Abstract
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 functionally inactive (Rexach, M., and Blobel, G. (1995) Cell 83, 683-692). Recycling of both factors for another round of function requires dissociation of the RanGTP-karyopherin beta complex. Here we show using BIAcoreTM, a solution binding assay, and GTP hydrolysis and exchange assays, with yeast proteins, that karyopherin beta and RanGTP are recycled efficiently in a reaction that involves karyopherin alpha, RanBP1, RanGAP, and the C terminus of the nucleoporin Nup1. We find that karyopherin alpha first releases RanGTP from karyopherin beta in a reaction that does not require GTP hydrolysis. The released RanGTP is then sequestered by RanBP1, and the newly formed karyopherin alphabeta binds to the C terminus of Nup1. Finally, RanGTP is converted to RanGDP via nucleotide hydrolysis when RanGAP is present. Conversion of RanGTP to RanGDP can also occur via nucleotide exchange in the presence of RanGEF, an excess of GDP, and if RanBP1 is absent. Additional nucleoporin domains that bind karyopherin alphabeta stimulate recycling of karyopherin beta and Ran in a manner similar to the C terminus of Nup1.
Highlights
Transport of proteins that contain a nuclear localization signal (NLS)1 into the nucleus of the cell requires energy, mobile transport factors, and nuclear pore complexes (NPC) in the nuclear envelope
Estimates from the inhibition of RanGAP by karyopherin b indicate an affinity below 1 nM for the RanGTP-karyopherin b interaction2 (0.3 nM for mammalian proteins [39])
As RanGAP is synthetically lethal with the C terminus of the nucleoporin Nup1 [45] and with karyopherin b [46], we investigated whether the C terminus of Nup1 and RanGAP are involved in the disassembly of the RanGTP-karyopherin b complex
Summary
Protein Expression and Purification—Yeast Ran and RanGAP were expressed and purified as described [1]. Solution Binding Assay—For each experiment, an E. coli lysate containing GST-C-Nup was incubated for 20 min at 4 °C with glutathione-agarose beads (Sigma) (2 mg of C-Nup per 10 ml of beads) in 0.5 ml of binding buffer (20 mM Hepes, pH 6.8, 150 mM KOAc, 2 mM Mg(OAc) mM dithiothreitol, 0.1% Tween 20). 15 nM Ran-[g32P]GTP and 25 nM karyopherin b were incubated for 10 min at 21 °C, and RanGAP, karyopherin a, C-Nup, and RanBP1 were added as indicated in the figure legends. The exchange reaction was quenched by adding 20 mM MgCl2, and unbound nucleotide was removed on a Nap spin column (Pharmacia) This resulted in Ran containing ;39% GMPPCP, ;60% GDP, and less than 1% GTP, determined as described [1]. A RanGMP-PCP surface was generated as described for Ran
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