DISASSEMBLY OF RANGTP-KARYOPHERIN-BETA COMPLEX, AN INTERMEDIATE IN NUCLEAR-PROTEIN IMPORT

We previously showed that RanGTP forms a 1:1 complex with karyopherin beta that renders RanGTP inaccessible to RanGAP (Floer, M., and Blobel , G. (1996) J. Biol. Chem. 271, 5313-5316) and karyopherin beta functi onally inactive (Rexach, M., and Blobel, G. (1995) Cell 83, 683-692). Recycling of both factors for another round of function requires disso ciation of the RanGTP-karyopherin beta complex, Here we show using BIA core(TM), a solution binding assay, and GTP hydrolysis and exchange as says, with yeast proteins, that karyopherin beta and RanGTP are recycl ed efficiently in a reaction that involves karyopherin alpha, RanBP1, RanGAP, and the C terminus of the nucleoporin Nup1. We find that karyo pherin alpha first releases RanGTP from karyopherin beta in a reaction that does not require GTP hydrolysis, The released RanGTP is then seq uestered by RanBP1, and the newly formed karyopherin alpha beta binds to the C terminus of Nup1. Finally, RanGTP is converted to RanGDP via nucleotide hydrolysis when RanGrAP is present, Conversion of RanGTP to RanGDP can also occur via nucleotide exchange in the presence of RanG EF, an excess of GDP, and if RanBP1 is absent. Additional nucleoporin domains that bind karyopherin alpha beta stimulate recycling of karyop herin beta and Ran in a manner similar to the C terminus of Nup1.