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 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 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...

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