encodes a polypeptide of -24 kDa. Many of the other proteins that bind Ran in the overlay assay are >100 kDa in size. Here we report the cloning of two additional RanBPs from a human hippocampal cDNA library and the identificaThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. tion of a highly conserved sequence motif that functions as a Ran-binding domain (RanBD).t The isolated domain stabilizes the GTP-bound state of Ran and acts as a costimulator of Ran GTPase activity in the presence of Ran-GTPaseactivating protein (GAP). A previously unidentified gene, sfol, from Saccharomyces cerevisiae possesses one potential RanBD (J. Trueheart and J. Thorner, personal communication), and a Caenorhabditis elegans gene could encode a protein that posseses two RanBDs. This latter gene additionally shows similarities to nucleoporins NUP153p, NSP1, and NUP255p (also known as the protooncogene CAN) (18, 19). MATERIALS AND METHODS Expression Cloning. A human hippocampus cDNA expression library in Lambda ZAPII (Stratagene) was screened with [a-32P]GTP-Ran, essentially as described by Coutavas et at (16). Recombinant Ran protein loaded with [a-32P]GTP and the complex was stabilized by addition of 10 mM MgCl2 (15). Unincorporated nucleotide was removed by passage over a Pharmacia PD-10 column. Plaques were lifted after induction for 4 hr with isopropyl f3-D-thiogalactoside and renatured overnight in 20 mM Hepes, pH 7.5/25 mM potassium acetate/10 mM magnesium acetate/5 mM dithiothreitol/50 ,tM GTP/50 ,uM GDP/4% nonfat dried milk/0.25% Tween 20. This was replaced with a similar buffer containing 0.05% Tween-20 and [a-32P]GTP-Ran (106 cpm/ml), and filters were incubated at 4°C for 2 hr and then washed five times in 150 mM NaCl/20 mM Tris, pH 7.4/10 mM magnesium acetate. pBluescript SK plasmids were excised from tertiary plaques according to the manufacturer's instructions (Stratagene). As a final test for clones that express Ran-binding proteins, bacteria containing plasmids with putative positive inserts were incubated with isopropyl P3-D-thiogalactoside to induce protein expression, and then bacterial extracts were run on SDS/ PAGE and transferred to nitrocellulose. RanBPs were detected by a Ran overlay assay as described (15). Two independent clones were identified that expressed RanBPs of -50 kDa (clone AB1) and 90 kDa (clone AB2) (data not shown). Inserts were completely sequenced in both directions by using an ABI automated sequencer. Expression and Analysis of the AB1 RBD. The putative Ran-binding domain of clone AB1 (390 bp, residues PHFE ... CKFE) was amplified by PCR and subcloned into pGEX-2T. The resulting glutathione S-transferase (GST) fusion protein was expressed in Escherichia coli and purified over glutathiAbbreviations: GST, glutathione S-transferase; RanBD, Ran-binding domain; RanBP, Ran-binding protein; GAP, GTPase-activating protein. *To whom reprint requests should be addressed at: Department of Pathology, Medical Alumni Building, Room A143, University of Vermont, Burlington, VT 05405-0068. tThe sequences reported in this paper have been deposited in the GenBank data base (accession nos. U19240 and U19248).