THE RAN TC4 GTPASE-BINDING DOMAIN - IDENTIFICATION BY EXPRESSION CLONING AND CHARACTERIZATION OF A CONSERVED SEQUENCE MOTIF/

Ran/TC4 is an essential, nuclear GTPase implicated in the initiation o f DNA replication, entry into and exit from mitosis, and in nuclear RN A and protein transport through the nuclear pore complex. This diversi ty of functions suggests that Ran interacts with a large number of dow n-stream targets. Using an overlay assay, we detected a family of puta tive target proteins that associate with GTP-bound Ran. The sequence o f only one such protein, HTF9a/RanBP1, is known. We have now cloned tw o additional Ran-binding proteins, allowing identification of a distin ctive, highly conserved sequence motif of approximate to 150 residues. This motif represents a minimal Ran-binding domain that stabilizes th e GTP-bound state of Ran. The isolated domain also functions as a coac tivator of Ran-GTPase-activating protein. Mutation of a conserved resi due within the Ran-binding domain of HTF9a protein drastically reduced Ran binding. Ran-binding proteins coimmunoprecipitated with epitope-t agged Ran from cell lysates, suggesting that these proteins may associ ate in vivo. A previously uncharacterized Caenorhabditis elegans gene could encode a protein (96 kDa) possessing two Ran-binding domains. Th is open reading frame also contains similarities to nucleoporins, sugg esting a functional link between Ran and nuclear pore complexes.